Proceeding Book

Proceeding Book

Proceeding book of the 49th Pokjanas TOI International Seminar

Proceeding Book: The 49th Pokjanas TOI International Seminar ISBN: 978-602-72418-2-4 Published : 2016

Advisory Team Rector of Pancasila University Prof. Dr. rer. nat. Wahono Sumaryono, Apt. Dean of Pharmacy Faculty Pancasila University Prof. Dr. Shirly Kumala, M.Biomed., Apt.

Editor Chief Yesi Desmiaty, S.Si., M.Si., Apt.

Editorial Board Member Prof. Dr. rer. nat. Wahono Sumaryono, Apt. Prof. Dr. Shirly Kumala, M.Biomed., Apt. Prof (ris). Swasono R. Tamat, M.Sc., Ph.D., Apt. Prof (ris). Dr. Partomuan Simanjutak, M.Sc., APU Prof. Dr. Syamsudin, M.Biomed., Apt.

Redactional Board Member Sesilia Andriani Keban, MSi., Apt. Mita Restinia, M.Farm., Apt Retno Ayu Pratiwi, S.Si.

Publisher Faculty of Pharmacy, Pancasila University Srengseng Sawah, Jagakarsa, Jakarta 12640 Phone/ Fax (021)7864727-28/ 23

PREFACE The 49th Pokjanas TOI International seminar has been scheduled organized by the Faculty of Pharmacy University Pancasila in collaboration with Pokjanas TOI Organization at Jakarta, Indonesia, 21-22 October 2015. Which is aimed to share information, findings and collaboration between researches, pharmacists, institution and natural product industries. Finally we were able to publish the proceedings and it is now ready for circulation among the researchers, industries, and scientists. This proceeding is consisted of 43 titles manuscripts which were presented as oral and poster in seminar. The topic of manuscript contain many fields including natural product chemistry, analytical technique in phytochemistry, biological activity, pharmacological study, herbal drugs and formulation.. The Organizing Committe gratefully acknowledges the Rector of University Pancasila, Pokjanas TOI Organization, as well us all sponsors in bringing forth this seminar. Furthermore, personally, I would like to express my deep apreciation to the members of the Organizing Committee, for the good teamwork and their great effort to bring success to the seminar.

Jakarta, November 2015 Chairman of Committee Dr. Ratna Djamil M.Si., Apt

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DAFTAR ISI Halaman Citotoxicity and Radical Scavenging Activity Test of Gambir (Uncaria gambir (HUNTER) ROXB.) In Vitro Sri Ningsih, Churiyah, Fahri Fahrudin, Rini Damayanti, Eriawan Rismana …………………...

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The Biological Activity of Eurycomanone Derivatives On T47d, MCF-7, HELA, and WIDR Cancer Cells Hanifah Yusuf, Darma Satria, Zulkarnain ……………………………………………….......... Antibacterial Activities of Dayak Paser Medicinal Plants Against Escherichia Coli Septina Asih Widuri, Noorcahyati ………………………………………………………...........

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Isolation of Anticancer Active Compound From Trigonella Foenumgraecum Leading by MCF7 Cytotoxicity Kurnia Agustin, Sriningsih, Julham Effendi ……………………………………………………

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Issues of Halal Standardization of Food, Drug and Cosmetic for the Implementation the Mandatory of Halal Certification According to Halal Product Guarantee M. Yanis Musdja ……………………………………………………………………………….. Virtual Screening Compounds in Fabaceae Plants as Ligands on Alfa Estrogen Receptor (ER-α) Esti Mumpuni, LH Gulo ……………………………………………………………………….... Preparation of Standardized Aqueous Extract of Annona Muricata Linn. Leaf, Its Potency as Antioxidant, and Total Flavonoid Content Assay Yesi Desmiaty, Deni Rahmat, Nilam Sari Maulidina …………………………………….............. Phytochemical Screening and Toxicity Test BSLT of 70 % Ethanol Extract of Gaharu Leaves (Aquilaria beccariana Tiegh.) Ahmad Musir, Wiwi Winarti, Siti Hasnah P. Siregar …………………………………………...... Optimization of Production of Β-Carotene and Astaxanthin from Microalgae Chlorella pyrenoidosa and Its Potential as an Antioxidant Ni Wayan Sri Agustini …………………………………………………………………………... Antioxidant Compound Isolated from Bioproduction of Endophytic Fungi of Turmeric (Curcuma longa L.) Hindra Rahmawati, Partomuan Simanjuntak …………………………………………………....... Analysis of Beta-Carotene In Green Melon and Orange Melon (Cucumis melo L. var. sky rock and var. cantaloupe) by TLC-Densitometry Rifa Rizkiyah, Zuhelmi Aziz …………………………………………………………………....... Spectrophotometric Method Precision to Assay of Lycopene in Tomatoes Fruit (Solanum lycopersicum Lam.) Liliek Nurhidayati, Wening Ariwanty …………………………………………………....………. Optimization and Validation of High Performance Liquid Chromatography for Determination of Coffein In White Tea Zuhelmi Aziz, Dhiah Resti …………………………………………………………………....…... The Effect of Extraction Method on Total Alkaloid Levels of Jembirit Leaves (Tabernaemontana sphaerocarpa BL) with Spectrofotometric Method Nina Salamah, Miftahul Rozak ………………………………………………………………....… Integration of Herbal or Traditional Medicine through Evidence Based Practice Anny Lumban Toruan, Galih Ajeng Kencana Ayu …………………………………….................

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Identification of Soursop Seeds (Annona muricata L.) Extract as a Candidate Against the Aedes aegypti L. Musquito Vector Control DBD Sarah Zaidan, Ratna Djamil, Siti Nuraini ……………………………………………………...... Antimicrobial and Biology Activity from Parasite Soursop (Dendropthoe pentandra L.) Extract Herbs Erlindha G, Lia Kartika Sari …………………………………………………………..………...... Antioxidant, Cytotoxic and Apoptotic Induction Activity of Ethanolic Extract of Andrographis paniculata on MCF-7 Cancer Cell Line Churiyah, Kurnia Agustini, Siska Andrina Kusumastuti ................................................................. In Vitro α-Glucosidase Inhibition Activities Test from Standardized Sambung Nyawa (Gynura procumbens (Lour.) Merr.) Leaves Extract Wiwi Winarti, Ratna Djamil, Sarah Zaidan, Raymond …………………………………….....….. Identification of Sugar-Apple Seeds (Annona squamosa L.) Extract as a Candidate Against the Aedes aegypti L. Musquito Vector Control DBD Ratna Djamil, Sarah Zaidan,Siti Nuraini ……………………………………………………......... Anticancer Activity of Jatropha SP. on Breast Cancer Cells and Cervix Cells Siti Rofida, Nailis Syifa, Nurkhasanah, Laela Hayu Nurani …………………………………........ Antihyperlipidemia Effect of Red Cabbage Juice (Brassica Oleracea VAR CAPITATA L. FORMA RUBRA) in Mice Lestari Rahayu, Yati Sumiyati, Desti Dwi Nandini ……………………………………………..... Hepatoprotective Study of Cosolvent Solution from Mangosteen (Garcinia mangostana L.) Rind in Rats Ros Sumarny, Liliek Nurhayati, Yati Sumiyati, Astri Yuliastri Permana ………………............... Immunomodulatory Activitiy of Lutein Extract from Sweet Corn Seeds (Zea mays L.) Through In Vivo Measurement of Activity and Phagocytic Capacity of Peritoneal Macrophage Cells of Mice Kusmiati, Yudha Prasetya, Erlindha Gangga …………………………………………………....... Effect of Ethanolic Extract of Bawang Tiwai (Eleutherine bulbosa (Mill.) Urb.) in Monosodium Urate-Induced Inflammation in Rat Hind Paw Dian R. Laksmitawati, Siti R.Rani ……………………………………………………................... Acute Toxicity of Ethanolic Extract of Fenugreek Seeds (Trigonella foenum-graecum L.) on White Rats Kurnia Agustini, Sriningsih, Julham Effendi ................................................................................... Antihypertensive and Diuretic Effects of the Ethanol Extract of Colocasia esculenta (L.) Schott. Leaves Rini Prastiwi, Siska, Ervina Bhakti Utami, Gigih Pangestu Witji ……………………………....... The Antioxidant Activity of Ethanol Extract of White-Oyster Mushroom in Decrease MDA and Increase the Activity of Catalase in Mice Hypercholesterolemia Vera Ladeska, Priyanto,Juju Jumiati …………………………………………………………........ Soursop Leaf (Annona muricata L.) Infusion in Lipid Profile of Hyperlipidemic Mice Ni Made Dwi Sandhiutami, Neni Anggraini ……………………………………………….…....... Xanthine Oxidase Inhibitory Activity of Secang (Caesalpiniasappan L.), Tempuyung (Sonchusarvensis L.), and Kepel (Stelechocarpusburahol) Extracts Pertamawati, Mutia Hardhiyuna, Shelvi Listiana, Rian Triana ………………………………....... Potency of Curcuma Mangga Val Rhizome Extract as a Selective Anti-Proliferative Agent on Breast Cancer Cell Line MCF-7 Siska Andrina, Churiyah, Nuralih ……………………………………………………………….... Assessment of Antibacterial Activity of Herbal Toothpastes to the Bacteria Causing Halitosis Syarmalina, Syahdu A. Ekowati dan Dwi A. Maulana ………………………………………......

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Formulation and the Antioxidant Activity of Green Cincau Leaves (Cyclea barbata L.MIERS) from the Ethanol Extract 70% Yunahara Farida, Erlindha Gangga, Kartiningsih, Arsila …………………………….................... Accelerated Stability Test and Antioxidant Activity of Ethanol Extract Green Cincau Leaves (Cyclea barbata L.Miers) with Gelling Agent HPCM AND HPMC Kartiningsih, Erlindha Gangga, Yunahara Farida, Maria Ulfah …….....………………….……… Capsule Formulation of Standardized 70% Ethanol Extract Johar Leaves (Senna siamea (Lam.) Irwin and Barneby) as α-Glucosidase Inhibitor Risma Marisi Tambunan, Kartiningsih, Everly Hendra ………………..................………….…… Formulation and Evaluation of Herbal Tablets Containing Voacanga foetida (Bl.) K.Schum Extract Fahleni, Yandi Syukri, Novelta Femmy Rischa, Adriani Susanty ………………….................….. Optimization of Patchouli Oil and Tea Tree Oil Emulgel Formulation Yuslia Noviani, Teti Indrawati, Shelly Taurhesia …………………………………….................... Formulation of Liquorice Extract (Glycyrrhiza glabra L) as Skin Whitening Cream Siti Umrah Noor, Faridah, Michico …………………………………………….………................ Accelerated Stability Test of Liquorice Extract (Glycyrrhiza glabra L) Cream Faridah, Siti Umrah Noor, Sulih Probo Sindi ………………………………….................………. The Variation of Tofu’s Wastewater Concentrations as Culture Medium to the Protein, Lipid and Chlorophyl Contents from Microalgae Nannochloropsis sp. Rian Nurul Hidayat, Sudjaswadi Wiryowidagdo, Ni Wayan Sri Agustini ……….……................. Utilization of Corn Husk Waste to Produce Cellulase Enzymes by Trichoderma viride FNCC 6013 Mira Andam Dewi, Ririn Puspadewi, Sylvia Heryanti ……………………………….................. Clinical Trials Efficacy Of Hyperglycemia Herbs Formula Agus Triyono, Zuraida Zulkarnain ……………………………………………….……................. Antihiperkolesterolemia Jamu Formula Effect on Plasma Cholesterol Levels in Patients with Mild Hypercholesterolemiam in Rumah Riset Jamu 'Hortus Medicus' Tawangmangu Zuraida Zulkarnain, Agus Triyono ……………………………………………….……................

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ISSUES OF HALAL STANDARDIZATION OF FOOD, DRUG AND COSMETIC FOR THE IMPLEMENTATION THE MANDATORY OF HALAL CERTIFICATION ACCORDING TO HALAL PRODUCT GUARANTEE NUMBER 33 YEAR 2014 Dr. Muhammad Yanis Musdja, M.Sc Email : [email protected] Former of Head of Study Program of Pharmacy, Islamic State University, Jakarta Former Dean of School of Pharmacy Muhammadiyah, Tangerang Chairman of Indonesian Halal Products Foundation ABSTRACT INTRODUCTION: Indonesian parliament has ratified law of the Halal Products Guarantee (LHPG) number 33 year 2014 (Undang-undang Jaminan Produk Halal No. 33 Tahun 2014) on September 25, 2014 ago. The basic principles on LHPG No. 33 Year 2014 is the change of halal certificate from voluntary becomes mandatory for food, drug and cosmetic, which will begin in 2017 and will be implemented gradually in Indonesia. For the implementation of halal certification mandatory, the Indonesian government does not yet have halal standardization, especially for drugs and cosmetics, and already there are some standardization of halal for food made by Majelis Ulama Indonesia (MUI). OBJECTIVES: How to prepare Halal Standardization for the implementation of LHPG No. 33 Year 2014, so that mandatory halal certification for food, drug and cosmetic can be implemented in Indonesia. ANALYSIS ISSUES AND CHALLENGES: Halal standardization must exist in order to implement of LHPG No. 33 Year 2014. To be able to make halal Standardization, necessary Muslim pharmacists and other experts that so much know halal Standardization. Based on the SWOT theory, it can be said briefly about: STRENGTHS; Islamic concept for consuming Halalanthoyyiban food is the best concept not only for Muslims but for all mankind. WEAKNESS: Until now, Indonesia does not have halal standardization of food, drug and cosmetic. OPPORTUNITIES: Make halal standardization is a good opportunity to make the food, drug and cosmetic that qualified in accordance with halalanthoyyiban concept in Islam. For make halal standardization can do verification food ingredients that exist on the Codex Alimentarius, Pharmacopoeia and standards books for food, drugs and cosmetics. THREATHENS: On Article 56 and 57 of this LHPG, there are severe legal sanctions for Industry Player and everyone that involved in the implementation of process Halal Products Guarantee shall be punished with imprisonment for a maximum of 5 (five) years or a fine of Rp 2,000,000,000.00, (two billion rupiah), if there is no halal standardization, the article No. 56 and 57 of LHPG No. 33, year 2014 can not be executed. CONCLUSION: Halal standardization should be made as soon as possible, if it is not exist. LHPG No. 33, year 2014 can not be implemented. The easiest way to create a standardization of halal with a faster time, low cost and easily understood and accepted by the international community is with do clarification about halal, mubaah, makruh or haram of material content in the book Codex Alimentarius for food and the book Martindale & Indonesia Pharmacopoeia for drugs and cosmetic. Key Words: Halal food standardization; Halal drugs standardization; Halal cosmetics standardization; LHPG No. 33 year 2014; Muslim pharmacists law experts.

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INTRODUCTION As it is said in the Quran on surah Al Baqarah verse 168, “O mankind, eat from whatever is on earth that is lawful and good (Halalan thayyiban) and do not follow the footsteps of Satan. Indeed, he is to you a clear enemy”. Verse in the Qur'an mentioned above, emphasize, that consuming of halal food and good (Halalanthoyyiban) is an obligation not just for Islamic people but for all mankind. As has been shown, according to evidence based medicine, that due to consuming of Haram foods, such as, alcohol, pork, beast animal, blood, disgusting animal and animals were slaughtered in a way not true, all of this haram food will have an adverse effect to the people who had consumed it. On the other hand, in Quran, Surah Al A’raaf Verse 31, “O Children of Adam, take your adornment at every place of worship, and eat, and drink, but do not overdoses. Certainly, He (Allah) loves not the extravagant” The meaning of eat and drink do not overdoses in this verse is Standardization. Therefore, eat food, drugs and cosmetics are not standard is dangerous and may disturb of health. Because of standardization of food, drugs and cosmetics are mandatory in Islam, then this provision is set in law of the Halal Products Guarantee (LHPG) number 33 year 2014 (Undang-undang Jaminan Produk Halal No. 33 Tahun 2014) On the other hand at Article 56 of LHPG No. 33 Year 2014: Industry Player that does not keep halal products have gained the Halal Certificate referred to in Article 25 letter b shall be punished with imprisonment for a maximum of 5 (five) years or a fine of Rp 2,000,000,000.00 (two billion rupiah ). At article 57 of LHPG No. 33 Year 2014: Everyone involved in the implementation of process Halal Products Guarantee that is not maintain the confidentiality of formulas that contained in information is submitted by industry player as referred to in Article 43 shall be punished with imprisonment for a maximum 2 (two) years or a maximum fine Rp. 2,000,000,000.00 (two billion Rupiah). Due to the fairly heavy sanctions to industry players and Everyone involved in the implementation of process Halal Products Guarantee, then, there will be many disputes in the implementation of mandatory Halal in the future. To be able to resolve the dispute with justice certainly needed Halal Standardization for food, drugs and cosmetics. If standardization of halal for food, drugs and cosmetics can not be determined, the implementation of the LHPG number 33 year 2014 will fail. On one side, standards are essential tools for local and international businesses, which shape the contribution of economic progress through industry development and trade, as well as, a guideline in the assurance of consumer protection. On the other hand, standards are able to be eliminators of trade barriers, which means that, they play a critical role to facilitate goods and services exchange across borders. Orriss and Whitehead (2000), On the other hand, Halal logistics is a new phenomenon, driven by the halal industry to extend halal from source to the point of consumer purchase, to ensure the integrity of the halal product for the endconsumer and export markets. The large discussion group shows that the conventional logistics handling of halal products does not provide sufficient assurance for the Muslim consumer in both Muslim and non-Muslim countries. Tieman, M. (2011).


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METHODS Make a compilations or attachment to the standard book for food, drugs and cosmetics by doing the clarification of these materials are halal, mubaah, makruh or haram with make the selection of a few books, to obtain book which more appropriate for clarification. Clarification of these materials carried out by Majelis Ulama Indonesia (MUI) in cooperation with experts of Pharmacy and other experts who are familiar with standardization of halal. RESULTS Results of the main choice of books to make a compilation or an appendix on halal standards are: 1. Codex Alimentarius new edition for food 2. Martindale new edition and Indonesian Pharmacopoeia new edition for drugs and cosmetics DISCUSSION Make books for the standardization of halal food, drugs and cosmetics are a very difficult job, need a long time and need very expensive cost. If we take a lesson from the history of the making of books Codex Alimentarius, the present, it takes about 52 years to become a book as it is now. The making of this book are result of synergy of several countries. Which is regulated by the Food Administraion Organization (FAO) The Codex Alimentarius (Latin for "Book of Food") is a collection of internationally recognized standards, codes of practice, guidelines and other recommendations relating to foods, food production and food safety. The Codex Alimentarius covers all foods, whether processed, semi-processed or raw. In addition to standards for specific foods, the Codex Alimentarius contains general standards covering matters such as food labeling, food hygiene, food additives and pesticide residues, and procedures for assessing the safety of foods derived from modern biotechnology. It also contains guidelines for the management of official i.e. governmental import and export inspection and certification systems for foods. Advantages the using of result clarification (halal, mubaah, makruh and haram) ingredients of Codex Alimentarius book as Halal Standart for food:  Indonesia can make a book that integration Halal Standart with Thayyiban Standart become Halalanthayyiban Standart  Readily accepted by every country and the international community, because the Codex Alimentarius is the main book used as a standard book for food by countries that are members of FAO and WHO.  Provide substantial economic benefits, because Indonesia does not need to spend a huge cost to conduct research by spending big to establish halal Standart  Indonesia only needs to perform the clarification of ingredients which foods are halal, mubaah, makruh and haram in the Codex Alimentarius and add it as a compilations or attachment in the book.of codex alimentarius.  Halal standart that exist on compilations or attachment on the codex alimentarius will easily be used as a manuals halal standards by any countries in the world, therefore the book codex alimentarius is the worldwide for food standard thayyiban


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Advantages the using of result clarification (halal, mubaah, makruh and haram) ingredients of Martindale book as Halal Standart for drugs and cosmetics, because of Martindale book contains :  Monographs on drugs and ancillary substances, listing over 6,000 monographs arranged in 49 chapters based on clinical use with the corresponding disease treatment reviews. Monographs summarize the nomenclature, properties, and actions of each substance. A chapter on supplementary drugs and other substances covers some 1190 monographs on new drugs, those not easily classified, herbals, and drugs no longer clinically used but still of interest. Monographs of some toxic substances are also included.  Preparations - including over 180,000 items from 43 countries and regions, including China.  Directory of Manufacturers listing some 20,000 entries.  Pharmaceutical Terms in Various Languages: this index lists nearly 5,600 pharmaceutical terms and routes of administration in 13 major European languages as an aid to the non-native speaker in interpreting packaging, product information, or prescriptions written in another language.  General index: prepared from 175,000 entries it includes approved names, synonyms and chemical names; a separate Cyrillic section lists nonproprietary and proprietary names in Russian and Ukrainian. Advantages the using of result clarification (halal, mubaah, makruh and haram) ingredients of Indonesia Pharmacopoeia as Halal Standart for drugs and cosmetics, because of Indonesia Pharmacopoeia book :  Indonesia Pharmacopoeia is book standard used by the Indonesian people for drugs and cosmetics.  Indonesia Pharmacopoeia using Indonesian language. therefore it is easily understood by the people of Indonesia. On the other hand, at this time. There is one institution that made under Organization for Islamic Cooperation (OIC). The name of institution is Standardization and Metrology of Industry for Islamic Country (SMIIC). Headquarters of SMIIC is located in Istanbul turkey. Because of this new institution was founded in 2011 ago. then they also have not been able to make halal standardization manual that can be used as a standard for food, drugs and cosmetics. Then, Indonesia has not been able to use the halal standard from the SMIIC. To be able to do the work above, should be started immediately. Therefore clarification halal, mubaah, makruh and haram for thousands of food drugs and cosmetics is not easy. Therefore, there are some ingredients of food, drugs and cosmetics that are difficult to set their status.

5. CONCLUSION 1. To be successful implementation of law of the Halal Products Guarantee (LHPG) number 33 year 2014, where its implementation will begin in early 2017. Then, Halal standardization should be made as soon as possible, if it is not exist. LHPG Number 33, year 2014 can not be implemented. 2. The easiest way to create a standardization of halal with a faster time, low cost and easily understood and accepted by the international community is with do clarification about halal, mubaah, makruh or haram of material content in the book Codex Alimentarius for food and the book Martindale & Indonesia Pharmacopoeia for drugs and cosmetic. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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REFERENCES 1.

2. 3. 4.

5. 6. 7. 8. 9.

Yanis Muhammad, Musdja, Majalah SWARA FBN, Edisi 1, Juni – Juli 2015, ISSN: 24431982,Bahaya Mengkonsumsi Makanan Haram Ditinjau dari Aspek Ilmiah Medis (Harmful of Consuming Food Based on Evidence Based Medicine) Law of the Halal Products Guarantee (LHPG) number 33 year 2014 (Undang-undang Jaminan Produk Halal No. 33 Tahun 2014). Al Quran, Terjemahan Kementerian Agama RI 2003. Orriss, D. G. and J.A. Whitehead, 2000. Hazard Analysis and Critical Control Point (HACCP) as a Part of an Overall Quality Assurance System in InternationalFood Trade. Food Control, 11: 345351 Tieman, M. (2011). The application of Halal in supply chain management: in -depth interviews. Journal of Islamic Marketing, 2(2), 186 – 195. CODEX Alimentarius: Understanding Codex". FAO and WHO. 1999. Retrieved 6 September 2012 Martindale : The Complete Drug Reference Thirty-eighth edition. Farmakope Indonesia edisi V, Tahun 2014. OIC/SMIIC 2:2011, “Guidelines for Bodies Providing Halal Certification” (with the references of ISO/IEC 17020, ISO/IEC 17021, ISO/IEC 17025, ISO/TS 22003 + Islamic Fiqh Rules),


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THE BIOLOGICAL ACTIVITY OF EURYCOMANONE DERIVATIVES ON T47D, MCF-7, HELA, AND WIDR CANCER CELLS 1

Hanifah Yusuf, 2Darma Satria, 3Zulkarnain

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Departement of Pharmacology and Therapeutic Faculty of Medicine University of Syiah Kuala, Tanoh Abee Street, Darussalam, Banda Aceh, Indonesia 2 Departement of Pathology Anatomi Faculty of Medicine University of Syiah Kuala, Tanoh Abee Street, Darussalam, Banda Aceh, Indonesia 3 Departement of Physiology Faculty of Medicine University of Syiah Kuala, Tanoh Abee Street, Darussalam, Banda Aceh, Indonesia Email: [email protected]

ABSTRACT Eurycomanone from the roots of Eurycoma longifolia Jack, has been reported to exhibit anticancer activity. Four of ester eurycomanone derivatives: eurycomanone dibutyrate, eurycomanone monovalerate, eurycomanone dimethoxybenzoate and eurycomanone disuccinate were synthesized for knowing their potencies on cancer cell lines T47D, MCF-7, Hela, WIDR and normal cells (Vero cells). The activity of eurycomanone derivatives as anticancer were evaluated by MTT colorimetric assay method. The results showed that eurycomanone has anticancer activity on T47D, MCF-7, Hela, WIDR cancer cells with IC50 values (1.17 ± 0.09; 3.96 ± 0.02; 2.95 ± 0.08;1.45 ± 0.01 µg/mL), respectively and no toxic to Vero cells (IC50 609.89± 29.77 µg/mL). Eurycomanone derivatives namely: eurycomanone dibutyrate have anticancer activity on T47D, MCF-7, Hela, WIDR cancer cells with IC50 values (25.16± 2.25; 21.56 ± 4.55; 29.32 ± 1.25; 149.42 ± 12.50 µg/mL), eurycomanone monovalerate (25.59 ± 1.31; 22.48 ± 1.25; 30.14 ± 1.89; 91.88 ± 8.90 µg/mL), eurycomanone dimethoxybenzoate ( 102.77 ± 2.56; 38.83 ± 2.55; 66.65 ± 1.90; 51.61 ± 2.37 µg/mL), eurycomanone disuccinate (218.94 ± 9.30; 198.87± 5.50; 166.67 ± 12.34; 145.39 ± 6.67 µg/mL)respectively. Eurycomanone dibutyrate, eurycomanone monovalerate and eurycomanone dimethoxybenzoate are safe to Vero cells with selectivity index (IS) more than 3, besides that eurycomanone disuccinate toxic to Vero cells. Keywords: Anticancer, Eurycomanone, Eurycoma longifolia Jack, Synthesis

INTRODUCTION Natural compounds from plants have been proven as a source of lead compounds for developing of new drugs (1,2). Eurycomanone is a natural pentacyclic quassinoid obtained from the roots of Eurycoma longifolia Jack (3) in the family Simaroubaceae. Eurycoma longifolia Jack (ElJ) or commercially known as Tongkat Ali in Malaysia, Pasak Bumi in Indonesia, Tung Saw in Thailand and Cay Ba Binh in Vietnam (4) is very popular plant as aphrodisiac and usually taken after 4 years of cultivation for preparing pharmaceutical products(5). Various natural compounds have been isolated and characterized from ElJ, mostly from the roots. Natural compounds from ElJ have been reported to have a wide pharmacological activities such as antimalarial (6,7,8), anticancer (6,7,8,9,10), antipyretic (10) and anti ulcer (11). Eurycomanone is one of the major natural quassinoids isolated from the roots of ElJ and has exhibited cytotoxic activities against selected cancer cell lines (12,13). Its pharmacological potency has been proven in numerous in vitro and in vivo experimental laboratory. But until now very limited efforts to develope this compound for obtaining its derivatives. Therefore this investigation was conducted to examine the anticancer activity of synthesized eurycomanone derivatives on selected cancer cells line. In this study, design and synthesis of eurycomanone derivatives were done by using eurycomanone natural compound and esterified without using protecting agent by using pharmacophore agents such as butirylchloride, valeroylchloride, para methoxy benzoyl chloride and succinate anhydride. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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METHODS Materials: The plant and roots of E. longifolia Jack were taken after 4 years of cultivation and identified by a specialist. Pharmacophore agents such as butirylchloride, valeroylchloride, para methoxy benzoyl chloride and succinate anhydride (p.a, Merck). Chloroform, ethyl acetate, methanol, pyridine, all chemical substances and selective cancer cell lines for study of anticancer activity Extraction: The roots (10kg) of E. longifolia were cleaned with tap water and then dried in the oven at 400C. After cutting in small pieces, the dried roots were ground into crude powder and stored in the desiccators. Then the crude powder was soaked in 30L methanol at room temperature and stirred regularly. The liquid extract was filtered and concentrated in rotary evaporator at 400C to produce methanolic extract. Isolation of eurycomanone Before isolation the methanolic extract of ElJ was subjected to vacum liquid chromatography by using stationary phase silica gel and the mixed mobile phase chloroform: methanol: water in ratio (5:5:1; 3:7:1: 1:9:1). Fractions with similar Rf values on thin layer chromatography (TLC) which were monitored at UV lamp at 254 nm, then pooled and used for isolation of eurycomanone as starting material for synthesizing its derivatives. Isolation of eurycomanone was done by preparative thin layer chromatography (PTLC) using silica gel PF254 as stationary phase and the mixed mobile phase ethylacetate: methanol : water in ratio 80: 20: 1. Repeated isolation, purification and crystallization were done to get the pure compound. The structure and its purity were confirmed by comparison with detailed spectroscopic data in published reports (UV, IR, NMR, LCMS-ESI positive ion, DEPT, COSY, HMQC and HMBC). Synthesis of eurycomanone derivatives Synthesis of eurycomanone derivatives can be performed by simple esterification without using protecting agent. Eurycomanone which is isolated from the E. longifolia roots (50 mg, 0,1225 mmol) was dissolved in cold pyridine (1 mL) and pharmacophore agent (butiryl chloride, valeroyl chloride, para methoxy benzoyl chloride and succinate anhydride 0,49 mmol, respectively) dissolved in cold chloroform. The solution of pharmacophore agent was added slowly to the eurycomanone solution at 0 0C and the reaction mixture was stirred for 1 hour in ice bath. After that, the reaction mixture was refluxed and stirred using magnetic heat stirrer for 6-8 hour and every 2 hours checked the product by TLC. After esterification process ended, the mixture was extracted three times with 10 ml of cold ethyl acetate. The ethyl acetate layer is washed three times with 10 mL cold water and then dried with sodium sulfate anhydrate. After filtration and drying, the cooled precipitate is poured in methanol and preparing for detailed spectroscopic analysis. Testing of Anticancer activity The anticancer activity test of eurycomanone and its derivatives on Vero cells and cancer cell lines (T47D, MCF-7, Hela, WIDR) were carried out by MTT Colorimetric Assay Methods (Mosman, 1983). The tested compounds were used at concentration 25; 12,5; 6,25; 3,125; 1,57625, 0,78125 µg/mL and prepared from the substock solutions by serial dilution of media to give a volume of 100 µL in each microtitre plate well. The concentration of tested compounds were prepared in triplicate. As standard drug used doxorubicine and 5 fluorouracil in same concentration. Then each well was added with 100 µL of 104/ mL of cells in complete growth media, respectively. As controls were used the cells and media that were placed into 96 well microplate then incubated for 24 hours at 370C, 5% CO2 and 90% humidity. After incubation, the media was removed and 100 µL of new medium and 10 µL MTT was added. Then, it was incubated again for 4 hours and next the media was aspirated and 100 µL SDS 10% in 0,001N HCl added. The microplate was re-incubated for 24 hours in room temperature and its absorbance was read at λ 405 nm (Vero cells) and 595 nm (cancer cell lines) by ELISA reader. The IC 50 value on Vero cells and cancer cell lines were determined by probit or linear regression analysis. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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RESULTS The result of eurycomanone isolation Investigating of new anticancer compound was originated from pasak bumi roots (E. longifolia, Jack.), we macerated the powdered of pasak bumi roots with methanol and after evaporation the liquid extract in vacum condition gave ± 6% solid extract. Fractionation were done to the extract by vacum liqiud chromatography (VLC) for obtaining the concentrated eurycomanone and yielded ± 2,5%. Isolation of eurycomanone from this fraction was performed by preparative thin layer chromatography (TLC) and yielded ± 0,04%. The result of eurycomanone derivatives synthesis Eurycomanone is the potential quasinoid anticancer was structurally esterified by using acyl chloride and carboxyclic anhydride to influence their activity and cytotoxicity to cancer cell lines. Synthesis of its derivatives by esterification is attempted with the aim of increasing activity, decreasing toxicity, or improving other pharmacological profiles. In finding new anticancer with better activity than previous compound, it was esterificated OH group in eurycomanone structure by butiryl chloride, valeryl chloride, para-methoxybenzoyl chloride and succinic anhydride. The result of esterification gave, eurycomanone dibutyrate (60,35%), eurycomanone monovalerate (55,10%), eurycomanone dimethoxybenzoate (60,10%) and eurycomanone disuccinate (65,25%). The result of identifying eurycomanone and its derivatives by spectroscopic analysis The chemical structure of all tested compounds had been analyzed by spectroscopic analysis. Eurycomanone as starting material has formula C20H24O9 (MW 408.02; m.p 2540-2570C) and its derivatives eurycomanone dibutyrate C28H36O11 (MW 548,94; m.p 241-2430C), eurycomanone monovalerate C25H32O10 (MW492,8; m.p 235-2370C), eurycomanone dimethoxybenzoate C36H36O13 (MW 676,13; m.p 225-2280C) and eurycomanone disuccinate C28H30O15 (MW 606,86, m.p 251-2540C). The result of anticancer activity test The evaluation of tested compounds on Vero cell is aimed for knowing the safety of these compounds on normal cells. In addition, these compounds are also used for examining their potencies on growth inhibition of cancer cell lines. The test was performed by MTT colorimetric assay method which was modified (14,15). The IC50 and selectivity index values of these compounds on cancer cell lines and Vero cells are showed in Table 1 and 2. DISCUSSION In vitro screening of anticancer activity of eurycomanone and its derivatives is based on the ability of the compounds to inhibit the growth of cancerous cel lines in medium culture. Several derivatives of eurycomanone: eurycomanone dibutyrate, eurycomanone monovalerate, eurycomanone dimethoxybenzoate and eurycomanone disuccinate were synthesized. Previous studies showed the anticancer activity of eurycomanone on various cancer cell lines has IC50 value on MCF-7 is 4.40 ± 0.42 µg/mL (16); 3.63 ± 0.11 µg/mL(17); and less than 2.5 µg/mL (9); 1,1µg/mL (7). The anticancer activity of eurycomanone on Hela cells has IC50 value 2.13 ± 0.09 µg/mL (17). The result of the test toward four semisynthesized compounds showed that eurycomanone more potent than monoacetylated and diacetylated eurycomanone to selected cancer cell lines above. Some structural requirements, like an α, β-unsaturated ketone in the A ring, an oxymethylene bridge in the C ring and an ester function in C-15 in the D ring are considered very important for the anticancer activity and antimalarial activity presented by quassinoids (18,19).


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Table 1. The IC50 of eurycomanone and its derivatives on selected cancer cell lines and Vero cells Tested compounds IC 50 Values (µg/mL) Vero WIDR Eurycomanone 1.45 ± 0.01 E. dibutyrate 149.42 ± 12.50 E. monovalerate 91.88 ± 8.90 E dimethoxy benzoate 2.37 E. disuccinate 145.39 ± 6.67 Doxorubicine 41.81 ± 2.25 5-Fluouracil 5.41 ± 1.33

T47D

609.89 ± 29.77 219.29 ± 11.38 92.40 ± 132.22± 6.98

7.51

1.17 ± 0.09 25.16 ± 2.25 25.5 ± 1.31

102.77± 2.56

MCF-7

Hela

3.96 ± 0.02

2.95 ± 0.08

21.56 ± 4.55

29.32 ± 1.25

22.48 ± 1.25

38.83 ± 2.55

30.14 ± 1.89

66.65 ± 1.90

12.75 ± 2.88

218.94 ± 9.30

3.54 ± 0.64

1.97 ± 0.05

4.6 8 ± 0.10

3.51

4.03 ± 0.23

3.16 ± 0.11

1.99 ± 0.0 1

280.54 ± 10.11

198.87 ± 5.50

51.61 ±

Table 2. The selectivity index of eurycomanone and its derivetives on selected cancer cell lines Tested compounds Selectivity Indexs T47D MCF-7 Hela WIDR Eurycomanone 521.27 154.00 206.54 420.20 E. dibutyrate 8.72 10.17 7.4 8 1.47 E. monovalerate 3.61 3.41 3.07 1.00 E dimethoxy benzoate 128.70 340.61 198.44 E. disuccinate 0.05 0.06 0.08 0.09 Doxorubicine 1.80 0.76 1.01 0.08 5-Fluouracil 69.61 88.78 140.97 52.86

166.67 ± 12.3 4 ± 0.05

256.27

CONCLUSION The data suggest that eurycomanone has anticancer activity more potential than its derivatives on selected cancer cell lines (T47D, MCF-7, Hela and WIDR) and safe to normal Vero cells. Eurycomanone dibutyrate, eurycomanone monovalerate and eurycomanone dimethoxybenzoate are safe to Vero cells with selectivity index (IS) more than 3, besides that eurycomanone disuccinate toxic to Vero cells. ACKNOWLEDGMENTS This study was supported by Ministry of Education and Culture of Indonesia. The authors would like to thank University of Syiah Kuala for this grant. The authors are grateful to Indonesian Institute of Sciences (LIPI) for analyzing our synthesis compounds (Mrs.Sofa Fajriah for NMR analysis and Mrs. Puspa Dewi for helping in LC-MS analysis).


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REFERENCES 1.

Newman, D.J., Cragg, G.M., Snader, K.M. 2003. Natural products as sources of new drugs over the period 1981–2002. J. Nat Prod. 66(7):1022-1037. 2. Kinghorn, A.D., Farnsworth, N.R., Soejarto, D.D., Cordell, G.A., Pezzuto, J.M., Udeani, G.O., Wani, M.C., Wall. M.E., Navarro, H.A., Kramer, H.A., Menendez, A.T., Fairchild, C.R., Lane, K.E., Forenza, S., Vyas, D.M., Lam, K.S., Shu, Y.Z. 1999. Novel strategies for the discovery of plant-derived anticancer agents. Pure Appl Chem, 71:1611-1618.) 3. Darise, M., Kohda, H., Mizutani, K., Tanaka, O. 1982. Eurycomanone and eurycomanol, quassinoids from the roots of Eurycoma longifolia. Phytochemistry. 21:20912093 4. Kuo, P.C., Shi, L.S., Damu, A.G., Su, C.R., Huang, C.H., Ke, C.H. 2003. Cytotoxic and antimalarial betacarboline alkaloids from the roots of Eurycoma longifolia. J Nat Prod. 66:13241327. 5. Chan, K.L., Lee, S.P.,Yuen, K.H. 1995. Antipyretic activity of quassinoids from Eurycoma longifolia Jack. Planta Medica : 219 . 6. Chan, K.L., O’Neill, M.J., Phillipson, J.D., and Warhurst, D.C. 1986. Plants as Source of Antimalarial Drugs. Part 3. Eurycoma longifolia. Planta Medica 52(2): 105 – 107. 7. Kardono, L.B.S., Angerhofer, C.K., Tsauri, S., Padmawinata, K., Pezzuto, J.M., and Kinghorn, A.D., 1991. Cytotoxic and Antimalarial Constituents of The Roots of Eurycoma longifolia. Journal of Natural Product. 54: 1360-1367 8. Jiwajinda, S., Santisopasri, V., Murakami, A., Kawanaka, M., Kawanaka, H., Gasquet, M. 2002. In Vitro Anti-tumor promoting and Anti-parasitic Activities of the Quassinoids from Eurycoma longifolia, a Medicinal Plant in Southeast Asia. J. Ethnopharmacol. 82: 55–58. 9. Kuo, P.C., Damu, A.G., Lee, K.K., and Wu, T.S. 2004. Cytotoxic and Antimalarial Constituent from The Roots of Eurycoma longifolia. Journal of Bioorganic Medicinal Chemistry. 12: 537 -544. 10. Tee, T.T., Cheah, Y.H., Hawariah, L.P. 2007. F16, a fraction from Eurycoma longifolia Jack extract, induces apoptosis via a caspase9independent manner in MCF7 cells. Anticancer Res. 27:34253430. 11. Tada, H., Yasuda, F., Otani, K., Dotenchi, M., Ishihara, Y., and Shiro, W. 1991. New Antiulcer Quassinoids from Eurycoma longifolia. European Journal of Medicinal Chemistry. 26: 345 – 349. 12. Wong, P., Cheong, W., Shu, M., Teh, C., Chan, K. and Bakar, S. A. 2012. Eurycomanone Suppresses Expression of Lung Cancer Cell Tumor Markers, Prohibitin, Annexin 1 and Endoplasmic Reticulum Protein 28. Phytomedicine. 19: 138–144 13. Zakaria Y, Rahmat A, Pihie AH, Abdullah NR, Houghton PJ (2009) Eurycomanone induce apoptosis in HepG2 cells via upregulation of p53. Cancer Cell Int 9:16 14. Mosman, T. 1983. Rapid Colorimetric Assay for Celluler Growth and Survival Application to Proliferation and Cytotoxicity Assays. Journal of Immunology Method. 65: 55 – 63. 15. Tada, H., Shiho, O., Kuroshima, K., Koyama, M., and Tsukamoto. 1986. An Improved Colorometric Assay for Interleukin-2. Journal of Immunology Method. 93 (2): 157-165. 16. Tee, T.T., and Hawariah, L.P. 2005. Induces of Apoptosiss by Eurycoma longifolia Jack Extract. Anticancer Res. 25 : 2205 -2214. 17. Nurhasanah, M., Hawariah L.P. 2007. Eurycomanone Induces Apoptosis Through The Up Regulation of p53 in Human Cervical Carcinoma Cells. Paper Research International Coference on Chemical Sciences (ICCS-2007), Yogyakarta, Indonesia. 18. Kupchan, S.M., Britton, R.W., Lacadie, J.A., Ziegler, M.F., and Siegel, C.W.J. 1975. The Isolation and Structural Elucidation of Bruceantin and Bruceantinol, New Potent Antileukemic Quassinoids from Bruceae antidysenterica. Journal Organic Chemistry 40: 648 – 654. 19. Kupchan, S.M., Fessler, D.C., Eakin, M.A., and Giacobbe, T.J. 1970. Reaction of Alpha Methylen Lactone Tumor Promoter Inhibitors with Model Biological Nucleophiles. Science 168, 376 – 377.


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ANTIBACTERIAL ACTIVITIES OF DAYAK PASER MEDICINAL PLANTS AGAINST Escherichia coli Septina Asih Widuri* and Noorcahyati* * Research Institute for Natural Resources Conservation and Technology, Ministry of Environment and Forestry, Republic of Indonesia Jl. Soekarno Hatta Km 38 Samboja PO Box 578 Balikpapan 76112, East Kalimantan, Indonesia [email protected] ABSTRACT Increasing resistance of infectious microorganisms to antibiotics leads to a challenge to develop new and more effective antibacterial agents. Traditional medicine is a potential source of antibacterial agents derived from screening ethnomedicinal plants. This study examined the antibacterial activities of five Dayak Paser medicinal plants from Paser, East Kalimantan namely Spatholobus ferrugineus, Melicope glabra, Ardisia serrata, Gonocaryum calleryanum, and Neonauclea gigantea against Escherichia coli by a disc diffusion method. All the ethanol extracts of these plants exhibited antibacterial performance at concentrations of 5000 ppm and 10000 ppm. The diameter of inhibition zone ranged between 7.17-10.65 mm at a concentration of 5000 ppm and 8.00-15.05 mm at a concentration of 10000 ppm. Melicope glabra showed the highest activity at both of concentration. Keywords: Antibacterial, Spatholobus ferrugineus, Melicope glabra, Ardisia serrata, Gonocaryum calleryanum, Neonauclea gigantea, Escherichia coli INTRODUCTION Infectious diseases caused by pathogenic bacteria have been considered as a major cause of morbidity and mortality in humans not only in Indonesia but also worldwide (1). Consequently, a number of new antibiotics have been produced but resistance of infectious microorganisms to antibiotics has also increased(2). This circumstance eventually leads to a challenge to develop new and more effective antibacterial agents. Natural products have been used in traditional medicine all over the world for thousands of years. Plants have been investigated as sources of many bioactive compounds. Tropical rain forests of Kalimantan have a vast diversity of bioactive potential plants. Tribes living around the forests have depended on forests for their needs especially for food and medicine. The local tribes’ knowledge about traditional medicine is a potential source of antibacterial agents derived from screening the plants. Dayak Paser is one of local tribe in East Kalimantan. Traditionally, they use plants from forest around them to treat some diseases such as diarrhea, dysentery, cold, toothache, wound, even stamina booster, high blood pressure and diabetic (3). The objective of this study was to examine antibacterial activities from selected Dayak Paser medicinal plants used for treatment of diseases that mostly caused by microorganisms. The phytochemicals and antibacterial properties from the plants which were focused in this study have not been widely reported. METHODS Plants material and extraction The plants material was collected from Petangis village, Batu Engau district, Paser, East Kalimantan in September 2014. The information about the plants and their traditional use were obtained by interviewing traditional healer and people at Petangis village. Spatholobus ferrugineus, Melicope glabra, Gonocaryum calleryanum and Neonauclea gigantea were selected in this study due to their properties in Dayak Paser 11 International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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traditional medicine to treat bacterial infection symptoms while Ardisia serrata was subjected to represent traditional medicine to treat non bacterial infection symptoms. They were further identified at the Herbarium of Research Institute for Natural Resources Conservation and Technology at Samboja, East Kalimantan. Roots, stem, stem barks, and leaves (Table 1) were dried at room temperature (27 oC) and sent to LPPM Biofarmaka Laboratory, Bogor for further analysis. The dried samples were extracted using maceration with 70% ethanol. The concentration of extracts used in all assays was 5000 ppm and 10000 ppm. Table 1. Summary of plants, part of plants and medicinal properties based on Dayak Paser traditional medicine. Species Family Local name Traditional usea Part of plantsb Spatholobus ferrugineus Benth. Leguminosae Rayak akar mouth ulcer roots Melicope glabra (Blume) T.G. Rutaceae Kotep stomachache leaves Hartley Ardisia serrata (Cov.) Pers. Primulaceae Tetung ngrengat arthritis stem bulu Gonocaryum calleryanum Stemonuraceae Kembayan wound leaves (Baill.) Becc. bintang Neonauclea gigantea (Valeton) Rubiaceae Memberatan wound Stem bark Merr. a based on interview with traditional healers at Petangis village b Part of plant used in antibacterial activity determinations

Antibacterial activity determinations Extracts were screened against Escherichia coli (Biofarmaka IPB collection). It was maintained on Liquid Broth (LB) slant cultures and kept at 37oC. The turbidity of liquid culture for use in the assay was adjusted to 1x108 CFU/mL (4). The antibacterial activity of plant extracts was measured using a standard disc diffusion assay(5). 100µL of liquid E coli culture was spread onto plates using a sterile technique and 10 µL of each concentration of extract was pipetted onto a 6 mm sterile filter paper disc. Ampicillin 10000 ppm served as a positive control and DMSO served as a negative control. Each extract was tested on two replicate plates. The plates were incubated at 37oC for 24 hours before zone of inhibition calculated. RESULTS All the ethanol extracts of the selected plants exhibited antibacterial performance at concentrations of 5000 ppm and 10000 ppm. The zone of inhibition ranged between 7.17-10.65 mm at a concentration of 5000 ppm and 8.00-15.05 mm at a concentration of 10000 ppm (Figure 1).


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Figure 1. Mean diameter of inhibition zones at concentrations of 10000 ppm and 5000 ppm

DISCUSSION The results of present investigation indicated that the ethanol extract of the plants inhibited the growth of E coli. Antibacterial activities vary with the species of plants (Figure 1). These differences could be due to the difference in the chemical composition of these plant secondary metabolites that affected antibacterial activity(6). The increase in concentration of the extracts increased the diameter of inhibition zones due to the higher concentration of the extracts contained the higher secondary metabolites compounds(7). Ethanol extract of M. glabra leaves showed the highest diameter of inhibition zone in this study (15.05 mm and 10.65 mm) as seen at Figure 1. Dayak Paser tribe has used M. glabra leaves to treat stomachache. The leaves of M. glabra were heated above the flame then bandaged over the stomach. Not only used by Dayak Paser, M. glabra has also been used by Dayak Banuaq tribe at Kutai Barat as medicinal plant but for different diseases such as influenza(8). Regardless of how the treatment was performed by Dayak Paser tribe, scientifically, M. glabra indicate efficacy as medicine. An experiment study (9) reported that the barks of M. glabra have a high phenolic content which were also thought to contribute to antibacterial activity. This report(9) also summarized that various Melicope species revealed the presence of alkaloid, flavonoids and terpenoids and some of these compounds have antibacterial activities. However, phytochemical and antibacterial activities from M. glabra leaves specifically have not been reported. The roots of S. ferrugineus were used by Dayak Paser tribe to treat mouth ulcer by drinking its boiled water. Mouth ulcer usually related to microorganisms contamination on mouth. The ethanol extracts of S. ferrugineus roots delivered 9.11 mm and 8.07 mm diameter of inhibition zone (Figure 1). The barks extract of S. ferrugineus contained alkaloid, flavonoid, and terpenes(10). These chemical compounds played a role in antibacterial activities(9). The roots were expected contain the chemical compound as the barks, thus the phytochemical screening of S. ferrugineus roots need to be done. Dayak Paser tribe has used the boiled water of G. calleryanum leaves to treat a wound inside the body and utilized the stem barks of N. gigantea to heal a wound on skin. These plants exhibited antibacterial activities (Figure 1). In fact, G. calleryanum possessed flavonoids(11) meanwhile chemical compounds of N. gigantea have not been reported yet . In accordance with their indications, G. calleryanum and N. gigantea were assumed to have tannins which were reported to have antibacterial 13 International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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activities against E. coli (12). Tannins also could accelerate the wound healing through several cellular mechanism(13). A. serrata was used by Dayak Paser tribe to cure arthritis. They boil the stem and drink the water. Although it was non-bacterial symptom, A. serrata exhibited antibacterial activities against E coli. This plant was expected to have bioactive compounds that have a role in either antiinflammatory, analgesic or antibacterial activities. Therefore, it becomes important to investigate the phytochemical and bioactivities of this plant since there are no reports about it. The result obtained from this study showed that medicinal plants used by Dayak Paser to treat bacterial and non bacterial infection symptoms exhibited antibacterial properties. This in vitro antibacterial determination was the first step towards to development of new antibacterial agents from ethnomedicinal plants. Although these selected plants produced less activity than positive control against E coli, however they were still potential for further investigation. Phythochemical screening, identification for the specific bioactive compounds and antibacterial activities determination of these plants against various pathogen bacteria are needed. CONCLUSION Ethanol extracts of S. ferrugineus, M. glabra, G. calleryanum, N. gigantea and A. serrata exhibited antibacterial performance against E. coli with zone of inhibition ranged between 7.17-10.65 mm at a concentration of 5000 ppm and 8.00-15.05 mm at a concentration of 10000 ppm. M. glabra showed the highest activity. They are potential as source of antibacterial agents. ACKNOWLEDGMENTS This work was part of the research program of Research Institute for Natural Resources Conservation and Technology, Ministry of Environment and Forestry, Republic of Indonesia. REFERENCES 1. 2.

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Nathan C. Antibiotics at the crossroads. Nature 2004, 431:899-902. Adwan G, Mhanna M. Synergistic effects of plant extracts and antibiotics on Staphylococcus aureus strains isolated from clinical specimens. Middle East J Sci Res [Internet]. 2008 [cited 2015 Sep 28]; 3:134-139. Available from: www.idosi.org/mejsr/mejsr3(3)/5.pdf. Noorcahyati, Widuri SA, Sitepu BS, Mediawati I, Arifin Z, Wibisono Y. Kajian etnobotani dan uji fitokimia jenis bahan baku obat kurang dikenal. Laporan Hasil Penelitian. Balai Penelitian Teknologi Konservasi Sumberdaya Alam. Tidak diterbitkan. 2014. Frey FM, Meyers R. Antibacterial activity of traditional medicinal plants used by Haudenosaunee peoples of New York State. BMC Complement Altern Med [Internet]. 2010 Nov [cited 2015 Jan 25]; 10(64):1-10. Available from: http://www.biomedcentral.com/1472-6882/14/122. Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Amer J Clin Pathol [Internet]. 1966 Aug [cited 2014 Des 12]; 45:493-496. Available from: garfield.library.upenn.edu/classics1985/A1985ANC2900001.pdf. Noumedem J, Mihasan M, Lacmata S, Stefan M, Kuiate J, Kuete V: Antibacterial activities of the methanol extracts of ten Cameroonian vegetables against Gram-negative multidrug resistant bacteria. BMC Complement Altern Med [Internet]. 2013 Jan [cited 2015 Sep 28];13(26):[about 5pp]. Available from: http://www.biomedcentral.com/1472-6882/13/26. Setha B, Laga A, Mahendradatta M, Firdaus. Antibacterial activity of leaves extracts of Jatropha curcas, Linn against Enterobacter aerogenes. International Journal of Scientific & Technology Research [Internet]. 2014 Jan [cited 2015 Oct 1];3(1):129-131. Available from: http://www.ijstr.org/final-print/jan2014/Antibacterial-Activity-Of-Leaves-Extracts-Of-JatrophaCurcas-Linn-Against-Enterobacter-Aerogenes.pdf.


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Falah F, Sayektiningsih T, Noorcahyati. Keanekaragaaman jenis dan pemanfaatan tumbuhan berkhasiat obat oleh masyarakat sekitar Hutan Lindung Gunung Beratus, Kalimantan Timur. Jurnal Penelitian Hutan dan Konservasi Alam. 2013;10(1): 1-18. Kassim NK, Rahmani M, Ismail A, Sukari MA, Ee GC, Nasir NM, Awang K. Antioxidant activity-guided separation of coumarins and lignan from Melicope glabra (Rutaceae). Food Chem [Internet]. 2013 Aug [cited 2015 Sep 28];139(1-4):87-92. Available from: www.ncbi.nlm.nih.gov/pubmed/23561082. Marliana E. Analisis senyawa metabolit sekunder dari batang Sptaholobus ferrugineus (Zoll&Moritzi) Benth yang berfungsi sebagai antioksidan. Jurnal Penelitian MIPA [Internet]. 2007 Dec [cited 2015 Sep 27];1(1):23-29. Available from: http://repository.usu.ac.id/bitstream/123456789/21203/1/kpm-des2007-1%20(2).pdf. Kaneko T, Sakamoto M, Ohtani K, Ito A, Kasai, R, Yamasaki K. Secoiridoid and flavonoid glycosides from Gonocaryum calleryanum. Phytochemistry [Internet]. 1995 May [cited 2015 Oct 6];39(1):115-120. Available from: http://www.researchgate.net/publication/222585377_Secoiridoid_and_flavonoid_glycosides_fro m_Gonocaryum_calleryanum Oboh G. Antioxidant and antimicrobial properties of ethanolic extract of Ocimum gratissimum leaves. J Pharmacol Toxicol [Internet]. 2010 [cited 2015 Sep 18]; 5(7):396-402. Available from: www.doi:10.3923/jpt.2006.47-53. Sheikh AA, Sayyed Z, Siddiqui AR, Pratapwar, Sheakh SS. Wound healing activity of Sesbania grandiflora Linn flower ethanolic extract using excision and incision wound model in wistar rats. International Journal of PharmTech Research [Internet]. 2011 April-June [cited 2015 Oct 6];3(2):895-898. Available from: sphinxsai.com/vol3.no2/pharm/pharmpdf/PT=43(895898)AJ11.pdf.


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CITOTOXICITY AND RADICAL SCAVENGING ACTIVITY TEST OF GAMBIR (Uncaria gambir (HUNTER) ROXB.) IN VITRO Sri Ningsih1*, Churiyah1, Fahri Fahrudin1, Rini Damayanti2, Eriawan Rismana3 1 Center of Phamaceutical and Medical Technology – Agency for the Assessment and Application of Technology - LAPTIAB 610-611 Bld. Kawasan Puspiptek Serpong , Tangerang Selatan, Banten, Indonesia 2 Indonesian Research Center for Veterinary Science - Jl. RE Martadinata 30 Bogor, West Java, Indonesia 3 BPPT *Corresponding author : [email protected] ABSTRACT Gambir (Uncariagambir(Hunter) Roxb.) is a native Indonesian medical shrub and used as traditional medicine for treating various diseases because of its highly polyphenol content. The aim of this studies were to evaluate the toxic effect of some gambir extracts on normal cell line in vitro and to determine their antioxidant activity. The samples were consist of 5 kinds of extracts, namely, gambir, ethanolic 50%extract of gambir, ethanolic 96% extract of gambir, ethanolic 50% extract of gambir leaves and ethanolic 96% extract of gambir leaves. Cytotoxicity assay was conducted on Vero normal cell line by 4,5-dimethylthiazol-2yl (MTT) assay, and showed that the Inhibitory Concentration 50(IC50) values of the 3samples of gambir extract were more than 1000 ppm resulted in the absent of proliferative effect, as well as both gambir leaves extracts were less than 1000 ppm. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities of all samples measured at 4 ppm final concentration had range of 29.5%49.7% with standard Vitamin C value of 53.1%. The activities decreased in the following order: ethanolic 96% extract of gambir>ethanolic 96% extract of gambir leaves>gambir >ethanolic 50% extract of gambir>ethanolic 50% extract of gambir leaves, respectively. These results showed that all gambir extract were categorized as non-cytotoxic with highly antioxidant activities. Keywords: Gambir (Uncariagambir(Hunter) Roxb.), cytotoxicity assay, radical scavenging, DPPH, antioxidant. INTRODUCTION Gambir or Uncaria gambir (Hunter) Roxb. belonging to Rubiaceae family, is a native plant especially found in Sumatera inland and Malaysia peninsula (Hussin MH. et al, 2011). Indonesia produce very high gambir that fulfill almost 80% of the worldwide need (Dhalimi A., 2006). The polyphenol compound of gambir is sufficiently high with flavonoid (+)-catechin content as the major compound. (+)Catechin content is almost 40-80% of dried water extract depending on the preparation process (Hayani E. et al, 2003). According to the natural compounds, gambir demonstrated some pharmacological benefits such as antiflatulence, antibacterial, skin tanning, remedies for diarrhea and sore throat and pesticides properties (Kassim MJ. et al., 2011).These polyphenol also exhibit antioxidant activity in some in vitro test (Widiyarti G. et al., 2011; Amir M., 2012, Anggraini T. et al., 2011). According to The National Agency of Drugs and Food Control (NA-DFC) regulation, the development of new medicines, including traditional medicines, cosmetics, and products complement as well as food and hazardous materials, beside of the efficacy assessment, it is also necessary to perform a set of toxicity evaluation (Anonim, 2014). Cell culture technique is frequently used as the first tool for estimating toxic effect of new material (Anussavice KJ., 2003).This methods is conducted in normal cells such as fibroblast (Graidist P. et al., 2015) and Vero(Pour BM, et al., 2011)cells. Toxicity levels is related to the cell viability as exposure to material tested in which the number of living cells is measured with MTT colorimetric (Graidist P. dkk., 2015). The studies were conducted to demonstrate the viability of Vero cells and the radical scavenging activity after exposing of some gambir extracts.


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METHODS Preparation of gambir extracts Fresh gambir simplisia (leaves and twigs) were collected from Limapuluh Kota – West Sumatera Province on February 2013. The shrubs was identified in Bogoriense Herbarium Research Center for Biology Indonesian Institute of Science (LIPI) Bogor, before being processed. The samples tested were gambir, ethanolic 50% extract of gambir, ethanolic 96% extract of gambir, ethanolic 50% extract of gambir leaves and ethanolic 96% extract of gambir leaves. Gambir was prepared based on Farmakope Herbal Indonesia with modification. Briefly, 1 kg of fresh gambir was steam for 60minutes and then pressed until gambir gum collected. Gum was separated from water with decantation for 10-12 hours followed by drying the obtained semisolid sediment in oven at temperature of 45-500C for overnight.Then, it was powdered by electric blender. Both ethanolic extracts of gambir were prepared with the agitating maceration technique at room temperature for 16-18 hours using ethanolic 96% and ethanolic 50%, respectively. Each collected filtrate was then separately evaporated under vacuum at 450C to get dried mass. The similar method was applied to obtain both extracts of gambir leaves in which the final extracts were semisolid mass. 4,5-dimethylthiazol-2yl (MTT) assay Cytotoxicity effect of gambir extract was estimated through the MTT assay. It is a colorimetric assay for assessing cell metabolic activities, conducted based on protocol test developed by Laboratory of Center of Phamaceutical and Medical Technology – Agency for the Assessment and Application of Technology. Vero cells was maintained in RPMI-1640 medium supplemented with 10% FBV, 1% penicillin-streptomicin, and 0.2% NaHCO3. The cells were cultured at 300C in humidified 5% CO2 incubator. Vero cells were diluted with Roswell Park Memorial Institute (RPMI) medium to 5x105 cells/mL and aliquots (5x104 cells/0,1mL) were placed in individual wells in 96-well micro plate. After attaching into the wells with incubating at CO2 5%, 370C for overnight, cells were treated with a diluted 2-fold series of concentration of each sample ranged from 62.5 to 1000 ppm in final solution. Samples were dissolved in medium with maximal dimethyl sulphoxide (DMSO) 0.1% each well. Next, the cells were incubated at the above conditions for 24 h and then their viability was determined by MTT color. The MTT solution (0.5 mg/mL in medium, 100 uL each well) was added to each well and incubated for 4 h. The 10% Sodium dodecyl sulphate (SDS) solution in 0.1 N HCl was added to each well to dissolve the formed formazan crystals, followed by incubating of the plate for 24 hours at room temperature for completing dissolution process. The absorbance was read at 570 nm on a microplate reader after shaking at 120 rpm for 15 minutes. Blank and control absorbance were prepared similar to samples treatment by using reagent only and reagent plus cells without samples, respectively. The tests were performed in triplicate. Percent viability was calculated with this equation

% viability = [ a – b ] x 100% [c–b] a: sample absorbance, b: blank absorbance, c: control absorbance 1,1-diphenyl-2picrylhydrazyl (DPPH) radical scavenging assay The antioxidant activity that is measured from free radical scavenging activity of the extracts was measured by using the stable DPPH free radical based on previous paperwith modifications (Hanani E. et al., 2005). Briefly, into 5 mL glass tube, it put in 3000 uL samples solution, 150 uL DPPH solution, consecutively. The concentration of sample solution was prepared by dissolving each extract with DMSO and methanol to get 4 ppm of final solution and DPPH solution was 0,004% in methanol as well. The absorbance was measured at 517 nm after being incubated at room temperature for 30 minutes. Ascorbic International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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acid was used as standard. The control absorbance was prepared from similar to the above reaction without sample, and blank absorbance was used methanol. The experiments were done in triplicates. The activity of radical scavenging was calculated with this equation.

% radical scavenging activity = [ a – b ] x 100% [c–b] a: sample/standart absorbance, b: blank absorbance, c: control absorbance Data Analysis Microsoft excel program was applied to calculate IC50 value of percent proliferation from proliferative percentage figure (concentration caused 50% Vero cells proliferate). T-test for two independent samples by SPSS 14.00 software was to analyze intergroup difference of free radical scavenging activity each extract against to standard vitamin C. RESULTS Cytotoxicity test The toxicity tests of all samples were conducted on kidney pig normal cell line, Vero cells, followed by MTT assay was depicted at Figure 1. The cytotoxicity level stated as IC50 value of percent viability was performed at Table 1. The IC50 value was indicated as concentration of tested sample that caused 50% of cell population survived.

Figure 1. Percent viability of Vero cells after extract treatment Table 1. The IC50 value of percent viability Vero cells after treated with gambir extracts Samples Gambir

IC50 (ppm) > 1000

Ethanolic 96% extract of gambir leaves

692

Ethanolic 50% extract of gambir leaves

716

Ethanolic 96% extract of gambir

> 1000

Ethanolic 50% extract of gambir

> 1000

1,1-diphenyl-2picrylhydrazyl (DPPH) radical scavenging activity The antioxidant activity of all samples was tested using DPPH free radical scavenging method. The level of antioxidant activity of each sample was compared to standard vitamin C, which the results were as shown in Table 2. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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Table 2. Percentage free radical scavenging activity of samples Radical p value Compared to scavenging Vit C Samples activity Gambir 40,2% 0,040* 76% Ethanolic 96% extract of gambir leaves 43,1% 0,018* 81% Ethanolic 50% extract of gambir leaves 29,5% 0,001* 56% Ethanolic 96% extract of gambir 49,7% 0,264 93% Ethanolic 50% extract of gambir 37,1% 0,003* 70% VIT C 53,1% -100% * p < 0,05 versus Vitamin C. Experiment was conducted at concentration 4 ppm of final solution

DISCUSSIONS Cytoxicity test To get the complete solution, preparation extracts were by added DMSO before diluting with RPMI media. Dimethyl sulfoxide (DMSO) is a polar aprotic solvent that dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvent and water. Therefore, DMSO is claimed as an excellent and selective solvent for many organic compound contained in some type of plant derived extracts (Martin HD. et al., 1967). It can give beneficial effect when used at optimal concentration. In culture cell experiments, it may not influence cell mortality when used at the concentration less than 2%. However, at high concentration, DMSO demonstrated cytotoxic effect. Studies by Anguilar JS. et al., 2002 claimed that incubation of Vero cells with 5% DMSO for 24 hours caused in mortality of about 20% by sulforhodamine B assay. In this research, for eliminating false negative due to toxic effect, DMSO was used at 1% of final solution in each well. Figure 1 showed the percentage of Vero cells viability after being treated with each gambir extract. The extract showed dose-dependent inhibition of cell viability in Vero cells. Among the extract, ethanolic 96% extract of gambir leaves caused cell proliferation when treated at low concentration. This was also occurred with gambir which had slight proliferative effect. However, both ethanolic extracts of gambir (ethanol96% and ethanol50%) did not initiate cells proliferation. Hence, extraction with ethanol could separate compounds that induced proliferation selectively. Furthermore, each extract at high concentration showed different effect toward cell viability. Both extract that prepared from gambir leaves (ethanolic96% and ethanolic50% extracts) demonstrated cytotoxic effect, but there were not demonstrated by both extracts of gambir. These results were also confirmed by the IC50 value of percent viability as illustrated at Table 1. However, based on earlier study, all of the samples were categorized as non-toxic category. It stated that non-toxic category when the IC50 value of some extract against to normal cells was more than 80 ppm (Graidist P. et al., 2015). Hence, the viability effect of the whole extract toward Vero cells could be attributed to the presence of a variety of compound. 1,1-diphenyl-2picrylhydrazyl (DPPH) radical scavenging activity The results of antioxidant evaluation using DPPH method demonstrated that extract ethanolic 96% of gambir exhibited the highest activity, 49.7% at concentration 4ppm as stated at Table 2. This antioxidant activity did not differ compared to standard Vitamin C significantly. Antioxidant activity was caused by donating hydrogen atom to free radical stable, DPPH, to form a stable DPPH-H molecule (Kassim MJ. et al., 2011) in which was characterized by reduced color intensity from purple into yellow. These results were in line with other previous studies in which gambir performed antioxidant activity (Widiyarti G. et al., 2011, Amir M., 2012, Anggraini T. et al., 2011). The activity of the radical DPPH scavenging activities varied among studies depend on the sources of gambir, sample preparation andthe analyzing method(Ningsih S. et al., 2014). Antioxidant activity of gambir was mainly caused by polyphenol compounds (Kassim MJ. et al., 2011). It had been reported that there were some antioxidant mechanisms of polyphenol compounds such as donating hydrogen atom and single electron toward radical compound, complexion with metal ion that played as oxidative inducer (Sugihara N. et al., 2001). International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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It was concluded that all of gambir extracts prepared in the study demonstrated radical scavenging activity and non-toxic properties to normal Vero cells, therefore this advantages can be thoroughly studied more deeply as potential traditional herbs. ACKNOWLEDGMENT Authors would like to deeply thank toward Insentif Sinas Ristek 2014 Program for providing this research funding. REFERENCES 1.

2.

3.

4.

5. 6. 7. 8.

9. 10.

11. 12.

13. 14.

Aguilar JS, Roy D, Ghazal P, Wagner EK. Dimethyl sulfoxide blocks herpes simplex virus-1 productiveinfection in vitro acting at different stages with positivecooperativity. Application of micro-array analysis. BMC Infectious Diseases2002;2(9):1-10. Amir M,Mujeeb M, Khan A, Ashraf K, Sharma D, Aqil M, Phytochemical analysis andin vitroantioxidant activity ofUncaria gambir. International Journal of Green Pharmacy 2012;6(1):6772. Anggraini T, Tai A, Yoshino T, Itani T. Antioxidative activity and catechin content of four kinds of Uncaria gambir extracts from West Sumatra Indonesia. African Journal of Biochemistry Research 2011;5(1):33-8. Anonim, Peraturan Kepala Badan Pengawas Obat Dan Makanan Republik Indonesia Nomor 7 Tahun 2014 tentang Pedoman Uji Toksisitas Nonklinik secara in vivo, Kementrerian Kesehatan Republik Indonesia 2014, Jakarta. Anussavice KJ. Phillips' science of dental materials. 11st ed. Elsevier Science (USA) Saunders; 2003:172–94. Dhalimi A. Permasalahan gambir (Uncaria gambir L.) di Sumatera Barat dan alternatif pemecahannya. Perspektif 2006;5(1):46-59. Graidist P, Martla M, Sukpondma Y. Cytotoxic activity of Piper cubebaextract in breast cancer cell lines. Nutrients 2015;7:2707-18. Hanani E, Mun’im A dan Sekarini R. Identifikasi senyawa antioksidan dalam spons Callyspongia sp dari kepulauan seribu. Majalah Ilmu Kefarmasian. 2005;2(3):127 – 133Sugihara N, Ohnishi M, Imamura M, Furuno K. Differences in Antioxidative Efficiency ofCatechins in Various MetalInduced Lipid Peroxidations in Cultured Hepatocytes. Journal of HealthScience 2001;47(2):99-106. Hussin MH, Kassim MJ. The corrosion inhibition and adsorption behavior ofUncaria gambir extract on mild steel in 1 M HCl. Materials Chemistry and Physics 2011;125(3):461–8. Kassim MJ, Hussin MH, Achmad A, Dahon NH, Suan TK, Hamdan HS. Determination of total phenol, condensed tannin and flavonoid contents and antioxidant activity of Uncaria gambir extracts. Majalah Farmasi Indonesia 2011;22(1):50–9. Martin HD, Weise A, Niclas HJ. The solvent dimethyl sulfoxide. Angewande Chemie 1967;6(4):318-334 (abstract). Ningsih S, Fachrudin F, Rismana E, Purwaningsih EH,Sumaryono W, Jusman SWA. Evaluation of antilipid peroxidationactivity of gambir extract on liver homogenat in vitro. International Journal of PharmTech Research 2014;6(3):982-9. Pour BM, Latha LY, Sasidharan S. Cytotoxicity and oral acute toxicity studies of Lantana camaraleaf extract. Molecules 2011;16:3663-74. Widiyarti G, Sundowo A, Hanafi M. The free radical scavenging and anti-hyperglycemic activities of various gambiers available in Indonesian market. Makara Sains 2011;15(2):129-34.


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Acute Toxicity of Ethanolic Extract of Fenugreek Seeds (Trigonella foenum-graecum L.) on White Rats KURNIA AGUSTINI*, SRININGSIH, JULHAM EFFENDI Center for Pharmaceutical and Medical Technology, Agency for the Asessment and Application of Technology, BPPT, Jakarta. correspondence author: [email protected] Abstract: Fenugreek seed or biji klabet (Trigonella foenum-graecum L.) was known having activity to handle some of degenerative diseases such as diabetes mellitus, hypercholesterolemia and also postmenopausal symptoms. This study was conducted to investigate the safety of ethanolic extract of biji klabet on white rat, especially to count the value of Lethal Concentration 50 (LC50). This in-vivo assay referred to WHO protocols for toxicity assay of natural medicines. We used Spraque dawley white rats, female and male, 6 weeks age, which divided into one group normal and five treatment groups (1g/kgBW, 4g/kgBW, 8g/kgBW, 12g/kgBW, 16g/kgBW). Sample was given once orally then animal were monitored for two weeks. Observation of toxic effect e.g physical symptom of central nerve system, autonom nerve system and digestive system. All lethality animal were observed and LC50 were counted. Result showed that there was no toxic effect and no lethal animal until 16g/kgBW dose. We can conclude that ethanolic extract of Fenugreek is practically non toxic. Keywords : Fenugreek seeds, Trigonella foenum-graecum L., acute toxicity.

INTRODUCTION Fenugreek seed or Foenigraeci semen is dried seed from Trigonella foenum-graecum L., Leguminosae(1) (WHO, 2007). In Indonesia, it calls Biji Klabet. Empirically, biji klabet was used for hemorrhoids, asthma, ulcers, muscle pain and often used as a preventative hair loss and skin softener. Many studies showed its activity as antidiabetic, anticancer and for hypercholesterolemia handling(2) (Mills, 2000). Biji Klabet has antiandrogen activities, due to its active compounds as beta-sitosterol, palmitic-acid and stearic-acid, and also has the ability to decrease of total cholesterol, LDL, VLDL cholesterol and triglycerides significantly. The anti-hyperglycemic and anti-inflammatory properties investigated in fenugreek are additional benefit. Agustini’s study (2007) showed that ethanolic extract of Biji Klabet have estrogenic effect on ovariectomized and immature female Wistar rats(3). Biji Klabet contains some sapogenin steroid ingredients, e.g. diosgenin, precursor for sexual hormone(4) (Evans, 2002), its isomer Yamogenin, gitogenin, tigogenin, and trigoneoside(5) (Dewick, 1997). Biji Klabet contains diosgenin in free base form 0.8 – 2.2 %(6) (Wiryowidagdo, 2000). Biji Klabet also contains fatty oil 20-30%, alkaloids (trigonelline, an alkaloid pyridine, gentianin and karpain), flavonoids e.g. vitexin in glycoside or ester form, isovitexin, orientin, vicenin, quercetin and luteolin, essential oil, saponine, nicotinamide, choline, bitter compound and mucilage(4). This study was carried out to investigate safety effect of ethanolic extract of Fenugreek in white rat. Acute toxicity assay also known as short term toxicity assay. Sample are given once in various grade of doses and observation are carried out for two weeks. All physical symptoms and lethal animal were analysed then compare to normal group. This acute toxicity study was meant to count the value of Lethal


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Dose 50% (LD50) from sample. LD50 is a dose that can cause 50% lethality of animal test. Sample can categorized as safe material when they have LD50 value bigger than 15g/kgBW(7) (Lu, 1995).

MATERIAL AND METHODS Sampel preparation. Biji klabet were obtained from Tawangmangu, Central Java, Indonesia. Seed were dried and grind, then were extracted with ethanol 96% food grade. Crude extracts were suspense with Carboxy Methyl Celulose (CMC) 0.5%. Animal preparation. Experimental animals used in this study were 30 males and 30 females Spraque Dawley (SD) white rats, 5-6 weeks age, obtained from Indonesian Food and Drug Administration (FDA/BPOM). The animal were kept in the animal room (25 + 20C) under 12 h light/dark cycle and fed with standard diet of pellet rat diet and free access to distilled water prior to the start of the study. Animal were kept for acclimatization for one week. Animal were maintained and handled according to ethical committee which approved the design of the animal experiment. Ethical clearance was approve by Ethical Committee of Indonesian Agency for Health Research and Development (Badan Penelitian dan Pengembangan Kesehatan/Balitbangkes). Animal treatment. Animals were divided into one group normal and five treatment groups (1g/kgBW, 4g/kgBW, 8g/kgBW, 12g/kgBW, 16g/kgBW), each 5 males and 5 females. Sample was given once orally then animal were monitored for two weeks. Observation of toxic effects were done, e.g. physical symptom of central nerve system, autonomy nerve system and digestive system. Body weights were weighing at day 1, 6,9,12,14. After two weeks all animal were autopsied. Table 1. Group of animal treatment. Treatment

No.

Groups

N

1.

N

Normal Diet + CMC Na 0,5% suspense

5 Male + 5 Female

2.

D1

Normal Diet + Sample 1 g/kgBW

5 Male + 5 Female

3.

D2


5 Male + 5 Female

4.

D3


5 Male + 5 Female

5.

D4

Normal Diet + Sample 12g/kgBW

5 Male + 5 Female

6.

D5


5 Male + 5 Female

RESULT AND DISCUSSION All animal treated with dose 1 (1g/kgBW) until dose 5 (16g/kgBW) showed no toxic effect significantly. Normally, after orally given sample treatment, all animal showed decrease of motoric activity for some minutes. But after 30 minutes, all activity back to normal. This spontaneous effect is normal after orally gavage treatment. The results of observation of physical symptom of central nerve system, autonom nerve system and digestive system observation, can be seen on Table 2.

Table 2. Physical toxic effect observation. Groups Observation N D1 D2 D3 Central Nerve System 1. Sedasi 2. Motoric Activity +/+/+/+/-


D4

D5

+/-

+/-

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Observation 3. Convulsion 4. Tremor Autonom Nerve System 1. Open eye 2. Salivation 3. Urination Breath Rate Heart Rate Digestive System 1. Diarrhea 2. Constipation 3. Bloody Fesses Stress hair Note: : No symptom + : There are symptom +/- : Normal

N -

D1 -

Groups D2 D3 -

D4 -

D5 -

+/+/+/-

+/+/+/-

+/+/+/-

+/+/+/-

+/+/+/-

+/+/+/-

-

-

-

-

-

-

Body weight analysis from all animal, both male and female, with treatment dose 1g/kgBW until 16g/kgBW gives no significant difference compare to normal control. Both male and female rats in all groups gives increasing of body weight almost similar to normal control for 14 days.

Figure 1. Body weight of male rats from all group for 14 days observation.


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Figure 2. Body weight of female rats from all group for 14 days observation.

There was no lethality case of animal found in all group after 14 days observation. Therefor this assay should be repeat using higher level doses than 16g/kgBW. But technically it’s so difficult to treat orally to the animal. Beside that, regulation from Indonesian FDA says that if until dose 15g/kgBW no lethality case occur, then its not necessary to repeat the assay. The sample can be categorized having LD50 higher than 15g/kgBW or categorized practically non toxic. The data about lethality case can be seen on Table 3.

Groups

n

Table 3. Lethality case of animal from all groups. Male Female Total Lethal Lethal n Lethal

% Lethality

Normal

5

0

5

0

0

0%

D1

5

0

5

0

0

0%

D2 D3 D4

5 5 5

0 0 0

5 5 5

0 0 0

0 0 0

0% 0% 0%

D5

5

0

5

0

0

0%

Table 4. Catagorization of Toxic Effect(8). (Lu,FC, 1996)

Toxic Category Super toxic Very hard toxic Very toxic Medium Toxic Mild Toxic Practically Non Toxic

LD50 Value < 5 mg/kgBB 5-50 mg/kgBB 50-500 mg/kgBB 0,5-5 g/kgBB 5-15 g/kgBB > 15 g/kgBB

CONCLUSION


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No delayed toxic effect and lethality was observed in all rats during fourteen days of recovery period. Orally treatment of Fenugreek within this range and treatment duration would not cause any severe toxic effects and organ damages in rats. In conclusion, ethanolic extract of Fenugreek have pseudo LD50 and categorized as practically non toxic.

REFERENCES 1. Agustini, K, Sumali W., Dadang K. 2007. Estrogenic Effect of Fenugreek (Trigonellafoenumgraecum L.) on White Female Rats. Conference Proceedings "Women's Health and Traditional Medicine", International Medicine and Medicinal Plants, Surabaya. 2. Agustini, Kurnia, Sumali W., Dadang K. 2005. Pengaruh Pemberian Biji Klabet (Trigonella foenum-graecum L.) terhadap Kadar Hormon Estradiol dan FSH Plasma Tikus Putih Betina Galur Wistar yang Diovariektomi. Prosiding Seminar Nasional Penggalian Potensi Sembilan Tanaman Obat Unggulan Indonesia, Purwokerto. 3. Agustini, Kurnia, Sumali W., Dadang K. 2005. Efek Estrogenik Biji Klabet (Trigonella foenumgraecum L.) Terhadap Perkembangan Uterus Tikus Putih Betina. Jurnal Bahan Alam Indonesia, Perhimpunan Peneliti Bahan Obat Alami (PERHIPBA), Vol.4, No.2, Juli 4. Anonim. 2000. Pedoman Pelaksanaan Uji Klinik Obat Tradisional. BPOM Departemen Kesehatan RI, Jakarta: vi + 47p 5. Anonim. 2000. Parameter Standar Umum Ekstrak Tumbuhan Obat. BPOM Departemen Kesehatan RI, Jakarta: viii + 68p 6. Dewick PM. Medicinal Natural Products. A Biosynthetic Approach. New York: John Wiley & Sons; 1997. 7. Evans CW. Pharmacognosy. 15th edition. London: W.B. Saunders; 2002. 8. General guideline for methodologies on research and evaluation of traditional medicine. 2000. Geneva: World Health Organization. 9. Guyton, C.Arthur. 1995. Fisiologi manusia dan mekanisme penyakit. Translate from Human Physiology and mechanism of disease, by Petrus Andrianto. EGC, Jakarta: xii + 821p. 10. Lu F.C., Toksikologi dasar, asas, organ sasaran dan penilaian resiko. [Translate by Edi Nugroho]. Ed. 2, Jakarta:UI Press; 1995. 11. Mills, Simon & K. Bone. 2000. Principles and Practice of Phytoterapy. Modern Herbal Medicine. Churcill Livingstone, Edinburgh: xx + 643p. 12. Wiryowidagdo S. Kimia dan Farmakologi Bahan Alam. Jakarta: Universitas Indonesia. 2001; 318-328


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VIRTUAL SCREENING COMPOUNDS IN FABACEAE PLANTS AS LIGANDS ON ALPHA ESTROGEN RECEPTOR (ER-α) Gulo LH1, Mumpuni E1* 1

Faculty of Pharmacy Pancasila University Jakarta [email protected]; [email protected] ABSTRACT Family Fabaceae has about 730 genera and 19.400 species. Some plant of family Fabaceae are known to have activity as an anti-breast cancer. This study does a series of computational chemistry method in virtual or in silico screening to compounds in the plant of family Fabaceae that are Abrus schimperi, Caesalpinia bonduc, Dalbergia vacciniifolia, Eriosema robustum, Erythrina falcata,Flemingia macrophylla, Genista saharae, Trifolium pratense L., Pachyrhizus erosus, Pissum sativum and dan DNP (Dictionary of Natural Products). The aim of this study is to find candidates of compounds as active ligands on estrogen receptor alpha (ER-α) by in silico and elucidating the amino acids contained in the binding site of compounds by using virtual screening validated Anita et al (2012) protocol. This protocol uses operating system LINUX Ubuntu LTS 14.04 with integrated applications such as SPORES, PLANTS 1.2, BKChem, Open Babel, R Computational Statistics and PyMOL, ZINC 01914469 as comparator compound, 4-[4-hydroxy-3-(prop-2-en-1-yl) fenil]-2-(prop2-en-1-yl) (dimer compound number 11) as reference compound and 4-hidroksitamoksifen as positive control. The results of virtual screening conducted on 60 compounds from ten plant of family Fabaceae obtained 24 compounds be active and 38 inactive compounds in the binding pocket of ER-α. The important amino acids to affinity compounds with estrogen receptor alpha (ER-α) that is GLU353, ARG394, ASP351 and THR347. Key words: Fabaceae, Virtual Screening, Estrogen Receptor Alpha (ER-α). INTRODUCTION In an era new drug design, drug plants were interested as materials of new drug design. One of strategy to developing molecule design a new drug is utilization of computational chemistry methods by virtual screening or in silico screening. Virtual screening can be reduced, cost and time can be more efficient. This study does a series of virtual screening to compounds in the plant of family Fabaceae. Isoflavon was contained in family Fabaceae has known as anti breast cancer. The most of breast cancers are selective on estrogen receptor alpha (ER-α). The important of breast cancer treatment is to inhibit the activity of estrogen on estrogen receptor alpha (ER-α). In this study using virtual screening validated Anita et al (2012) protocol. The protocol used to identify ligands can be active on estrogen receptor. MATERIALS Tools: Hardwere: A computer TOSHIBA with processor Intel(R) Core(TM) i5-4200M CPU @ 2.50GHZ, NVIDIA 2GB, RAM 4GB, HDD 750GB. Software: LINUX Ubuntu operating system (version 14.04), screening virtual applications (SPORES, PLANTS, BKChem, Open Babel, PyMOL), Statistical analysis application R i386 3.1.3.


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Materials: The 2D structure of compounds: Abrus schimperi, Caesalpinia bonduc, Dalbergia vacciniifolia, Eriosema robustum, Erythrina falcata,Flemingia macrophylla, Genista saharae, Trifolium pratense L., Pachyrhizus erosus, Pissum sativum and DNP (Dictionary of Natural Products) The 2D structure of comparison ligand: ZINC 01914469 The 2D structure of reference compound: 4-[4-hidroksi-3-(prop-2-en-1-y1)fenil]-2-(prop-2-en-1-y1) (senyawa dimer no.11) The 2D structure of positive control: 4-hidroksitamoksifen METHODS This study using a protocol of screening virtual validated Anita et al., for the screening virtual in silico compounds in the plant of family Fabaceae that areAbrus schimperi, Caesalpinia bonduc, Dalbergia vacciniifolia, Eriosema robustum, Erythrina falcata, Flemingia macrophylla, Genista saharae, Trifolium pratense L., Pachyrhizus erosus, Pissum sativum and DNP (Dictionary of Natural Products), which is used as a test compounds. BKChem application for drawing the structure of the test compound in the form of 2-dimensional (2D), and then type your converted files by using applications Open Babel. Ligands and receptors were prepared using Spores application. PLANTS application is used to simulate the docking of test compound to ER-α at least 3 times replication. Virtual screening results were analyzed using a statistical test one-tailed paired t-test (to see the match data between pairs of samples tested). PyMOL application used to display the active compounds representative of the test compound in the form of three-dimensional (3D) and the elucidation of amino acids in the binding pocket of ER-α. RESULTS Table 1. Compounds Score and In Silico Activity

Compound Caesalpinia bonduc Asam asetat 5-hidroksi-4,4,7,11b-tetrametil9-okso-1,2,3,4,4a,5,6,7,9,11,11a,11bdodekahidro-6aH-10-oksasiklopenta[b]fenantren-10a-il ester Dalbergia vacciniifolia 7-[6-(3,4-Dihidroksimetil-tetrahidro-furan-2iloksimetil)-3,4,5,-trihidroksi-tetrahidropyran-2-iloksi]-5-hidroksi-3-(5-hidroksi-2,4dimetoksi-fenil) 7-[6-(3,4-Dihidroksimetil-tetrahidro-furan-2iloksimetil)-3,4,5,-trihidroksi-tetrahidropiran-2-iloksi]-6-metoksi-3-(5-hidroksi-2,4dimetoksi-fenill) Eriosema robustum 5,7-Dihidroksi-6-(3-metil-but-2-enil)-2-fenil Erythrina falcata Pokjanas TOI International Seminar Faculty of Pharmacy Pancasila University

Score Chem PLP ± SD

p-value

Ligand activity as estrogen receptor alfa (ER-α) (in silico)

-87,6484 ± 0,3821

0,9989

Active

-99,2499 ± 1,8347

0,9949

Active

-92,4449 ± 0,5732

0,9953

Active

-85,8764 ± 0,6143

0.9991

Active

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5,7-Dihidroksi-2-(4-hidroksi-fenil)-8-(3,4,5trihidroksi-6-hidroksimetil-tetrahidro-piran-2il)-6-(3,4,5-trihidroksi-6-hidroksimetiltetrahidro-piran-2-il) 5,7-Dihidroksi-2-(4-hidroksi-fenil)-8-(3,4,5trihidroksi-6-hidroksimetil-tetrahidro-piran-2il) 5,7-Dihidroksi-2-(4-hidroksi-fenil)-6-(3,4,5trihidroksi-6-hidroksimetil-tetrahidro-piran-2il) 5,7-Dihidroksi-2-(3-hidroksi-4-metoksifenil)-6-(3,4,5-trihidroksi-6-hidroksimetiltetrahidro-piran-2-il) Flemingia macrophylla 2-(3,7-3-Dihidroksi-2,2-dimetil-6-il)-5hidroksi-8,8,dimetil-10(3-metil-but-2-enil)2,3-dihidro-8H-piranol[3,2-g] 5,7-Dihidroksi-3-(4-hidroksi-fenil) 5,7-Dihidroksi-3-[4-hidroksi-2-(3-metil-butenil)-fenil] 4-(4-Metoksi-7,7-dimetil-7H-furo[3,2-g] -2il)-benzen-1,3-diol 5-Hidroksi-2-(2-hidroksi-4-metil-fenil)-8,8dimetil-10-(3-metil-but-2-enil)-2,3-dihidro8H-pirano[3,2-g]-2-il) 5-Hidroksi-2-[2-hidroksi-5-(3-metil-but-2enil)-fenil]-8,8-dimetil-10-(3-metil-but-2enil)-2,3-dihidro-8H-pirano[3,2-g] 5-Hidroksi-2-[2-hidroksi-5-(3-metil-but-2enil)-fenil]-8,8-dimetil-10-(3-metil-but-2enil)-2,3-dihidro-8H-pirano[3,2-g] 2-(2,4-Dihidroksi-fenil)-5,7-dihidroksi-6,8bis-(3-metil-but-2-enil) 2-(2,4-Dihidroksi-fenil)-5,7-dihidroksi-6,8bis-(3-metil-but-2-enil) Genista saharae 3-(3,4-Dihidroksi-fenil)-5,7-dihidroksi Dihidroalpinumisoflavon Trifolium pratense L. 5,7-Dihidroksi-3-(4-hidroksi-fenil) 5,7-Dihidroksi-3-(4-metoksi-fenil) Pachyrhizus erosus 2-(3,4,5-Trihidroksi-fenil) -3,5,7-triol Pissum sativum 2-(4-Hidroksi-fenil)- 3,5,7-triol 2-(3,4-Dihidroksi-fenil)-,5,7-triol


-84,5962 ± 0,1870

0,9997

Active

-84,7865 ± 0,5025

0,9984

Active

-85,1969 ± 0,2934

0,9991

Active

-84,5379 ± 0,2995

0,998

Active

-99,4763 ± 0,1407

0,9966

Active

0,9991

Active

0,9975

Active

0,9941

Active

-111,2990 ± 0,6015

0,9743

Active

-103,3447 ± 0,4218

0,997

Active

-90,3197 ± 0,2126

0,9998

Active

0,9998

Active

0,997

Active

1

Active

0,9996

Active

0,9978

Active

0,9971

Active

0,9999

Active

0,9997

Active

0,9989

Active

-86,6118 ± 0,0303 -88,4177 ± 0,5095 -93,6756 ± 0,6518

-90,5774 ± 0,1828 -87,6588 ± 0,2543 -84,7146 ± 0,0203 -84,5062 ± 0,1480 -95,0540 ± 0,0032 -85,5595 ± 0,0503 -85,7861 ± 0,0055 -87,3650 ± 0,0174 -84,7334 ± 0,0543

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Figure A. 5-Hydroxy-2-[2-hydroxy-5-(3-methyl-but-2-enyl)-phenyl]-8,8dimetyhl-10-(3-methyl-but-2-enyl)-phenyl)-2,3-dihydro-8H-pyrano[3,2-g] chromen-2-yl)-chromen-4-one. A. representative active compound from Fleminga macrophylla plantss visualized 3D and showed the compound position in binding pocket ER-α.

Figure B. Pentanoic acid. A. representative inaktive compound from Eriosema robustum plantss visualized 3D and showed the compound position in binding pocket ER-α.

DISCUSSION Based on the score Chem PLP from the docking simulation of 60 compounds in the plants of family Fabaceae with using reference ligand 4-[4-hidroksi-3-(prop-2-en-1-yl) fenil]-2-(prop-2-en-1yl) (dimer compound number 11) obtained 24 active compounds (Table.1) on binding pocket ER-α by in silico. Figure A and B is a 3D visualized from active and inactive representative compounds using PyMOL application. The compounds were elucidated using PyMOL to explore some active amino acid residues. CONCLUSION 1. The results of virtual screening conducted on 60 compounds from ten plant of family Fabaceae obtained 24 compounds be active and 38 inactive compounds in the binding pocket of ER-α. 2. The important amino acids to affinity compounds with the protein that is GLU353, ARG394, ASP351 and THR347. REFERENCE Anita Y, Radifar M, Kardono LBS, Hanafi M, Enade P. Structure-Based Design Of Eugenol Analogs As Potential Estrogen Receptor Antagonists. 2012. p. 8(19). [2] Huang, Niu. Schoichet BKIJ. Benchmarking Sets for Molecular Docking. JMed Chem: 2006. p. 801. [3] Pranowo Dwi Harno HA. Pengantar Kimia Komputasi. Bandung: Lubuk Agung. 2011. 4-5:118. [4] Molares S, Ladio A. The Usefulness of Edible and Medicinal Fabaceae in Argentine and Chilean Patagonia : Environmental Availability and Other Sources of Supply. 2012.

[1


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Preparation of Standardized Aqueous Extract of Annona Muricata Linn. Leaf and Its Potency as Antioxidant Yesi Desmiaty, Deni Rahmat, Nilam Sari Maulidina Faculty of Pharmacy, University of Pancasila, Srengseng Sawah, Jagakarsa, South Jakarta, 12460 [email protected] ABSTRACT Extracts from various parts of Soursop leaves (Annona muricata Linn.) are widely used medicinally in all over the world for the management, control and/or treatment of cancer. Based on in vitro and animal studies, the presence of exogenous antioxidant has been shown to prevent free radicals activities associated with cancer development. Soursop leaves is one of medicinal herbals containing flavonoid compounds and has a potency as an antioxidant. Flavonoids are polyphenolic compounds and generally present as constituents of flowering plants, particularly dietary plants. The role of dietary flavonoids in cancer prevention has been widely discussed. The present study was carried out to investigate the intensity of free-radical scavenging activity, pharmacognosy characteristics and phytochemical screening of soursop leaves extract. The results of extract quality showed that water soluble extract of 45.30%, ethanol soluble extract of 39.99%, water content of 8.99%, loss on drying of 9.69%, total ash content of 5.53%, acid insoluble ash content of 1.14%, total plate count of 0.389 x 10 4 colony/g, yeast plate count of 0.9970 x 103 colony/g, and total flavonoid of 3.40%. From the study of antioxidant activity in vitro the extract has a powerful antioxidant with IC50 value of 76.91 ppm. Keywords: Soursop leaf, Annona muricata Linn., DPPH, radical scavenging activity, flavonoid. INTRODUCTIONS Various degenerative diseases such as diabetes, cancer, tissue inflammation, immune disorders, cardiac infarction and premature aging are caused by high levels of free radicals in the body. Free radicals that damage the body can be neutralized by antioxidants. Antioxidants are compounds that can inhibit reactive oxygen and free radicals in the body. These antioxidant compounds will share one or more electrons to free radicals to make a normal form of the molecule back and prevent the harmful reactions. One of the compounds containing a potential as antioxidants is flavonoid (1). Based on research Ni Putu, et al, n-butanol extract of soursop leaves has a value of DPPH free radical reduction of 91.10% (2). While Kunthi, et al, demonstrated the results of the phytochemical screening indicated that the leaves contain alkaloids, flavonoids, saponins, essential oils, quinones, coumarins, steroids/triterpenoids and tannins. The antioxidant activity of 70% ethanolic extract of the leaves displays the IC50 value of 22.25 ppm (3). According to the study, the leaves have an antioxidant activity and the potential as a standardized herbal medicine. Accordingly, the leaves can be developed as a preparation containing standardized herbal medicine which has been proven its safety and efficacy scientifically with preclinical trials and standardization of raw material (4). Therefore, in this research, the determination of extract quality (specific and non-specific parameters) was carried out to guarantee their consistency. To prove its efficacy in vitro, the antioxidant activity of the extract was performed using the method of scavenging of DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical.


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MATERIALS AND INSTRUMENTS Materials Dry simplicia of soursop leaves (Annona muricata L.), DPPH (1,1-difenil-2-pikrilhidrazil), and standard reference of quercetin.. INSTRUMENTS Spectrophotometer (Shimadzu UV-Visible Spectrophotometer), furnace (Nabertherm-Germany), oven (Memmert), and air Karl-Fischer (Metrohm 870 KF Titrino plus). METHODS 1. Collecting of dry soursop leaves Soursop leaves used in this study was obtained from Balitro, Cimanggu, Bogor and the determination was conducted in the Laboratory of Biological Research Center, Institute of Sciences Research Indonesia, Cibinong. 2. Preparation of soursop leaves infusion A total of 80 g of soursop leaves simplicia was poured into an infusion pot and added 1.6 L of water. Afterwards, the pot was heated in water bath for 15 minutes at the temperature of 90 °C, while stirring occasionally. The infusion was filtered through a flannel. The whole process was repeated for 7 times. 3. Phytochemical screening Phytochemical screening of the extract included alkaloids, falvonoid, saponins, tannins, essential oils, quinones, coumarin, and steroid/triterpenoid. 4. Determination of extract parameter The study included the determination of specific parameters (organoleptic, water soluble compounds, ethanol soluble compounds) and non-specific parameters (loss on drying, moisture content, total ash content, acid-insoluble ash content and microbial contamination). 5. Determination of total flavonoid 1) The test solution unless otherwise stated the amount of the concentrated extract equivalent to 200 mg of simplicia was carefully weighed and poured into a round-bottom flask. 1 ml HMT solution, 20 ml acetone and 2 ml of hydrochloric acid were added successively and refluxed for 30 minutes. The resulting extract was filtered through the cotton. The filtrate was collected into a 100 ml flask. The residue was refluxed with 20 ml acetone for 30 minutes and filtered. The filtrate was also collected into the same flask. Acetone was added up to the mark. 20 ml of the mixture was put into a separating funnel, added 20 ml of water and extracted three times, each time using 15 ml of ethyl acetate. Ethyl acetate phase was put into 50 ml flask, added ethyl acetate up to the mark. 2) Dilution of test solution Pipette 10 ml of test solution into a 25 ml flask; add 5% v/v of glacial-acetic acid solution in methanol up to the mark. 3) Measurement of total flavonoid Pipette 10 ml of the test solution into a 25 ml flask; add 1 ml aluminum chloride solution in 5% v/v glacial-acetic acid solution in methanol up to the mark. In different flask, 10 ml of 0.001% quercetin solution was added 1 ml aluminum chloride solution. The measurements were performed after incubation time of 30 minutes using a spectrophotometer at the wavelength of 429.5 nm. The total flavonoid content is calculated as indicated in the monograph by the formula : F (%) =

X 1.25 X 100 ml/ W (g)

Where, F = Total flavonoid Cp = Concentration of standard solution (ppm) Au = Absorbance of test solution with aluminum chloride International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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Abu Ap Abp

= Absorbance of test solution without aluminum chloride = Absorbance of standard solution with aluminum chloride = Absorbance of standard solution without aluminum chloride

DPPH-Radical Scavenging Activity The free radical scavenging activity of the fractions was measured in vitro by 2,20- diphenyl-1picrylhydrazyl (DPPH) assay. The stock solution was prepared by dissolving 8 mg DPPH with 50 ml methanol and stored at 20°C in the dark. A 1 ml aliquot of this solution was mixed with the sample solution in methanol and added methanol to get various final concentrations (5 - 100 μg/ml) in 5 ml flask. The reaction mixture was shaken well and incubated in the dark for 15 min at room temperature. Vitamin C served as standard and was treated in the same manner with the final concentration in the range of 2 – 10 μg/mL. Both the sample and the standard solution were incubated at temperature of 37 oC for 30 minutes. Afterwards, the absorbance was measured at 517 nm. The control was prepared in the same manner excluding any sample. The scavenging activity was estimated based on the percentage of DPPH radical scavenged as the following equation: Inhibition (%) = (Blank absorbance – Sample absorbance)/ Blank absorbance x 100% RESULTS 1.

Phytochemical Screening Table 1. Phytochemical Screening Secondary metabolite Result Alkaloid + Flavonoid + Saponin + Polyphenol + Quinone + Steroid/triterpenoid +/Volatile oil + Qoumarin +

2.

Quality Parameter and Total Flavonoid Table 2. Quality Parameter and Total Flavonoid Soursop leaves extract Requirement (Materi Medika Indonesia) Water soluble content 45.30% ≥ 18% Ethanol soluble content 39.99% ≥ 12.5% Water content 8.99% ≤ 10% Loss on drying 9.69% Total ash content 5.53% ≤ 6% Acid-insoluble ash content 1.14% ≤ 1.5% Total plate count 0.3888x104 colony/g ≤ 104 colony/g 3 Yeast plate count 0.9970x10 colony/g ≤ 103 colony/g Total flavonoid 3.40% Parameter

3. Antioxidant Activity Study The antioxidant activity was expressed in the relationship between concentration (x) versus % inhibition (y) by soursop leaves extract.


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Figure 1. Inhibition Percentage of DPPH Free Radical by The Extract Table 3. IC50 and Antioxidant Intensity of Soursop Leaves Extract Sampel Soursop leaves extract

IC50 76,91

Intensity Strong

CONCLUSION Phytochemical screening displayed that soursop leaves extract (Annona muricata L.) possesses alkaloid, flavonoid, saponin, polyphenol, quinon, steroid, volatile oil and qoumarin. The results of the extract evaluation demonstrated that the extract was fulfill all the requirement as mentioned in Materia Medica Indonesia where water soluble content of 45.30%; ethanol soluble content of 39.99%; water content of 8.99%; loss on drying 9.69%; total ash content of 5.53%, acidinsoluble ash content of 1.14%; total plate count of 0.389 x 104 colony/g; yeast plate count of 0.997 x 103 colony/g; and total flavonoid of 3.40%. Soursop leaves extract has a potential as a strong antioxidant with 1C50 value of 76.91ppm. ACKNOWLEDGEMENT This research was supported by Hibah Bersaing Ristek Dikti 2015. REFERENCES 1.

2. 3. 4.

S Adewole, J Ojewole , Protective effects of Annona muricata linn. (Annonaceae) leaf aqueous extract on serum lipid profiles and oxidative stress in hepatocytes of streptozotocin-treated diabetic rats, African Journal of Traditional, Complementary and Alternative Medicines. Vol 6, No 1 (2009) ISSN: 0189-6016. Putu N, Wahjuni S, Dwijani W. Ekstrak daun sirsak (Annona muricata L.) sebagai antioksidan pada penurunan kadar asam urat tikus wistar. 2012 (2);1-2. Marisi R, Desmiaty Y, Wida K. Uji pendahuluan aktivitas sitotoksik dan antioksidan ekstrak etanol daun sirsak (Annona muricata L.) dan batang brotowali (Tinospora crispa). 2012;1-2. Badan Pengawas Obat dan Makanan RI. Ketentuan pokok pengelompokan dan penandaan obat bahan alam Indonesia. Taken from: http://www.pom.go.id/ pom/hukum_perundangan/pdf/penandaan_oai.pdf. Accessed on 11 Desember 2014.


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PHYTOCHEMICAL SCREENING AND TOXICITY TEST BSLT OF 70 % ETHANOL EXTRACT OF GAHARU LEAVES (Aquilaria beccariana Tiegh.) 1

Ahmad Musir1, Wiwi Winarti1, Siti Hasnah P. Siregar1 Faculty of Pharmacy, University of Pancasila, Jakarta 12640 Jagakarsa E-mail: [email protected]

ABSTRACT Gaharu or agarwood (Aquilaria beccariana Tiegh.) is a forest plant in Indonesia that has been developed with the cultivation by the public and can be used as anti-tumor drugs, anti-cancer, diarrhea and others This research aimed to determine the toxicity of 70% ethanol extract of gaharu leaves using Brine Shrimp Lethality Test (BSLT). The study was conducted on the phytochemical screening and toxicity testing of ethanol extract of gaharu leaves is not inoculated and inoculated. Results of phytochemical screening of the powder and condensed extract of gaharu leaves to non-inoculated and inoculated showed the presence of compounds flavonoids, saponins, catechuic and gallic tannins , quinons, coumarins, steroids/ triterpenoids, but on the inoculated also showed the presence of volatile oil compounds. The toxicity test of 70% ethanol extract of non inoculated gaharu leaves that does have a LC50 value of 113.73 ppm and 60.60 ppm LC50 inoculated. Key words: Agarwood, Aquilaria beccariana Tiegh, BSLT INTRODUCTION Plants gaharu (Aquilaria beccariana Tiegh ) is a forest plant that has been developed with the cultivation by the public and can be used as a diarrhea medication, anti-tumor, anti-cancer, and others. Agarwood is also commonly used as fragrances, body by burning (fumigation) at religious events. Currently no less than 46 (forty six) types of plants that can produce agarwood, plants most frequently used is (Aquilaria beccariana Tiegh ) from thymeleaceae family, where one of the benefits as anticancer. Broadly speaking, the process of formation of aloes consists of 2 kinds: natural or artificial and not at inoculation or inoculation. The process of formation of gaharu inoculation include: hurt / trunk section, include the injection of a microorganism of the genus Fusarium sp that play a role in the formation of agarwood. Thus aloes contain secondary metabolites are thought to have anticancer activity. Based on this crude drug powder in this study were extracted by the method of kinetic maceration with 70% ethanol, and the extracts obtained, further toxicity tests with the method BSLT (Brine Shrimp Lethality Test). MATERIALS AND METHODS MATERIALS Gaharu leavest powder (Aquilaria beccariana Tiegh ) that is not in the inoculation and inoculation used were obtained from the Center for Conservation and Rehabilitation Research Development and the Ministry of Forestry in the Ministry of Forestry determined Forestry Research and Development Agency Bogor, after the wood is dried and pulverized. Chemicals: ethanol 96%, Dragendorf LP, LP Mayer, p HCl, amylalcohol, ether, anhydrous acetic acid, FeCl3 1% , H2SO4 cons., chloroform, ammonia 10% LP, water, salt NaCl, shrimp larvae (Artemisia salina Leach). Instruments: vacuum rotary evaporator (Buchi 205), analytical balance (Sartorius) ,macerator kinetic (Heidolph), glass tools. 18 watt fluorescent lamp, a magnifying glass, bottles vials and micro pipette. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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METHODS Preparation of extracts. A total of approximately 250 grams of gaharu leaves is pulverized dried macerated using 70 % ethanol (by means of kinetic maserator) and filtered, then filtered. Remaceration until all secondary metabolites extracted perfectly. The filtrate obtained was collected, further concentrated by vacuum rotary evaporator, then evaporated on a water bath at a temperature of 40 ° C to obtain a viscous extract. Phytochemical screening : Performed by identifying classes of secondary metabolites, compounds contained in the leaves powder and extracts Biological activity test using larval shrimp Artemia salina Leach a. Hatching eggs Artemia salina Leach. Prepare by dissolving synthetic sea water (38 g Sodium Chloride (NaCl) in 1000 mL of water) and filtered with Whatman paper. Brooders sealed vessel that has two sides of the room, which is open and closed sides. Then enter the egg Artemia salina Leach. into the incubator vessel which already contains the synthetic sea water and irradiated with 18 watt fluorescent lamp. After 24 hours the eggs that have hatched into nauplii were transferred to another place, 24 hours after the nauplii is ready to be used as test animals. b. Preparation of test solutions Extract solution of each sample were made in 9 vials for three concentration is 10 ppm, 100 ppm, 1000 ppm and one vial for control. The mother liquor is made by weighing 20 mg of extract, dissolved in 2 ml of sea water that has been filtered if it is poorly soluble samples added dimethylsulfoxide (DMSO) 1% as much as 0.1 to 50.0 mL to increase the solubility. c. Toxicity tests Pipetted mother liquor of 500, 50 and 5 mL in a row is inserted into the vial and then evaporated to dryness. Each concentration was made with three repetitions, then into each vial inserted + 3 ml of sea water, if samples of poorly soluble in sea water, then add dimethyl sulfoxide (DMSO) 1% as much as 0.1 to 50.0 mL. The solution was stirred until homogeneous and enter 10 nauplii tail, sea water is then added to 5 mL. For each concentration performed three repetitions. Determination of LC50 in mg/ mL performed using probit analysis. RESULTS AND DISCUSSION Gaharu wood extraction . Extraction by maceration kinetic gaharu leaves pulvered was not inoculated and inoculated each with 70 % ethanol, producing a viscous ethanol extract of gaharu leaves 54,40 grams not inoculated with a yield of 21.76 % and condensed ethanol extract of gaharu leaves were inoculated 59.00 grams with a yield of 23.58 %. The content of chemical powder and extract of gaharu leaves The study of the content of the extract of gaharu leaves (Aquilaria beccariana Tiegh ) both powder and ethanol extracts of gaharu leaves that was not inoculated and inoculated showed a class of compounds flavonoids, saponins,catechuic and gallic tannins, steroids / terpenoids, quinons and

coumarins, but on the inoculated also showed the presence of volatile oil compounds. Content of the test results can be seen in Table 1.


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Table 1. Result of phytochemical screening the leaves powder and extract Result

No.

Class of Compounds

Powder of Gaharu leaves non inoculation

Extract of Gaharu leaves non inoculation

Powder of Gaharu leaves in inoculation

Extract of Gaharu leaves in inoculation

1

Alkaloids

-

-

-

-

2

Flavonoids

+

+

+

+

3

Saponins

+

+

+

+ +

Catechuic Tannins

+

+

+

Gallic tannins

+

+

+

+

5

Quinons

+

+

+

+

6

Steroids/ triterpenoids

+/+

+/+

+/+

+/+

7

Coumarins

+

+

+

+

8

Essential oils

-

-

+

+

4

Description : + -

= positive results = negative results

BSLT biological activity test (Brine Shrimp LethalityTest) Biological activity test with metod (BSLT), 70 % ethanol extract of gaharu leaves turns inoculation showed the toxic with LC50 values of 60.60 ppm, while the ethanol extract of gaharu leaves not inoculated at 113.73 ppm LC50, Based on this it can be said that the ethanol extract of 70 % ethanol of inoculated gaharu leaves are toxic and ethanol extracts were not inoculated gaharu leaves is less toxic. The data can be seen in Table 2 and Table 3. Table 2. Ethanol extract toxicity test results were inoculated gaharu Concentration (ppm)

Log C (X)

Dead

Life

MR

NL

MR/T

1000

3

27

3

53

3

53/56

% of Mortality (Y) 94.64

100

2

20

10

25

15

25/40

64.50

10

1

7

23

7

37

7/44

15.91

LC50 (ppm)

60.60


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120

Graph LC50 Of 70 % Ethanol Extract Agarwood Inoculation

% of mortality

100 80 60 40 20 0 1

2

3

4

Log D Figure 1 Graphs the relationship between the concentration (ug/ ml) and mortality 70% ethanol extract of the inoculated agarwood Table 3. Ethanol extract toxicity test results were not inoculated agarwood Concentration Log C Dead Life MR NL MR/T % of (ppm) (X) Mortality (Y) 1000

3

23

8

43

8

43/51

(%) of

LC50 (ppm)

84.31 113.73

100

2

13

17

20

25

20/46

45.87

10

1

7

23

7

47

7/54

12.96


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Graph LC50 Of 70% Ehanol Extract Agarwood non Inoculation 100

90

% of mortality

80

70 60 50

40 30 20

10 0 1

2

3

4

Log D Figure 2. Graphs the relationship between the concentration (ug/ ml) and ethanol extract of agarwood were not inoculated

mortality (%) of 70 %

An extract contains compounds that are included in the category of very active or are highly toxic if LC50 values ≤ 30 ppm, a plant extract said to be toxic if its LC50 value is 30 - 1000 ppm. results test showed that the extract Agarwood leaves so have the cytotoxic activity as antitumor or anticancer potential. CONCLUSION The survey results revealed that 70 % ethanol extract of Agarwood leaves not inoculated and inoculated class of compounds containing flavonoids, saponins, catechuic and gallic tannins , steroids / triterpenoids, quinons and coumarins, but in addition it also inoculated existence of this class of compounds containing volatile oil. In BSLT biological activity test showed that ethanol extract of agarwood leaves inoculated with LC50 values of 60.60 ppm had higher active of ethanol extract of agarwood leaves is not inoculated with LC50 values of (113.73 ppm).


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1.

2. 3.

4.

REFERENCES Kartasubrata J. 2010. Successful cultivation of medicinal plants. Bogor: IPB Press. Department of Forestry in 2004, Utilization profile (cultivation) Agarwood. Jakarta. Forestry Extension Development Center. Jakarta; p. 41-45 Farnsworth NR, 1966. Biological and phytocemycal of plant. J. Pharm, Sci; . p. 224 264. Novriyanti Eka 2008. Role of Extractive Substances In Formation Agarwood on Aquilaria crassna Pierre ex Lecomte and Aquilaria microcarpa Baill. Bogor Agricultural University. Meyer BN, et al. 1982 . Brine shrimp a convenient general bioassay for active plant constituent, Planta Medika 45; things. page 31-34


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OPTIMIZATION OF PRODUCTION OF Β-CAROTENE AND ASTAXANTHIN FROM MICROALGAE Chlorella pyrenoidosa AND ITS POTENTIAL AS AN ANTIOXIDANT Ni Wayan Sri Agustini Research Centre for Biotechnology-LIPI e-mail : [email protected] ABSTRACT Astaxanthin and -carotene is a carotenoid pigment that is known to be useful as an antioxidant. One microorganism producing Astaxanthin and -carotene is Chlorella pyrenoidosa. Increased content of pigments in microalgae cells are affected by the availability of nutrients in the growth medium, based on that research is conducted with the optimization of production is -carotene and astaxanthin in Chlorella pyrenoidosa were cultivated in various kinds of foliar fertilizer and its potential as an antioxidant. Type of foliar fertilizer used are Gandasil D, Growmore and Hyponex and Technical medium (Urea, TSP and ZA) as a control. Analysis of astaxanthin and -carotene using extraction method based Hua Bin Li (2002), while the antioxidant activity using of reduction of free radicals (DPPH) and vitamin C as a positive control. The results obtained showed that Hyponex is the best fertilizer that can optimize the production of -carotene and astaxanthin in C. pyrenoidosa is 679.780 ppm ( -carotene) and 217.444 ppm (astaxanthin), while its antioxidant activity, namely the LC50 values 111.417 ppm. Based upon these results the carotenoid from C. pyrenoidosa containing astaxanthin and -carotene can be as an alternative source of antioxidant natural. Keywords: Chlorella pyroneidosa, -carotene, astaxanthin, an antioxidant INTRODUCTION Some species of microalgae are a source of carotenoids include Chlorella pyrenoidosa, Spirulina platensis, Dunaliella salina, and Nannocloropsis sp. Carotenoids are a group of yellow or orange pigment. Carotenoids can have a positive influence to health because it has potent antioxidant activity, also potentially reduces the chances of the formation of cancer cells (1). Carotenoid are divided into two derivative, namely carotene and xantofil. Carotene is a carotenoid derivative of the compound that does not contain elements oxygen such as ɑ-carotene, -carotene, -carotene and ε-carotene. While xantofil is a derivative of a compound containing the oxygen such as lutein, rubixantin, astaxantin, zeaxantin and violaxatin(2). -carotene is widely used in the food industry, pharmaceuticals, and cosmetics. so many of research to finding sources of -carotene derived from a variety of biological resources (3). While astaxanthin is derived xantofil, which is one of the most important properties is an antioxidant with a stronger effect because it has 10 methyl groups and two hydroxy groups which allows the esterification. A drink containing astaxanthin has been shown to prevent arteriosclerosis, ischemic heart disease or ischemic encephalopathy(4). The beneficial effects of astaxanthin for heart health by reducing inflammation associated coronary heart disease(5). In addition astaxanthin has anti-cancer activity in several studies conducted by Tanaka et al (1995) reported the inhibition of bladder carcinogenesis in rats chemically by astaxanthin. Astaxanthin was also found to be effective in protecting mice against azometane induction in colon cancer(6). Based on the above and will need astaxanthin and -carotene continues to increase, it is necessary to optimize the cultivation of -carotene and astaxanthin produced by microalgae. Microalgae cultivation depends on a sufficient supply of nutrients to the growing medium and a light source to perform photosynthesis. The addition of nutrients to grow the right media can promote the growth of microalgae in inorganic synthetic medium(7). International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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Cultivation of microalgae in laboratory scale, the type of fertilizer frequently used type of fertilizer such as Conwy and Knops. Economically, this type of fertilizer has a price that is quite expensive, so it is necessary to find a replacement fertilizer price is relatively cheaper, but it is expected the fertilizer is capable of producing -carotene and astaxanthin maximum. Fertilizers such as Gandasil D, Hyponex and Growmore fertilizer which is technical and has some elements of nutrients such as nitrogen, phosphorus, sulfur is needed in culture and are cheaper and easily available in the market. Based on the above, then do research on the effect of several types of synthetic fertilizers on the production of -carotene and astaxanthin in microalgae Chlorella pyrenoidosa, and its potential as an antioxidant METHODOLOGY a. Cultivation of Microalgae Stock cultures of microalgae Chlorella pyrenoidosa cultivation in a 2-liter bottle size. The composition of media of stock culture consists of urea (1 g / L), TSP (0.3 g / L) and ZA (0.8 g / L) (technical media ). After reaching the logarithmic phase of stock cultures are harvested by means of centrifuge. Biomass of C. pyrenoidosa cultivated again in 4 bottles that have added 4 different kinds of fertilizers are Gandasil D, Hyponex, Growmore and Medium Control (Urea 1 g/L, TSP 0,3 g/, and ZA 0,8 g/L). The concentration of fertilizer used is 1 g / L. Cultivasi performed in a 2-liter bottle size.C. pyrenoidosa cultivated again to 4 bottles in a 2 -litre bottle size. . Cultivation is carried out with the addition of continuous aeration using a blower, the light intensity of 2500 lux and a neutral pH 7. Sampling was done when the culture reaches logarithmic and stationary phases b. Analysis β-carotene and astaxanthin(8) Extraction use of Hua Bin Li (2002). 0.5 gram of wet biomass is suspended with 4 ml of 10 M KOH then heated above the bath at 60°C for 10 minutes, then cooled at room temperature. Once cool, plus 2.6 ml dichloromethane were homogenized with a homogenizer for 5 min and centrifuged at a speed of 3500 rpm for 10 minutes. The resulting filtrate is collected and re-extracted sludge treatment as above to obtain a pale yellow filtrate. once collected, centrifuged and the filtrate evaporated at 40 ° C. Rest of evaporation added 10 ml of ethanol and 10 ml of n-hexane Furthermore, inserted into the separating funnel, shaken and allowed to separate and form a second layer of the phase of ethanol and n-hexane phase, the measured absorption at a wavelength of 455 nm (beta carotene) and 479 nm (astaxanthin) using a UV-Vis spectrophotometer. Uptake value obtained plotted on a standard that was created regression equation c. Antioxidant Activity of Free Radicals Reduction Method Using 1,1-Diphenyl-2Picrylhydrazyl (DPPH). Free radical scavenging activity of extracts carotenois from C. pyrenoidosa were measured by 1, 1diphenyl-2-picryl hydrazyl (DPPH). In brief, 0,4 mM solution of DPPH in methanol was prepared. Pipette stok solution as much as 25, 50, 125, 250 and 500 mL. DPPH solution was added 1.0 mL into each tube and added with methanol pro analysis up to add 5 mL, to obtain the concentration of the sample 5, 10, 25, 50 and 100 mg / mL. The mixture was shaken vigorously and allowed to stand at room temp for 30 min. then, absorbance was measured at 517 nm. by using spectrophotometer (UV-VIS Shimadzu). Reference standard compound being used was ascorbic acid and experiment was done in triplicate. The IC50 value of the sample was calculated using following equation: DPPH scavenging effect (%) or Percent inhibition = A0 - A1/ A0× 100. Where A0 was the Absorbance of control reaction and A1 was the Absorbance in presence of test or standard sample (Taylor


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RESULT AND DISCUSSION a.

GROWTH of Chlorella pyrenoidosa

(A) (B) Figure 1. Optical dencity (A) and Biomass (B) of Chlorella pyrenoidosa cultivated in Various of Media Cultivation(Gandasil D, Hyponex, Growmore and Control).

Media for the growth of microalgae Chlorella pyrenoidosa using technical media with the addition of various types of fertilizers with of fertilizers concentration of each 1 g / L. Fertilizers used in this cultivation is Gandasil, Hyponex and Growmore while the technical media as a control. The third composition of this fertilizer has the same nutrients are nitrogen, phosphorus and potassium, with different concentrations. The function of the nitrogen is known as a major component of cell protein which is a basic part of life of all organisms. Phosphorus is needed for the formation of protoplasm and the cell nucleus. Phosphorus is the base material forming nucleic acids, phospholipids, enzymes and vitamins. The function of potassium is one of the main organic in the cell and a cofactor for several coenzyme (Fogg, 1975; Becker, EW., 1994). Cultivation of Chlorella pyrenoidosa growth observed from day 1 to day 13. The growth curve was made using turbidimetry method based on the value of the absorption optical density or OD in a spectrophotometer with a wavelength of 680 nm. Based on the above curve on day three to nine Chlorella pyrenoidosa cells show logarithmic phase, while the day 10 to 11 shows the stationary phase. Chlorella pyrenoidosa growth is best generated by fertilizer Hyponex. This dimugkinkan because the composition ratio between nitrogen, phosphorus and potassium from fertilizer Hyponex better than other fertilizers. On the composition of Hyponex there is the content of N: P: K as many (20%: 20%: 20%), Growmore (32%: 10%: 10), Gandasil D (4.2%: 1%: 16%). Growmore has a higher nitrogen content and the Gandasil D have a lower nitrogen content compared to Hyponex, but the growth of C. pyrenoidosa lower than Hyponex, this suggests that a deficiency or excess nitrogen content in the media can be a limiting factor for metabolism and biosynthetesis processes microalgae cells (Healey, 1973). In Figure 1, seen biomass obtained in the stationary phase is higher than when logarithmic, this suggests that the higher the growth of microalgae (OD) the higher the biomass obtained. b. β-CAROTENE -Carotene is a strongly colored red-orange pigment abundant in plants, fruits, microorganism like is microalgae. It is an organic compound and chemically is classified as a hydrocarbon and specifically as a terpenoid (isoprenoid), reflecting its derivation from isoprene units.


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Figure 2. β-carotene of Chlorella pyrenoidosa cultivated in Various of Media Cultivation (Gandasil D, Hyponex, Growmore and Control).

Β-carotene in microalgae cells to function to absorb light energy for use in photosynthesis and also serve to protect chlorophyll from damage caused by light(9).In this study the content of -carotene logarithmic phase current is smaller than the stationary phase. The highest beta carotene obtained in C. pyrenoidosa were cultivated on Hyponex that is equal to 697.716 ppm, followed Growmore in the amount of 389.945 ppm and the lowest in the media Gandasil D is 194.065 ppm (Figure 2). It is also consistent with the results obtained in this study that the maximum achievable beta carotene when the cells undergo stationary phase. According Fogg, 1975 when the stationary phase cell density reaches a maximum so that frequent self-shading among cells, chlorophyll will suffer destruction was replaced by secondary carotenoid pigments as photosynthetic pigments. And it is known that the Beta carotene is a carotenoid part of the maximum will accumulate in the cell when the stationary phase(10). b. ASTAXANTHIN Astaxanthin, unlike some carotenoids and the other carotenoids are known, are not converted to vitamin A (retinol) in the human body. As with beta-carotene, astaxanthin in microalgae cells to function as a secondary photosynthetic pigments so that the accumulation of the pigment astaxanthin in the cells associated with the growth and cell density. In this study, the maximum content of astaxanthin obtained during the stationary phase is 47.722 ppm on Gandasil D, 217.444 ppm in Hyponex, 119.018 ppm on Growmore and 80.870 ppm on Control. Visually visible as yellowish stationary phase cells and showed that chlorophyll suffered destruction and was replaced by carotenoids to perform photosynthesis

Figure 3. Astaxanthin of Chlorella pyrenoidosa cultivated in Various of Media Cultivation (Gandasil D, Hyponex, Growmore and Control).

C. ANTIOXIDANT ACTIVITY DPPH method measures the ability of an antioxidant compound in capturing free radicals. Radical scavenging ability relates to the ability of the compound components in donating electrons or hydrogen. Each molecule that can donate electrons or hydrogen will react and will dilute DPPH. DPPH color intensity will change from purple to yellow by electrons derived from antioxidant compounds. DPPH concentration at the end of the reaction depends on the initial concentration and structural components catcher compound radical(11). In this study was obtained, with a concentration of 150 ppm carotenoid extract containing beta carotene and astaxanthin have a value of inhibition of 55.66%, whereas vitamin C (positive control) has a value of 49.83% inhibition at a concentration of 8 ppm IC50 is a number that indicates the extract concentration (ppm), which is able to inhibit the oxidation process by 50%. The smaller the IC50 value means the higher the antioxidant activity. Specifically a International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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compound said to be a very powerful antioxidant if the IC50 value of less than 50 ppm, strong for the IC50 value-50-100 ppm, while if it is worth 100-150 ppm, and weak if the IC50 value worth 151-200 ppm (Anonymous, 2005). Based on the results obtained carotenoid extract of C. pyrenoidosa including weak category because it had LC50 values of 111.42 ppm . Nevertheless these carotenoid can be used as an alternative as natural antioxidant. CONCLUSION C. pyrenoidosa can grow well in growth media used Hyponex and resulted in -carotene and Astaxanthin respectively 697.716 ppm and 217.444 ppm on the stationary phase. The antioxidant activity of the carotenoid extract of C. pyrenoidosa at 111.417 ppm so that it can be used as asource of natural antioxidant REFERENCES 1. Leach G, G Oliveira, and R Morais. Production of a caretonoid-rich product by Alginate en modern trapment and fluid-bed drying of Dunaliella salina. J Sci Food Agric. 1998. 76:298-302. 2. Becker EW. Biotechnology and Microbiology, 1st edition. New York: Cambridge University Press; 1994. p. 9-12,51-58,253. 3. Vega PJ, MO Balaban, CA Sims, SF O’Keefe, and JA Cornell. 1996. Supercritical carbon dioxide extraction efficiency for carotene 4. Miki, W., K Hosoda, K Kondo, and Itakura, H. Astaxanthin containing drink. 1998. Patent abstract JP10155459 5. Tracy, R. P. Inflamantion Markers And Coronary Heart Disease. Current Opinion In Lipidologi . 1999. 10, 435-441 6. Tanaka, T., T Kawamori., M Ohnishi., H Makita., H Mori,. K Satoh, and A Ha. Suppression of azomethan-induced rat colon carcinogenesis by dietary administration of naturally occurring xanthophylls astaxanthin and canthaxanthin during the postinitiation phase. Carcinogenesis 1995. 16,2957-2963 7. Teresa M, Mata., Antonio A, Martins., Nidia, S, Caetano. Microalga for Biodiesel Production And Other Applications. 2010. Portugal. 8. Li Hua-Bin, Jiang You and Cheng Feng. Isolation and purification of lutei from the microlaga Chlorella vulgaris by extraction after saponifonification. Journal of agricultural and food chemistry. 2002, p. 1070-1072. 9. Armstrong GA, Hearst JE. Carotenoids 2: Genetics and molecular biology of carotenoid pigment biosynthesis. FASEB J. 1996. 10 (2): 228–37. PMID 8641556 10 Fogg GE. Algal Culture and Phytoplankton Ecologi. London: The University of Wisconsin Press from carrot by RSM. J. Food Sci. 1975. 61(4):757 11 Naik, G.H., Priyadarsini, K.I., Satav, J.G., Banavalikar, M.M., Sohoni, D.P., Biyani, M.K., and Mohan H. Comparative antioxidant activity of individual herbal components used in ayurvedic medicine, Phytochemistry. 2003. 63(1): 97-104


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ANTIOXIDANT COMPOUND ISOLATED FROM BIOPRODUCTION OF ENDOPHYTIC FUNGI OF TURMERIC (Curcuma longa L.) Hindra Rahmawati1, Partomuan Simanjuntak1,2 1 Faculty of Pharmacy, Pancasila University, Jl. Srengseng Sawah, Jagakarsa, Jakarta 12640, Indonesia 2 Research Centre for Biotechnology - Indonesian Institute of Sciences (LIPI), Jl. Raya Bogor Km 46 Cibinong 16911, Indonesia Email: [email protected] ABSTRACT Endophytic fungi are microorganisms that live in the plants and do mutualism symbiosis with the hosts in order to produce chemical compounds which are similar to what the hosts do. Five endophytic fungi isolates were successfully obtained from various parts of turmeric (Curcuma longa L.) from Cibinong. Bioproduction of those five isolates were tested for their antioxidant activity by free radical scavenging method using 1,1-diphenyl-2-picrylhydrazyl (DPPH). One of the isolates, K.CI.Cb.U.1, produced compound(s) with highest antioxidant activity. Bioproduction of K.CI.Cb.U.1 in large amount was obtained by fermentation method using Potato Sucrose Broth (PSB) medium. Fermentation result showed that the ethyl acetate extract of the biomass had higher antioxidant activity than that of the filtrate. The biomass extract was fractionated by gradient column chromatography (silica gel 60; n-hexane-ethyl acetate 10:1 to 2:1). Three combined fractions were obtained and their IC50 values were determined by DPPH. Fraction no. 2 had the highest IC50 value (67.18 ppm), and was selected for further purification using preparative TLC method to produce isolate X. Based on FTIR and GC-MS spectra of isolate X, it could be estimated that the bioproduction compound of K.CI.Cb.U.1 endophytic fungi isolate was a glycoside with one saccharide molecule and mass weight of 578, and its aglycone was supposed to be 3,7-Dimethyl-7-(4-methyl-3-pentenyl)-8-(2,6,10-trimethyl-1,5,9-undecartrienyl)bicyclo[4.2.0]oct-2ene. Keywords: antioxidant, bioproduction, endophytic fungi, turmeric (Curcuma longa L.) INTRODUCTION The level of pollution in the world becomes higher from day to day. Indonesia has become one of the countries that have pretty high levels of pollution. Pollution is a source of free radicals that can cause dangerous diseases, therefore we need antioxidants that can counteract and prevent the effect of pollution. Antioxidants are substances which can prevent oxidation process by scavenging free radicals that attack our body cells. Biodiversity of medicinal plants in Indonesia reveals high potency to discover new antioxidant compound. One of the plants that can be a source of antioxidants is turmeric (Curcuma longa L.). Endophytic fungi are microorganisms that live in the plants and do mutualism symbiosis with the hosts in order to produce chemical compounds which are similar to what the hosts do. The plants provide the nutrition for the endophytes and this microorganism convert the nutrition into secondary metabolite compounds (1,2). In this study, endophytic fungi of turmeric was fermented to produce antioxidant compound(s). The bioproduction obtained was then isolated and identified.


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METHODS Material Five endophytic fungi isolates, with codes K.Cl.Cb.Bu.1, K.Cl.Cb.Bu.2, K.Cl.Cb.U.1, K.Cl.Cb.U.2 and K.Cl.Cb.B.1, were successfully obtained from various parts of turmeric from Cibinong. These isolates were collection of Natural Product Laboratory, Research Centre for Biotechnology - Indonesian Institute of Sciences (LIPI), Cibinong. The filtrates and biomasses extracts of their bioproductions were examined their antioxidant activities and revealed that the biomass extract of the isolate K.Cl.Cb.U.1 had the highest antioxidant activity with Inhibition Concentration value of 86.41%. This isolate was selected to be examined further to isolate its bioproduction antioxidant compound. Bioproduction The endophytic fungi isolate K.Cl.Cb.U.1 was inoculated in Potato Dextrose Agar (PDA) medium in Petri dish and incubated for seven days at room temperature. The morphology of the fungi was performed at Figure 1. The culture was then transferred into Potato Sucrose Broth (PSB) medium and fermented for 14 days while being shaken using the rotary shaker at room temperature.

(a)

(b)

Figure 1. Endophytic fungi of K.Cl.Cb.U.1; top side (a); bottom side (b)

Extraction Procedure The fermentation yield was filtered to seperate the biomass from the filtrate. The biomass, which had higher antioxidant activity than the filtrate, was dried and extracted using ethyl acetate as solvent and evaporated under reduced pressure using rotary evaporator. Antioxidant Activity Test The extracts of the filtrates and the biomasses of the five fungi isolates, the biomass extract of isolate K.Cl.Cb.U.1 and its fractions were tested for their antioxidant activity by free radical scavenging method using 1,1-diphenyl-2-picrylhydrazyl (DPPH)(3). Series of concentration of the extracts and fractions were made and each solution was added with one mL of 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution (0.4 mM in methanol) and diluted until 5 mL with methanol. After being homogenized, the solutions were incubated for 30 minutes at 37oC. The absorptions were measured spectrometrically at 517 nm. The IC50 value of each extract or fraction was calculated using its linear regression curve (4). The ascorbic acid was used as standard. Fractionation and Purification Fractionation and purification procedure was carried out using column chromatography, followed by preparative TLC to get pure substance(s). The column chromatography procedure was performed using silica gel 60 with gradient solvent system of n-hexane-ethyl acetate 10:1 to 2:1. TLC monitoring was carried out during the fractionation using silica gel GF254 plate developed in n-hexane-ethyl acetate 2:1. The spots were examined visually after being sprayed with cerium sulphate solution (1% in 10% sulphuric acid) and heated electrically until the spots were appeared. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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Purification was performed by preparative TLC using silica gel GF254 plate and n-hexane-ethyl acetate 2:1 as mobile phase. The band was extracted with ethyl acetate solvent, and after filtering the extract was evaporated until the pure substance was obtained. RESULT Three combined fractions obtained from column chromatography were tested their antioxidant activity and indicating that the fraction 2 was the most active fraction with IC 50 value of 67.18 ppm compared to vitamine C as positive standard with IC50 value of 4.07 ppm. Further purification by preparative TLC revealed single band of substance. After being extracted, the compound of the isolate (isolate X) was identified using FTIR Spectrophotometer and Gas Chromatography-Mass Spectrometer.

Figure 2. FTIR Spectrum of the isolate X

Figure 3. GC-MS spectrum of the isolate X The peak with Retention Time (tR) 25.346 was the highest peak

Figure 4. The mass spectrum of the isolate X

DISCUSSION


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The FTIR spectrum performed by Figure 2, indicated that the isolate X had stretching vibrations of hydroxyl group at 3538.17 cm-1, aliphatic C-H bonding at 2950.89, 2920.03 and 2853.49 cm-1, and carbonyl group which appeared at 1733.89 cm-1 . GC-MS analysis revealed several peaks at the chromatogram, indicating that the isolate was not pure enough although the preparative TLC performed only a single band. The GC-MS spectrum at Figure 3 indicated that the peak with Retention Time (tR) 25.346 was the highest peak and had the biggest % area (24.00% ). This peak that had molecular weight (MW) 578 was supposed to be the most active compound, and its mass spectrum could be seen at Figure 4. According to Willey09th.L database used in GC-MS instrumentation this highest peak was indicated as 3,7-Dimethyl-7-(4-methyl-3-pentenyl)-8-(2,6,10-trimethyl-1,5,9-undecartrienyl) bicyclo[4.2.0]oct-2-ene . Because of its low similarity (42 % quality and MW = 408) it was predicted that the substance had one sugar moiety (MW about 170), indicating that it was a glycoside compound with one saccharide molecule. The chemical structure of the aglycone of the isolate could be seen at Figure 5.

Figure 5. The chemical structure of the aglycone of the isolate X

CONCLUSION Based on FTIR and GS-MS spectra, the compound of isolate X produced by bioproduction of endophytic fungi isolate K.Cl.Cb.U.1 that had antioxidant activity was predicted as a glycoside with one saccharide molecule and mass weight of 578, and its aglycone was supposed to be 3,7-Dimethyl-7-(4-methyl-3pentenyl)-8-(2,6,10-trimethyl-1,5,9-undecartri- en yl)bicyclo[4.2.0]oct-2-ene. ACKNOWLEDGEMENTS The authors wish to thank Ms. Felicia Chikita Fredi for her assistance, and Research Centre for Biotechnology - Indonesian Institute of Sciences (LIPI) for financial support and laboratory facility. REFERENCES 1. 2.

3. 4.

Strobel G. Endophytic fungi: New sources for old and new pharmaceuticals. Pharmaceutical News.1996. (3)6:7-9. Simanjuntak P, Parwati T, Kurnia N, Rahmat J, Rosalinda N. Investigation of bioactive compounds from microbial resources in Indonesia: Bioactive metabolites from endophytic microbes of “Kina” Cinchina spp. J Pharm Sci; 1999. Molyneux P. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J Sci Technol. 2004. Mar-Apr; (26)2:212-219. Rohman A, Sugeng R, Diah. Antioxidant activities, total phenolic and flavonoid contents of ethyl acetate extract of mengkudu (Morinda citrifolia L.) fruit and its fractions. Yogyakarta: Fakultas Farmasi Universitas Gadjah Mada, 2005.


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ANALYSIS OF BETA-CAROTENE IN GREEN MELON AND ORANGE MELON (Cucumis melo L. var. Sky Rock and var. Cantaloupe) BY TLC-DENSITOMETRY Rifa Rizkiyah1, Zuhelmi Aziz2 Fakultas Farmasi UniversitasPancasila, Jakarta [email protected] ABSTRACT Two kinds of melon are ubiquitously found in the market nowadays which are green flesh melon (var. Sky Rock) and orange flesh melon colour (var. Cantaloupe). Melon contains natural coloring pigment which one of them is beta-carotene. The presence of beta-carotene contributes to the redness and orangeness of a fruit or vegetable. An experiment was conducted to analyze the content of beta-carotene in green melon and orange melon through thin layer chromatography — densitometry method. Beta-carotene in melon was extracted using chloroform and the analyzed through TLC using silica gel GF 254 as stationary phase and n-hexane-ethyl acetate (8:2) as mobile phase.The beta-carotene was determined by densitometre on maximum wavelength of 457 nm. The content of beta-carotene in green melon is 0.22 mg/ 100 g, with % recovery of 98.76% and RSD of 1.50%, and the content of beta-carotene in orange melon is 2.05 mg/100 g, with % recovery of 98.81% and RSD of 1.14%. The result shows beta-carotene content in green and orange melon significantly differ; orange flesh melon contained higher beta-carotene than green flesh melon. INTRODUCTION Melon (Cucumis melo L.) is a fruit that comes from Cucurbitaceae . Currently in the market is found melons with green (var. Sky Rock) and orange flesh (var. Cantaloupe) (1). Orange-flesh melon has a sweeter taste than the green-flesh melon. Fruits as a melon in addition it contains many nutrients also contain pigments such as beta-carotene that make it more colorful. The color of fruits which is contain beta-carotene usually orange-red or may have different colors caused by other pigments that blend together with beta-carotene so as to form a unique color. (2) Beta-carotene is the pigment carotene which is a precursor of vitamin A. It is consisting of two groups of retinol, and when it was in the dehydrogenation will decompose to retinol and retinoic acid or vitamin A. In accordance with literature, melon contained vitamin A total 2140.00 SI / 100 grams. (3) and vitamin A has antioxidant activity. The content of vitamin A in melon indicate the presence of beta-carotene in the melon. The difference flesh color in the two types of melon varieties strengthen the suspicion of beta-carotene in the melon. Research on determination of beta-carotene in fruits and vegetables has previously been carried out as in carrots, papaya, Cantaloupe, pumpkin, and spinach, using spectrophotometry and high performance liquid chromatography (HPLC). In the previous studies have been conducted assay of beta-carotene in the fruit and vegetables carried out as in carrots, papaya, Cantaloupe, pumpkin and spinach. The method used is a spectrophotometric method and high performance liquid chromatography (HPLC), and the determination of beta-carotene in honey kapok (Ceiba pentandra) and longan honey (Nephelium longata L.) has been also carried out by TLC-densitometry.(4) In this research, analysis of beta-carotene in fruits green and orange melon (Cucumis melo L. var. Sky Rock and var. Cantaloupe) with TLC-densitometry method with reference to the condition of the research I Purwata Adi M. Oka, K. Ratnayani, and Ana Listya by changing the composition of the mobile phase into n-hexane-ethyl acetate (8: 2). (4) International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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METHODOLOGY Green and orange melon (Cucumis melo L. var. Sky Rock and var. Cantaloupe) from supermarkets in Depok area were used as sample. Chloroform (pa), n-hexane (distilled) , ethyl acetate (distilled), reference standard of beta-carotene (Sigma Aldrich). Analysis was performed by Densitometer Camag-TLC scanner 3, Digistore Camag-Reprostar 3, chamber, separating funnel, rotary evaporator, analytical balance, silica gel GF254 TLC plate, vial, linomat, capillary tube, filter paper, water bath, set of glassware used in laboratory. Sample Processing and Extraction: green and orange flesh melon (Cucumis melo L. var. Sky Rock and var. Cantaloupe) cleaned and peeled as generally for consumption, and extracted with chloroform 20 ml disposed cloroformnya phase. The extraction process be repeated 4 times with each 20 mL of chloroform, disposed of all phases of chloroform in a 100-mL volumetric flask and diluted with chloroform until the line mark. Chloroform phase concentrated by rotary evaporator to obtain a concentrated extract. Preparation reference standard of beta-carotene solution at concentration 200 ppm, and sample solution of green melon extract 10mg/2.0 mL of chloroform and sample solution of orange melon extract 10mg/5.0 mL of chloroform Optimization of TLC-Densitometry performed with selection of the mobile phase based on the mobile phase used by previous studies (4), which is a mixture of chloroform - ethyl acetate (7: 3) with a various composition, besides that it also performed the mobile phase composition of n-hexane - ethyl acetate with a various composition. It also carried out optimization the distance of migration mobile phase were used 8, 10 and 15 cm using silica gel GF254 plate, and determining the maximum wavelength by densitometer at specific wavelengths (200-700 nm) Qualitative Analysis was performed usde TLC by comparing Rf spot in sample with Rf reference standard of beta-carotene. It were observed visually, and a 254 nm UV lamp. Linearity test was performed to determine whether there is an influence of the sample matrix on the relationship between the concentration with peak area using a series solution consisting a minimum 5 samples of different concentrations and then the data is processed by using linear regression. Spotted a certain number of samples with 5 different concentration levels side by side, then eluated using the best mobile phase, then spotting gained broad peak measured using a densitometer at maximum wavelength of beta-carotene. Quantitative Analysis of Beta-Carotene was performed by spotted reference standard of betacarotene and samples solution each 20 µL on the plates side by side chromatographs, then eluated using the best mobile phase in a chamber . The plates were dried at room temperature and then measured peak area of beta-carotene with a densitometre at maximum wavelength 457 nm. Precision and accuracy test were performed by standard addition method, the determination of precision accuracy test performed 3 times. RESULT AND DISCUSSION Optimization Mobile Phase and Distance of Migration The selection starts with a mobile phase of chloroform-ethyl acetate in various compositions with the distance of migration mobile phase 8 cm, obtained Rf is too high. Then try to reduce the polarity of the mobile phase, replacing the mobile phase with a mixture of n-hexane -ethyl acetate in various compositions. Best separation was obtained from the mobile phase n-heksan- ethyl acetate (8: 2) with successive Rf value for reference standards of beta-carotene and analyte in orange melon extract: 0.77 and 0.75. Analysis of Beta-Carotene In Green And Orange Melon (Cucumis melo L. Var. Sky Rock And Var. Cantaloupe) By Thin Layer Chromatography 50 International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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In the qualitative analysis, the chloroform melon extract tested TLC using the mobile phase n-hexane ethyl acetate (8: 2) with a distance of migration of 10 cm, yielding one real orange patches on a sample of orange melon (var. Cantaloupe) with Rf 0.73 which is equal to the value of Rf reference standard of beta-carotene 0.73. Whereas in the samples of green melon (var. Sky Rock) produced three spots. First spots yellow with Rf 0.20, second spot dark green with Rf 0.39, and the third orange with Rf 0.725 approaching the value of Rf reference standard of beta-carotene. Results of identification by TLC can be seen in Fig 1. and Table 1. The results of the qualitative analysis showed the green and orange melon contain beta-carotene. Table 1. Rf value of beta-carotene Spot Reference Standar Beta-carotene

Rf 0.73

Green melon (var. Sky Rock) Orange melon (var. Cantaloupe)

0.725 0.73

Figure 1. TLC Chromatogram of green and orange melon

Determination of the Maximum Wavelength of Beta-Carotene Determination of the maximum wavelength is done by making spectrum reference standard of betacarotene in the range 200-700 nm. The results of determination can be seen in Figure 2, the maximum absorption is obtained at a wavelength of 457 nm.

Figure 2. Spectrum reference standard of beta-carotene


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Linearity test The linearity test showed a linear relationship between spot area with the concentration, linearity test results can be seen in Figures 3 and 4.

Figure 3. Linearity test curve of green melon (var. Sky Rock)

Figure 4. Linearity test curve of orange melon (var. Cantaloupe)

Data from linearity test is used to calculate the limit of quantitation (LOQ) of green melon and orange, respectively for 89.83 (mg / mL); 32.81 (mg / mL) Determination of Beta-carotene on green dan orange melon (Cucumis melo L. Var. Sky Rock Dan Var. Cantaloupe) by TLC-Densitometry The result of determination of beta-carotene in green and orange melon can be seen in Table 2. Table 2. Quantitative Analyze Result of Beta-carotene on Green and Orange Melons Material Green Melon (var. Sky Rock)

Orange Melon (var. Cantaloupe)

Content (mg/100g) 0.22 0.22 0.23 2.04 2.11 2.00

Average content (mg/100g) 0.22

2.05

The average content of beta-carotene in green melon is 0.22 mg/ 100 g and orange melon is 2.05 mg/ 100 g. Meanwhile according to literature, content of beta-carotene in the melon is 1.284 mg/100 g. Based on these results showed that there is a correlation between the colour of the fruit with higher levels of International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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beta-carotene contained in it. The more orange-red color of the fruit, the higher the content of betacarotene. Accuracy Precision Test The result of recovery test obtained by comparing the difference in the total weight of beta-carotene contained in the sample after the addition of reference standard of beta-carotene solution with the weight of beta-carotene contained sample to the reference standard weight of beta-carotene which is actually added. Precision test results obtained by calculating the relative standard deviation (RSD) from third 3% recovery of the sample solution is added a solution of reference standard beta-carotene. The test results reacquisition and precision green melon (var. Sky Rock) and orange melon (var. Cantaloupe) can be seen in Table 3. Table 3. The Results of Accuracy Precision Test in Green and Orange Melons Material Recovery (%) RSD (%) Melon hijau (var. Sky Rock) 98.76 1.50 Melon jingga (var. Cantaloupe) 98.81 1.14

The method used to meet the requirements of linearity with R2 = 0.9955 for green melon and 0.9854 for orange melon . The precision of the method is shown by the coefficient of variation of less than 2%, while accuracy indicated by the value recovery of 98.76% - 98.81% and limit of quantitation of green melon and orange, respectively for 89.83 (mg / mL); 32.81 (mg / mL). The method has been successfully to determine of beta-carotene in green melon is 0.22 mg/ 100 g and orange melon is 2.05 mg/ 100 g. CONCLUSION 1. The content of beta-carotene in green melon is 0.22 mg/ 100 g, with % recovery of 98.76% and RSD of 1.50%, and the content of beta-carotene in orange melon is 2.05 mg/100 g, with % recovery of 98.81% and RSD of 1.14%. 2. The result shows beta-carotene content in green and orange melon significantly differ; orange flesh melon contained higher beta-carotene than green flesh melon. REFERENCES 1. 2. 3. 4.

5.

Tim Redaksi Agromedia. Budi Daya Melon. Jakarta: Agromedia Pustaka; 2007. p. 1-14 Beta-karoten [Internet]. [cited 2015 May 5]. Available from: http://www. bbppbinuang.info/news17-beta-karoten.html. Rukmana R. Melon Hibrida. Yogyakarta: Kanisius; 1995. p 14 Parwata IM; OA, K. Ratnayani, Ana L. Aktivitas Antiradikal Bebas Serta Kadar Betakaroten Pada Madu Randu (Ceiba pentandra) dan Madu Kelengkeng (Nephelium longata L.). Bukit Jimbaran, Bali : Jurnal Fakultas Kimia FMIPA Universitas Udayana; 2010 International Conference On Harmonization (ICH). (2005). Validation of analytical procedure of: text and methodology.


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SPECTROPHOTOMETRIC METHOD PRECISION TO ASSAY OF LYCOPENE IN TOMATOES FRUIT (Solanum lycopersicum Lam.) Liliek Nurhidayati, Wening Ariwanty Faculty of Pharmacy, Pancasila University, Jakarta [email protected] ABSTRACT The main active substance in tomatoes fruit (Solanum lycopersicum Lam.) which found in large quantities was lycopene. Lycopene was one of the natural carotenoid antioxidants, as well as a pigment which give the red color in tomatoes. In this study, lycopene was extracted using Low Volume Hexane Method by addition of butylated hydroxytoluene. The mixture was filtered, and the absorbtion of n-hexane phase was measured at ±503 nm. Lycopene content in ripe fruits was 6.15 mg/100g , This method met the precision requirements with the relative standard deviation of 1.03%. Keywords : lycopene, tomatoes, visible spectrophotometry, precision INTRODUCTION Tomatoes (Solanum lycopersicum Lam) is one of the Solanaceae plant. The main active compound in tomatoes fruit was lycopene. Lycopene was good for health because of its antioxidant activity(1). Lycopene was one of potential antioxidant with the ability to reduce singlet oxygen twofold better than beta-carotene and tenfold better than the tocopherol (2). Lycopene content in tomatoes were interesting to be studied. It could be extracted using Fish et al, method. Lycopene was cool macerated using aceton, ethanol of 95%, and n-hexane and added with butylated hydroxytoluene (BHT) as antioxidant in aceton (3). Since it was a color pigment so the absorbance of lycopene could be measured in visible region . It has 11 conjugated double bond and the absobance could be measured at the longer wavelength (λmax 444, 470, 502 nm in n-hexane) (4). Before applied to assay the lycopene in tomatoes fruit this methods must be validated. In this research, the precision of visible spectrophotometry was tested. METHODS Ripened tomatoes (± 3.5 months) fruit was obtained from a local fruit farm at Gunung Cibiuk, Sukabumi. Taxonomic determination was performed by Herbarium Bogoriense LIPI, Bogor. Lycopene working standard was obtained from PT. Soho Industri Farmasi. Spectroscopic analysis was carried out on Shimadzu 1700 double beam UV/Visible spectrophotometer. Determination of maximum wavelength of lycopene Absorbtion spectrum of working standard lycopene solution (2.5 ppm in n-hexane) were recorded over the wavelength range of 300 to 700 nm against solvent blank, in quartz cuvetts with 1 cm diameters. Absorbance stability Absorption of working standar 2.5 ppm lycopene solution were recorded on 502.5 nm during 60 minute against solvent blank, in quartz cuvetts with 1 cm diameters. Calibration curve Preparation of calibration curve was performed by measuring the absorption of lycopene working standard solution of 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0 and 4.5 ppm in 502.5 nm. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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Precision test of spectrophotometry methods for determination of lycopene in tomatoes. Fresh tomatoes was cut into small pieces, blended. About 0.2 g of tomatoes juice was poured into Erlenmeyer, then added of 0.05% (w/v) BHT in 5 ml of acetone, 5 ml of 95% ethanol, 10.0 ml of nhexane. Put the Erlenmeyer inside the beaker glass with ice and placed on orbital shaker, rotated 180 rpm for 15 minutes. After 15 minutes of agitation, 3 ml aquademineralisata neutral was added, and shake again for 5 minutes. Filtered, the filtrate was transferred to a separating funnel and then allowed to stand 5 minutes at room temperature. Take n-hexane phase. The absorption of sample solution was measured in 502.5 nm against n-hexane as blank . Lycopene content was calculated using calibration curve equation. It was conducted six times to test the precision method. To identify of lycopene in tomatoes, absorption spectrum of sample solution were recorded over the wavelength range of 300 to 700 nm against solvent blank, in quartz cuvetts with 1 cm diameters. RESULTS Report from research Center for Biology, Indonesian Institute of Sciences mentioned that tomatoes has species name that is Solanum lycopersicum Lam. The spectrum of lycopene standard solution was showed in Fig.1.

Fig. 1. Absorption spectra of lycopene working standard solution in n-hexane

Absorbance stability Fig 2. Showed the result of absorbance stability test.

Fig. 2. The absorbance stability of lycopene solution in 502.5 nm


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Qualitative analysis The spectrum of solution extracted from tomatoes was shown in Fig 3.

Fig 3. Spectrum of sample solution I n-hexane Determination of lycopene content in tomatoes fruit Determination of lycopene content was conducted five times to assay the precision of this methods. The results was showed in Table 1. Table 1. The results of lycopene content determination Sample weight (g)

Absorption

Content of lycopene (mg/100g)

0.2783

0.3545

215.1

0.2746

0.3534

225.5

0.2871

0.3585

210.4

0.2850

0.3573

211.2

0.2823

0.3567

214.2

=6.15 SD= 0.0636 RSD = 1.03

DISCUSSION Determination of maximum wavelength was performed to choose the wavelength where the absorbance of both standard solution and samples were measured. Fig. 1 shows that the maximum wavelength of lycopene were 444.0 nm; 470.5 nm; and 502,5 nm. The highest absorption was in 502.5 nm. This wavelength was choosen to conducted the lycopene analysis. In this wavelength there was no disturbance of the other carotenoid(4). Absorbance stability test was performed to determine the correct time to measure the absorbance solution. The lycopene solution had stable absorbance during minute of 15 until 40. Calibration curve of International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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lycopene that performed using seven different consentration of lycopene showed that the equation of regression line was y = 0.0866 + 0.1557x with correlation coeffisient of 0.9913. This equation was used to calculate lycopene content. The solution spectrum of samples (Fig 3) was similar with the spectrum of lycopene working standard (Fig 1). It indicated that there were lycopene in ripe tomatoes. Detemination results with five replication showed that the tomatoes fruit has lycopene content of 6.15 mg/100 g with RSD of 1.03%. RSD is not allowed more than 2.0% (5) or based on the analyte content not more than 11.31% (6). The precision of this methods fulfill both of them. CONCLUSION The visible spectrophotometry using n-hexane as solvent in 502.5 nm fulfill the precision requirement to determination of lycopene in tomatoes fruit.

REFERENCES 1. 2. 3. 4. 5. 6.

Kailaku SI, Dewandari KT, Sunarmani. Potensi likopen dalam tomat untuk kesehatan. Buletin Teknologi Pascapanen Pertanian Volume 3 2007. pp .50-8 Sanjiv A. Rao AV. Tomato lycopene and its role in human health and chronic disease. Canadian Medical Association Journal. Vol. 163 (6). 2000. p.734-44 Fish WW, Veazie PP, Collins JK. A quantitative assay for lycopene that utilizes reduced volumes of organic solvents. J. Food Comp. and Anal. 15; 2002. p.309-17 Delia B, Amaya R, Mieko K. Harvestplus handbook for carotenoid analysis second ed. Washington DC and California: Harvestplus; 2004. p.4, 13-20 United States Pharmacopeial Convention. The United States Pharmacopeia 35-The National Formulary 30. Rockville: United States Pharmacopeial Convention. 2012. pp. 877-81. Harmita. Petunjuk Pelaksanaan Validasi Metode dengan Cara Perhitungannya. Majalah Ilmu Kefarmasian. Vol 1No.3 Desember 2004.h.119


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OPTIMIZATION AND VALIDATION OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR DETERMINATION OF CAFFEINE IN WHITE TEA Zuhelmi Aziz*, Dhiah Resti Fakultas Farmasi Universitas Pancasila Srengseng Sawah, Jagakarsa, Jakarta 12640 email: [email protected] ABSTRACT White tea comes from the terminal buds and leaves of Camellia sinensis, L which are still unopened and covered with a fine silvery-white hairs. Thence, white tea has more caffeine than green and black tea because active of biosynthesis of caffeine occurs in young tea leaves. In accordance with United State Pharmacopeae (USP) 35th, reverse phase high performance liquid chromatography (HPLC) is used to determination of caffeine. HPLC method has been optimized and validated for determination of caffeine in white tea. Optimum conditions for the HPLC in the experiment are as follows: stationary phase; Waters C18 (150 mmx 3.9 mm), column temperature 40oC, mobile phase; methanol - water (30:70) with a flow rate of 1.0 mL / min and UV detector with the maximum wavelength 273 nm. The result of validation as follow: the linearity test showed a linear relationship with a correlation coefficient; r = 0.9989, the accuracy and precision test appropriate ICH requirements. The experiment found that the contain of caffeine in white tea which is determine by HPLC in the optimum condition is 3.84%, with precision indicated by RSD value ≤ 2%, and accuracy indicated by recovery value 98.97% -99.72% with t-score which is less than the corrresponding value on the t-table (DoF=4, p=0.05) Keywords: Caffeine, High Performance Liquid Chromatography, White tea, Validation INTRODUCTION White tea comes from the terminal buds and leaves of Camellia sinensis, L which are still unopened and covered with a fine silvery-white hairs. White tea has a higher polyphenol and has more caffeine than green and black tea, because active of biosynthesis of caffeine occurs in young tea leaves. Therefore, it is necessary to the determination of caffeine in white tea. In accordance with United State Pharmacopeae (USP) 35th, reverse phase high performance liqiud chromatography (HPLC) is used to determination of caffeine . In this study, HPLC method has been optimized and validated for determination of caffeine in white tea. Optimization of HPLC performed with determining the maximum wavelength and composition of the mobile phase. Validation methode of HPLC performed on several parameters such as: linearity test, accuracy and precision test and sensitivity according to the International Conference On Harmonization (ICH). (8,9) METHODOLOGY Commercial Products white tea A and B were used as sample, reference standard of caffeine, methanol pro-HPLC, distilled water, aquabidest, chloroform, ammonium 30%. Analysis was performed by HPLC (Shimadzu Autosampler LC-20AD), Spectrophotometer Shimadzu 1800, analytical balance (Mettler AB 204), thermometer (Jena), ultrasonic cleaner (Elma LC 30 H), filter Millipore, syringe (special HPLC), rotary evaporator (IKA RV-10), and a glass tools that are commonly used in laboratory analysis (Pyrex Iwaki Glass). Optimization of HPLC performed with determining the maximum wavelength and composition of the mobile phase. The linearity test were used reference standard of caffeine solution at concentrations of 40, 60, 80, 100, 120, 140, and 160 ppm and each of which are added of the sample solution. Limit of detection and 58 International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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quantitation were determined based on linear line equation. Precision and accuracy test were performed by standard addition method. RESULTS AND DISCUSSION Optimum conditions for the HPLC in the experiment are as follows: stationary phase; Waters C18 (150 mmx 3.9 mm), column temperature 40oC, mobile phase; methanol - water (30:70) with a flow rate of 1.0 mL / min and UV detector with the maximum wavelength 273 nm. The standard curve equation is y = -8239.90 + 56507.23 x, with coefficient correlation (r) of 0.9986. While the result of the linearity test showed that the equation is y = 3306287.62 + 27932.56 x with coefficient correlation (r) of 0.9989 and limit of detection and quantitation of the method were 7.4 and 22.3 ppm.

Figure 1: The Curve of Linearity Test ; relationship between concentration(X) and peak area (Y).

Accuracy of the method can be showed by the value of recoveries of with the addition of reference standards caffeine 400 ug, 600 ug and 800 ug in white tea. The experiments were performed three times repetitions for each analyte concentration. Method is precise and accurate with the relative standard deviation (RSD) of ≤ 2% and precent recoveries of 98.97 – 99.72% are included in the range of requirements 97-103% with t-score which is less than the corrresponding value on the t-table. (DOF = 4, p = 0 05. The result of precision and accuracy test of caffeine in white tea can be seen in Table 1. Table 1: Results of precision and accuracy test of caffeine in white tea.

The method used to meet the requirements of linearity with r = 0.9989. The precision of the method is shown by the coefficient of variation of less than 2%, while accuracy indicated by the value recovery of 98.97% -99.72% and the t value smaller than t table in the degrees of freedom (df) = 4 and p = 0 05. The value of the limit of detection of 7.4 ppm and 22.3 ppm limit of quantitation. 59 International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University

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The method has been successfully to determine caffeine in white tea by HPLC in the optimum condition with the result is 3.84%. CONCLUSION Reverse phase HPLC method with in condition of: a stationary phase; Waters C18 (150 mmx 3.9 mm), column temperature 40oC, mobile phase; methanol - water (30:70), flow rate of 1.0 mL / min and UV detector with the maximum wavelength 273 nm met the requirements of ICH for determination of caffein in white tea, with limit of detection and quantitation of the method were 7.4 and 22.3 ppm. The content of caffeine in white tea is 3.84%.


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REFERENCES 1. Dalimartha, Setiawan. Atlas Tumbuhan Obat Indonesia. Jilid I. Jakarta: Trubus Agriwidya; 1999. h. 150-3. 2. Kubota, H. Patterns of Adenine Metabolism and Caffeine Biosynthesis in Different Parts of Tea Seedlings. Plant Physiology; 1986. h. 275-281. 3. Tiurmaida, Yuliana. Penetapan Kadar Kofein dalam Minuman Teh Kemasan secara Kromatografi Cair Kinerja Tinggi (Essay). Jakarta: Fakultas Farmasi Universitas Pancasila; 2012. 4. Komes, D. et. al. Determination of Caffeine Content in Tea and Mat Tea by using Different Methods. Czech J. Food Sci (journal) 2009; vol. 27:p.S213-6. 5. Skoog, D. A. Principles of Instrumental Analysis. Sixth Edition. Canada: Standford University; 2007. h. 818-48. 6. Harmita. Petunjuk Pelaksanaan Validasi Metode dan Cara Perhitungannya. Majalah Ilmu Kefarmasian. 2004; 1(3).h. 117-34. 7. Is White Tea Better Than Other Teas as a Potential Anticarcinogen?. Diambil dari: http://lpi.oregonstate.edu/news/whitetea.html. Diakes: 1 Februari 2014. 8. International Conference On Harmonization (ICH). (2005). Validation of analytical procedure of: text and methodology. 9. United States Pharmacopeial Convention (USP) 35th. (2012). The United States Pharmacopeia The National Formulary 32 27. Rockville: The United States Pharmacopeial Convention Inc., pp. 734-6.


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THE EFFECT OF EXTRACTION METHOD ON TOTAL ALKALOID LEVELS OF JEMBIRIT LEAVES (Tabernaemontana sphaerocarpa BL) WITH SPECTROFOTOMETRIC METHOD Nina Salamah, Miftahul Rozak Pharmacy Faculty, Universitas of Ahmad Dahlan Yogyakarta Jl. Prof. Dr. Soepomo, Janturan, Yogyakarta, phone 0274 – 385123 Email: [email protected]

ABSTRACT One of the plants which contain the alkaloid is a plant jembirit (Tabernaemontana sphaerocarpa BL. ). Sap and leaves from this plants have been used to treat skin diseases and sprain. Alkaloid from jembirit plants showed potent cytotoxicity against various human cancer cell. The goal of this research is to find out the influence of the extraction method against the level of total alkaloid jembirit leaves. Jembirit leaves were extracted by maceration method and extraction with Soxhlet apparatus used ethanol 70 % as a solvent. Standardization of extracts conducted by test of ash content, test of moisture content, and extract yield. Qualitative analysis conducted by alkaloid test. Determination of total alkaloid was analyzed with visible spectrophotometry method using Bromocresol green as complexing agent. The results showed that jembirit leaves contained alkaloid compounds. The determination resulted the levels of total alkaloid of maceration was 0.727% ± 0.0032, levels of total alkaloid extraction with Soxhlet apparatus was 0.666% ± 0.0022. The stastitical analysis showed significance differences of total alkaloid levels between maceration method and extraction with Soxhlet apparatus viewed from siginificancy value (0.0010.05) between groups, mean a normal and homogeneous data. One-way ANOVA test showed no significant different for total cholesterol (p=0.640) and trigyceride (p=0.840) concentration. Measurement of total cholesterol and triglyceride concentration on day 21 was conducted to determine effect of the treatment. One-way ANOVA showed significance difference between groups for total cholesterol and triglycerides (p= 0.00) thus LSD test was performed to explore further and compare the mean of one group with the mean of another. Figure 1 a,b showed mean of total cholesterol and trigyceride concentration on day 0, 14 and 21.

International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 109

Triglyceride conc

Total cholesterol conc (mg/dL)

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Figure 1a. Mean of total cholesterol concentration.

Normal control Negative control Positive control Low dose Medium dose High dose

Figure 1b. Mean of triglyceride concentration.

Summary of LSD test can be found in Table 1 and 2. Tabel 1. Summary of LSD test for total cholesterol concentration. Group Mean I II III IV V VI I 87,60 II 148,40 * III 92,80 * IV 114,40 * * * V 100,60 * * VI 94,20 * * Note: * no significantly different. Tabel 2. Summary of LSD test for triglyceride concentration. Group Mean I II III IV V I 59,20 II 79,60 * III 64,00 * IV 77,20 * * V 69,20 * * * VI 68,60 * * * Note: * no significantly different.

VI

The Tables showed that low, medium and high dose groups had lower mean concentration of total cholesterol and triglycerides compared to negative control. Medium and high dose groups showed no significant different compare to positive controls, mean red cabbage juices had the same ability with simvastatin in lowering total cholesterol and triglyceride concentrations. Total cholesterol concentration close to normal levels. Lower concentration of total cholesterol and triglyceride after treatment of red cabbage juice allegedly because Anthocyanin- cyanidin which has hypolipidemic effect. The underlying mechanism possibly by increasing the activity of LDL receptors in the liver so that activated LDL catabolism. A decrease in total cholesterol and triglyceride concentration may be associated with a decrease in the concentration of plasma LDL in which the two compounds are constituent of LDL molecules. Anthocyanin is suspected to inhibit the absorption of cholesterol in the gastrointestinal tract or inhibit cholesterol synthesis in the liver(3). High fiber contained in red cabbage can also be able to lower total cholesterol concentration(8). International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 110

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CONCLUSION Red cabbage juice can decrease concentration of total cholesterol and triglyceride in hyperlipidemia mice; Reduction level of medium and high dise as effective as simvastatin, close to normal levels for cholesterol where dosage II is the most effective for lowering the concentration of total cholesterol and triglycerides. ACKNOWLEDGMENTS The Authors are Grateful to Pharmacy Faculty of Pancasila University for Incentive Research Funding.

REFERENCES 1. 2. 3.

4.

5. 6. 7.

8.

Murray RK. Biochemistry harper. XXII Edition. Translated by A Hartono. EGC. Jakarta: 1995. p. 252-93, 308-10. Kelompok kerja ilmiah. Penapisan farmakologi, pengujian fitokimia dan pengujian klinik. Jakarta: Yayasan Pengembangan Obat Bahan Alam Phyto Medica; 1993. h.191. Davalos A, Fernandez-Hernando C, Cerrato F, Martinez-Botas J, et al. Red grape juice and polyphenols alter cholesterol homeostasis and increase LDL-receptor activity in human cells in vitro. J Nutr, 2006. 136. 1766-1773. Middleton E, Kandaswami C, Theoharides CT. The effect of plant flavonoids on mammalian cells: implication for inflammation, heart disease, and cancer. Pharmacological Reviews, 52 (4).2000.673751. Huang D J, Lin C D, Chen H J, Lin Y H. Antioxidant and antiproliferative activities of sweet potato (Ipomoea batatas L. Lam Tainong 57) constituents. Bot Bull Acad. Sin, 45: 179-186. Kwon SH, et al. Anti-obesity and hypolipidemic effects of black soybean anthocyanins. JM Food. 2007. 10 (3).552-556. Fox GJ, Barthold WS, Davisson TM, Newcomer EC, Quimby WF, Smith LA. The mouse in biomedical research: normative biology, husbandry, and model. 2nd edition. UK: Academy Press; 2007. P. 188. El mowafy, A. Maha. Treatment effect of red cabbage and cystein against paracetamol induced hepatotoxicity in experimental rats. Journal of Applied Science Research. 2012; 8(12): 5852-5859.


ISBN : 978-602-72418-2-4

Hepatoprotective Study of Cosolvent Solution From Mangosteen (GARCINIA MANGOSTANA L.) Rind in Rats ROS SUMARNY*, LILIEK NURHAYATI, YATI SUMIYATI, ASTRI YULIASTRI PERMANA Faculty of Pharmacy, Pancasila University, Jakarta, Indonesia. Email: [email protected]

Abstract: Garcinia mangostana L. rind consist of -mangostin, an active compound that considerably can protect the liver -mangostin which prepared as cosolvent solution and ethanolic extract suspension. This was experimental study where hepatoprotective effect was assessed using Aspartate transminase (AST) and Alanine transminase (ALT). Twenty five rats were divided into 5 groups: negative (I) and positive controls (II); cosolvent (III), extract (IV) and commercial extract® suspensions (V) of 33,1 mg/kg BW -mangostin. Rats were treated for 14 days, and then induced by carbon tetrachloride to produce trichlorometylperoxyl, a free radical which cause lipid peroxidation. AST and ALT levels were analyzed by using kinetic UV method. Kruskall Wallis and Mann Whitney test were used in statistical analysis. AST levels were 207.08±9.17; 111.98±29.90; 134.54±30.00; 128.10±21.84 and 138.84± 16.62 UI/L meanwhile ALT levels were 79.18±17.29; 28.02±5.17; 37.1±5.25; 46.34±13.19 and 34.5±4.09 UI/L for group I, II, III, IV, V respectively. There were no significant differences between groups II with group III, IV and V (p0.05) for ALT and AST, meaning cosolvent, extract and extract ® has comparable ability to inhibit enzymes increment. Keywords: α-mangostin, Garcinia mangostana L, AST, ALT, hepatoprotective. INTRODUCTION Mangosteen (Garcinia mangostana L.) is a tropical fruit where mangosteen rind mostly thrown away as a waste. The rinds previously has been utilized only for tanning leather, anti-rust and textile dyes. Research showed that mangosteen rind contains many compounds that are beneficial to health such as anthocyanins, tannins, polyphenols, epicatechin and xanthone, including α-, -, -mangostin(1). Mangosteen rind extract were marketed in capsule dosage form, meanwhile it is known that mangostin has very low solubility in water (1:>10.000). Enhancement of α-mangostin solubility has been studied by Fatimah who formulates oral solution of mangosteen rind extract by using cosolvency technique(2). Liver has complex role of metabolism in the body. Hepatotoxic compounds such as carbon tetrachloride, is metabolized by liver which produce free radicals that bind unsaturated fatty acids cause lipid peroxidation and lead to liver damage(3). Antioxidant activity of mangosteen rind cosolvent solution has similarities mechanism in preventing free radicals that cause liver damage, presumed mangosteen rind has an activity as hepatoprotector. Thus, to investigate in vivo hepatoprotective activity of cosolvent mangosteen rind solution, a comparative study against mangosteen rind extracts suspension was conducted. There were two types of extracts: the same extracts of cosolvent solution (hereinafter referred as extract) and marketed extract from capsule dosage form (hereinafter referred as extract®). Levels of α-mangostin in cosolvent solution and extracts is equated, 33.1 mg/kg. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 112

ISBN : 978-602-72418-2-4

METHODS Materials. Mangosteen rind extract solution with 8,94% α-mangostin; mangosteen rind extract capsule with 15,32% α-mangostin; curcumin; PEG 400, glycerin, aqua demineralization, Diagnostic kit for AST and ALT, alcohol 70, 80, 90 and 100%; formalin 40%; glacial acetic acid; solid paraffin; xylol; hematoxylin 2%; hydrochloric acid. Equipment. Analytical balance (Kern KB 3600-2N), laboratory glassware, rotary evaporator (Buchi R-205), rat cage, animal scales, syringes (Terumo), oral sonde, surgical tools, eppendorf tube, microcentrifuge (Kubota m6800), micropipette (Transferpette), MicroLab 300, microscope (Olympus BH2), object and cover glass. Cosolvent Solution and Extracts Suspension Preparation. Determination of mangosteen rind was performed at “Herbarium Bogoriense”, Indonesian Institute of Sciences - Cibinong. One kg of mangosteen rind powder was extracted by maceration using 70% ethanol (1:4) for 3x24 hours with stirring. The filtrate is evaporated at 400C and 120 mBar, and evaporated on a water bath to obtain a viscous extract. Solution is made from the extract by cosolvency techniques. Extract and extract® suspension were suspended into CMC. Animals. Twenty five of Sprague-Dawley strain rats were adapted to the laboratory environment for a week and divided into 5 groups: group I (negative control) were given cosolvent, group II (positive control) were given curcumin dose of 75 mg/kg bw, group III (cosolvent solution), group IV (extract suspension), group V (extract® suspension). Treatments were given for 14 days. CCl4 (dose of 1 mL/kg bw) were induced on day 14, two hours after the last administration of preparation. Blood was collected on day 16. AST/ALT measurement. 50 uL of serum was added to 500 uL mixture of R1-R2 AST/ALT (1:4); After Incubation for a minute, measurement was performed by using Microlab 300.

RESULTS Average of AST and ALT concentration is shown at Table 1 and 2. Table 1. Summary of Mann Whitney Test for AST (IU/L). Group I (negative ) II (positive) III (cosolvent) IV (extract solution) V ( extract® solution)

Average of AST(UI/L) 207.08 ± 9.17a 111.98 ± 29.90bc 134.54 ± 30.00bcd 128.10 ± 21.84bcde 138.84 ± 16.62bcde

Note:

Different sign at the same column showed significant different (p α 0.05 and homogeneity test results obtained significance value 0.676 > α 0.05. ANOVA table of the results of the activity of catalase significance value 0,000 < α 0.05. This shows the significant difference between treatments. It can be concluded that the effect of the test preparation to the decrease of catalase activity. Catalase activities for Tukey test showed that the first dose group there was no significant difference in dose group II and III dose there was no significant difference in the positive control group. Table 2. Results of measurement of catalase activity. Group Average (Unit/mL) ± SD Normal

119,835 ± 7,46 a

Negatif

22,415 ± 2,97 b

Positif

94,615 ± 5,61 c

Dosis I

54,067 ± 5,67 d

Dosis II

65,902 ± 4,09 d

Dosis III

88,567 ± 4,80 c,e


ISBN : 978-602-72418-2-4

Table 3. Results of measurement of total cholesterol levels.

Group

Normal

Negatif

Positif

Dosis I

Dosis II

Dosis III

Measurement of Total Cholesterol Levels Repeat

Total cholesterol

1

73.22

2

67.9

3

84.86

4

79.8

1

249.23

2

229.75

3

248.68

4

269.7

1

232.14

2

270.93

3

251.45

4

236.95

1

260.33

2

237.14

3

227.49

4

245.85

1

244.03

2

241.91

3

209.44

4

218.95

1

247.05

2

229.19

3

257.23

4

217.42

Average (mg/dl) ± SD

76.445 ± 7.427

249.34 ± 16.317

247.867 ± 17,428

242. 703 ± 13.940

238.582 ± 13.466

247.723 ±13.270


ISBN : 978-602-72418-2-4

Table 4. Results measurement MDA levels. Measurement MDA levels

Group Repeat

Normal

Negatif

Positif

Dosis I

Dosis II

Dosis III

Absorbansi

Average (µg/ml) ± SD

MDA levels

1

0.227

1.255

2

0.263

1.454

3

0.266

1.471

4

0.254

1.405

1

0.642

3.550

2

0.675

3.733

3

0.742

4.103

4

0.673

3.722

1

0.328

1.814

2

0.293

1.620

3

0.316

1.747

4

0.302

1.670

1

0.457

2.527

2

0.427

2.361

3

0.484

2.677

4

0.414

2.289

1

0.361

1.996

2

0.382

2.112

3

0.367

2.030

4

0.378

2.090

1

0.327

1.808

2

0.289

1.598

3

0.311

1.720

4

0.334

1.847

1.396 ± 0.098

3.777 ± 0.233

1.713 ± 0.085

2.464 ± 0.173

2.057 ± 0.054

1.743 ± 0.110


ISBN : 978-602-72418-2-4

Table 5. Results of measurement of catalase activity. Measurement of Catalase Activity Group

Normal

Negatif

Positif

Dosis I

Dosis II

Dosis III

Average (Unit/mL) ± SD Repeat

Activity Catalase

1

123.89

2

118.2

3

127.14

4

110.11

1

23.88

2

20.12

3

19.77

4

25.89

1

102.34

2

94.76

3

89.32

4

92.04

1

51.97

2

48.03

3

54.78

4

61.49

1

63.14

2

68.05

3

61.87

4

70.55

1

89.19

2

81.72

3

92.82

4

90.54

119.835 ± 7.46

22.415 ± 2.97

94.615 ± 5.61

54.067 ± 5.67

65.902 ± 4.09

88.567 ± 4.80

CONCLUSION Based on the results of this study concluded that 70% ethanol extract of white-oyster mushroom (Pleurotus ostreatus (Jacq.) P. Kumm at a dose of 42 mg/ kg BW, 84 mg / kg BW, and 168 mg / kg BW was able to reduce levels of MDA and increased activity catalase. Dose of 168 mg / kg BW was able to reduce levels of MDA and increase catalase activity comparable to atorvastatin dose of 5.2 mg / kg BW.


ISBN : 978-602-72418-2-4

REFERENCES 1. 2. 3.

4. 5. 6.

7. 8.

9.

Trilaksani W. 2003. Antioksidan: jenis, sumber, mekanisme kerja dan peran terhadap kesehatan. Tesis. Institut Pertanian Bogor, Bogor. Hlm.12. Priyanto. Toksikologi, mekanisme, terapi antioksidan dan penilaian resiko. Editor Hadi Sunaryo. Leskonfi, Depok. 2009. Hlm.73-86. Mutiasari IR. Uji Aktifitas antioksidan jamur (pleurotus ostreatus) dengan metode DPPH dan identifikasi golongan senyawa kimia dari fraksi teraktif. Skripsi. Fakultas FMIPA Universitas Indonesia, Depok. 2012 .Hlm.41. Nawawi RH. Uji aktivitas, stabilitas fisik dan keamanan sediaan gel pencerah kulit yang mengandung ekstrak jamur tiram. Tesis. FMIPA Universitas Indonesia. Depok. 2012. Hlm. 94 Suniarti FRT. Aktifitas antioksidan jamur tiram putih (Pleurotus ostreatus) rebus, panggang dan goreng pada tikus sparague dawley hiperkolesterolemia. Tesis. UGM, Yogyakarta. 2014. Abstrak. Asri DS. Aktivitas antihiperkolesterolemia fraksi etanol ekstrak daun kelor (Moringa Oleifera Lam.) Berdasarkan kadar kolesterol total dan LDL kolesterol. Skripsi. Fakultas Farmasi dan Sains. UHAMKA, Jakarta. 2014. Hlm. 17. Soewoto, H. dkk. Biokimia eksperimen laboratorium. Widya Medika. Jakarta. 2001. Hlm. 153. Priyanto. Status oksidan dan antioksidan serta pengaruh pemberian kombinasi vitamin (E + C) pada Polantas di kota besar dan polisi di pedesaan. Tesis. Program Pasca Sarjana Bidang Ilmu Kesehatan. Universitas Indonesia. Depok. 1999. Hlm. 36. Mayes PA, Botham PA. Cholesterol synthesis, transport, and excretion. McGraw-Hill. New York. 1996. Hlm 26: 219-230.


141

Soursop Leaf (Annona muricata L.) Infusion in Lipid Profile of Hyperlipidemic Mice NI MADE DWI SANDHIUTAMI*, NENI ANGGRAINI Faculty of Pharmacy, Pancasila University. Email : [email protected]

Abstract: The increase of blood lipid levels can be leaded into atherosclerosis. Soursop leaf (Annona muricata L.) contains flavonoid, that can be used as anti hyperlipidemia. This study aims to determine the effect of soursop leaf (Annona muricata L.) infusion for the reduction of Total Cholesterol, Triglyceride, LDL, and also the elevation of HDL. In this experiment, male mice (DDY strain) were induced by 80% yolk, 65% sucrose solution 15%, and 5% animal-fats two times/day, for 14 days. 30 mice were divided into 6 groups of 5 mice each: a normal group (I), a negative control group (II), a positive control group treated with simvastatin (III), and a final group had been treated with soursop leaf infusion (1.4 g/kg BW, 2.1 g/kg BW, and 2.8 g/kg BW) (IV). The preparation given at 15th – 21st day and were observed on 0, 14th, and 21st days with a photometer 4010. On the 21st day, the measurement of Total Cholesterol, Triglyceride and LDL decreased to 33.12%, 35.95%, and 46.05%, while HDL increased to 36.36% in mice that had been given soursop leaf infusion dose 2.8 g/kg BW. The results showed that a soursop leaf infusion dose 2.8 g/kg BW is equal to simvastatin dose 1.3 mg/kg BW. Keywords: Soursop leaf, Annona muricata L., flavonoid, hyperlipidemic.

INTRODUCTION Nutritious food is needed as an additional health factor. One of the effects potentially experienced if a lot of food high in saturated fat is consumed is hyperlipidemia. Hyperlipidemia is an increase in the total level of cholesterol, LDL, triglyceride, or several of them, or a decrease in HDL (1). Hyperlipidemia can lead to atherosclerosis, which in turn can lead to coronary heart disease(2). The blood lipid level can be reduced with hypolipidemic drugs, one of which is klofibrat. Klofibrat was withdrawn from circulation because increased mortality had been documented in users of the drug(2). The potential of natural material for treatment is being investigated because it is cheap and has a smaller risk than chemical treatment. Soursop (Annona muricata L.) has long been known as a medicinal plant; its leaves, flowers, fruits, seeds, roots and bark can be used for medical purposes(3). The results showed that the ethanol extract from the soursop bark (Annona muricata L.) has antihyperglycemic and hypolipidemic activity, especially in lowering the state of hypertriglyceridemia and hypercholesterolemia induced by alloxan in rats(4). Soursop leaves contain flavonoid compounds that can be used as agents for antihiperlipidemia. Commonly, soursop leaves are used to lower blood cholesterol levels by boiling them in water. Given the hypolipidemic activity found in the bark of the soursop, the public assumption that the leaves of the soursop lower cholesterol levels requires further testing. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 141

ISBN : 978-602-72418-2-4

MATERIAL AND METHODS MATERIAL : Soursop leaf (Annona muricata L.), simvastatin, inducer of hyperlipidemia, and reagents kits (Biolabo). Tools. Oral probes, syringes, animal scales, analytical scales, glasses, surgical instruments, cottons, eppendorf tubes, centrifuge, and the photometer 4010. Animal preparation : Mice, DDY strain, male, 2-3 months old, weight 25-30 grams. Preparation of the soursop leaf infusion (Annona muricata L.) Fresh leaves were collected and washed in tap water to remove feces, and then drained, also dried, however do not dry it in direct sunlight. Soursop leaf infusion was made by boil 11.2 grams soursop leaf simplicia (equivalent to 35 sheets soursop leaf simplicia) with 125 mL water. Heating begins at 900 C for 15 minutes. Infusaion was filtered while hot with flannel. Infusion volume was obtained 100 mL, So that, the concentration of soursop leaf is 11.2%. Administration volume was given according to dose 1.4 g / kg, 2.1 g / kg, and 2.8 g / kg. Preparation of Hyperlipidemia Inducers. Animal-fat was obtained from chicken skin. It weighed in 100 grams, then heated it in a non-stick frying pan until all the oil come out. 65% sucrose solution was prepared by dissolve 65 grams of sugar in 100 ml hot water. Yolk were used as an emulsifier. Each ingredients is were mixed according to its weight and volume. inducers was made every day and given it orally two times/day. Preaparation of simvastatin suspense dose 1.3 mg/kg. 0.5% CMC Na solution was made by weigh in 0.5 gram CMC Na in hot water with volume 20 times of weight of CMC Na, that is 10 mL and let it stand for approximately 30 minutes until its expands. 20 tablets simvastatin which has been weighed, and crushed into powder and converted into the dose used in mice. 0.1 grams simvastatin powder is mixed with CMC Na which has expanded and crushed until homogeneous, then added to 100 mL distilled water. Soursop leaf (Annona muricata) Infusion in Lipid Profile of Hyperlipidemic Mice. Animals were divided into 6 groups of 5 mice each: a normal group, a negative control group, a positive control group treated with simvastatin dose 1.3 mg/kg, and a final group had been treated with soursop leaf infusion (1.4 g/kg BW, 2.1 g/kg BW, and 2.8 g/kg BW) The lipid levels was measured in all groups on day 0 and furthermore given a hyperlipidemia inducers dose 17.5 g/kg BW two time/day for 14 days. Remeasurement of lipid levels and given the preparation test for 7 days. RESULTS AND DISCUSSION Soursop leaf infuse (Annona muricata L.) positively contains flavonoids in the form of yellow color in amyl alcohol layers. Flavonoid compounds can be used as anti hiperlipidemia which affect total cholesterol, triglycerides, LDL, and HDL blood levels. The average total cholesterol levels (mg/dL)

Average of total cholesterol levels (mg/dL)

200 150 Normal Control Negative Control Positive Control Low Dose

100 50 0 0

14

21

Experimental time (days)

Average total cholesterol levels in mice (mg/dL).


ISBN : 978-602-72418-2-4

The total cholesterol levels in mice on day 0 is in normal range. According to the literature, the levels in normal mice is between 55-128 mg/dL(5)). On the 14th day 14 the total cholesterol levels increased in all groups, except a normal group. Measurements on day 21 showed that a moderate dose (2.1 g/kg) and high dose (2.8 g/kg) were able to reduce total cholesterol compared to a negative control group. A decrease of total cholesterol levels were significantly occurred in a positive control group. It had a total cholesterol levels of 92.40 ± 11.82 mg/dL on day 21. A high doses of 2.8 g/kg body weight show no significant difference to positive control group with a percentage 33.12%, so that it has proven its effectiveness in lowering total cholesterol levels. The average triglyceride levels (mg/dL) Average of triglyceride levels (mg/dL)

150 100

Normal Control

Negative Control

50

Positive Control Low Dose

0

Medium Dose

0

14

21 Experimental time (days)

High Dose

Average triglyceride levels in mice (mg/dL).

Based on the chart above that triglyceride levels in mice on day 0 are in the normal range. According to the literature, the levels in normal is between 13-67mg / dL(5). The triglyceride levels in all groups increased On day 14, except a normal group. However, the triglyceride levels decreased on day 21. A decreased significantly in a positive control group, in low-dose (1.4 g / kg), moderate-dose (2.1 g / kg) and high-dose (2.8 g / kg). A positive control group has the higher levels of triglycerides 60.20 ± 7.22 mg / dL on day 21. Low-dose (1.4 g / kg), medium-dose (2.1 g / kg) and high-dose (2.8 g / kg) have no significant difference with a positive control group, so that, all of three doses is are effective in hypertriglyceridemia. High-dose 2.8 g / kg could reduce triglyceride levels to 35.95%. The average LDL levels (mg/dL) Average of LDL levels (mg/dL)

150 100

Normal Control

50

Negative Control Positive Control Low Dose

0 Medium Dose

0

14

21 Experimental time (days)

Average mice LDL levels (mg/dL).

The average LDL levels in mice has changed during study. On day 14, an increased in LDL cholesterol levels show any significant in all groups compared to a normal control group. A decreased in LDL levels at low-doses (1.4 g / kg) and low-dose (2.1 g/kg) do not show any significant difference to a negative control group. A decreased in LDL levels at high-dose (2.8 g/kg) does not show significant different with a positive control group and normal group, so that, high-dose (2.8 g / kg) is able to reduce LDL levels and it effect is similar to simvastatin with precetage is 46.05%.


ISBN : 978-602-72418-2-4

Average HDL levels in mice (mg / dL).

The chart above shows the changes in HDL levels in mice during study, especially on days 0, 14, and 21. On Day 0, the range of HDL levels in all groups is between 27.60 ± 3.58 mg/dL ─ 30 60 ± 2.97 mg/d. On day 14, a decreased HDL levels in mice occurred in the group II, III and IV. On day 21, an increased HDL levels in low-dose (1.4 g / kg) and moderate-dose (2.1 g / kg) do not show any significant difference compared to a normal group and a negative control group. High-dose (2.8 g / kg) could elevate the HDL levels which is has no significant difference to a positive control groups and normal controls group. So that, high-dose (2.8 g / kg) could increase the HDL levels with percentation is 36.36%, and this effect is similar to simvastatin. Results of in vitro studies indicate that flavonoids work as inhibitors of HMG-CoA reductase enzyme that decreases cholesterol synthesis(6). Flavonoids also increases the activity of lipoprotein lipase, and therefore contributes to a decrease in blood triglyceride levels(7). In addition, flavonoids can affect the metabolism of LDL cholesterol by increasing LDL ability to bind to its receptor. LDL bound to the receptor to be metabolized into cholesterol ester in the network. HDL binds cholesterol esters contained in the network and then excreted into the small intestine(8). CONCLUSION Soursop leaf infuse a dose of 2.8 g/kg BW can decreases total cholesterol, triglycerides, and LDL, also increase HDL levels. REFERENCES 1. Wells BG, Dipiro JT, Schwinghammer TL, Dipiro CV. Pharmacotherapy handbook seventh edition. The McGraw-Hill Companies; 2009. p. 98, 102-103. 2. Gunawan SG, Setiabudy R, Nafrialdi, Elysabeth. Pharmacology and therapeutics 5th edition (reprinted with improvements). Jakarta: Department of Pharmacology and Therapeutics Faculty of Medicine, University of Indonesia; 2008. p. 373-77, 379-88. 3. Mardiana L, Ratnasari J. Herb and efficacy of soursop. Jakarta: Penebar Organization; 2011. p. 17, 38-40. 4. Ahalya B, Shankar KR, Kiranmayi GVN. Exploration of anti-hyperglycemic and hypolipidemic activities of Ethanolic extract of Annona muricata bark in alloxan induced diabetic rats. Int J Pharm Sci Rev Res. 2014; 25 (2): 21.26. 5. Fox JG, Barthold SW, MT Davisson, Newcomer CE, Quimby FW, Smith AL. The mouse in biomedical research: normative biology, husbandry, and models, second edition.UK: Academy Press; 2007. p. 188. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 144

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6. Prahastuti S, Tjahjani S, Hartini E. Effect bay leaf infusion (Syzygium polyanthum (Wight) Walp) against a decrease in total blood cholesterol levels of rat models of dyslipidemia wistar strain. Medical journal Planta. 2011; 1 (4): 27-9. 7. Arauna Y, Aulanni 'am, Oktavianie DA. Study triglyceride levels and histopathological picture liver animal model rat (Rattus norvegicus) hypercholesterolemia treated with aqueous extracts of mango parasites (Dendrophthoe petandra). Malang: Study Program of the University of Brawijaya Veterinarian; 2012. p. 2. 8. Rofida S, Firdiansyah A, Fitriyastuti E. Activities antihiperlipidemia ethanol extract of leaves of Annona squamosa L. J Pharm Sci Pharm Pract. 2015; 2 (1): 1-3.


ISBN : 978-602-72418-2-4

Xanthine Oxidase Inhibitory Activity of Secang (Caesalpiniasappan L.), Tempuyung (Sonchusarvensis L.), and Kepel (Stelechocarpusburahol) Extracts PERTAMAWATI*, MUTIA HARDHIYUNA, SHELVI LISTIANA, RIAN TRIANA

Center for Pharmaceutical and Medical Technology – LAPTIAB – BPPT PUSPIPTEK Area – Serpong – Banten. e-mail : [email protected]

Abstract: As we all know that xanthine oxidase is an enzyme that act as catalyst in the process of oxidizing hypoxanthine to become xanthine and then into uric acid. Uric acid is the product of metabolism of purine that settles in the joints and form crystal that sparks great pain and stiffness, also an enlargement and protrusion of swollen joints. As synthetic drug commonly used to overcome uric acid is allopurinol. Allopurinol work by inhibiting the formation of uric acid precursor (xanthine and hypoxanthine), however allopurinol have few side effects, sometimes occurs in gastrointestinal toxicity and increase gout attack acute at the beginning of therapy. Hence, many people use medicinal plants as anti uric acid because it has less side effects, easy to get and are relatively inexpensive as opposed to synhesis medicine. Bark of secang, tempuyung leaves and kepel leaves have the capability to inhibit of the activity of the xanthine oxidase until 56,473%, 20,154% and 12,071% while allopurinol capable of inhibiting the activity of the xanthine oxidase until 87,474%. The result of this research proves that bark of secang, tempuyung leaves and kepel leaves having activity to inhibit of xanthine oxidase, so that it can be used as traditional medicines for anti uric acid. Keywords : Xanthine oxidase, secang, tempuyung, kepel, inhibitory activity.

INTRODUCTION Xanthine oxidase is an enzyme acting as catalyst in the oxidation process ofhypoxanthine to xanthine and later became uric acid. Xanthine oxidation reduces O2 to H2O2 in cytosol and is expected to be a mayor factor ischemia injury, especially on cells of the intestinal mucosa. Xanthine oxidase is homodimer catalytic subunit independent, an enzyme that catalyzes hipoxanthine to xanthine and xanthine to uric acid, which is the relegation of purine. Xanthine Oxidase oxidizes oxypurine to xanthine and hypoxanthine to become uric acid. So everything septal on the metabolism of purine will produce uric acid and will cause deposit of sodium hydrogen urate monohydrate crystal. In an individual with the lack of O2, the body will form Xanthin Oxidase through degradation of ADP. In a person with high intake of purine can produced uric acid more easily. If alcaloida is present at high level in the blood, the existence of enzyme Xanthine Oxidase will form uric acid. Uric acid is the metabolism product of purine that settles at joints and form small crystals that may caused great pain and stiffness, also an enlargement and protrusion joints that swollen. In certain conditions, elevated levels of uric acid can occur in the blood exceeding normal limits called hiperurisemia(1). Hiperurisemia can be caused by excess production level of uric acid, the excretion of uric acid through the kidneys that diminished or combination both of them(2). Hiperurisemia can develop International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 146

ISBN : 978-602-72418-2-4

into gout. Gout is a type of metabolic disease. Its existence is quite popular among the community as pirai(3). A synthetic drug commonly used to overcome uric acid is allopurinol. Allopurinol is an analogous of uric acid, works by inhibiting the formation of uric acid from their precursor (Xanthine and Hipoxanthine) by inhibiting the activity of the Xanthine Oxidase(3). However allopurinol have few side effects such as redness of the skin, leukopenia, sometimes toxic in gastrointestinal and increase acute attack in early therapy(4). Therefore, nowaday people use medicinal plant as a traditional remedy because it has relatively small side effects, easy to get and are relatively inexpensive as opposed to synthesis medicine. Bark of secang (Caesalpinia sappan L.), has been empirical used as material for the treatment of uric acid disease. Various substance contained in the bark secang include brazilin, an alkaloid, flavonoid, saponin, tannin, phenyl propane and terpenoid . It also contain gallic acid , brasilein, delta-a phellandrene, oscimene, resins and resorin(5). Perceived research is weak in terms of effect anti-hiperurisemia. Because of the great biology potential of secang needs to be surveyed as agent of anti hiperurisemia. Tempuyung (Sonchus arvesnsis) is a herb found in the wild that grows in the mois areas that have high enough intensity of rainfall. The chemical content contained in this plant are mineral ions include silica , potassium , magnesium , sodium and compound organic kind of flavonoid (kaemferol, luteolin-7o-glukosida and apigenin-7-o-glukosida), coumarin (scepoletin) , taracsasterol , inositol , and acid fenolat (cinnamic , cumarat and vanilat). In a report, the total flavonoid content in the tempuyung leaves is 0,1044 %. Whereas tempuyung root contain a total alkaloid of more or less 0,5 % and the highest flavonoid is apigenin-7-0-glukosida. Apigenin-7-0-glukosida is one of the flavonoid that have the potential to hinder the activity of Xanthine Oxidase and superoxidase, so that the formation of uric acid can be constrained or reduced(6). Kepel or burahol (Stelechocarpus burahol)l is a tree producing the fruit Kepel fruit is popular with women because is believed to make fragant scented sweat and make water odorless. For medicine, meat this fruit serves as deciduous urine, prevent inflamation of the kidney and cause sterility (temporary) in women. Its timber is suitable to wrench households and reported to last 50 years. The purpose of this experiment is to find out the Xanthine Oxidase inhibitory effect of three extracts of secang bark (Caesalpinia sappan L.), tempuyung (Sonchus arvesnsis) and kepel (Stelechocarpus burahol). Data processing acquired is carried out a statistical analysis of the variants and methods to test the difference between the average values of the two treatment used the methods of Multiple Region Test Duncan. The results of the experiment are expected to provide information to improve health by developing Obat Herbal Terstandar/ Standardisea Herbal Medicine (OHT). This research was carried out in April until June 2014 in the laboratory of Center of Pharmaceutical and Medical Technology – LAPTIAB - TAB - BPPT. MATERIAL AND METHODS MATERIAL. Bark of secang, leaves of tempuyung and leaves of kepel are mashed into small pieces (approximately 1 mm). Chemicals. DMSO (Sigma), K2HPO4 (Sigma P-0662), NaOH (Sigma 221465), HCl (Merck), Xanthine (X 0626), Xanthine Oxidase (Sigma X 4375), ethanol 96%, aquadest free CO2, Allopurinol. Equipment. Percolator (BuchiPump Controller C-610 and Buchi Pump Module C-610: UVVIS1700 Spectrophotometer Shimadzu PharmaspecGlassware. METHODS. Plant Identification. The plants were identified in Research Center for Biology, Indonesian Institute for Science (LIPI). Preparation of a solvent extraction simplisia.The solvent for simplisia are ethanol 96% as much as 30%, 50% and 70%. and for the comparison of simplisia are 1:10; 1:15 and 1:20. Preparation of Extract. The percolation used as usual, with material secang herbs, tempuyung herbs and kepel herbs. Percolation run for 2 hour until the expected extract is obtained. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 147

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Preparation of Solutions. Potasium phosphat dapar solution pH 7.5 (50 mM). Substrat solution 0.75 mM (Xanthine) make it fresh. Enzyme solution. Enzyme solution stock. Solution of enzyme 0.3 unit/ml. Sampel solution : - Stock solution (LS); Working solution (LK) Preparation of Comparison Solution. Allopurinol tablets crushed to smooth and put into squash measure. Add phosphat buffer and sonification for 5 minutes at room temperature. Move it into small Ependorf flask and centrifuge to get supernatan. Nex, the addition of phosphat buffer to achive 1000 ppm concentration. Implementation of Xanthine Oxidase Inhibitory Activity. The process of inhibition Xanthine Oxidase testing is performed using spectrophotometer UV-VIS with 3ml quatz cuvette at 290 nm wavelength.

Table 1.Composition reagent on measuring inhibition xanthine oxidase. Control Enzyme

Blanco Enzyme

Sampel/ Allopurinol

Sampel/allopurinol bp xantin

Blanco Sampel

-100 800 840 700 160 160 160 -----incubation at 370C for 5 minutes XO 40 -40 -----incubation at 370C andmeasuring absorbance every minute of the 4 minutes in λ mak 290 nM

100 740 160 --

Data analysis. The data collected of the measurement result absorption in UV spectrophotometri namely ΔA/minutes is used to calculate activity Xanthine Oxidase with the formula (7) (Anonymous, 1994). The data is used to calculate the percentage of Xanthine Oxidase inhibitory with the formula. The value of IC50 inhibitor (concentration that produces inhibitory Xanthine Oxidase avtivity worth (50%) can be determine with linear regression analysis between compound concentration test against the percentage of inhibitory Xanthine Oxidase activity, then continue with statistical test by test with confidence level of 95%. RESULT AND DISCUSSION The Xanthine Oxidase inhibitory activity of secang, tempuyung and kepel extracts conducted triplo using equipment UV-VIS spectrophotometry. The results obtained written in Table 2 as follows : Table 2. The xanthine oxidase inhibitory activity of secang, tempuyung and kepel extracts. No. 1 2 3 4 5 6 7 8 9 10

Extract Secang (1) Secang (2) Secang (3) Tempuyung (1) Tempuyung (2) Tempuyung (3) Kepel (1) Kepel (2) Kepel (3) Allopurinol (positive control) 1000 ppm

% Inhibition 56.473 55.808 58.922 20.154 16.760 16.200 7.138 12.071 4.059 87.474

From the table 2, it can be showed that percentage of inhibition of secang extract is the highest (52.922%), followed by percentage of inhibition of tempuyung extract (20.154%) and percentage of inhibition of kepel extract (12.072%). Meanwhile, the percentage of inhibition of Allopurinol is 87.474%. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 148

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From the test, it was found that secang extract has higher Xanthine Oxidase percentage inhibition compared with Xanthine Oxidase percentage inhibition of tempuyung and kepel extracts.The percentage inhibition level of Allopurinol extract is the highest because Allopurinol is a synthetic drugs that decreased uric acid level. The Xanthine Oxidase inhibitory activity of secang, tempuyung and kepel extracts were described in Graphic 1 as follow.

Graphic 1. Xanthine oxidase percentage (%) inhibition of secang, tempuyung, kepel extracts and Allopurinol (+ controle 1000 ppm).

The Xanthine Oxidase inhibitory activity of ethanol extracts of secang showed the greatest inhibition among two other extracts, while Allopurinol as synthetic drugs with anti-uric acid capability has the highest percentage inhibition (87.474%). The results obtained from this research indicated that secang could be developed as anti uric acid. It can be used in the medicinal herbs industry with a relatively cheap and affordable price. The Xanthine Oxidase percentage inhibition of Allopurinol (as positive control) aimed to determine the trust calculated statistically. The results reflected in Graphic 2. From Graphic 2, it can be seen that the higher concentration of Allopurinol (1000 ppm) gives the higher Xanthine Oxidase percentage inhibition (87.474%). To reach 100% percentage inhibition, Allopurinol must be tested with more than 1000 ppm of concentration.

Graphic 2. Xanthine oxidase percentage (%) inhibition of different concentration of Allopurinol as a positive control


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CONCLUSION 1.

2.

Secang, tempuyung and kepel extracts are potential as anti uric acid with the inhibitory percentage of each extract are 58.922%, 20.154% and 12.071% respectively compared with the Allopurinol as a synthetic drugs with 87.474%. This study was restricted to the preliminary screening of Xanthine Oxidase inhibitory activity from secang, tempuyung and kepel extracts. Further structural elucidation and characterization methodologies have to be carried out in order to identify the bioactive metabolites in the extracts. REFERENCES 1. Walker R, &Edward C. Clinical pharmacy and therapeutics. Edisi3. USA: Churchill Livingstone. 2003. 2. Wibowo S. Asam Urat, http://suryo_wibowo.blogspot.com. 2006. Diakses pada tanggal 9 September 2015 3. Price S & Wilson L. Patofisiologi: Konsep klinis proses-proses penyakit. Edisi 6. EGC, Jakarta. 2005. 4. Joseph T DiPiro, Robert L, Talbert Gary C, Yee Gary R, Matzke Barbara G, Wells L, Michael Posey. Pharmacotherapy: A pathophysiological approach (Eds). 7th edition. 2005. 5. Xu H, Zhou Z, Yang J. Chemical constituents of caesalpiniasappan L. ZhongguoZhongyaoZazhi. 1994. 19, (8) 485-486. 6. Chairul. Tempuyung untuk menghadang asam urat. http://digilib.itb.ac.id/ files/disk1/615/jbptitbpp-gdl-drchairula-30748-1-tempuyun-t.pdf Accessed on October 12th 2015. Chapter 100. Acne Vulgaris : Treatment : Acne Vulgaris Accesspharmacy. http://www.accesspharmacy.com/content.aspx?aID=3212123 http://www.agrobisnisinfo.com/2015/07/daun-tempuyung-tanaman-obat-herbal-Accessed on 9th September 2015. https://id.wikipedia.org/wiki/Kepel. Accessed on 10th September 2015.. http://tyasistiqomah.blogspot.co.id/2011_02_01_archive.html xanthine oxidase Accessed on September 10th 2015.

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Potency of Curcuma Mangga Val Rhizome Extract as a Selective AntiProliferative Agent on Breast Cancer Cell Line MCF-7 SISKA ANDRINA*, CHURIYAH, NURALIH Center of Pharmaceutical and Medical Technology, Agency for the Assessment and Application of Technology. Corresponding Author Email: [email protected]

Abstract: Curcuma Mangga Val is original plant from Indo-malesian which is distributed in Indonesia, Taiwan, Thailand, China, Pacific and North Australia. In Indonesia, Curcuma Mangga have been used for traditional medicine as an antioxidant, anti-diarrhea, and anti-pyretic. In this study, we investigate potency of ethanolic extract of Curcuma Mangga Rhizome as an anti-proliferative agent against Breast Cancer cell line MCF-7. We also measured the cytotoxicity effect of extract on Human Dermal Fibroblast as a normal cell to determine its selectivity. Rhizome of Curcuma Mangga Val was macerated by 70% ethanolic and tested for cytotoxicity using MTT cell viability assay on MCf-7 and Human Dermal Fibroblast. The result showed selectively inhibited of ethanolic extract of Curcuma Mangga Rhizome on MCF-7 Cell with IC50 6.05 ppm and less sensitivity on Human Dermal Fibroblas with IC50 31,9 ppm. We also observed apoptotic mechanism of Curcuma Mangga Val extract on MCF-7 by double staining using etidium bromide and acridin orange. The figure showed that half of cell was dead because of apoptotic and necrotic mechanism either in 7 ppm. This study demonstrate the potential of Curcuma Mangga Val Ethanolic Extract as a selective antiproliferative agent on breast cancer cell line MCF-7. Keyword: Curcuma Mangga Val, anti-proliferative, MCf-7, Human Dermal Fibroblast, apoptotic INTRODUCTION Cancer is a major public health problem in the worldwide. Cancer is one of non-communicable disease which is the first leading causes of death all over the world. In 2012, WHO reported 8.2 billion of people in the world having cancer and it would be increasing up to 22 billion of cases in next two decades. In Indonesia, Ministry of Health has reported that breast cancer is the top leading cause of cancer deaths among Indonesian women in 2014. Chemotherapy is one of the commonly-used strategies in breast cancer treatment. This therapy is usually associated with adverse side effects, ranging from nausea to bone marrow failure(2) and development of multidrug resistance (MDR)(3). In a few last decades, herbal plants have been widely used for diseases treatment and immunological enhancement. The increasing trend of herbal application in traditional herbal industry is mainly due to numerous beneficial effects of natural sources compared to single synthetic drug. Natural herbal medicines usually offer less undesirable side effect, more efficiency and less toxic to patient. Therefore, finding natural compounds from plants may provide an alternative cancer treatment. Curcuma Mangga Val is original plant from Indo-malesian which is distributed in Indonesia, Taiwan, Thailand, China, Pacific and North Australia. Curcuma manga Val is a member of the family Zingiberacae. It is also known by its common Indonesian name Temu Mangga, because of its mango-like smell when fresh rhizomes are cut. In Indonesia, Curcuma Mangga Val have been used as traditional medicine for relieving stomachic complaint, gastric ulcer, chest International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 151

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pain, fever, hepato-protective and anti-allergic. Rhizome of Curcuma Mangga has been known containing phenolic, alkaloid, saponin and also natural antioxidant curcuminoid. As we know that those of substance have potential activity as a cancer killer from herbal plants In this study, we investigated the potential activity of ethanolic extract of Curcuma manga val as a cytotoxicity agent on breast cancer cell line MCF-7 and its apoptotic mechanism using staining assay. We also evaluated extract selectivity against human dermal fibroblast as a normal cell. Selectivity index between IC50 on breast cancer cell line and normal cell will inform its selectivity as an anti-proliferative agent against breast cancer cell. MATERIAL AND METHODS MATERIAL. Plant Material and Chemicals. Rhizome of Curcuma mangga Val, Ethanol 70% Analytical Grade, Cell line MCF.7, Human Dermal Fibroblas Cell, Roswell Park Memorial Institute medium (RPMI), Dulbecco’s Modified Eagle Medium Low Glucose (DMEM-LG), Fetal Bovine Serum (FBS) 10% v/v (Gibco), penisilin-streptomisin 1% v/v (Gibco), Dimethyl sulfoxide (DMSO), and 3-(4,5dimethylthiazol-2yl)-2,5-diphenyltetrazoliu bromide (MTT) , Sodium Deodecyl Sulfate (SDS), Phosphate Buffer Saline (PBS). Sample Preparation. Rhizome of Curcuma manga Val cut and dried in the oven at 60o C, macerated all of dried sample in ethanol 70% for a night. Evaporated filtrate of ethanol using high pressure evaporation until viscous extract was obtained. Cell Line Culture. MCF-7 was obtained from Gadjah Mada University Faculty of Pharmacy Yogyakarta and Human Dermal Fibroblast was original cells from Center of Pharmacy and Medical Technology BPPT. MCF-Cells were cultured in RPMI 1640 and Human Dermal Fibroblas were cultured in DMEM-LG and both of medium were supplemented with 10% foetal bovine serum, glutamine (2mM) and 1% penicillin-streptomycin in static 75 cm T-Flask (Nunc, Denmark). The cells were incubated in a humidified atmosphere with 5% CO at 37 oC. Cell Cytotoxicity Assay. Cells were plated in a 96-well-plate with 50.000 cells/well of concentration. The cells were left to adhere for 24 hours before exposed to the plant extracts (1-50 µg/ml) administered in media containing 10% of FBS and returned to the incubator for 24 hrs. Subsequently, MTT reagent (0.5 mg/mL in sterile PBS) was added directly to the wells. Cells were returned to the incubator for 4 hrs. The formation of insoluble purple formazan from yellowish MTT by enzymatic reduction was dissolved in SDS. Incubate for a night and the optical density of solution was measured at 570 nm using a microplate reader. Analyze the absorbance to obtain percentage of inhibition Apoptotic Observation on MCF-7. Cells were plated in a 24 well plate with 100.000 cells/well growing in RPMI medium supplemented 10% of FBS and 1% Penicillin-Streptomycin. Incubate the cells for a night to adhere then add the extract with IC50 concentration. Incubating for 24 hours and observe the morphology and staining of the cell after acridin orange and etidium bromide (1:1) was added under fluorescence microscope 100x. RESULT The Cytotoxicity Effect of Ethanolic Extract of Curcuma Manga Val Rhizome on Breast Cancer Cell Line MCF-7. MTT assay is a rapid and high accuracy colorimetric approach that widely used to determine cell growth and cell cytotoxicity, particularly in the development of new drug. It measures cell membrane integrity by determining mitochondrial activity through enzymatic reaction on the reduction of MTT to insoluble formazan salt. Ethanolic Extract of Curcuma mangga Val Rhizome was measured its potency to inhibit MCF-7 cell growth using MTT assay. This method will provide the percentage of cell proliferation inhibition and also Proliferation inhibition concentration 50 (IC50). The result showed that all concentration of extract inhibit the proliferation of cell with dose dependent manner. It means that increasing of concentration will give higher inhibition of proliferation cells (Figure 1). That graph represented IC50 of extract with values 6.05 ppm International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 152

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Rhizome of Curcuma Mangga Val. Ethanolict Extract % Cell proliferation Inhibition

120.000 100.000 80.000 60.000 40.000 20.000 0.000 0

0.2

0.4

0.6 0.8 1 (ug/mL) 1.2 Log Concentration

1.4

1.6

1.8

Fig 1. Cell proliferation inhibition of ethanolic etract of curcuma manga val rhizome on breast cancer cell line MCF-7 in 5 concentration of extract.

The Cytotoxicity Effect of Ethanolic Extract of Curcuma manga Val Rhizome on Human Dermal Fibroblast Cell. Human Dermal Fibroblast cells as a normal cell were used to determine extract selectivity. Index selectivity between IC50 on HDF and MCF-7 more than 2 means that the extract has selectivity inhibiting cancer cells and does not has impact on normal cell. The result showed that Ethanolic Extract of Curcuma manga Val Rhizome has higher inhibition of percentage compared than cytotoxicity on MCF-7 (Figure 2). Extract has IC50 31.93 ppm and index selectivity 5 (Table 1). It means that Ethanolic Extract of Curcuma manga Val Rhizome has selectivity inhibiting MCF-7 breast cancer cell proliferation.

% Proliferation Inhibition

Comparison of inhibition proliferation on MCF-7 and HDF 120.000 100.000 80.000 60.000

MCF-7

40.000

HDF

20.000 0.000 -20.000

0.194

0.796

1.398

1.699

Log Concentration Figure 2. Comparison of cell proliferation inhibition on MCF-7 and human dermal fibroblast cell. Table 1. IC50 of ethanolic extract of curcuma manga val rhizome on MCF-7; human dermal fibroblast and index selectivity of extract. Extract IC50 value (ug/mL) Index Selectivity MCF-7 HDF Ethanolic Extract of Curcuma 6.05 31.93 5 manga Val Rhizome

Apoptotic Observation of Ethanolic Extract of Curcuma manga Val Rhizome on MCF-7. The apoptotic mechanism of the treated cells has been observed under an inverted microscope using ethidium bromide and acridine orange (EB/AO) as staining agent. The apoptotic cells are stained in orange with lighter in nucleus or green, the live cells are stained in green and the necrotic cells are stained in orange. The figure showed that early apoptotic, late apoptotic and necrotic was found in all treated cell as presented in figure 3. It means that in IC50 concentration of extract, cells were died because of apoptotic mechanism. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 153

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(a)

(b)

Fig 3. Double staining of ethanolic extract of curcuma manga val rhizome on MCF.7 in (a) control cell; (b) extract at 7 ppm.

CONCLUSION Ethanolic Extract of Curcuma Mangga Val has potency as cytotoxic agent on breast cancer cell line MCF-7. Ethanolic Extract of Curcuma Mangga Val Rhizome has selective proliferation inhibition due to value of index selectivity on HDF.Ethanolic Extract of Curcuma Mangga Val Rhizome has cytotoxicity effect through apoptotic mechanism. REFERENCE 1. Castell J V, and Lechon MG. In vitro methods in pharmaceutical research. Academic Press, San Diego, California. 1997. 2. Da’i M. Uji aktivitas antiproliferatif pentagamavunon-o terhadap sel raji, sel hela, dan sel myeloma. Tesis. Fakultas Farmasi UGM. Yogyakarta. 2003. 3. Doyle A, and Griffiths JB. Cell and tissue culture for medical research. John Willey and Sons Ltd, New York.2000. 4. Fox M. Cancer deaths declining in U.S. Reuters Health, United States. 2007. 5. Franks LM, and Teich NM. Introduction to the cellular and molecular of cancer. Third Ed.,Oxford University Press, New York.1997. 6. Freimoser, F.M., Jakob, C.A., Aebi, M, and Tuor, U, 1999, The MTT [3-(4,5-Dimethylthiazol-2-yl)2,5-Diphenyltetrazolium Bromide] Assay Is a Fast and Reliable Method for Colorimetric Determination of Fungal Cell Densities, Applied and environmental microbiology, 65(8), 3727-3729. 7. Hanahan D, and Weinberg RA. The hallmark of cancer. Cell, 100, 57-70. 2000. 8. Harborne J B. Metode fitokimia, penuntun cara modern menganalisis tumbuhan. Penerbit ITB, Bandung. 1987. 9. Haridas V, Mujoo K, Hoffmann JJ, Wachter GA, Hutter LK, Lu Y, et.al. Triterpenoid saponins from Acacia victoriae (Bentham) decrease tumor cell proliferation and induce apoptosis.Cancer Research, 61, 5486–5490. 2001. 10. Hartwell JL. Plant used against cancer. a Survey, Lioydia. 1971. 30-34. 11. Jacobz. Tumbuhan berkhasiat obat sebagai obat. KTO Karyasari, Jakarta. 2003. 12. King RJB. Cancer biology, 2nd ed., Pearson Education Limited, London.2000. 13. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods,1983. 65 (1-2): 55-63 cit. 14. Itagaki, H et.al. Validation study on five cytotoxicity assay. JSAAE-VII details of the MTT. 1997.1-12.


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Assessment of Antibacterial Activity of Herbal Toothpastes to The Bacteria Causing Halitosis SYARMALINA*, SYAHDU A. EKOWATI DAN DWI A. MAULANA Faculty of Pharmacy,Pancasila University Jalan Srengseng Sawah, Jagakarsa,Jakarta Selatan 12640, Indonesia. Email:[email protected]

Abstract: The use of toothpaste is now not only as a cosmetic, but also as a treatment of halitosis. The content of the herbal in the toothpaste can help halitosis therapy because of its antibacterial activity. The purpose of the study is toobtain pure isolates from saliva of halitosis patients and to test antibacterial activity of toothpaste to microbes causing halitosis. Microbial limit test of toothpaste, isolation of microbial testing from saliva of halitosis patients and antibacterial activity test of herbal toothpaste to microbes were accomplished. Microbial limit test toothpaste MYC (mold yeast count) and TPC (total plate count) values obtained toothpaste A, B and D are eligible not more than 103 colonies/g TPC is between 3.6x102-5.48x102 colonies/g; MYC between 0.05. Keywords: accelerated stability test, cincau hijau leaves extract, gel, HPC-m, HPMC, antioxidant.

INTRODUCTION Stability is ability of a product or cosmetic to be able to last or stay in standard specification range during storage time and use to ensure the identity, strength, quality dan purity of the product. Medicine product of each preparation has different stability quality, therefore, each preparation or product has to undergo stability test to ensure the medicine product manufucturing and storaging. Stability test proceed as instructed in Good Manufacturing Practice for medicine products (CPOB) such as testing method and storaging condition that must qualify the standard. In this paper, stability test will be held for three months in month 0, 1, 2, and 3 in room temperature 27 ± 20C dan temperature of 45o-50oC/RH 75% in climatic chamber. Green grass jelly leaf extracts have the most active chemical content of alkaloids and flavonoids. Flavonoids acts as natural antioxidant to decrease the number of free radical in body, make it able to reduce /prevent aging process, therefore, not only the stability test is held, but also the antioxidant activity test. There are two formulas with 4% HPCm gel base and 5% HPMC base under stability test in room temperature 27 ± 20C dan temperature of 40o RH 75% in climatic chamber for three months and then then the gel be chemically and physically evaluated, inculding organoleptic, homogenity, viscosity and rheology, spreadability, pH and antioxidant activity test. MATERIALS AND METHODS Materials. Fresh green grass jelly leaf extract (Cyclea barbata L.Miers), Hydroxypropyl cellulose (HPCm), Hydroxypropylmethyl cellulose (HPMC), Propylene glycol, sodium benzoate, sodium metabisulfite,


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disodium EDTA, ethanol 96%, ethanol 70%, distilled water, DPPH (1,1-difenil-2-pikrilhidrazil), methanol pro analysis, dan vitamin C. Tools. Analytic scale, rotary evaporator, Brookfield viscometer RV type, homogenizer, water bath, laboratory glass tools, digital pH meter, gel spreadability tester, spectrofotometer, and climatic chamber Methods Grass jelly leaves concentrated extract production. Fresh grass jelly leaves are collected, washed until clean, cut and crushed, and then macerated with ethanol 70% with kinetic maceration technique with pedal for three hours. After that, filtered and concentrated using rotary evaporator until concentrated extract retrieved. The concentrated extract is physically tested. Grass jelly leaves extract gel preparation. Two formulas are made with Hydroxy propyl cellulose medium (HPC-m), Hydroxy propyl methyl cellulose (HPMC). HPC-m dispersed with ethanol96%&stayed still for 24 hours. HPMC dispersed with distilled water temperature of 60700C&stayed for 24 hours. Sodium benzoate, propylene glycol , dinatrium EDTA, Na.metabisulfite dissolved with distilled water. The mixture are homogenized and added with Green grass jelly leaf extract. The gel is physically evaluated and Accelerated stability test in room temperature and temperature of 400C. Evaluation. Organoleptics. The color and odor of the gels were observed. Viscosity and Rheology. The viscosity of different samples gel formulations were determinated at room temperature and 400C (Brookfield). Spreadability. The spreadability of different samples of gel formulations were determinated with spreadability tester at room temperature and 400C. pH. pH meter was submerged in gel until stable pH value obtained at room temperature and 400C. RESULTS AND DISCUSSION

12 10 8 6 4 2 0

Room temperature

Batch 1 0

200000

400000

F (dyne/cm2)

Batch 2

12 10 8 6 4 2 0

RPM

RPM

Green grass jelly leaves extract gel which was proven for having flavonoids that have antioxidant activity. Antioxidant activity analysis with DPPH submersion method showed that Green grass jelly leaves extract gel has strong antioxidant activity with IC50 value of 60,73 – 67,47 µg/mL.

Batch 1 Batch 2 0

500000

1000000

F (dyne/cm2)

Graph 1. Rheogram green grass jelly leaf extract gel preparation of Fomula 1.


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Viscosity stability evaluation. Rheogram green grass jelly leaf extract gel preparation of Fomula 1 was shown in graph 1. Gel preparation has stable rheogram for three months, which is plastic. Plastic flow of the gel preparation did not change after 3 months storage. Spreability Stability evaluation. Spreability Stability results of formula 1 was shown in table 1. Spreadability of the gel decreased after 3 months storage due to the increase of viscosity which increased each month. pH stability evaluation. pH stability evaluation results were shown in table 2. pH value of room temperature and 400C of the gels were decreased each month. pH is affected by time. In room temperature 3 months storage, the pH decreased from 6,31 ± 0,02 to 6,04 ± 0 and in 400C 3 months storage, the pH decreased from 6,02 ± 0,01 to 5,76 ± 0,03. Table 1. Spreadability Stability results of green grass jelly leaves extract gel preparation Formula 1. Temperature Room

40oC

Time (months) 0 1 2 3 1 2 3

1 D (mm) 66,80 67,00 67,10 67,30 65,20 62,80 55,00

2 F (mm2) 3502,86 3523,87 3534,39 3555,49 3337,07 3095,91 2374,63

D (mm) 66,50 66,50 66,70 66,90 65,60 61,00 53,30

F (mm2) 3471,47 3471,47 3492,38 3513,35 3378,14 2920,99 2230,10

D (mm) 67,10 67,10 67,30 67,50 65,00 61,70 53,00

3 F (mm2) 3534,39 3534,39 3555,49 3576,66 3316,63 2988,41 2205,07

Table 2. pH stability test results of green grass jelly leaves extract gel preparation. Temperature Time (Months) Batch 1 1 2 3 Mean ± SD 6,29 6,33 6,30 6,31 ± 0,02 0 Room 6,24 6,25 6,26 6,25 ± 0,01 1 6,10 6,12 6,12 6,11 ± 0,01 2 6,04 6,04 6,04 6,04 ± 0 3 6,01 6,02 6,03 6,02 ± 0,01 1 40oC 5,90 5,76 5,80 5,82 ± 0,07 2 5,78 5,73 5,77 5,76 ± 0,03 3

CONCLUSION Different gelling agent in Formula 1 HPC-m and Formula 2 HPMC have different physical quality. In Formula 1, green grass jelly leaf extract gel preparation has more stable physical quality after 3 months storage time in room temperature 30oC ± 2oC and temperature of 400C / RH 75% which is a gel with greenish yellow gel, specific odor, homogeneous. Statistic Analysis with two way ANVA on viscosity test, spreadability, and pH test. Gel preparation Formula 1 Batch 1 dan 2 in room temperature 30oC ± 2oC and 40oC for 3 months storage time have P value =0,00 > 0,05, Means that there is no effect on viscosity, spreadability and pH Formula 1 and 2 during storage time of 3 months. ACKNOWLEDGEMENT Special thanks are devote for Directorate General of Higher Education of Indonesia which has given grants in this research.


ISBN : 978-602-72418-2-4

REFERENCES 1. Lieberman HA, Rieger, Martin M, Banker, Gilbert S. Pharmaceutical Dosage Forms Dieperse System. Vol 2. USA. Marcel Dekker, Inc. 1996. P. 403 2. Carstensen JT. Drug Stability Principles and Practices. Third Edition. New York. Marcel Dekker, Inc. 2000. P. 516 3. Ansel HC. Pharmaceutical Dosage Forms and Drug Delivery System. Eight Edition. Lippincott Williams and Wilkins, 2010. 4. Aulton, ME. Pharmaceutical The Science of Dosage Form Design. Second Edition. New York: Churchill Livingstone. 2007.


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Capsule Formulation of Standardized 70% Ethanol Extract Johar Leaves (Senna siamea (Lam.) Irwin and Barneby) as Α-Glucosidase Inhibitor RISMA MARISI TAMBUNAN, KARTININGSIH, EVERLY HENDRA Faculty of Pharmacy, Pancasila University. Srengseng sawah, Jagakarsa, South Jakarta 12640. Email: [email protected] Abstract : Standardized 70% ethanol extract of johar leaves showed that the extract has the consistency viscous, fulfill the requirements parameters of quality extract and has inhibitory activity α-glucosidase at 225 ppm is 90,88% equivalent to acarbose 95,21%. In this study, dried extract of johar leaves was formulated into capsule dosage form for practical use as an oral antidiabetic. The extract was dried with freeze drying using maltodextrin 10%. Dried extract was formulated into capsule dosage form using three variety diluents, namely dibasic calcium phosphate, lactose and avicel pH 102. Based on the evaluation results obtained that avicel pH 102 as the optimal formula that showed weight uniformity 0,4234 g and disintegration time 7 minutes 2 second with inhibitory activity α-glucosidase 85,88%. Keywords: Johar leaves, capsule, diluent INTRODUCTION Johar leaves (Senna siamea (Lam.) Irwin and Barneby) is a plant that people using empirically as a antidiabetic drug (1). 70% ethanol extract of johar leaves known to inhibits α-glucosidase at 225 ppm is 85,88% equivalent to acarbose 77,49%. In this study, 70% ethanol extract of johar leaves was made into capsule dosage form for practically use and cover the bitter taste of johar leaves. 70% ethanol extract of johar leaves was dried by freeze drying using maltodextrin 10%. Dried extract was made into capsule dosage form using variation diluent to obtain formula of capsule dosage form with good characteristics. Capsule dosage form was evaluated physical quality, ie weight uniformity and disintegration time (2, 3, 4). MATERIALS AND METHODS Materials Crude drug of johar leaves (Senna siamea (Lam.) Irwin dan Barneby), ethanol 70%, maltodextrin, pvp, dibasic calcium phosphate, lactose and avicel pH 102, aquadest, hard gelatin capsule no. 0, α-glucosidase (G5003-100UN, Sigma), p-nitrophenyl-α-D-glucopyranoside (N1377-1G, Sigma), bovine serum albumin (Sigma), acarbose (Bayer), phosphate buffer pH 7,0, dimethyl sulfoxide, sodium carbonate, sodium hydroxide. Tools Analytical scales, glass tools, vacuum rotary evaporator, desiccators, mortar and stamfer, water bath, freeze dryer (LABCONCO), moisturemeter karl fischer, flow rate tools (LES-AUTONICS), stopwatch, sieve shaker (shive shakers of BBS product, BCL 601), bulk density Omron H3CR Tester, oven (Memmert), capsule filler, disintegration time tools (Guoming BJ-2), micropipette, absorbance microplate reader Elx 800. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 166

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Methods Determination of Extract Quality Parameters. Performed of determination of parameters quality extract includes determination of the ash content, the determination of ash content acid insoluble, water soluble extract assay, content of ethanol soluble extract, loss on drying, water content, residual solvent, contamination of metals (Pb and Cd), Microbial contaminations, and total flavonoid content From the determination of parameter quality extract johar leaves by methods “Parameter standart mutu ekstrak tumbuhan obat” results can be seen in Table 1. Table 1. Result of determination of parameters quality extract. Pharameters extract Result 65,27 Water soluble extract (%) 72,60 Content of ethanol soluble extract (%) 13,27 Loss on drying (%) 10,45 Water content (%) 0,48 Ash content (%) 0,05 Ash content acid insoluble (%) 0,10 Residual solvent (%) Contamination of metals (mg/kg) 0,0301 Pb 0,003 Cd Microbial contaminations (colonies/g) 9,1x103 ALT 3,98x102 AKK 8,44 Total Flavonoids content (%)

Preparation of Capsules Dried extract of johar leaves is the active ingredients. Additional materials (excipients) ie avicel pH 102, lactose, dibasic calcium phosphate as diluent. Three formula was made into capsule dosage form using hard gelatin capsule no. 0 (table 2). Dried extract of johar leaves added diluent and mixed until homogeneous. Homogeneous mass inserted into the capsule shell using capsule filler tools. Table 2. Capsule formulation. Formulation Weight (g) Materials I II III 35,08 35,08 35,08 Dried extract 8,06337 CaHPO4 8,9357 Lactose 4,4736 Avicel pH 102

Evaluation Of Capsule Weight Uniformity A number of 20 capsules were weighed. Then one by one capsule weighed and removed its contents. Empty capsule shells then weighed. Calculated weight of the contents of each capsule and the average weight of the content. Capsule weight uniformity requirement is there should not be any deviation capsules larger percentage of column A (±7,5%) and there should be no more than 2 capsules a greater percentage deviation from column B (±15%).


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Disintegration time Six capsules are put into the basket on the medium water-temperature 37⁰C ± 2⁰C as much as 1000 mL, then run the tool. Automatically, the basket will be up and down on a regular basis. When the basket fell, the basket into the medium of water. For each capsule, which was disintegrated, note the time. The capsule was disintegrated if there is no part of the capsule remains, except parts of the outer shell of the capsule. When 2 capsules not disintegrated completely, repeat the test with 12 other capsules with terms no less 16 of 18 capsules tested must be crushed completely. Terms of the time disintegrated is not more than 15 minutes (4). α-Glucosidase Inhibitory Activity of Johar Leaves Capsule Dosage Form The same concentration of each capsule formula and acarbose as positive control were conducted to αglucosidase inhibitory activity using p-nitrophenyl-α-D-glucopyranoside as substrate. α-glucosidase activity was determined by kawanishi method at 405 nm wave length. 200 mg bovine serum albumin and 1,0 mg αglucosidase were diluted in buffer phosphate pH (7,0). The mixing solution contains 250 µl solution of 2 mM p-nitrophenyl-α-D-glucopiranoside, 400 µl buffer phosphate (pH 7,0), and series of 10 µl, 30 µl, 50 µl, 70 µl dan 90 µl sample. Then, it was pre-incubated at 37⁰C for 5 minutes. Reaction was started by adding 250 µl αglucosidase, then incubated it at 37⁰C for 15 minutes. The reaction was stopped by adding 1000 µl Na2CO3 solution. The amount of p-nitrophenol obtained was measured at 405 nm wave length. The percentage of the inhibitory activity was counted by using this formula :

Where C = absorbance of enzyme activity without inhibitor (absorbance of DMSO), and S = absorbance of enzyme activity with sample examined. RESULTS AND DISCUSSION Determination of extract quality parameters The result determination of parameters quality specific extract of (Senna siamea (Lam.) Irwin and Barneby) leaves obtained shows that the extract has the consistency viscous, brown, and distinctive smell. Assay of compounds that are dissolved in two solvents was conducted to compare the number of secondary metabolites compound who extracted in a solvent of water and ethanol . Assay of compounds dissolved in a solvent of water shows the amount of inorganic compounds contained in extracts . While compounds assay dissolved in ethanol shows the amount of organic compounds present in the extract. From the results of the determination showed that the concentration of substances dissolved in ethanol (72,60%) and compounds that are dissolved in water (65,27%). Determination of total ash and ash insoluble in acid the conducted to determine the presence of trace elements in extracts who known as inorganic matter or ash. In the process of combustion in furnaces with a temperature of 4500C , organic materials in the extract can be burned but not for inorganic substances, namely ash. Ash total is the ash produced from a number of extracts which incandescent in the furnace. Ash total can be use to determine the mineral content of both physiological compounds such as K, Mg, Ca, or non-physiological such as pollutants, dust, soil contained in the extract. Results of the determination of total ash content obtained by 0,48%. While the levels of acid insoluble ash conducted to determine heavy metals compounds that do not dissolve in acid such as Hg, Pb, Cd and silicates. Acid insoluble ash content of 0,05% were obtained. These results meet the requirements of acid insoluble ash content of the extract is generally less than 1%. Assay of compounds that are dissolved in two solvents was conducted to compare the number of secondary metabolites compound who extracted in a solvent of water and ethanol . Assay of compounds dissolved in a solvent of water shows the amount of inorganic compounds contained in extracts. While compounds assay dissolved in ethanol shows the amount of organic compounds present in the extract . From International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 168

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the results of the determination Showed that the concentration of substances dissolved in ethanol (72,60%) and compounds that are dissolved in water (65,27%). Loss on drying was conducted to determine the water content and the compounds in the extract evaporated after the drying process in oven at 1050C. Obtained loss on drying is 13,27%. Water content of extract is 10,45%. These results meet the requirements of the general extract moisture content of less than 10%. Low water levels can guarantee the stability of the extract to long period. If the moisture content is too high then extract will not be stable for a long period of time because it is vulnerable over grown microbes. Results of the determination of residual solvent ethanol with gas liquid chromatography in extracts obtained 0,10% ethanol content. The determination results still meet the requirements of the maximum threshold of residual solvent in the extract is less than 1%. The content of Pb and Cd in Extract can comes from the Environment And Crop Production Process. Heavy metal Pb and Cd in the body are limited in number because it is dangerous to health . Pb metal can cause nerve damage , urogenetal , reproduction and hemopoitik. Cd metal can cause poisoning and organ damage and kidney damage of the research showed Pb content in the extract 0,0301 mg/kg while the level Cd 0,003 mg/kg. From the result obtained the microbial contamination with total plate count 9,1x103 colonies/g, number of mold and yeasts 3,98x102colonies/g, and total flavonoids 8,44%. Results of The Evaluation of Weights Uniformity Capsule weight uniformity evaluation results can be seen in Table 3 below. Table 3. Result of weight uniformity evaluation. No. Formulation Weight uniformity (g) SD I 462,3 3,57 1. II 466,3 3,31 2. III 423,4 1,14 3.

The results of the evaluation of weight uniformity of formula I, II and III quality ie weight of the capsule of the formula I, II and III do not deviate much from column A (±7,5%) and column B (±15%) of the average weight each capsule. This is caused by the flow properties of powders are eligible. If the flow properties good then process of filling into the shell, particles flowing continuously and uniformly, so that will be produced capsules with uniform weight or the smaller the coefficient of variation. Evaluation Results of Disintegration Time Evaluation results of disintegration time results can be seen in Table 4 below. Table 4. Evaluation results of disintegration time results. No. Formulation Disintegration time I 6 minute 10 second 1. II 7 minute 28 second 2. III 7 minute 02 second 3.

Terms of the time disintegrated is not more than 15 minutes. The evaluation results indicate that the disintegration time of the three formulas are eligible, ie less than 15 minutes even though used three different diluents. The Result of α-Glucosidase Inhibitory Activity The result of α-glucosidase inhibitory activity can be seen in Table 5 and Graphic 1 below. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 169

ISBN : 978-602-72418-2-4 Table 5. The results of α-glucosidase inhibitory activity. Inhibitor Inhibitory (%) 95,21 Acarbose 77,49 Quercetin 90,88 70% ethanol extract of johar leaves 86,00 Dried extract 84,42 Formulation 1 75,68 Formulation 2 85,88 Formulation 3

Graphic 1. Graphic % inhibitor of johar leaves capsule dosage form.

The result of α-glucosidase inhibitory activity by formula 3 at concentration of 25 µg/mL was 36,47%, the concentration of 75 µg/mL was 48,83%, the concentration of 125 µg/mL was 65,44%, the concentration of 175 µg/mL was 72,65% and a concentration of 225 µg/mL was 85,88%. Thus the IC50 of the formula 3 was 76,67 ppm. Based on the evaluation, avicel pH 102 showed the highest α-glucosidase inhibitory activity indicating that the capsule dosage efficacious as an antidiabetic. CONCLUSION Examination of specific parameters of quality extract ethanol 70% of (Senna siamea (Lam.) Irwin and Barneby) leaves showed that the extract has the consistency viscous, brown, and distinctive smell. Water soluble extract content of 65,27% and content of ethanol soluble extract of 72,60%. Examination of nonspecific quality parameters obtained loss on drying 13,27%, water content of 10,45%, total ash of 0,48%, acid insoluble ash content of 0,05%, residual solvent of 0,10%, Pb 0,0301 mg/kg and Cd levels of 0,003 mg/kg, microbial contamination with total plate count 9,1x103 colonies/g and number of mold and yeasts 3,98x102 colonies/g, and total flavonoids 8.44%. Based on the evaluation results abtained that avicel pH 102 as the optimal formula that showed weight uniformity 0,4234 g and disintegration time 7 minute 2 second with inhibitory activity of α-glucosidase 85,88%. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 170

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REFERENCES 1. Syamsuhidayat SS, Hutapea JR. Inventaris tanaman obat Indonesia. Edisi I. Jakarta: departemen Kesehatan Republik Indonesia Badan Penelitian dan Pengembangan Kesehatan; 1991. 2. Aulton M. E., 2007, Pharmaceutics; the science of dosage form design, New York; Churchill Livingstone, p. 517-523. 3. Lachman L, Lieberman HA, Kanig JL. Teori dan Praktek Farmasi Industri Edisi II Vol 1 & II. Diterjemahkan oleh Suyatmi S. Jakarta: UI Press; 1989, p. 795-822. 4. Swarbrick J, Boylan JC. Encyclopedia of Pharmaceutical Technology vol.4 Newyork: Marcel Dekker Inc; 1991. p. 37-76.


ISBN : 978-602-72418-2-4

Formulation and Evaluation of Herbal Tablets Containing Voacanga foetida (Bl.) K.Schum Extract FAHLENI1*, YANDI SYUKRI2, NOVELTA FEMMY RISCHA3, ADRIANI SUSANTY3 1)

Faculty of Pharmacy Pancasila University, Jakarta. 2) Islamic University of Indonesia, Yogyakarta. 3) Sekolah Tinggi Ilmu Farmasi Riau. Email : [email protected]

Abstract : Ethanolic extract of leaves of Voacanga foetida (Bl.) K.Schum has been formulated using wet granulation method. The aim of this study was to obtain the optimal concentration of starch as binder with varying concentrations; F1 (1%), F2 (2%) and F3 (3%). Evaluation of granules including true specific density, tapped density, apparent density, compressibility, Hausner factor, porosity, water content and angle of repose. Tablets were evaluated for weight variation, thickness, hardness, friability, in vitro desintegrating time. The results of angle of repose, Carr’s Index and Hausner ratio of all formulas indicated that the powder mixtures possess good flow properties and good packing ability. The physical properties tablets met the requirements according to Indonesian Pharmacopoeia 3th edition. It can be concluded that F1 was the best formula with the hardness 6,51 ± 0,62 kg/cm2, friability 0,99 ± 0,16%, and disintegration time 9,38 ± 0,28 minutes. The hardness and disintegration time increased with increasing concentration of cassava starch as binder employed. Keywords : Voacanga foetida (Bl.) K.Schum, cassava starch, herbal tablets, binder INTRODUCTION Tampa Badak (Voacanga foetida (Bl.) K.Schum), belongs to the family called Apocynaceae, have traditionally been used by people in Indonesia to cure skin infection, headache and abdominal pain(1). This plant also has hypotensive and sedative effect(2). Its leaves contain alkaloids vobtusin, vobtusina lactone, desoxy vobtusinlakton, voafolin, voafolidin, isovoafolin and isovoafolidin. In traditional medicine, the leaves of Voacanga foetida is usually soaked in water and unspecified quantities of the decoction are ingested. The ethanolic extract of the leaves of plants Voacanga foetida (Bl.) K.Schum at a dose of 300 mg / kg in mice have potential and specifically inhibit cancer cell growth and safe to use with IC50 < 50 µg/mL(3). In spite of their efficacy, herbal medicinal products have been widely criticized due to lack of standardization and poor-quality presentation. However, to improve patient compliance and acceptance, there is need to formulate the extract of leaves of Voacanga foetida (Bl.) K.Schum into tablet dosage form. Based on the description above, the authors were interested in doing the research to make a tablet of the ethanol extract of the leaves using wet granulation method with various concentrations of cassava starch paste as binder. Binder intended to provide compatibility and durability(4). Starch paste has good binding properties, especially for active ingredients that are insoluble and are in significant amounts. MATERIALS AND METHODS The leaves of Voacanga foetida (Bl.) K.Schum were collected from Anai Valley, Padang Panjang, West Sumatra. Extraction of the Powdered leaves The leaves were washed thoroughly in water, chopped and airdried at 35 to 40°C. The dried leaves were milled severally in an electric grinder. 4.6 Kg of the powdered sample was exhaustively extracted three International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 172

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times with absolute ethanol for 5 days by maceration. The solvent was removed at 30°C under reduced pressure and then evaporated. Preparation of Granules V.foetida extract was dried with dibasic calsium phospate (1:4) in mixer kneader. Primojel and dibasic calsium phospate were added into the dry-mixed of extract for 15 minutes then moistened with 1, 2 and 3% w/w concentration of binder solution (cassava starch mucilage). The wet masses were granulated by passing them manually through a No. 12 mesh sieve, dried in hot air oven for 18 hours at 50 0C, and then resieved through a No. 16 mesh size. Evaluation of mixed blends was carried out for all the formulations for angle of repose, bulk density, tapped density, % compresibility and flowability. Table 1. Formulations of the tablets. Ingredients

Formula I

Formula II

Formula III

Extract of Voacanga foetida (Bl.) K.Schum leaves

200 mg

200 mg

200 mg

Dibasic calcium phospate

800 mg

800 mg

800 mg

1%

2%

3%

112,5 mg 15 mg 357,5 mg

112,5 mg 15 mg 342,5 mg

112,5 mg 15 mg 327,5 mg

Cassava starch (mucilage amyli 10% b/v) Primojel Magnesium stearat Dibasic calcium phospate

Preparation of Tablets Tablets (final weight - 1500 mg) were prepared from the granules by compressing the materials using single punch tablet machine (Korsch type EKO) Friability Tablet friability was measured as the percentage of weight loss of 20 tablets randomly selected from each batch tumbled in friability apparatus (Erweka type T-200). After 4 minutes of rotation at 25 rpm, the dust of tablet was removed and the percentage of weight loss calculated. Tablet Hardness Twenty tablets randomly selected from each batch were used for the test. Erweka automatic hardness tester was employed. Disintegration Test

The disintegration times (DT) of the tablets were determined in distilled water at 37±0.50C using an Erweka type ZT-502 disintegration testing apparatus. Six tablets randomly selected from each batch were used for the test. RESULTS AND DISCUSSION The recommended dose of dry extract of Voacanga foetida (Bl.) K.Schum leaves according to Susanty (2008) was 300 mg/kg for producing required therapeutic response in mice. When converting this dose into a tablet form, this dosage regimen should not be altered. Thus, a tablet containing as high as 200 mg of the dry extract is formulated, because in order to make dry powder there were large portion of dibasic calcium phospate needed.


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Table 2. Pre-compression parameters of formulations. Physical properties Formula I Formula II Formula III Tapped Density (g/mL) Bulk Density (g/mL) Flow rate (second) Angle of repose (º) Compresibility index (%) Moisture content (%) Hausner ratio

0,7431 0,6192 6,17 ± 0,15 28,62 ± 0,55 16,67 ± 0,58 2,91 ± 0,40 1,20

0,6749 0,5647 5,97 ± 0,06 29,41 ± 0,29 16,33 ± 0,58 2,90 ± 0,09 1,19

0,6845 0,5659 5,63 ± 0,25 28,88 ± 1,32 17,33 ± 2,31 2,64 ± 0,30 1,21

Figure 1. Pictures of tablets Formula I, II and III (respectively) Table 3. Tablets properties. Physical properties Formula I Formula II Weight variations (%) 0,82 1,52 6,07±0,07 5,90±0,22 Thickness (mm) Diameter (mm) 15,06±0,13 15,15±0,04 2 Hardness (kg/cm ) 6,51 ± 0,62 6,42±0,91 Friability(%) 0,99 ± 0,16 0,41 ± 0,01 Disintegration time (min) 9,38 ± 0,28 13,31 ± 0,47

Formula III 2,39 6,15±0,03 15,11±0,03 7,26±1,01 0,34 ± 0,01 14,17 ± 0,17

Results obtained from the micromeritic studies are presented in Table 3. The results showed that granules exhibited good flowability and the values obtained fell within the acceptable range for good powder flow. Values of angle of repose below 35°, which showed that the granules had low interparticle cohesion and hence good flowability. Hausner’s ratio less than or equal to 1.25 indicates good flow, while Hausner’s ration greater than 1.25 indicates poor flow. Therefore, the granules were within the specified limits for good flow. Also, Carr’s index of 5 to 16 indicates good flow, while 18 to 23 shows fair flow(5,6). The results of compressibility index indicate that the prepared granules had good flowability and consolidation properties. When the CI and HR are adequate, the powder flows at minimum bulk density. A high bulk density, that is, a low porosity, will result in a low deformation potential, a lack of space for deformation during compression will cause less intimate contact between the particles within the tablets, resulting in weaker tablets(6). The results showed that the granules had low bulk and tapped densities and hence, exhibited good properties required for the production of good quality tablets. The maximum weight variation of the tablets was ± 2.39%, which fulfilled the acceptable weight variation range of ± 5%, hence the tablets of all batch passed the weight variation test. Hardness for tablets of all batches was in the range of 6.42 to 7.26 kg/cm², which falls above the limit of not less than 3.0 kg/cm². Friability value for parameters observed are given in table 2. None of the tablets of the batches has friability more than 1%. Therefore, the tablets can comfortably withstand handling, packaging and transportation without compromisingthe properties of the tablets. The thickness of the tablets of all the batches was found in the range of 5.90- 6.15 mm2 and 15.06 – 15.15 mm in diameter indicating fairly acceptable tablets. Disintegration time is an important parameter of tablet. An ideal tablet should disintegrate within 15min. The tablets of all the batches disintegrated less than 15 minutes. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 174

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CONCLUSION Tablets made from ethanolic extract of Voacanga foetida (Bl.) K.Schum leaves were produced by wet granulation method using cassava starch mucilage as binder. Increase in binder concentration caused an decrease in friablity and increase disintegration time of tablets. ACKNOWLEDGMENT The authors thank to Faculty of Pharmacy Islamic University of Indonesia, Yogyakarta for research fasilities.

REFERENCES 1. Valkenburg, V.J.L and Bunyapraphatsara. Medicinal and Poisonous Plants, 2008:584. 2. Rahmanudin. Uji Efek Hipotensif Ekstrak Akar Voacanga foetida (Bl.) K.Schum pada Tikus Putih Jantan.1988. Universitas Andalas.Padang. 3. Susanty, A. Uji Toksisitas dan Daya Antikanker Ekstrak Etanol Daun Tampa Badak (Voacanga foetida (Bl.) K.Schum).2008. Andalas University. Padang. 4. Voigt, R. Buku Pelajaran Teknologi Farmasi, Edisi V, diterjemahkan oleh Dr.Soedani Noerono. Penerbit Gadjah Mada University Press: Yogyakarta, 1994. 5. Aulton ME. Pharmaceutics. The Science of dosage form design, 3rd Ed. Churchill Living Stone, Edinburgh. 2007:197-210. 6. Yüksel N, Türkmen B, Kurdoğlu AH, Basaran B, Erkin J, Baykara T. Lubricant efficiency of magnesium stearate in direct compressible powder mixtures comprising cellactose® 80 and pyridoxine hydrochloride. FABAD. J. Pharm. , 2007, Sci. 32:173-183.


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Optimization of Patchouli Oil and Tea Tree Oil Emulgel Formulation YUSLIA NOVIANI1*, TETI INDRAWATI, SHELLY TAURHESIA1,2 1)

Faculty of Pharmacy University of Pancasila, Srengseng Sawah Jagakarsa Jakarta. 2) Amway Indonesia, Jakarta. [email protected]

Abstract : Emulgel preparations is created in order to be easier and convenient used. The objective of the study to obtain an emulgel preparation which has containing natural active ingredients as anti-bacteria that cause acne. There are 3 factors which influence this study, therefore 23 factorial design. The factor are concentration of carbopol 940 (0.5% - 1%), oils (7.5% - 10%), and emulsifying agent (1.5% - 2.5%). Evaluation of physical, chemical, and effectiveness in vitro are conducted to emulgel preparation. Data were analyzed using Minitab 16 to determine the effects of these factors and their interactions to the response and determine the optimum formula. Keywords : optimization, emulgel, patchouli oil, tea tree oil, patchouli oil and tea tree oil emulgel

INTRODUCTION Emulgels are emulsion, either of the oil-in-water or water-in-oil type, which are galled by mixing with a gelling agent. They have a high patient acceptability since they possess the previously mentioned advantages of both emulsions and gels. Therefore, they have been recently used ad vehicles to deliver various drugs to the skin(1). The aim of this work was to develop an emulgel formulation of patchouli oil and Tea Tree Oil (TTO) with the oil-in-water type emulsion, washable and has a good spread on the skin. The design of the formula in this work is using a factorial design 2 3 with 3 factors thought to play an important role on the physical, chemical, and effectiveness of the emulgel preparations, there are carbopol 940 as gelling agent, olive oil as the oil component, and the combination of tween 20 and Span 20 as emulsifying agent. Each material used in high and low concentrations that can be known optimum concentrations of each factor to result the good, effective, and stable emulgel preparations as anti bacteria that cause acne. MATERIALS AND METHODS Experimental Design. Eight emulgel formulations were prepared according to a 23 factorial design employing the qualitative factors and levels shown in Table 1. Table 1. Factors and levels for the 23 factorial design. Factors A = gelling agent concentration B = oils concentration C = emulsifying agent concentration

Low Levels (%)

High Levels (%)

0,5 7,5 1,5

1 10 2,5

Preparation of emulgel formulations. The composition of emulgel formulations is shown in Table 2. The gel formulations was prepared by dispersing carbopol 940 in purified water with constant stirring at a moderate speed, then the pH was adjusted to 6 – 6,5 using TEA. The oil phase of the emulsion was prepared by dissolving span 20 and BHT in olive oil, pathchouli oil and TTO while the aqueous phase International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 176

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was prepared by dissolving tween 20 in purified water. Methyl and propyl parabens were dissolved in propylene glycol, and both solutions were mixed with the aqueous phase. Both the oily and aqueous phases were separately heated to 70°C, then the oily phase was added to the aqueous phase with continous stirring until cooled to room temperature. Table 2. Quantitative composition of emulgel formulations. % (w/w) Ingredient F1 F2 F3 F4 F5 F6

F7

F8

Patchouli oil

2,5

2,5

2,5

2,5

2,5

2,5

2,5

2,5

TTO

2,5

2,5

2,5

2,5

2,5

2,5

2,5

2,5

Olive oil

2,5

2,5

5

2,5

5

2,5

5

5

Carbopol 940

0,5

1

0,5

0,5

1

1

0,5

1

Tween 20

0,6

0,6

0,6

1

0,6

1

1

1

Span 20

0,9

0,9

0,9

1,5

0,9

1,5

1,5

1,5

Propylene glycol

5

5

5

5

5

5

5

5

Methyl paraben

0,01

0,01

0,01

0,01

0,01

0,01

0,01

0,01

Prophyl paraben

0.03

0.03

0.03

0.03

0.03

0.03

0.03

0.03

BHT

0,01

0,01

0,01

0,01

0,01

0,01

0,01

0,01

Purified water

100

100

100

100

100

100

100

100

Physical Examination. The prepared emulgel formulation were inspected visually for their color, homogeneity, emulsion type and spreadability. The pH values of 1% aqueous solutions of the prepared emulgels were measured by a pH meter. Rheological Studies. The viscosity of the different emulgel formulations was determined using Viscometer Brookfield RV with spindel 6. Microbiological Assay. Ditch plate technique was used. Previously prepared Nutrient Agar dried plates were used. Three grams of the emulgel were placed in a ditch cut in the plate. Freshly prepared culture loops were streaked accros the agar at a right angel from the ditch to the edge of the plate. The commercial gel was used for comparison. Control plates containing plain emulgel bases were also prepared. After incubation at 35 - 37°C for 18 – 24 hours (Staphylococcus aureus) and for 24 – 48 hours (Propionibacterium acnes), the bacterial growth was observed and the percentage inhibition was measured as follows: % inhibition = L1/L2 x 100 (2) where L1 = total length of the streaked culture, and L2 = length of inhibition .

RESULTS AND DISCUSSION The result of the emulgel formulations are shown in Table 3.


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Table 3. Result of the examination of emulgel formulations. Examination Physical Color Homogeneity Emulsion type Spreadability pH Rheological Studies Microbiological assay - S. aureus (%) - P. acne (%) -

F1

F2

F3

F4

F5

F6

F7

F8

C1

C2

white good O/W good 5,49 plasti s










100 78,67

100 78,14

100 78,38

100 78,62

100 77,90

100 78,33

100 79,77

100 78,52

100 100

89,29 61,67

Where F1 – F8 = formula 1 to 8, C1 = commercial gel 1 that containing clindamycin 1%, C2 = commercial emulgel that containing TTO. Physical Examination. The prepared emulgel formulation were white viscous creamy preparation with a smooth and homogeneous appearance. There were easily spreadable with acceptable bioadhesion and fair mechanical properties. The pH values of all the prepared formulations range from 5,04 to 5,84, which is considered acceptable to avoid the risk of irritation upon application to the skin.3,4 Rheological Studies. Figure 1 show the entire rheograms (shear stress vs shear rate) of emulgel formulations. As seen in the figures, the emulgel formulations has thixotropy rheological properties and has the yield value of 276,568.6443 dyne/cm2. Thixotropy is a desirable characteristic in pharmaceutical preparations. Thixotropy, or time dependent flow, occurs because the gel requires a finite time to rebuild its original structure that breaks down during continuous shear measurements.5

F

Figure 1. Rheograms of emulgel formulation.

Microbiological Assay. The use of control plates showed that the plain emulgel bases were microbiologically innert toward the tested S. aureus and P. acne. The antibacteria activity of in its different emulgel formulations as well as in its commercially available (C1) for S. aureus. Percentage inhibition was taken as a measure of the antibacteria activity. The greatest activity was observed with formula F7, where the percentage inhibition reached up 79,77%, while the lowest activity was found with F5, where the percentage inhibition was 77,90%. CONCLUSION The composition of optimum formula for the emulgel formulation are 0,635% of karbopol 940 concentration, 8,75% oils, and 1,5% of emulsifying agent.


ISBN : 978-602-72418-2-4

ACKNOWLEDGMENT Faculty of Pharmacy University of Pancasila, Srengseng Sawah Jagakarsa Jakarta. REFERENCES 1. Magdy IM. Optimization of chlorphenesin emulgel formulation. The AAPS journal. 2004;6(3). 2. Hugo WB, Russell AD. Pharmaceutical Microbiology. Oxford, UK: Blackwell Scientific Publications; 1977:190. 3. Clearly GW. Transdermal controlled release systems. In: Larger RS, Wise DS, eds. Medical Applications of Controlled Release. Vol 1. Boca Raton, FL: CRC Press; 1984:204-251. 4. Lucero MJ, Vigo J, Leon MJ. A Study of shear and compression deformarions on hydrophilic gels of tretionin. Int J Pharm. 1994; 106:125-133. 5. Klich CM. Jels and Jellies. In: Swarbrick J, Boylan JC, eds. Encyclopedia of Pharmaceutical Technology. Vol 6. New York, NY: Marcel Dekker Inc; 1992:415-439.


ISBN : 978-602-72418-2-4

Formulation of Liquorice Root Extract (Glycyrrhiza glabra L.) as Skin Whitening Cream SITI UMRAH NOOR*, FARIDAH, MICHICO Faculty of Pharmacy Pancasila University, Jakarta 12640. [email protected]

Abstract : Liquorice root extract (Glycyrrhiza glabra L.) contains glabridin isoflavane as a tyrosinase inhibitors to inhibit melanin synthesis under the skin so that potentially as a skin whitener. The aim of this research are to determine the tyrosinase inhibition activity in liquorice root extract as skin whitening and formulated into a cream with a variety of 0.1%, 0.5% and 0.9% of emulsifier glyceryl monostearate to determine the effect of emulsifier on the physical quality and effective as a skin whitener. Liquorice root extract made by kinetic maceration using ethanol 96%, invitro tyrosinase inhibition assay was then conducted with kojic acid as positive control using 96-well microtiter plate and microplate reader, sample incubated at temperature 37°C for 20 minutes, and its absorbance measured at 490 nm wavelength. The physical quality parameters of cream were evaluated includes organoleptic, homogeneity, type of emulsion, viscosity, flow properties, spreadability, droplet size, centrifugation, pH and the inhibition activity of the cream. The research results that tyrosinase inhibition activity of liquorice root extract (IC50) was 126.75 µg/mL. Creams of 1.01 % liquorice root extract were yellowish white, aromatics odours, soft textures, homogeneous and segregation did not occur with O/W emulsion type and plastic thixotropic rheological properties, viscosity of (2800±0.00) – (4000±0.00) Ps, spreadability of (3029.72±0.81) – (3531.79±6.15)mm2, droplet size of (60.00±0.00) – (65.12±0.01)μm, pH of (4.55±0.03)–(4.63±0.04) with (10.14 -19.30)% tyrosinase inhibition value of cream. It can be concluded that the formula with concentration of 0.1% of glyceryl monostearate was the best formula that conforms physical quality test and potentially as a skin whitening cream. Keywords: liquorice root extract, glyceryl monostearate, tyrosinase inhibitor, skin whitening cream INTRODUCTION Increased production and accumulation of Melanin locally and unadequately can cause local pigmentation or black spot on certain parts of the face. The production of melanin occurs with the help of biocatalis tyrosinase enzymes and was further accelerated by the UV light. One of the ways to prevent or inhibit the production of melanin is to inhibit the activity of tyrosinase. The concept of "from nature to cosmetic" concept will produce natural cosmetics. Besides more secure, cosmetics made from natural ingredients have been proven to have a better effectiveness which is good for health, beauty and eco-friendly. One of the plants that can inhibit the activity of tyrosinase is liquorice (Glycyrrhiza glabra L.). Licorice contains glycirrhizin (10-25%), liquiritin, liquiritigenin, isoliquiritigenin, isoliquiretin, glizirhizat, glabrene acid and glabridin. Glabridin as a phenolic compounds (isoflavan) contained in the root of liquorice and can act as an antioxidant, neuroprotective agent, anti inflamation agent, cure eczema, pruiritis, other dermatitis symptoms, as well as an effective whitening agent. According to Yokota (1998), inhibitory effects of glabridin against inflammation and melanogenesis in B-16 melanoma cell culture of and guinea pig skin showed that glabridin inhibited the enzyme tyrosinase activity at 0.1 -1,0 μg/ml concentration without affect DNA synthesis. The research of Zheng (2011) also stated that Glabridin has the stronger activity than kojic acid. Depigmentation effect of Glabridin known 15 times better than kojic acid and the wider activity than arbutin which is a natural compound that is widely used in skin whitening cosmetic.


ISBN : 978-602-72418-2-4

Based on journal "Extraction of glycyrrhizic Acid and Glabridin from Licorice", the optimum Glabridin extraction conditions of liquorice obtained by using 96.0% ethanol for 240 minutes at a temperature below 50°C. Viscous extract was formulated into cream preparations. The formulation of the O/W mediumbased cream use the concentration of Liquorice Root Ethanol Extract from invitro tyeosinase inhibition activity assay results. The cream is made with nonionic surfactants polysorbate 80 combined with coemulgator cetyl alcohol, cetearyl alcohol and glyceryl monostearate. The variation of glyceryl monostearate concentration as co-emulgator based on research that had been done before. The addition of 0.5-1% glyceryl monostearate will improve the stability and appearance of creams by making them not easily creaming. Glyceryl monostearate is “emollient lipophilic thickening agent and stabilizer" that commonly used in emulsion and cream, it is included in the fatty alcohol group that can improve the consistency or as the stiffening agent so that increase the stability of the cream. MATERIALS AND METHODS Tyrosinase inhibition activity: Liquorice Root Extract (Glycyrrhiza glabra L.) (Simplicia from CV. Herbaltama Yogyakarta), Kojic acid (Thornhill, Canada), L-DOPA and Tyrosinase from Mushroomlyophilized powder (SIGMA-Aldrich, USA), 0.1 M Phosphate Buffer pH 6.8, Dimethyl Sulfoxide1%. Liquorice whitening cream: Liquorice Root Extract, triple pressed stearic acid (Shanghai FuXin, RRC), cetyl alcohol (BASF, Germany), cetearyl alcohol (Ecogreen, Singapore), anhydrous lanolin (WuXi, RRC), propylene glycol (Dow Chemical Pacific, Canada), parafin liquid (Sonneborn, Netherland), polysorbate 80 (KAO Indonesia Chemical, Indonesia), glyceryl monostearate (Danisco, RRC), methyl and propyl paraben (UENO Fine Chemical, Germany), butylated hidroxy toluene (Sterlitamak Petrochemical Plant, Russia), perfumes, aquadest. Reagen Sudan III, methylene blue Equipments and instruments : Microplate reader (BioTek, ELx800), 96-well microtiter plate (Bio-RAD), micropippete (transferpette), kinetic macerator (IKA, RW20), rotary vacuum evaporator (Heidolph, Laborota 4000), water bath (Memmert, WNB400), microbalance (Mettler, MT5), analytical balance (KERN, ABT 1205DM), homogenizer (IKA, RW20), oven (Memmert, U-110), viscometer (Brookfield RV type), pH meter (HANNA, HI 2211), microscope (Olympus, CH20), centrifugator (Kokusan, K-103N), incubator (Memmert, IN-55), refrigerator (LG).  Organoleptic  Solvent miscibility  pH

Liquorice Root Extraction with 96% ethanol Condensed with vacuum rotavapor

Tyrosinase inhibition activity invitro

Liquorice extract

Introduction test:  Incubation time  Wavelength  Enzyme concentration  Substrate concentration

IC50 Oil

Water

Cream base Tyrosinase Inhibition Activity invitro

Licorice Whitening Cream

 Mixing speed  Mixing time  Organoleptic (Colour, Odour, Texture)  Homogeneity  Cream type  Viscosity & Rheological Properties  Spredability  Globule size  Centrifugation  pH test

Figure 1. Methods in scheme.


ISBN : 978-602-72418-2-4

Simplicia manufacture and extraction Part of the root of the liquorice plant is harvested in the form of cylindrical rods with a length of up to 1 m and diameter in 0.5 cm to 3 cm, yellowish brown to dark brown, and wrinkle. All the cleaned simplicia then dried by sunlight indirectly for 1-3 days. Then the simplicia mashed using a blender and sifted with no. 4/18 mesh. 500 g of liquorice powder were macerated kinetically using 5 L of ethanol (96%) for 4 hours. In 10 repeated process, each process using 4 L of solvents. Then the collected filtrate was evaporated in the rotary vacuum evaporator to obtain Liquorice Root Ethanol Extract at a temperature of ± 40°C, 180 mmHg pressure, and speed of 60 rpm up to gained a thick extract of ethanol. It was packed in a seal opaque container and stored in the refrigerator. Inhibition of tyrosinase activity invitro This assay was performed using methods as described earlier with modification (Zhang, (2011); Batubara, et.al., (2010) ; Juwita, N.K. (2011). Extract were dissolved in DMSO to a final concentration of 20 mg mL-1. This extract stock solution was then diluted to 25-4000µg mL-1 in 100 mM phosphate buffer (pH 6.8). Kojic acid, whick was used as positive control was also tested at concentrations 2,5-50 µg mL-1. In a 96-well plate 80 μL phospate buffer (0,1 M, pH 6.8) was combined with 40 μL L-DOPA (5mM phospate buffer) in triplicate, 40 μL of each sample dilution. After 5 minutes, add 40 μL tyrosinase ( 310 Units mL-1 in phospate buffer) to each well. Incubation commenced for 20 minutes at 37°C. Optical densities of the wells were then determined at 490 nm. Each samples done with blank samples which tyrosinase solution not added. The tyrosinase inhibition activity was calculated according to the following formula: B : control absorbance – control blank absorbance (B1-B0) S : sample absorbance – sample blank absorbance (S1-S0) Inhibitory activity of the sample can be determined by calculating the IC50, namely the concentration in which the sample inhibit tyrosinase activity by 50% by using the linear regression equation. Concentration of the sample (in logarithms) as the x-axis and the percent inhibition (% inhibition) as the y-axis. Liquorice cream formulation Cream made by mixing the oil phase (Stearic acid, Cetyl alcohol, Cetearyl Alcohol, Lanolyn anhydrous, Paraffin Liquid, Glyceryl monostearate, BHT) to the aqueous phase (Aquadest 70°C added Propylene Glycol, Polysorbate 80, Methyl paraben, Propyl paraben) which has been heated at a temperature of 70°-75°C. Liquorice extract certain concentration test results inhibitory activity mixed into a cream base in warm temperature (50°C). Table.1 Formula of cream. FORMULA (%) b/v Ingridients Blank I Blank II Blank III I 1.01 Liquoorice Root Ethanol Extract Sodium metabisulfite (to extract) 0,1 Stearic acid 2,0 2,0 2,0 2,0 Cetyl alcohol 3,0 3,0 3,0 3,0 Cetearyl alcohol 6,0 6,0 6,0 6,0 Lanolin anhydrous 4,0 4,0 4,0 4,0 Paraffin liquid 10,0 10,0 10,0 10,0 Glyceryl monostearate 0,1 0,5 0,9 0,1 Polysorbate 80 6,0 6,0 6,0 6,0 Propylene glycol 15,0 15,0 15,0 15,0 Methyl paraben 0,15 0,15 0,15 0,15 Propyl paraben 0,05 0,05 0,05 0,05 Butyl hydroxy toluene 0,05 0,05 0,05 0,05 Perfumes qs qs qs qs Aquadest ad 100 100 100 100

II 1.01 0,1 2,0 3,0 6,0 4,0 10,0 0,5 6,0 15,0 0,15 0,05 0,05 qs 100

III 1.01 0,1 2,0 3,0 6,0 4,0 10,0 0,9 6,0 15,0 0,15 0,05 0,05 qs 100


ISBN : 978-602-72418-2-4

Physical quality parameter Evaluation of the physical quality of cream were done, include: organoleptic (colour, odour, and texture); homogeneity by using a glass object; cream emulsion type with microscopic methods; viscosity and rheological properties by using a Brookfield viscometer type RV; spreadability by using a teflon ring; globule size is measured using an optical microscope; centrifugation for 5 hours at a speed of 3800 rpm; cream pH using pHmeter. Liquorice cream Inhibition of tyrosinase activity invitro Liquorice cream inhibition of Tyrosinase Activity were done with a microplate reader. This assay was performed using methods as described earlier after the cream extracted with a solvent and centrifuged for 15 minutes. Statistical analysis Data of physical parameter assay were expressed with ANOVA one ways without replication, such as viscocity, spreadability, globule size and pH with p = 0.05 significance level RESULTS AND DISCUSSION Liquorice Extract and Kojic Acid Tyrosinase Inhibition Activity Assay

Figure 2. Liquorice extract tyrosinase inhibition activity assay results.

Figure 3. Kojic acid tyrosinase inhibition activity assay results.

Inhibition of tyrosinase enzyme activity in vitro carried out on 96% ethanol extract of the Liquorice root of and kojic acid as a positive control using enzyme concentration of 310 U / mL, pH 6.8, incubation time of 20 minutes, 5 mM substrate concentration and temperature of 37ºC in accordance with preliminary test done. Kojic acid chosen as a positive control because of kojic acid is one of the active substances that are commonly used in skin whitening preparations with a mechanism of tyrosinase inhibitory activity as non-competitive in the oxidation of L-DOPA into the pigment melanin. IC50 value of kojic acid obtained in this study was 20.88 mgmL-1. When compared with the positive control kojic acid, inhibitory activity shown by the liquorice root ethanolic extract lower at 126.76 mgmL-1. In the previous study revealed that Glabridin activity 15 times more potent than kojic acid, but in this study the activity of the liquorice root ethanolic extract showed a lower activity. It caused by the crude extract of liquorice ethanol extract, so there was still many other component which disturb the inhibition activity assay. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 183

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Physical quality parameter of Cream

Parameters

Organoleptic

Blank I Milky white, aromatic odours, soft textures

Homogeneity

Table 2 Physical quality parameter of cream. Formula Blank II Blank III I II Milky Milky Yellowish Yellowish white, white, white, white, aromatic aromatic Aromatics Aromatics odours, soft odours, less odours, Soft odours, Soft textures soft textures textures textures Homogeneity

III Yellowish white, Aromatics odours, less Soft textures

O/W Type of cream

Viscosity (Ps) Yield value(dyne/cm)

4000

4400

4600

2800±0,00

3200±100

4000±50

83817,27-149957,53

Rheological Properties

Rheological : Tyxotrophy plastic Spreadability (mm2) Globule size (μm)

3053.41

2927.69

2885.98

3531,79±6,15

3394,77±24,95

3029,72±0,81

60.81

58.21

56.67

65.12±0,01

63.72±0,02

60.00±0,00

4.39

4.38

(-) segregation did not occur 4.35 4.63 ±0,04 19.30

4,55±0,03 10.14

4.59±0,03 -17.78

Distribution Curve

Centrifugation pH Activity (%)

The results of physical quality parameter studies showed Formula I with 0,1% glyceryl monostearate gave soft textures so it is the most convenient in usage and spread easily when applicated, and had the higher activity of tyrosinase inhibitory activity. The variation of Gylceryl monostearate concentration in cream formula related with the softness textures of cream, viscosity spreadbility, globule size, pH and inhibition activity. The higher concentrations of Glyceryl monostearate made the cream less soft, higher visocity, lower spreadability and globule size also lower to acid pH value. This is due to the function of glyceryl monostearate that can improve the cream consistency. The addition of stearyl alcohol also can improve the texture and adds hardness cream that can be softened with cetyl alcohol so cream remained soft. Glyceryl monostearate as coInternational Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 184

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emulsifier can reduce the size of the oil globules and can reinforce the coating film that is formed thus increasing the cream consistency. O/W cream emulsion type can be determined by the addition of sudan III, which gained colorless outer phase and inner phase in the form of red globules, while the addition of methylene blue obtained blue outer phase and colorless inner phase in the. The formula viscocity had a lower value than blank formula. This is due to the addition of liquorice extract which tends to be acidic in formula, the interaction between the extract containing polyphenols and saponins compounds with a base of cream, causing a decrease viscosity. The ability to spread decreases with increasing concentration of glyceryl monostearate is used, which is an oil phase of a solid with a large molecular weight, the greater the molecular weight of a substance will be smaller then spreadability, so that the higher the concentration, the lower spreadability. Results of the evaluation showed that the increased concentration of glyceryl monostearate may reduce the ability to spread the cream. Most globules obtained in the range of 31-41 μm and decreases with increasing diameter. Globule size distribution showed normal distribution, if the curve did not meet the normal distribution law can lead to distability of an emulsion system according to Stokes law, which is expected to sedimentation. Globule size can also be influenced by various factors that occur during the manufacturing process such as stirring or mixing. It can be concluded that the concentration of glyceryl monostearate used can affect the liquorice cream globule size distribution. All formulas showed no sedimetation occured after centrifugation for 5 hours at a speed of 3800 rpm, so it can be said cream can be stable during storage year. Cream to be unstable by observing the separation of the dispersed phase that occurs as a result of centrifugation. The principle is that if globules meet the same liquid then tends to form a globule with a larger size. From the evaluation it can be concluded that the concentration of emulsifier in cream enough to form a monomolecular layer on the surface of the oil globules of water so as to prevent coalescence. Liquorice extract containing polyphenols and saponins compounds that big amount of OH- causing the pH of the dosage decreased to acid. The results of the pH approached liquorice extract and also get into the skin's normal pH range is 4.5-6.5. With this pH range, we expected to remain stable and do not irritate the skin because too acidic or alkaline dossage forms can irritate the skin or damage the acid mantle skin can cause skin unprotected against invading microorganisms. However, when compared, % inhibition decreased with the increasing glyceryl monostearate concentrations used in cream. More concentrations of glyceryl monostearate used, then the closer film formed and the closer matrix were available, made it more difficult to extarct the compound dispersed in there. High consistency of cream also produced a complex matrix that currently extracted by the solvent and put in a plate in a certain amount to make the conditions in the plate becomes complex and made the inhibition reaction more difficult for the compound. Based on previous research, liquorice extract contained glycosides, saponins, flavonoids and tannins that had a different stability. The total content of these compounds, especially total polyphenols and total flavonoids affect the activity of the extract in inhibiting the enzyme tyrosinase. Cream formulation using crude extract led to many constituents who might be able to disrupt the activities. Inhibitory activity of formula III result was negative, this is due to the amount of substrate and enzyme were less in favor of the course of the enzymatic reaction. In addition, the compounds Glabridin identified as a whitening agent is a flavonoid. Flavonoids in the plant as a sugar bound to aglycone glycosides and flavonoids that may be in some form of glycosides combination. In order that, the flavonoids are usually better examine the aglycone contained in extracts of plants through the process of hydrolysis before to be its glycon and aglycon. In general, groups of a flavonoid aglycone more active than as a single form of flavonoids. CONCLUSSION Liquorice (Glycyrrhiza glabra L.) ethanol extract which contains glabridin is potential to be whitening agent through invitro tyrosinase inhibition activity assay with IC50 value of 126, 75 ppm. Liquorice Ethanol extract 1.01% can be formulated into creams which pass physical quality test and pH as well as effective in inhibiting tyrosinase enzyme. Formula with 0.1% gliceryl monostearate concentration (Formula I) as the best formula. ACKNOWLEDGEMENT Special thanks are devote for Directorate General of Higher Education of Indonesia which has given grants in this research. International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 185

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REFERENCES 1. Aulton”s Pharmaceutics; The Design and Manufacture of Medicines; 3thed; Edinburgh; Churchill Livingstone Elsevier. 2007; p. 42-58; 70-97; 384-405. 2. Lloyd HW, Jenna N, Kammer BA. Treatment of Hyperpigmentation. Semin Cutan Med. Surg.2011;30:171-175. 3. Fais A, Corda M, Era B, et al. Tyrosinase Inhibitor Activity of Coumarin-Resveratrol Hybrids. Molecules.2009;14:2514-2520. 4. Parvez S, Kang M, Chung HS, et al. Survey and Mechanism of Skin Depigmenting and Lightening Agent. Phytother. Res.2006;20(11):921-934. 5. Draelos ZD, Thaman LA. Cosmetic Formulation of Skin Care Products. London: Taylor&Francis; 2006. p.203, 206, 214. 6. Smith N, Vicanova J, Pavel S. The hunt for natural skin whitening agents. Int. J. Mol. Sci. 2009;10:2440-2475 7. Vinayak BR, Mary SR. Natural ingredients for creating food textured cosmetics. USA: Cosmetic Science Technology Journal. 2007. p.33. 8. Qiushi C. Evaluate the effectiveness of the natural cosmetic compared to chemical-based product. International Journal of Chemistry.2009;1:57-59. 9. Yokota T, Nishio H, Kubota, et al. The inhibitory effect of Glabridin from Licorice Extracts on Melanogenesis. Pigment Cell Res. 1998; 11:955-961. 10. Ramsden CA, Patrick AR. Mechanistic Studies of Tyrosinase Inhibitors in Cultures of Pityrosporum. The Journal of Investigative Dermatology. 2010;71:205-208. 11. Jennifer C, Stephie CM. A review on skin whitening property of plant extracts. Bangalore. Int J Pharm Bio Sci. 2012; 3(4):332 – 347. 12. Schrader K, Domsch A. Cosmetology Theory and practice Vol. III. Germany: Cassler Druck and Medien;2005.p.17,28,32,44-53. 13. Ansels HC. Pharmaceutical Dosage Forms and Drug Delivery Systems 8th, Philadelphia; Lippincott Williams & Wilkins; 2005.p.276-297. 14. Wilkinson JB, Moore RJ. Harry’s Cosmeticology. 8th ed. London: George Godwin;2005.p. 226, 707726. 15. Batubara I, Darusman LK, Mitsunaga T, et al. Potency of Indonesian medicinal plants as tyrosinase inhibitor and antioxidant agent. J. Bio. Sci., 2010;10(2): 138-144.


ISBN : 978-602-72418-2-4

Accelerated Stability Test of Liquorice (Glycyrrhiza glabra L.) Extract Cream FARIDAH, SITI UMRAH NOOR, SULIH PROBO SINDI Faculty of Pharmacy, Pancasila University. Abstract : The accelerated stability test of Liquorice extract as a whitening agent has been done. The test was conducted for three months at a room temperature and at a temperature of 40°C with 75% RH in climatic chamber for 2 formulas with different concentrations of the co-emulsifier glyceryl monostearate 0.1% and 0.5%. Cream was evaluated include organoleptic test, homogenity, cream type, viscosity and flow properties, the ability to spread, the size of the globules, centrifugation, and the pH test. The results was obtained by these two formulas have cream-colored, flavored green tea, soft textured, homogeneous, O/W type, thixotropy-pseudoplastis,no undergo separation after centrifugation test (3800 rpm for 5 hours), as well as pH which decreased at both room temperature storage at pH formula I (4,56 to 4,30) and formula II (4,54 to 4,33) and at 40˚C / 75% RH storage both of formula I (4,49 to 4,31) and formula II (4,49 to 4,31). The spreadability cream at room temperature storage was reduced in formula I (4416,7 mm2-2237,3 mm2) and the formula II (2734 mm2-1666,9 mm2). In contrast, at 40˚C / 75% RH storage was increased in formula I (4217,6 mm2-5360,9 mm2) and formula II (2886,2 mm2-4287,8 mm2). Viscosity was measured by the speed of 0.5 rpm at room temperature storage has increased in formula I (172000 cPs to 228667 cPs) and formula II (195333 to-269333 cPs), but at 40˚C / 75% RH storage, the viscosity has decreased in formula I (172667 cPs to131333 cPs) and formula II (185333 cPs-158 000 cPs) .The globule sizes of cream at room temperature storage was decreased in formula I (62.26 µm -55.64 µm) and formula II (56,66 µm to 47.81 µm). While at 40˚C / 75% RH storage, was increased in formula I (63,75 µm to 69,03 µm) and formula II (61,55 µm -64,90 µm). It can be concluded that both liquorice cream formula with various glyceryl monostearate concentration was stable for 1 year storage at room temperature. Key words : accelerated stability test, liquorice, Glycyrrhiza glabra l.

INTRODUCTION Stability is defined as the ability of a product to keep the specification limits set throughout the period of storage and use to ensure the identity, strength, quality and purity of the product and keep the characteristics product that has been made. There are five types of stability that is generally known are the stability of the chemical, physical, microbiological, therapeutic and toxicology(1). Cream stability test is designed to assess the stability characteristics, appropriate storage conditions and the expired date(1). the cream that has been made must be qualified in general criteria, which is physically and chemically stable. as well as effective and safe when it used. Stability cream is a period in which the cream is stored in a certain period of time will have a constant level, no changes in shape, color, and other changes that can be determined by physical or chemical(2). To obtain the value of the stability of the cream in a short time, can be carried out by accelerated stability test. This test is intended to obtain the desired information in the shortest possible time by keep the sample in designed conditions to accelerate the changes that normally occur in normal conditions. If the test results cream in an accelerated test for 3 months obtained stable results, it shows that the cream is stable at room temperature storage for one year(3). Based on the recommendations of WHO documents, for products International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 187

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that are marketed globally tested under conditions of climatic zone IV (hot and humid), in which Indonesia is zone IV claim. According to ICH and FDA, a country located in the climatic zones III and IV with the temperature and humidity, is 400 C ± 20C and 75% ± 5% RH. In nature, there are various plantswhich has efficacy as bleaching or whitening agent, such as yam bean, chamomile and liquorice. In the previous studies, the efficacy of liquorice (Glycyrrhiza glabra L.) as an inhibitor of the enzyme tyrosinase has been proved and formulated into a cream dosage forms with varying concentrations of glyceryl monostearate as a co-emulsifier and stiffening agent(4.5). Liquorice extract cream should be required the criteria of stability, so that the formulation is designed to consider some related issues, among physico chemical properties of substances forming such as glabridin which contained in liquorice extracts. . Glabridin is stable at storage temperatures of 2-10˚C in a dry place and protected from light(6). The selected cream base in the formula is a medium cream. This base has a pH of 4.40 to 4.50 so that it has a similarity with the active substance Glabridin, is 4.54. Using the non ionic surfactant as emulgator, such as polysorbat 80 and some co-emulgator as a stiffening agent such as cetyl alcohol, stearyl alcohol, monostearic glyceryl that will enhance the stability of cream. MATERIALS AND METHODS Ingredients Ethanol extract of liquorice (Glycyrrhiza glabra L.), stearic acid (Wilfarin, China), stearyl alcohol, lanolin anhydrous (Wuxi, China), propylene glycol (Dow Chemical Co., Japan), paraffin Liquidum (Sonneborn, Japan), cetyl alcohol , glyceryl monostearate, polysorbate 80, methyl paraben, propyl paraben, BHT, distilled aqua and green tea perfume. Tools Cup vaporizer, analytical balance, spatula, stir bar, mortar, pestle, stirrer, water bath, viscometer, glass objects, glass cover, pH meter, optical microscope, spreadebility tester, centrifuges and climatic chamber (Memmert). Procedure 1. Extraction Process Liquorice powder macerated with ethanol 96% until perfectly extracted. The filtrate was evaporated with a rotary evaporator to become thick extract. 2. Identification of Flavonoids Extract was boiled with hot water for 5 minutes, then filtered (solution A), to 5 ml of filtrate was added magnesium powder and 1 ml of concentrated HCl, add 2 ml of amyl alcohol, shaked vigorously and allow to separate. The presence of the flavonoid was showed by the formation of red, yellow, or orange in the lining of amyl alcohol. 3. Cream Basic Formulation The cream consist of liquorice extract 0,2% with the medium base : Stearyl alcohol, stearic acid, lanolin, squalene, paraffin liquidum,glyceryl monostearic, polysorbat 80, propylenglycol, methylparaben, propylparaben, BHT, green tea parfume and aquadest. 4. Evaluation of the Cream a. Organoleptic Observed the cream appearance is the color and odor visually. b. Homogenity The cream is applied on the objects glass then clenched with another object glass, and then observed the homogenity, look at the surface. It should be smooth and homogen c. Examination of cream type


ISBN : 978-602-72418-2-4

Tests carried out using the color method by mixing the cream base with a few drops of a methylene blue solution on the glass object, and then observed with a microscope, if the external phase is blue and the internal phase is transparant drops, so the type of emulsion is oil in water emulsion. On the addition of sudan III, the external phase is red and the internal phase is transparant drops. So, the emulsion type is an oil in water. d. Viscosity and flow properties Determination of the viscosity is carried out by using a Brookfield viscometer RV with a capacity 18000000 Cps. Cream was placed in a container and the appropriate spindle is inserted to the prescribed limit, then rotated at a certain speed until the needle viscometer showed a constant scale. e. Spreadability test The cream is filled on the 15 mm inner diameter of Teflon ring and spread the cream with spatula until there is no bubble. Remove the ring carefully, then covered with a glass plate, then press with a 200 grams load, allow for 3 minutes, then measured the diameter of cream surface then calculated by the following formula : F = π x r2 (mm2) Description: F = Spreadability; π = 3,14; r = radius (mm) f. Globule size Analysis of globul size was done by measuring the average diameter of globule using microscope. Globul diameter on the object glass was measured using a calibrated micrometer. The amount of calculated globule is about 300 – 500. g. Mechanical tests (centrifugal test) Samples was put into a test tube and then inserted into sentrifugator at a speed of 3800 rpm for 5 hours, then observed the separation of oil and water. h. pH cream pH cream measurement was obtained using a calibrated pH meter. Data analysis Stability test results data were analyzed by 2-way ANOVA to find out the effect of temperature and self life to the response. RESULTS AND DISCUSSION A. Extract Characteristics Test Extract Characteristic

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Result A thick , blackish brown color , aromatic odor and a sweet taste Yield = 8,99 % DER – Native = 11,13 % Flavonoids = + pH = 4,54

B. Stability Test Result Organoleptic Test Organoleptic observation until the first month either at room temperature or at 40 ° C / 75% RH, do not change the color, odor and texture cream. Yellowish white to cream color due to the addition of liquorice extract. The Color changes can occur due to high temperature might accelerate chemical reactions, because each 10˚C rise could accelerate chemical reactions 2 to 3 times. The cream uses green tea International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 189

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perfume so the odor produced is the scent of green tea. Cream texture changes started in the second month. This is caused by the differences in the concentration of glyceryl monostearate which act as a stiffening agent, where the presence of temperature changes, it will cause changes in the globules size of the cream, so that at room temperature, cream texture tends to be more coarse and at 40˚C creams tend to be softer. Besides of the texture, the odor of the cream is also change in 40˚C storage temperature, this is because the perfume evaporates at high temperatures. The odor changes often referred to rancidity can be caused by oxydation of the oil or fat. The effect of light is a catalyst for the onset of rancidity, that is the combination of these two factors can cause accelerated fat oxidation. Viscocity of cream

Rheology of cream


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Spreadibility of cream

Globule size

pH of cream

The results was obtained by these two formulas have cream-colored, flavored green tea, soft textured, homogeneous, O/W type, thixotropy-pseudoplastis,no undergo separation after centrifugation test (3800 rpm International Seminar Pokjanas TOI Faculty of Pharmacy Pancasila University 191

ISBN : 978-602-72418-2-4

for 5 hours), as well as pH which decreased at both room temperature storage at pH formula I (4,56 to 4,30) and formula II (4,54 to 4,33) and at 40˚C / 75% RH storage both of formula I (4,49 to 4,31) and formula II (4,49 to 4,31). The spreadability cream at room temperature storage was reduced in formula I (4416,7 mm 22237,3 mm2) and the formula II (2734 mm2-1666,9 mm2). In contrast, at 40˚C / 75% RH storage was increased in formula I (4217,6 mm2-5360,9 mm2) and formula II (2886,2 mm2-4287,8 mm2). Viscosity was measured by the speed of 0.5 rpm at room temperature storage has increased in formula I (172000 cPs to 228667 cPs) and formula II (195333 to-269333 cPs), but at 40˚C / 75% RH storage, the viscosity has decreased in formula I (172667 cPs to131333 cPs) and formula II (185333 cPs-158 000 cPs) .The globule sizes of cream at room temperature storage was decreased in formula I (62.26 µm -55.64 µm) and formula II (56,66 µm to 47.81 µm). While at 40˚C / 75% RH storage, was increased in formula I (63,75 µm to 69,03 µm) and formula II (61,55 µm -64,90 µm). Cream-type examination is performed to determine the suitability of the composition of the proportion of each phase, the dispersed phase and the dispersing phase associated with comfort during use. Results of the evaluation of the type of cream made from moon-0 until the 3rd month, either at room temperature or a temperature of 40 C showed that the cream has type O/W.. Cream Type O/W more consumer demand for more convenient to use and have better penetration capability compared to type W/O.

CONCLUSION Both formulations liquorice cream type O/W with the variation of glyceryl monostearate stable in storage at temperatures of 400C / 75% RH for 3 months or at room temperature (20˚C-25˚C) for a year. REFERENCES 1. Joshita D. Cosmetic stability. Departemen Farmasi Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia. Depok: 2004 2. United State Pharmacopea. Volume 2. United State: 2009 3. Departemen Kesehatan Republik Indonesia. Farmakope Indonesia. Edisi IV. Jakarta: 1995 4. Jens T. Cartensen, C.T. Rhodes. Drug Stability principles and practices, 3rd ed. New York: 1990 5. Connors K.A, Gordon LA, Valentino JS, Stabilitas kimiawi sediaan farmasi. Edisi II, New York: 1986 6. Tim Padfield. A climate chamber for simulating a temperature and humidity gradient across a wall of roof. Technical University of Denmark: 2000 7. Mitsui T. New cosmetic science Edisi I. Amsterdam: 1997 8. B. Eckmann. Prediction of emulsion properties from binder/ emulsifier characteristic. Barcelona: 2000 9. Chang TS, An update review of tyrosinase inhibitors. Taiwan: 2009 10. Yokota T, Nishio N, Kubata Y, Mizoguchi M. The inhibitory effect of glabridin from licorice extract on melanogenesis and inflamatory [abstract]. Pigmen cell research, 2008 11. Raymon CR, Paul JS, Marian EQ. Handbook of pharmaceutical excipients. 7th edition. London: 2009


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