Acetylcholinesterase Inhibition Activity of Portuguese ...

i JEOBP JOURNAL OF ESSENTIAL OIL- BEARING PLANTS

(ISSN Print: 0972-060X) (ISSN Online: 0976-5026)

VOLUME 14 NUMBER 2 March/April 2011

Founder Late Mr. H.K.L. Bhalla

Publisher

Har Krishan Bhalla & Sons 7/1/2C, Prem Nagar, P.O. Prem Nagar Dehra Dun- 248007 (Uttaranchal) INDIA Web site: www.jeobp.com E-mail:

Cited in: Analytical Abstracts, Cambridge Scientific Abstracts, Chemical Abstracts, Elsevier SCOPUS, EMBiology, Science Citation Index Expanded (Thomson ISI), Forestry Abstracts, Medicinal and Aromatic Plant Abstracts

ii JEOBP JOURNAL OF ESSENTIAL OIL- BEARING PLANTS EDITORS AND EDITORIAL BOARD Hony. Chief Editor Prof. C.S. Mathela

Emeritus Professor Chemistry Department of Chemistry Kumaun University, Nainital, India-263002 (India) Managing Editor Arvinder Singh Bhalla 7/1/2C, Prem Nagar, P.O. Prem Nagar, Dehradun- 248007 (India) e-mail:

EDITORIAL BOARD Prof. E.A. Aboutable Cairo University (Egypt)

Prof. Mitsuo Miyazawa Kinki University, Osaka (Japan)

Prof. Chlodwig M. FRANZ University of Veterinary Medicine Vienna Vienna (Austria)

Prof. J.C. Chalchat University Blaise Pascal de clermont 63177 Aubiere Cedex (France)

Prof. C. Menut Universite Montpellier II, (France)

Prof. Jose Guilherme Maia DEQAL - UFPA, Belem, PA, (Brazil)

Prof. Gerhard Buchbauer University of Vienna, (Austria)

Prof. Naoharu Watanabe Shizuoka University (Japan)

Prof. Victor David Univ. of Bucharest (Romania)

Dr. Leopold Jirovetz University of Vienna (Austria)

Prof. Pat Sandra Research Institute of Chromatography Kennedypark 20, 8500 Kortrijk (Belgium)

Dr. Jorge A. Pino Instituto de Investigaciones para la Industria Alimentaria, La Habana (Cuba)

Prof. William N. Setzer University of Alabama in Huntsville Huntsville, AL 35899 (USA)

Prof. Fatemeh Sefidkon Res. Inst. of Forests and Rangelands Tehran (Iran)

Dr. Gopal Rao Mallavarapu A-602, Renaissance Temple Bells, Yeshwanthapur, Bangalore (India)

Prof. Jeno Bernath Corvinus University of Budapest Budapest (Hungary)

iii Prof. Farid CHEMAT Université d’Avignon Avignon (France)

Dr. Chandi C. Rath North Orrisa University Takatpur, Baripada, Orrisa (India)

Prof. Zrira Saadia Institut Agronomique et Veterinaire Hassan II, Rabat (Morocco)

Prof. Giuseppe Alonzo Univ. delgi studi di Palermo Palermo (Italy)

Prof. N.Y. Osee Muyima University of Fort Hare Alice (South Africa)

Prof. M. Ali Jamia Hamdard (Hamdard University) New Delhi (India)

Prof. Mehmet Musa ÖZCAN Selcuk University Konya (Turkey)

Dr. Danuta Kalemba Inst. of General Food Chemistry Technical Univ. Lodg (Poland)

Dr. Igor Jerkovic University of Split, 21000 Split (Croatia)

Prof. Dr. Azza Amin Ezz El - Din National Research Center Dokki, Cairo, Egypt

Prof. Gurdip Singh DDU, Gorakhpur University Gorakhpur (India)

Prof. Bruno Marongiu Università degli Studi di Cagliari Monserrato (Cagliari), Italy

Dr. Nebojsa Simic Norwegian University of Science and Technology, Trondheim (Norway)

Dr. V.K. Varshney Forest Research Institute Dehra Dun (India)

Dr. Inder Pal Singh National Institute of Pharmaceutical Education and Research (NIPER), Mohali (India)

Dr. Lee Seong Wei Universiti Malaysia Terengganu Terengganu (Malaysia)

Dr. Hema Lohani Centre for Aromatic Plants Dehradun (India)

Dr. B.R. Rajeswara Rao Central Institute of Medicinal and Aromatic Plants, Hyderabad (India)

iv JEOBP JOURNAL OF ESSENTIAL OIL- BEARING PLANTS Volume 14 Number 2

March - April 2011 CONTENTS

1.

Thujone-Rich Essential Oils of Artemisia rutifolia Stephan ex Spreng. Growing Wild in Tajikistan by Farukh S. Sharopov and William N. Setzer (Tajikistan, USA).

136

2.

Acetylcholinesterase Inhibition Activity of Portuguese Thymus Species Essential Oils by Susana A. Dandlen, Maria G. Miguel, João Duarte, Maria L. Faleiro, Maria J. Sousa, Ana S. Lima, Ana C. Figueiredo, José G. Barroso, Luís G. Pedro (Portugal).

140

3.

Influence of Summer Savory Essential Oil (Satureja hortensis) on Decay of Strawberry and Grape by Neslihan Dikbas, Fatih Dadasoglu, Recep Kotan, Ahmet Cakir (Turkey).

151

4.

Volatile Constituents from Leaves of Justicia pectoralis Jacq. var. tipo by Jorge A. Pino (Cuba).

161

5.

Effect of Ionizing Irradiation on Origanum Leaves (Origanum vulgare L.) Essential Oil Composition by Juan J. Elizalde , Mónica Espinoza (Chile).

164

6.

GC-MS Analysis of Ammoides atlantica (Coss. et Dur.) Wolf. from Algeria by Tarek Boudiar, Chawki Bensouici, Javad Safaei-Ghomi, Ahmed Kabouche and Zahia Kabouche (Algeria, Iran).

172

7.

Assessment of the Preservative Activity of Some Essential Oils to Reduce Postharvest Fungal Rot on Kiwifruits (Actinidia deliciosa) by Habib Shirzad, Abbas Hassani, Ali Abdollahi, Youbert Ghosta, Seied Rasool Finidokht (Iran).

175

8.

Chemical Composition and Antimicrobial Study of Essential Oil from the Leaves of Curcuma haritha Mangaly and Sabu by Gopan Raj, Nediyaparambu S. Pradeep, Mathew Dan, Mathur G. Sethuraman and Varughese George (India).

185

9.

Composition and Bioactivity of Essential Oils from Leaves and Fruits of Myrtus communis and Eugenia supraxillaris (Myrtaceae) Grown in Egypt by E.A. Aboutabl, K.M. Meselhy, E.M. Elkhreisy, M.I.Nassar and R. Fawzi (Egypt).

192

10. Chemical Composition of the Essential Oil from Aerial Parts of Haplophyllum acutifolium (DC.) G. Don from Iran by Javad Asili, Maryam Rajae Fard, Ali Ahi and Seyyed Ahmad Emami (Iran).

201

11. Chemical Composition and Antimicrobial Activity of the Essential Oil of Mentha pulegium L. by K. Morteza-Semnani, M. Saeedi and Mohammad Akbarzadeh (Iran).

208

12. The Effect of Microwaves on Essential oils of White and Black Pepper (Piper nigrum L.) and their Antioxidant Activities by Magda A. Abd El Mageed, Amr F. Mansour, Khaled F. El Massry, Manal M. Ramadan, Mohamed S. Shaheen (Egypt).

214

v 13. Volatile Constituents of Chromolaena odorata (L.) R.M. King & H. Rob. Leaves from Benin by Cosme Kossouoh, Mansour Moudachirou, Victor Adjakidje, Jean-Claude Chalchat, Gilles Figuérédo, Pierre Chalard (Benin, France).

224

14. Bioconversion of Essential Oil from Plants with Eugenol Bases to Vanillin by Serratiamarcescens by A. Khanafari, M. Seyed Jafari Olia F. Sharifnia (Iran).

229

15. Essential Oil Composition and Antibacterial Activity of Nepeta glomerulosa Boiss from Iran by Azizollah Nezhadali, Mahboobeh Masrornia, Hossein Bari, Mina Akbarpour, Mohammad Reza Joharchi and Mahboobeh Nakhaei -Moghadam (Iran).

241

16. Chemical Composition of Essential Oil from Doum Fruits Hyphaene thebaica (Palmae) by Nahla A. Ayoub, Omayma A. Eldahshan, Abdel-Nasser B. Singab and Mohamed M. AlAzizi (Egypt).

245

17. Comparison of Essential Oils from Ferula ovina (Boiss.) Aerial Parts in Fresh and Dry Stages by Hossein Azarnivand, Marzieh Alikhah-Asl, Mohammad Jafari, Hossein Arzani, Gholamreza Amin, Seyed Saeed Mousavi (Iran).

250

18. In vitro Antibacterial and Antifungal Activity of Salvia multicaulis by Taran Mojtaba, Ghasempour Hamid Reza, Safoora Borzo, Najafi Shiva, Samadian Esmaeil (Iran).

255

Jeobp 14 (2) 2011 pp 136 - 139

136

Journal of Essential Oil Bearing Plants ISSN Print: 0972-060X Online: 0976-5026 www.jeobp.com

Thujone-Rich Essential Oils of Artemisia rutifolia Stephan ex Spreng. Growing Wild in Tajikistan Farukh S. Sharopov 1 and William N. Setzer 2* 1

V. I. Nikinin Institute of Chemistry, Tajik Academy of Sciences, Ainy St. 299/2, Dushanbe, 734063, Tajikistan 2 Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA Received 27 September 2010; accepted in revised form 18 January 2011

Abstract: The essential oil from the aerial parts of Artemisia rutifolia Stephan ex Spreng., collected from two different regions of Tajikistan, were obtained by hydrodistillation and analyzed by GC-MS. A total of 77 compounds were identified in the oils, accounting for 98.6 % and 99.2 % of the two oils. Both essential oils were dominated by α-thujone (20.9 % and 36.6 %) and β-thujone (47.3 % and 36.1 %) with lesser amounts of 1,8-cineole (3.2 % and 11.7 %) and germacrene D (2.8 % and 1.8 %). Key words: Artemisia rutifolia, essential oil composition, thujone, Tajikistan. Introduction: Artemisia rutifolia Stephan ex Spreng. is a member of the Asteraceae (Compositae) and is distributed in Afghanistan, China, India, Kazakhstan, Kyrgyzstan, Mongolia, Nepal, Pakistan, Russian Federation and Tajikistan 1. The plant is an important traditional medicine. An infusion of the herb is taken to relieve painful urination; the fresh herb is used as an analgesic for toothache; the dried herb is used to treat excessive sweating; a decoction of the herb is gargled for treatment of angina, stomach problems, and heart problems. A. rutifolia essential oil has antibacterial, antifungal and anthelmintic activity 2. The essential oil of A. rutifolia growing in the Pamirs had been previously investigated by Goryaev in 1962, who reported the main constituents to be 1,8-cineole, α- and β-thujone, camphor, αand β-pinene, camphene, and limonene 3. Shavarda examined A. rutifolia oil from the Mongolian People’s Republic and identified 15 components: 1,8-cineole (35.0 %), camphor (18.0 %), α- and βthujone (11.0 %), terpinen-4-ol (7.0 %), α-terpineol (5.0 %), α-pinene, β-pinene, camphene, limonene, β-phellandrene, p-cymene, 4-phenylbutan-2-one, 4-phenyl-butan-2-ol, and 4-phenylbut-2-yl acetate 4. Experimental Plant material: Aerial parts of Artemisia rutifolia were collected from two regions of Tajikistan: Sample #1, the Khonaobod village, Muminobod region (38.107547 N, 69.966431 E, 1200 m above sea level), on 2 May 2010; Sample #2, the Chormaghzak village, Yovon region, (38.417502 N, 69.172175 E, 1300 m above sea level), on 25 July 2010. The plant was identified by V.A. Sulaimanova, *Corresponding author (William N. Setzer) E-mail: < [email protected] >

© 2011, Har Krishan Bhalla & Sons

William N. Setzer et al. / Jeobp 14 (2) 2011 136 - 139

137

and a voucher specimen (TJ2010-040) has been deposited in the herbarium of the Chemistry Institute of the Tajikistan Academy of Sciences. The air-dried samples (300 g each) were crushed and hydrodistilled using a Clevenger apparatus for 3 h to give the yellow essential oils, which were stored at 4°C until analysis. Gas chromatographic-Mass spectral analysis: The essential oils of Artemisia rutifolia were analyzed by GC-MS using an Agilent 6890 GC with Agilent 5973 mass selective detector [MSD, operated in the EI mode (electron energy = 70 eV), scan range = 45-400 amu, and scan rate = 3.99 scans/sec], and an Agilent ChemStation data system. The GC column was an HP-5ms fused silica capillary with a (5 % phenyl)-polymethylsiloxane stationary phase, film thickness of 0.25 μm, a length of 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 48.7 kPa and a flow rate of 1.0 mL/min. Injector temperature was 200°C and detector temperature was 280°C. The GC oven temperature program was used as follows: 40°C initial temperature, hold for 10 min; increased at 3°C/min to 200°C; increased 2°/min to 220°C. A 1 % w/v solution of the sample in CH2Cl2 was prepared and 1 μL was injected using a splitless injection technique. Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes, and by comparison of their mass spectral fragmentation patterns with those reported in the literature 5 and stored on the MS library [NIST database (G1036A, revision D.01.00)/ChemStation data system (G1701CA, version C.00.01.080)]. The percentages of each component are reported as raw percentages based on total ion current without standardization. The essential oil composition of A. rutifolia is summarized in Table 1. Results and discussion: The yellow essential oils of Artemisia rutifolia were obtained in 0.5 % yield for sample #1 (Muminobod region) and 0.8 % yield for sample #2 (Yovon region). A total of 77 compounds were identified in the A. rutifolia essential oils accounting for 98.6 % and 99.2 % of the compositions, respectively. The essential oils were dominated by oxygenated monoterpenoids, chiefly α-thujone (20.9 % and 36.6 %, respectively, for the Muminobod sample and the Yovon sample) and βthujone (47.3 % and 36.1%, respectively). Other notable components included 1,8-cineole (3.2 % and 11.7 %, respectively) and germacrene D (2.8% and 1.8 %, respectively). The A. rutifolia essential oils from Tajikistan, as revealed in this study, clearly belong to a thujone-rich chemotype, and differ markedly from the cineole/camphor-rich chemotype previously reported from Mongolia 4. Acknowledgments: FSS is grateful to the Fulbright Program for a generous research/travel grant. WNS is grateful to an anonymous private donor for the gift of the GC-MS instrumentation. We thank Dr. Bernhard Vogler for technical assistance with GC-MS data collection.

1. 2. 3. 4. 5.

References Tropicos.org. (2010). Missouri Botanical Garden. 26 Sep 2010. http://www.tropicos.org/Name/ 2727691 Llere6Hble TpaBbl (Healing herbs). (2007). http://medherb.if.ua/art_ru.htm Goryaev, M.I., Pliva, I. (1962). Methods of Investigating Essential Oils [in Russian], Izv. Akad. nauk KazSSR, Alma Ata, p. 680. Shavarda, A.L. (1976). Essential oils of Mongolian plants. A study of the essential oil of Artemisia rutifolia. Chem. Nat. Comp. 12: 42-45. Adams, R.P. (2007). Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry, 4th Ed. Allured Publishing, Carol Stream, IL, USA.


138

Table 1. Chemical compositions of the essential oils of Artemisia rutifolia Stephan ex Spreng. from Tajikistan RIa 852 919 935 941 953 976 978 981 993 1004 1016 1024 1031 1037 1048 1058 1088 1106 1119 1130 1137 1141 1145 1151 1154 1158 1162 1165 1174 1177 1184 1185 1190 1194 1195 1203 1206 1226 1228 1236 1243 1246 1255 1261

Compound (2E)-Hexenal Santolina triene α-Thujene α-Pinene Camphene Sabinene β-Pinene 1-Octen-3-ol Myrcene α-Phellandrene α-Terpinene p-Cymene 1,8-Cineole Santolina alcohol (E)-β-Ocimene γ-Terpinene Terpinolene α-Thujone β-Thujone Chrystanthenone iso-3-Thujanol trans-p-Menth-2-en-1-ol Camphor p-Menth-3-en-8-ol Menthone Sabina ketone Pinocarvone Borneol cis-Pinocamphone Terpinen-4-ol Thuj-3-en-10-al p-Cymen-8-ol α-Terpineol cis-Piperitol Myrtenol γ-Terpineol trans-Piperitol m-Cumenol nor-Davanone Pulegone Carvone Carvotanacetone cis-Piperitone epoxide cis-Chrysanthenyl acetate

Percent #1b Composition #2c 0.1 0.1 0.2 0.1 0.3 0.1 0.1 2.8 0.5 0.2 1.8 3.2 0.4 tr 0.5 20.9 47.3 0.1 0.3 0.9 0.9 0.1 0.9 0.2 0.1 0.2 0.6 0.2 0.1 0.4 tr 0.5 0.1 1.0 0.9 0.1 2.0 0.2

0.1 tr 0.4 0.1 0.3 0.1 0.2 0.9 11.7 0.4 0.1 36.6 36.1 0.8 0.1 0.5 0.2 0.2 0.3 0.2 0.4 0.1 1.2 0.1 0.3 0.3 0.2 0.1 0.1 0.3 0.1 0.1 0.9 tr


139

table 1. (continued). RIa 1265 1279 1283 1285 1289 1292 1301 1317 1336 1343 1366 1375 1399 1418 1458 1467 1476 1482 1487 1497 1509 1514 1524 1578 1583 1589 1592 1603 1640 1654 1670 1685 1694

Compound iso-3-Thujanol acetate neoiso-3-Thujanol acetate 1-Phenyl-2,4-pentadiyne Bornyl acetate p-Cymen-7-ol Thymol Carvacrol (Z)-Patchenol cis-Piperitol acetate Piperitenone Piperitenone oxide α-Copaene (Z)-Jasmone (E)-Caryophyllene (E)-β-Farnesene (2E)-Dodecenal β-Chamigrene Germacrene-D (E)-®-Ionone Bicyclogermacrene β-Bisabolene Davana ether δ-Cadinene Spathulenol Caryophyllene oxide Davanone Viridiflorol Ledol Germacrene-D 1,10-epoxide α-Cadinol Phloroacetophenone 2,4-dimethylether Germacra-4(15),5,10(14)-trien-1α-ol 4-Cuprenen-1-ol Compounds Identified Monoterpene hydrocarbons Oxygenated monoterpenoids Sesquiterpene hydrocarbons Oxygenated sesquiterpenoids Miscellaneous compounds

a

Percent #1b Composition #2c 0.1 0.1 0.7 0.9 0.1 0.1 1.4 0.1 0.1 0.4 0.2 0.2 0.1 2.8 0.1 0.5 0.2 0.1 0.7 0.2 0.4 0.1 0.3 0.1 0.3 0.1 -

0.1 0.1 0.1 tr tr 0.2 0.4 0.2 0.1 0.1 tr tr 0.3 0.1 0.1 1.8 tr 0.8 0.1 tr 0.2 0.1 1.3 0.1 tr

98.6 6.5 85.0 4.4 1.9 0.8

99.2 2.3 91.9 2.8 1.8 0.5

RI = Retention Index, determined with reference to a homologous series of normal alkanes on an HP-5ms column b Sample # 1: collected from Muminobod region c Sample # 2: collected from Yovon region

Jeobp 14 (2) 2011 pp 140 - 150

140


Acetylcholinesterase Inhibition Activity of Portuguese Thymus Species Essential Oils Susana A. Dandlen 1, Maria G. Miguel 1,2,*, João Duarte 1, Maria L. Faleiro 3, Maria J. Sousa 4, Ana S. Lima 2, Ana C. Figueiredo 2, José G. Barroso 2, Luís G. Pedro 2 1

Universidade do Algarve, Faculdade de Ciências e Tecnologia, DQF, Edifício 8, Campus de Gambelas 8005-139 Faro, Portugal 2 Universidade de Lisboa, Faculdade de Ciências de Lisboa, Dep. de Biologia Vegetal, Instituto de Biotecnologia e Bioengenharia, Centro de Biotecnologia Vegetal, C2, Campo Grande, 1749-016 Lisbon, Portugal 3 Universidade do Algarve, Faculdade de Ciências e Tecnologia, Edifício 8, Instituto de Biotecnologia e Bioengenharia, Centro de Biomedicine Molecular e Estrutural, Campus de Gambelas 8005-139 Faro, Portugal 4 Instituto Politécnico de Bragança, Escola Superior Agrária, Dep. de Biologia e Biotecnologia, Campus de Santa Apolónia - Apartado 1172, 5301-855 Bragança, Portugal Received 07 November 2010; accepted in revised form 14 January 2011

Abstract: Thymus species are commonly known in Portugal as thyme and they are currently used as culinary herbs, as well as for ornamental, aromatizing and traditional medicinal purposes. The essential oils isolated from the Portuguese Thymus caespititius (Sect. Micantes), T. camphoratus and T. capitellatus (Sect. Thymus, Subsect. Thymastra), T. carnosus, T. zygis subsp. sylvestris and T. zygis subsp. zygis (Sect. Thymus, Subsect. Thymus) were evaluated for acetylcholinesterase inhibition capacity. A great variability in activity was detected in the assayed essential oils, even among oils isolated from a same species with different geographical origins, such as T. caespititius. T. zygis subsp. zygis essential oil showed the strongest acetylcholinesterase inhibition capacity with an IC50 = 1.1μg/ml. Key words: Acetylcholinesterase inhibition; Thymus; Essential oils; Carvacrol; Borneol; 1,8Cineole. Introduction: Ethnobotanical studies have revealed that Portuguese Thymus species are predominantly used in the treatment of circulatory and digestive problems. They can also be used for apoplexy, as expectorant, to alleviate cough and toothache, externally as antiseptic, rubefacient, parasiticide, in the treatment of falling hair and cutaneous eruptions 1. More recently, and for the first time, Mata et al. 2 demonstrated the capacity of the Portuguese wild thyme oil (T. serpyllum) for inhibiting acetylcholinesterase, with the consequent possibility of its application in the treatment of Alzheimer’s disease. The main goal of the present work was to evaluate

*Corresponding author (Maria G. Miguel) E-mail: < [email protected] >


Maria G. Miguel et al. / Jeobp 14 (2) 2011 140 - 150

141

the antiacetylcholinesterase activity of the essential oils of T. caespititius (Sect. Micantes), T. camphoratus and T. capitellatus (Sect. Thymus, Subsect. Thymastra), T. carnosus, T. zygis subsp. sylvestris and T. zygis subsp. zygis (Sect. Thymus, Subsect. Thymus) collected on different regions of Portugal. Material and methods Plant material: Sections and Subsections of Thymus species, Portuguese common names and harvesting places of the investigated plants are reported in Table 1. Voucher samples are deposited in the Herbarium of the Departamento de Biologia e Biotecnologia, Escola Superior Agrária de Bragança (BREZA) and in the Herbarium of the Museu, Laboratório e Jardim Botânico de Lisboa (LISU). Isolation of essential oils: Essential oils were isolated by hydrodistillation for 3h in a Clevengertype apparatus according to the European Pharmacopoeia 3. The essential oils were stored at -20°C in the dark prior to analysis. Chemical analysis of essential oils Gas chromatography (GC): Gas chromatographic analyses were performed using a Perkin Elmer Autosystem XL (Perkin Elmer, Shelton, Connecticut, USA) gas chromatograph equipped with two flame ionization detectors (FIDs), a data handling system and a vaporizing injector port into which two columns of different polarities were installed: a DB-1 fused-silica column (30 m x 0.25 mm i. d., film thickness 0.25 μm) (J & W Scientific Inc., Rancho Cordova, CA, USA) and a DB-17HT fused-silica column (30m x 0.25mm i. d., film thickness 0.15 μm) (J & W Scientific Inc.). Oven temperature was programmed, 45-175°C, at 3°C/min, subsequently at 15°C/min up to 300°C, and then held isothermal for 10 min; injector and detector temperatures, 280°C and 300°C, respectively; carrier gas, hydrogen, adjusted to a linear velocity of 30 cm/s. The samples were injected using split sampling technique, ratio 1:50. The volume of injection was 0.2 μl of a pentane-oil solution. The percentage composition of the oils was computed by the normalization method from the GC peak areas, calculated as mean values of two injections from each oil, without using correction factors. Gas chromatography-mass spectrometry (GC-MS): The GC-MS unit consisted of a Perkin Elmer Autosystem XL (Perkin Elmer, Shelton, Connecticut, USA) gas chromatograph, equipped with DB-1 fused-silica column (30 m x 0.25 mm i.d., film thickness 0.25 μm) (J & W Scientific, Inc.), and interfaced with a Perkin-Elmer Turbomass mass spectrometer (software version 4.1, Perkin Elmer, Shelton, Connecticut, USA). Injector and oven temperatures were as above; transfer line temperature, 280°C; ion trap temperature, 220°C; carrier gas, helium, adjusted to a linear velocity of 30 cm/s; split ratio, 1:40; ionization energy, 70 eV; ionization current, 60 μA; scan range, 40-300 amu; scan time, 1 s. The identity of the components was assigned by comparison of their retention indices, relative to C9-C19 n-alkane indices and GC-MS spectra from a home made library, constructed based on the analyses of reference oils, laboratory-synthesised components and commercial available standards. Acetylcholinesterase (AChE) inhibition: The enzymatic activity was measured according to Mata et al. 2, with minor modifications. Briefly, in a total volume of 1 ml, 415 μl of Tris-HCl buffer 0.1 M (pH 8), 100 μl of ethanolic dilutions of essential oils with different concentrations and 25 μl of enzyme solution containing 0.28 U/ml were incubated for 15 min at room temperature. 75 μl of a solution of acetylthiocholine iodide (AChI) 1.83 mM and 475 mL of 5,5’-dithiobis[2-nitrobenzoic acid] (DTNB) 3 mM were added and the final mixture incubated, for 30 min, at room temperature. Absorbance of the mixture was measured at 405 nm in a Shimadzu 160 UV spectrophotometer. The


142

percentage inhibition of enzyme activity was calculated by comparison with the negative control: % = [(A0 – A1) / A0] * 100 where A0 was the absorbance of the blank sample and A1 was the absorbance of the sample. Tests were carried out in triplicate. Sample concentration providing 50 % inhibition (IC50) was obtained plotting the inhibition percentage against essential oil concentrations. Results and discussion: The four main components of the tested essential oils of Thymus species from several regions of Portugal were already reported 4 and are depicted in Table 1 as well as the antiacetylcholinesterase activities of the oils, for a better correlation of the results obtained. The detailed composition of the thirty-two essential oils tested is presented in Table 2. Acetylcholine (ACh) is delivered in the synapses and is rapidly hydrolyzed into choline and acetate by acetylcholinesterase (AChE). The deficit of ACh causes diseases that are treated with AChE inhibitors, which enhance its level in the synapses by hampering acetylcholine hydrolysis. Alzheimer’s disease therapy, like other diseases with cognitive decline and mental deterioration symptoms, involves the increasing of acetylcholine levels. Some essential oils components that are able to improve the performance and overall quality of memory in healthy adults also possess a strong AChE inhibition capacity 5. The tested essential oils showed a great variability in their AChE inhibitory activity (IC50 = 112448 μg/ml), even within the same species and no straight correlation could be found between essential oil main components and AChE inhibition activity. It is noteworthy the strong AChE inhibition capacity of T. zygis subsp. zygis essential oil (IC50 = 1μg/ml). Of the 32 essential oil samples tested, 16 showed an IC501500 μg/ml) were predominantly found in those oils mainly dominated by α-terpineol (T. caespititius from Caramulo, Covide, Lordelo, Óbidos, Outeiro, Terras de Bouro and Vilarinho das Furnas) or by p-cymene (T. zygis subsp. sylvestris from Condeixa and Duas Igrejas). The discrepancy of results reported in the present work can be attributed to the chemical complexity of the essential oils (Table 2), since such products are constituted by dozens of compounds and each one can exert synergistic or antagonistic effects. In addition, chiral compounds exert diverse biological effects that might also influence our results. Nevertheless it is also important to point out that even pure compounds tested by different authors, using similar methods, also revealed diverse AChE


143

inhibition effects, which turns difficult to compare data and to draw any conclusions. For example, the IC50 values reported for antiAChE activity of carvacrol show some variation: 63 μg/ml 6 and 115 μg/ ml 2. Thymol, an isomer of carvacrol, was reported as being 10 times less active than carvacrol 6, or as being inactive against AChE 7. Nevertheless, it is clear that thymol is a much weaker AChE inhibitor than carvacrol, showing the importance of the position of the hydroxyl group in their molecular structures. For 1,8-cineole, IC50 values also show a remarkable variation: 15 μg/ml 8 and 60 μg/ml 9. Borneol was found to be inactive against AChE 9. In conclusion, among the tested essential oils, practically all carvacrol, borneol or 1,8-cineolerich oils present AChE inhibitory effects, suggesting that these compounds are important for their bioactivity. Nevertheless, in the same group of oils, there are others that showed almost no inhibitory effect on AChE, suggesting that, despite the importance of those compounds, the presence of other minor components or chiral isomers in the essential oils, providing a synergistic or antagonistic effect, is determinant for the AChE inhibition activity. T. zygis subsp. zygis from Rebordãos excels comparatively to the remaining samples as AChE inhibitor. It is important to note that this essential oil showed the closest activity to galantamine, a compound used pharmacologically that showed an IC50 = 0.38 μg/ml 8, determined by the same procedure. This Thymus species from Rebordãos deserves, therefore, some attention in further studies in the determination of the factors responsible for such ability because its oil can be helpful as therapeutic remedies for Alzheimer’s disease. Acknowledgment: This study was partially funded by the Fundação para a Ciência e a Tecnologia (FCT) under research contract PTDC/AGR-AAM/70136/2006.

1.

2.

3. 4.

5.

6.

7.

8. 9.

References Figueiredo, A.C., Barroso, J.G., Pedro, L.G., Salgueiro, L., Miguel, M.G. and Faleiro, M. L. (2008). Portuguese Thymbra and Thymus species volatiles: chemical composition and biological activities. Curr. Pharm. Des. 14: 3120-3140. Mata, A.T., Proença, C., Ferreira, A.R., Serralheiro, M.L.M., Nogueira, J.M.F. and Araújo, M.E.M. (2007). Antioxidant and antiacetylcholinesterase activities of five plants used as Portuguese food spices. Food Chem., 103: 778-786. Council of Europe (COE) - European Directorate for the Quality of Medicines (2007). European Pharmacopoeia 6th Edition. Strasbourg. Dandlen, S.A., Lima, A.S., Mendes, M.D., Miguel, M.G., Faleiro, M.L., Sousa, M.J., Pedro, L.G., Barroso, J.G. and Figueiredo, A.C. (2010). Antioxidant activity of Portuguese thyme species. Flavour Fragr. J. 25: 150-155. Moss, M., Cook, J., Wesnes, K. and Duckett, P. (2003). Aromas of rosemary and lavender essential oils differently affect cognition and mood in healthy adults. Int. J. Neurosci., 113: 1538. Jukic, M., Politeo, O., Maksimovic, M., Milos, M. and Milos, M. (2007). In vitro acetylcholinesterase inhibitory properties of thymol, carvacrol, and their derivatives thymoquinone and thymolhydroquinone. Phytother. Res., 21: 259-261. Orhan, I., Senol, F. S., Gülpinar, A.R., Kartal, M., Sekeroglu, N., Deveci, M., Kan, Y. and Sener B. (2009). Acetylcholinesterase inhibitory and antioxidant properties of Cyclotrichium niveum, Thymus praecox subsp. caucasicus var. caucasicus, Echinacea purpurea and E. pallida. Food Chem. Toxicol., 47: 1304-1310. Dohi, S., Terasaki, M. and Makino, M. (2009). Acetylcholinesterase inhibitory activity and chemical composition of commercial essential oils. J. Agric. Food Chem., 57: 4313-4318. Savalev, S.U., Okello, E.J. and Perry, E.K. (2004). Butyril- and acetyl-cholinesterase inhibitory activities in essential oils of Salvia species and their constituents. Phytother. Res., 18: 315-324.

Thymus caespititius Brot.* / MP: Tormentelo, serpol-do-monte; M: alecrim da serra, hisopo; A: erva-úrsula

Species / Common name

Thymus / Thymus camphoratus Hoffmanns. Thymastra & Link♣/Tomilho-do-mar

Micantes

Section / Subsection

4 Main Components (%)

IC50b

Caramulo (MP)

α-Terpineol (40.3), p-cymene (13.8), γ terpinene (5.4), τ-cadinol (5.2) 5898.0±235.4 Covide (MP) α-Terpineol (35.2), p-cymene (17.3), γ terpinene (9.1), τ-cadinol (6.2) 4647.2±26.1 Lordelo (MP) α-Terpineol (23.5), p-cymene (15.9), γ terpinene (11.7), τ-cadinol (6.9) 1766.5±18.3 Óbidos (MP) α-Terpineol (51.5), p-cymene (14.5), γ terpinene (6.5), τ-cadinol (6.2) 12448.0±517.6 Outeiro (MP) α-Terpineol (40.5), p-cymene (13.7), γ terpinene (8.7), β-caryophyllene (2.7) 3000.6±125.3 Pico (A) Carvacrol (50.7), carvacryl acetate (18.7), p-cymene (5.7), γ-terpinene (3.8) 567.4±12.8 Pico Verde (A) Carvacrol (32.2), thymol (23.0), carvacryl acetate (7.0), p cymene (5.9) 181.4±4.2 Planalto Central (A) Carvacrol (61.9), carvacryl acetate (11.5), α-terpineol (3.0), p-cymene (2.6) 158.9±2.4 Ponta dos Rosais (A) Thymol (24.9), α-terpineol (19.1), p-cymene (11.5), γ-terpinene (9.6) 3601.6±13.4 Serra do Cume (A) Thymol (34.8), carvacrol (12.7), p P-cymene (8.2), thymyl acetate (7.9) 139.1±2.5 Terras de Bouro (MP) α-Terpineol (24.1), γ-terpinene (13.8), p-cymene (12.3), γ-eudesmol (6.2) 2579.1±2.3 Vilarinho das Furnas(MP) α-Terpineol (42.4), p-cymene (13.5), γ-terpinene (6.2), β-caryophyllene (3.7) 4219.5±37.2 Atalaia (MP) 1,8-Cineole (46.7), linalool (12.2), linalyl acetate (8.8), α-pinene (4.3) 182.0±11.4 Boca Rio (MP) 1,8-Cineole (26.5), borneol (15.0), α-pinene (12.3), camphene (11.6) 123.5±34.4

Harvesting place

μg/ml) Table 1. Four main components and acetylcholinesterase inhibition ability of Thymus species essential oils expressed as IC50a (μ

Maria G. Miguel et al. / Jeobp 14 (2) 2011 140 - 150 144

Thymus / Thymus


Thymus carnosus Boiss.♣ / Tomilho-das-praias

Thymus capitellatus Hoffmanns. & Link¨ /Tomilho-do-mato, erva-ursa


table 1. (continued). 4 Main Components (%)

Cabo de S. Vicente (MP) 1,8-Cineole (37.0), α-pinene (10.1), terpinen-4-ol (9.8), borneol (4.3) Espartal (MP) Borneol (23.2), camphor (19.1), camphene (17.2), linalool (9.5) Alcácer do Sal (MP) 1,8-Cineole (35.0), borneol (16.2), α-pinene (12.4), camphene (11.5) Carvalhal (MP) Borneol (20.3), camphene (18.2), camphor (17.8), α-pinene (12.4) Santiago do Cacém (MP) 1,8-Cineole (33.7), borneol (16.9), α-pinene (13.6), camphene (11.2) Sines, Grândola (MP) Borneol (22.4), 1,8-cineole (21.1), camphene (16.9), camphor (10.5) Tróia (MP) 1,8-Cineole (25.8), borneol (21.0), camphene (12.9), α-pinene (11.1) Carvalhal (MP) Borneol (22.9), camphene (21.1), terpinen-4-ol (11.1), α-pinene (9.7) Melides (MP) Borneol (22.9), terpinen-4-ol (18.3), camphene (16.9), bornyl acetate (6.0) Praia do Barril (MP) Borneol (26.0), camphene (18.7), terpinen-4-ol (11.1), bornyl acetate (10.2) Quinta do Lago (MP) Camphene (22.5), borneol (20.1), α-pinene (10.0), bornyl acetate (9.6) Tróia (MP) Borneol (21.1), camphene (19.8), terpinen-4-ol (13.6), bornyl acetate (8.0) Vila Real de S. António Borneol (24.8), camphene (23.7), (MP) bornyl acetate (9.5), terpinen-4-ol (8.1)

Harvesting place

159.0±3.5

1022.9±6.1

136.0±1.3

86.9±2.7

117.9±2.8

130.1±7.3

148.9±3.8

178.5±10.1

125.1±2.2

561.4±59.6

450.3±60.6

195.3±7.7

115.0±9.5

IC50b


4 Main Components (%)

Carvacrol (31.8), p-cymene (23.5), γ-terpinene (7.7), borneol (6.4) p-Cymene (35.9), thymol (24.2), γ-terpinene (7.1), borneol (6.4) sargacinha, erva-santa, erva-das-azeitonas, Covão do Coelho (MP) Carvacrol (34.6), p-cymene (24.6), serpão, tomilho-branco borneol (9.7), camphene (5.3) Duas Igrejas (MP) p-Cymene (39.4), thymol (21.6), γ-terpinene (10.7), linalool (4.3) Thymus zygis Loefl. ex L. subsp. zygis Rebordãos (MP) Carvacrol (43.6), p-cymene (24.1), ♣ /Serpão-do-monte, tomilhinha γ-terpinene (15.8), linalool (3.2)

Harvesting place

Thymus zygis Loefl. ex L. subsp. sylvestris Alcanena (MP) (Hoffm. & Link) Brot. ex Coutinho ♣ / Erva-de-Santa-Maria, marganiça, Condeixa (MP)


* Iberian, Madeira and Azores endemism ♣ Iberian endemism ♦ Portuguese endemism A: Azores M: Madeira MP: Mainland Portugal a Concentration providing 50 % inhibition b Average ± SD from three experiments


table 1. (continued).

1.1±0.0

1766.6±48.9

1479.3±25.5

1352.5±45.0

980.3±14.5

IC50b


Tricyclene α-Thujene α-Pinene Camphene Thuja-2,4(10)-diene Sabinene β-Pinene 3-Octanol β-Myrcene α-Terpinene p-Cymene β-Phellandrene 1,8-Cineole Limonene trans-β-Ocimene γ-Terpinene trans-Sabinene hydrate cis-Linalool oxide trans-Linalool oxide Terpinolene cis-Sabinene hydrate Linalool Camphor trans-Verbenol Borneol Terpinen-4-ol α-Terpineol Linalyl acetate Bornyl acetate Thymol Carvacrol Thymyl acetate

Components

A2

A3

Thymus caespititius A4 A5 A6 A7 A8 A 9 A 10 A 11 A 12

921 0.2 0.1 0.1 0.1 0.1 t t t 0.2 t 924 1.6 2.1 2.3 1.2 1.7 1.7 1.7 0.9 1.8 0.9 2.4 1.7 930 2.1 1.5 1.3 0.8 1.3 0.6 0.6 0.3 0.6 0.3 2.1 0.9 938 - 1.3 1.2 0.3 t 0.1 - 3.3 940 3.2 - 1.5 t t 0.1 - 0.6 958 0.6 0.7 1.0 1.2 1.1 0.6 2.8 t 0.2 0.1 3.9 0.5 963 0.5 0.3 1.3 0.3 0.5 0.1 t 0.1 0.1 0.1 1.5 0.2 974 1.0 1.3 2.2 0.5 1.3 t t t 0.4 1.0 975 1.0 1.3 1.4 0.8 1.3 0.2 t 0.1 t 0.2 0.2 1.0 1002 0.4 0.4 0.1 t 0.7 0.3 0.6 0.5 0.9 0.7 1.5 0.5 1003 13.8 17.3 15.9 14.5 13.7 5.7 5.9 2.6 11.5 8.2 12.3 13.5 1005 t t t t t 0.1 0.1 0.1 t 0.1 t t 1005 1009 2.1 2.2 1.5 1.3 2.0 2.4 0.8 1.1 0.9 1.2 0.4 1.8 1027 t t t t t t t t t 0.2 t 1035 5.4 9.1 11.7 6.5 8.7 3.8 2.6 2.0 9.6 3.8 13.8 6.2 1037 t - 0.1 t t 0.5 0.4 t 0.2 0.2 0.2 t 1045 1059 1064 0.1 0.3 0.4 0.1 0.2 0.4 0.5 0.7 0.1 0.8 0.2 0.2 1066 t t t t t t t t 0.1 t t 1074 t t t t t 0.3 t t t 0.2 t 1095 - 0.4 1114 - 0.7 1134 3.2 1.6 1.7 0.7 2.4 t t t t 4.4 0.8 1148 1.5 0.4 0.9 1.4 1.3 0.6 0.1 0.8 0.5 0.5 2.5 0.8 1159 40.3 35.2 23.5 51.5 40.5 2.9 0.8 3.0 19.1 4.3 24.1 42.4 1245 1265 1.6 0.4 0.2 0.3 0.5 t t t t 1.3 0.1 1275 t t t t 0.4 23.0 2.4 24.9 34.8 t t 1286 t - 0.2 t t 50.7 32.2 61.9 1.7 12.7 0.2 t 1330 - 0.9 4.4 0.9 2.1 7.9 -

RI* A 1 t t 4.3 0.1 0.2 1.5 1.1 t 0.2 46.7 0.7 0.7 0.1 t 2.2 t 1.2 0.1 12.2 11.1 1.9 0.8 0.4 3.6 8.8 t t -

0.6 0.1 1.1 t 0.1 0.0 12.3 10.1 7.1 11.6 2.5 17.2 0.4 1.1 0.5 2.2 2.8 0.2 1.6 0.7 0.7 0.2 2.4 0.2 1.0 3.6 0.7 26.5 37.0 0.8 1.4 0.8 0.0 t t 0.0 0.7 4.1 0.3 0.7 1.3 0.8 0.2 0.4 2.0 0.1 0.3 1.3 t 0.9 0.2 0.5 0,1 1.1 0.7 9.5 2.2 19,1 2.6 0,8 15.0 4.3 23.2 2.1 9.8 1.1 0.4 1.2 1.4 t t 3.1 0.6 1.2 1.1 t t t -

Thymus camphoratus B1 B2 B3 B4

Table 2. Percentage composition of the essential oils isolated from thirty-two populations of five Portuguese Thymus species


- 18.7 2.7 0.1 0.5 t 0.9 1.4 0.9 0.7 0.9 0.2 0.1 0.6 0.1 1.3 t 0.6 0.8 0.9 0.9 2.7 0.9 0.1 2.7 2.1 0.6 t 0.6 t 1.7 0.9 0.5 1.1

7.0 11.5 - 0.1 0.1 t 1.1 0.4 0.8 0.5 0.2 0.4 0.5 0.2 t 0.6 0.2 2.3 1.2 0.1 0.3 2.8 1.9 - 0.2 - 0.2 1.9 1.0 0.8 0.3

t 0.1 0.1 2.0 2.0 0.6 0.9 0.2 1.1 4.4 0.4 6.6 0.3 0.3 1.4 1.4

2.4 t 0.2 1.6 1.3 0.2 0.6 t 0.7 2.7 0.1 4.7 t t 3.3 1.0

0.5 0.4 0.4 1.5 0.9 0.7 1.5 2.2 1.3 6.2 1.4 t t 1.9 2.9

3.7 1.7 0.9 0.9 0.9 0.3 0.1 0.1 1.0 1.2 1.2 1.7 3.1 1.2 1.2 3.0 0.8

A 9 A 10 A 11 A 12 0.2 1.5 0.3 2.0 3.8 -

0.2 1.3 0.4 1.9 0.4 -

0.1 1.5 0.4 0.1 t -

t 0.7 0.1 1.3 0.4 -

Thymus camphoratus B1 B2 B3 B4

A 1: Caramulo; A 2: Covide; A 3: Lordelo; A 4: Óbidos; A 7: Pico Verde; A 8: Planalto Central; A 9: Pta dos Rosais; A 10: Serra do Cume; B 1: Atalaia; B 2: Boca Rio; B 3: Cabo S. Vicente; *Relative to C9-C17 n-alkanes on a DB-1 column; t = trace (


Jorge A. Pino et al. / Jeobp 14 (2) 2011 161 - 163

162

distillation. In this way, all the distillate continuously passed across the layer of the organic solvent during the distillation. The extract was dried over anhydrous Na2SO4 and concentrated to 0.6 mL in a Kuderna-Danish evaporator with a Vigreux column and then to 0.2 mL with a gentle nitrogen stream. A Konik 4000A GC equipped with a DB-5 fused silica column (30 m x 0.25 mm x 0.25 μm) and a flame ionization detector (FID) was used. Injector and detector temperatures were both 250°C. Oven temperature was held at 60°C for 2 min and then raised to 250°C at 4°C/min and held for 10 min. Carrier gas (hydrogen) flow rate was 1 mL/min. Linear retention indices were calculated against those of C8-C24 n-paraffins. Quantitative determinations were carried out by the normalization method, without consideration of calibration factors for all compounds. GC-MS was performed on a Shimadzu QP 500 equipped with an XTI-5MS fused silica column (30 m x 0.25 mm x 0.25 μm) was used. The chromatographic conditions were the same as those described for GC (FID). Carrier gas (helium) flow rate was 1 mL/min. The detector operated in electronic ionization mode (70 eV) at 230°C. Detection was performed in the scan mode between 30 and 400 Daltons. Constituents were identified by comparison of their mass spectra with those of authentic standards (FLAVORLIB library) or those in NBS and NIST libraries, and confirmed in many compounds by their relative retention indices (KI). Mass spectra from the literature 11 were also compared. Results and discussion: The volatile composition of the leaves from J. pectoralis are presented in Table I. Thirty-two constituents were identified, which represented 100 % of the total isolated compounds. This composition is characterized by many aliphatic compounds from C6 to C10, and only one terpene (limonene) was found. We must stress that dihydrocoumarin and umbelliferone were not detected in the isolated oil, even though we specifically looked for them. The most prominent compounds were nonanal (45.9 %), 1-octen-3-ol (8.4 %) and coumarin (7.4 %). The characteristic sweet and herbal odor of the leaves is derived from these compounds 12. Acknowledgements: Special thanks to Lianet Marrero for providing the plant material.

1. 2. 3.

4. 5. 6.

7.

8.

References Roig, J.T. (1974). Plantas Medicinales, Aromáticas o Venenosas de Cuba. Ciencia y Técnica, La Habana. Más, R, Menéndez, R., Garatoix, A., Fernández, L. (1987). Justicia pectoralis: efectos anticolinérgicos en neuronas centrales de moluscos. Rev. Ciencias Biológicas 17: 91-93. Fernández, L., Más, R., Pérez-Saad, H., Biscay, R., Galán, L. (1989). Evaluación preliminar de los efectos neurofarmacológicos de Justicia pectoralis. Rev. Cubana Farm. 23 (1-2), 161166. Rodríguez, C., Virués, T., Alemán, C.L. (1989). Estudio preliminar de la Justicia pectoralis sobre el EEG de adultos normales. Rev. Cubana Farm. 23 (3): 302-308. González, L., Más, R., Alonso, A. (1992). Justicia pectoralis: unión con los receptores cerebrales de los aminoácidos exitatorios. Rev. Cubana Farm. 22 (3): 99-103. Lino, C.S., Taveira, M.L., Viana, G.S.B., Matos, F.J.A. (1997). Analgesic and antiinflammatory activities of Justicia pectoralis Jacq. and its main constituents: coumarin and umbelliferone. Phytotherapy Res. 11: 211-215 de Vries, J.X., Tauscher, B., Wurzel, G. (2005). Constituents of Justicia pectoralis Jacq. 2. gas chromatography/mass spectrometry of simple coumarins, 3-phenylpropionic acids and their hydroxy and methoxy derivatives. Biomed. Environ. Mass Spect. 15: 413-417. Moreno, E., Valero, M., Herrera, P. (1994). El uso de plantas mágicas y medicinales por las parteras tradicionales cubanas. Fontqueria 39: 219-241.

Jorge A. Pino et al. / Jeobp 14 (2) 2011 161 - 163 9. 10.

11. 12.

163

Hernández-Cano, J., Volpato, G. (2004). Herbal mixtures in the traditional medicine of Eastern Cuba. J. Ethnopharm. 90: 293-316. Fuentes, V. (1984). Sobre la medicina tradicional en Cuba. Boletín de Reseña de Plantas Medicinales. Centro de Información y Documentación Agropecuaria, La Habana, vol. 10, pp. 1-39. Adams, R.P. (2001). Identification of Essential Oil Components by Gas Chromatography/ Quadrupole Mass Spectroscopy. Allured Publishing Co., Carol Stream, IL. Arctander, S. (1969). Perfume and Flavor Chemicals. Det Hoffensbergske Etablissement, Copenhagen. Table I. Volatile composition of the leaves from Justicia pectoralis Compound 2-Furfural (E)-2-Hexenal (Z)-3-Hexenol 1-Hexanol Nonane Heptanal Benzaldehyde 1-Heptanol 1-Octen-3-one 1-Octen-3-ol 3-Octanone 2-Octanone 3-Octanol Octanal Limonene Phenylacetaldehyde (E)-2-Octenal 1-Octanol 2-Nonanone Nonanal (E)-2-Nonenal 1-Nonanol 2-Decanone Safranal Decanal (E)-2-Decenal Undecanal Nonanoic acid (E)-β-Damascenone Ethyl decanoate Dodecanal Coumarin tr: < 0.1%

RI

%

836 855 859 871 900 904 960 967 979 982 985 991 993 999 1030 1042 1057 1068 1090 1101 1162 1169 1192 1197 1202 1264 1307 1271 1385 1396 1410 1434

0.4 0.5 1.0 0.7 0.3 4.4 2.1 tr 1.1 8.4 tr 0.7 1.7 4.7 0.7 1.2 0.3 1.9 0.2 45.9 0.6 0.9 3.2 0.3 1.5 2.7 0.3 2.7 0.3 3.7 0.3 7.4

Jeobp 14 (2) 2011 pp 164 - 171

164


Effect of Ionizing Irradiation on Origanum Leaves (Origanum vulgare L.) Essential Oil Composition Juan J. Elizalde *, Mónica Espinoza Cramer Research Area, Lucerna 4925, Cerrillos, Santiago, Chile Received 19 June 2010; accepted in revised form 26 November 2010

Abstract: The composition of essential oils depends among others on the characteristics of the starting material and production process. In this work, dried Origanum vulgare L. leaves were subjected to gamma irradiation and then processed by steam distillation to obtain an essential oil, which was analyzed by GC-MS and HPLC-DAD. The composition of the essential oil was determined, and changes in the aromatic profile were analyzed by changes in both chromatographic profile and compound content. Among the aromatic compounds of the oregano essential oil, a significant direct relation with irradiation dose was observed for four compounds (Myrcene, p-cymene, carvacryl methyl ether and l-borneol), whereas important changes were observed for carvacrol and thymol in those irradiated samples when compared with the non-irradiated control sample oil. Key words: Origanum vulgare, essential oil, chemical composition, gamma irradiation, chromatography. Introduction: Origanum leaves are widely used as flavouring agent for various culinary applications. The essential oil obtained from the plant is used at industrial stage as flavouring, mainly for seasonings and also as ingredient for fragrances and cosmetics 1-6. Origanum vulgare essential oil main components are basically the same found in other herbs from the Labiatae family. They include phenols (as carvacrol and thymol), p-cymene, β-caryophyllene, linalool and other terpenoids 7. There are clear differences in the composition of essential oils obtained from different starting materials, e.g. different genus species 8 or even same plants from different geographic origin. The amount and proportion of constituents of an essential oil may influence the sensorial profile, and thus, lead to problem standardization on the product when not taken into account. It has been demonstrated that different processing methods (e.g. distillation, drying and extraction) during production of essential oil from different herbaceous species affect the essential oil composition 7,9,10. According to this, in order to obtain a standard quality essential oil, care must be taken to have a controlled and uniform production process, from the starting material to the end product. A common procedure used at industrial level to extend shelf life of food products is the application of ionizing gamma radiation. Many products such as seasonings, dehydrated fruits and vegetables, frozen foods and herbs are treated with this type or irradiation in order to sterilize or reduce microbiological content, finally leading to reduced pathogenicity and enhanced storage life. Ionizing radiation penetrates products without leaving residues, *Corresponding author (Juan J. Elizalde) E-mail: < [email protected] >


Juan J. Elizalde et al. / Jeobp 14 (2) 2011 164 - 171

165

but producing changes and breaking DNA chains in bacteria and other microorganisms causing its elimination or avoiding its reproduction. Cramer uses ionizing radiation as a routine process to sterilize many food (especially herbs) products. Through an external service company, gamma radiation from Co60 is applied at standard conditions to spices and herbs. Since every step of essential oil production has an influence on the final result of the product, the question arises if the irradiation of the starting material using ionizing radiation could possibly affect the composition of the essential oil obtained when distilling the herb. The aim of this study was to test the influence of irradiation of the dried herb of Origanum vulgare L. on the composition of the essential oil obtained after steam distillation. Experimental Plant material: Dried leaves of Origanum vulgare L., were used. The variety is local and grows wild near Santiago Valley. A 30 Kg sample of oregano leaves was kept without the irradiation process normally used for preservation and storage. 1 Kg proportions of this lot were then separated, stored in paper bags and subjected to different doses of gamma irradiation. The process of irradiation was performed by CCE (Chilean Company of Sterilization), according to standard certified procedures.. Doses of gamma radiation from Co60 applied to plant material were 1 KGy, 2 KGy, 3 KGy, 5 KGy, 10 KGy, and 15 KGy. A portion of non-irradiated plant material was kept and used as a control. Three independent replicates of the treatment were performed and analyzed in each case. Extraction of essential oils: Steam distillation was used as the extraction method of the essential oil of oregano. 50 g dried oregano leaves were placed in a still, and water steam distilled for 20 minutes. The obtained essential oil was received in a column of water and then separated and stored for GC and HPLC analysis. Gas chromatography - Mass spectrometry : The composition of oregano essential oil obtained by steam distillation with and without ionizing irradiation was analyzed by Gas Chromatography coupled to Mass Spectrometry detection. GC analyses were performed using an Agilent 7890A GC System and an Agilent 5975C inert Mass Spectrometer Detector, equipped with a HP-5 column (60m x 0.25 mm i.d.; film thickness 0.25 μm). Carrier gas used was helium, with a linear velocity of 25 cm/ s. Split ratio was 1:29. Oven temperature was held at 30°C for 8 min and then programmed to 300°C at a rate of 30°C/min. Temperature was held at 300°C for 20 min. Mass spectra were taken at 70 eV, mass range was from 20 to 350 amu. High performance Liquid chromatography (HPLC): The actual concentration of carvacrol and thymol was determined by HPLC coupled to a Diode Array Detector. HPLC analyses were performed with a Merck Hitachi LaChrom HPLC System with a L-7100 HPLC quaternary pump, combined with a 10 mm pre-column and a 100-4.6 mm Merck Chromolith Performance RP-18e analytical column, and a L-7450 photodiode array detector (DAD). The system was controlled with HPLC D-7000 software with a D-7000 data interface. A binary MeCN-H2O acidified gradient was used for elution. Solvents A and B were MeCN-H2O-H3PO4 (30 %:69.75 %:0.25 %) and MeOH respectively. At a flow of 1.5 mL/min, the eluent consisted of 100 % A during the first 12 min, then the percent of B was increased to 100 % at 12.1 min, and remained at this percentage during the next 3 min. At 15.1 min the percent of solvent A was again increased to 100 %, where it remained the last 2 min of run time. Carvacrol and thymol detection was made at 274 nm. With this method the retention times were 11.5 min and 13.2 min for carvacrol and thymol respectively. Results and discussion: The oils obtained from different dose-treated oregano leaves by steam distillation were found to be yellow liquids with no significant differences in yield. After distillation,


166

essential oils were first analyzed by GC-MS, in order to obtain the aromatic compound profile. The main peaks obtained in the chromatographic profile were identified and the amount expressed as relative percentage of the total peaks determined. Under chromatographic conditions previously described, approximately 40 peaks were clearly identified, representing nearly 95 % of the total area detected in each chromatogram. The remaining 5 % of total area of the chromatogram was distributed between small peaks, which were not possible to identify by means of the mass spectra obtained for them. Figure 1 shows the GC chromatogram obtained for non-irradiated oregano essential oil. Table 1 shows the main compounds found and identified, and the relative amount of each of them. TIC: SINIRRAD.D 4.4e+07 4.2e+07 4e+07 3.8e+07 3.6e+07 3.4e+07 3.2e+07

Abundance

3e+07 2.8e+07 2.6e+07 2.4e+07 2.2e+07 2e+07 1.8e+07 1.6e+07 1.4e+07 1.2e+07 1e+07 8000000 6000000 4000000 2000000 16.00

18.00

20.00

22.00

24.00

26.00

28.00

30.00

32.00

34.00

Time Figure 1. GC-MS chromatogram of non-irradiated oregano essential oil When analyzing the chromatographic profiles of the irradiation treated oregano oils, a clear dose-dependent number of peaks increase was observed. The number of unidentified peaks was higher for higher dose-irradiated oregano essential oil, thus, the relative total area of identified peaks decreased with an increase of irradiation dose on the oregano used as starting material for the oil. Figure 2 shows the difference in the chromatographic profile in the zone of 32 to 50 minutes, between the non-irradiated oregano essential oil, and the 5 KGy irradiated oregano leaves essential oil. The increase in the number of unidentified peaks was observed mainly in the last part of the chromatograms; thus, correspond to higher molecular mass compounds. These peaks could represent reaction products of the aromatic compounds contained in oregano leaves and then extracted in the essential oil. Although when treating oregano with ionizing radiation there was no change in the identified aromatic compound oil composition, there was a change in the proportion of some of the aromatic compounds found in it. When comparing non-irradiated with irradiated oregano oil composition, it was observed an increase in the relative amount of some compounds, while a decrease in the relative amount was observed for some other components. Table 2 shows the aromatic compounds that have an increase in relative amount in those irradiation-treated samples compared with non-irradiated oregano oil. Compounds showing a decrease in relative amount oil are included in Table 3. As can be seen from Tables 2 and 3, some of the compounds show a non-dose-dependent change in their relative amount, while some other show a clear dose-dependent effect is observed for four aromatic compounds. Myrcene, p-cymene and carvacryl methyl ether relative amount increases when


167

increasing the ionizing radiation dose up to 10 KGy. L-Borneol on the opposite seems to decrease with an increase in radiation dose. The observed trend is confirmed by regression statistical analyses, with positive values for variable x (slope) with P of 0.0166, 0.0055 and 0.019 for myrcene, p-Cymene and carvacryl methyl ether respectively. A negative slope is confirmed by statistical analysis for l-Borneol, having a P value of 0.00030 At 15 KGy, both increase and decrease observed for the mentioned compounds seem to have stop, and get to stabilization in relative amount of each of them. TIC: SINIRRAD.D TIC: IRR5KGY.D

1000000

900000

800000

Abundance

700000

600000

500000

400000

300000

200000

100000

32.00

34.00

36.00

38.00

40.00

42.00

44.00

46.00

48.00

Time Figure 2. Chromatogram overlay of non-irradiated oregano essential oil (lower line) and 5 KGy irradiated oregano essential oil (upper line). The zone of 32 to 50 minutes of the chromatograms is shown On the other hand, Carvacrol and Thymol, two important aromatic compound that confer the characteristic oregano profile, seem to be influenced by the irradiation process of the starting material. Figures 3 and 4 show the observed trend for carvacrol and thymol respectively. As observed in Figures 3 and 4, both carvacrol and thymol seem to increase linearly with irradiation dose up to 5 KGy, and then stars to decrease from 5 KGy to 15 KGy. Possibly they continue to increase in the higher doses of irradiation, but they are consumed in the formation of some reaction products, as carvacryl methyl ether and thymyl methyl ether. The former has a clear increase in irradiated samples, the latter shows a small increase with increased irradiation dose, but it is not statistically significant, so more data are needed to confirm this trend. In brief, the irradiation process effects on the final essential oil produced are reflected in: a) an increase in the number of compounds of the essential oil, being this increase due mainly to the emergence of unidentified compounds (probably high molecular mass terpenes, according to their retention time in the chromatographic analysis); b) an effect (increase or decrease) in some of the identified compounds of the oil (Tables 2 and 3), being this effect not necessarily linear with increasing irradiation dose, c) a linear effect with irradiation dose on myrcene, p-cymene, carvacryl methyl ether and l-borneol; and finally, d) a linear effect of irradiation dose on carvacrol and thymol content of the oil up to 5 KGy. Clearly, being the irradiation process based on gamma (ionizing) radiation, it not only acts in sterilizing the material, but it also provides the energy for producing changes in the composition of the essential oil contained in the oregano leaves. The final effect on different compounds probably responds to a complex series of mechanisms, and a possible explanation to the observed effects need further research.


168

Taking into account that there is no change in the oil yield but rather in the proportion of some of the aromatic compounds, including the characteristic oregano compounds carvacrol and thymol, the irradiation of oregano could lead to a change in the herb, and thus in the sensory profile of the essential oil. Carvacrol content in oregano essential oil at increasing irradiation does 4.8 4.6

Carvacrol (%)

4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 0

2

4

6

8

10

12

14

16

Irradiation doses (KGy) Figure 3. Carvacrol content determined in oregano essential oil, at increasing gamma-irradiation doses. Percentages were measured by HPLC-DAD. Values correspond to averages of three independent measurements, with bars showing the dispersion of data Thymol content in oregano essential oil at increasing irradiation does 19.0

Thymol (%)

18.0 17.0 16.0 15.0 14.0 13.0 0

2

4

6

8

10

12

14

16

Irradiation doses (KGy) Figure 4. Thymol content determined in oregano essential oil, at increasing gamma-irradiation doses. Percentages were measured by HPLC-DAD. Values correspond to averages of three independent measurements, with bars showing the dispersion of data


169

Acknowledgement: We thank Innova Chile Program of CORFO through project number 08IE16078, for financial support.

1.

2. 3. 4. 5. 6. 7.

8. 9.

10.

11.

References. Vági, E., Simándi, B., Suhajda, Á., Héthelyi, E. (2005). Essential oil composition and antimicrobial activity of Origanum marjoram L. extracts obtained with ethyl alcohol and supercritical carbon dioxide. Food Research International, 38: 51-57. Aburjai, T., Natsheh, F.M.. (2003). Plants used in cosmetics. Phytotherapy Research, 17: 987-1000. Pybus, D., Sell, C. (1999). In The Chemistry of Fragrances. The Royal Society of Chemistry. Leung and Foster (1995). In Encyclopedia of Common Natural Ingredients: Used in Food, Drugs, and Cosmetics. Wiley-Interscience, New York, Calkin, R.R., Jellinek, J.S. (1994). In Perfumery: Practice and Principles. Wiley-Interscience, New York. Hay, R.K.M., Waterman, P.G. (1993). In Volatile Oil Crops: Their Biology, Biochemistry and Production. John Wiley & Sons, New York. Sefidkon, F, Abbasi, K., Bakhshi Khaniki, B. (2006). Influence of drying and extraction methods on yield and chemical composition of the essential oil of Satureja hortensis. Food Chemistry, 99: 19-23. Nickavar, B., Mojab, F., Dolat-Abadi, R. (2005). Analysis of the essential oils of two Thymus species from Iran. Food Chemistry, 90: 609-611. Durling, N., Catchpole, O., Grey, J., Webby, R., Mitchell, K., Yeap Foo, L., Perry, N. (2007). Extraction of phenolics and essential oil from dried sage (Salvia officinalis) using ethanolwater mixtures. Food chemistry, 101: 1417-1424. Sefidkon, F., Abbasi, K., Jamzad, Z., Ahmadi, S. (2007). The effect of distillation methods and stage of plant growth on the essential oil content and composition of Satureja rechingeri Jamzad. Food Chemistry, 100: 1054-1058. Adams, R.P (2001). The Identification of Essential Oil Components By Gas Chromatogaphy/ Quadrupole Mass Spectroscopy. Allured Publishing Corporation, Carol Stream, Illinois.


170

Table 1. Oil composition of non-irradiated Oregano Data shown correspond to average of three replicates RI 939 954 975 979 991 1003 1003 1017 1025 1029 1031 1031 1050 1060 1089 1098 1122 1132 1144 1169 1177 1189 1196 1197 1216 1235 1243 1245 1257 1270 1290 1299 1419 1423 1455 1466 1506 1563 1578 1583

Compound

(%)

α-Pinene Camphene Sabinene β-Pinene Myrcene α-Phellandrene β-Phellandrene α-Terpinene p-Cymene Limonene δ-3-Carene δ-4-Carene β-Ocimene γ-Terpinene Terpinolene trans-Sabinene hydrate cis-p-2-Menthen-1-ol α-Ocimene cis-β-Terpineol l-Borneol Terpinen-4-ol α-Terpineol Piperitol 2-Decen-1-al Linalyl Propionate Thymyl Methyl Ether δ-Carvone Carvacryl Methyl Ether Linalyl Acetate n-Decanol Thymol Carvacrol β-Caryophyllene Linalyl Butyrate α-Humulene trans-2-Dodecenal β-Bisabolene Nerolidol Spathunellol Caryophyllene Oxide Unidentified

Data corresponds to average of three replicates;

0.48 0.04 3.65 0.34 1.49 0.84 0.52 4.32 3.69 1.52 0.04 1.38 0.13 7.30 1.68 3.68 2.03 0.56 16.49 0.31 10.24 4.35 0.79 0.06 0.83 0.41 0.11 1.89 3.92 0.04 13.26 3.35 2.67 0.00 0.35 0.02 1.13 0.05 0.56 0.33 5.15

RI: Kovats Index by Robert Adams 11


171

Table 2. Compounds with increased relative amount in irradiated oregano oil compared with non-irradiated oregano oil. Compounds marked with (*) were quantified by HPLC-DAD. Data shown in table correspond to averages of three replicate analyses Compound

Non 1 KGy irradiated

α-Pinene 0.48 Sabinene 3.65 Myrcene 1.49 p-Cymene 3.69 Ocimene 0.56 cis-β-Terpineol 16.49 Carvacryl pethyl ether 1.89 Linalyl propionate 0.83 Thymol (*) 13.26 Carvacrol (*) 3.35 Spathunellol 0.56 Caryophyllene oxide 0.33

0.64 4.59 1.88 3.73 0.79 23.81 2.13 2.39 15.12 3.83 1.02 0.61

2 KGy

3 KGy

5 KGy

0.60 4.40 1.95 3.88 0.80 22.06 2.40 2.75 16.99 4.11 0.98 0.56

0.57 5.01 2.02 4.03 0.92 23.26 2.56 2.13 17.42 4.36 1.10 0.65

0.64 5.01 2.13 4.38 0.92 22.17 2.65 2.78 18.36 4.55 1.10 0.62

10 KGy 15 KGy

0.66 5.20 2.23 4.41 0.93 19.37 2.77 3.69 17.13 4.52 1.27 0.80

0.70 5.31 2.21 4.57 0.88 22.04 2.57 2.61 16.66 4.40 1.05 0.59

Table 3. Compounds with decreased relative amount in irradiated oregano oil compared with non-irradiated oregano oil. Data shown in table correspond to averages of three replicate analyses Compound

Non 1 KGy irradiated

β-Phellandrene 0.52 α-Terpinene 4.32 γ-Terpinene 7.30 trans-Sabinene hydrate 3.68 Terpinolene 1.68 cis-p-2-Menthen-1-ol 2.03 δ-4-Carene 1.38 l-Borneol 0.31 Terpinen-4-ol 10.24 Piperitol 0.79

0.24 2.20 4.95 3.18 0.65 0.94 0.53 0.27 4.29 0.49

2 KGy

3 KGy

5 KGy

0.25 2.27 5.80 2.96 0.66 0.73 0.35 0.26 3.49 0.35

0.23 1.89 5.39 2.65 0.46 0.59 0.41 0.19 3.02 0.31

0.25 1.99 5.28 2.78 0.57 0.61 0.31 0.22 2.85 0.33

10 KGy 15 KGy

0.27 2.18 5.68 2.49 0.61 0.69 0.38 0.20 2.94 0.40

0.26 2.13 5.37 2.78 0.60 0.61 0.30 0.19 3.04 0.31

Jeobp 14 (2) 2011 pp 172 - 174

172


GC-MS Analysis of Ammoides atlantica (Coss. et Dur.) Wolf. from Algeria Tarek Boudiar 1, Chawki Bensouici 1, Javad Safaei-Ghomi 2, Ahmed Kabouche 1 and Zahia Kabouche 1* 1

Laboratoire d’Obtention de Substances Thérapeutiques (L.O.S.T), faculté des sciences, Université Mentouri-Constantine, Campus Chaabat Ersas, 25000 Constantine, Algeria 2 The Essential Oil Research Center, University of Kashan, 51167 Kashan, Iran Received 18 September 2010; accepted in revised form 15 February 2011

Abstract: The essential oil of fresh aerial part of Ammoides atlantica Coss. et Dur. (Apiaceae), obtained by hydrodistillation in a Clevenger-type apparatus, was analyzed by GC-MS. Twenty compounds were characterized representing 97.9 % of the essential oil with safranal (17.9 %), endo-borneol (17.6 %), chrysanthenone (15.5 %), filifolone (12.1 %) and camphor (11.8 %) as main components. Key words: Ammoides atlantica (Coss. et Dur.) Wolf., Apiaceae, Essential oil, Safranal, endoBorneol, Chrysanthenone, Filifolone, Camphor. Introduction: Two Ammoides (Apiaceae) species, A. pusella and A. atlantica are found in Algeria 1. These species are used in folk medicine as antibacterial, anti-diarrhoea, anti-fever, antiinfluenza and to treat vitiligo in addition of their use as condiments 2. The aqueous extract of A. pusilla is used in Eastern Morocco to treat diabetes 3. Antimicrobial and antibacterial activities have been recently established for the essential oils of A. pusella and A. atlantica (Coss. et Dur.) Wolf. from Setif, respectively 4,5. Our work concerns the GC-MS analysis of the essential oil of the species A. atlantica (Coss. et Dur.) Wolf. growing at Jijel, locally used to treat children fever. Experimental Plant material: Ammoides atlantica (Coss. et Dur.) Wolf. was collected in May 2008 at Jijel. The plant has been gathered by Mr. Kamel Kabouche and authenticated by Prof. Gérard De Belair (Annaba University, Algeria). Voucher specimen (ZKLOST Aa05/08) was deposited at the herbarium of the Laboratory of Substances Therapeutic (LOST), Faculty of Sciences, Mentouri-University, Constantine, Algeria. Essential oils extraction: The hydrodistillation of 200 g of fresh leaves of Ammoides atlantica (Coss. et Dur.) Wolf. for 3 h using a Clevenger-type apparatus, yielded 1.2 % (w/w) of a yellowish oil which was stored until analyzed. *Corresponding author (Zahia Kabouche) E- mail: < [email protected] >


Zahia Kabouche et al. / Jeobp 14 (2) 2011 172 - 174

173

Gas chromatography-Mass spectrometry: GC analysis was performed on a Shimadzu GC17A gas chromatograph equipped with a cross-linked DB5-MS column (40 m × 0.18 mm, film thickness 0.18 μm). The oven temperature was programmed as isothermal at 60°C for 5 min, then raised to 275°C at 5°C/min and held at this temperature for 5 min. Helium was used as the carrier gas at a rate of 1 ml/min. GC-MS was performed using a Shimadzu QP5050 mass selective detector. Operating conditions were the same as for the analytical GC. The MS operating parameters were as follows: 0.1 mL of crude oil was mixed with diethyl ether (40 %); ionization potential, 70 ev; ionization current, 2 A; ion source temperature, 200°C; resolution, 1000. scan time, 5 s; scan mass range, 40-400 amu; split ratio, 1:50; linear velocity, 30.0 cm/sec. Relative percentage amounts were calculated from peak area without the use of correction factors. Essential oil components were identified based on their retention indices (determined with reference to a homologous series of normal alkanes), and by comparison of their mass spectral fragmentation patterns with those reported in the literature 6,8 and with authentic compounds. Results and discussion: The hydrodistillation of aerial parts of Ammoides atlantica (Coss. et Dur.) Wolf. yielded 1.2 % of a yellowish oil. Twenty compounds were characterized representing 97.9 % of the essential oil with with safranal (17.9 %), endo-borneol (17.6 %), chrysanthenone (15.5 %), filifolone (12.1 %) and camphor (11.8 %) as main components (Table 1). It appears that the composition of our oil is quite different from the previously reported essential oil of A. atlantica collected from Djebel Meghress in the province of Setif 5, mainly represented with thymol (44.5 %), γ-terpinene (32.9 %) and ρ-cymene (13.5 %) (Table 1). Acknowledgments: We are grateful to the ANDRS and MESRS (DG/RSDT) for financial support.

1. 2. 3.

4.

5.

6. 7.

References Quezel, P. and Santa, S. (1963). Nouvelle Flore de l’Algérie et des Régions Désertiques Méridionales. C.N.R.S., Paris, France. Bellakhdar , J. (1997). La pharmacopée marocaine traditionnelle. Ibiss press, Paris, France Bnouham, M., Merhfour, F.Z., Legssyer, A., Mekhfi, H., Maallem, S. and Ziyya, A. (2007). Antihyperglycemic activity of Arbutus unedo, Ammoides pusilla and Thymelaea hirsute. Pharmazie, 62: 630-632. Laouer, H, Zerroug, M.M, Sahli, F., Chaker, A.N., Valentini, G., Ferretti, G., Grande, M. and Anaya, J. (2003). Composition and antimicrobial activity of Ammoides pusilla (Brot.) Breistr. essential oil, Journal of Essential Oil Research, 15: 135-138. Laouer, H., Boulaacheb, N., Akkal, S., Singh, G., Marimuthu, P., De Heluani, C., Catalan, C. and Baldovini, N. (2008). Composition and antibacterial activity of the essential oil of Ammoides atlantica (Coss. et Dur.) Wolf. Journal of Essential Oil Research, 20: 266-269. Adams, R.P. (2007). Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th Ed. Allured Publishing Co. Carol Stream, Illinois. Mclafferty, F.W, Stauffer, D.B. (1991). The Important Peak Index of the Registry of Mass Spectral Data. John Wiley & Son, New York.

Zahia Kabouche et al. / Jeobp 14 (2) 2011 172 - 174

174

Table 1. Chemical compositions of Ammoides atlantica (Coss. et Dur.) Wolf. essential oil

a

No

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 14 18 19 20

Camphene p-Cymene cis-Sabinene hydrate Filifolone α-Thujone Chrysanthenone Camphor endo-Borneol Terpenin-4-ol Safranal Verbenone Pulegone E-Ocimenone Bornyl acetate Thymol α-Copaene Ar-Curcumene Germacrene D δ-Cadinene Viridiflorol

RIa

Percentage composition

954 1025 1070 1080 1102 1128 1146 1169 1177 1197 1205 1235 1238 1289 1290 1377 1481 1485 1523 1593

2.4 0.1 1.8 12.1 1.6 15.5 11.8 17.6 3.5 17.9 0.8 2.4 2.4 0.8 1.8 0.8 0.9 1.2 0.6 1.9

RI = retention indices as determined on DB-5MS column using homologous series of nalkanes

Jeobp 14 (2) 2011 pp 175 - 184

175


Assessment of the Preservative Activity of Some Essential Oils to Reduce Postharvest Fungal Rot on Kiwifruits (Actinidia deliciosa) Habib Shirzad 1*, Abbas Hassani 1, Ali Abdollahi 2, Youbert Ghosta 3, Seied Rasool Finidokht 1 Department of Horticultural Sciences, Faculty of Agriculture, Urmia University, Urmia, P.O. Box: 165, Iran 2 Department of Medicinal Plants, Faculty of Agriculture and Natural Resource, Sistan and Baluchestan University, Saravan, P.O. Box: 99515-143, Iran 3 Department of Plant Protection, Faculty of Agriculture, Urmia University, Urmia, P.O. Box: 165, Iran 1

Received 03 March 2010; accepted in revised form 18 September 2010

Abstract: The aim of this study was to find a natural alternative for synthetic agrochemicals currently used in preservation of kiwifruits during postharvest stage. Different concentrations (0, 250, 500 and 750 μl/l) of thyme (Thymus vulgaris L.), ajowan (Carum copticum L.), fennel (Foeniculum vulgare Mill.) and summer savory (Satureja hortensis L.) essential oils were sprayed on kiwifruits. Treated fruits were kept in storage (0 - 1°C) for 90 days. Accordingly, T. vulgaris and S. hortensis oils showed a good antifungal activity compared with C. copticum and F. vulgare. Evaluation of sensory parameters showed that total soluble solids content (TSS), titrable acidity (TA) and vitamin C content were decreased under essential oil treatment while, fruit firmness and weight loss were not affected significantly. In addition, TSS, TA, TSS/TA and vitamin C content in kiwifruits treated with T. vulgaris oil were higher than other oils. GC and GC-MS analysis showed that β-ocimene (12.6 %), thymol (63.0 %), trans-anethole (64.7 %) and carvacrol (54.1 %) were the main compounds identified in T. vulgaris, C. copticum, F. vulgare and S. hortensis oils, respectively. Key words: Essential Oil, Kiwi fruit, Antifungal, Postharvest, Fungal decay. Introduction: The storage period and marketing life of fruits and vegetables are generally lost via some fungal diseases. Kiwi fruit [Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson var deliciosa] is a climacteric and susceptible fruit sensitive to decay caused by several phytopathogenic fungi, including Botrytis cinerea, Botryosphaeria dothidea, Colletotrichum, Alternaria alternata, Sclerotinia, Phoma and Diaporthe actinidiae. Stem-end rot caused by B. cinerea is the most important postharvest disease of kiwifruit in many areas of the world 1. Application of synthetic fungicides is a primary way to reduce postharvest fungal diseases in kiwifruit 2. However, postharvest use of synthetic fungicides is becoming ineffective due to development of fungicide resistant strains of postharvest fungi and also consumers awareness on the side-effects of chemical fungicides 3. In addition, recently, postharvest use of chemical fungicides considered forbidden *Corresponding author (Habib Shirzad) E- mail: < [email protected] >


Habib Shirzad et al. / Jeobp 14 (2) 2011 175 - 184

176

in European Union countries 1. Therefore, in response to these challenges, several biological and physical approaches such as application of plant and animal derived substances i.e. table grape volatiles 1 salicylic acid 2 and chitosan 5; biological control agents 6, modified atmosphere 7, controlled atmosphere 8 and ozone 9 were evaluated for preservation of postharvest quality parameters in kiwifruit. But, researches on the application of plants derived essential oils for this purpose are limited. Essential oils are complex natural mixtures of volatile secondary metabolites, commonly isolated from plants by steam distillation and cold expression. The main constituents of essential oils (mono and sesquiterpenes), along with carbohydrates, alcohols, ethers, aldehydes and ketons are responsible for fragrant and biological properties of aromatic and medicinal plants. Essential oil bearing plants have widely been used as flavouring and pharmaceutical agents in food and drugs since recorded history and it is established that many of essential oils have a wide range of antifungal, antiviral, antibacterial and insecticidal actions 10, 11, 12, 13. The literature review showed several examples of previous trials conducted on the antifungal properties of various essential oils against postharvest fungi. Antifungal activity of essential oils has mostly been tested in in-vitro conditions. For example, the antifungal activity of thyme (Thymus vulgaris), ajowan (Carum copticum), fennel (Foeniculum vulgare) and summer savory (Satureja hortensis) against B. cinerea was reported previously 14, 15, 16. But only a few studies conducted on the antifungal/preservative activities of essential oils on fruits and vegetables. Thanassoulopoulos and Yanna 17 studied the antifungal activity of essential oils from origanum, sweet basil and thyme on gray mold rot in kiwifruits. Antifungal activity of T. vulgaris essential oil has been investigated against B. cinerea and A. alternata rots in strawberry and cherry tomatoes as well 18, 19. Furthermore, antifungal activity of S. hortensis essential oil has been confirmed against Aspergillus flavus grown on lemon fruits 20. Similarly Tripathi et al. 21 showed that essential oils from Prunus persica, Ocimum sanctum and Zingiber officinale inhibited the growth of B. cinerea on table grapes and increased the storage life of oil treated grapes by 4, 5 and 6 days, respectively. Plant essential oils posses a number of highlighted advantages over traditional chemical fungicides which make their future applications promising. In general, they are considered safe to human, biodegradable and environmentally friendly, non phytotoxic and very low risk that pathogens will develop resistance to the mixture of components that make up the oils with their apparent diversity of antifungal mechanisms 15. The objective of our research was to apply the essential oils obtained from T. vulgaris, C. copticum, F. vulgare and S. hortensis as antifungal/preservative agents to maintain fresh kiwifruit quality through storage. Experimental Essential oil preparation: Four locally available aromatic plants were selected in order to extract their essential oils. Fruits of ajowan (Carum copticum L.), fennel (Foeniculum vulgare Mill.) and aerial parts of thyme (Thymus vulgaris L.) and summer savory (Satureja hortensis L.) harvested from plants growing in the Research Garden of the Agricultural College of Urmia University and identified by Dr. Abbas Hassani from Department of Horticultural Sciences, Urmia University were used for extraction of essential oils by hydrodistillation during 3 hrs using a Clevenger-type apparatus. The oils were separated, dried over anhydrous sodium sulphate and kept in air tight sealed dark glasses at 4°C until used. GC and GC-MS analysis: The GC analyses were carried out on a Shimutzu 17A gas chromatograph equipped with a non-polar DB-5 (95 % dimethyl polysiloxane) capillary column (30 m × 0.25 mm; 0.25 μm film thickness). The oven temperature was held at 30°C for 3 min then programmed at 280°C. Other operating conditions were as follows: carrier gas He, with a flow rate of 2.1 ml/min; injector temperature 230°C; detector temperature 250°C; split ratio, 50:1 GC-MS analyses were performed on a Shimadzu 17A GC coupled to a Shimutzu QGD5050 Mass Spectrometer. The operating conditions were the same as described above. Mass spectra were taken at 70 eV. Mass range was from


177

m/z 50-450 amu. The constituents of the oils were identified by calculation of their retention indices under temperature-programmed conditions based on co-injection of homologous n-alkanes (C6-C24) on DB5 capillary column. Compounds were identified by comparison of their mass spectra with those of the internal reference mass spectra library (NIST 98 and Wiley 5.0) or with authentic compounds or with those reported in the literature 22, 23. Percentage amounts of individual components were obtained from FID area without using the correction factors. Treatment of kiwifruits with essential oils: Kiwifruits (Actinidia deliciosa (A. Chev.) C.F. Liang and A.R. Ferguson var. deliciosa ‘Hayward’) were obtained from Urmia wholesale market in Urmia, Iran. Fruits were selected for uniformity in size, appearance, ripeness and the absence of physical defects. The selected fruits were randomized before being used for treatment with essential oils. Fruits (100-125 g) were randomly distributed into plastic boxes (1.5 L) with four replicates of eight fruits per treatment. The different concentrations (0, 250, 500 and 750 μl/l) of essential oil (Tween 80 was used as surfactant) were sprayed on fruits by using a hand-sprayer until fruits were enough wet to runoff. Treated fruits were placed on absorbent pad in plastic boxes, and then were sealed immediately to minimize vaporization. Boxes were stored in a cold room at 0-1ºC and 90% RH (90 days).

Assessment of quality parameters Evaluation of fruits decay, fruit flavour, fruit firmness and weight loss: At the end of storage period, disease severity on total fruits evaluated, the surface of fruit divided to 10 part and percentage of decayed fruits were estimated. Flavour analyses were carried out to compare the flavour quality of treated and control kiwifruits by 6 trained panellists. A questionnaire was used to record the data for each treatment for the following characteristics: visual aspect (general aspect), firmness, sweetness, juiciness, sourness and crunchiness, on a 5-point scale: (1) very low; (2) low; (3) medium; (4) high and (5) very high. Flesh firmness of all fruits was destructively measured. Firmness reading was taken by using a penetrometer (FT 327, International Ripening Company, Alfonsine, Italy) fitted with a flat-8 mm diameter tip. The tip was pushed toward pulp after skin removal, at the fruit equator, in opposite sides to a depth of 7 mm. Weight loss was calculated by weighting the fruit before treatment and reweighting at the end of the storage period. Weight loss percentage was calculated as percentage loss of initial weight. Evaluation of TSS, TA, TSS/TA and vitamin C content: A random sample of fruits (8 fruit) was sampled per replicate, juiced, and filtered to get a clear sample. Total soluble solids content (TSS) was determined by means of digital Refractometer (Atago, Tokyo, Co. Ltd, Japan) and results were expressed in Brix. Titrable acidity (TA) content was measured by titration of fruit juice with 0.1 N sodium hydroxide (NaOH) to an end point of 8.1 and expressed as percentage of citric acid. The ratio between TSS and TA was calculated. Ascorbic acid concentration (Vitamin C) was determined using the 2, 6 dichlorophenol indophenol titration method 24. Statistical analysis: Data were analyzed based on completely randomized design (CRD) with 4 replications representing 8 fruits per replication. Statistical analysis of the data was performed with MSTATC version 4.00/EM program 25. Data were subjected to ANOVA analysis. Mean differences were separated by Duncan’s multiple range test (P < 0.05). Results Essential oil analysis: The chemical compositions of essential oils used in the present study


178

are listed in table 1. The major compounds found in T. vulgaris oil were β-ocimene (12.6 %), thymol (10.6 %) and carvacrol (6.9 %). The oil of C. copticum was particularly rich in thymol (63.2 %), ρ-cymene (21.4 %) and γ-terpinene (13.8 %). trans-anethole (64.7 %), fenchone (14.6 %) and methyl chavicol (6.7 %) were the main components of F. vulgare. At the same time, S. hortensis oil contained carvacrol (54.1 %), terpinolene (20.6 %) and α-phellandrene (5.3 %) as main components. Effect of essential oils on fruits decay, flavour, firmness and weight loss: Evaluation of disease severity at the end of storage period showed that T. vulgaris oil had the highest influence on the prevention of disease incidence followed by S. hortensis, F. vulgare and C. copticum, respectively (Fig. 1). But there was no significant difference between several essential oil concentrations (Table 2). Assessing the effect of essential oils on flavour of kiwifruits showed that T. vulgaris and S. hortensis oils had significant effect on fruits flavour compared with control. On the other hand, F. vulgare oil treated fruits had off-flavour followed by C. copticum (Table 3). Essential oil treatments had no effect on the firmness and weight loss percentage of kiwifruits (Table 2).

Figure 1. Effect of Thymus vulgaris, Carum copticum, Foeniculum vulgare and Satureja hortensis essential oils on disease severity in treated kiwifruits * Different letters on bars shows significant difference based on Duncan’s multiple range test (P < 0.05). Effect of essential oil treatments on TSS, TA, TSS/TA and vitamin C content in fruits: Assay of essential oil treatment impacts on TSS, TA and TSS/TA showed that the highest and the lowest amounts for those traits belonged to kiwifruits treated with T. vulgaris and C. copticum, respectively (Figs. 2-4). However, no significant differences were found among different essential oil concentrations (Table 2). In addition, the vitamin C content in fruit treated with T. vulgaris was the highest, and the other oils had no significant effect on its content (Fig. 5).

Figure 2. Effect of Thymus vulgaris, Carum copticum, Foeniculum vulgare and Satureja hortensis essential oils on total soluble solids (TSS) in treated kiwifruits. Different letters on bars shows significant difference based on Duncan’s multiple range test (P < 0.05).


179

Figure 3. Effect of Thymus vulgaris, Carum copticum, Foeniculum vulgare and Satureja hortensis essential oils on titrable acidity (TA) in treated kiwifruits. Values followed with unlike letters differ significantly according to Duncan’s multiple range test (P < 0.05)

Figure 4. Effect of Thymus vulgaris, Carum copticum, Foeniculum vulgare and Satureja hortensis essential oils on TSS/TA in treated kiwifruits. Values followed with unlike letters differ significantly according to Duncan’s multiple range test (P < 0.05)

Figure 5. Effect of Thymus vulgaris, Carum copticum, Foeniculum vulgare and Satureja hortensis essential oils on vitamin C in treated kiwifruits. Values followed with unlike letters differ significantly according to Duncan’s multiple range test (P < 0.05). Discussion: The results of our experiment revealed that the essential oil of T. vulgaris followed by S. hortensis had appreciable antifungal activity compared with C. copticum and F. vulgare oils. The antifungal property of T. vulgaris oil has been reported on some important postharvest diseases such as Alternaria and Penicillium rot in tomato fruits 16 as well. In addition, essential oils from two clonal types of T. vulgaris (Laval-1 and Laval-2) were reported to be highly effective against B. cinerea and Rhizopus stolonifer in strawberry fruits 18. Furthermore, S. hortensis oil has showed high antifungal activity against Aspergillus flavus in lemon fruits 20. However, Thanassoulopoulos and


180

Yanna 17 stated that thyme and oregano oils had no efficiency in reduction of gray mold rot in kiwifruits. Expression of antifungal activity of essential oils is often very clear, but their mechanism(s) and action(s) are not completely understood. There is overwhelming consensus that prevalent components of essential oils such as phenols, aldehydes and alcohols exert most antifungal activity and among those compounds, special attention has been focused on phenolics such as carvacrol, thymol and their related precursors ρ-cymene and γ-terpinene 26. It is confirmed that cell wall and cell membrane are the main target of essential oils 27. An important characteristic of essential oils is their lipophilic nature which enables them to interact with lipids of the fungi cell membrane and mitochondria and to disturb their structure and function 28. Other authors attributed this function of essential oils to especially phenolic compounds and their deteriorative interaction with bio-membranes 29. In addition, essential oils may affect the essential metabolic pathways in microorganisms. Conner and Beuchat 30 suggested that the antimicrobial activity of essential oils could be a result of disturbance in enzymes involved in energy production and synthesis of structural components of microorganisms. Although, the antimicrobial property of essential oils is attributed mainly to their principal constituents however, the synergistic effect of some minor components commonly available in essential oil composition has to be considered as well. For example, T. vulgaris oil contains lower than 1/3 phenolic compounds thymol (10.6 %) and carvacrol (6.6 %). However in the present work it was proved to be the most effective in reduction of fungal diseases in kiwifruits. According to these observations, it can be speculated that the high occurrence of antifungal activity of T. vulgaris could be related to thymol itself and /or its synergistic effects with other essential oil components. These finding are in well accordance with previous reports 31. Evaluation of the effect of essential oil treatment on sensory parameters in kiwifruits showed that essential oil treatment decreased the amount of TA, TSS and vitamin C content compared to controls. These results were in agreement with previous works 32, 33. Tzortzakis 33 showed that eucalyptus and cinnamon essential oils increased the TSS in cherry tomato. On the other hand, these essential oils had not affected the TA, pH, TSS/TA, firmness and weight loss in strawberry and main crop tomatoes. Also, in the present study it was determined that F. vulgare essential oil treatment decreased the flavour of kiwifruits, but Ranasinghe et al. 32 reported that cinnamon and clove essential oil treatment had no affect on the physicochemical and organoleptic attributes of banana fruits. In contrast with many reports on the antifungal property of essential oils under in vitro and in vivo conditions, only a few studies have been carried out to evaluate the effect of essential oil treatment on sensory parameters of fruits and the reason of differences among several essential oils is indeterminate. Conclusion: It can be concluded that the plant essential oils especially T. vulgaris oil may be promising natural agent in postharvest disease control of kiwifruits. On the other hand, increasing consumer demand for naturally preserved fruits and vegetables attracts the special attention on essential oils as natural inexpensive natural preservative tools for storage of fruit crops. However further investigations are needed to elucidate the preservative action of these phytochemicals. This will be the subject of future study. 1. 2. 3.

References Kulakiotu, E.K., Thanassoulopoulos, C.C. and Sfakiotakis, E.M. (2004). Postharvest biological control of Botrytis cinerea on kiwifruit by volatiles of ‘Isabella’ grapes. Phytopathol. 94: 1280-128. Eckert, J.W. and Ogawa, J.M. (1988). The chemical control of postharvest diseases: Deciduous fruit, berries, vegetables and root/tuber crops. Annu. Rev. Phytopathol. 26: 433-469. Tripathi, P. and Dubey, N.K. (2004). Exploitation of natural products as an alternative strategy to control postharvest fungal rotting of fruits and vegetables. Postharvest Biol. Technol. 32: 235-245.

Habib Shirzad et al. / Jeobp 14 (2) 2011 175 - 184 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

181

Poole, P.R. and McLeod, L.C. (1994). Development of resistance to picking wound entry Botrytis cinema storage rots in kiwifruit. New Zealand. J. Crop Hort. Sci. 22: 387-392. Du, J.M., Gemma, H. and Iwahori, S. (1997). Effects of chitosan coating on the storage of peach, Japanese pear, and kiwifruit. J. Japan Soc. Hort. Sci. 66: 15-22. Cook, D.W.M., Long, P.G. and Ganesh, S. (1999). The combined effect of delayed application of yeast biocontrol agents and fruit curing for the inhibition of the postharvest pathogen Botrytis cinerea in kiwifruit. Postharvest Biol. Technol. 16: 233-243. Lagopodi, A.L., Cetiz, K., Koukounaras, A. and Sfakiotakis, E.M. (2009). Acetic acid, ethanol and steam effects on the growth of Botrytis cinerea in vitro and combination of steam and modified atmosphere packaging to control decay in kiwifruit. J. Phytopathol. 157: 79-84. Antunes, M.D.C. and Sfakiotakis, E.M. (2002). Ethylene biosynthesis and ripening behaviour of ‘Hayward’ kiwifruit subjected to some controlled atmospheres. Postharvest Biol. Technol. 26: 167-17. Hur, J-S., Oh, S-K., Lim, K-M., Jung, J.S., Kim, J-W. and Koh, Y.J. (2005). Novel effects of TiO2 photocatalytic ozonation on control of postharvest fungal spoilage of kiwifruit. Postharvest Biol. Technol.35: 109-113. Holley, R.A. and Patel, D. (2005). Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiol. 22: 273-292. Mendonça-Filho, R.R. (2006). Bioactive phytocompounds: New approaches in the phytosciences. in: Modern Phytomedicine. Turning Medicinal Plants into Drugs. Ahmad, I. Aqil, F. and Owais, M. (eds.) WILEY-VCH Verlag Gmbh and Co. KGaA, Weinheim. Abdolahi, A., Hassani, A., Ghosta, Y., Bernousi, I. and Meshkatalsadat, M.H. (2010). In vitro efficacy of four plant essential oils against Botrytis cinerea Pers.:Fr. and Mucor piriformis A. Fischer. J. Essent. Oil Bearing Plants. 13: 97-107. Linde, J.H., Combrinck, S., Regnier, T.J.C. and Virijevic, S. (2010). Chemical composition and antifungal activity of the essential oils of Lippia rehmannii from South Africa. South African J. Botany 76: 37-42. Boyraz, N. and Özcan, M. (2006). Inhibition of phytopathogenic fungi by essential oil, hydrosol, ground material and extract of summer savory (Satureja hortensis L.) growing wild in Turkey. Int. J. Food Microbiol. 107: 238-242. Viuda-Martos, M., Ruiz-Navajas, Y., Fernández-López, J. and Pérez-Álvarez, J.A. (2007). Antifungal activities of thyme, clove and oregano essential oils. J. Food Saf. 27: 91-101. Abdolahi, A., Hassani, A., Ghosta, Y., Javadi, T. and Meshkatalsadat, M.H. (2010). Essential oils as control agents of postaharvest Alternaria and Penicillium rots on tomato fruits. J. Food Saf. 30: 341-352. Thanassoulopoulos, C.C. and Yanna L. (1997). Biological control of Botrytis cinerea on kiwifruit cv. Hayward during storage. Acta Hort. 444: 757-762. Reddy, M.V.B., Angers, P., Gosselin, A. and Aru, l.J. (1998). Characterization and use of essential oil from Thymus vulgaris against Botrytis cinerea and Rhizopus stolonifer in strawberry fruits. Phytochemistry. 47: 1515-1520. Feng, W. and Zheng, X. (2007). Essential oils to control Alternaria alternata in vitro and in vivo. Food Control 18: 1126-1130. Dikbas, N., Kotan, R., Dadasoglu, F. and Sahin, F. (2008). Control of Aspergillus flavus with methanol and extracts of Satureja hortensis. Int. J. Food. Microbiol. 124: 179-182. Tripathi, P., Dubey, N.K. and Shukla, A. (2008). Use of some essential oils as postharvest botanical fungicides in the management of grey mould of grapes caused by Botrytis cinerea. World J. Microbiol. Biotechnol. 24: 39-46. Davies, N.W. (1990). Gas chromatographic retention indices of monoterpenes and sesquiterpenes

Habib Shirzad et al. / Jeobp 14 (2) 2011 175 - 184 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

182

on methyl silicon and carbowax 20M phases. J. Chromatogr. 503: 1-24. Adams, R.P. (1995). Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Publishing Corp, Carol Stream, USA. Tian, S., Xu, Y., Jiang, A. and Gong, Q. (2002). Physiological and quality responses of longan fruit to high O2 or high CO2 atmospheres in storage. Postharvest Biol. Technol. 24: 335-340. Freed, R., Eisensmith, S.P., Goetz, S., Reicosky, D., Small, V.W. and Wolberg, P. (1991). User’s Guide to MSTATC. Michigan State University, East Lansing, MI. Ultee, A., Bennik, M.H.J. and Moezelaar, R. (2002). The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Appl. Environ. Microbiol. 68: 1561-1568. Rasooli, I. and Owlia, P. (2005). Chemoprevention by thyme oils of Aspergillus parasiticus growth and aflatoxin production. Phytochemistry. 66: 2851-2856. Kumar, A., Shukla, R., Singh, P., Prasad, C.S. and Dubey, N.K. (2008). Assessment of Thymus vulgaris L. essential oil as a safe botanical preservative against postharvest fungal infestation of food commodities. Inno. Food Sci. Emerg. Tech. 9: 575-580. Veldhuizen, E.J., Tjeerdsma-van Bokhoven, J.L., Zweijtzer, C., Burt, S.A. and Haagsman, H.P. (2006). Structural requirements for the antimicrobial activity of carvacrol. J. Agri. Food Chem. 54: 1874-1879. Conner, D.E. and Beuchat, L.R. (1984). Effects of essential oils from plants on growth of food spoilage yeasts. J. Food Sci. 49: 429-434. Daferera, D., Ziogas, B. and Polissiou, M. (2003). The effectiveness of plant essential oils on the growth of Botrytis cinerea, Fusarium sp. and Clavibacter michiganensis subsp. michiganensis. Crop Prot. 22: 39-44. Ranasinghe, L., Jayawardena1, B. and Abeywickrama, K. (2005). An integrated strategy to control post-harvest decay of Embul banana by combining essential oils with modified atmosphere packaging. Int. J. Food Sci. Technol. 40: 97-103. Tzortakis, N.G. (2007). Maintaining postharvest quality of fresh produce with volatile compounds. Inno. Food Sci. Emerg. Tech. 8: 111-116.


183

Table 1. The chemical compositions (%) of essential oils extracted from Thymus vulgaris, Carum copticum, Foeniculum vulgare and Satureja hortensis No

Compound a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

α-Pinene Limonene β-Pinene Camphene γ-Terpinene α-Phellandrene β-Ocimene ρ-Cymene Fenchone Terpinolene α-Terpinolene Camphor Linalool Nerol oxide Borneol trans-Anethole Linalyl acetate Carvacrol Thymol Geranyl acetate α-Copaene β-Caryophyllene Germacrene D δ-Cadinene Spathulenol Widdrol Caryophyllene oxide Docosane a

RI b

T. vulgaris C. copticum F. vulgare

936 964 971 988 996 997 1030 1040 1051 1075 1080 1083 1090 1103 1107 1240 1253 1285 1289 1352 1358 1420 1462 1517 1550 1569 1586 2300

5.2 3.8 2.6 8.5 12.6 1.2 4.2 3.2 2.3 1.3 6.9 10.6 2.0 1.4 6.1 1.9 2.1 1.7 2.1 5.7 2.8

13.7 21.4 63.2 -

3.4 1.5 1.4 14.6 1.5 64.4 -

The compounds that present lower than 1% are not presented;

b

S. hortensis 5.3 3.6 20.6 54.1 -

Retention indices

Table 2. Means squares for the effects of Thymus vulgaris, Carum copticum, Foeniculum vulgare and Satureja hortensis essential oils on quality parameters of treated kiwifruits Treatments Disease Flavor severity EO a Con b EO×Con

** ns ns

* ** *

Firmness

Weight loss

TSS

TA

TSS/TA

Vitamin C content

ns ns ns

ns ns ns

** ** ns

* ** ns

** ns ns

** ** ns

b EO: Essential oil; Con: Essential oil concentration. Treatments with an associated asterisk were statistically significant (*, **, and ns = P < 0.05, 0.001, and not significant, respectively). a


184

Table 3. Effect of different concentration of Thymus vulgaris, Carum copticum, Foeniculum vulgare and Satureja hortensis essential oils on flavor of kiwifruits Essential oil

T. vulgaris C. copticum F. vulgare S. hortensis

Flavor 0

250

500

μl l-1) 750 (μ

4.15 a * 3.68 ab 4.27 a 4.28 a

3.62 abc 1.21 e 2.45 cde 3.28 ad

4.12 a 1.56 e 2.11 de 2.09 de

3.03 a-d 2.09 de 2.31 de 2.84 bcd

* Different letters in columns shows significant difference based on Duncan’s multiple range test (P < 0.05). Table 4. Effect of different concentrations of Thymus vulgaris, Carum copticum, Foeniculum vulgare and Satureja hortensis essential oils on titrable acidity (TA), total soluble solids (TSS) and vitamin C content of kiwifruit μl l-1) Concentration (μ Control (0) 250 500 750

TA 1.63 b * 1.51 a 1.54 a 1.54 a

TSS

Vitamin C content

12.94 b 11.78 a 11.87 a 12.14 a

37.84 b 29.41 a 29.52 a 29.48 a

* Different letters in columns shows significant difference based on Duncan’s multiple range test (P < 0.05).

Jeobp 14 (2) 2011 pp 185 - 191

185


Chemical Composition and Antimicrobial Study of Essential Oil from the Leaves of Curcuma haritha Mangaly and Sabu Gopan Raj 1, Nediyaparambu S. Pradeep 2, Mathew Dan 3, Mathur G. Sethuraman 4 and Varughese George 1* 1

Phytochemistry and Phytopharmacology Division, Tropical Botanic Garden and Research Institute, Pacha - Palode, Thiruvananthapuram - 695 562, Kerala, India 2 Plant Systematics and Evolutionary Science Division, Tropical Botanic Garden and Research Institute, Pacha - Palode, Thiruvananthapuram - 695 562, Kerala, India 3 Plant Genetic Resources Division, Tropical Botanic Garden and Research Institute, Pacha - Palode, Thiruvananthapuram - 695 562, Kerala, India 4 Department of Chemistry, Gandhigram Rural Institute, Gandhigram - 624 302, Tamil Nadu, India Received 23 June 2010; accepted in revised form 18 December 2010

Abstract: The essential oil obtained from the fresh leaves of Curcuma haritha by hydrodistillation was analysed by gas chromatography and gas chromatography-mass spectrometry. Forty one constituents, representing 97.0 % of the analysed oil constituted mainly by oxygenated mono and sesqui terpenes. Curdione (18.3 %), 1,8-cineole (11.8 %), camphor (11.8 %), furanogermenone (8.6 %) and furanodiene (8.9 %) were identified as the major constituents in C. haritha leaf oil. The in vitro antimicrobial activity of the leaf oil was studied by agar disc diffusion technique. The leaf essential oil was tested against both Gram-positive and Gram-negative bacterial strains and pathogenic fungal species of Candida. The leaf oil at 1:1 dilution with DMSO was found to be more active against fungal strains than bacterial strains. Antibacterial activity was significant only against Gram positive bacterium, Staphylococcus aureus. Key words: Curcuma haritha, Zingiberaceae, leaf essential oil composition, curdione, furanodiene, antibacterial activity and antifungal activity. Introduction: Genus Curcuma, belonging to the family Zingiberaceae, comprises over 80 species is widely distributed in the tropics of Asia to Africa and Australia 1. About 41 species are known to occur in India, of which 10 species are endemic to the Indian subcontinent 2. Twenty species and one variety of Curcuma are reported from south India 1. Many species belonging to the genus Curcuma have commercial value as medicines and spices. They have traditionally important role as a coloring agent in food, cosmetics, and textiles. The rhizomes and roots of Curcuma are commonly used as traditional drugs 3. Generally leaves of Curcuma species are a waste product during post-harvest operations. Despite intensive studies on the chemical composition of rhizome essential oil of Curcuma species, only few Curcuma species are studied for their chemical constitutes in the leaf oil. *Corresponding author (Varughese George) E-mail: < [email protected] >


Varughese George et al. / Jeobp 14 (2) 2011 185 - 191

186

Curcuma haritha Mangaly and Sabu is an aromatic rhizomatous herb with large rhizome, yellowish grey inside, many sessile tubers and fleshy roots. Leaves are distichous and petolate 1. It is endemic to southern Western Ghats region in India and is distributed in the coconut groves in the coastal region of Kerala and rarely found in high altitude grasslands 1. Rhizome is expectorent, astringent and useful in diarrhoea 4. Locally, rhizomes are used as substitute of arrowroot by natives in different parts of Kerala, India 2. The rhizome ‘paste’ of C. haritha is used by the natives of Parambikulam forest range of Palakkad district in Kerala for repelling blood sucking leeches 5. Chemical composition of the rhizome oil of C. haritha was reported with camphor (36.0 %), isoborneol (10.6 %), 1,8-cineole (13.9 %), curdione (10.5 %) and furanogermenone (6.4 %) as the major compounds 6. Antimicrobial 7 and pharmacognostic5 studies of C. haritha have been reported. To our knowledge, there is no published report in the literature about the composition and antimicrobial activity of C. haritha leaf oil. The present study reports chemical composition, antibacterial and antifungal activities of the leaf essential oil from the wild relative of turmeric C. haritha. Experimental Plant material: C. haritha leaves were collected from the campus of Tropical Botanic Garden and Research Institute (TBGRI) in June 2006. Voucher specimen No. 51813 of C. haritha was deposited at the Herbarium of TBGRI. Essential oil isolation: Essential oil from fresh leaves of C. haritha was obtained by hydrodistillation for 4 hrs. in a Clevenger type apparatus. The oil was dried over anhydrous sodium sulphate and stored at 4°C until analyzed. The physical parameters of the oil were determined. Refractive index was determined using J257 Refractometer (Rudolph Research Analytical, USA) and specific rotation was determined using Autopol IV Polarimeter (Rudolph Research Analytical, USA). GC-FID Analysis: The volatile oil obtained from the fresh leaves was analyzed by GC-FID using a gas chromatograph (Nucon model 5765 series) fitted with SE-30 (10 %) chromosorb-W packed stainless steel column (2m x 2mm) with FID detector. Nitrogen was used as the carrier gas at flow rate of 40 mL/min. Oven programme: 80°-150°C (8°C/min.), 150°-230°C (5°C/min), 230°C (10 min), injector temperature 220°C, detector temperature 250°C. Relative percentages of components were calculated from the peak area-percent report of volatiles from GC-FID data. GC-MS Analysis: GC-MS analysis of the leaf oil was performed by splitless injection of 1.0 μL of the oil on a Hewlett Packard 6890 gas chromatograph fitted with a cross-linked 5% PH ME siloxane HP-5 MS capillary column, 30m x 0.32mm, 0.25μm coating thickness, coupled with a model 5973 mass detector. GC-MS operation conditions: injector temperature 220°C; transfer line 290°C; oven temperature programme 60°-246°C (3°C/min); carrier gas - helium at 1.4mL/min. Mass spectra: Electron Impact (EI+) mode 70eV, ion source temperature 250°C. Centroid scan of mass range from 30-300 amu. Individual compounds in the oil were identified by WILEY 275.L database matching, comparison of mass spectra with published data 8 and by comparison of their LRIs 9,10. Linear Retention Indices of constituents were determined on the HP-5 column using C5-C30 straight chain alkanes as standards 9,10. Antimicrobial studies: Antimicrobial activity of the oil was studied using standard strains [Microbial Type Culture Collection (MTCC)] of Gram-positive and Gram-negative bacteria and pathogenic fungal strains obtained from the Institute of Microbial Technology, Chandigarh, India. Test micro organism used in the study were the Gram- positive bacteria Bacillus subtilis (MTCC No. 441), Bacillus cereus (MTCC No. 430) Staphylococcus aureus (MTCC No. 2940) and Salmonella


187

typhi (MTCC No. 733); Gram- negative bacteria Proteus vulgaris (MTCC No. 426), Escherichia coli (MTCC No. 443), Serratia marcescens (97), Pseudomonas aeruginosa (MTCC No. 741) and Klebsiella pneumoniae (MTCC No. 109) and the fungi included three strains of Candida albicans (MTCC -227, 1637 and 3017) and C. glabrata (MTCC 3049). In vitro antimicrobial assay of the leaf oil was carried out according to disc agar diffusion method 11,12 using Whatman No.1 filter paper discs of 6 mm diameter. The bacterial strains were cultured in Mueller-Hinton agar medium and fungal strains in modified Sabouraud,s agar medium. Aliquots of 10 μL of the leaf oil at 1:1 and 1:2 dilutions in an inert solvent dimethyl sulfoxide (DMSO) were impregnated on the discs and then aseptically applied to the surface of the agar plates at wellspaced intervals. The plates were incubated at 37°C for 24 h and observed inhibition zones including the diameter of the discs were measured. Control discs impregnated with 10 μL of the solvent DMSO and streptomycin (10 μg/disc), reference for bacteria and fluconazole (2 μg/disc), reference for fungi were used alongside the test discs in each experiment. Both the assays were performed in triplicate and the results are the mean values. The activities are expressed in mm diameter of the inhibition zones of bacterial growth including the 6 mm disc diameter. Results and discussion: Fresh leaves (365 g) of C. haritha were cut into small pieces and hydrodistilled using a Clevenger-type apparatus for four hours to obtain 0.4 mL of clear violet coloured oil with a camphoraceous odour at 0.14 % (v/w, fresh weight basis) yield. The physical parameters of the oil were refractive index (nD25) 1.4960, specific rotation [a]D25 +13.09 (c=1, CHCl3) and specific gravity 0.8981. Forty-one out of 48 constituents in C. haritha leaf oil were identified (Table-1) by a combination of GC-MS, linear retention indices, database and literature comparison of data. Oxygenated mono (33.4 %) and sesquiterpenes (48.3 %) constituted major portion of the analysed leaf oil. Camphor (11.8 %) and 1,8-cineole (11.8 %) are the major monoterpenes. Germacrane-type sesquiterpenes viz., curdione (18.3 %), furanogermenone (8.6 %), germacrone (7.5 %) and furanodiene (8.9 %) are the major sesquiterpenes in the leaf oil. Sesquiterpene hydrocarbons (11.0 %) in the oil contained germacrene A (2.8 %) germacrene B (1.5 %) and germacrene D (1.2 %) as the major constituents. Monoterpene hydrocarbons characterized by camphene (1.6 %) and limonene (1.3 %) represented low percentages (4.3 %) in the leaf oil. Earlier high amount of curdione and other germacrane compounds were reported from the leaf oils of C. indora 13, C. harmandii 14, C. cochinchinensis 15 and C. aeruginosa 16. Morphologically, C. haritha is related to C. aromatica, but they differ in their chemical compositions. Leaf oil of C. aromatica contained p-cymene (25.2 %) and 1,8-cineole (24.8 %) as principal constituents 17. Comparison with the rhizome oil of C. haritha 6 showed that both the oils have several common constituents. The major components of the leaf oil viz. camphor, 1,8-cineole, isoborneol, camphene, borneol, curdione, furanogermenone and germacrone were also reported as major components of its rhizome oil. 1,8-Cineole, was found in both the leaf and rhizome oils as the second major constituent. It is reported from many Curcuma species as major constituent 18-20. Furanodiene, the anti-inflammatory sesquiterpene from Curcuma zedoaria 21 was found significantly in the leaf oil but not detected in the rhizome oil. Results of antibacterial activity (Table-2) showed that most of the bacterial strains were resistant to leaf oil and the oil inhibited the growth of only the Gram positive bacterium, Staphylococcus aureus (MTCC No 96). Results on antifungal activity showed that the growth of fungal species was significantly inhibited by the essential oil (Table-3). The leaf oil at 1:1 dilution with DMSO exhibited good activity against the fungal strains Candida albicans (MTCC 227 and 3017) and Candida glabrata. Previous reports showed that essential oil from shade dried aerial parts of C. haritha was active against the fungi Aspergillus parasiticus, Rhizoctonia oryzae sativae and bacterium Escherichia coli 7. It is reported that essential oils containing oxygenated monoterpenes such as 1,8-cineole, linalool, α-terpineol,


188

nerolidol, spathulenol in high proportions exhibit antibacterial and antifungal activities 22. 1,8-Cineole is present in both the oils in significant amount. Detailed analysis on chemical composition of essential oil from the leaf of Curcuma haritha, showed that this endemic Curcuma species can be exploited as a source of biologically active constituents curdione and furanodiene. The results on antimicrobial studies indicate that leaf oil of C. haritha could be used as a natural antimicrobial agent for human and infectious fungal diseases and in food preservation. Acknowledgements: The authors express their sincere thanks to the Director TBGRI for providing Laboratory facilities and to the Director (Laboratories), Textiles Committee, Ministry of Textiles, Govt. of India for GC-MS analysis.

1. 2. 3. 4. 5.

6.

7.

8. 9.

10. 11.

12. 13. 14.

15.

References Sabu, M. (2006). Zingiberaceae and Costaceae of South India, Indian Association for Angiosperm Taxonomy: Calicut. 126-186. Sabu, M. and Mangaly, J.K. (1996). Taxonomic revision of south Indian Zingiberaceae. Proc. 2nd Symp. Family Zingiberaceae. Botany 2000-Asia China., pp: 15-22. Sasikumar, B. (2005). Genetic resources of Curcuma: diversity, characterization and utilization. Plant Genet Resour, 3: 230-251. Srivastava, S., Chitranshi, N., Dan, M., Rawat, A.K.S. and Pushpangadan, P. (2006). Pharmacognostic evaluation of Curcuma haritha Linn. J. Sci. Ind. Res., 65: 916-920. Dan, M., George, V. and Pushpangadan, P. (2002). Studies on the essential oils of Curcuma haritha Mangaly and Sabu and C. raktakanta Mangaly and Sabu. J. Spices and Aromatic Crops., 11: 78-79. Gopan Raj, Sabulal, B., Thaha, A.R.M., Dan, M., George, V. (2008). Volatile constituents from the rhizomes of Curcuma haritha Mangaly and Sabu from southern India. Flav. Fragr. J. 23: 348-352. Umesh, B.T., Sree Ranjini, K., Leela, L., Sandhya, K.K., Betty, K.P. and Thoppil, J.E. (2003). Microbicidal potential of essential oil of Curcuma haritha Mangaly & Sabu. J. Nat. Remed., 3: 199-201. Adams, R.P. (2001). Identification of essential oil components by gas chromatography/ quadrupole mass spectroscopy. Allured publishing corporation, Carol Stream, Illinois. Dool, H.V.D. and Kratz, P.D. (1963). A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatogr., 11: 463471. Davies, N.W. (1990). Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases. J. Chromatogr. A., 503: 1-24. Bershe, D.A.V. and Vlietnick, A.J. (1991). Screening methods for antibacterial and antiviral agents from higher plants, In methods in plant biochemistry. Dey, P.M., Harborne, J.B. (Eds.) Academic press: London, VI, 47-69. Cappucino, J.G. and Sherman, N. (1999). Microbiology: A laboratory manual, 5th edition, Benjamin Cumming Science Publishing: Menlo Park, CA, p 254. Malek, S. N., Seng, C. K., Zuriati, Z., Ali, N. A., Halijah, I. and Jalil, M. N. (2006). Essential oil of Curcuma inodora aff. Blatter from Malaysia. J. Essent. Oil Res, 18: 281-283. Dung, N.X., Truong, P.X., Ky, P.T. and Leclercq, P.A. (1997). Volatile constituents of the leaf, stem, rhizome, root and flower oils of curcuma harmandii Gagnep from Vietnam. J. Essent. Oil Res., 9: 677-681. Dung, N.X., Truong, P.X., Ky, P.T. and Leclercq, P.A. (1996). Chemical Composition of the


16.

17.

18. 19.

20.

21. 22.

189

Essential Oils of Curcuma cochinchinensis Gagnep. from Vietnam. Chem. Res. Commun, 5: 11-16. Jarikasem, S., Thubthimthed, S., Chawananoraseth, K., Suntorntanasat, T. and Brophy, J.J. (2005). Essential oils from three Curcuma species collected in Thailand. Acta hortic., 677: 37-41. Singh, G., Prakash Singh, O., Menut, C. and Bessiere, J. M. (2004). Studies on essential oils part 34: Chemical and biocidal investigations on leaf volatile oil of Curcuma aromatica; J. Essent. Oil Bearing Plants, 7: 258-263. Behura, S. and Srivastava, V.K. (2004). Essential Oils of Leaves of Curcuma Species. J. Essent. Oil Res., 16: 109-110. Jirovetz, L., Buchbouer, G., Puschmann, C., Shafi, M.P. and Nambiar, M.K.G. (2000). Essential oil analysis of Curcuma aeruginosa Roxb. leaves from South India. J. Essent. Oil Res., 12: 47-49. Chalchat, J.C., Garry, R.P., Menut, C., Lamaty, G., Malhuret, R. and Chopineau, J. (1997). Correlation between chemical composition and antimicrobial activity. VI Activity of some African essential oils. J. Essent. Oil Res., 9: 67-75. Makabe, H., Maru, N., Kuwabara, A., Kamo, T. and Hirota, M. (2006). Anti-inflammatory sesquiterpenes from Curcuma zedoaria. Nat. Prod. Res., 20: 680-685. Yoshihiro, I., Akiko, S., Toshiko, H., Kazuma, H., Hjime, H. and Jingoro, S. (2004). The antibacterial effects of terpene alcohols on Staphylococcus aureus and their mode of action. FEMS Microbiol. Lett., 23: 325-331. Table 1. Chemical composition of the leaf oil of Curcuma haritha Component

LRI a

% Composition

Methods of Identification

Camphene Sabinene β-Pinene Myrcene Limonene 1,8-Cineole cis-Ocimene trans-Ocimene γ-Terpinene Linalool Camphor Camphene Hydrate Isoborneol Borneol Terpinen-4-ol α-Terpineol δ-Elemene β-Eemene β-Bourbonene β-Caryophyllene Aromadendrene

944 968 979 986 1027 1028 1037 1047 1056 1099 1143 1146 1160 1168 1176 1190 1334 1388 1379 1412 1443

1.6 0.3 0.1 0.6 1.3 11.8 0.1 0.2 0.1 2.4 11.8 1.6 1.8 1.1 0.7 2.2 0.1 1.0 0.2 0.4 0.2

a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c


190

table 1. (continued). Component

LRI a

% Composition

Methods of Identification

α-Humulene Germacrene D δ-Selinene α-Selinene Curzerene Germacrene A β-Curcumene δ-Cadinene Germacrene B Caryophyllene oxide Guaiol trans-β-Elemenone Humulene-1,2-Epoxide Curcumol Furanodiene Germacrone Curdione Neocurdione Furanogermenone Germacrone-4,5-Epoxide

1447 1474 1478 1488 1494 1498 1514 1519 1550 1576 1581 1596 1602 1613 1687 1690 1717 1747 1790 1835

1.4 1.2 0.1 0.2 0.9 2.8 0.4 0.6 1.5 0.2 0.5 0.2 0.5 0.8 8.9 7.5 18.3 2.5 8.6 0.3

a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c

Total Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others (unidentified) a

97.0 4.3 33.4 11.0 48.3 3.0

Linear retention index on HP-5 column WILEY 275.L database matching, c Comparison of mass spectra with published data ; Adams (2001) b


191

Table 2. In vitro Antibacterial activities of the leaf oil of Curcuma haritha Diameter in mm of the inhibition zone excluding the diameter of the disc Bacteria (MTCC No.)

Gram-postive bacteria Bacillus cereus (430) Bacillus subtilis (441) Staphylococcus aureus (96) Gram-negative bacteria Serratia marcescens (97) Pseudomonas aerugenosa (741) Klebseilla pneumoniae (109) Proteus vulgaris (426) Escherichia coli (443) Salmonella typhi (733)

Leaf oil

Streptomycin (10 μg/disc)

1:1

1:2

12 ± 0.9

10± 1.3

8 ± 1.2 12 ± 1.4 9 ± 1.1

7 ± 1.0 9 ± 0.2 8 ± 0.4 7 ± 1.3

7 ± 0.5 7 ± 1.5

17.5 ± 0.8 19 ± 1.0 10 ± 1.0 15 ± 1.2 12 ± 1.5 14 ± 1.6

Experiments were done in triplicate and results were mean values Oil dilution - 1:1 and 1:2 in DMSO Table 3. In vitro Antifungal activities of the leaf oil of Curcuma haritha Diameter in mm of the inhibition zone excluding the diameter of the disc Fungi (MTCC No.)

Candida albicans (227) Candida albicans (1637) Candida albicans (3017) Candida glabrata (3049)

1:1

Leaf oil 1:2

Fluconazole (2 μg/disc)

10 ± 0.4 7 ± 1.0 20 ± 0.6 10 ± 0.6

8 ± 1.2 18 ± 0.8 7 ± 1.1

9 ± 1.0 7 ± 1.3 25 ± 1.1 9 ± 0.5

Experiments were done in triplicate and results are mean values Oil dilution - 1:1 and 1:2 in DMSO

Jeobp 14 (2) 2011 pp 192 - 200

192


Composition and Bioactivity of Essential Oils from Leaves and Fruits of Myrtus communis and Eugenia supraxillaris (Myrtaceae) Grown in Egypt E.A. Aboutabl 1*. K.M. Meselhy 1, E.M. Elkhreisy 2, M.I.Nassar 2 and R. Fawzi 2 1

Department of Pharmacognosy and Medicinal Plants, Faculty of Pharmacy, Cairo University, Kasr-el-Aini Street, 11562 Cairo, Egypt 2 Department of Natural Products Chemistry, National Research Centre, El-Bohouth Street, Dokki, Cairo, Egypt Received 19 August 2010; accepted in revised form 11 December 2010

Abstract: Comparative investigation of hydro-distilled essential oils from leaves (L) and fruits (F) of Myrtus communis (M) and Eugenia supraxillaris (E) (Fam. Myrtaceae) grown in Egypt was carried out; including: yield, physical characteristics, chemical composition and certain bioactivities. GC-FID and GCMS analyses revealed that the oil samples differ in composition and percentages of certain components. The total number of identified constituents was 23, 21, 21 and 17 in EL, EF, ML and MF oil samples; representing: 98.6 %, 98.5 %, 90.3 % and 91.8 % of the total oil composition, respectively. Oxygenated compounds were found dominant in EF, ML and MF oil samples, being 81.4 %, 61.5 % and 71.5 %; respectively, and 13.5 % in EL sample. All the oil samples appeared dominated by monoterpenoids, among which limonene (21.8 %), eugenol (35.5 %), 1,8-cineole (27.2 % and 29.6 %) are major in EL, EF, ML and MF; respectively. Sesquiterpenes are minors in all samples, comprising mainly: α-humulene in the 2 leaf samples EL and ML; while β-selinene and trans-caryophyllene recorded the highest sesquiterpenoid percentile for EF and MF samples; respectively. Certain bioactivities including: cytotoxicity against different tumor cell lines, antimicrobial, and antiwormal activity against Allolobophora caliginosa were evaluated and found variable in the leaves and fruits of the two plants. Key words: Myrtus communis, Eugenia supraxillaris, Myrtaceae, essential oil composition, α-pinene, β-pinene, eugenol, cytotoxic activity, antimicrobial activity, antiwormal activity. Introduction: Myrtaceae is a dicotyledonous plant family which comprises approximately 3000 species in 130 genera, widely distributed in warm regions of the world, with the greatest concentration of species in Australasia. Myrtaceae species are mostly woody essential oils-bearing plants, with evergreen, alternate (rarely opposite), simple leaves, usually with entire margin 1-3. The genus Myrtus comprises approximately 16 species of evergreen shrubs or small trees. Myrtus communis known as true myrtle and in arabic as mirsin, grows spontaneously in countries bordering the mediterranean area (Spain, France, Tunisia, Algeria and Morocco) and in west Asia. On the other hand, the genus Eugenia, with approximately 1000 species, has a worldwide distribution in tropical and subtropical regions. Most species are evergreen trees and shrubs, grown as ornamental plants for their attractive *Corresponding author (E.A. Aboutabl) E-mail: < [email protected] >


E.A. Aboutabl et al. / Jeobp 14 (2) 2011 192 - 200

193

glossy foliage and a few produce edible fruits that are eaten fresh or used in jams and jellies. Chemical composition of essential oils from certain Myrtus species 4-16, as well as certain Eugenia species 17-29 was studied. In addition, phenolics and other phytochemicals were investigated (30-38). Several bioactivities were reported for essential oils, as well as extracts of certain Myrtus 39-42 and Eugenia 4347 species. This study aimed at evaluating certain bioactivities of the essential oils hydro-distilled from leaves, as well as fruits of Myrtus communis and Eugenia supraxillaris grown in Egypt, in order to support the possibility of their uses as a natural resource in therapeutics and to demonstrate the correlation between chemical composition and bioactivity. In this respect, cytotoxic, antiwormal, and antimicrobial activities of the different oils were evaluated. Moreover, a comparative chemical investigation of oil samples was carried out. Experimental Plant material: Samples of leaves and fruits of Myrtus communis and Eugenia supraaxillaris (Fam. Myrtaceae) were collected in May 2006, from El-Orman Garden, Giza, Egypt and kindly authenticated by Taxonomist Triessa Labib. Voucher specimens are kept in the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University. Preparation of the oils: The fresh leaves, as well as fruits of both Eugenia supraaxillaris and Myrtus communis (each sample, 500 g) were subjected to hydrodistillation. The oils obtained were, separately, dried over anhydrous sodium sulphate and stored in a refrigerator till analysis. Analysis of the oils: GC-MS analysis was performed with a Varian 3400 chromatograph coupled to mass selective detector. The capillary column used was DB-5, 30 m x 0.25 mm, 0.5 μm film thickness. Operating conditions: injector and ion source temperatures, 220°C; interface temperature, 270°C; maximum temperature, 260°C; oven temperature program: 3 min. isothermal at 50°C, then programmed from 50°-150°C at 10°C/min and from 150°-270°C at 5°C/min, followed by 3 min. isothermal at 270°C; carrier gas: 0.88 mL He/min.; ion source, 70 ev. For quantitation (area %), the GC analyses were performed on an apparatus fitted with a FID detector with conditions similar to that of GC-MS (carrier gas was helium; injector temperature, 220°C while detector temperature, 280°C) (tables 1, 2). The different components of the oils were identified by their mass spectra, retention indices and/ or retention times, which were compared with reference compounds and published data 48. Evaluation of potential cytotoxicity by SRB assay: Potential cytotoxicity of the oil samples was tested at the National Cancer Institute of Egypt adopting the method of Shaken et al 49. Cells were plated in a 96-wells plate (104cells/well) for 24 hrs before treatment with the oil sample to allow attachment of the cells to the wall of the plate. Different concentrations of each of the oil samples under test (0, 1, 2.5, 5 and 10 μg/ml) were added to the cell monolayer. Triplicate wells were prepared for each individual dose and were incubated for 48 hrs at 37°C in an atmosphere of 5 % CO2. After 48 hrs, cells were fixed, washed and stained with sulphorodamine B stain. Excess stain was washed with acetic acid and attached stain was recovered with Tris-EDTA buffer. Colour intensity was measured in an ELISA reader. The survival curves of each of the tumor cell lines (cervices, colon, larynx, liver and breast) were plotted and IC50 was calculated for each of the oil samples tested (table 3). Antimicrobial activity screening: The disc diffusion method 50,51 was adopted with slightly modification using filter paper discs (0.5 cm in diameter). Each of the oils under investigation was diluted with DMSO at 20 % v/v concenteration and 20 ml placed on the filter paper disc. The control


194

disc contained 20 ml of DMSO. Ofloxacin and Amphotricin B were used as standards. Tested microorganisms were: Escherichia coli (ATCC 10536), Proteus vulgaris (NCTC 4175), Pseudomonas aeruginosa (CNCM A21), Staphylococcus aureus (ATCC 4175), Sarcina lutea (Laboratory collection strains), Bacillus subtilis (NCTC 6633), Mycobacterium phlei (Laboratory collection strains), Candida albicans (ATCC 60193) and Candida tropicalis (Laboratory collection strains). Zones of inhibition and minimum inhibitory concentration were measured and results are compiled in (table 4). Testing for antiwormal activity: The antiwormal effect of the four oil samples was evaluated using earth worms (Allolobophora caliginosa) not less than 10 cm long as experimental animals 52. 0.1 and 0.2 % dilutions of each sample, prepared in 1 % Tween 80, were tested. A group of three worms was dipped in 10 ml of each of the tested solutions in addition to a negative control consisting of 1 % Tween 80. The activity of the treated worms was observed and the time required for complete inhibition of the response of the worms to external stimuli (indicating paralysis or death) was recorded and taken as a measure for antiwormal activity. Results are compiled in (table 5). Results and discussion: Eugenia supraxillaris leaves (EL), Eugenia supraxillaris fruits (EF), Myrtus communis leaves (ML) and Myrtus communis fruits (MF) yielded on hydrodistillation 2.4, 2.7, 3.5 and 2.9 % v/ w, respectively, of colourless to pale amber oils with fragrant spicy-scented odour (calculated on dry weight basis). Specific gravity of the samples (determined at 25°C) ranged from 0.94 to 0.98 and they were all readily soluble in 70 % ethanol GC-MS analytical results of the oil samples compiled in (tables 1, 2) revealed qualitative and quantitative variation in chemical composition. Among the fifty five components identified in appreciable amounts in the oils (relative % > 0.01), only two were common in all the samples viz., α-pinene and αterpineol. The total number of identified constituents was 23, 21, 21 and 17 in EL, EF, ML and MF oil samples; respectively (representing: 98.6 %, 98.5 %, 90.3 % and 91.8 % of the total oil composition). Oxygenated compounds were found dominant in EF, ML and MF oil samples, being (81.4 %, 61.5 % and 71.4 %; respectively) compared with EL sample (13.5 %). The overall chromatographic profiles of all the oil samples (EL, EF, ML and MF) appeared dominated by monoterpenoid constituents (tables 1, 2) among which limonene and β-pinene are major in EL sample; eugenol, methyl eugenol and myrcene are main monoterpenes in EF sample. Meanwhile, the predominant monoterpenoids in ML sample were 1,8-cineole and α-pinene, while 1,8-cineole and menthyl acetate were the major monoterpenes of MF sample. Sesquiterpenes are minors in all samples (tables 1, 2); comprising mainly: α-humulene in the two leaf samples EL and ML; while β-selinene and trans-caryophyllene being the major sesquiterpenoids in the EF and MF samples; respectively. The difference in oil composition of each of Eugenia supraxillaris and Myrtus communis is obviously influenced by the organ type. The oil obtained from Eugenia supraxillaris could be graded as rich in eugenol, if only hydrodistilled from the fruits rather than leaves which are poor in oxygenated compounds. Furthermore, the yield and composition of myrtle oils (Myrtus communis) depend upon the organ type. Although the two samples of myrtle were rich in 1,8-cineole (27.2 and 29.6 %, respectively), α-pinene, was only major in leaf sample (25.5 %). The EF, ML and MF oil samples could find valuable applications in both aromatherapy and fragrance industry, due to their high content of oxygenated constituents. The cytotoxic, antimicrobial, and antiwormal bioactivities evaluated for the aforementioned oils (tables 3-5) revealed variable and significant efficacy and potency for all the samples in comparison with standards; suggesting their potential incorporation in phytomedicines after necessary clinical trials. The bioactivities of the oil samples appeared influenced by type of organ, which may be attributed to the difference in chemical composition. All oil samples exhibited high potency as cytotoxic (table 3) against tumor cell lines (cervices, colon, larynx, liver and breast) which may be attributed to the


195

presence of limonene, cineole, eugenol and methyl eugenol. The fruit oil samples (EF and MF) containing the highest relative percentage of phenolic compounds (eugenol) and oxides (cineole) exhibited significant antimicrobial activity (table 4) against gram +ve bacteria, gram -ve bacteria and acid-fast bacilli and, hence, can offer a valuable antibacterial and antituberculosis phytomedicine. These effects support the previously reported use of these oils as mucous membrane stimulant. All oil samples exhibited significant antiwormal activity against the earthworm Allolobophora caliginosa (table 5). Under experimental conditions, 0.1 % and 0.2 % suspensions of the oil sample induced paralysis in 3 to 7 minutes. The response to external stimuli was of irreversible nature, even after leaving the worms into recovery bath (fresh water bath). The recorded observations offer a support to recommendations of the possible utility of the oil samples as antiwormal agents. It is obvious that EL sample recorded high percentages of pinene and limonene (17.4 % and 21.8 %), respectively, which explains its significant activities as fungicidal and antiwormal. In general, this is the first report about the effect of organ type on the chemical composition of the oils from both species and their bioactivities.

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

7. 8. 9. 10.

11. 12. 13.

14.

15.

References: Hora, F.B. (1997). “Flowering Plants of the World” Oxford University Press, London, UK Evans, W.C. (2002). Trease and Evans. Pharmacognosy. 15th Edition, WB Saunders Company Limited, London, UK Roth, L.S. (2010). “Mosby’s Handbook of Herbs & Natural Supplements”, 4th ed.,Mosby, Elsevier. Fernandes, C.A. and Cardoso, D.V.J. (1952). Essential oil of Myrtus communis. Noticias Farmaceuticas 18: 194-200. Frazao, S., Domingues, A. and Sousa, M.B. (1974). Characteristics and composition of Portuguese essential oil of Myrtus communis. Essent. Oils, [Pap.], 112-113 . Gauthier, R., Gourai, M. and Bellakhdar, J. (1988). The essential oil of Myrtus communis L. var. italica and var. baetica harvested in Morocco. II. Yields and composition in relation to extraction method; comparison with various sources. Biruniya 4(2): 117-32. El Hossary, G.A. and Tadros, S.H. (1989). Phytochemical study of the leaves of Myrtus communis L. grown in Egypt. Bull.Fac.Pharm. Cairo Univ., 27(1): 101-3. Akgul, A. and Bayrak, A. (1989). Essential oil content and composition of myrtle (Myrtus communis L.) leaves. Ormancilik Dergisi, 13(2): 143-7. Boelens, M.H. and Jimenez, R. (1992). The chemical composition of Spanish myrtle oils. Part I. J. Essent. Oil Res. 4(4): 349-53. Fujita, S., Kawai, K. and Nogami, K. (1992). Miscellaneous contributions to the essential oils of plants from various territories. Part III. On the components of the essential oils of myrtle (Myrtus communis). Joshi Daigaku Kiyo, Kaseigakubu-hen, 40: 33-7. Chalchat, J., Garry, R. and Michet, A. (1998). Essential oils of Myrtle (Myrtus communis L.) of the Mediterranean littoral. J. Essent. Oil Res. 10(6): 613-617. Asllani, U. (2000). Chemical composition of Albanian myrtle oil (Myrtus communis L.). J.Essent. Oil Res. 12(2): 140-142. Jerkovic, I., Radonic, A. and Borcic, I. (2002). Comparative study of leaf, fruit and flower essential oils of Croatian Myrtus communis (L.) during a one-year vegetative cycle. J. Essent. Oil Res. 14(4): 266-270. Jamoussi, B., Romdhane, M., Abderraba, A., Ben, H.B. and El Gadri, A. (2005). Effect of harvest time on the yield and composition of Tunisian myrtle oils. Flav. Fragr. J. 20(3): 274277. Aidi, W.W., Mhamdi, B. and Marzouk, B. (2009). Variations in essential oil and fatty acid


16.

17. 18. 19. 20. 21. 22.

23. 24.

25. 26.

27.

28.

29.

30. 31. 32.

33.

34.

196

composition during Myrtus communis var. italica fruit maturation. Food Chemistry 112(3): 621-626. Pezhmanmehr, M., Dastan, D., Nejad Ebrahimi, S. and Hadian, J. (2010). Essential oil constituents of leaves and fruits of Myrtus communis L. from Iran. J. Essent. Oil Bearing Plants, 13(1): 123-129. Arrillaga, N.G. (1939). Distillation of the leaves of Eugenia buxifolia yields an oil rich in cineole. Puerto Rico Agr. 29: 378. Deyama, T. and Horiguchi, T. (1971). Components of the essential oil of clove (Eugenia caryophyllata). Yakugaku Zasshi, 91: 12 Craveiro, A.A., Andrade, C.H.S., Matos, F.J.A., Alencar, J.W. and Machado, M.I.L. (1983). Essential oil of Eugenia jambolana. J. Nat. Prod. 46(4): 591-592. Bello, A., Rodriguez, M.L., Castineiras, N., Urquiola, A., Rosado, A. and Pino, J.A. (1995). Major components of the leaf oil of Eugenia banderensis Urb. J. Essent. Oil Res. 7(6): 697-8. El-Shabrawy, A.O. (1995). Essential oil composition and tannin contents of the leaves of Eugenia uniflora L. grown in Egypt. Bull. Fac. Pharm. Cairo Univ. 33(3):17-21. De Morais, S.M., Craveiro, A.A., Machado, M.I.L., Alencar, J.W. and Matos, F.J.A. (1996). Volatile constituents of Eugenia uniflora leaf oil from northeastern Brazil. J.Essent. Oil Res. 8(4): 449-451. Alves, L., Alegrio, L., Castro, R. and Deoliveira, G.R. (2000). Essential oil of Eugenia speciosa Camb. (Myrtaceae) from Rio de Janeiro, Brazil. J. Essent. Oil Res.12(6): 693-694. Bello, A., Pino, J.A., Marbot, R., Urquiola, A., Aguero, J. and Garcia, J. (2001). Volatile components of plants of Eugenia genus from western Cuba: E. axillaris (Sw.) Willd., E. cristata Wr. and E. rocana Britt. and Wils. Ciencias Quimicas 32(3): 135-138. Bello, A., Urquiola, A., Aguero, J. and Marbot, R. (2003). Fruit volatiles of Cayena cherry (Eugenia uniflora L.) from Cuba. J.Essent.Oil Res.15(1): 70-71. Apel, M.A., Sobral, M., Schapoval, E.E.S., Henriques, A.T., Menut, C. and Bessiere, J. (2004). Essential oil composition of Eugenia florida and Eugenia mansoi. J.Essent.Oil Res.16 (4): 321-322. Apel, M.A., Sobral, M., Schapoval, E.E.S., Henriques, A.T., Menut, C. and Bessiere, J. (2005). Volatile constituents of Eugenia mattossi Legr (Myrtaceae). J.Essent.Oil Res. 17(3): 284-285. Duarte, A.R., Costa, A.R.T., Santos, S.C., Ferri, P.H., Paula, J.R. and Naves, R.V. (2008). Changes in volatile constituents during fruit ripening of wild Eugenia dysenterica DC. J.Essent. Oil Res., 20(1): 30-32. Villanueva, H.E., Haber, W.A. and Setzer, W.N. (2009). Chemical Composition of the Leaf and Fruit Essential Oils of Eugenia monteverdensis from Monteverde, Costa Rica. J. Essent. Oil Bearing Plants,, 12: 4 El Sissi, H.I. and El Ansary, M.A.I. (1967). Tannins and polyphenolics of the leaves of Myrtus communis. Planta Med. 15(1): 41-51. Bhargava, K.K., Dayal, R. and Seshadri, T.R. (1974). Chemical components of Eugenia jambolana stem bark. Current Science 43(20): 645-6. Bannon, C.D., Eade, R.A. and Simes, J.J.H. (1976). Extractives of Australian timbers. XVI. The constituents of the wood of Eugenia crebrinervis (syn. Syzigium crebrinerve) and Eugenia gustavioides (syn. Cleistocalyx gustavioides). Austral. J. Chem. 29(5): 1135-41. Diaz, A.M. and Abeger A. (1986). Study of the polyphenolic compounds present in alcoholic extracts of Myrtus communis L. seeds Ethyl acetate-soluble fraction. Anales de la Real Academia de Farmacia 52(3):541. Diaz, A.M. and Abeger, A. (1986). Further contribution to the study of polyphenols present in


35. 36. 37. 38.

39.

40.

41. 42.

43. 44.

45.

46.

47.

48. 49. 50. 51. 52.

197

alcoholic extracts of Myrtus communis L. seeds.water-soluble fraction. Anales de la Real Academia de Farmacia 52(2): 355-64. Diaz, A.M. and Abeger, A. (1987).. Phenolic compounds of Myrtus communis L. seeds. Plantes Medicinales et Phytotherapie 21(4): 317-22. Daulatabad, C.J.D., Mirajkar, A.M., Hosamani, K.M. and Mulla, G.M.M. (1988). Epoxy and cyclopropenoid fatty acids in Syzygium cuminii seed oil. J. Sci. Food Agr. 43(1): 91-94. Appendino, G., Bianchi, Federica,M.A.S.O., Ballero, M. and Gibbons, S. (2002). Oligomeric acylphloroglucinols from myrtle (Myrtus communis). J. Nat. Prod. 65(3): 334-338. Cakir, A. (2004). Essential oil and fatty acid composition of the fruits of Hippophae rhamnoides L. (sea buckthorn) and Myrtus communis L. from Turkey. Biochem.Systematics Ecol. 32(9): 809-816. Davidyuk, L.P., Degtyareva, A.P., Dmitriev, L.B., Drozd, V.N. and Grandberg, I.I. (1989). Structure of biologically active substances from Myrtus extracts. Izvestiya Timiryazevskoi Sel’skokhozyaistvennoi Akademii (6): 164-9. Hayder, N., Kilani, S., Abdelwahed, A., Mahmoud, A., Meftahi, K., Ben, Chibani, J., Ghedira, K. and Chekir, G.L. (2003). Antimutagenic activity of aqueous extracts and essential oil isolated from Myrtus communis. Pharmazie 58(7): 523-524. De Laurenti, N., Rosato, A.; Gallo, L, Leone, L., Milillo, M.A. (2005). Chemical composition and antimicrobial activity of Myrtus communis. Rivista Italiana EPPOS, 39: 3-8. Hayder, N., Bouhlel, I., Skandrani, I., Kadri, M., Steiman, R., Guiraud, P., Mariotte, A. and Ghedira, K. (2008). In vitro antioxidant and antigenotoxic potentials of myricetin-3-Ogalactoside and myricetin-3-O-rhamnoside from Myrtus communis: Modulation of expression of genes involved in cell defence system using c DNA microarray. Toxicology in Vitro, 22(3): 567-581. Consolini, A.E. and Sarubbio, MG. (2002). Pharmacological effects of Eugenia uniflora (Myrtaceae) aqueous crude extract on rat’s heart. J. Ethnopharmacol., 81: 57. Consolini, A.E., Baldini, O.A.N. and Amat, A.G. (1999). Pharmacological basis for the empirical use of Eugenia uniflora L. (Myrtaceae) as antihypertensive J. Ethnopharmacol., 66: 33. Chaieb, K., Hajlaoui, H., Zmantar, T., Ben, K.A., Rouabhia, M., Mahdouani, K. and Bakhrouf, A. (2007). The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae). Phytother. Res., 21(6): 501506. Costa, T. R., Fernandes, O.F.L., Santos, S.C., Oliveira, C.M.A., Liao, L.M., Ferri, P.H., Paula, J.R., Ferreira, H.D., Sales, B.H.N. and Silva M.D.R.R. (2000).Antifungal activity of volatile constituents of Eugenia dysenterica leaf oil. J. Ethnopharmacol., 72(12): 111-117. El Alfy, T.S., Ibrahim, T.A. and Sleem, A.A. (2003). Phytochemical and biological study of the essential oils and petroleum ether extract of the leaves and flowers of Eugenia uniflora L. grown in Egypt [Part I]. Bull.Fac.Pharm.Cairo Univ., 41(2): 83-92. Adams, R.P. (1995). Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Publ. Corp., Carol Stream, IL. Skehan, P. and Strong, R. (1990). New colourimetric cytotoxicity assay for anticancer drug screening. J.Nat.Cancer Inst., 82: 1107-1112. Colee, J.G. (1976). Applied medical microbiology, p. 93, Blackwell Science Publications, London. Sleigh, J.D. and Timburg, M.C. (1981) Notes on medical bacteriology, p. 43, Churchill, Livingston, London. Watkins, T.I. (1958). The chemotherapy of helminthiasis. J. Pharm. Pharmacol., 10: 211.


198

Table 1. Identified components in the hydrodistilled oils obtained from the leaves and fruits of Myrtus communis and Eugenia supraxillaris grown in Egypt KI

931 964 969 988 1002 1022 1029 1057 1071 1094 1109 1149 1169 1179 1180 1221 1226 1235 1239 1254 1259 1261 1280 1289 1290 1294 1293 1351 1354 1361 1374 1376 1381 1389 1396 1398 1406 1433 1448 1472 1479

Components α-Pinene β-Pinene Sabinene Myrcene α-Phellandrene Limonene 1,8-cineole γ-Terpinene Linalool oxide Linalool Fenchyl alcohol Borneol α-Terpineol P-allyl anisole cis-pinocarveol Citronellol Nerol Cuminaldehyde Myrtenyl acetate Chavicol Geraniol Linalyl acetate Anethole P-menth-1-enol trans-Pinocarvyl acetate Sabinyl acetate Menthyl acetate α-Terpinyl acetate Eugenol Neryl acetate α-Copaene β-Maaliene β-Bourbonene β-Cubebene (Z)-Caryophyllene Methyl eugenol trans-caryophyllene β-Gurjunene α-Humulene Chamigrene Germacrene D

Eugenia leaf

Eugenia fruit

0.1 17.4 0.7 0.9 21.8 0.5 0.1 0.9 0.8 0 1.6 8.3 5.4 8.7 7.7

0.1 0.2 12.8 0.2 7.4 1.0 2.3 1.4 0.9 0.2 0.2 0.3 35.5 0.3 0.2 32.8 -

Myrtus leaf 25.5 1.6 27.2 0.5 11.8 0.2 0.7 0.4 0.4 4.2 0.7 3.4 7.0 0.2 1.4 0 2.9 0.5 0.5 0.6 -

Myrtus fruit 3.3 29.6 3.6 0.3 1.3 2.0 9.2 3.1 13.4 2.5 0.7 10.5 1.2 3.2 1.4 -


199

table 1. (continued). KI

Components

Eugenia leaf

β-selinene α-Elemene α-selinene α-Murrolene γ-cadinene δ-Cadinene Eugenyl acetate Spathulenol Viridiflorol Carotol δ-cadinol γ-cadinol β-Eudesmol Palusrol

1482 1488 1490 1491 1510 1519 1521 1574 1589 1591 1631 1652 1653 2314

Total

Eugenia fruit

Myrtus leaf

Myrtus fruit

1.5 2.4 6.6 1.2 1.4 0.7 0.8 5.7 3.4 -

1.0 0.5 0.8 0.1 0.4

0.4 0.3 -

4.9 1.6 -

98.6

98.5

90.3

91.8

Table 2. Variability of different classes of constituents in essential oils from leaves and fruits of Myrtus communis and Eugenia supraxillaris grown in Egypt Compound

Oxygenated Unoxygenated

Monoterpenes Sesquiterpenes E.L E.F. M.L. M.F. E.L E.F. M.L. M.F.

E.L

Total E.F. M.L. M.F.

1.4 81.0 61.3 65.0 40.9 14.4 27.1 3.3

13.5 85.1

81.4 17.1

12.1 44.2

0.4 2.7

0.3 1.6

6.5 17.0

61.6 28.7

71.5 20.3

Table 3. Cytotoxic activity of essential oils from the leaves and fruits of Myrtus communis and Eugenia supraxillaris grown in Egypt Tumor cell lines E. supraxillaris Leaves Cervices Colon Larynx Liver Breast

0.62 0.43 0.54 0.40 0.40

μl) IC50 (μ E. supraxillaris M. communis Fruits Leaves 1.30 0.43 0.87 0.38 1.40

0.42 0.43 0.54 0.36 1.30

M. communis Fruits 0.87 0.69 0.72 0.40 1.30

10 10 8 10 8 10 12

>200 25 50 50 100 100 100 25

E. supraxillaris Leaves DIZ MIC μl/ml) (mm) (μ 24 15 10 10 12 15 20 9

25 50 50 50 50 12.5 25 100

E. supraxillaris Fruits DIZ MIC μl/ml) (mm) (μ 8 10 11

>200 >200 100 >200 100 >200 >200 100

M. communis Leaves DIZ MIC μl/ml) (mm) (μ 22 15 10 9 10 15 22 9

25 50 50 50 100 12.5 25 100

M. communis Fruits DIZ MIC μl/ml) (mm) (μ 29 38 29 32 30 36 25 -

DIZ (mm) 2 2 4 1 1 0.5 2 -

MIC μl/ml) (μ

Ofloxacin

25

DIZ (mm)

1

MIC μl/ml) (μ

Amphotricin B

Control 1% (aqueous Tween 80) Eugenia supraxillaris Leaves Eugenia supraxillaris Fruit Myrtus communis Leaves Myrtus communis Fruit

Oil Sample tested

7 9 8 5

4 7 3 4

Time elapsed before worm death (min.) after treatment with 0.1% of tested samples 0.2% of tested samples

Table 5. Antiwormal effect of essential oils from the leaves and fruits of Myrtus communis and Eugenia supraxillaris grown in Egypt

DIZ (mm) : Diameter of inhibition zone in mm MIC (μl/ml) : Minimum inhibitory concentration in (μ/ml)

E. coli P. vulgaris P. aeruginosa S. aureus S. lutea B. subtilis M. phlei C. albicans

Microorganisms

Table 4. Antimicrobial activity of essential oils from the leaves and fruits of Myrtus communis and Eugenia supraxillaris grown in Egypt

E.A. Aboutabl et al. / Jeobp 14 (2) 2011 192 - 200 200

Jeobp 14 (2) 2011 pp 201 - 207

201


Chemical Composition of the Essential Oil from Aerial Parts of Haplophyllum acutifolium (DC.) G. Don from Iran Javad Asili, Maryam Rajae Fard, Ali Ahi and Seyyed Ahmad Emami * Department of Pharmacognosy, Faculty of Pharmacy, Mashhad University of Medical Sciences, 1365-91775, Mashhad, I.R. Iran Received 11 August 2009; accepted in revised form 14 September 2010

Abstract: The oil of aerial parts of Haplophyllum acutifolium was analyzed by GC and GC-MS. Ninety two components, representing 97.7 % of the total components were identified. The major compounds were α-cadinene (25.1 %), β-cedrene (19.1 %), sabinene (8.1 %), 8,14-cedranoxide (5.5 %) and terpin-4-ol (5.7 %). Key words: Haplophyllum acutifolium, Rutaceae, essential oil composition, α-cadinene, βcedrene, sabinene. Introduction: The genus Haplophyllum A. Juss. belongs to the family Rutaceae contains about 70 species in the Mediterranean countries 1. These species are perennial herbs with numerous erect stems, usually somewhat woody to suffruticose below, furnished with pale or dark punctate glands. Leaves alternate, entire, lanceolate to elliptic or linear (more rarely broadly ovate) or trisect with lanceolate or linear segments. Inflorescence cymose, bracteate. Sepals and petals 5. Petals concave, entire, yellow or reddish, with a ± distinct thickened dorsal keel. Stamens 10, The filaments usually pilose to villous in the lower half. Capsule 5-lobed usually dehiscent, loculi 2-4(-10)-ovulate. Styles fused. Seeds reniform, usually transversely rugose, more rarely interruptedly longitudinally rugose 2. Around thirty species of the genus found in Iran which 14 of them are endemic to the country 3-5. Several biologically active secondary metabolites especially alkaloids 6-8, coumarins 9-10, flavonoids 1113 and lignanes 14-15 have been isolated from various Haplophyllum species in the world. Experimental Plant materials: Aerial parts of Haplophyllum acutifolium (DC.) G. Don [syn: H. perforatum (M. Beib.) Kar. & Kir.; H. sieversis Fisch.; H. suavolens Lbd.; Ruta divaricata Siev.; R. acutifolia DC.; R. perforate M. Beib.] were collected during the flowering stage from campus of Ferdowsi University of Mashhad, Razavi Khorasan province in northeast of Iran in Jun 2006. Voucher specimen (11867) was deposited in Herbarium of Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Oil Isolation: Aerial parts (150 g) of Haplophyllum acutifolium were subjected to hydro*Corresponding author (Seyyed Ahmad Emami) E-mail: < [email protected] or [email protected] >


Seyyed Ahmad Emami et al. / Jeobp 14 (2) 2011 201 - 207

202

distillation using a Clevenger-type apparatus for 3 h. After decanting and drying over anhydrous sodium sulfate, the slightly yellow colored oil was recovered in yield of 0.1 % (v/w). Gas chromatography: The GC analyses were performed using a Perkin-Elmer (UK) 8500 gas chromatograph equipped with flame ionization detector (FID). Separations were achieved on a DB-5 fused silica column (30 m 0.25 mm i.d., 0.25 μm film thickness); temperature programming (mostly), 60-275°C at 5°C/min; injector temperature (split: 1/25) 250°C; detector temperature, 280°C; carrier gas, N2 at 12 psi. Gas chromatography-Mass spectrometry: The GC-MS analysis were performed using a Thermoquest-Finnigan Trace gas chromatograph apparatus fitted with a DB-5 fused silica column (60 m 0.25 mm i.d., 0.25 μm film thickness) interfaced with a Thermo-Finnigan quadrapule mass detector and a computer equipped with Wiley 7 and Nist 1.7 spectra libraries; column temperature, 60-250°C at 5°C; injector temperature 250°C; volume injection, 0.1 μl; split ration, 1:50; carrier gas, He at 1.1 ml min ; ionization potential, 70 eV; ionization current, 150 μA; ion source temperature, 250°C; mass range, 35-465 mui. The oil components were identified from their GC retention indices obtained with reference to nalkenes series on DB-5 column, comparison of their mass spectra and fragmentation patterns reported in literature 16 and by computer matching with Wiley 7 and Nist 1.7 Mass Spectral Database for GCMS (Table 1). Results and discussion: The percentage of composition of the oil and their grouped components are given in Table 1. The components are listed in order of their retention indices on DB-5 column. Ninety two components, representing 97.7 % of the total components were identified. Considering the quantitative results, the pattern of main grouped components in essential oil was: sesquiterpene hydrocarbons 52.0 %, monoterpene hydrocarbons 16.8 %, oxygenated sesquitepenes 15.7 % and oxygenated monoterpenes 13.1 %. α-cadinene (25.1 %), β-cedrene (19.1 %), sabinene (8.1 %), 8, 14cedranoxide (5.5 %) and terpin-4-ol (5.7 %) were the main components identified in the oil. The first study on chemical composition of obtained oil from aerial parts of Iranian Haplophyllum species was date back to 2000. In this year Yari et al. examined the composition of the oil of H. tuberculatum (Forrssk.) A. Juss. collected from Gachsaran (Kohgylouyeh-Buyer Ahmad Province). The yield of this oil was 0.35 % (w/w). The oil has 18 compounds representing 84.0 % of the compounds. Main components of this oil were α-pinene (21.9 %) and limonene (27.3 %) 17. In similar study 18 in the United Arab Emirates (a neighbor of Iran, located in the southeast of the Persian Gulf), on H. tuberculatum, the composition of three sample oils isolated in different seasons differed significantly from that one reported for the H. tuberculatum collected in Iran 17. The United Arab Emirates samples were collected in May 1997 and 2001. Both samples were similar in α-phellandrene (10.7-32.9 %) as the major component. This study reports linalool (15.0 %), linalyl acetate (10.6 %), β-caryophyllene (9.7 %) and α-terpineol (6.7 %) as the major components of the sample collected in April 1998. In 2004 Masoudi and her colleagues studied the composition of H. robustum aerial parts oil. The plant was harvested from Kerman (Kerman Province) and the yield of its oil was 0.5 % (w/w), with total of 23 compounds representing 86.1 % of the components. Sabinene (3.05 %), β-pinene (18.2 %) and limonene (12.1 %) were as the main compounds of this oil 19. Another investigation on this species showed that leaves, stems, flowers and fruits of H. robustum collected from Kashan (Isfahan Province) yielded 1.1, 0.39, 1.1 and 2.1 % (w/w) of volatile oil respectively 20. Total identified compounds of these oils were 13 (82.8 %), 12 (82.7 %), 11 (89.2 %) and 12 (83.4 %) respectively. Main components of the leaf oil were: cis-sabinene hydrate (27.7 %),


203

1,8-cineole (19.1 %) and γ-terpinene (10.3 %). The stem oil of the species contained 1,8-cineole (27.7 %); γ-terpinene (12.2 %), cis-sabinene hydrate (11.5 %) and limonene (11.1 %) while the obtained oil from the flower of the same species contained: 1,8-cineol (45.1 %), limonene (12.3 %), cis-sabinene hydrate (12.0 %) as main components. The main components of the obtained oil from the fruits of this plants were: 1,8-cineole (28.4 %), limonene (13.8 %), cis-sabinene hydrate (12.2 %) and γ-terpinene (10.1 %). In the other report, the obtained oil from aerial parts of H. robustum collected from Aran and Bidgol (Isfahan Province) with 0.5 % yield, had 30 compounds representing 99.2 % of identified components and its main components were myrcene (10.7 %), α-pinene (8.5 %), 4-terpineol (7.0 %) and sabinene (6.2 %) 21. In 2006, the aerial parts of H. tuberculatum collected in Shiraz (Fars Province) was reported 0.02 % (w/w) volatile oil 22. This oil had 40 compounds representing 91.8 % of identified components and main compounds were linalool (15.5 %), α-pinene (7.9 %) and limonene (5.3 %). Biniyaz et al. studied the composition of the aerial parts of H. furfuraceum Bge. ex Boiss. and H. virgatum Spach 23. The first species was collected from Kashmar (Khorasan Province) and the yield of its oil was 0.35 % (w/w) while the second species was harvested from Darab (Fars Province) and the yield of its oil was 0.2 % (w/w). The volatile oil of H. furfuraceum had 33 compounds representing 98.1 % of the components while the oil of H. virgatum had 25 compounds representing 90.5 % of identified components. The main components of first essential oil were elemol (11.8 %) and eudesmol (10.1 %) and the main compound of the second oil were 2-nononone (28.4 %) and 2-undecane (21.5 %) . Recently the obtained oils from two taxa of Iranian Haplophyllum were studied by Javidnia et al. 24. The yields of obtained oils from H. lissonotum C. Town. collected from Sarvestan (Fars Province) and H. buxbaumii (Poir.) G. Don. subsp. mesopotamicum (Boiss.) C. Town from Noor-Abad (Fars Province) were 0.23 % and 0.26 % (w/w) respectively. The oil of first taxon representing 88.5 % of total components. The main compounds of this oil were caryophyllene oxide (26.9 %), β-caryophyllene (12.2 %); humulene epoxide (8.3 %); α-humlene (7.2 %) and caryophylla-4 (14), diene-β-ol (7.1 %). The oil of the second taxon had 43 compounds representing 89.5 % of the components. This oil contained hexadecanoic acid (18.5 %); ethyl linoleate (14.0 %), phytol (9.9 %) and caryophyllene oxide (5.5 %) as main components. Acknowledgments: The authors would like to acknowledge the financial support (grant No. 84369) of the Research Council of Mashhad University of Medical Sciences, Mashhad, Iran. This work is done as a part of a pharm.D. thesis (No. 1243) in the School of Pharmacy, Mashhad University of Medical Sciences.

1. 2. 3.

4. 5. 6.

References Townsend, C.C. (1986). Taxonomic Revision of the Genus Haplophyllum (Rutaceae) Hooker’s Icon Plantarum 40 (1-3) Bentham-Maxon UK. Townsend, C.C. (1967). Haplophyllum in Flora of Turkey and the Aegean Islands Edit. Davis P.H. Edinburgh University Press Vol.2, spp. 469-506. Joharchi, M.R. (1998). Rutaceae in Flora of Iran Edits. M. Assadi, M. Khatamsaz, A.A. Maassoumi and V. Mozaffarian, Research Institute of Forests and Rangelands, Tehran, Iran No. 60, pp.8-75. (In Persian). Ghahraman, A. and Attar, F. (1999). Biodiversity of Plant Species in Iran Tehran University Publication, Iran. Vol. 1, p. 256. Emami, S.A. and Aghazari. F. (2008). Les Phanerogames Endemiques de la Flore d’Iran. Publication de l’ Universite d’ Iran des Sciences Medicales, Teheran, Iran. p. 297-300/ Ali, M.S., Fatima, S. and Pervez. M.K. (2008). Haplotin, a New Furanoquinoline from


7.

8.

9.

10.

11 12. 13. 14.

15. 16. 17. 18.

19. 20.

21.

22. 23.

24.

204

Haplophyllum acuitifolium (Rutaceae). Journal of the Chemical Society of Pakistan 30 (5): 775-779. Jansen, O., Akhmedjanova, V., Angenot, L., Balansard, G., Chariot, A., Ollivier, E., Tites, M. and Fredrich, M. (2006). Screening of 14 Alkaloids Isolated from Haplophyllum A. Juss. for Cytotoxic Properties. Journal of Ethnopharmacology 105 (1-2): 241-245. Akhmedzhanova, V.I., Rasulova, K.I., Bessonova, I.A., Shashkov, A.S., Abdullaev N.D. and Angeot, L. (2005). Folipidine, a New Type Quinoline Alkaloid from Plants of the Haplophyllum Genus. Chemistry of Natural Compounds 41: 60-64. Ivanova, A., Mikhova, B., Stambolijska, T. and Kostova, I. (2001). Liganens and Coumarin Glycosides from Haplophyllum suaveolens. Zeitschrift fur Naturforschung - Section C Journal of Biosciences 56 (5-6): 329-333. Filippini, R., Piovan, A., Innocenti, G., Caniato, R. and Cappelletti, E.M. (1998). Production of Coumarin Compounds by Haplophyllum patavinum in vivo and in vitro. Phytochemistry 49(8): 2337- 2340. Ulubelen, A. (1986). Flavonoids from Haplophyllum suaveolens and Haplophyllum buxbaumii. Fitoterapia 57(4): 274-275. Khalid, A. and Waterman, G. (1981). Alkaloid, Lignin and Flavonoid Constituents of Haplophyllum tuberculatum from Sudan. Planta Medica 43 (2): 148-152. Yuldashev, M.P., Batirov, E. Kh. and Malikov, V.M. (1987). Flavonoids of Some Plants of the Genus Haplophyllum. Chemistry of Natural Compounds, 23(3): 377-378. Lim, S., Grass, J., Akhmedjanova, V., Debiton, E., Balansard, G., Beliveau. R. and Barthomeuf, C. (2007). Reversal P-Glycoprotein-Mediated Drug Efflux by Eudesmin from Haplophyllum perforatum and Cytotoxicity Pattern Versus Diphyllin, Podophyllotoxin and Etoposide. Planta Medica 73(15): 1563-1567. Saglam, H., Golzar, T. and Golzar, B. (2003). A New Prenylated Arylnaphthalene Lignan from Haplophyllum myrtifolium. Fitoterapia 74(6): 564-569. Adams, R.P. (2004). Identification of Essential Oil Components by Gas Chromatography/ Quadrupole Mass Spectroscopy Allured Publishing Corporation, Carol Stream, Illinois USA. Yari, M., Masoudi, S. and Rustaiyan, A. (2000). Essential Oil of Haplophyllum tuberculatum (Forssk.) A. Juss. Grown Wild in Iran. Journal of Essential Oil Research 12(1): 69-70. Al Yousuf, H.M., Bashir, K.A., Veres, K., Dobos, Á., Nagy, G., Máthé, I., Blunden, G., Vera, J.R . (2005). Essential Oil of Haplophyllum tuberculatum (Forssk.) A. Juss. from the United Arab Emirates. J. Essent.Oil Research. 17(5), 519-521. Masoudi, S., Rustaiyan, A. and Azar, P.A. (2004). Essential Oil of Haplophyllum robustum Bge. from Iran. J. Essent.Oil Research. 16(6): 548-549. Bamonieri, A., Safaei-Ghomi, J., Asadi, H., Batooli, H., Masoudi, S., Rustaiyan, A. (2006). Essential Oils from Leaves, Stems, Flowers and Fruits of Haplophyllum robustum Bge. (Rutaceae) Grown in Iran. J. Essent.Oil Research. 18(4): 379-380. Rahimi-Nasrabadi, M., Gholivand, M and Batooli, H. (2009). Chemical Composition of the Essential Oil from Leaves and Flowering Aerial Parts of Haplophyllum robustum Bge. (Rutaceae). Digest Journal of Nanomaterials and Biostructures 4(4): 819-822. Javidnia, K., Miri, R., Banani, A. (2006). Volatile Oil Constituents of Haplophyllum tuberculatum (Forssk.) A. Juss. (Rutaceae) from Iran. J. Essent.Oil Research. 18 (4): 355-356. Biniyaz, T., Habibi, Z., Masoudi, S., Rustaiyan, A. (2007). Composition of the Essential Oils of Haplophyllum furfurceum Bge. ex Boiss. and Haplophyllum virgatum Spach. from Iran.J. Essent.Oil Research. 19 (1): 49-51. Javidnia, K., Miri, R., Soltani, M. and Varamini, P. (2009). Volatile Constituents of Two Species of Haplophyllum A. Juss. from Iran [H. lissonotum C. Town. and H. buxbaumii (Poir.) G. Don. subsp. mesopotamicum (Boiss.) C. Town.]. J. Essent.Oil Research. 21(1): 48-51.

Seyyed Ahmad Emami et al. / Jeobp 14 (2) 2011 201 - 207 Table 1. Percentage composition of the essential oil of aerial parts of Haplophylum acutifolium No

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

α-Thujene α-Pinene Camphene Benzaldehyde Sabinene 2-Octanone β-Myrcene α-Phellandrene α-Terpinene o-Cymene Limonene cis-Ocimene trans-Ocimene γ-Terpinene cis-Sabinene hydrate cis-Linalool oxide Terpinolene 2-Nonanone trans-Sabinene hydrate Linalool n-Nonanal 1,3,8-para-Menthatriene p-Menth-2-en-1-ol 3-iso-Thujanol trans-Sabinol trans-Verbenol 3-neo-Thujanol Sabina ketone β-Pinene oxide Borneol Terpin-4-ol Cymen-8-0l α-Terpineol cis-Piperitol Verbenone trans-Piperitol trans-Carveol Fenchyl acetate neo-iso-Dihydrocarveol Citronellol exo-Fenchyl acetate Myrtenyl acetate Carvone

RI* 928 934 949 959 973 983 988 1002 1014 1022 1027 1036 1047 1058 1065 1069 1086 1089 1094 1096 1100 1108 1118 1128 1136 1141 1146 1153 1155 1164 1175 1181 1187 1190 1199 1201 1214 1218 1224 1226 1231 1232 1238

Percentage 0.2 2.9 **t t 8.1 t 0.7 0.5 0.8 0.4 1.1 t 0.2 1.3 0.9 t 0.6 t 0.3 1.1 0.1 t 0.3 t 0.2 0.1 t t t 0.2 5.7 0.1 2.8 0.1 t 0.1 t 0.1 0.4 0.2 t t 0.1

205

Seyyed Ahmad Emami et al. / Jeobp 14 (2) 2011 201 - 207 table 1. (continued). No

Compound

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

Thymoquinone Linalool acetate Geraniol Citronellyl formate α-Terpinen-7-al Menthyl acetate Carvacrol Isomenthyl acetate trans-Dihydro-α-terpenyl acetate cis-Piperitol acetate α-Cubebene 2-Methyl undecanal α-Ylangene Bourbonene β-Cubebene Longifolene β-Caryophyllene β-Cedrene cis-Thujopsene α-Himachalene α-neo-Clovene cis-Muurola-4(14),5 diene allo-Aromadenderene γ-Gurjunene γ-Himachalene Germacrene D cis-β-Guaiene Viridiflorene Benzyl tiglate α-Muurolene α-Bisabolene(Z) γ-Cadinene 7-epi-α-Selinene δ-Cadinene Cadina-1,4-diene α-Cadinene 8,14-Cedranoxide Himachalene epoxide Spachulenol Viridiflorol Guaiol 10-epi-γ-Eudesmol γ-Eudesmol β-Eudesmol

RI* 1245 1253 1268 1274 1276 1291 1297 1307 1311 1327 1345 1362 1370 1378 1386 1398 1401 1415 1424 1446 1452 1454 1458 1469 1472 1481 1486 1491 1494 1496 1502 1511 1514 1521 1528 1548 1549 1572 1574 1589 1592 1608 1623 1644

Percentage t t t t 0.2 0.1 t t t 0.1 t t t 0.1 0.3 0.1 t 19.1 t 2.3 0.1 0.1 t 0.1 1.6 t 0.5 0.2 t t 0.2 0.9 1.0 0.1 0.2 25.1 5.5 0.6 1.0 t 0.1 0.5 2.8 2.0

206

Seyyed Ahmad Emami et al. / Jeobp 14 (2) 2011 201 - 207 table 1. (continued). No

Compound

RI*

88 89 90 91 92

α-Eudesmol 7-epi-α-Eudesmol Occidentalol acetate α-Bisabolol epi-α-Bisabolol Total

1647 1659 1676 1679 1684 97.7

Major Grouped Compounds Monoterpene hydrocarbons Oxygenated monoterpens Sesquiterpene hydrocarbons Oxygenated Sesquiterpens Miscellaneous

16.8 13.1 52.0 15.7 0.1

Percentage 1.1 2.1 t t t

*RI: The retention Kovats indices were determined on DB-5 capillary column **t: trace


K. Morteza-Semnani et al. / Jeobp 14 (2) 2011 208 - 213

209

Center of Agriculture and Natural Resources of Mazandaran. A voucher specimen (herbarium No. 191) was deposited at the Herbarium of the Department of Botany, Research Center of Agriculture and Natural Resources of Mazandaran. Extraction and isolation of the essential oil: The dried aerial parts (100 g) were subjected to hydrodistillation using a Clevenger-type apparatus for 3 h. The oil was dried over anhydrous sodium sulfate and kept at 4°C in a sealed brown vial until required. The oil was submitted to GC and GC-MS analysis. Gas chromatography: Gas chromatographic analysis was carried out on a Perkin-Elmer 8500 gas chromatograph with FID detector and a DB-5 capillary column (30 m x 0.25 mm; film thickness 0.25 μm). The operating conditions were as follows: carrier gas helium with a flow rate of 2 mL/min, split ratio was 1:30, the oven temperature was programmed 4 min, isothermal at 60°C and then 60°220°C at 4°C/min, injector and detector temperatures were set at 240°C. The percentage of composition of the identified compounds was computed from the GC peak areas without any correction factors and was calculated relatively. Gas chromatography-Mass spectrometry: Gas chromatography/mass spectrometry analysis was performed on Hewlett Packard 6890 series, using a DB-5 capillary column (30 m x 0.25 mm, film thickness 0.25 μm) which was programmed as follows: 60°C for 5 min and then up to 220°C at 4°C/ min. The carrier gas was helium at a flow rate of 2 mL/min; split ratio, 1: 40; ionization energy, 70 eV; scan time, 1 s; acquisition mass range, m/z 40-400. The components of the oil were identified by their retention time, retention indices relative to C9C28 n-alkanes, computer matching with the WILEY275.L library and as well as by comparison of their mass spectra with those of authentic samples or with data already available in the literature 14,15. The percentage of composition of the identified compounds was computed from the GC peak area without any correction factor and was calculated relatively. Antimicrobial assay: Bacillus subtilis PTCC 1023, Staphylococcus aureus PTCC 1112, Escherichia coli PTCC 1330, Salmonella typhi PTCC 1639, Pseudomonas aeroginosa PTCC 1074, Aspergilus niger PTCC 5011 and Candida albicans PTCC 5027 were used for testing the antimicrobial activity. Diffusion method using filter paper disk (6 mm) was used for the screening of oil antibacterial and antifungal activities 16-19. Bacterial and fungal strains were tested on Muller-Hinton agar and Sabouraud dextrose agar, respectively. Sterilized paper disks were loaded with different amounts of M. pulegium oil (250, 500, 1000, 2000, 4000 and 8000 μg /disk) and applied on the surface of agar plates. All plates were incubated at 37°C for 24 h for bacteria; at 25°C for 24 h for C. albicans; and at 25°C for 3 days for A. niger. Inhibition zone diameters were measured after conventional incubation period. Gentamycin (50 μg/disk), Amikacin (3 μg/disk) and Amphotericin B (100μg/disk) (obtained from Sigma) were used as positive reference standards. The estimation of the minimal inhibitory concentration (MIC) was carried out by the broth dilution method. Dilutions of essential oil from 6.4 to 0.1 mg/ml were used. MIC values were taken as the lowest essential oil concentration that prevents visible microbe growth after the incubation period (described above) 20,21. Gentamycin, Amikacin and Amphotericin B (128, 64, 32, 16, 8, 4 and 2 μg/ mL) were used as positive reference standards and control with no essential oil was used. Each experiment was made three times. Results and discussion: The hydrodistillation of the dried flowering aerial parts of M. pulegium gave light yellowish oil with yield of 0.6 % (w/w). As shown in Table 1, fifty-five components were


210

identified in this oil, which presented about 96.9 % of the total composition of the oil. The major constituents of the essential oil were pulegone (54.6 %) and menthone (15.1 %). The oil of M. pulegium comprised 30 monoterpenoids (91.8 %), 17 sesquiterpenoids (2.4 %) and 8 non-terpenoids (2.7 %). The essential oil of the flowering aerial parts of M. pulegium was rich in monoterpenoids. Tables 2 and 3 give a summary of the results of the antimicrobial screening of M. pulegium oil. The antimicrobial activity of M. pulegium oil was concentration-dependent on Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhi, Pseudomonas aeroginosa, Aspergilus niger and Candida albicans. The oil had considerable activity against Gram-positive bacteria and fungi. The oil was more active against Gram-positive bacteria than Gram-negative bacteria; the least activity was against Escherichia coli. There is a great number of studies on the essential oil composition of M. pulegium 1,5-12 and three chemotypes have been established: pulegone-type, piperitenone/piperitone-type and isomenthone/ neoisomenthol-type. In 2005, Agnihotri et al. reported pulegone (65.9-83.1 %) and menthone (8.3-8.7 %) as the major componenets of the oil of M. pulegium collected from different locations in the higher Himalayas, Jammu and Kashmir, India 1. Pulegone, piperitenone and isomenthone were reported as the main compounds of the oil of M. pulegium growing in Bulgaria 11. In 2008, piperitone (38.0 %) and piperitenone (33.0 %) were reported as the major compounds of oil of M. pulegium collected from Kazeron area (Fars Province, Iran); the oil showed a significant activity against microorganisms especially Gram-positive bacteria, whereas the least susceptible were Gram-negative bacteria especially Escherichia coli 12. The differences in oil composition established in our investigation and those reported by Mahboubi and Haghi 12 may be because of the collection time, chemotypes, drying conditions, mode of distillation, geographic and climatic factors. Since the antimicrobial activity of oil depends on its chemical composition, thus the antimicrobial effects of the oil of Mentha pulegium collected from different areas can be different.

1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

References Agnihotri, V.K., Agarwal, S.G., Dhar, P.L., Thappa, R.K., Baleshwar, Kapahi, B.K., Saxena, R.K. and Qazi, G.N. (2005). Essential oil composition of Mentha pulegium L. growing wild in the North-Western Himalayas India. Flavour Fragr. J., 20: 607-610. Mozaffarian, V. (1996). A Dictionary of Iranian plant names, Farhang Mo’aser Publishers, Tehran, Iran, pp. 344-345. Rechinger, K.H. (1982). Flora Iranica. Akademische Druck-U.Verlagsanstalt, Graz, Austria, No. 150, p. 570. Zargari, A. (1993). Medicinal Plants. Tehran University Publications, Tehran, Iran, Vol. 4, pp. 14-18. Topalov, V. and Dimitrov, S. (1969). Studies on the content and quality of the essential oil from some Peppermint species of the Bulgarian flora. Plant Sci., 6: 77-83. Lawrence, B.M. (1978, 1989, 1998). Progress in essential oils. Perfum. Flavor., 3: 40; 14: 7678; 23: 64-68. Baser, K., Kürkçüglu, M., Tarimcilar, G. and Kaynak, G. (1999). Essential oils of Mentha species from Northern Turkey. J. Essent. Oil Res., 11: 579-588. Reisvasco, E. Coelho, J. and Palavra, A. (1999). Composition of Pennyroyal oils obtained by supercritical CO2 extraction and hydrodistilation. Flavour Fragr. J., 14: 156-160. Chalchat, J., Gorunovic, M., Maksimovic, Z. and Petrovic, S. (2000). Essential oil of wild growing Mentha pulegium L. from Yugoslavia. J. Essent. Oil Res., 12: 598-600. Kokkini, S., Hanlidou, E. and Karousou, R. (2002). Variation of pulegone content in Pennyroyal (Mentha pulegium L.) plants growing wild in Greece. J. Essent. Oil Res., 14: 224-227

K. Morteza-Semnani et al. / Jeobp 14 (2) 2011 208 - 213 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21.

211

Stoyanova, A., Georgiev, E., Kula, J. and Majda, T. (2005). Chemical composition of the essential oil of Mentha pulegium L. from Bulgaria. J. Essent. Oil Res., 17: 475-476. Mahboubi, M. and Haghi, G. (2008). Antimicrobial activity and chemical composition of Mentha pulegium L. essential oil. J. Ethnopharmacol., 119: 325-327. Morteza-Semnani, K., Saeedi, M. and Akbarzadeh, M. (2006). The essential oil composition of Mentha aquatica L. J. Essent. Oil Bearing Plants, 9: 283-286. Davies, N.W. (1990). Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and carbowax 20M phases. J. Chromatogr., 503: 1-24. Adams, R.P. (2001). Identification of Essential Oil Components by Gas Chromatography/ Quadrupole Mass Spectroscopy. Allured Publishing Corp., Carol Stream, IL. Morteza-Semnani, K., Saeedi, M., Mahdavi, M.R. and Rahim, F. (2006). Antimicrobial studies on extracts of three species of Phlomis. Pharm. Biol., 44: 426-429. Saeedi, M. and Morteza-Semnani, K. (2007). Chemical composition and antimicrobial activity of essential oil of Origanum vulgare L. Int. J. Biol. Biotech., 4: 259-265. Saeedi M., Morteza-Semnani K., Mahdavi M.R. and Rahimi, F. (2008). Antimicrobial studies on extracts of four species of Stachys. Indian J. Pharm. Sci., 70: 403-406. Saeedi, M. and Morteza-Semnani, K. (2009). Chemical composition and antimicrobial activity of the essential oil of Heliotropium europaeum L. Chem.Nat. Compd., 45: 98-99. Hernandez, T., Canales, M., Teran, B., Avila, O., Duran, A., Garcia, A.M., Hernandez, H., Angeles-Lopez, O., Fernandez-Araiza, M. and Avila, G. (2007). Antimicrobial activity of the essential oil and extracts of Cordia curassavica (Boraginaceae). J. Ethnopharmacol., 111: 137141. Morteza-Semnani, K., Saeedi, M. and Akbarzadeh, M. (2009). Chemical composition and antimicrobial activity of the essential oil of Echium italicum L. J. Essent. Oil Bearing Plants, 12: 557-561. Table 1. The chemical constituents of the essential oil of Mentha pulegium L. No.

Components

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

α-Pinene 3-Methylcyclohexanone Sabinene β-Pinene 3-Octanone Myrcene 3-Octanol α-Terpinene Limonene 1,8-Cineole γ-Terpinene cis-Sabinene hydrate Terpinolene trans-Sabinene hydrate 3-Octanol acetate trans-p-Mentha-2,8-dien-1-ol cis-p-Mentha-2,8-dien-1-ol

RIa

GC area (%)

Method of identification b

941 953 977 981 985 992 993 1018 1030 1033 1062 1072 1090 1099 1124 1125 1139

0.4 0.2 0.3 0.4 0.1 0.3 1.3 0.1 2.2 0.3 0.3 0.1 0.3 0.1 0.6 0.1 0.1

MS, KI, CoI MS, KI MS, KI, CoI MS, KI, CoI MS, KI, CoI MS, KI, CoI MS, KI, CoI MS, KI MS, KI, CoI MS, KI, CoI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI


212

table 1. (continued). No.

Components

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

trans-Verbenol Menthone Isomenthone Isopulegone Menthol α-Terpineol γ-Terpineol trans-Piperitol trans-Carveol 4-Methylene-Isophorone Pulegone Piperitone cis-Piperitone epoxide trans-Piperitone epoxide Menthyl acetate Piperitenone α-Cubebene Piperitenone oxide β-Bourbonene β-Cubebene β-Caryophyllene α-Humulene Germacrene D γ-Cadinene δ-Cadinene Spathulenol Caryophyllene oxide Humulene epoxide II Alloaromadendrene epoxide epi-α-Muurolol α-Cadinol Guaia-3,10(14)-dien-11-ol Eudesma-4(15),7-dien-1-β-ol 14-Hydroxy-a-muurolene 6,10,14-Trimethyl-2-pentadecanone n-Eicosane n-Heneicosane n-Docosane Total

a

RI: Retention index on DB-5 column MS: mass spectroscopy CoI: co-injection

b

RIa

GC area (%)

Method of identification b

1146 1155 1164 1170 1173 1191 1200 1209 1218 1219 1238 1254 1255 1257 1296 1345 1353 1371 1389 1390 1421 1457 1487 1515 1525 1579 1585 1609 1642 1643 1655 1679 1689 1782 1844 2002 2102 2202

0.1 15.1 4.3 4.1 0.4 0.3 1.0 0.3 0.5 0.3 54.6 0.8 1.6 0.1 0.1 3.1 0.1 0.1 0.1 0.1 0.1 0.1 0.3 0.1 0.1 0.3 0.3 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.1 96.9

MS, KI MS, KI, CoI MS, KI MS, KI MS, KI, CoI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI, CoI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI

250 500 1000 2000 4000 8000 50 3 100

Conc. (μ μg/disc)

7.2 ± 0.4 8.1 ± 0.75 9.1 ± 1.4 9.9 ± 1.6 11.3 ± 1.8 29.8 ± 1.9 21.8 ± 1.55 -

Bacillus subtilis (G+) 7.25 ± 0.5 8.6 ± 0.75 9.8 ± 1.3 10.4 ± 1.6 37.3 ± 2.5 24.9 ± 3.1 -

7.3 ± 0.5 8.2 ± 0.6 31.6 ± 3.2 23.8 ± 2.5 -

7.2 ± 0.6 7.9 ± 0.5 8.9 ± 0.7 29.0 ± 2.5 16.8 ± 3.1 -

- no inhibition; ND: Not Determined.

a

Bacillus subtilis (G+) Staphylococcus aureus (G +) Escherichia coli (G -) Salmonella typhi (G -) Pseudomonas aeroginosa (G -) Candida albicans Aspergilus niger

Strains

7.6 ± 0.5 8.5 ± 0.7 9.6 ± 1.2 10.2 ± 1.5 11.1 ± 1.3 22.3 ± 2

0.4 0.8 3.2 1.6 1.6 0.4 0.8

Essential oil

32 × 10-3 8 × 10-3 16 × 10-3 32 × 10-3 32 × 10-3 ND ND

4 × 10-3 4 × 10-3 2 × 10-3 8 × 10-3 8 × 10-3 ND ND

MIC (mg/ml) Gentamycin Amikacin

ND ND ND ND ND 64 × 10-3 32 × 10-3

Amphotericin B

7.3 ± 0.5 8.7 ± 0.7 10.8 ± 1.1 11.9 ± 1.3 22.7 ± 2.1

Fungi Candida Aspergilus albicans niger

Table 3. Minimum inhibitory concentration (MIC) of essential oil of Mentha pulegium L.a

7.4 ± 0.4 8.2 ± 0.6 9.1 ± 1.1 31.02 ± 1.2 15.8 ± 1.12 -

Diameter (mm) of zone of inhibition (Mean ± S.D.) Bacteria Staphylococcus Escherichia Salmonella Pseudomonas aureus (G +) coli (G -) typhi (G -) aeroginosa (G -)

Values are inhibition zone (mm) and an average of triplicates; - no inhibition

a

Gentamycin Amikacin Amphotericin B

M. pulegium oil

Sample

Table 2. Antimicrobial activity of the essential oil of Mentha pulegium L.a

K. Morteza-Semnani et al. / Jeobp 14 (2) 2011 208 - 213 213

Jeobp 14 (2) 2011 pp 214 - 223

214


The Effect of Microwaves on Essential oils of White and Black Pepper (Piper nigrum L.) and their Antioxidant Activities Magda A. Abd El Mageed*, Amr F. Mansour, Khaled F. El Massry, Manal M. Ramadan, Mohamed S. Shaheen National Research Centre, Chemistry of Flavour and Aroma Department, Elbuhouth Street, Dokki, Cairo, Egypt Received 01 April 2010; accepted in revised form 19 October 2010

Abstract: White and black peppers were subjected to conventional roasting as well as microwave heating in order to study the effects of such techniques on the volatile constituents of each spice oil and its antioxidative activity. Hydrodistillation oil of each spice was analyzed using Gas Chromatography (GC) and Gas chromatography-Mass spectrometry (GC-MS). Twenty six compounds were identified in essential oil of white pepper, whereas, twenty compounds were detected in black pepper essential oil. δ-Carene, limonene, α- and β-pinene and β-caryophyllene were the major components in both. Due to heating treatments, increase in sesquiterpenes and drastic increase in oxygenated terpenes were observed in comparison to the raw samples. Such changes affected the antioxidant activity of the treated samples using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging as well as β-carotene bleaching test against tert-Butylhydroquinone (TBHQ). The strongest effect for reduction of DPPH radical was by white pepper microwave heated sample which exhibit 78.2 % ± 2.1), followed by conventionally roasted black pepper sample, (75.2 % ± 1.9) compared to TBHQ (98.8 % ± 2.1) at the same concentration 400 μg/ml. However, conventionally roasted black pepper recorded the highest inhibiting effect for the oxidation of linoleic acid and the subsequent bleaching of βcarotene (78.2 % ± 2.3), followed by white pepper microwave heated sample (76.2 % ± 1.9) in comparison to TBHQ (98.8 % ± 2.1). Key words: White and black pepper, Microwave, Volatile oils, Antioxidant activity. Introduction: Recently, the use of microwaves for the sanitization of food acquired an ever increasing importance. In particular, this treatment is very efficient for the sanitization of herbs and spices 1,2. A new technique developed by Cannamela **SpA for treating spices e.g. pepper, origanum, sage, basil and chili, depends on continuous supply of spices and herbs into pasteurization chamber, where microwave oscillators allow the sanitizastion with drastic reduction of the microbial contamination and maximum control of all relevant physical parameters. The treatment conditions are between 30 and 80 W/kg of spices for about 15 min depending on the kind of material. The effectiveness of the microwave treatment for spices depends on the high penetration power **Cannamela SpA-Via S. Lazzari, 6-40064 Ozzano dell'Emilia (Bologna, Italy) [E-mail: [email protected]://www.cannamela.it]. *Corresponding author (Magda A. Abd El Mageed) E-mail: < [email protected] >


Magda A. Abd El Mageed et al. / Jeobp 14 (2) 2011 214 - 223

215

of radiations in these products, that have low water content, usually 80-150 g/kg; which causes a moderate and uniform heating, while the higher humidity content of polluting agents leads to a higher heating, which is lethal 1,2 . Due to the extension of global demand on naturally occurring functional foods, researches have focused on herbs and spices, not only for their sensory properties, but also for their antioxidant activity. Pepper (Piper nigrum L.) is one of the world’s most important spices, used for both its aroma and pungency. The main constituent responsible for its aroma is the steam volatile oil, which should normally yield between 1 and 3 %. Black, green and white peppers are the main three different forms available in the market, although most of the pepper oil in commerce is distilled from black pepper 3. For more than a century many studies have been devoted to the chemical composition of pepper oil and these were reviewed by several authors 4,5,6. The monoterpene hydrocarbons account for as much as 70-80 % with smaller amounts of sesquiterpene hydrocarbons (20-30 %), which appear to possess the main desirable attributes of pepper flavour. Although the oxygenated terpenes are relatively minor constituents, comprising less than 4 %, they contribute to the characteristic odour of pepper oil 3 . Pepper essential oil plays an important role in the manufacture of perfumery and confectionery products. It has been already reported that spice volatile oils and aromatic plant extracts possess strong antioxidant activity 7,8,9,5,10. Generally, some spices are processed for microbial stability and removal of extraneous matter. Roasting is one of the important phases in the cooking process to release characteristic flavour volatiles and undesirable constituents 11,12. Hence, roasting of spices affects flavour quality, this study aimed to reveal the effect of microwave heating and conventional roasting on volatile components of white and black pepper compared with raw samples in addition to evaluate the antioxidant activity as well as the total phenolic content of their volatile oils. Materials and methods: Dry, clean, white and black pepper (Piper nigrum L.) were purchased from the local market (Cairo, Egypt). Authentic and standard n-paraffins (C8-C22) were purchased from Sigma-Aldrish Co, s (St. Louis, MN, USA), and Merck (Darmstadt, Germany). All other chemicals were of analytical grade. Processing of raw material: Two samples (100 gm each) of white and black pepper were separately subjected to microwave heating for 2 minutes (Daewoo DE Microwave, Mod: KoG-181G, 200-240V 50Hz. Microwave input power was 1400 W, Korea). Another 100 gm of each tested samples were separately roasted in a conventional electric oven at 150°C for 15 min. Isolation of volatile compounds: The raw and roasted samples of white and black pepper were separately subjected to hydrodistillation for 4 hours using Clevenger type apparatus. The obtained oil was dried over anhydrous sodium sulfate. Gas chromatographic analysis: GC analysis was performed by using Hewlett-Packard model 5890 equipped with a flame ionization detector (FID). A fused silica capillary column DB-5 (60m x 0.32 mm. id,) was used. The oven temperature was maintained initially at 50°C for 5 min., then programmed from 50 to 250°C at a rate of 4°C/min. Helium was used as the carrier gas, at flow rate of 1.1 ml/min. The injector and detector temperatures were 220 and 250°C, respectively. The retention indices (Kovats index) of the separated volatile components were calculated using hydrocarbons (C8C22, Aldrich Co.) as references. Gas chromatographic - Mass spectrometric analysis: The analysis was carried out by using a coupled gas chromatography Hewlett-Packard model (5890) / mass spectrometery Hewlett-PackardMS (5970). The ionization voltage was 70 eV, mass range m/z 39-400 amu. The GC condition was


216

carried out as mentioned above. The isolated peaks were identified by matching with data from the library of mass spectra (National Institute of Standard and Technology) and compared with those of authentic compounds and published data 13. The quantitative determination was carried out based on peak area integration. Antioxidant activity assay: Antioxidant activity of hydrodistilled essential oils of white and black pepper were determined by using: 1. Scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical: The potential antioxidant activity of oils were assessed according to Hatano, et al.,14, in comparison to synthetic antioxidant used in food industry, tert-butylhydroquinone (TBHQ). The absorbance was measured at 517 nm using spectrophotometer (Shimadzu UV-1601PC, Japan); all tests were run in three replicates and averaged. 2. β-Carotene bleaching assay: Antioxidant activity of the aqueous solution was determined by a β-carotene / linoleic acid system as described by Taga, et. al.,15, in comparison to TBHQ. The absorbance was measured at 470 nm, every 15 min up to 60 min. 3. Total phenolic content assay: Total phenolic compounds in aqueous extract from tested samples were determined by Folin-Ciocalteu reagent using gallic acid as the standard 16. The absorbance of the developed blue colour was determined at 700 nm. The amount of light absorbed is proportional to the amount of oxidisable material present and the results were expressed as milligrams of gallic acid equivalents (GAE) per gram extract. Results and discussion: The essential oils contents of white and black pepper recovered after 4 hrs of hydrodistillation (HD) decreased to (2.6 % ± 1.3, 2.8 % ±2 .4) for conventionally roasted and 2.8 % ± 1.4, 2.9 % ± 1.3 for microwave heated compared to (3 % ± 2.3, 3. 2% ±1.7) for raw samples, respectively. The total area percentages of the main chemical classes of the HD volatile oil of raw and roasted samples of white and black pepper are shown in (Fig.1). Twenty six compounds were identified in HD oil of white pepper while twenty compounds only were identified in HD oil of black pepper. All these compounds are listed with their area percentages in (Table 1,2). Identification of the volatile components were identified by Kovats index values and MS spectra 13. The volatile profile of raw HD oil of white pepper consisted mainly of β-caryophyllene (21.17 %) followed by δ-carene (20.23 %), limonene (17.64 %), β-pinene (14.02 %), α-pinene (7.16 %), myrcene (4.34 %), δ-elemene (3.15 %) and β-farnesene (2.16%) (Table 1). These results are in quite agreement with Plessi et al 4. Roasting caused a decrease in the total yield of monoterpenes, increase in sesquiterpenes and drastic increase in oxygenated terpenes recorded 49.49 %, 30.88 % and 16.15 %, respectively in comparison to their contents in the raw one 66.50 %, 29.27 % and 2.83 %. Same phenomenon appeared in microwaved samples, the above classes recorded 16.79 %, 53.71 % and 25.08 %, respectively in comparison to the raw (Fig. 1 ). The considerable increasing in sesquiterpenes contents of microwave treated sample was due to the increase in δ-elemene 5.53 %, α-copaene 1.31 %, β-cubebene 1.75 %, β-caryophyllene 37.66 %, β-farnesene 4.7 %, α-humulene 1.19 %, β-bisabolene 0.81 % and δ-cadinene 0.76 % (Table 1). These results are in accordance with Chacko et al 17 and Emam et al.1. In our study β-caryophyllene increased during microwave heating to almost two times (37.66 %) in comparison to its concentration in raw sample (21.17 %) (Table 1), whereas Chacko et al.,17 found that it is increased via microwave heating almost to three times on control value but decreased on further heating. Due to the presence of sesquiterpenes in a higher content, which are responsible for desirable pepper flavour attributes, microwave heated samples showed higher quality than conventionally roasted samples. The drastic


217

increase in oxygenated terpenes against decreasing in monoterpenes seems to be very interesting. Monoterpenes easily oxidized under heating conditions to generate oxygenated derivatives as well as alter the flavour profile. For example, limonene, the major terpene in most essential oils from citrus fruits, is easily oxidized into a mixture of isomeric hydroperoxides, which are converted into limonene oxides and the respective hydroxylic derivatives; carvone and carveol belong to other sensorically important products. Odour potencies were different in different limonene oxidation products, and other components of lemon oil 18. The volatile profile of raw HD oil of black pepper consisted mainly of δ-carene (27.85 %) followed by limonene (24.07 %), β-caryophyllene (15.96 %), sabinene (14.04 %), α-pinene (9.12 %) and β-pinene (2.11 %) (Table 2). These results are in accordance with those reported by Pino et al.3 Plessi et al.4 ; Singh et al.5 and Saad, et al.19. The effect of heat techniques on black pepper is similar to that on white pepper (Fig. 1). Monoterpenes decreased to 65.13 and 66.30 % in roasted and micrwaved samples in comparison to 79.07 % in the raw one, whereas, sesquiterpenes increased to 32.06 and 28.75 % for roasted and microwaved samples in comparison to 20.32 % in the raw one.

Figure 1. The total area percentages of the main chemical classes of the volatile oils of raw, conventionally roasted and microwave heated white and black pepper Concerning the total yield of oxygenated terpenes, roasted and microwaved samples comprised remarkable increase 2.80 %, 4.94 % compared to 0.58 % in raw black pepper sample. These compounds strongly contribute to the fragrance; therefore the microwave heated samples should be expected to give better reproduction of natural aroma than those of oven roasted samples. Additionally, oxygenated terpenes exhibited a higher antioxidant power in comparison to the other identified classes 20. The profile of scavenging activity on DPPH radical as well as evaluation of antioxidant activity using β-carotene/ linoleate assay are shown in (Fig. 2,3) for (raw, conventionally roasted and microwave heated) of both white and black pepper essential oils. The radical scavenging activity of both oils on DPPH increased with increasing concentration of both oils. The strongest effect for reduction of DPPH radical was by white pepper microwave heated sample which exhibit (78.2 % ± 2.1), followed by conventionally roasted black pepper sample, (75.2 % ± 1.9) compared to TBHQ (98.8 % ± 2.1) at the same concentration 400 μg/ml (Fig. 2). As shown in (Fig. 3), conventionally roasted black pepper


218

recorded the highest inhibiting effect for the oxidation of linoleic acid and the subsequent bleaching of β-carotene (78.2 % ± 2.3), followed by white pepper microwave heated sample (76.2 % ± 1.9) in comparison to TBHQ (98.8 % ± 2.1). Such small differences in antioxidant activity are revealed in (Fig. 4) which showed the total phenolic content for both pepper types treated by different heating techniques. GC-MS studies showed that black pepper contains α-pinene, β-pinene, camphene and camphor in considerable percentages, and they may contribute to the antioxidant activity of black pepper essential oil. Many studies deal with antioxidant activity of various herbs and spices but, to our knowledge there is little literature 21-24 concerning that of pepper. In addition, antioxidative activities observed in volatile oil and its extract could be the synergistic effect of many compounds that may be present in the system to produce a broad spectrum of antioxidative activities that create an effective defense system against free radical attack 25 .

100

90

80

70

Inhibition %

60

50

40

30

20

10

B.P (raw) B.P (oven) B.P TBHQ 0 W.P (raw) W.P (oven) W.P microwave) (microwave) Figure 2. Scavenging effect of white and black pepper volatile oils on DPPH radicals with different concentrations compared to TBHQ ± SD of the mean


219

100

90

80

70

Inhibition %

60

50

40

30

20

10

0 W.P (raw) W.P (oven) W.P B.P (raw) (microwave)

B.P (oven)

B.P TBHQ (microwave)

Figure 3. β-Carotene-linoleate assay of whithe and black pepper volatile oils with different concentrations compared to TBHQ ± SD of the mean Conclusion: Heating results in changes in relative composition of major components like limonene, pinenes, sabinene, caryophyllene etc., which may be responsible for the change in flavour profile. Both types of peppers showed a decrease in monoterpenes accompanied with either increase in sesquiterpenes or a drastic increase in oxygenated terpenes. Essential oils of white and black peppers as raw, roasted or microwaved samples exhibited antioxidative activity against DPPH radical or by using β-carotene / linoleate assay, which is proved by the phenolic content identified in all samples, however, still lower in activity than the synthetic ones.


220

250

mg gallic acid equi (G A E) / gm extract

200

1500

100

50

0 W.P (raw)

W.P (oven)

W.P (microwave)

B.P (raw)

B.P (oven)

B.P (microwave)

Figure 4. Total phenolic compounds of raw, conventionally roasted and microwave heated of white and black pepper

1. 2.

3. 4.

References Emam, O.A., Farage, S.A. and Aziz, N.H. (1995). Comparative effects of gamma and microwave irradiation on the quality of black pepper. Z. Lebensm. Unters. Forsch., 21: 557-561. Legnani, P.P., Leoni, E., Righi, F. and Zarabini, L.A., (2001). Effect of microwave heating and gamma irradiation on microbiological quality of spices and herbs. Italian J. of Food Science, 13: 337-345. Pino, J., Rodriguez-Feo, G., Borges, P. and Rosado, A. (1990). Chemical and sensory properties of black pepper oil. Nahrung 34: 555-560. Plessi, M., Bentelli, D. and Miglietta, F. (2002). Effect of microwaves on volatile compounds in white and black pepper. Lebensm. Wissn. Technol. 35: 260-264.

Magda A. Abd El Mageed et al. / Jeobp 14 (2) 2011 214 - 223 5.

6. 7. 8.

9.

10. 11. 12.

13. 14.

15. 16.

17. 18. 19.

20.

21. 22. 23. 24.

221

Singh, G., Marinuthu, P., Catalan, C. and de Lampasona MP. (2004). Chemical antioxidant and antifungal activities of volatile oil of black pepper and its acetone extract. J. Sci. Food Agric. 84: 1878-1884. Nisha, P., Singhal, R.S. and Pandit, A.B. (2009). The degradation kinetics of flavour in black pepper (Piper nigrum L) J. of Food Engineering 92: 44-49. Burits, M. and Bucar, F. (2000). Antioxidant activity of Nigella sativa essential oil. Phytother Res., 14: 323-328. Dessi, M.A., Dieana, M., Rosa, A., Pipredda, M., Cottigilia, F., Bon-signore, L., Deiddn, D., Pompei, R. and Corongiu, F. (2001). Antioxidant activity of extracts from plants growing in Sardinia. Phytother Res., 15: 511-518. Bandoniene, D., Venskutonis, P.R., Gruzdiene and Murkovic, M. (2002). Antioxidant activity of Sage (Salvia offcinalis L. ), Savory (Satureja hortensis L.) and Borage (Borago officinalis L.) extracts in rapeseed oil. Eur. J. Lipid Sci .Technol. 104: 286-292. Suhaj, M., Racova, J., Polovka, M. and Brezova, V. (2006). Effect of γ-irradiation on antioxidant activity of black pepper (Piper nigrum L.) Food chemistry 97: 696-704. Susheela, R.U. (2000). Handbook of Spices, seasonings and flavourings (pp.47-48). Lancaster, USA: Technomic Publishing. Suresh, D., Manjunatha, H. and Srinivasan, K. (2007). Effect of heat processing of spices on the concentrations of their bioactive principles: Turmeric (Curcuma longa), red pepper (Capsicum annuum) and black pepper (Pipper nigrum) J. Food Composition and Analysis 20: 346-351. Adams. R.P. (2001). Identification of essential oil components by gas chromatography/ quadrupole mass spectroscopy. Carol Stream IL, USA: Allured. Hatano, T., Kagawa, H., Yasuhara, T., Okuda, T. (1988). Two new flavonoids and other constituents in licorice root: their relative astringency and radical scavenging effects. Chem. pharm. Bull. 36: 1090-2097. Taga, M.S., Miller, E.E., Pratt, D.E. (1984). Chia Seeds as source of Natural lipid Antioxidants. J. Am. Oil Chem. Soc. 61: 928-931. Kahkonen, M.P., Hopia, A.I., Vuorela, H.J., Rauha, J.P., Pihlaja, K., Kujala, T.S. and Heinonen, M. (1999). Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem, 47: 3954-3962. Chacko, S., Jayalekshmy, A., Gopalakrishnan, M. and Narayanan, C.S. (1996). Roasting studies on black pepper (Piper nigrum L.) Flavour and Fragrance J. 11: 305-310. Schieberle, P. and Grosch, W. (1989). Potent odourants resulting from the peroxidation of lemon oil. Z. Lebensm. Unters. Forsch. 189: 26-31. Saad, R., Abd El Mageed, M.A., Fadel, H.M., Yasin, N.M.A. and Hassan, I.M. (2007). Preparation and flavour evaluation of high quality freeze dried seasoning blend. Arab-Univ. J. Agric. Sci 15: 71-87. Radonic, A. and Milos, M. (2003). Chemical composition and in vitro evaluation of antioxidant effect of free volatile compounds from Satureja montana L. Free Radical Research 37: 673679. Lee K.G. and Shibamoto T. (2002). Determination of antioxidant potential of volatile extracts isolated from various herbs and spices. J. Agric Food Chem., 50: 4947-4952. Takeoka G.R. and Dao L.T. (2003). Antioxdant constituents of almond [Pruns dulcis (Mill.) D.A. Webb] hulls. J. Agric Food Chem., 51: 496-501. Duenas, M., Hernandez, T., Estrella, I. and Rabanal, R. (2003). Phenolic composition and antioxidant activity of mocan seeds (Visnea mocanera L.) Food Chem., 82: 373-379. Lu, F. and Foo, L.Y. (2001). Antioxidant activity of polyphenols from sage (Salvia officinalis). Food Chem. 75: 197-202.

Magda A. Abd El Mageed et al. / Jeobp 14 (2) 2011 214 - 223 25.

222

Lu, F. and Foo, L.Y. (1995). Phenolic antioxidant component of evening primrose, In A.S.H. Ong, E. Niki, & L. Packer (Eds.), Nutrition, Lipids, Health, and Disease (pp. 86-95). Champaign: American Oil Chemists’ Society. Table 1. Volatile components isolated in the hydrodistilled oil of raw, conventionally roasted and microwave heated white pepper *

KIa

929 939 953 977 982 992 1009 1013 1033 1064 1088 1097 1180 1197 1247 1289 1292 1344 1376 1388 1437 1446 1454 1507 1530 1581

Components α-Thujene α-Pinene Camphene Sabinene β-Pinene Myrecene α-Phellandrene δ-Carene Limonene γ-Terpinene Terpinolene Linalool Terpinen-4-ol α-Terpineol Carvon Unknown Unknown δ-Elemene α-Copaene β-Cubebene β-Caryophyllene β-Farnesene α-Humulene β-Bisabolene δ-Cadinene Caryophyllene Oxide

Raw white Conventional roasting Microwave Methods of pepper 150°C for 15 min heating identification b 0.19 7.16 0.33 14.02 4.34 0.64 20.23 17.64 0.38 1.57 1.15 0.40 0.30 0.21 3.15 0.76 0.87 21.17 2.16 0.48 0.34 0.34 0.77

0.36 3.74 0.11 1.82 7.71 1.68 16.27 16.05 1.04 0.71 2.15 1.22 1.51 10.76 0.85 2.12 3.18 0.70 0.82 22.96 2.05 0.55 0.32 0.30 0.51

0.03 0.48 0.29 2.04 0.43 0.52 6.08 6.29 0.35 0.28 2.42 0.78 1.50 17.23 1.54 2.88 5.53 1.31 1.75 37.66 4.70 1.19 0.81 0.76 3.15

MS, KI ST, MS, MS, KI ST, MS, MS, KI MS, KI MS, KI MS, KI ST, MS, MS, KI ST, MS, MS, KI MS, KI MS, KI ST, MS, MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI MS, KI

KI KI

KI KI

KI

*Values expressed as relative area percentage to total identified components Compounds listed according to their elution on DB5 column. -: not detected a : Kovat index b : compounds identified by GC-Ms (MS) and / or Kovat index on DB5 (KI) and / or by comparison of MS and KI of standard compounds run under similar GCMS conditions.


223

Table 2. Volatile components isolated in the hydrodistilled oil of raw, conventionally roasted and microwave heated black pepper * KIa

929 939 953 977 982 992 1009 1013 1033 1064 1097 1344 1388 1402 1437 1446 1454 1507 1530 1581

Components α-Thujene α-Pinene Camphene Sabinene β-Pinene Myrecene α-Phellandrene δ-Carene Limonene γ-Terpinene Linalool δ-Elemene β-Cubebene β-Elemene β-Caryophyllene β-Farnesene α-Humulene β-Bisabolene δ-Cadinene Caryophyllene Oxide

Raw white Conventional roasting Microwave Methods of pepper 150°C for 15 min heating identification b 0.19 9.12 0.52 14.04 2.11 0.26 0.51 27.85 24.07 0.42 0.30 0.65 0.32 0.89 15.96 0.89 0.81 0.52 0.31 0.28

0.22 8.23 0.51 12.19 2.09 0.27 3.50 21.98 15.43 0.71 1.24 2.97 0.67 2.95 13.71 3.85 3.58 3.00 1.33 1.56

0.16 7.36 1.26 12.93 2.40 0.22 0.60 22.32 18.94 0.11 1.74 1.09 0.51 1.69 19.29 1.62 2.20 1.47 0.88 3.20

MS, KI ST, MS, MS, KI ST, MS, MS, KI MS, KI MS, KI MS, KI ST, MS, MS, KI ST, MS, MS, KI MS, KI MS, KI ST, MS, MS, KI MS, KI MS, KI MS, KI MS, KI

KI KI

KI KI

KI

*Values expressed as relative area percentage to total identified components Compounds listed according to their elution on DB5 column. -: not detected a : Kovat index b : compounds identified by GC-Ms (MS) and / or Kovat index on DB5 (KI) and / or by comparison of MS and KI of standard compounds run under similar GCMS conditions.

Jeobp 14 (2) 2011 pp 224 - 228

224


Volatile Constituents of Chromolaena odorata (L.) R.M. King & H. Rob. Leaves from Benin Cosme Kossouoh 1*, Mansour Moudachirou 1, Victor Adjakidje 2, Jean-Claude Chalchat 3, Gilles Figuérédo 3, Pierre Chalard 4 1

Laboratoire de Pharmacognosie et des Huiles Essentielles, 01 BP 188 I.S.B.A. Champ de Foire, Faculté des Sciences et Techniques, Université d’Abomey-Calavi, B.P. 526 Cotonou, Bénin 2 Département de Biologie Végétale, Laboratoire de Botanique Systématique et d’Ecologie Végétale, Faculté des Sciences et Techniques, Université d’Abomey-Calavi, B.P. 526 Cotonou, Bénin 3 Chimie des Huiles Essentielles, Université Blaise Pascal de Clermont, Campus des Cézeaux, 63177 Aubière Cédex, France 4 Clermont Université, ENSCCF, EA 987, LCHG, BP 10448, F-63000 Clermont-Ferrand, France Received 06 February 2010; accepted in revised form 14 November 2010

Abstract: Essential oils from fresh leaves of Chromolaena odorata (L.) were extracted by steam distillation. The oil from leaves (I) was obtained with a very low percentage. The extraction yield for the leaves (II) was 0.07 %. Analysis made by GC and GC-MS showed a total of 64 compounds identified. The main components of the oils were pregeijerene (29.9 %), germacrene D (21.6 %), β-caryophyllene (14.3 %), geijerene (10.1 %), α-pinene (8.0 %), sabinene (5.4 %). Key words: Chromolaena odorata (L.), Asteraceae, volatile constituents, essential oil composition, pregeijerene, germacrene D, β-caryophyllene, geijerene, α-pinene. Introduction: Chromolaena odorata (L.) King and Robinson (ex Eupatorium odoratum L.) (Asteraceae) is an annual, biennial and sometimes perennial ubiquitary aromatic herb (or small shrub) common in the Lesser Antilles. It is found in Central and Southern America from Florida to Argentina. It was introduced into the Old World tropics and then accidentally in Africa, where it has become a nuisance weed, invading and crowding out crops. It is found throughout the forest areas of West Africa. In Benin the juice of the fresh leaves is used as a body lotion to treat hyperthermia, muscle stiffness and mycosis. A decoction of the leaves is also used orally to help prevent spontaneous abortion 1 . The plant has exhibited allelopathic effects and has been reported to cause livestock death 2. Medicinally, the plant decoction is taken as remedy for coughs and colds or in baths to treat skin diseases 3.The chemical composition and insecticidal, insect repellent, antimicrobial fungicidal and acaricide activities of Chromolaena odorata essential oil have been studied by Iwu and Chiori 4, Bamba et al. 5, Chowdhury 6, Ling et al.7, Inya-Agha et al.8, Cui et al.9, Pisutthanan et al.10 , Owolabi *Corresponding author (Cosme Kossouoh) E-mail: < [email protected] >


Cosme Kossouoh et al. / Jeobp 14 (2) 2011 224 - 228

225

et al.11. and Tedonkeng E. Pamo et al.12. To our knowledge there are no literature reports to date concerning the volatile components of the leaves of Chromolaena odorata of Benin. Our main aim here was thus to study the chemical composition of essential oils extracted from fresh leaves of Chromolaena odorata (L.) of Benin, the variation of this chemical composition and extraction yields according to the season when the leaves were harvested. Experimental Plant material: Leaves of Chromolaena odorata (L.) were collected in the morning at the Abomey-Calavi University farm. The fresh leaves of Extract I were harvested in January 2007, a period of very hot weather, and those of Extract II in August 2007, a very cool period with occasional light rain. Essential oil isolation: Five hundred grams (500 g) of the same fresh leaves was steam distilled for 3 h in a Clevenger-type apparatus. The amount of oil obtained after the steam distillation of the leaves I was tiny (less than 0.001 mL) and had to be trapped in cyclohexane. The extraction yield for leaves oil II was 0.07 %. A voucher specimen of these leaves is conserved at the University of AdomeyCalavi Herbarium. Gas chromatography: The gas phase chromatography analysis was carried out using a Delsi DI 200 instrument equipped with a flame ionization detector and a DB5 column (25 m x 0.25 mm, df: 0.25 μm) with a split flow rate of 60 mL/min, nitrogen as carrier gas and temperature programming 5 min at 50°C and 3°C/min up to 220°C, injector temperature was 220°C and detector temperature was 235°C. Gas chromatography - Mass spectrometry: The oil was analyzed using a Hewlett-Packard gas chromatograph Model 6890 coupled to a Hewlett-Packard MS Model 5873 equipped with an HP5 column (30 m x 0.25 mm, df: 0.25 μm). The oven temperature was programmed from 50°C (5 min) to 300°C at 5°C/min, and 5 min hold. The carrier gas was helium (1.0 mL/min), injection was set in the split mode (1/10). Injector and detector temperatures were 250°C and 320°C, respectively. Ionization was by electron impact at 70 eV, electron multiplier was 2200 V, ion source temperature was 230°C. Mass spectral data were acquired in the scan mode in the m/z range of 33-450. Identification was carried out by calculating retention indices and comparing mass spectra with those in data banks; personal, Adams13, Mc Lafferty and Stauffer14. Results and discussion: The oils extracted from samples I and II were obtained in very small quantities with widely different yields (traces and 0.07 %, respectively). A total of 64 compounds were identified (Table I). Sesquiterpenes and hydroxyl derivatives predominated. Extract I (32 compounds) obtained from leaves harvested in the hot season was characterised by the presence of α-pinene (8.0 %), geijerene (10.1 %), pregeijerene (16.3 %), β-caryophyllene (14.3 %) and germacrene D (13.3 %) together with sabinene (5.4 %), β-cubebene (2.1 %), α-humulene (2.8 %), and δ-cadinene (4.0 %). Extract II obtained in the cold season (57 constituents) was characterised by a high concentration of pregeijerene (29.9 %), β-caryophyllene (7.3 %), germacrene D (21.6 %), along with α-pinene (4.1 %), β-pinene (3.0 %), (E)-β-ocimene (2.1 %), geijerene (3.5 %), α-humulene (1.9 %), bicyclogermacrene (2.8 %), δ-cadinene (3.3 %), viridiflorol (1.9 %) and α-cadinol (1.6 %). The concentrations of all the other constituents were less than 1 %. Each extract was thus characterised by known but different main compounds; for I, geijerene, pregeijerene, β-caryophyllene


226

and germacrene D, and for II (with levels twice higher), pregeijerene and germacrene D. If we compare our results with those of the literature we see that our oils contained very little bicyclogermacrene relative to plant material from Cameroon 12, unreported in Chinese 7, Ivorian 5, Nigeria 11 and Thailand 10 oils. The levels of geijerene were similar between some oils while pregeijerene was not found in others. Pregeijerene was thus characteristic of Extract II. The Chinese extract, the oils of Shillong (N.E. India) 6 and those studied by Inya-Agha et al.,8 did not apparently contain any geijerene-type derivatives, whereas the levels of geijerene from Ivorian Coast 5 and Thailand 10 were close to those reported in Extract II. In Extract II, the levels of β-caryophyllene was quite similar to those reported from Thailand oil and those studied by Inya-Agha et al.,8. The level of β-pinene in Extract I was quite similar from Thailand 10. Extract II differed sharply by the marked presence of two major components compared with the other oils studied. Extract I presented similar levels, between 10 % and 16 %, of geijerene, pregeijerene, β-caryophyllene and germacrene D. Also, β-caryophyllene, observed in the extracts from Benin, has been only reported in the extract from Nigeria and Thailand but in lower amount. References Adjanohoun, E.J., Adjakidje, V. (1989). Médecine traditionnelle et pharmacopée. Contribution aux études ethnobotaniques et floristiques en République Populaire du Bénin, p 121: ACCT, Paris. 2. Zachariades, C., Day, M., Muniappan, R. and Reddy, G.V.P. (2009). Chromolaena odorata (L.) King and Robinson (Asteraceae). In: Biological Control of Tropical Weeds Using Arthropods, eds: R. Muniappan, G.V.P. Reddy and A. Raman. Cambridge University Press, UK, pp 130160. 3. Morton, J.F. (1981). Atlas of Medicinal Plants of Middle America, Vol.II. Charles C. Thomas, Publisher, Springfield, Illinois, USA, pp 932-933. 4. Iwu, M.M. and Chiori, C.O. (1984). Antimicrobial activity of Eupatorium odoratum extracts. Fitoterapia, 55(6): 354-356. 5. Bamba, D., Bessière, J.M., Marion, C., Pélissier, Y. and Fouraste, I. (1993). Essential oil of Eupatorium odoratum L., Planta Medica, 59(2): 184-185. 6. Chowdhury, A.R. (2002). Essential oil of the leaves of Eupatorium odoratum L. From Shillong (N.E.), J. Essent. Oil Bearing Plants, 5(1): 14-18. 7. Ling, B., Zhang, M., Kong, C., Pang, X. and Linag, G. (2003). Chemical composition of volatile oil from Chromolaena odorata and its effect on plant, fungi and insect growth. Ying Yong Sheng Tai Xue Bao, 14(5): 744-746. 8. Inya-Agha, S.I., Oguntimein, B.O., Sofowora, A. and Benjamin, T.V. (1987). Phytochemical and antibacterial studies on the essential oil of Eupatorium odoratum. Int. J. Crude Drug Res. 25: 49-52. 9. Cui, S., Tan, S., Ouyang, G., Jiang, S. and Pawliszyn, J. (2009). Headspace solid-phase microextraction gas chromatography-mass spectrometry analysis of Eupatorium odoratum extract as an oviposition repellent. J. Chromatogr. B 877: 1901-1906. 10. Pisutthanan, N., Liawruangrath, B., Baramee, A., Apisariyakul, A., Korth, J. and Bremner, J.B. (2006). Constituents of the essential oil from aerial parts of Chromolaena odorata from Thailand. Nat. Prod. Res. 20: 636-640. 11. Owolabi, M.S., Ogundajo, A., Yusuf, K.O., Lajide, L., Villanueva, H.E., Tuten, J.A. and Setzer, W.N. (2010). Chemical Composition and Bioactivity of the Essential Oil of Chromolaena odorata from Nigeria. Rec. Nat. Prod. 4,: 72-78. 12. Tedonkeng E. Pamo, Zollo Amvam P.H, Tedonkeng, F., Kana J.R., Fongang, M. D. et Tapondjou L.A. (2004). Chemical composition and acaricide effect of the essential oils from 1.


13. 14.

227

the leaves of Chromolaena odorata (L.) king and Robins. And Eucalyptus saligna Smith., on ticks ( Rhipicephalus lunulatus Neumann) of West African Dwarf goat in west Cameroon. Livestock Research for Rural Development 16 (9)2004 Adams, R.P. (1995). Identification of Essential Oil Components by gas Chromatography / Mass Spectroscopy, Allured Publishing Corporation, Carol Stream, Il. McLafferty, F.W. and Stauffer, D.B. (1989). The Wiley NBS Registry of Mass Spectral Data, 2ème Edition, J. Wiley and Son, NY. Table I. Volatile compounds identified in the leaves essential oil of Chromolaena odorata (L.) from Benin Compounds α-Thujene α-Pinene Camphene Sabinene β-Pinene Myrcene α-Phellandrene α-Terpinene p-Cymene Limonene + β-Phellandrene 1,8-Cineole (Z)-β-Ocimene (E)-β-Ocimene γ-Terpinene Terpinolene Linalool 2-Methyl-6-methylen-octa-1,7-diene Perillene Geijerene isomer Geijerene Terpinen-4-ol Methyl salicylate α-Terpineol Isogeijerene C Isogeijerene C isomer Pregeijerene isomer Pregeijerene δ-Elemene α-Cubebene α-Ylangene α-Copaene β-Bourbonene β-Cubebene β-Elemene + b-Cubebene β-Elemene

KI

(I) (%)

II (%)

931 939 953 976 980 991 1005 1018 1026 1031 1033 1040 1050 1060 1088 1096 1104 1113 1128 1144 1177 1192 1195 1250 1261 1276 1287 1339 1351 1375 1379 1388 1388 1391 1391

8.0 5.4 1.1 tr tr tr 0.9 0.3 1.7 0.1 0.1 0.1 0.1 0.9 10.1 0.6 0.3 0.2 16.3 2.1 0.1

tr 4.1 tr 0.7 3.0 0.9 tr tr tr 0.5 tr 0.3 2.1 tr tr 0.1 0.2 0.2 3.5 0.1 tr 0.1 0.1 0.1 0.1 29.9 0.1 tr tr 1.3 0.1 0.4 -


228

table 1. (continued). Compounds β-Caryophyllene Pyrethrone β-Copaene Cadina-3,5-diene α-Humulene Cadina-1(6),4-diene γ-Muurolene Germacrene D Bicyclosesquiphellandrene Bicyclogermacrene Germacrene A γ-Cadinene δ-Cadinene trans-Calamenene Elemol Germacrene B Nerolidol Spathulenol Caryophyllene oxide Viridiflorol epi-Globulol epi-α-Eudesmol 1-epi-Cubenol γ-Eudesmol epi-α-Cadinol α-Muurolol α-Eudesmol α-Cadinol Phytol tr = traces (inferior or equal to 0.05 %)

KI

(I) (%)

II (%)

1419 1422 1430 1448 1454 1472 1477 1485 1487 1494 1503 1510 1523 1532 1549 1556 1564 1578 1583 1593 1599 1624 1629 1632 1640 1646 1654 1663 2104

14.3 2.8 13.3 4.0 0.5 0.2 1.5 0.2 0.6 0.3 0.7 -

7.3 0.4 0.1 0.2 1.9 0.3 0.3 21.6 0.6 2.8 0.3 0.2 3.3 0.2 1.0 0.2 0.3 1.9 0.1 0.4 0.4 0.9 0.2 1.6 1.3

Jeobp 14 (2) 2011 pp 229 - 240

229


Bioconversion of Essential Oil from Plants with Eugenol Bases to Vanillin by Serratia marcescens A. Khanafari *1, M. Seyed Jafari Olia 1 F. Sharifnia 2 1

Microbiology Department, Tehran North Branch, Islamic Azad University, Tehran, Iran 2 Biology Department,Tehran North Branch, Islamic Azad University Tehran, Iran Received 06 May 2010; accepted in revised form 14 February 2011

Abstract: Essential oils extraction from native or collective plants with eugenol basses such as Eugenia caryophyllata and Ocimum basilicum and bio-transformation to vanillin by Serratia marcescens (ATCC 13880) was investigated. The oils were obtained by steam distillation and were analyzed by UV and GC-MS. Essential oils with eugenol bases was added to resting phase of bacterium growth curve with final concentration of 20 gL-1 and incubated at 27°C and 150 rpm, for 24 hours to bioconversion to vanillin. Crystalline structure of vanillin was isolated and confirmed by GC-MS. Forthy-three and one hundred and fifteen compounds were identified in essential oil of Eugenia caryopyllata by using first and second methods and eugenol was determined as the major component with 88.205 % and 54.628 % respectively. Eugenol in essential oil of Ocimum basilicum was rare. The highest vanillin concentration, 0.6 gL-1, was obtained with 46 % purity from flower buds of Eugenia caryopyllata. Key words: Eugenia caryophyllata, Ocimum basilicum, eugenol, vanillin, bioconversion, Serratia marcescens. Introduction: Natural vanilla is the second most valuable flavoring in the food industry, various medical industries, perfumes and pharmaceuticals ($ 4,000/Kg of natural vanillin) 1,2. It is derived from the fruits of the tropical Orchid Vanilla planifolia. The mature green vanilla beans have no characteristic aroma and flavor develops during the post harvest processing of the beans (curing). Curing processes differ from country to country, consist of several steps, and are still rather traditional 3. Indonesia is the second largest producer of natural cured vanilla in the world after Madagascar. Vanillin (3- methoxy-4-hydroxybenzaldehyde) is the most important organoleptic component in vanilla and glucosides, such as glucovanillin are major aroma precursors in green vanilla beans 3. More than 12,000 tons of synthetic vanillin is produced each year from petrochemical and wood pulping industries 4. Isolated vanillin appears as white needle-like crystalline powder with an intensely sweet and very tenacious creamy vanilla-like odor 2. Strong market demand for natural and environmentally friendly products has spawned efforts to produce vanillin by microbial transformation from natural substrates, including phenolic stibenes 5, eugenol 6, 7 and ferulic acid 1, 8. Direct extraction from vanilla beans is expensive and limited by plant supply, which makes this compound a promising *Corresponding author (A. Khanafari) E-mail: < [email protected] >


A. Khanafari et al. / Jeobp 14 (2) 2011 229 - 240

230

target for biotechnological flavor production 9, 10, 11, 12, 13. Eugenol (C10H12O2) is an allyl chain-substituted guaiacol and is a member of the phenylpropanoids class of chemical compounds. It is a clear to pale yellow oily liquid extracted from certain essential oils especially from Eugenia caryophyllata Thunb., Geum urbanum L. and Ocimum basilicum L. that belong to Myrtaceae, Rosaceae and Lamiaceae families respectively 14. Eugenol is the main constituent of the essential oil of the clove tree Eugenia caryophyllata (Eugenia caryopyllata) and with a current market price of about US$ 5 Kg–1, it is a cheap, commercially available raw material for biotransformation processes 2. The conversion of natural eugenol and isoeugenol from essential oils into vanillin has been investigated using microbial and enzymatic biotransformation 9, 10, 11, 12, 13. Microorganisms have historically played an integral role in the elaboration of the flavor components of many different foods 15. Degradation cellulose and hemicellulose by microorganisms yields aromatic compounds 16, 17. In addition, microbial activities on cell wall compounds release ferulic acid that can be transformed via a large variety of bacteria and fungi into flavor compounds, such as vanillin and guaiacol 17. Microorganisms include Pseudomonas putida, Aspergillus niger, Corynebacterium glutamicum, Corynebacterium sp., Arthrobacter globiformis, Serratia marcescens, Klebsiella, Enterobacter, Bacillus and Arthrobacter species could convert eugenol or isoeugenol to vanillin 2, 18, 19, 20, 21, 22, 23, 24, 25, 26. Natural eugenol and isoeugenol from essential oil are more resourceful and economical for the production of vanillin by bioconversion specially using microbial and enzymatic conversion of isoeugenol to vanillin have been propounded 6, 7, 10, 12. The objective of this study was to extract essential oil from native or collective plants with eugenol basses such as Eugenia caryophyllata (clove tree) and Ocimum basillicum (basil) and biotransformation to vanillin by Serratia marcescens to achieve native flavor. Experimental Plants and bacteria samples and culture conditions: Flower buds of Eugenia caryopyllata (not endemic in Iran) were collected in May 2009 from cultivated specimen in Boushehr city (south of Iran) before the flowering stage in May 2009 and leaves of Ocimum basilicum (endemic in Iran) were collected from Shahriyar cultivated specimen, around of Tehran, in July 2009 after the flowering stage. Plants were identified and authenticated by a plant taxonomist. Voucher specimens were kept at the Herbarium of the Department of Plant Biology in Islamic Azad University, North of Tehran Branch, with numbers EC1 and OB1. Serratia marcescens (ATCC 13880) was originally obtained from Persian Type Culture Collection (PTCC), Tehran-Iran. Cultures were grown in shaking incubators at 27°C and 120 rpm for 24-48 h in capped 250-ml Erlenmeyer flasks containing 50 ml nutrient broth [100 gL-1 NaCl (DSMZ medium 372)]. The cell density in the culture was monitored at 600 nm with a photometric colorimeter 27. Culture was plated on nutrient agar medium (Merk) and classic physiological characteristics were tested according to Bergey’s Manual of Determinative Bacteriology 28. Essential oil extraction from Eugenia caryophyllata First Method: Essential oil was extracted from flower buds of Eugenia caryophyllata by Modified Jeffers method and Houghton Mifflin company method (1998) 29. 14-16 g flower buds powder was allowed to soak in the water for about 15 min and steam distilled. The milky distillate was collected and standing in ice water. Then dichloromethane was added (1:6). The organic layer was separated and extracted twice more by dichloromethane. Potassium hydroxide solution 5 % was added, gently was kept turning for 4-5 minutes. The aqueous solution was washed with a fresh 15 ml portion of dichloromethane and slowly was acidified to pH 1 using hydrochloric acid 5 % and extracted by fresh dichloromethane. The dichloromethane layer was saved and washed with half-saturated solution chloride


231

solution. In the final step, dichloromethane solution was evaporated using a hot water bath (approximately 40 -50°C) and a gentle steam of compressed air to give the product eugenol 30, 31. Second method: The distillation pot was charged with 1.1g of powder of flower buds of Eugenia caryophyllata. The distillate was extracted with dichloromethane (1:6) and evaporated using a hot water bath (approximately 40-50°C) and a gentle steam of compressed air 30, 31. Essential oil extraction from Ocimum basilicum: Dried leaves of basil (about 16 g) were cut into small pieces and subjected to hydro-distillation 32. The distillate was extracted with dichloromethane (1:6). Remaining aqueous solution was extracted twice more with dichloromethane 29. Dichloromethane solution was evaporated using a hot water bath (approximately 40-50°C) and gentle steam of compressed air 30, 31. Mass spectrometry: Oily components extracted from Eugenia caryophyllata flower buds and Ocimum basilicum leaves were identified by analytical gas chromatography (GC) was performed using GC-MS-Agilent USA, GC 68 goN Network GC system, MS 5973 Network to confirm eugenol. The sample was diluted with dichloromethane and 1μM was injected into the column. A fused silica Capillary column Hp5-MS (30 m × 250 mm × 0.25 μm) was used. Helium was the carrier gas, and a split 30:1 was used. The oven temperature was kept at 50°C for 1 min and programmed at 50°C per 5 min over the range 50-150°C at 8°C/min and 150-280°C at 8°C/min. The temperature of inlet was 275°C and the temperature of Aux was 285°C. The components were identified by comparing linear NIST library indices, their retention index (RI) and mass spectra with those obtained from the authentic samples and/or the MS library 32. Serratia marcescens growth curve: Serratia marcescens (ATCC 13880) is grown in nutrient broth (Merk) at 27°C for 24 hours. Equal volume of the culture was combined in the inoculums. Dilutions were made in TSB to obtain appropriate cell concentrations. The turbidity of the cultures was measured at 600 nm by UV-VIS scanning spectrophotometer (UV 2101 pc, SHIMADZU) every 2 hours. The data were collected in a Microsoft Excel spreadsheet 18. Preparation of resting cells and bioconversion of essential oil to vanillin: Bio-transformation of essential oil to vanillin was modified by Cintins, Marianne 1989. Serratia marcescens (ATCC 13880) was first incubated in extraction medium (10 g glycerol, 3.125 g Na2HPO4, 2.5 g KH2PO4, 2.5 g NH4Cl, 0.025 g FeSO4. 7H2O per 100 ml of water) at 27°C and 150 rpm on a rotating shaker. When the strain grew to stationary phase, essential oil was added directly to a final concentration of 20 gL-1. The bioconversions were conducted at 150 rpm, 27°C during the stationary phase. Time-course samples were taken and analyzed by UV and gas chromatography (GC-MS) after centrifugation at 10,000×g for 10 min and extracted by ethyl acetate solvent 18. Estimated residual of essential oil: In order to identify the residual of essential oil which unused by Serratia marcescens to biotransformation to vanillin, cells were harvested by centrifugation at 10,000×g for 10 min. The oily residue on the top of supernatant was taken and weigh and analyzed by GC-MS. Results and discussion: Bioconversion of essential oil was extracted from plants with eugenol basses such as Eugenia caryopyllata (clove tree) and Ocimum basilicum (basil) to vanillin was investigated. More than 80 % of the vanillin is produced by chemical synthesis employing a variety of precursors, such as lignin, guaiacol, eugenol or hydroxybenzaldehyde. Vanillin production by


232

biotechnological means has been widely studied and several substrates have been tested, such as ferulic acid, eugenol, isoeugenol, vanillic acid, lignin, phenolic stilbenes and even glucose 33. Microbial catalysis has many advantages such as relatively mild reaction conditions; high substrate or product specificity leading to only one product isomer and fewer environmental problems over chemical synthesis 33, 34, 35, 36 . In many biotransformation processes of eugenol only trace amounts of vanillin were accumulated (Table 1) 33. The concentrations of essential oils obtained by two methods in this study are listed in Table 2. The highest amount was 1 gL-1 by using first method that chose for bioconversion. According to Table 2, flower buds of Eugenia caryophyllata were better source for extracting of eugenol. However, concentrations of the desired compounds in plants are usually low but they are considered as environmentally friendly products 33. For example Eim et al. extracted 0.077 g of eugenol from 1.032 g of flower buds of clove tree by steam distillation 31. In the most researches, substrates which used to biotransformation to vanillin such as eugenol and isoeugenol had not botanical source. For example, isoeugenol (98%, cis- /trans-mixture) were applied by Shimoni et al.12, Zhao et al.22 and Hua et al.18 from Sigma Co., Ltd. Aldrich, Milwaukee, WI and Sigma-Aldrich, respectively. Forthy-three compounds were identified in essential oil of Eugenia caryopyllata by used first method. Phenol, 2-methoxy-4-(2-propenyl) eugenol (88.2%) was determined as the major component in 14.032 minutes (Table 3). Chemical analysis of Eugenia caryopyllata also carried out by Ayoola et al.45. Three peaks were identified from the GC-MS data. These major peaks were identified as eugenol, eugenol acetate and caryophyllene from the GC-MS database. The retention times (RT) for eugenol (12.5 min), caryophyllene (13.3 min) and eugenol acetate (14.7 min) was determined. In the other research, chemical compositions of the essential oil of Ocimum basilicum obtained by Ozcan 32. Fourty-nine constituents were identified in O. basilicum, representing 88.1 % of the oil methyl eugenol (78.0 %), α-cubebene (6.2 %), nerol (0.8 %), ε-muurolene (0.7 %), 3,7-dimethyloct1,5-dien-3,7-diol (0.3 %) and β-cubebene (0.3 %) were found as the major compounds. Isoeugenolepoxide and isoeugenol-diol were both identified in the biotransformation process by Bacillus pumilus strain S-1 and a metabolic pathway from isoeugenol to vanillin was proposed by Hua et al.18. In second method, one hundred and fifteen compounds were identified in essential oil of Eugenia caryopyllata. Pheno l, 2-methoxy-4-(2-propenyl)-eugenol (54.6%), was determined as the major component in 14.1 minutes. Forthy compounds were identified in essential oil of Ocimum basilicum. Benzene, 1,2 dimethoxy4-(2-propenyl) (0.54 %), caryophyllene(0.5 %), 5,6,7,8-tetrahydroquinoxaline (0.4 %) and azulene,1,2,3,3a,4,5,6,7-octahydro-1,4-dimethyl-7-(1-methylethenyl) (0.38) were obtained as main constituents. Eugenol was identified in 15.735 minutes as a minor compound (0.03%) (Table 4). According to these results the highest amount of eugenol was showed in essential oils of Eugenia caryopyllata by using first method. For bioconversion eugenol to vanillin, Serratia marcescens (ATCC 13880) was applied. Bacterium growth curve, biomass concentration and essential oil utilization were showed in Fig 1. According to growth curve, resting cells phase was determined at 96-120 h. The highest biomass concentration was 290 mgL-1 at 120 h in stationary phase. During 24 hours incubation after adding essential oil, the bioconversion ratio was calculated 60 %. However, vanillin was accumulated as the major product in the biotransformation of essential oil by Serratia marcescens. Essential oil residual which unused by Serratia marcescens to biotransformation to vanillin was estimated 35 %. Eugenol compounds were not observed in residual which analyses by GC-Mass. According to Hua et al.18, since isoeugenol is toxic to bacterial cells, directly adding to immature culture is unadvisable. Many microorganisms could conversion isoeugenol to vanillin isomers in resting cell phase. In the other hand, Isoeugenol is a starting material for both the synthetic and biotechnological


233

Fig 1. Growth curve, biomass concentration and essential oil utilization by Serratia marcescens (ATCC 13880) production of vanillin and vanillic acid. For example, Nocardia iowensis DSM 45197 (formerly Nocardia species NRRL 5646) resting cells catalyze the conversion of isoeugenol to vanillic acid, vanillin, vanillyl alcohol and guaiacol. Also Ramya Seshadri et al.46 used a variety of chemical, microbial and enzymatic approaches to probe the pathways used by N. iowensis in the oxidation of isoeugenol to these products. There were many reports about vanillin production by isoeugenol and eugenol biotransformation which summarized in Table 1. The highest vanillin concentration, 0.6 gL-1, was obtained at 150 h with 46 % purity from flower buds of Eugenia caryopyllata (Table 5). Identification of vanillin in samples was confirmed by GC-MS. The advantage of this research is that essential oil with eugenol bases also can use for bioconversion to vanillin. However, in most biotransformation processes the yield and the concentration of the products are typically too low but with consumers’ preferences for ‘natural’, ‘green’ and ‘health’ products in the market, flavour production by microbial biotransformation has been a research focus in recent years 47.

1. 2.

References Muheim, A. and Lerch, K. (1999). Towards a high-yield bioconversion of ferulic acid to vanillin. Appl. Microbiol. Biotechnol. 51: 456-461. Priefert, H., Rabenhorst, J. and Steinbuchel, A. (2001). Biotechnological production of vanillin. Appl. Microbiol Biotechnol. 56: 296-314.

A. Khanafari et al. / Jeobp 14 (2) 2011 229 - 240 3.

4. 5. 6. 7. 8. 9. 10.

11.

12. 13. 14. 15. 16. 17.

18.

19. 20.

21.

22. 23.

234

Ranadive, A.S. (1994). Vanilla-cultivation, curing, chemistry, technology and commercial products. In G. Charalambous (ed.), Developments in food science, Elsevier Science Publishers BV, Amsterdam, The Netherlands. 34: 517-577. Hagedorn, S. and Kaphammer, B. (1994). Microbial biocatalysis in the generation of flavor and fragrance chemicals. Annu. Rev. Microbiol. 48: 773-800. Yoshimoto, T., Samejima, M., Hanyu, N. and Koma, T. ( 1990). Dioxygenase for styrene cleavage manufactured by Pseudomonas. Japanese patent. 2: 195-871. Rabenhorst, J. (1996). Production of methoxyphenol type natural aroma chemicals by biotransformation of eugenol with a new Pseudomonas sp. Appl Microbiol Biotechnol. 46: 470-474. Washisu, Y., Tetsushi, A., Hashimoto, N. and Kanisawa, T. (1993). Manufacture of vanillin and related compounds with Pseudomonas. Japanese Patent. 5: 227-980. Labuda, I.M., Goers, S.K. and Keon, K.A. (1992). Bioconversion process for the production of vanillin. U.S. patent. 5: 128-253. Washisu, S., Aida, T. and Hashimoto, N. (1993). Production of vanillin and its related compound by fermentation. Patent application JP5227980. Overhage, J., Priefert, H., Rabenhorst, J. and Steinbüchel, A. (1999c). Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene. Appl. Microbiol Biotechnol. 52: 820-828. Rao, S.R. and Ravishankar, G.A. (1999). Biotransformation of isoeugenol to vanilla flavour metabolites and capsaicin in suspended and immobilized cell cultures of Capsicum frutescens: Study of the influence of β-cyclodextrin and fungal elicitor. Process Biochem. 35: 341-348. Shimoni, E., Ravid, U. and Shoham, Y. (2000). Isolation of a Bacillus sp. capable of transforming isoeugenol to vanillin. J. Biotechnol. 78: 1-9. Shimoni, E., Baasov, T., Ravid, U. and Shoham. Y. (2003). Biotransformations of propenylbenzenes by an Arthrobacter sp. and its t-anethole blocked mutants. J. Biotechnol. 105: 61-70. Zargari, A. (1993). Plant medicine, vol: 4, Tehran University Publishment. Longo, M.A. and Sanroman, M.A. (2006). Production of Food Aroma Compounds, Food Technol. Biotechnol. 44(3): 335-353. Ghosh, P. and Singh, A. (1993). Physicochemical and biological treatments for enzymatic/ microbial conversion of lignocellulosic biomass. Adv Appl. Microbiol. 33: 295-333. Rosazza, J.P.N., Huang, Z., Dostal, L., Volm, T. and Rousseau, B. (1995). Review: Biocatalytic transformations of ferulic acid: an abundant aromatic natural product. J Ind. Microbiol. 15: 457-471. Hua, D., Cuiqing, M., Shan, L., Lifu, S., Zixin, D., Zarao, M., Zhaobin, Z., Bo, Y. and Ping, X. (2007). Biotransformation of isoeugenol to vanillin in a newly isolated Bacillus pumilus strain: identification of major metabolites. J. Biotechnol. 130: 463-470. Yamada, M., Okada, Y., Yoshida, T. and Nagasawa, T. (2007). Biotransformation of isoeugenol to vanillin by Pseudomonas putida IE27 cells. Appl. Microbiol. Biotechnol. 73: 1025-1030. Zhang, Y., Xu, P., Han, S., Yan, H. and Ma, C. (2006). Metabolism of isoeugenol via isoeugenoldiol by a newly isolated strain of Bacillus subtilis HS8. Appl. Microbiol. Biotechnol. 73: 771779. Ryu, J.Y., Seo, J.Y., Lee, Y.S., Lim, Y.H., Ahn, J.H. and Hur, H.G. (2005). Identification of syn- and anti-anethole-2, 3-epoxides in the metabolism of trans-anethole by the newly isolated bacterium Pseudomonas putida JYR-1. J. Agric. Food Chem. 53: 5954-5958. Zhao, L.Q., Sun, Z.H., Zheng, P. and Zhu, L.L. (2005). Biotransformation of isoeugenol to vanillin by a novel strain of Bacillus fusiformis. Biotechnol. Lett. 27: 1505-1509. Zhao, L.Q., Zhi, H., Sun, P.U., Zheng, J.Y.H. (2006). Biotransformation of isoeugenol to vanillin by Bacillus fusiformis CGMCC1347 with the addition of resin HD-8. Process Biochem.

A. Khanafari et al. / Jeobp 14 (2) 2011 229 - 240 24. 25.

26.

27. 28.

29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

39. 40.

41.

42. 43.

235

41: 1673-1676. Jin, J., Mazon, H., van den Heuvel, R.H.H., Heck, A.J. and Fraaije, M.W. (2007). Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1. FEBS J. 274: 2311-2321. Kasana, R.C., Sharma, U.K., Sharma, N. and Sinha, A.K. (2007). Isolation and identification of a novel strain of Pseudomonas chlororaphis capable of transforming isoeugenol to vanillin (dagger). Curr. Microbiol. 54: 457-461. Plaggenborg, R., Overhage, J., Loos, A., Archer, J.A., Lessard, P., Sinskey, A.J., Steinbuchel, A. and Priefert, H. (2006). Potential of Rhodococcus strains for biotechnological vanillin production from ferulic acid and eugenol. Appl. Microbiol. Biotechnol. 72: 745-755. Baron, E.J. and Finegold, S.M. (1990). Diagnostic microbiology. 8th ed, USA, Bailey & Scott,s. Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T. and Williams, S.T. (1994). Bergey’s Manual of Determinative Bacteriology, 9 eddition. Williams & Wilkins Press, Baltimore, Maryland, 559-564. Extraction of Eugenol from Cloves. Eugenol is used in perfumery, for the manufacture of synthetic vanillin, as: C O. H vanillin. (vanilla beans). Copyright © Houghton Mifflin Company. Pavia, D., Lampman, G., Kriz, G. and Engel, R. (1999). Introduction to Organic Laboratory Techniques, Saunders College Publishing, Eim, A. ( 2005). Chemist Isolation of Eugenol from Cloves by Steam Distillation and its Identification by Infrared Spectroscopy. CHEM 303. Ozcan, M. and Chalchat, J.C. (2002). Essential oil composition of Ocimum basilicum L. and Ocimum minimum L. in Turkey. Czech J. Food Sci. 20: 223-228. Xu, P., Qiu, J.H., Zhang, Y.N. (2007). Efficient whole-cell biocatalytic synthesis of NacetylD-neuraminic acid. Adv. Synth. Catal. 349: 1614-1618. Vandamme, E.J. and Soetaert, W. (2002). Bioflavours and fragrances via fermentation and biocatalysis. J. Chem. Technol. Biotechnol. 77: 1323-1332. Schmid, A., Dordick, J.S., Hauer, B., Kiener, A., Wubbolts, M. and Witholt, B. (2001). Industrial biocatalysis today and tomorrow. Nature 409: 258-268. Mathew, S. and Abraham, T.E. (2006). Bioconversions of ferulic acid, anhydroxycinnamic acid. Crit. Rev. Microbiol. 32: 115-125. Rabenhorst, J. and Hopp, R. (1991). Process for the preparation of vanillin. US Patent 5, 017, 388. Abraham, W.R., Arfmann, H.A., Stumpf, B., Washausen, P. and Kieslich, K. (1988). Microbial transformations of some terpenoids and natural compounds. In: Schreier, P. (Ed.), Bioflavour’ 87, Analysis, Biochemistry, Biotechnology, Proc. Int. Conf. Walter de Gruyter, Berlin. 399-414. Chatterjee, T., De, B.K. and Bhattacharyya, D.K. (1999). Microbial conversion of isoeugenol to vanillin by Rhodococcus rhodochrous. Indian J. Chem. B. 38: 538-541. Furukawa, H., Morita, H., Yoshida, T. and Nagasawa, T. (2003). Conversion of isoeugenol into vanillic acid by Pseudomonas putida I58 cells exhibiting high isoeugenol-degrading activity. J. Biosci. Bioeng. 96: 401-403. Overhage, J., Steinbüchel, A. and Priefert, H. (2003). Highly efficient biotransformation of eugenol to ferulic acid and further conversion to vanillin in recombinant strains of Escherichia coli. Appl. Environ. Microbiol. 69: 6569-6576. Li, Y.H., Sun, Z.H., Zhao, L.Q. and Xu, Y. (2005). Bioconversion of isoeugenol into vanillin by crude enzyme extracted from soybean. Appl. Biochem. Biotechnol. 125: 1-10. Overhage, J., Steinbüchel, A. and Priefert, H. (2006). Harnessing eugenol as a substrate for production of aromatic compounds with recombinant strains of Amycolatopsis sp. HR167. J.

A. Khanafari et al. / Jeobp 14 (2) 2011 229 - 240 44.

45.

46. 47.

236

Biotechnol. 125: 369-376. Zheng, L., Zheng, P., Sun, Z., Bai, Y., Wang, J. and Guo, X. (2006). Production of vanillin from waste residue of rice bran oil by Aspergillus niger and Pycnoporus cinnabarinus Bioresource Technology. 98: 1115-1119. Ayoola, G.A., Lawore, F.M., Adelowotan, T., Aibinu, I.E., Adenipekun, E., Coker, H.A.B. and Odugbemi, T.O. (2008). Chemical analysis and antimicrobial activity of the essential oil of Syzigium aromaticum (clove). African Journal of Microbiology Research. 2:162-166. Ramya, S., Andrew, S.L., Arshdeep, K. and Rosazza J.P.N. (2008). Oxidation of isoeugenol by Nocardia iowensis. Enzyme and Microbial Technology, 43: 486-494. XU, P., Hua, D. and Cuiqing, Ma. (2007). Microbial transformation of propenylbenzenes for natural flavour production. TRENDS in Biotechnology, 25(12): 571-576.

32.5 1.36 1.36 tr tr 8.1 2.8 3.75 1.2 1 3.8 16.1

Isoeugenol Isoeugenol Isoeugenol Isoeugenol

3.80 0.08 1 0.61 0.9 tr 0.3 2.46

40.5 12.9 40.5 71

14.7 17.5 61.9

5.8

20.5 10 58 12.4 14.0 89.3 13.3

Vanillin amount Yeild (%) (g L-1)

Isoeugenol Isoeugenol Isoeugenol growing culture cell free extract Isoeugenol Eugenol Isoeugenol + Crude enzyme extracted from soybean Isoeugenol isoeugenol Isoeugenol Eugenol Eugenol Isoeugenol Ferulic acid

Substrate using for biotransformation process

Bacillus fusiformis SW B.subtilis strain HS8, Bacillus subtilis HS8 Rhodococcus opacus PD630 Amycolatopsis sp. HR167 Bacillus fusiformis CGMCC1347 Combination of Aspergillus niger CGMCC0774 and Pycnoporus cinnabarinus CGMCC1115 B. pumilus strain S-1 Pseudomonas chlororaphis Bacillus pumilus S Pseudomonas putida IE27

Serratia macescens DSM 30126, Aspergillus niger ATCC9142 Rhocodoccus rhodochrous MTCC 289 Bacillus subtilis strain B2, Bacillus subtilis strain B2, Pseudomonas putida I58 Pseudomonas sp. HR199 -

Microorganisms

Refernces

Hua et al. 2007 18 Kasana, R.C. et al. 2007 25 Hua, D. et al. 2007 18 Yamada, M. et al. 2007 19

Zhao, L.Q. et al. 2005 22 Zhang et al., 2006 20 Zhang, Y. et al. 2006 20 Plaggenborg, R. et al. 2006 26 Overhage, J. et al. 2006 43 Zhao, L.Q. et al. 2006 23 Zheng et al. 2006 44

Rabenhorst and Hopp, 199137 Abraham et al. 1988 38 Chatterjee et al. 1999 39 Shimoni et al. 2000 12 Shimoni et al., 2000 12 Furukawa, H. et al. 2003 40 Overhage, J. et al. 2003 41 Li, Yong-Hong, 2005 42

Table 1. Previous reports summarized of biotransformation substrate to vanillin

A. Khanafari et al. / Jeobp 14 (2) 2011 229 - 240 237


238

Table 2. The concentration of Eugenia caryopyllata (clove tree) and Ocimum basilicum (basil) essential oils Samples

method

Samples amount (g)

Oil extracted (gL-1)

Eugenia caryopyllata (flower buds) Eugenia caryopyllata (flower buds) Ocimum basilicum * (Leaves)

first

16

1

second

1.1

0.009

-

16

0.035

*Used method that mentioned in materials and methodsm Table 3. All component were identified in essential oil of Eugenia caryopyllata by GC-MS spectrum analyses (first method) Compound

(%)

RI*

Benzene,methyl-(CAS)Toluene Chavicol Eugenol Tetradecane, 2,6,10-trimethyl Hexadecane Heptadecane,3-methyl-(CAS) Octadecane n-hexadecanoic acid 1-Eicosene.alpha.-Eicosene cetyl ethylene Eicosane(CAS)n-Eicosane Eicosane 7,9-di-tert-butyl-1-0xaspiro[4.5]deca-6,9-diene 1-Docosene Octadecanoic acid Docosane Tetracosane Pentacosane Tetracosane,3-ethyl-(CAS)-3-Ethyltetracosane 1-Hexacosene Heptacosane Eicosane,9-octyl-9-n-Octyleicosane Octacosane (2,3-Diphenylcyclopropyl)Methyl phenyl sulfoxide Nonacosane Triacontane Hexacosane, 9-octyl-9-n-Octylhexacosane Tetratriacontane [N-Benzyl-O-[4-Nitrobenzyl]]Aspartylglycine ethyl

0.04 0.51 88.2 0.06 0.46 0.05 0.63 1.01 0.14 0.1 0.75 0.04 0.2 0.55 0.68 0.54 0.17 0.09 0.22 0.13 0.17 0.2 0.18 0.48 0.1 0.08 0.08 0.1

794 1203 1392 1519 1612 1746 1810 1968 1999 2009 2009 2081 2129 2167 2208 2407 2506 2542 2596 2705 2740 2804 2835 2904 3003 3337 3401 3626

* Retention index


239

Table 4. All components were identified in essential oil of Ocimum basilicum by GC/MS spectrum analyses Compound Heptane,3-methyl Furfural Ethylbenzene O-Xylene 5-Hepten-2-one,6-methyl 1-Octen-3-ol Benzaldehyde Benzyl Alcohol Benzene,1-methyl-4-(1-methylethyl) 1,6-Octadien-3-ol,3,7-dimethylBicyclo[3.1.1]hept-3-en-2-one,4,6,6-trimethyl Phenylethyl Alcohol Bicyclo[2.2.1]heptan-2-ol,1,3,3-trimethyl 5,6,7,8-Tetrahydroquinoxaline Cyclohexanol,5-methyl-2-(1-methylethyl Isopropyl phenyl ketone Benzene,1-methoxy-4-(1-propenyl) Acetophenone,4-methoxy Copaene 2,6-Octadien-1-ol,3,7-dimethyl Thymol Phenol,2-methl-5-(1-methylethyl) Benzenemethanol,4-(1-methylethyl) Tridecane α-Cubebene 1-Undecanol Benzene,1,2-dimethoxy-4-(2-propenyl) Pyridine,3-phenyl Eugenol Phenol,2-methoxy-4-(1-propenyl) 2-Buten-1-one,1-(2,6,6-trimethyl-1,3-cyclohexadien-1-yl) Azulene,1,2,3,3a,4,5,6,7-octahydro-1,4-dimethyl-7-(1-methylethyl) Naphthalene,1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl) Naphthalene,1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl) Azulene,1,2,3,4,5,6,7,8-octahydro-1,4-dimethyl-7-(1-methylethenyl) Caryophyllene 1-Tridecanol Caryophyllene oxide Phenol,2-methoxy-4-(2-propenyl)-acetate α-Bisabolol * Retention index

(%)

RI*

0.02 0.001 0.01 0.02 0.1 0.03 0.04 0.04 0.003 0.06 0.06 0.15 0.04 0.4 0.08 0.02 0.03 0.03 0.03 0.11 0.05 0.01 0.01 0.24 0.03 0.01 0.54 0.001 0.03 0.03 0.01 0.38 0.04 0.04 0.23 0.5 0.005 0.26 0.2 0.05

752 831 893 907 938 969 982 1036 1042 1082 1119 1136 1138 1154 1164 1164 1190 1218 1221 1228 1262 1262 1284 1313 1344 1357 1361 1361 1392 1410 1440 1461 1469 1474 1490 1494 1556 1507 1552 1625


240

Table 5. Vanillin concentration and its purity with two eugenol natural sourses Substrate Flower buds of Eugenia caryopyllata Leaves of ocimum basilicum

Amount (gL-1) Purity (%) Formula

Molar mass(g/mol)

0. 6

46

C8H8O3

152

0.04

9.1

C8H8O3

152

Jeobp 14 (2) 2011 pp 241 - 244

241


Essential Oil Composition and Antibacterial Activity of Nepeta glomerulosa Boiss from Iran Azizollah Nezhadali *1, Mahboobeh Masrornia 2, Hossein Bari 1,2, Mina Akbarpour 1 , Mohammad Reza Joharchi 3 and Mahboobeh Nakhaei -Moghadam 2 1

2

Department of Chemistry, Payame Noor University (PNU), Mashhad, Iran, Faculty of Science, Department of Chemistry, Islamic Azazd University-Mashhad Branch, Iran 3 Ferdowsi University of Mashhad, Mashhad, Iran Received 01 March 2010; accepted in revised form 26 November 2010

Abstract: The chemical compositions of the essential oil of Nepeta glomerulosa Boiss aerial parts, grown in Iran were determined by GC-MS. Fifty-two compounds (97.2 %) were determined. The major compounds were geranyl acetate ( 17.0 %), limonene (12.0 %), eucalyptol (5.8 %), (bornyl acetate (5.3 %), citronellal (4.9 %), spathulanol (4.2 %), sabinene (3.9 %), β-ocimene (3.9), β-sesquiphellandrene (2.8 %), neryl acetate ( 2.5 %), α-humulene (2.4 %), α-pinene (2.3 %), humulene oxide (2.2 %), norsolanadione ( 2.1 %) and terpinen-4-ol (2.0 %). The yield of the oil was 1.1(v/w) %. The essential oil showed antibacterial activity for Staphylococcus aureus. Key words: Nepeta glomerulosa Boiss, essential oil, hydrodistillation, geranyl acetate. Introduction: The genus Nepeta (Lamiaceae) comprises 280 species that are distributed over a large part of central and southern Europe, West, central, and Southern Asia. About half of the existing species are recorded in Iran. The genus Nepeta is represented in Turkey by 33 species and altogether 38 taxa, 17 of these being endemic in Turkey 1. Nepeta species are widely used in folk medicine because of their antispasmodic, diuretic, antiseptic, antitussive, antiasthmatic, ethnobotanical effect, diaphoretic, vulneary, antispasmodic, tonic, febrifuge 2-5. The feline attractant properties of several Nepeta species have been known for a long time. The compounds of essential oil of Nepeta are considered to be responsible for the feline attractant activity of Nepeta species 6-7. As far as our literature survey, there are no reports on the chemical compositions of essential oil and antibacterial activity of the essential oil of N. glomerulsa Boiss. Thus, this study is the first report on this plant. The aims of this work are to identify of the chemical compositions and a brief study of antibacterial activity of essential oil of N. glomerulsa Boiss obtained by using a Clevenger distillation apparatus. The chemical compositions of the essential oil were evaluated by using gas chromatographymass spectrometry (GC-MS). Experimental Plant material: The aerial parts of Nepeta glomerulosa Boiss was collected during August *Corresponding author (Azizollah Nezhadali) E-mail: < [email protected] >


Azizollah Nezhadali et al. / Jeobp 14 (2) 2011 241 - 244

242

2008 from Zhoshke Mountain , Mashhad, Iran. The plant was identified at FUMH Herbarium, Ferdowsi University of Mashhad, Iran, and a Voucher specimen is kept at FUMH Herbarium ( 27628 FUMH ). Isolation procedure: The air dried leaf of specimens (70g) were extracted by hydrodistillation using Clevenger-type apparatus for 4 h. The oil was dried over anhydrous sodium sulfate. The corresponding oils were isolated in yield of 1.1% (v/w) . Identification of oil components: The essential oil was analyzed by gas chromatography mass spectrometry (GC-MS) . The GC-MS analysis was carried out on a Shimadzu GC-MS model QP 5050. The capillary column was DB-5 (30 × 0.2 mm , film thickness 0.32 μm). The initial temperature of column was 60°C (held 1 min) and then heated to 200°C with a 3°C/min rate and then heated to 250°C and kept constant for 2 min. The flow rate of Helium as carrier gas with (1.7 mL/ min). The analysis uses split ratio 1/28. The injector and detector temperatures were both at 280°C ; volume injected 0.1 μl of the essential oil and ionization potential 70 eV. The same condition of temperature programming used for n-alkenes mixture to calculate the retention index (RI). Identification of components in the oil was based on the retention index (RI) , Wiley computer library and literature survey 8 . The relative percentage of the oil constituent was calculated. Antibacterial activity: Antibacterial activity by disc diffusion method and determination of inhibition zones at different oil dilutions were done for Staphylococcus aureus. Results and discussion: The compositions of essential oil and antibacterial activity of aerial parts of Nepeta glomerulosa Boiss are shown in Table 1 and 2, respectively. Fifty-two constituents, representing 97.2% of the total components in the oil , have been identified in the essential oil extracted from the aerial parts of this plant. The essential oil with major compositions of geranyl acetate (17.0 %), limonene (12.0 %) , eucalyptol (5.8 %), bornyl acetate (5.3 %), showed moderate antibacterial activity and inhibited the growth of the tested bacteria. Due to the high amount of geranyl acetate (17.0 %), limonene (12.0 %) , eucalyptol ( 5.8 %), and other terpenoids in the oil of Nepeta glomerulosa Boiss, it can be concluded that the herb and essential oil of Nepeta glomerulosa Boiss can be used as flavoring agents in food and also in the medicinal and perfume industries. These main components have been reported in the literatures for Nepeta genus 9-11. The results indicating that Nepeta glomerulsa Boiss has potential use in phytotherapy. Acknowledgments: The authors thank Payame Noor University (PNU) research Council for financial support

1. 2.

3.

4. 5.

References Davis, P. H. (1982). Flora of Turkey and the East Aegean Islands, Edinburgh University press. Ghannadi, A., Aghazari, F., Mehrabani, M., Mohaghzadeh, A., Mehregan, A. (2003). Quantity and Composition of the SDE prepared essential oil of Nepeta macrosiphon Boiss., Iranian J. Pharm. Sci., 2, 103-105. Gkinis, G., Tzakou, Q., IIiopoulou, D., Roussis, V.Z (2003). Chemical composition and biological activity of Nepeta parnassica oils and isolated Nepetalactons, Naturforsch., 58: 681686. Dorman, H.J., Deans, S.G. (2000). Antibacterial activity of plant volatile oils, J. Appl Microbiol., 88: 308-316. Zenasni, L., Bouidida, H., Hancali, A., Boudhane, A., Amzal, H., Idrissi, A., Aouad, R.,


6. 7.

8. 9. 10. 11.

243

Benjouad, Y. (2008). The essential oils and antimicrobial activity of four nepeta species from Morocco, J. Med Plants Res., 2: 111. Dabiri, M., Sefidkon, F. (2003). Chemical composition of Nepeta crassifolia Boiss. Flavour and Fragrance J., 18: 225-227. Hussain, J., Jamila N., Gilani, S.A., Abbas ,G., Ahmed, S. (2009). Platelet gregation, antiglycation, cytotoxic, phytotoxic and antimicrobial activities of extracts of Nepeta juncea, Afr. J. Biotechnol., 8: 935-940. Adams, R.P. (1995). Identification of essential oil components by Gas chromatography- Mass spectrometry, Allured publishing, Illinois. Rustaiyan, A., Monfared, A., Masoudi, Sh. (1999). Composition of the essential oil of Nepeta astero- trichus Rech. from Iran. J. Essent. Oil Res. 11: 229-230. Sefidkon, F., Dabiri, M., Alamshahi A. (2002). Analysis of the essential oil of Nepeta fissa C. A. Mey. from Iran. Flavour Fragr. J. 17: 89-90. Rustaiyan, A., Nadji, K. (1999). Composition of the essential oils of Nepeta ispahanica Boiss., and Nepeta binaludensis Jamzad from Iran. Flavour Fragr. J., 14: 35-37. Table 1. Chemical composition of Nepeta glomerulosa Boiss essential oil No.

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

α-Thujene α-Pinene Camphene β-Pinene β-Myrcene Octan-3-ol α-Terpinene Limonene Cyclooctyne β-Ocimene Sabinene Sabinene hydrate Terpinolene Linalool 1-Pentylallyl acetate Thujone Camphor Citronellal Borneol Terpinen-4-ol α-Terpineol Chrysanthemal iso-Borneol Nerol D-Pulegone Citronellol Geraniol β-Farnesene

RI

%

927 935 950 979 994 1002 1021 1038 1045 1057 1069 1072 1090 1105 1120 1138 1143 1163 1170 1182 1193 1211 1225 1235 1237 1244 1267 1272

0.2 2.3 1.1 0.5 1.9 0.2 0.3 12.0 0.2 3.9 4.0 0.5 0.1 0.2 0.4 0.3 0.5 4.9 1.4 2.0 0.7 0.1 0.1 0.2 1.4 0.4 1.4 0.2


244

table 1. (continued). No.

Compound

29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

Bornyl acetate Limonene oxide Eucalyptol Citronellyl acetate Neryl acetate Geranyl acetate Caryophylene Bergamotene α-Humulene Hotrienyl acetate Nerolidol β-Bisabolene β-Sesquiphellandrene Norsolanadione Spathulanol iso-Caucalol Caryophyllene oxide Nepetalactol Humulene oxide β-Elemene Lancifold Citral Terpinyl acetate Iso-isopulegol acetate Total

RI

%

1290 1314 1359 1361 1372 1396 1407 1432 1441 1466 1476 1501 1518 1537 1546 1568 1581 1586 1591 1607 1613 1675 1692 1732

5.3 2.0 5.8 0.7 2.5 17.0 0.7 1.3 2.4 0.1 1.4 0.5 2.8 2.1 4.2 1.1 1.2 1.1 2.2 0.2 0.1 0.2 0.1 0.8 97.2

Table 2. Antimicrobial activity of Nepeta glomerulosa Boiss essential oil

Microorganism

Staphylococcus aureus a

Inhibition zone [mm]a The ratio of oil dilutions (with methanol) Standard antibiotics 1 1/2 1/4 1/8 1/16 Ampicillinb Tetracyclinec 19.5*

16

12.5*

9

7

13

includes diameter of the disc (6mm) and the ranges are: (7-13) moderatelyactive; (>14) highly active . The results are average of two experiments b tested at 10 μg/disc c tested at 30 μg/disc a similar inhibitory type of activity of the oil to that of standard antibiotics

20

Jeobp 14 (2) 2011 pp 245 - 249

245


Chemical Composition of Essential Oil from Doum Fruits Hyphaene thebaica (Palmae) Nahla A. Ayoub*, Omayma A. Eldahshan, Abdel-Nasser B. Singab and Mohamed M. Al-Azizi Department of Pharmacognosy, Faculty of Pharmacy, Ain - Shams University, Cairo, Egypt Received 11 August 2009; accepted in revised form 14 November 2010

Abstract: Results of GC and GC-MS analysis of essential oil isolated from doum fruits (Hyphaene thebaica) revealed the presence of fifty-seven compounds. Fifty of them could be identified with monoterpenes represent 15.97 % including compounds such as sabinene (0.82 %), β-pinene (1.98 %), limonene (2.42 %), terpinen 4-ol (1.77 %) %), α-terpineol (0.95 %). While diterpenes represent 40.49 %, of which incensole (17.52 %) and incensole acetate (19.81 %) were found to be the main components. Oxygenated compounds constituted 66.78 % of the total compounds identified which indicated the economical value of this oil. The scent of doum oil fruits may be attributed to the presence of volatile diterpenes as cembrene A, cembrene C, incensole and incensole acetate; these compounds are reported here for the first time in family Palmae. Key words: Doum fruits; Hyphaene thebaica; essential oil composition; cembrene A, cembrene C; incensole; incensole acetate. Introduction: The doum palm, Hyphaene thebaica (L.) Mart. Family Palmae (nom. Alter. Arecaceae) is growing wild throughout the dry regions of tropical Africa, the Middle East and Western India 1, 2. In Egypt, Hyphaene thebaica grows commonly in the Nile Valley, especially in Aswan and Qena Provinces 3. The aqueous extract of the fruits revealed an interesting antihypertensive activity 4 as well as an antifungal activity against a wide range of fungal isolates 5. Also, prominent antibacterial activities of doum fruits were reported against gram positive and gram negative bacteria 6. The aqueous extract of doum fruits showed an antioxidant activity; this is more likely due to the substantial amount of their water-soluble phenolics content 7 while the aqueous ethanolic extract of doum leaves has the potency to scavenge the reactive oxygen species 8. It was reported that the glycan isolated from doum showed hypoglycemic and hypolipidemic effects where glycan could significantly reduce plasma glucose, total cholesterol and triglyceride concentration and at the same time markedly increase lipoprotein cholesterol (HDL-c) levels 9. The effect of prolonged administration of un-saponifiable matter (USM) of doum fruits on liver and kidney functions was also studied 10. USM improved the level of sex hormones except estradiol and ameliorated the liver and kidney functions 10. Although doum fruits were known to Ancient Egypt, considered sacred and the palm pictured on the tombs in different situations, nothing could be traced in literature concerning the chemical composition of doum oil. Therefore, the present study is the first one to deal with the chemical composition volatile components of doum from palm fruits. *Corresponding author (Nahla A. Ayoub) E-mail: < [email protected] >


Nahla A. Ayoub et al. / Jeobp 14 (2) 2011 245 - 249

246

Experimental Plant material: Fruits of the doum were collected from Orman garden in Giza, Egypt. The fruits of doum palm were identified by Prof. Dr. Abdel Salam El-Nowaihi, Professor of taxonomy, Faculty of Science, Ain-Shams University, Egypt. Voucher specimens were deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Cairo, Egypt. Isolation of volatile constituents: Volatile constituents were isolated by hydrodistillation in a modified Karlsurher apparatus 11 for 4 hours. This device maintains the essential oil during the period of distillation in cold condition. Fresh plant materials (500 g) under investigation were loaded into a 2 L round bottom flask and covered with 1.5 L distilled water, using heating mantel at 100°C and npentane was used as a collecting solvent. The use of n-pentane ensures the isolation of water free oil and prevents the loss of highly volatile components. Oil was stored in dark glass bottles at 4°C in refrigerator until analysis was commenced. Gas chromatography: An Orion Micromat 412 double column instrument with 25 m fused silica capillaries column with CPSil 5 and CPSil 19 (Chrompack) was used. Operating conditions: Linear temperature program from 50°C to 230°C /min; injector and detector temperatures 200°C and 250°C respectively; split injection, flam ionization detection, and carrier gas hydrogen at a flow rate of 0.5 bars. GC-MS system: Electron impact (70 eV) GC-MS measurements were carried out on a HewlettPackard HP 5890 gas chromatography equipped with a 25 m polydimethylsiloxane (Chrompack) CP Sil 5 CB) capillary column and coupled to a VG analytical VG 70-250 S mass spectrometer. Helium is used as a carrier gas at a flow rate of 0.5 ml/sec. Linear temperature program from 80°C to 270°C at 10°C/min; injector temperature 220°C, transfere line 230°C, Ion source 220°C. The identification was performed by computer search of the Wiley/NBS Registry of mass spectral data. Mass Finder spectral data, and a user generated library with retention indices mass spectral data of authentic reference substances. Results and discussion: No references could be traced in the available literature concerning the investigation of volatile constituents of the title plant. Therefore, steps were taken to explore the nature of volatile constituents of fresh fruits. The essential oil of Hyphaene thebaica was isolated from 500 g., of freshly collected fruits by hydrodistillation in a modified Karlsruher apparatus 11. The oil was subjected to different physical property measurements including the oil yield, color, odor, specific gravity and refractive index. Hyphaene thebaica yielded a yellowish color volatile oil with a fragrant aromatic odor at a yield of 0.5 % (fresh wt). Physical constants measured include: specific gravity (0.168) and refractive index (1.383). The result of analysis of essential oil of doum by GC and GC-MS techniques revealed the presence of a total fifty-seven compounds. Monoterpenes represent 15.97 % including compounds such as sabinene (0.82 %), β-pinene (1.97 %), limonene (2.42 %), terpinen 4-ol (1.77 %), α-terpineol (0.95 %), sesquiterpenes (3.2 %), diterpenes represent 40.49 %, of which incensole (19.81%) and incensole acetate (17.52 %) were found to be the main components, non-terpenoidal components amount to 15.21 % of which octylacetate (9.38 %) was found to be the major and fatty acid (8.55 %) with the main component palmitic acid (5.90 %). Oxygenated compounds constituted 66.78 % of the total compounds identified which indicated the economical value of this oil. Fruit of doum oil was found to contain volatile diterpenes especially cembrene A which showed cytotoxic activity 12, and this revealed the medical importance of the volatile oil of doum which could be utilized medicinally.


247

CH3 HO O H 3C

CH3

Cembrene A Incensole

CH3 AcO O CH3

H3C

Cembrene C

Incensole acetate

The result of the analysis of the essential oil by GC and GC/MS techniques are presented in table (1). Acknowledgement: The authors are grateful to late Prof. W. König for hosting the GC and GC and GC-MS at his lab. And also for his valuable scientific comments and advise.

1. 2. 3. 4.

5. 6.

7.

8.

9.

References Fanshawe, D.B. (1966). The doum palm-Hyphaene thebaica (Del.) Mart. East Afr. Agric. For. J., 32: 1108-116. Ledin, R.B. (1961). Cultivated Palms. Amer. Hort. Mag., 40 (1): 189. Täckholm, V. (1974). Student’s Flora of Egypt. Cairo University, Cairo, Egypt. Sharaf, A., Sorour, A., Gomaa, N. and Youssef, M. (1972). Comparative studies on hypocholesterolemic effect of different fractions of Hyphaene thebaica (Doum) in experimental animals. Qual. Plant. Mater. Veg. XXII, 1: 83-92. Irobi, O.N. and Adedayo, O. (1999). Antifungal activity of aqueous extract of dormant fruits of Hyphaene thebaica (Palmae). Pharm-Biol, 37 (2): 114-117. El-egami, A.A., Almagboul, A.Z., Omar, M.E.A. and El-Tohami, M.S. (2001). Sudanese plants used in folkloric medicine: screening for antibacterial activity. Part X. Fitoterapia, 72(7): 810-817. Cook, J.A., VanderJagt, D.J., Dasgupta, A., Glew, R.S., Blackwell, W. and Glew, R.H. (1998). Use of the trolox assay to estimate the antioxidant content of seventeen edible wild plants of niger. Life-Science, 63 (2): 106-110. Eldahshan, O. A., Ayoub, N.A., Singab, A.B. and Al-Azizi, M.M. (2008). Potential superoxide anion radical scavenging activity of doum palm (Hyphaene thebaica) leaves extract. Rec. Nat. Prod. 2(3): 83-93. El-Badrawy, E.E.Y. (2006). Hypoglycemic and hypolipidemic effects of glycan isolated from

Nahla A. Ayoub et al. / Jeobp 14 (2) 2011 245 - 249 10.

11. 12.

248

doum, (Hyphaen thebaica L.), VDLUFA-Schriftenreihe, 61: 310-317. El Zalabani, S.M., Hetta, M.H. and Yassin, N.Z. (2007). Bioactive lipoids and phenolics from fruits of Hyphaene thebaica (Doum). Bull. Fac. Pharm. (Cairo University). 45(3): 161172. Sprecher, E. (1963). Rücklaufapparatur zur erschöpfenden Wasserdamfdestillation ätherischen Öls aus voluminösem Destillationsgut, Deutsche Apotheker Zeitung. 103: 213-215. Duh, C.Y., Wang, S.K., Weng, Y.L, Chiang, M.Y., and Dai, C.F. (1999). Cytotoxic terpenoids from the Formosan soft coral Nephthea brassica. J Nat. Prod. 62(11): 1518-1521. Table 1. Results of GC-MS analysis of volatile constituents of Doum fruits

Compound

(%)

RT

Tricycline + α-Thujene + α-Pinene [M]+ = 126 Methyl 2-methylbutyrate Sabinene β-Pinene 2-Pentylfuran cis-Hex-3-enylacetate Phenyl acetaldehyde m-Cymene Limonene (Z)-β-Ocimene

2.98

0.53 0.15 0.82 1.98 0.29 0.15 0.23 0.07 2.42 0.23

927 932 936 943 954 973 978 981 1002 1012 1013 1025 1029

E-2-Octenal [M]+ = 84 1-Octanol n-Nonanal

0.12 0.18 3.36 0.30

1034 1048 1063 1076

Linalool [M]+ = 105 [M]+ = 162 Terpinene-4-ol

0.29 0.33 0.30 1.78

1086 1130 1149 1164

Estragol α-Terpineol Octylacetate Carvone p-Isopropylbenzaldehyde Piperitone Trans-Anethol + 2-allyl-4-Methyl phenol Nonanoic acid Safrol Thymol

0.24 0.95 9.38 1.00 0.59 0.12 0.09

1175 1176 1188 1214 1220 1226 1262

0.11 0.22 0.41

1263 1265 1267

Fragmentation 136[M]+(100),121(20),105(10),93(100), 27 (26) 136[M]+(12),124(16),93(100),79(48), 53 (25) 136[M]+(7),105(14),93(100),77(35),53(13) 126[M]+(10),111(15),79(56) 85[M]+(26),75(100),41(100),53(21),41(20) 136[M]+(10),121(5),93(100),77(35),41(26) 136[M]+(13),121(13),93(100),79(23),69 (26) 138[M]+(11),95(3),81(100),53(21),41(20) 82[M]+(41),67(66),54(8),43(100) 120[M]+(15),91(100),65(23),51(8),51(8),39(18) 136[M]+(3),121(1), 93(5),69(6),41(100) 136[M]+(9),121(11),107(11),93(53),79(3),68(100) 154[M]+(26),136(11),121(6),111(51),93(63),71(100), 43(77) 126[M]+,97(3),83(31),7(36),55(51),41(100) 84[M]+(36), 69(45),56(85),41(100) 112[M]+(1),84(80),70(85),56(100),56(100),1(58) 124[M]+(3),14(5),98(28),82(26),70(36),57(66),41 (100) 136[M]+(5),121(18),93(61),71(100),55(46),41(78) 105[M]+(5),83(40),70(55),55(58),41(100) 162[M]+(30),100(75),73(35),60(50),41(100) 154[M]+(26),136(11),121(6),111(51),93(63),71(100), 43(7) 148[M]+(100),133(21),121(38),105(20),77(25). 136[M]+(26),121(35),93(45),81(31),59(100),43(40) 112[M]+(6), 84(25),70(30),61(250,56(35),43(100) 150[M]+(7),108(28),93(35),82(100),54(55) 148[M]+(63),133(93),105(100), 77(58),51(35) 152[M]+(9),137(15),110(66),82(100),54(16),9(31) 148[M]+(100),133(30),117(28),105(36),91(23), 77(45).+148[M]+(100),133(66),105(41),91(28),77(26) 158[M]+(1),129(21),115(28),73(87),60(100)41(69) 162[M]+(100),131(46),104(75),77(58),51(60) 150 [M]+(25),135(100),115(8),91(9),77(8)


249

table 1. (continued). Compound

(%)

RT

Carvacrol Menthyl acetate Eugenol Dihydroca-rveolacetate + Chavibetol [M]+ = 182 [M]+ = 170 β-Elemene

0.07 0.10 0.46 0.15 1.97 1.92 2.72

1278 1280 1330 1342 1346 1356 1375 1389

(E)-β-Caryophyl-lene Allo-aromadendrene Ar-Curcumene Germacrene-D γ-Humulene γ-Patchoulene (E-E)-α-Farnesene δ-Cadinene n-Dodecanoic acid Caryophyl-lenoxide 4(14)-Salvialene-1-one γ-Eudesmol α-Turmerone Pogostol n-Tetradecanoic acid n-Hexadecanoic acid Cembrene A

0.31 0.17 0.21 0.55 0.39 0.39 0.41 0.23 1.56 0.14 0.08 0.23 0.13 0.24 1.09 5.90 1.22

1421 1462 1473 1479 1483 1497 1498 1520 1545 1578 1592 1618 1643 1647 1746 1951 1962

[M]+ =290

9.08

1958

Cembrene C

1.94

2023

Incensole

17.52

2187

Incensole acetat

19.81

2192

Fragmentation 150[M]+(26),135(100),115(6),107(6),91(16) 138[M]+(40),123(35),95(100),81(56),43(85) 164[M]+(100),149(37),137(28),103(58),77(86),51(63) 136[M]+(43)121(50),107(51),93(70),68(46),43(100) +146[M]+(100),149(45),137(16),103(31),77(36) 182[M]+(25),139(35),125(20),111(43),41(100) 170[M]+(100),141(43),115(13), 77(45),51(45) 204[M]+(30),189(21),161(23),147(31),133(21), 107(51),81(100) 204[M]+(6),161(20),133(55),93(75),69(76),41(100) 204[M]+(23),161(55),133(38), 105(51),41(100) 202[M]+(21),145(23),132(70),119(100),105(50) 204[M]+(8),161(100),147(3),119(26),105(43),91(36) 204[M]+(28),189(20),161(36),133(51),93(85)41(100) 204(55)[M]+,161(75),147(25),121(63),41(100) 204[M]+(5),161(11),107(41),93(100),69(68) 204[M]+(45),189(16),161(100),134(60),105(41), 91(25)

200[M]+(18),129(45),73(100),60(94),55(58) 220[M]+,205(1),121(16),93(46),79(58),41(100) 220[M]+(1),177(11),123(100),107(16),81(63) 222[M]+(1),204(53),189(100), 161(51),133(55) 216[M]+(16),201(16),132(10),119(63),83(100) 204[M]+(26),189(41), 14(40), 107(100),81(93) 228[M]+(30),129(44),73(100),60(81),55(58) 256[M]+(35),227(1),213(9),129(21),73(79),43(100) 272[M]+,229(1),215(1),147(11),133(15),121(40),107 (25),93(61),68(100) 290[M]+,227(1),213(10),129(23),73(86),60(81),43 (100). 272[M]+,229(8),187(4),161(11),136(60),121(100),93 (80) 306[M]+,238(10)156(11),136(8),125(23),107(11),43 (100) 348[M]+,227(0.8),219(1),187(3),150(23),121(21), 107(18),71(70),43(100)

Jeobp 14 (2) 2011 pp 250 - 254

250


Comparison of Essential Oils from Ferula ovina (Boiss.) Aerial Parts in Fresh and Dry Stages Hossein Azarnivand 1, Marzieh Alikhah-Asl 2*, Mohammad Jafari 1, Hossein Arzani 1, Gholamreza Amin 3, Seyed Saeed Mousavi 4 1

Rehabilitation of Arid and Mountainous Regions Department, Faculty of Natural Resources, University of Tehran, Karaj, Iran 2 Natural Resources and Environmental Engineering Department, Faculty of Agricultural Sciences, Payame Noor University, Tehran, Iran 3 Pharmacognosy Department, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. 4 Agronomy and Plant breeding Department, Faculty of Agriculture, University of Buali Sina, Hamedan, Iran Received 25 March 2010; accepted in revised form 21 December 2010

Abstract: The oils obtained by hydrodistillation from fresh and dried aerial parts of Ferula ovina at the flowering stage were analyzed by GC and GC-MS to investigate the variations of oil yields, oil components along with their percentages in fresh and dry stages. The yields of oils were 0.4 % for fresh and 0.25 % for dried aerial parts. Forty-two and twenty-one components were identified in the oil of fresh and dried aerial parts, respectively. Limonene (16.9 %), α-pinene (15.2 %), β-myrcene (7.7 %), cis-β-ocimene (6.1 %), isosylvestrene (5.1 %), β-pinene (4.4 %) were identified in fresh aetial parts. The oil of dried aerial parts was mainly comprised of α-pinene (20.2 %), spathulenol (9.6 %), germacrene D (6.3 %), β-caryophyllene (5.1), α-terpineol (5.0 %) and caryophyllene oxide (4.4 %). There was quantitative difference between the oils of fresh and dried aerial parts. β-myrcene, γ-elemene and dill apiole as major compounds in the fresh stage oil were absent in the oil extracted from dried aerial parts. The oil of dry stage in comparison with fresh stage oil, contained lower quantity of iso-sylvestrene, cis-β-ocimene and trans-β-ocimene. Key words: Ferula ovina, essential oil, fresh and dry stage, GC-MS. Introduction: The genus Ferula belongs to the Umbelliferae family and comprises 53 species that grow wild in Iran 1 and popular Persian name of this genus is “Koma” 2. Ferula ovina is distributed in different regions of Iran 3. Previous studies demonstrated anti-spasmodic, anti-cholinergic and smooth muscle relaxant properties of Ferula ovina 4-5. According to a previous study 6 the volatile components from the aerial parts obtained by hydrodistillation of Ferula ovina were investigated. The main constituents were carvacrol (9.0 %), α-pinene (8.2 %), geranyl isovalerate (7.2 %), limonene (6.7 %) and carotol (6.5 %). Another study 7 inspected the oil obtained by hydrodistillation from the dried *Corresponding author (Marzieh Alikhah-Asl) E-mail: < [email protected] >


Marzieh Alikhah-Asl et al. / Jeobp 14 (2) 2011 250 - 254

251

fruits of F. ovina. The major components were α-pinene (37.4 %), β-phellandrene (10.8 %), isobornyl acetate (9.2 %), α-fenchene (8.9 %), β-myrcene (5.8 %), γ-elemene (4.6 %) and β-pinene (4.1 %). The previous study 6 reported the compounds of F. ovina aerial parts’ oil (representing 86.7 % of the total oil components) at the vegetative stage (non-flowering). In current work, the compounds of aerial parts oil (representing 91.1 % of the total oil components) at the flowering stage are identified. Furthermore, we compare the oil yields and volatile component percentages of the aerial parts of F. ovina, in 2 stages (fresh and dry) to determine their variations. Experimental Source: The aerial parts of the plant were collected from Taleghan, 110 km northwest of Tehran, Iran, in June 2008, at the flowering stage. The herbarium specimen # 6689-TEH was identified by Dr. Gholamreza Amin and deposited in the herbarium of Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. Plant part: The aerial parts of the plant in 2 stages; once when they were fresh and next time, when they were air-dried at ambient temperature, were hydrodistilled for 4 h in a Clevenger-type apparatus. The distillated oils were dried over anhydrous sodium sulfate and stored in sealed vials at 4°C before analyzing. Qualitative and quantitative analysis: The oil was investigated by capillary GC (Agilent 6890), carried out using fused silica capillary DB-5 column (30 m × 0.25 mm, film thickness 0.25 μm) by a temperature program of 50-240°C at 3°C min-1, injector and detector temperatures of 290°C. GC-MS analysis was carried out using Agilent 5973. EI mass spectra were measured with ionization voltage of 70 eV; ion source temperature 220°C equipped with a HP-5MS capillary column (30 m × 0.25 mm, film thickness 0.25 μm) with helium as a carrier gas; flow rate, 0.8 ml min-1. Retention indices were calculated using retention times of n-alkanes injected under the same chromatographic conditions. Identification of the components was made by comparison of their mass spectra and relative retention indices (RRI-HP-5) with those given in the literature or with authentic samples 8. For quantitative analysis, relative percentages of individual components were based on the peak areas obtained by GC without the use of correction factors. Results and discussion: The essential oils isolated by hydrodistillation from fresh and dried aerial parts of Ferula ovina were obtained in yields of 0.4 % and 0.25 % (w/w), respectively. Fortytwo components were identified in the fresh aerial parts oil of this species, representing 95.0 % of the total oil’s compounds. The main components of the oil were limonene (16.9%), α-pinene (15.2 %), βmyrcene (7.7 %), cis-β-ocimene (6.1 %), iso-sylvestrene (5.1 %), β-pinene (4.4 %), γ-elemene (4.3 %), dill apiole (3.9 %), germacrene D (3.3 %) and trans-β-ocimene (3.2%). Twenty-one components were identified in the oil obtained from dried aerial parts of F. ovina, representing 91.1 % of the total oil’s components. The main components of the oil were α-pinene (20.2 %), spathulenol (9.6 %), germacrene D (6.3 %), β-caryophyllene (5.1 %), α-terpineol (5.0 %), caryophyllene oxide (4.4 %), limonene (4.3 %), α-cadinol (3.7 %), β-pinene (3.3 %) and bicyclogermacrene (3.1 %). The essential oil of dried F. ovina is composed of Monoterpene hydrocarbons (38.5%), Sesquiterpene hydrocarbons (22.5%), Oxygenated Sesquiterpenes (15.3%), Sesquiterpene alcohols (7.1%), Oxygenated Monoterpenes (5.0%) and Other (2.7%). According to our results, the oil yield of fresh plant (0.4 %) was higher than that of dried plant (0.25 %). As demonstrated by a comparison between the major volatile components of fresh and dried aerial parts; the percentages of β-pinene and limonene found in the oils of both stages were higher in fresh stage oil than in dry stage oil (Table 1). Moreover, contrary to the oil of dried aerial parts, fresh


252

stage oil contained the lower percentages of α-pinene, germacrene D and sabinene. The other major components of fresh stage oil were completely different from those of dried aerial parts oil. For example, the content of spathulenol in fresh plants oil reached 9.6 % but it was not identified in the dry plants oil as a main component. According to Mozaffarian 3, fresh Prangos uloptera and Ferula ovina (Umbelliferae) are poisonous plants if taken up as fresh, but they are consumed as valuable forage if dried, accumulated and then fed to livestock. As cited in literature; among the chemical compounds the percentages of which decreased in F. ovina dry stage oil, β-myrcene and limonene have harmful biological effects. Based on previous reports about mentioned compounds; β-myrcene, a monoterpene, causes skeletal alterations in mice fetuses from females fed on this substance during pregnancy 9. Paumgartten et al. 10 reported that as far as the digestive system is concerned, β-myrcene is toxic for the stomach and liver after its oral administration to mice. It is also highly irritant to the peritoneum, and deaths after intraperitoneal injection in rats and mice are possibly due to drug-induced chemical peritonitis 10. Limonene and its oxidation products are skin irritants, and limonene-1,2-oxide (formed by aerial oxidation) is a known skin sensitizer. Most reported cases of irritation have involved long-term industrial exposure to the pure compound 11. There are various reports confirming the noxious effects of plant volatile oils 9-12-13-14. However, other compounds such as proteins and enzymes may be present in the plant and act as harmful factors. These compounds could be denatured during the storage of the plant. Based on a previous study 6, α-pinene (8.2 %) and limonene (6.7 %) were the major components of F. ovina. Our results showed higher percentage of α-pinene (20.2 %) and the lower percentage of limonene (4.3 %) in the oil composition. Other main components were not the same in two mentioned studies. Iranshahi and Hassanzadeh-Khayyat 7 showed that the content of α-pinene in the volatile oil of F. ovina fruits reached 37.4 %. Furthermore, β-phellandrene, isobornylacetate and α-fenchene were the main constituents of the fruits oil, while based on our investigation and the previous study 6, they were absent in the oil of aerial parts. These variations may be due to the different climatological factors, nutritional status, genetic, and especially the locality and growth stage of the plants. Acknowledgements: The authors would like to thank the Research Institute of Medicinal Plants, Jahade daneshgahi (University of Tehran) for their collaboration. We are grateful to Dr. MohammadAli Zare-Chahouki, the assistant professor of Natural Resources Faculty, University of Tehran, for his helps.

1. 2. 3. 4. 5. 6. 7.

References Rechinger, K.H. (1982). Umbelliferae in: Flora Iranica. Rechinger KH (Ed). Vol. 68, Akademische Druck-u, Verlagsanstalt, Graz, 387. Mozaffarian, V. (2006). A Dictionary of Iranian Plant Names. Farhang Moaser Publishers, Tehran, Iran, 228. Mozaffarian, V. (1983). The Family of Umbelliferae in Iran, Keys and Distribution. Research Institute of Forests and Rangelands Press, Tehran, pp. 114-116. Al-Khalili, S., Aqel, M., Afifi, F. and Al-Eisawi, D. (1990). Effects of an aqueous extract of Ferula ovina on rabbit and guinea pig smooth muscle. J. Ethnopharmacology. 30(1): 35-42. Aqel, M., Al-Khalili, S. and Afifi, F. (1992). Relaxing effect of Ferula ovina extract on uterine smooth muscle of rat and guinea pig. Int. J. Pharmacognosy. 30(1): 76-80. Ghannadi, A., Sajjadi, S.E. and Beigihasan, A. (2002). Composition of essential oil of Ferula ovina (BOISS.) BOISS. from Iran. J. Daru. 10(4): 165-167. Iranshahi, M. and Hassanzadeh-Khayyat, M. (2008). Chemical composition of the volatile oil from Ferula ovina (Boiss.) Boiss. Rech. fruits. J. Essent. Oil Bearing Plants. 11(4): 350355.

Marzieh Alikhah-Asl et al. / Jeobp 14 (2) 2011 250 - 254 8. 9.

10.

11. 12.

13. 14.

253

Adams, R.P. (2004). Identification of Essential Oil Components by Gas Chromatography/ Quadrupole Mass Spectroscopy. Allured Publishing Corporation, Carol Stream, Illinois. 456. Delgado, I.F., Carvalho, R.R., Nogueira, A.C., Mattos, A.P., Figueiredo, L.H., Oliverira, S.H., Ghahoud, I. and Paumgartten, F.J. (1993). Study on embryo-foetotoxicity of β-myrcene in the rat. J. Food and chemical toxicology. 31: 31-35. Paumgartten, F.J., Delgado, I.F., Alves, E.N., Nogueira, A.C., De-Farias, R.C. and Neubert, D. (1990). Single dose toxicity study of β-myrcene, a natural analgesic substance. Brazilian J. Medical and Biological Research. 23: 837-839. IARC (1999). Monographs on the Evaluation of Carcinogenic Risks to Humans. Lyon, France. 73: 307-327. Abramov, A.Y., Zamaraeva, M.V., Hagelgans, A.I., Azimov, R.R. and Krasilnikov, O.V. (2001). Influence of plant terpenoids on the permeability of mitochondria and lipid bilayers. J. Biochimica et Biophysica Acta (BBA)/Biomembranes. 1512(1): 98-110. De-Olivera, A.C., Ribeiro-Pinto, L.F., Otto, S.S., Goncalves, A. and Paumgartten. F.J. (1997). Induction of liver monooxygenases by beta-myrcene. J. Toxicology. 26: 135- 140. Zeinsteger, P., Romero, A., Teibler, P., Montenegro, M., Rios, E., Ciotti, E.M., Acosta De Perez, O. and Jorge, N. (2003). Toxicity of volatile compounds of Senecio grisebabachii baker (margarita) flowers in mice. J. RIA, 32(2): 125-136. Table 1. Percentage composition of the oil of Ferula ovina fresh and dried aerial parts Components α-Thujene α-Pinene Camphene Verbenene Sabinene β-Pinene β-Myrcene α-Phellandrene iso-Sylvestrene ortho-Cymene Limonene (Z)- β-Ocimene (E)- ®-Ocimene γ-Terpinene Terpinolene Linalool trans-Verbenol Terpinen-4-ol α-Terpineol Myrtenol iso-Dihydro carveol endo-Fenchyl acetate Bornyl acetate Myrtenyl acetate

RI (fresh) 932 941 954 959 979 983 995 1009 1016 1030 1037 1042 1053 1064 1094 1104 1153 1189 1199 1205 1214 1228 1294 1308

RI (dry) 936 979 991 1012 1031 1038 1049 1195 -

% in oil

% in oil

fresh

dry

0.5 15.2 1.2 0.1 1.2 4.4 7.7 0.4 5.1 0.4 16.9 6.1 3.2 0.4 1.0 0.6 0.2 0.3 0.1 0.1 0.1 0.8 0.5 0.2

20.2 5.1 3.3 1.9 4.3 2.3 1.4 5.0 -


254

table 1. (continued). Components

RI (fresh)

cis-Piperitol acetate 1328 1-Tetradecene 1377 α-Copaene 1388 β-Elemene 1403 β-Caryophyllene 1436 γ-Elemene 1446 α-Humulene 1471 β-Acoradiene 1485 Germacrene D 1498 Bicyclogermacrene 1513 δ-Cadinene 1536 (E)- γ -Bisabolene 1552 Germacrene B 1576 Spathulenol 1597 Caryophyllene oxide 1604 Humulene epoxide II Dill apiole 1639 α-Cadinol 1674 epi-α-Bisabolol 1697 10-nor-Calamenen-10-one -

Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated Sesquiterpenes Alkenes Phenylpropanoids Sesquiterpene alcohols Other Total

RI (dry)

1385 1399 1432 1467 1493 1509 1533 1593 1599 1626 1668 1692 1702

% in oil

% in oil

fresh

dry

0.5 2.5 0.6 1.2 1.9 4.3 0.8 0.3 3.3 2.7 0.7 1.0 1.4 0.5 0.3 3.9 1.0 1.4 -

1.4 2.1 5.1 2.5 6.3 3.1 2.0 9.6 4.4 1.3 4.0 3.1 2.7

63.8 3.4 18.2 0.8 2.5 3.9 2.4

38.5 5.0 22.5 15.3 7.1 2.7 91.1

95.0

Jeobp 14 (2) 2011 pp 255 - 259

255


In vitro Antibacterial and Antifungal Activity of Salvia multicaulis Taran Mojtaba 1*, Ghasempour Hamid Reza 2, Safoora Borzo 2, Najafi Shiva 2, Samadian Esmaeil 1 1

Microbiology Lab., Biology Department of Razi University, Kermanshah, Iran 2 Plant Tissue Culture, Biotechnology and Physiology Lab., Biology Department of Razi University, Kermanshah, Iran Received 09 February 2010; accepted in revised form 29 November 2010

Abstarct: Some aromatic plants such as Salvia are used for medical purposes. Compounds such as germacrene, linalool, 1,8-cineole (eucalyptol), borneol, α-pinene, β-pinene, camphor and camphene are found in the genus Salvia. Salvia multicaulis is one of 17 Salvia species endemic to Iran. In this study, antimicrobial activity of essential oil and ethyl acetate and ether extracts of S. multicaulis were examined against some species of bacteria and fungi. The essential oil of the aerial part of S. multicaulis was examined by GC and GC-MS. In the essential oil of S .multicaulis 27 Compounds have been identified. Benzyl benzoate (60.3 %), n-hexyl benzoate (16.7 %), Amyl benzoate (5.2 %) and 2- octyl benzoate (4.2 %) were the main components of the essential oil. The essential oil analysis showed greatest antimicrobial activity against Staphylococcus epidermidis (5.3 μg/ml) and S. cerevisiae (9.3 μg/ml). The ethyl acetate showed greatest antimicrobial activity against Bacillus subtilis (106.7 μg/ml), Candida albicans (5.3 μg/ml) and ether extract showed greatest antimicrobial activity against Klebseilla pneumoniae (10.7 μg/ml) and Saccharomyces cerevisiae (10.7 μg/ml). In conclusion, we suggest that the antimicrobial activity of S. multicaulis may be due to its content of germacrene and linalool. Key words: Salvia multicaulis, antibacterial activity, antifungal activity. Introduction: In many developing countries, some plant materials play an important role in PHC (Primary Health Care) 1,2. Some materials produced by aromatic plants such as essential oils have antimicrobial activity. Despite the chemical drugs with antimicrobial activity are developing, but some herbal drugs are used for the prevention and treatment of infectious diseases in some countries 3,4. In the essential oil of aromatic plants, there are many compounds such as monoterpenes, sesquiterpenes, alcohols, aldehydes, phenols, esters, and ether, sulphurous and nitrogenous substances 5,6. The genus Salvia belongs to laminaceae plant family 7. Salvia comprises about 900 species world wide, of which 17 species are endemic to Iran 8. S. multicaulis is an aromatic perennial woody sub-shrub of these species that are found in their natural in Iran. S. multicaulis is considered one of the most important Salvia species for medical (antimicrobial and antipestic) purposes 9. Several reports *Corresponding author (Taran Mojtaba) E-mail: < [email protected]; [email protected] >


Taran Mojtaba et al. / Jeobp 14 (2) 2011 255 - 259

256

have been published in recent years on the antimicrobial activity of some extracts and essential oils obtained from some species of Salvia 5, 10, 11, 12. The plants of the genus Salvia have been used since antiquity in food, drug, cosmetics, and perfumery 13. The literature on the chemical constituents of the genus Salvia gave information for compounds such as germacrene, linalool, 1,8-cineole (eucalyptol), borneol, α-pinene, β-pinene, camphor and camphene 5,11 .There are no data concerning the essential oil composition and antimicrobial activity of S. multicaulis ( endemic in Kermanshah, Iran). The aim of this paper is to demonestrate the compounds and antimicrobial activities of essential oil, ethyl acetate and ether extract of S. multicaulis endemic in Kermanshah, Iran. Experimental Plant material: The aerial parts of S. multicaulis were collected from wild plant in Kermanshah city (west of Iran). The voucher specimen (No.2468) is deposited in the Herbarium of Agricultural, Faculty of Razi University, Kermanshah, Iran. The aerial parts were cut into pieces and air-dried for 10 Days at room temprature.70 g aerial parts were powderd, mixed with 500 ml of distilled water and the essential oil hydrodistilled in a Clevenger apparatus according to the British method for 3h. Ethyl acetate and ether extracts of S. multicaulis were obtained using 40 gr of powdered aerial part and150 ml of each solvent (ethyl acetate, ether) by maceration method. Gas chromatography: GC analysis of the oil was conducted using a Thermoquest-Finnigan Trace GC instrument equipped with a DB-1 fused silica column (60 m x 0.25 mm i.d., film thickness 0.25 μm). Nitrogen was used as the carrier gas at the constant flow of 1.1 mL min-1. The oven temperature was held at 60°C for 1 min, then programmed to 250°C at a rate of 4°C min-1 and then held for 10 min. The injector and detector (FID) temperatures were kept at 250 and 280°C, respectively. Gas chromatography-Mass spectrometry: GC-MS analysis was carried out on a ThermoquestFinnigan Trace GC-MS instrument equipped with a DB-1 fused silica column (60 m x 0.25 mm i.d., film thickness 0.25 μm). Helium was used as a carrier gas at a flow rate of 1 mL/min. The oven temperature was raised from 60 to 250°C at a rate of 5°C min-1 and then held at 250°C for 10 min.; transfer line temperature was 250°C. The quadrupole mass spectrometer was scanned over the 45-465 amu with an ionizing voltage of 70 eV and an ionization current of 150 μA. Identification of the components was based on comparison of their retention indices (RI), obtained using n-alkanes(C6-C25), and comparison of their mass spectra with those of Wiley library spectra and literature data. Additionally, the identity of all compounds was confirmed by comparison of the expected molecular weights with the results of available authentic samples. Microorganisms: Five strains of bacteria and two strains of fungi were studied including Bacillus subtilis (ATCC 127111), Enterococcus faecalis (ATCC 29737), Staphylococcus aureus (ATCC 29737), Staphylococcus epidermidis (ATCC 12229), Klebsiella pneumoniae (ATCC 10031), Candida albicans (ATCC 10231) and Saccharomyces cerevisiae (ATCC 9763). Bacterial strains were cultivated on Mueller Hinton broth (Merck, Germany) and fungi were cultivated on Sabouraud liquid Medium broth (Merck, Germany). The minimum inhibitory concentration (MIC) of essential oil and extracts against the bacteria and fungi was determined using broth microdilution method. Result and discussion: The essential oil of the aerial part of S. multicaulis was examined by GC and GC-MS. The constituents of the oil are showed in table 1. In the essential oil, 27 Compounds have been identified. Benzyl benzoate (60.3 %), n-hexyl benzoate (16.7 %), Amyl benzoate (5.24 %), 2- octyl benzoate (4.2 %) were the main components of the essential oil. According to the data shown in table 2, the essential oil of S. mluticaulis had antimicrobial activity against all tested microorganisms.


257

The MIC ranged from 5.3 to 298.7 μg/ml with highest inhibition values observed against S. epidermidis, B. subtilis (5.3 μg/ml) and S. cerevisiae (9.3 μg/ml) among bacteria and fungi. The antimicrobial activity against S. epidermidis (5.3 μg/ml) and S. cerevisiae (9.3 μg/ml). B. subtilis (170.7 μg/ml) and C. albicans (85.3 μg/ml) showed the greatest level of resistance to essential oil. The essential oil showed more antimicrobial activity on bacteria and fungi than ethyl acetate and ether extract. The ethyl acetate showed greatest antimicrobial activity against B. subtilis (5.3 μg/ml), C. albicans (106.7 μg/ml) and ether extract showed greatest antimicrobial activity against K. pneumoniae (10.7 μg/ml) and S. cerevisiae(10.7 μg/ml). S. epidermidis(149.3 μg/ml) and S. cerevisiae (298.7 μg/ml) showed the highest resistance to ethyl acetate extract and S. epidermidis(106.7 μg/ml) and C. albbicans (149.3 μg/ml) showed the highest resistancce to ether extract. The observed antimicrobial (antibacterial and antifungal) effect of S. multicaulis against Bacteria and fungi is according to previous reports for other species of Slavia 5,10,11,12. In the literature, the antimicrobial activity of some genus Salvia essential oils against microbes has been reported 5,15. It is due to the presence of chemical components that have antimicrobial activity such as germacrene, Linalool. Germacrene is an important sesquiterpenes that occur widely in nature 16 . Antibacterial and antifungal activities of germacrene have been reported in previous studies 16,17. Linalool is a major constituent of essential oil of some plants such as genus Lavandula. This componund has been used as an antimicrobial agent in some countries such as Greece 18,19. The antibacterial and antifungal activity of terpenes such as linalool and germacrene has not been exactly understood but these compounds may cause disruption in membrane by lipophylic reactions 6. It is very difficult to contribute antimicrobial activity of the essential oils to a few compounds because those contain different chemical compounds 20. In conclusion, we suggest that the antimicrobial activity of S. multicaulis may be due to its content of germacrene and linalool.

1. 2. 3.

4.

5.

6. 7. 8. 9. 10. 11.

References Zakaria, M. (1991). Isolation and characterization of active compounds from medicinal plants. Asian Pacific J. Pharmacol. 6: 15-20. Sokmen, A., Jones, B.M. and Erturk, M. (1999). The in vitro antibacterial activity of Turkish medicinal plants. J. Ethnopharmacol. 67: 79-86. Inouye, S.H., Yamaguchi, H. and Takizawa, T. (2001). Screening of the antibacterial effects of a variety of essential oils on respiratory tract pathogens, using a modified dilution assay method. J. Infect Chemother. 7: 251-254. Federspil, P., Wulkow, R. and Zimmermann, T. (1997). Effects of standardized Myrtol in therapy of acute sinusistis. Results of a double-blind randomized multicenter study compared with placebo. Laryngorhinootologie. 76: 23-7. Delamare, A.P.L., Moschen-Pistorello I.T., Artico, L., Atti-Serafini, L., Echeverrigaray, S. (2007). Antibacterial activity of the essential oils of Salvia officinalis L. and Salvia triloba L. cultivated in South Brazil. Food Chem. 100: 603-608. Cowan, M.M. (1999). Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12: 564582. Lu, Y. and Yeap, F.l. (2002). Polyphenolic of Saliva-areview. Phytochem. 59: 117-140. Nickavar, B., Mojab, F. and Asgharpanah J. (2005). Voltile composition of the essential oil of Slavia hypolueca Benth. Int. J. Aromsth. 15: 51-53. Ghasempour, H.R., Kahrizi, D. and Borzo S. (2007). Plant micropropagation in wild sage (salvia multicaulis). Plant Sci. 269: 947.950. Jalsenjak, V., Peljnajk, S. and Kustrak, D. (1987). Microcapsules of sage oil, essential oils content and antimicrobial activity. Pharmazie. 42: 419-420. Tepe, B., Daferera, D., Sokmen, A., Sokmen, M., and Polissiou, M. (2005). Antimicrobial


12.

13. 14.

15.

16. 17.

18.

19. 20.

258

and antioxidative activities of the essential oils and various extracts of Salvia tomentosa Miller (Lamiaceae). Food Chem. 90: 333-340. Khalil, M., Hassawi, D.S. and Kharma, A (2005). Genetic relationship among Salvia species and Antimicrobial activity of their crude extract against pathogenic bacteria. Asian J. Plant Sci. 4: 544-549. Ozcan, M., Tzacou, O. and Couladis, M.(2003). Esential oil composition of Turkish herbal tea (Salvia aucheri Bentham var.canescens Boiss Á Heldr.).Flavour and Fragr. J. 18: 325-327. Ghasempour, H., Shirinpour, E. and Heidari, H. (2007). Analysis by Gas Chromatographymass Spectrometry of Essential Oil from Seeds and Aerial Parts of Ferulago angulata (Schlecht.) Boiss Gathered in Nevakoh and Shahoo, Zagross Mountain, West of Iran. Pakistan J. Biol. Sci., 10: 814-817. Hayouni, E.A., Chraief, I., Abedrabba, M., Bouix, M., Leveau, J.Y., Mohammed, H., Hamdi, M. (2008). Tunisian Salvia officinalis L. and Schinus molle L. essential oils: Their chemical compositions and their preservative effects against Salmonella inoculated in minced beef meat. Int. J. Food Microbiol. 125: 242-251. Adio, A.M. (2009). Germacrenes A-E and related compounds: thermal, photochemical and acid induced transannular cyclizations.Tetrahedron. 65: 1533-1552. Luna-Herrera, J., Costa, M.C., González, H.G., Rodrigues, A.I. and Castilho, P.C. (2007). Synergistic antimycobacterial activities of sesquiterpene lactones from Laurus spp. The Journal of antimicrobial chemotherapy. 59: 548-52. Sato, K., Krist, S. and Buchbauer, G. (2007). Antimicrobial effect of vapours of geraniol, (R)(–)-linalool, terpineol, γ-terpinene and 1,8-cineole on airborne microbes using an airwasher Flavour Fragr. J. 22: 435437. Lis-Balchin, M., Deans, S.G. And Eaglesham, E. (1998). Relationship between bioactivity and chemical composition of commercial essential oils. Flavour Fragr J. 13: 98-104. Ben-Amor, I.L., Neffati, A., Sgaier, M.B., Bhouri, W. , Boubaker, J., Skandrani, I., Bouhlel, I., Kilani, S., Ammar, R.B., Chraief, I., Hammami, M., Ghoul, M., Chekir-Ghedira, L. and Ghedira, K. (2008). Antimicrobial Activity of Essential Oils Isolated from Phlomis crinita Cav. ssp. mauritanica Munby. J. Am. Oil Chem. Soc. 85: 845-849.


259

Table 1. Chemical composition of identified compounds in the oil of Salvia multicaulis No

Component

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Benzyl aldehyde Linalool 2- Methyl butyl isovalerate 2- Methyl propanoate Acetic acid octyl ester 2- Methyl butanoate Hexyl isovalerate Benzyl isobutanoate Isobutyl benzoate n- Butyl isovalerate n- octyl isobutirate n- Butyl benzoate Benzyl valerate Benzyl isovalerate Amyl benzoate Isopentyl benzoate Germacrene D E,E- α-farnesene Benzyl hexanoate 2-Allylpent-4, enoic acid, benzyl ester n- Hexyl benzoate β-Eudesmol α-Eudesmol Tert-butyl benzoate Benzyl benzoate 2- ocetyl benzoate Bergamotol

%

RI

0.1 2.2 0.1 0.5 0.1 1.0 1.2 0.3 0.3 0.1 0.1 0.4 0.3 1.1 5.2 0.2 0.4 0.5 0.2 0.1 16.7 0.4 0.1 0.4 60.3 4.2 2.1

932 1082 1089 1128 1187 1217 1221 1267 1301 1318 1323 1345 1357 1363 1411 1446 1479 1490 1510 1513 1553 1639 1644 1651 1738 1756 1814

RI: Linear retention indices on column Table 2. The minimum inhibitory concentration (MIC, μg/ml) of essential oil, ether, ethyl acetate extract of Salvia multicaulis against microorganisms

Microorganism Enterococcus faecalis Staphylococcus aureus Staphylococcus epidermidis Bacillus subtilis Klebsiella pneumoniae Candida albicans Saccharomyces cerevisiae

Essential oil 106.7 85.3 5.3 170.7 125 85.3 9.3

The results are the conclusion of three replicates

Extract Ether

Ethyl acetate

74.7 37.3 106.7 85.3 10.7 149.3 10.7

42.7 21.3 149.3 5.3 74.7 106.7 298.7

JEOBP

JOURNAL OF ESSENTIAL OIL- BEARING PLANTS

SUBSCRIPTION and ADVERTISEMENT RATES Subscription (India)

Online only USD 550,00 Print Only USD 550,00 + (60 Postal charge) Online and Print USD 700,00 + (60 Postal charge) Personal Rates USD 450,00 + (60 Postal charge)

Advertisement page

Four colours (INR) 1 Issue 6 Issue

Front cover back and First insert page front Inside back cover, 1st Insert page back Back cover Full page (8.5 X 6 inch) Half page (4 X 6 inch) Insert

12,000 10,000 15,000 6,000 4,000 8,000

Four colours (Foreign USD) 1 Issue 6 Issue

60,000 50,000 80,000 30,000 20,000 42,000

300 250 400 200 150 250

1500 1200 2000 1000 700 1200

CONSENT FORM Please publish our advertisement in Journal of Essential Oil Bearing Plants Placement: Front Cover back / First insert page front / Inside back cover / First insert page back / Last insert page / Back Cover/ Text : Full page / Half page Issue No(s):

First / Second / Third / Fourth / Fifth / Sixth / All

Draft / Cheque no. ……………………………Date ……………………..Amount ………………………......... Amount in words: …………………………………………………………………….......................................... Bank: ……………………………………………………………………………………..................................... Drawn in favour of : Har Krishan Bhalla and Sons

Name : ……………………………………………………………Position:…………………………………........ Name of company: ………………………………………………………………………………………………… Address: …………………………………………………………………………………………………………… ...................................................................................................................................................................... Phone: Off: ………………………………………………… E-mail: ……………………………………………..

Please send form with DD to : Har Krishan Bhalla and Sons 7/1/2C Prem Nagar, Dehradun - 248007, Uttarakhand Mobile + 91 07417016166 09219506760