Table3.1: solvent polarity ratio of chloroform-methanol for isolation of catechins. .... 4.2 HPLC Standard curves of Polyphenols, Catechins & Caffeine. 55 .... that it is a cultural norm tea to offer tea to guests and visitors instead of alcoholic drinks. ... However this type of tea does not appeal to all and sundry, it takes some.
“Extraction, Purification, Identification and Estimation of Catechins from Camellia sinensis” A dissertation submitted to
INSTITUTE OF HIMALAYAN BIORESOURCE AND TECHNOLOGY PALAMPUR, HIMACHAL PRADESH In partial fulfillment for the award of BACHELOR OF TECHNOLOGY IN CHEMICAL ENGINEERING
By ASHRAY GUPTA
Guide
Submitted to
Dr. Ashu Gulati Scientist, Hill Area Tea Science Division Institute of Himalayan Bioresource Tech.
Prof R.K. Sharma
Palampur Himachal Pradesh i
ATPO Dept of Chemical Engineering, Beant College Of Engg & Technology, Gurdaspur Punjab
ABSTRACT Tea is the most consumed beverage all around the world after water. The present study evaluates the extraction, purification and isolation of the most important compound in tea, the Catechins. Tea shoots (2 and a bud) were dried, crushed, extracted with solvents (methanol and acetone) and purified by column chromatography using different resins. Different methods were devolped for TLC for the detection of catechins. Individual catechins were isolated using C-18 as the stationary phase in column chromatography. Five major catechins i.e. EGCG , ECG, EGC,EC and catechin hydrate were purified which were confirmed by TLC and HPLC profiles. The antioxidant potential of catechins in terms of DPPH radical scavenging activity was also studied.
ii
ACKNOWLEDGEMENT
A journey is easier when you travel together. This project report is the result of six months of work whereby, I accompanied and supported by many people and it’s the time to express my gratitude for all of them. Fore most, I would like to thank God for His grace and blessings. The first person I express my heartiest thanks to Dr. Ashu Gulati, Scientist, Tea Technology Lab, HATS Division and Dr Arvind Gulati HOD HATS Division, CSIR-IHBT, Palampur, for providing me excellent laboratory facilities, valuable guidance, untiring help during my project work and constant encouragement during the whole period of my project work. I wish to express my sincere gratitude to Dr. P.S Ahuja, Director, CSIR-IHBT, Palampur for giving me an opportunity to carry out my project work in such a prestigious institute. A special thanks to my senior Mr. Robin Joshi for providing an invaluable help at various stages during the training at CSIR-IHBT. I owe my gratitude to Prof. P.K Yadav, H.O.D., and Prof R.K. Sharma, ATPO, Deptt. Of Chemical Engineering, Beant College of Engineering and Technology, Gurdaspur (Pb) for their kind support throughout the course. Finally, I express my gratitude to my family members for their blessings and constant support.
Ashray Gupta Chemical Engineering, Final year, Beant college of Engg & Tech, Gurdaspur
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About the Institute The CSIR-Institute of Himalayan Bioresource Technology is located in picturesque town of Palampur (32 degree N,76 degree E, and 1300m above sea level) perched in the lap of majestic snow clad Dhauladhar range of Himalayas in the state of Himachal Pradesh, India, with an annual mean temperature of 19 degree Celsius and an annual mean rainfall of over 250 cm. Amongst the 39 labs of Council of Scientific and Industrial Research (CSIR), Institute of Himalayan Bioresource Technology holds a prestigious position.
Council of Scientific and Industrial Research (CSIR) established in 1942, is an autonomous body and India's largest Research and Development (R&D) organization, with 39 laboratories and 50 field stations or extension centers spread across the nation, with a collective staff of over 17,000. Although CSIR is mainly funded by the Ministry of Science and Technology, it operates as an autonomous body registered under the Registration of Societies Act of 1860.
The research and development activities of CSIR includes various fields such as aerospace engineering, structural engineering, ocean sciences, molecular biology, metallurgy, chemicals, mining, food, petroleum, leather, and environment. Dr. Samir K. Brahmachari is the present Director General of CSIR. In late 2007, the then Minister of Science and Technology, Sh. Kapil Sibal in reply to a question in the Question Hour session of the Parliament told that CSIR has developed over 1,300 technologies/knowledgebase during the last decade of 20th century.
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In the year 1983 the foundation stone of this National lab was laid as CSIR complex Palampur by Prof. Nurul Hasan former Vice President of CSIR, with Chief Minister of HP chairing the function. The Co-ordinating Director was appointed in February 1984. Since then the Institute is working relentlessly on developing technologies for sustainable utilization of Himalayan bioresources, and in the area of tea, floriculture, bamboos and medicinal and aromatic plants. Looking at the mission of the Institute ,the quantum of work undertaken and milestones achieved it was rightly conceived to rename the Institute from CSIR Complex Palampur to Institute of Himalayan Bioresource Technology at a high level meeting. Finally the Institute received its new name Institute of Himalayan Bioresource Technology (IHBT) in 1997.
The institute headed by its director Dr P.S Ahuja, is committed to provide R&D services on economic bioresources in western Himalayan region leading to value added plants, products and processes for industrial, societal and environmental benefits.
The major thrust areas include:
Biodiversity mapping and conservation
Bioprospection of Himalayan Bioresource
Tea Sciences
Genomics, proteomics and metabolomics
Adaptation biology
Natural health management
Nan biology v
DECLARATION I hereby declare that the work being presented to this dissertation entitled “Extraction, Purification, Identification and Estimation of Catechins from Camellia sinensis” submitted for partial fulfillment of the award of degree of Bachelors of Technology in Chemical Engineering from ‘Beant College of Engg and Technology’, Gurdaspur(Pb), is an authentic record of my work carried out under supervision and able guidance of Dr. Ashu Gulati, Scientist, Hill Area Tea Science Division,
at ‘ CSIR-Institute of Himalayan
Bioresource and Technology’, Palampur(H.P)
The matter embodied in this project report has not been submitted by me in any other institute for any other degree.
Date: Place: Palampur
Ashray Gupta Chemical Engineering, Final year, Beant college of Engg & Tech, Gurdaspur
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List of tables: Table 2.1: Chemical composition of a typical two and a bud of assamica and sinensis varieties Table3.1: solvent polarity ratio of chloroform-methanol for isolation of catechins. Table 3.2: Gradient solvent system for HPLC Table 4.1: % Concentration of polyphenols in different leaf parts Table 4.2: major catechins in tea by HPLC Table 4.3:Total catechins of different leaf parts Table 4.4: % Purity of catechins from different resins Table 4.6:Major catechins purified by XAD-16 Table 4.6: Comparison of catechins before and after purification. Table 4.7:Areas of different standards with respect to concentration Table: A.1 Absorbance of Gallic acid as standard Table: A.2 Absorbance of Catechin hydrate as standard Table A.3: Areas of different standards with respect to concentration
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LIST OF FIGURES: Fig 1.1:Tea leaves Fig 1.2: Withering of tea leaves Fig1.3: Rolling of tea leaves Fig 1.4: Drying of tea leaves Fig 2.1: Structures of Six major Catechins from tea Fig3.1: Column chromatography Fig3.2: Shimadzu UV-VIS 2450 Spectrophotomoter Fig3.3: Waters 717 HPLC with quaternary solvent system,auto sampler and PDA detector Fig 3.4: Working of HPLC Fig 3.5: Image of Rheodyne injector Fig 3.6: Internal diagram of Rheodyne injector during loading and injection. Fig 4.1: Comparison of total polyphenols in different leaf parts Fig 4.2: Comparison of total catechins in different leaf parts Fig 4.3: % Yield comparison of different resins Fig 4.4: % Purity comparison of different resins Fig 4.5: Catechin profile of crude using HPLC Fig 4.6: Catechin profile of purified catechins using HPLC.
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Fig 4.7: Scavenging effect of catechins on DPPH radical and each value is expressed as mean of triplicate. Fig 4.8: TLC plate of standard and crude extract Fig4.9: TLC plate of different fractions against standard catechins Fig 4.10: HPLC profile of purified ECG Fig 4.11: HPLC profile of purified EGCG Fig4.12: HPLC profile of purified EGC Fig 4.13: HPLC profile of purified EC Fig4.14: HPLC profile of purified Catechin Hydrate Fig:A.1 Gallic acid standard curve Fig:A.2 Catechin Hydrate standard curve Fig:A.3 Gallic acid standard curve by HPLC Fig:A.4 Catechin Hydrate standard curve by HPLC Fig:A.5 Caffeine standard curve by HPLC Fig:A.6 EC standard curve by HPLC Fig:A.7 ECG standard curve by HPLC Fig:A.8 ECGG standard curve by HPLC Fig:A.9 CG standard curve by HPLC Fig A.10: Catechin profile of standard mix. ix
CONTENTS CHAPTER NO.
1.
PAGE
Abstract
ii
Acknowledgement
iii
Profile of the Institute
iv
Declaration
vi
List of Tables
vii
List of Figures
viii
INTRODUCTION4 1.1
2.
TITLE
General Introduction of tea
4
1.1.1 History
5
1.1.2 The History Of tea drinking in India
5
1.1.3 Tea Drinking in India
7
1.1.4 Tea Industry in India
8
1.1.5 Economic importance
10
1.2
Tea processing
12
1.3
Different types of teas
14
1.4
Health advantages of tea
16
1.5
Why this project
18
REVIEW OF LITERATURE
19
2.1
19
Tea varieties
2.2 Chemical composition of tea
19 1
3.
2.3 Advancements in Tea Research
22
MATERIAL AND METHODS
30
3.1
Material
30
3.2
Methods
31
3.2.1
Extraction and Purification of Catechins
31
3.2.2
Extraction of each component of tea plant
31
3.2.3
Purification by column chromatography
32
3.2.3.1 Extraction of material for selection of column resin
35
3.2.3.2 Bulk extraction and purification using XAD-16
36
3.2.3.3 Isolation of individual catechins using C-18 column
37
Methods of Analyses and Instrumentation
38
3.2.4.1 Thin layer chromatography(TLC)
38
3.2.4
3.2.4.1.1
Method for identification of Catechins
39
3.2.4.2 Spectrophotometer
40
3.2.4.2.1
Preparation of stock solution
42
3.2.4.2.2
43
3.2.4.2.3
Preparation for standard curve of polyphenol Estimation of total polyphenols in tea
43
3.2.4.2.4
Preparation for standard curve of catechin
43
3.2.4.2.5
Estimation of total catechins in tea
44
3.2.4.2.6
Estimation of total catechins in purified Catechins
44
3.2.4.3 High Performance Liquid Chromatography (HPLC) 2
45
3.2.4.3.1
Preparation of standard curves
51
3.2.4.3.2
Study of catechin profile of extracted tea Samples
52
Study of catechin profile of purified Catechins
52
3.2.4.3.3
3.2.4.4 DPPH radical-scavenging activity 4.
5.
52
RESULTS AND DISCUSSIONS
54
4.1
Spectrophotometer
54
4.1.1 Standard curve for total polyphenols
54
4.1.2 Standard curve for total Catechins
54
4.2
HPLC Standard curves of Polyphenols, Catechins & Caffeine
55
4.3
Total polyphenols of tea leaves
56
4.4
Total polyphenols of different parts of leaf parts
56
4.5
Total catechins of tea leaves
57
4.6
Total catechins in different parts of leaf
58
4.7
Comparative estimation of Catechins of purified Catechins from XAD-4, XAD-7 and XAD-16
60
4.8
Total purified Catechins by XAD-16
61
4.9
DPPH radical –scavenging activity
63
4.10
Isolation of individual Catechins by column Chromatogpraphy
64
CONCLUSIONS AND FUTURE SCOPE OF TRAINING
70
5.1
Conclusion
70
5.2
Future perspective
71
APPENDIX
72
REFERENCE
77
3
1. INTRODUCTION 1.1 General Introduction:
Fig 1.1: Tea shoots
Tea is non-alcoholic beverage prepared by pouring boiling hot water over processed leaves of Camellia sinensis plant. The term also refers to the plant itself. After water, tea is the most widely consumed beverage in the world. It has a refreshing, astringent flavour which many people enjoy. Camellia sinensis, the evergreen plant that we all know as the tea bush, was originally named Thea sinensis, as designated by the Swedish botanist Linnaeus in the mid 1750's. However, a few months later he also named the same plant Camellia sinensis, creating much 4
confusion. After some years of debate and several further name changes, finally, in 1905 the debate was settled and the plant would be known as a member of the family Camellia.
1.1.1 History Tea first originated in the Chinese province of Yunnan and its neighbouring area of Assam in India. The history of tea can be traced back to 2737 B.C. According to a Chinese legend, tea was discovered accidentally by a Chinese emperor Shen Nung when wild tea leaves fell into a pot of boiling water. He drank the resulting infusion and felt energetic. Ever since then, tea has been influencing art, literature, religion and daily life all over the world. Tea is currently produced in about 30 countries, ranging from Soviet Georgia (latitude 42 degrees North) to South Africa (latitude 29 degrees South). The largest producers are China, India, Sri Lanka, Kenya, Japan, Indonesia, Turkey and Malawi. The distribution of the tea plant extends from the foothills of the Himalayas, from northeast India to southwest China with subtropical monsoon climate characterizing wet and hot summers and relatively cold and dry winters, to near the equator. India is the largest producer and consumer of tea today, with over two million people employed in the industry. About 50% of all the tea produced in India is grown in Assam. It is the largest black tea producing region in the world.
1.1.2 The History of Tea Drinking in India The documented evidence according to the history of tea drinking in India dates back to 750 BC. Tea in India is generally grown in the North Eastern regions and the Nilgiri Hills. Having evolved since those early days, tea drinking in India has now come a long way. Today this nation is proud to be one of the largest tea producers in the world. Buddhist 5
monks in India have used tea for its medicinal values since thousands of years. According to a very interesting legend, the history of tea drinking in India began with a saintly Buddhist monk almost about 2000 years ago. It so happened that this monk who later became the founder of Zen Buddhism, decided to spend seven sleepless years contemplating the life and teachings of Buddha. While he was in the fifth year of his contemplation and prayer, he almost fell asleep. He took some leaves from a nearby bush and began chewing them. These leaves revived him and enabled him to stay awake as he chewed them whenever he felt drowsy. Thus he was able to complete his penance for seven years. These leaves were of the wild tea plant. As per the history of tea drinking in India, local people used to brew and drink extract of the leaves of the wild native tea plants. Since that time, different varieties of tea have emerged including the most famous Darjeeling tea. The commercial production of tea in India was started by the British East India Company and vast tracts of land were exclusively developed into tea estates which produce various types of tea. In the 16th century, the people of India prepared a vegetable dish using tea leaves along with garlic and oil, while the boiled tea leaves were also used to prepare a drink as well. The first tea garden was established by the British East India Company by the end of the 19th century after the Company took over tea cultivation in Assam. One of the most popular snippets related to the history of tea drinking in India dates back to the 19th century when an Englishman noticed that the people of Assam drank a dark liquid which was a type of tea brewed from a local wild plant. In the year 1823, a Singpho King offered an English Army Officer tea as a medicinal drink. Tea drinking has evolved in 6
different ways over the years in India and differs from region to region. First thought as the drink of the Royals, tea has now become the favorite of the common man as India leads the world in tea drinking. From the humble roadside tea stalls and the railway platforms to the boardrooms of corporate India, tea is easily available. The cup of sweet and refreshing ‘chai’ available in teashops or train stations to the ‘masala teas’ of North India, the variety of brews available is numerous. According to the records, Assam tea is named after the region from where the tea comes and has revolutionized the tea drinking habits of the Indians. Most Indians drink tea with milk and sugar. Traditionally, a guest in any Indian home is welcomed with a cup of tea
1.1.3 Tea Drinking in India In India, tea is a very popular beverage and Indians love their cup of tea. The importance of tea drinking in India cannot be confined to words, Indians have to have their cup of hot steaming tea first thing in the morning in order to stimulate their senses and refresh themselves. In fact some even argue that tea as a beverage enjoys much more preference as compared to coffee in India. Tea is like a comfort drink for many Indians, especially on rainy days, you can always catch them savoring a steaming cup of tea along with ‘pakoras’. India is the world’s largest producer of tea and most of the tea production in India takes place in Assam, West Bengal in northeast and Nilgiris in south. Tea is popular all over the country mainly as an evening or breakfast drink but nothing complements a family get-together or a college reunion more than an endless supply of tea throughout the day. In fact it is so popular that it is a cultural norm tea to offer tea to guests and visitors instead of alcoholic drinks. Although in recent years many people have shifted to imbibing tea liquor, the age-old 7
tradition is to blend the tea liquor with a little bit of milk, add sugar in accordance with one’s taste and then sip it contentedly. Almost everywhere you go you are sure to find tea or ‘chai’. Although there are lots of different types of tea available, the most common variety has to be the ‘railway tea’ type hands down. This one is basically a cheap version (Rs.2-Rs.5 per cup) which is sweet and uniquely refreshing once you develop a liking for it. This type of tea is made by brewing CTC tea leaves along with milk and sugar and serving it steaming hot. Another famous variety of tea in India is the ‘Masala chai’ which has spices added to tea during its preparation. The commonly used spices are ginger, cardamom, cinnamon and black pepper. However this type of tea does not appeal to all and sundry, it takes some time before you cultivate a taste for it. Masala tea is popular in Central as well as Northern India only because the people in Eastern India like their tea without any spices.
1.1.4 Tea Industry in India Tea isn’t simply tea in India but it is like a staple beverage here and a day without it is impossible and incomplete. Indians prefer their steaming cup of tea because for them it acts as an energy booster and is simply indispensable. This popular beverage has a lot of health benefits too as its antioxidants help to eliminate toxins and free radicals from the blood. Originally tea is indigenous to the Eastern and Northern parts of India, but the tea industry has expanded and grown tremendously over the years, making India the largest grower and producer of tea in the world. The tea production in India was 979,000 tonnes as of 2009. In terms of consumption, export and production of tea, India is the world leader. It accounts for 31% of the global production of tea. India has retained its leadership over the tea industry for the last 150 years. The total turnover of this industry is roughly Rs.10, 000 crores. Since 8
1947, the tea production in India has increased by 250% and the land are used for production has increased by 40%. Even the export sector of India has experienced an increase in the export of this commodity. The total net foreign exchange in India is roughly Rs.1847 crores per annum. The tea industry in India is labor intensive, an depends heavily on human labor instead of machines. This industry provides employment to more than 1.1 million Indian workers and almost half the workforce constitutes of women. There is a wide variety of tea offered by India; from Green Tea to CTC black tea to the aromatic orthodox black tea, the range of tea available in India is unparalleled. Indians take a lot of pride in their tea industry because of the pre-eminence of the industry as a significant earner of foreign exchange and a significant contributor to India’s GNP. The three prominent tea-growing regions in India are Darjeeling, Assam and Nilgiri. While Darjeeling and Assam are located in the Northeast regions, Nilgiri is a part of the southern region of the country. A visit to these regions is made truly memorable by the endless rolling carpets in green which are the tea gardens and one cannot but help feeling enthralled and captivated at the sight of the huge tea estates. Majority of the tea factories are located within the premises of the tea estates and this is what accounts for the freshness of the tea. The process of tea production has a series of procedures. The process starts with the plucking of tea leaves in the tea estates by women employees carrying a basket over the head. There are mainly two ways of producing tea in India namely the CTC black tea production and Orthodox black tea production. CTC is an acronym for crush, tear and curl. The tea produced by this method is mostly used in tea bags. The orthodox tea is a whole leaf type tea. The production method for both consists of five stages, namely withering, rolling, fermentation, 9
drying and finally storing. It is not possible to compare the two as they are processed using different varieties because their quality depends on factors such as rainfall, soil, wind and the method of plucking of tea leaves and both possess a unique charm of their own. As the primary producer of an assortment of tea, India is the ideal destination for all tea enthusiasts. The only tea region in India that comprises exclusively China, or China-hybrid, tea bushes is the valley of Kangra. Kangra district is situated in the North-West Indian state of Himachal Pradesh. This is mountain district, very much part of the Himalayas with dramatic landscapes ranging from pine tree-covered slopes to frozen high-altitude deserts and deep gorges with bubbling streams that flow into the Ganges river of India. The craggy Dhauladhar range towers over the Kangra Valley, where in the foothills lies India’s smallest tea region with its own tea town of Palampur. Kangra Valley tea is quite naturally a specialty tea. The China leaf, when processed according to quality norms, yields a distinctive brew that’s golden in colour, with a sweet undertone and none of the astringency associated with Darjeeling teas. Kangra Valley tea appeals to the Western palate as it is best when taken neat, without milk or sugar. That’s a downside for local Indian markets, but an upside for consumers in Europe, US and Japan.
1.1.5 Economic importance The economic importance of the genus Camellia is primarily due to the use as tea. Tea was initially used as a medicine and subsequently as a beverage. Now it is proven well by research that tea has the potential as an important future raw material for the pharmaceutical and nutraceutical industries. Apart from being used as a beverage, green tea leaves are also used as vegetable such as ‘leppet tea’ in Burma and ‘meing tea’ in Thailand.
10
Though the oil of tea seeds is used as lubricant, yet extraction from seeds is not economical (Islam et al., 2005). Additionally, cakes of tea seed contain saponins, which have poor value as fertilizer and are unfit for animal feed due to low nitrogen, phosphorus and potassium content. However, these can be used successfully in the manufacture of nematocide (Islam et al., 2005). Tea leaves have more than 700 chemical constituents, among which flavanoids, amino acids, vitamins (C, E, K), caffeine and polysaccharides are important to human health. Importantly, the vitamin C content in leaves is comparable to that of lemon. Tea drinking is now being associated with cell-mediated immune responses of the human body and reported to improve the growth of beneficial microflora in the intestine. Tea also reported to impart immunity against intestinal disorders, protect the cell membranes from oxidative damages, prevent dental caries due to presence of fluorine, normalize blood pressure, prevent coronary heart diseases due to lipid depressing activity, reduce the blood-glucose activity and normalizes diabetes (Chen, 1999). Tea also possesses germicidal and germ static activities against various gram-positive and gram-negative human pathogenic bacteria such as Vibrio cholera, Salmonella sp., Clostridium sp. (Chengyin et al., 1992). Both green and black tea infusions contain a number of antioxidants like catechins, myrcetin, quercetin that have been reported to have anticarcinogenic, antimutagenic and anti-tumorous properties.
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1.2 Tea processing The most common misperception is that the different types of tea come from different tea plants. Black, Green and Oolong teas are all derived from the same plant Camellia sinensis. The different methods of manufacture of tea account for the marked differences in the chemical compositions of green, oolong and black teas. Quality of made tea is determined by the chemical composition of the shoot, the environment and process conditions. Plucking is a first and very important step in tea manufacture. The tender apical bud and the subtending 2 leaves are used to make the commercial tea.
Withering
Fig 1.2: Withering of tea leaves Newly plucked leaves are thinly spread to dry during this process. Heated air is forced over the leaves if the climate is not suitable. The main goal of this process is to reduce the water content. By the end of this process, the leaves should be pliable enough to be rolled 12
Rolling From the withering racks, the leaves are now twisted and rolled so that the leaf cellular structure are broken up. Sometimes shaking is done as well.
Fig1.3: Rolling of tea leaves Volatile components are released with this rolling process that give the tea its distinctive aroma. The leaves can be rolled with machinery or by hand. The juices that are released remain on the leaf; a chemical change will occur shortly.
Oxidation This is the chemical process where oxygen is absorbed. This process began once the leaf membranes are broken during the rolling process. Oxidation causes the leaves to turn bright copper in color by the oxidation of flavanols by the enzyme polyphenol oxidase. This process is the main deciding factor whether we have Green, Oolong or Black tea.
13
Drying or Firing
Fig 1.4: Drying of tea leaves In this stage the leaves are dried evenly and thoroughly without burning the leaves. Firing the leaves stops the oxidation process. The moisture level in the final product is brought down to 3 per cent.
1.3 Different types of teas Black tea The Black tea process goes through the most stages. Once the leaves are picked, they are left to wither for several hours. After the leaves are rolled, volatiles from the leaves are brought to the surface. The last step consists of placing the leaves in an oven with temperatures reaching up to 190-200 ° C. When the leaves are 80%, the leaves complete their drying over wood fires. The resulting product is brownish (sometimes black) in color and is sorted accordingly to size, the larger grade is considered "leaf grade," and smaller "broken grade" are usually used for tea bags.
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Oolong tea Oolong goes through a similar process that black tea goes through. The first two steps are withering and rolling. Withering is done for a shorter period and under the Sun. Followed by a short indoor withering and rolling. The oxidation period for oolong is very short and the leaves are dried after rolling. This prevents the full oxidation of catechins to theaflavins and thearubigins. The infusion is light yellow because of theaflavins formation but no thearubigins. The oxidation process is stopped by firing. For oolong tea, the leaves are heated at a higher temperature so that they can be kept longer, due to the lower resulting water content. Green tea The process for making green tea is the shortest. Inactivation of enzyme polyphenol is done to prevent the oxidation of catechins at the first instance. The leaves are cooled and dried to dry any extra moisture. This is followed by rolling mainly to give shape to the product. The moisture in the leaves is sequentially dried with intermittent rolling. The green tea leaves usually remain green. White tea This is the rarest variety of tea. Apical buds are picked and harvested before they are fully open and the buds still have a covering of white hairs on them. White tea undergoes the least processing and is also not fermented. It has a light and sweet flavor and contains less caffeine and more antioxidants than any other type of tea.
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1.4 Health advantages of tea •Antioxidant Powerhouse: Researchers have found that green tea was the best antioxidant scavenger of deadly free radicals. Free radicals are very powerful oxidants, which cause intense cell damage. When exposed to oxygen cell tissues are vulnerable to free radical attachment, causing an effect much like that of rust. Over time this may lead to cancer or cardiovascular diseases. Antioxidants in tea are able to neutralize the damaging effects of oxygen and free radicals that are present in the body. Antioxidants slow or prevent cell damage from exposure to oxygen by creating a barrier around cell tissue. A single dose of tea with or without milk increases plasma antioxidant activity in humans. •Cancer prevention: Green Tea has been found to inhibit the growth of esophageal and stomach tumors in mice. Green and black tea could inhibit the development of pre-cancerous lesions as well. •White tea health benefits: Recent studies show that the polyphenols found in green tea appear in greater concentrations in white tea helping to destroy bacteria and other organisms that cause disease. •Improved cardiovascular health and reduced risk of stroke: Flavonoids are theorized to improve the lining of blood vessels, accounting for the decrease. Studies show that drinking black tea helps to prevent narrowed or clogged arteries that lead to ischemic heart disease, heart attack, or stroke. •Reduces “bad” cholesterol: Black tea was shown to reduce LDL-cholesterol (“bad cholesterol”) 16
•Oral health and prevention of tooth decay and bad breath: Green tea, specifically flavonoids, mainly catechins, found in green tea have exhibited inhibitor effects on the growth of cariogenic bacteria by inhibiting the adherence and growth of plaque bacteria at the tooth surface. Polyphenols found in both green and black tea can block bacteria from producing foul-smelling compounds such as hydrogen sulfide in the mouth •Reduced risk of kidney stones: The study on women, part of the 81,093 subjects in the long-term Nurses' Health Study, reported in the "Annals of Internal Medicine" in 1998 that tea drinking decreased the risk for kidney stones by 8 percent. The study on men, published in the "American Journal of Epidemiology" in 1996, involved 45,289 men and found tea drinkers in the group had a 16 percent reduced risk of forming stones. •Retards the aging process: It has been shown that Green tea reduces infection and the stresses of bacteria on the system thus significantly retarding the aging process. •Immune booster: Blood cells from tea drinkers respond 5 times faster to germs than those of coffee drinkers.
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1.5 WHY THIS PROJECT?
The main reason for picking the topic “Purification, Estimation and Analysis of Catechins from Tea Leaves” is that catechins are water soluble and hence not easy to separate. Moreover research has proven various health benefits of the catechins present in the tea leaves making it an extremely useful component having a wide range of medicinal applications.
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2. REVIEW OF LITERATURE 2.1 Tea varieties There are two main varietals of Camellia sinensis, Camellia sinensis var. sinensis, better known as China bush, and Camellia sinensis var. assamica, also known as Assam bush. However, all together, there are more than a thousand sub varieties of Camellia sinensis. Camellia sinensis var. assamica - The Assam variety predominantly grown in the Assam region or plainer areas. It is a small tree (single stemmed) with large leaves. In the wild it reaches a height of 6 to 20 meters (20–65 feet) and is native to north-east India, Myanmar, Vietnam, and South China. In tea estates it is kept trimmed to just above waist level (1m). It is a lowland plant that requires high rainfall but good drainage. It does not tolerate extreme temperatures. All teas from Assam, Kenya and majority from Sri Lanka are based on this variety. The Assam variety produces malty, earthy strong infusions. Camellia sinensis var. sinensis - This is the China variety characterized by small leaved bush with multiple stems that reaches a height of about 3 meters. It is native to South-East China. All teas from Darjeeling, major proportions from Japan, China and some from Sri Lanka are based on this variety. The China variety produces flowery mild infusions. bush growth of sinensis, assamica resembles a small tree reaching heights of 35 to 50 feet.
2.2 Chemical composition of tea Chemical composition of a ‘two and a bud’ of sinensis and assamica varieties is given in 19
Table1.1 The assamica variety is richer in Catechins compared to the sinensis variety. The Catechins in assamica variety are attached to mono- or di-glycosides while in sinensis they are attached to more glycosides (triglycosidic). The sinensis variety has higher per cent of volatile substances like linalool while the assamica variety possesses lower percent of volatile substances.
Table2.1: Chemical composition of a typical two and a bud of assamica and sinensis varieties Component Polyphenols Catechins (Flavan-3-ols): EGCG ECG EGC EC C Flavonols and their glycosides Caffeine Lipids and fatty acids Sugars Theanine and amino acid Volatiles (High b.p. terpenoids)
Content 10-34%
Sinensis 10-12%
3-5% 7% 7% 2-5% traces
Assamica 13-29%
8.59 1.35 2.38 1.13 0.07 Triglycosides
12.10 3.35 0.35 1.44 0.02 Mono and di-glycosides
Higher
Lower
Tea shoots are extremely rich in polyphenolic compounds, the largest group being the catechins which constitute up to 30% on dry weight basis. Several catechins are present in significant quantities and these are Epicatechin (EC), epigallocatechin (EGC), Epicatechin gallate (ECG), epigallocatechin gallate (EGCG) and catechin (C) and gallo catechins (GC).
20
EC
EGC
ECG
EGCG
C
GC
Fig2.2.Structures of Six major Catechins from tea. 21
2.3 Advancements in Tea Research:
Tea extracts are of great interest as food antioxidants due to beneficial effects on human health. Many great researches have been done in this field in past few years.
Studies done by Kempler et al. (1977) and Onishi et al. (1981) suggested that a diet supplemented with green tea may beneficial in dental caries management.
The use of Liquid Chromatography for the determination of tea constituents was first reported by Hoefler et al. (1976). The report demonstrated identification of five catechins (C, EGC, EC, EGCG and ECG) directly in green tea infusion. The separation was lacking in resolution, but represented an early and significant achievement towards the identification and measurement of catechins in tea.
Roedig-Penman et al. (1997) reported antioxidant properties of catechins and green tea extracts in Model Food Emulsions stored for longer periods, which are more relevant to normal food systems.
Zijp et al (2000) studied the effect of tea on human health. Iron binding ability of Catechins was highlighted. Because of the gallyol group in their structure, they tend to bind with the iron on foods, thus inhibiting its absorption in the body. This factor is considered when using Catechins in foods that are iron rich , such as cocoa, liver, kidney, dried fish, sesame seed and shellfish.
22
Ryoko Kuruto-Niwa et al. (2000) studied the effects of tea catechins on the ERE-Regulated Estrogenic Activity. Tea catechins exert many biological effects, including anticancer and antibacterial activities. Also, it is reported that some plant flavonoids exhibit estrogenic activity. The estrogenic or antiestrogenic activities of catechins in HeLa cells transiently transfected with an estrogen response element (ERE)-regulated luciferase reporter and an estrogen receptor (ER) α or ERβ expression vector. Catechins alone did not induce luciferase (luc) activity in either of the ERs. Addition of 17β-estradiol (E2) plus epicatechin gallate (ECG) or epigallocatechin gallate (EGCG) at 5 × 10-6 M resulted in significant decreases in the ERα-mediated luc activity compared with that of E2 alone. On the contrary, lower concentrations significantly increased the E2-induced luc activity. Similar effects were observed with tamoxifen. The ERβ-mediated estrogenic activities were stimulated by catechins. In conclusion, some catechins, particularly EGCG, were antiestrogenic for ERα at higher doses and co-estrogenic for ERα at lower doses and for ERβ. The lower doses were found in human plasma after tea-drinking. Different effects of catechins on 6-hydroxydopamine-induced apoptosis in PC12 cells were studied by Jin et al. (2001). PC12 cells were pretreated with the five catechins for 30 min before exposure to 250 μM 6-OHDA. MTT results showed that the five catechins had different effects: EGCG and ECG had obvious concentration-dependent protective effects at 50−400 μM; EC and (+)-C had almost no effects; and EGC especially decreased cell viability. Catechins also had different effects on apoptotic morphology. Only 200−400 μM EGCG and ECG kept cells adhering well. When pretreated with other catechins at any concentration, PC12 cells became round and some of them were detached as when treated with 6-OHDA. Typical apoptotic characteristics of PC12 cells determined by fluorescence 23
microscopy, flow cytometry, and DNA fragment electrophoresis showed significant inhibitory effects against PC12 cell apoptosis after the cells were treated with 250 μM 6OHDA for 24 h or pretreated with catechins before it. The preincubation was done with 200−400 μM EGCG and ECG. The other catechins had little protective effect. Therefore, at 200−400 μM, the classified protective effects of the five catechins were in the order ECG > EGCG EC > (+)-C > EGC. The data also indicated that EGCG and ECG might be potent neuroprotective agents for Parkinson's disease.
Baumann et al. (2001) studied an efficient protocol for the preparative purification of the major tea catechins specially (−)-epicatechin gallate and epigallocatechin gallate, employing liquid
liquid
partitioning,
high-speed
countercurrent
chromatography,
and
gel
chromatography for final purification.
Inhibition of cancer cells by tea catechins was studied by Morre et al (2002). The study suggested that the anticancer properties of tea catechins are most frequently attributed to the principal catechin the (-)-epigallocatechin-3-gallate (EGCg). The amount of EGCg required to inhibit cancer–associated cells was reduced 10 times by combination of inactive catechins such as (-)-epicatechin (EC), (-)-epigallocatechin (EGC) or (-)-epicatechin-3-gallate (ECG). EGCg exhibited significant activity with an EC50 for inhibition of HeLa growth of ca. 2 mM (1 mg/ml). Sing et al. (2002) studied the effect of EGCg on human endothelial cells. He reported that EGCg acts as an angiogenesis inhibitor by modulating protease activity during endothelial morphogenesis .Angiogenesis is a crucial step in the growth and metastasis of cancer.
24
Zuo et al. (2002) developed a simple and fast HPLC method for simultaneous determination of four major catechins, gallic acid and caffeine using a photo diode array detector. After multiple extractions with aqueous methanol and acidic methanol solutions, tea extract was separated using a methanol–acetate–water buffer gradient elution system on a C18 column.
Cabrera et al. (2003) developed a simple and fast HPLC method using PDA for simultaneous determination of four major cCatechins, gallic acid and caffeine. After multiple extractions with aqueous methanol and acidic methanol solutions, tea extract was separated within 33 min using a methanol-acetate-water buffer gradient elution system on the C18 reversed-phase packaging column (4.5 mm 25 cm, 5 ím). The wavelength was set in the range of 200-400 nm. A gradient elution was performed by varying the proportion of solvent A (water-acetic acid, 97:3 v/v) to solvent B (methanol), with a flow rate of 1 mL/min. The mobile phase composition started at 100% solvent A for 1 min, followed by a linear increase of solvent B to 63% in 27 min. Antioxidant property of the tea components was also studied and analyzed.
Cabrera et al. (2003) also studied different tea components with antioxidant activity. Levels of essential elements with antioxidant activity, as well as catechins, gallic acid, and caffeine levels, in a total of 45 samples of different teas were evaluated. Chromium, manganese, selenium, and zinc were determined in the samples mineralized with HNO3 and V2O5, using ETAAS as the analytical technique. The four major catechins [(−)-epigallocatechin gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), and (−)-epicatechin (EC)], gallic acid (GA), and caffeine were simultaneously determined by a simple and fast HPLC method using a photodiode array detector.
25
Chan et al(2003) reported the use of capillary electrophoresis(CE) for the determination of theanine, caffeine and catechins in fresh tea leaves and oolong tea. CE separated these tea polyphenols from three other tea ingredients,namely, caffeine, theophylline, and theanine, within 8 min. The young leaves (apical bud and the twoyoungest leaves) were found to be richer in caffeine, (-)-epigallocatechin gallate (EGCg), and (-)-epicatechin gallate (ECg) than old leaves (from 5th to 7th leaves). On the other hand, the old leaves(from 8th to 10th leaves) contained higher levels of theanine, (-)-epigallocatechin (EGC), and (-)-epicatechin (EC). Results from a comparison of fresh young tea and oolong tea compositions indicated oolong tea contained more theanine and catechins than fresh young tea.
Sharma et al. (2004) studied the effect of manufacturing procedure and temperature of infusing water on the extractability of catechins. Teas were infused at Catechins, especially EGCG, EGC, and EC, showed marked differences when extracted at different temperatures. Considerable amounts of catechins and caffeine can be extracted each time when the same leaf is infused repeatedly 4–5 times. Unno et al. (2005) developed a rapid method for extracting tea catechins from human plasma samples with a solid-phase extraction technique and to subsequently measure their concentrations using an HPLC system. The method was successfully applied to measure tea catechin concentrations in the plasma of two healthy subjects who received a single ingestion of a green tea beverage. The proposed method enables the rapid and accurate quantitation of plasma tea catechins and might prove useful for the evaluation of beneficial health effects of tea consumption.
26
Friedman et al. (2006) studied the structure activity relationships of tea compounds against Human Cancer Cells. Aqueous and 80% ethanol/water extracts of the same tea leaves were evaluated for their ability to induce cell death in human cancer and normal cells using a tetrazolium microculture (MTT) assay. Compared to untreated controls, most catechins, theaflavins, theanine, and all tea extracts reduced the numbers of the following human cancer cell lines: breast (MCF-7), colon (HT-29), hepatoma (liver) (HepG2), and prostate (PC-3) as well as normal human liver cells (Chang). Statistical analysis of the data showed that (a) the ant carcinogenic effects of tea compounds and of tea leaf extracts varied widely and were concentration dependent. 80% ethanol/water extracts with higher levels of flavonoids determined by HPLC were in most cases more active than the corresponding water extracts; and flavonoid levels of the teas did not directly correlate with anticarcinogenic activities. Vyas et al. (2007) investigated O-acyl derivatives of (−)-epigallocatechin-3-gallate as antitumor agents. The partially purified catechin fraction isolated from tea extract was treated with a variety of acylating agents (acyl anhydrides/chloride) to obtain EGCg O-acyl derivatives. These compounds were evaluated for their antitumor activity by use of a twostage
carcinogenesis
model
in
7,12-dimethylbenz[a]anthracene
(DMBA)/12-O-tetra
decanoylphorbol 13-acetate (TPA)--induced cancer in Swiss albino mice. The study showed that there was a significant decrease in the antitumor activity with the increase in size and branching of the chain length of acyl groups. The results indicated that O-acyl derivatives of (−)-EGCgG have the potential to be developed as cancer chemopreventive agents. Sharma et al. (2007) studied the health benefits of tea consumption. In its review article, he highlighted the antioxidant property of tea and reported its use in the management of colon, 27
esophageal, lung cancers, urinary stones and dental caries. He suggested that the polyphenols and the catechins present in the tea can decrease the risk factor of specific type of cancers by including phase I and phase II metabolic enzymes that increase the formation and excretion of detoxified metabolites of carcinogens. Yoshinori Uekusa et al. (2007) developed an isocratic HPLC procedure for simultaneous determination of six catechins, gallic acid, and three methylxanthines in tea water extract. A baseline separation was achieved on a Cosmosil C18-MS packed column with a solvent mixture of methanol/doubly distilled water/formic acid (19.5:80.2:0.3, v/v/v) as mobile phase. A gradient HPLC procedure was also provided for the separation of these tea components. Minor catechins such as EGC, EC, and (−)-gallocatechin 3-gallate were found to be higher in Japanese green tea products, whereas ECg, gallic acid, theophylline, and theobromine were found to be higher in Chinese green tea products. Oolong tea products possessed lower levels of catechins, whereas pu-erh tea products contained negligible amounts of these constituents.
Yuzo Mizukami et al. (2007) developed a high-performance liquid chromatography-based method for simultaneous analysis of nine catechins, gallic acid, strychnine, caffeine, and theobromine in green tea by using catechol as an internal standard. Although the high cost and instability of the catechin reference standards limit the application of this method, the addition of ascorbic acid to the standard stock solution preserved the stability of the reference standards in the solution for 1 year when stored at −30 °C. This method proved to be appropriate for quantification and yielded good correlation coefficients, detection levels, repeatability, reproducibility, and recovery rates. 28
Sirk et al. (2008) performed the molecular dynamics simulation to study the interactions of bioactive catechins (flavonoids) with lipid bilayer. Experimental studies rationalized catechins having anticarcinogenic, antibacterial, and other beneficial effects in terms of physicochemical molecular interactions with the cell membranes. Findings show that the seven tea catechins evaluated have a strong affinity for the lipid bilayer via hydrogen bonding to the bilayer surface, with some of the smaller catechins able to penetrate underneath the surface. EGCg showed the strongest interaction with the lipid bilayer based on the number of hydrogen bonds formed with lipid headgroups. Muzolf et al. (2008) investigated the effect of pH on the radical scavenging capacity of green tea catechins. pH-dependent increase in radical scavenging activity of the catechins is due to an increase of electron-donating ability upon deprotonation. The data also reveal that the radical scavenging activity of the catechins containing the pyrogallol (or catechol) and the galloyl moiety over the whole pH range is due to an additive effect of these two independent radical scavenging structural elements. Wang et al. (2008) applied the high-speed countercurrent chromatography for the separation of theaflavins and catechins. The chromatography run was carried out with a two-phase solvent system composed of hexane–ethyl acetate–methanol–water–acetic acid. Pure theaflavin, theaflavins-3-gallate, theaflavins-3′-gallate and theaflavin-3,3′-digallate were obtained from crude theaflavins sample and black tea and structures were positively confirmed by 1H NMR and 13C NMR, MS analysis, HPLC data and TLC data.
29
3. Material and Methods: 3.1 Materials: The tea shoots used in all type of analysis in this report was plucked from the Banuri Tea Ex[perimental Farms of CSIR-IHBT. All the shoots used were freshly plucked on the day of analysis only. Solvents used such as acetone, methanol, chloroform, ethanol were all purchased from Merck, India. The seven standards (+)-catechin (C), (−)-epicatechin (EC), (−)-epicatechin gallate (ECg), (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCg), (-)catechin galate(CG), ECG, EGC, catechin hydrate, caffeine and gallic acid were obtained from Sigma-Aldrich, India. XAD-4, XAD-7 and XAD-16 resins used for purification were bought from Sigma Aldrich, India. DPPH (2,2-diphenyl-1-picrylhydrazyl) used for analysis of redical scavenging activity was purchased from Sigma Aldrich , India. C-18 used for isolation of individual Catechins was purchased from Biotage, India.
30
3.2 Methods: 3.2.1 Extraction and Purification of Catechins: Extraction in chemistry is a separation process consisting in the separation of a substance from a matrix. Extraction of fresh tea leaves was done with 60% methanol and 70% acetone for respective analysis by HPLC and Spectrophotometer. The extracts were centrifuged at 4°C to enhance cell breakage and to separate the particles from the extract. In case of bulk material the tea leaves were homogenized in a homogenizer along with the solvent and maceration was allowed to take place in order to obtain maximum yield. The extraction process was repeated at least 3 times to avoid any loss of essential components.
3.2.2 Extraction of each component of tea shoot: Tea is originally made from the apical bud of the bush along with the subsequent 2 leaves which is known as “2 and bud”. Extraction of these parts was done in order to check the varying concentration of catechins from the bud to the 3rd stem in a tea bush. Freshly plucked leaves from the tea gardens were divided into seven parts starting from the bud, first Leaf, stem between bud and first leaf, second leaf, stem between second and third leaves, stem after third leaf. These parts were separately dried in the microwave and crushed to make powder. 100 mg of each sample was extracted with 70% methanol which was accompanied by vortexing and centrifuged at 4°C for 10 min to enhance the separation. The extraction process was repeated 3 times with a total extract of 5ml. 31
3.2.3 Purification by column chromatography: Column chromatography: Column chromatography is liquid chromatography technique which is used to purify individual chemical compounds from a mixture of compounds. It is often used for preparative applications on scales from micrograms up to kilograms. In column chromatography, the stationary phase is a solid adsorbent mostly silica, alumina or any resin which is placed in a vertical glass (usually) column (5mm to 50mm dia) and the mobile phase, a liquid, is added to the top and flows down through the column (by either gravity or external pressure). Column chromatography is generally used as a purification technique or isolation of desired compounds from a mixture.
Fig3.1: Column chromatography. 32
The mobile phase or eluent is either a pure solvent or a mixture of solvents. It is chosen so that the retention factor value of the compound of interest is roughly around 0.2 0.3 in order to minimize the time and the amount of eluent to run the chromatography. The eluent has also been chosen so that the different compounds can be separated effectively. The liquid solvent (the eluent) is poured on the top and passed through the column by gravity or by the application of air pressure. An equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. As the different components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation will be achieved. The individual components or eluents, are collected as the solvent drips from the bottom of the column. There is an element of trial and error involved in selecting a suitable solvent and column stationary material for the purification and separation of individual constituents from a mixture. XAD-4 resin: Amberlite XAD-4 are polymeric white translucent beads, sometimes with faint yellow cast and are synthetic in nature. The nonpolar XAD resins are generally used for adsorption of organic substances from aqueous systems and polar solvents. It is hydrophobic polyaromatic in nature (dipole movement 0.3) and are used for the removal of hydrophobic compounds up to 40,000 mw. It has pore volume of 0.98 ml/g and mesh size varies from 20 to 60 nomimal. With a pore dia of 50 Angstroms it has a surface area of 725 sqm/g. in case of polyphenols they act as a good absorbents and are generally used for purification purposes. XAD-7 resin: Amberlite XAD-7 are also polymeric white translucent beads, sometimes with faint yellow cast and are synthetic in nature with a chemical nature of an acrylic ester it 33
is used to adsorb molecules up to MW 60,000. With a pore volume of 1.14 ml/g its mesh size varies from 20 to 60. It has pore dia of 90 Angstroms its area is 450 sqm/g. XAD-16 resin: Amberlite XAD-16 are hydrophobic polyaromatic white translucent beads and are synthetic in nature. These are used to remove hydrophobic compounds up to 40,000 MW. With the highest pore volume of 1.82 mL/g its pore dia is 100 Angstroms. It gives the surface area of 900 sqm/g and a dry density of 1.08 g/mL. In case of polyphenols it has a better holding capacity and is more efficient than XAD-4 and XAD-7. C-18 silica: Silica gel is a granular, vitreous, porous form of silicon dioxide made synthetically from sodium silicate. Silica gel is tough and hard; it is more solid than common household gels like gelatin or agar. It is a naturally occurring mineral that is purified and processed into either granular or beaded form. In chemistry, silica gel is used in chromatography as a stationary phase. C-18 is the most preferred silica gel for compound isolation. It has 18 carbon atoms attached in a chain and the particle size varies from 40-63 µm. it has pore size of 90 Å and a total surface area of 400m2/g.it is mainly used for reversed phase in HPLC, but can be used in reversed as well as normal phase in column chromatography.
3.2.3.1 Extraction of material for selection of column resin: The material of tea leaves (two and a bud) was increased from mg to gm. Tea leaves were plucked fresh from the tea gardens (250gm), were macerated, extracted and concentrated to pass it through the column. This was done for the selection of appropriate resin required for purification of catechins.
34
Fresh Tea leaves (2 and a bud) Camellia sinensis var. assamica variety) 250 gm fresh tea leaves were dried in microwave till constant weight and crushed to powder using pestle and mortar
Extraction was done with 60 % acetone (500 ml x 3) and concentrated to remove acetone
The concentrated extract was passed through 3 resin columns for comparative study of purification
Resin 2: XAD-7(35gm)
Resin 1: XAD 4(35 gm) The resin had less carrying capacity and it easily eluted with water
The chloroform was dried and analyzed with HPLC-PDA and GC-FID and collected as a Bi-Product.
The resin was capable of holding the catechins to an extent
Resin 3: XAD-16(35gm) The resin had the highest holding capacity and could retain maximum catechins
The column was run in reverse phase and the impurities were removed with the help of water and caffeine was eluted as a bi-product with Chloroform
The column was eluted with Methanol and the fractions were collected which were tested with TLC
The final caffeine free fractions were collected freeze- dried and analyzed with 35 HPLC-PDA and UV to get purity of the total Catechins.
3.2.3.2 Bulk extraction and purification using XAD-16.
3kg of Fresh Tea leaves (2 and a bud) Camellia sinensis mixed variety were plucked from the gardens of the institute.
The extraction was done on wet basis and the material was homogenized with the help of a lab homogenizer .The extraction was done with 60% acetone(800 ml x 6 x 500gm of material ). The extraction process was repeated thrice with a total solvent of 10 lt
The chloroform was dried and analyzed with HPLC-PDA and GC-FID and collected as a Bi-Product.
Partitioning was done with chloroform and hexane to remove respective caffeine and fats.
Concentrated and acetone free extract was passed through resin for purification.
Resin: XAD-16 (130gm) The resin had the highest holding capacity and could retain maximum Catechins
The column was run in reverse phase and the impurities were removed with the help of water .
The column was eluted with Methanol and the fractions were collected which were analyzed with TLC
The final caffeine free fractions were collected freeze- dried 36 and analyzed with HPLC-PDA and UV to get purity of the total Catechins.
10 ml of crude sample was dried, powdered and kept for analysis.
3.2.3.3 Isolation of individual catechins using C-18 column Individual catechins were isolated by from the purified catechins (XAD-16) using column chromatography with C-18 column. The C-18 being non-polar in nature was first rinsed with water followed by methanol. After methanol washing it was then brought to chloroform and was allowed to stand overnight in the same solvent. Purified and powdered extracts were dissolved in chloroform in mg/ml concentration and loaded in the column. The column was eluted with increasing concentration of methanol in a methanol–chloroform solvent system Table 3.1. About twenty to twenty five fractions each of 2ml were collected and analyzed for individual Catechins by TLC and HPLC. Table 3.1: solvent polarity ratio of chloroform-methanol for isolation of catechins. Methanol (%) 2 5 7 10 14 18 20 26 30
Chloroform (%) 98 95 93 90 86 82 80 74 70
37
3.2.4 Methods of Analyses and Instrumentation 3.2.4.1 Thin Layer Chromatography (TLC) Thin Layer Chromatography (TLC) is a commonly used analytical technique that allows for rapid and inexpensive analysis of various mixtures. TLC setup has a stationary phase and a mobile phase. In the stationary phase the supporting material is a glass plate, a plastic sheet or a piece of metal foil. A thin layer of stationary phase i.e. aluminum, C-18 reverse phase, cellulose, ion exchange resins, and many more. is laid over this inert support which may be as thin as 250 µm for analytical separations and as thick as 2-5 mm for preparative separations. A binding agent such as calcium sulphate or gypsum may be incorporated into the chromatographic media to facilitate firm adhesion of adsorbent to the plate. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action. TLC is used to support the identification of a compound in a mixture when the Rf (retention value) of a compound is compared with the Rf of a known compound (both run on the same TLC plate).
Advantages of TLC:
TLC is an inexpensive and a rapid separation technique in which the result can be achieved within an hour
TLC is very sensitive and relatively lower conc. of compounds can be detected and separated.
There are very little materials needed for TLC (chambers, watch glass, capillary, plate, solvent, pencil) 38
Once the best solvent is found, it can be applied to other techniques such as High Performance Liquid Chromatography
Identification of most compounds can be done simply by checking R f literature values.
3.2.4.1.1 Method for identification of Catechins: A TLC method was developed for polyphenols (catechins) to identify catechins in crude tea extract. The individual catechins separated were also detected and analyzed on TLC. MOBILE PHASE: Chloroform: Methanol: Acetic Acid (9:3:3) Identification by Iodine: The plate was heated at 100ºC for 2 min, and then kept in Iodine chamber for few minutes. Evaluation was performed under white light and desired catechins were identified by a yellow spot.
39
3.2.4.2 Spectrophotometer:
Fig3.2: Shimadzu UV-Vis 2450 Spectrophotomoter A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector Working: It is capable of focusing a beam of light of a specified wavelength or wavelength range on a sample containing a constituent that absorbs the light. The instrument often measures the absorbance of light by splitting the light beam (using optics) and comparing the amount of light transmitted through a sample that doesn’t contain the light absorbing constituent (a blank). 40
The basic law that governs the absorption of light by molecules is Beer-Lambert law: log (Io/I) = εcl Io is the intensity of incident light I is the light transmitted through the sample solution c is the concentration of the solute l is the path length of the sample ε is the molar absorptivity log (Io/I) is the absorbance (A) of the solution formerly called the Optical Density (OD). There are two major classes of devices: single beam and double beam. Double Beam: A double beam spectrophotometer compares the light intensity between two light paths, one path containing a reference sample and the other the test sample. It is easy to operate and very stable. Single Beam: A single beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted. Single beam instruments can have a larger dynamic range and are optically simpler and more compact. Depending upon the detection parameters there are majorly two classes:
41
UV-Visible Spectrophotometer: The most common spectrophotometers are used in the UV and visible regions of the spectrum, and some of these instruments also operate into the nearinfrared region as well. Visible region of 400–800 nm is used for detection. Traditional visible region spectrophotometers cannot detect if a colorant or the base material has fluorescence.
IR Spectrophotometer: Spectrophotometers designed for the main infrared region are quite different because of the technical requirements of measurement in that region. One major factor is the type of photosensors that are available for different spectral regions, but infrared measurement is also challenging because virtually everything emits IR light as thermal radiation, especially at wavelengths beyond about 5 μm.
3.2.4.2.1 Preparation of Stock solution Catechin reagent (Reagent A): Catechin reagent or diazotized sulfanilamide stock solution (0.1% w/v) was prepared in 10ml of acetone and stored in a well stoppered dark colored bottle at 4 °C in the refrigerator. Dilute Hydrochloric Acid (30%) (Reagent B): Concentrated hydrochloric acid was diluted (30:70) with distilled water. Catechin standard (mg/ml): Dissolved 0.1 gm catechin hydrate in and made up the volume to 100ml with purified distilled water. Gallic acid (mg/ml): Dissolved 0.1 gm gallic acid in
42
3.2.4.2.2 Preparation for standard curve of polyphenols Aliquots of 0.1% Gallic acid stock solution containing 200-500µl of gallic acid were dispensed into triplicate sets of 25ml volumetric flasks. 500μl of folin ciocalteau (diluted 1:1 with distilled water) reagent followed by 100μl of 35% Na2CO3 were added to the flasks and mixed. The volume is made upto 25 ml mark by distilled water and allowed to react at room temperature for 30 min. The blue color developed is read against reagent blank at 730 nm in a UV-Vis (Shimadzu UV-2450) spectrophotometer. The gallic acid concentration was plotted against absorbance.
3.2.4.2.3 Estimation of total polyphenols in tea : 100μl of the extracted samples were placed in the triplicate sets of 25ml volumetric flasks in place of gallic acid standard solution and total phenols estimated as described above. The absorbance was then read at 730 nm against the reagent blank. The concentration of the total polyphenols was estimated against the standard curve of the gallic acid.
3.2.4.2.4 Preparation for standard curve of catechin Aliquots of catechin stock solution (mg/ml) containing (0.1-0.5ml) of catechin hydrate as a standard were dispensed into triplicate sets of 25ml volumetric flasks. Modified method of Brey et al .(1954) was used in which 1ml of reagent A (1% diazotized sulfanilamide in acetone, w/v) followed by 1ml of reagent B (HCl 30 % v/v) was subsequently added to the aliquots and allowed to react at room temperature for 1 hr. The reagent blank was included. At the end of incubation period the volumes were made up to the 25 ml mark with purified distilled water, shaken well and the absorbance read at 425 nm in a UV-VIS (Shimadzu UV43
2450) spectrophotometer against the reagent blank. The catechin concentration was plotted against absorbance.
3.2.4.2.5 Estimation of total catechins in tea : 100μl of the prepared sample were placed in the triplicate sets of 25ml volumetric flasks in place of catechin hydrate standard solution and total catechins estimated as described above. The absorbance was then read at 425 nm against the reagent blank. The concentration of the total catechins was estimated against the standard curve of the catechin hydrate standard..
3.2.4.2.6 Estimation of total catechins in purified Catechins Different aliquots of purified catechins by XAD-4 , XAD-7 and XAD-16 (mg/ml) containing 0.1 ml of catechin were dispensed into triplicate sets of 25ml volumetric flasks. Method by Singh et al. (1999)One ml of reagent A (1% diazotized sulfanilamide in acetone, w/v) followed by 1ml of reagent B (HCl 30 % v/v) was subsequently added to all the aliquots and allowed to react at room temperature for 1 hr. The reagent blank was included. At the end of incubation period the volumes were made up to the 25 ml mark with purified distilled water, shaken well and the absorbance read at 425 nm in a UV-VIS (Shimadzu UV-2450) spectrophotometer against the reagent blank. The catechin concentration was plotted against absorbance.
44
3.2.4.3 High Performance Liquid Chromatography (HPLC):
Fig3.3 :Waters 717 HPLC with quaternary solvent system,auto sampler and PDA detector High-performance liquid chromatography (HPLC) is an advanced form of liquid chromatography used in separating the complex mixture of molecules encountered in chemical and biological systems, in order to understand better the role of individual molecules. In liquid chromatography, a mixture of molecules dissolved in a solution (mobile phase) is separated into its constituent parts by passing through a column of tightly packed solid particles (stationary phase). The separation occurs because each component in the mixture interacts differently with the stationary phase. Molecules that interact strongly with the stationary phase will move slowly
45
through the column, while the molecules that interact less strongly will move rapidly through the column. This differential rate of migration facilitates the separation of the molecules. Normal-phase chromatography: When stationary phase is polar (e.g. PEG) and the mobile phase is non polar, the process is normal phase chromatography. Reversed-phase chromatography: When stationary phase is non-polar (e.g. ODS) and mobile phase is polar, the process is reversed-phase chromatography. Basis of separation of different components by HPLC: 1) Partition 2) Adsorption 3) Ion exchange 4) Size exclusion
Working of HPLC Solvent delivery systems (Quaternary): It consists of 4 pumps capable of delivering a pulse-less flow of mobile phase at pressures up to 6000 psi. Flow rates up to 2 ml/min with increments of 0.01 ml/min are typical according to column dimensions. In the isocratic mode, where a mobile phase of constant composition is used throughout the run. The mobile phase needs to be prepared externally by mixing the liquids in the required proportion and degassing it by an inline degasser. In the gradient mode, the composition of the mobile phase is constantly changed during the chromatographic run as set by the user to the method. Sample injection system: Sample injection can be done both by autosampler and manual Rheodyne injector: 46
Fig 3.4: Working of HPLC Auto sampler injector: In an auto sampler an automatic needle injects the sample into the column (1 to 100µl) according to the requirement and column capacity. The samples are kept in vials (1ml) and placed as accordingly in the sampler tray for injection. Rheodyne injector: Rheodyne injector is a manual injector system in which the sample is loaded manually into the column with the help of an syringe which loads the sample into the Rheodyne valve. A clockwise rotation of the valve rotor places the sample-filled loop into the mobile-phase stream, with subsequent injection of the sample onto the top of the column through a low-volume, cleanly swept
Fig 3.5:Image of Rheodyne injector
47
channel (Fig 3.6).
Fig 3.6:Internal diagram of Rheodyne injector during loading and injection. Column: The solvent system along with the sample is injected into the column at high pressure. Stationary phases for modern liquid chromatography column typically consist of an organic phase chemically bound to silica or other materials. Particles are usually 3, 5 or 10μm in diameter. Column polarity depends on the polarity of the bound functional groups, which range from relatively non-polar octadecyl silane to very polar nitrile groups. The percent carbon load (amount of bonded phase material loaded on to the silica support, measured as weight percentage of bulk silica) is important in determining the polarity of reversed phase columns. On increasing carbon load and chain length, the polarity of the column is reduced. .
48
End capping of columns (a chemical process that reduces the number of free silanol groups attached to the base silica support material) minimizes competing mechanisms. Normal phase column: Normal phase silica is polar in nature and the mobile phase used in non-polar in nature eg. chloroform, DCM etc Reversed phase column: Reversed phase column silica is non-polar and the mobile phase used is polar in nature eg. Methanol, water, etc Guard column: A guard column protects and extends the life of analytical column. It retains the non-polar substances and any particulate matter in the sample. Guard columns in different sizes (0.5 – 5.0 cm in length) are available for both normal and reversed phase columns. Columns are housed in column housing with a thermostatic system to control the temperature. Columns may be heated to give more efficient separations, but only rarely are they used at temperatures above 60° because of potential stationary phase degradation or mobile phase volatility (Certain resin based columns e.g. carbohydrate columns are heated to 90° to achieve efficient separation of sugars).
DETECTORS: The common detectors used in LC analysis are: 1) UV-VIS detector (PDA): Photo-diode array detector utilizes deuterium or xenon lamp that emits light over UV-Vis spectrum range. Light from lamp is focused by means of an achromatic lens which flows the sample cell and onto a holographic grating. Dispersed light 49
from grating is arranged to fall on linear diode array. Resolution of detector will depend on the no. of diodes in the array and also on range of wavelength covered. Array may contain many hundreds of diodes and output from each diode is regularly sampled by a computer and stored on a hard disc. At the end of run, output from any diode can be selected and chromatogram produced using UV-Vis wavelength that was falling on that particular diode. PDA is used for the detection of compounds detected in the wavelength range of 200 – 800 nm. 2) Evaporating light scattering detector (ELSD): It utilizes spray of nebular gas that atomizes column eluent into small droplets. These droplets are allowed to evaporate leaving solute as fine particulate matter suspended in the atomizing gas. Atomizing gas can be air, if necessary an inert gas(nitrogen). Suspended particles pass through light beam and scattered light viewed at 45 to the incident light and sensed by photomultiplier. The intensity of the light scattered from solid suspended particles depends on their particle size. Therefore, the response is dependent on the solute particle size produced. The output is electronically processed and passed to computer data acquisition system. Theoretically, detector responds to all solutes that are not volatile and as the light dispersion is largely Rayleigh scattering, response should be proportional to the mass of solute present. 3) Fluorescence detector: fluorescence detector is is used specifically for the measurement of specific fluorescent species in samples The fluorescence sensitivity is 10 -1000 times higher than that of the UV detector for strong UV absorbing materials. Fluorescence detectors are very specific and selective among the others optical detectors. When compounds having specific functional groups are excited by shorter wavelength energy and 50
emit higher wavelength radiation which called fluorescence. Usually, the emission is measured at right angles to the excitation.
3.2.4.3.1 Preparation of standard curves Standards of polyphenols i.e gallic acid, catechin hydrate, epicatechin (EC), epigallocatechin gallate (EGCG), catechin gallate (CG), epicatechin gallate (ECG) & caffeine were prepared in 70% methanol (HPLC Grade) and analyzed on concentration range for standard equations was mg/ml (0.05 to 1). All the samples were filtered through hypodermic glass syringe assembly using Millipore membrane filter (0.45μ). Samples prepared were analyzed by method developed by Sharma et al. (2005) on a Waters 717 HPLC using a C-18 reverse phase 250x4.0mmx5µm column fitted with a C-18 guard column with a quaternary solvent manager using a gradient elution employing acetonitrile (A) and 0.1% orthophosphoric acid (B) as the mobile phase(Table 3.2). The mobile phases were degassed before use and area and the retention time of the standard peaks were recorded at 280 nm using Waters 2998 PDA detector. Table 3.2: Gradient solvent system for HPLC TIME (MINUTES) 0 10 12 15 18 20 24 28 51
A%
B%
10 20 35 36 36 30 20 10
90 80 65 64 64 70 80 90
3.2.4.3.2 Study of catechin profile of extracted tea samples: The methanol extracted tea sample (5mg in 100ml 50% methanol) was filtered through hypodermic glass syringe assembly using Millipore membrane filter (0.45 μ). 20 μl injection was used for analysis. The area and the retention time of the analyte peaks in samples were compared with those of respective standards at 280nm (PDA).
3.2.4.3.3 Study of catechin profile of purified catechins : Purified catechin extract was prepared in methanol (mg/ml) concentration. 20 μl injection was used for analyses. The retention time and areas were observed at 280nm (PDA) and compared with those of the respective standards.
3.2.4.4 DPPH radical-scavenging activity: DPPH is a well-known radical generator and a trap ("scavenger") for other radicals. Therefore, rate reduction of a chemical reaction upon addition of DPPH is used as an indicator of the radical nature of that reaction. Because of a strong absorption band centered at about 517 nm, the DPPH radical has a deep violet color in solution, and it becomes colorless or pale yellow when neutralized. DPPH radical-scavenging activity was performed by the method described by Akter et al. (2010). For determination, the purified catechins from XAD-16 (mg/ml) were diluted to a series (200-1000µg/ml) with 60% (v/v) ethanol. Aliquots of different concentration (20µl – 100µl) were mixed with ethanolic solution of DPPH (1.5ml, 0.05mM). The mixtures were shaken vigorously and incubated at 37°C for 30 minutes in dark. At the same time control 52
containing 50% (v/v) ethanol (100 µl) and ethanolic solution of DPPH (1.5ml, 0.05mM) was run. The absorbance was measured at 517nm against ethanol as a blank. The percentage of DPPH scavenging was calculated as follows: DPPH radical scavenging activity (%) = [(Abscontrol – Abssample)/Abs control] x 100 The percentage of DPPH scavenging versus concentration of samples was plotted. The concentration of the sample necessary to decrease the DPPH concentration by 50% was obtained by interpolation from linear regression analysis and denoted IC50 value.
53
4.RESULTS AND DISCUSSION 4.1Spectrophotometer standard curves 4.1.1 Standard curve for total polyphenols Aliquots of Gallic acid were mixed with 500 μl of FR reagent(1N) followed by 100μl of 35% Na2CO3 and the volume was made upto 25 ml with water after the incubation of 30 minutes. The absorbance was taken at 730 nm with an increase in the absorbance value with the concentration(Table A.1). An absorbance versus concentration graph was plotted and the standard curve equation was calculated. Gallic acid standard curve equation: Y=0.097x+0.2888 (R2=0.990) ………………………….. (4.1) The Gallic acid equation was used as a standard curve equation for the estimation of total polyphenols in different samples. In the standard curve equation, y denotes the absorbance and x denotes the concentration of the compound.
4.1.2 Standard curve for total Catechins Aliquots of catechin hydrate (mg/ml) used as a standard were mixed with 1ml of Catechin reagent and 1ml of 30%HCl. After an hour of incubation the yellow colored aliquots were made up to the mark of 25ml with water and the absorbance was noted at 425 nm. The absorbance tends to increase as the concentration is increased (Table A.2). Absorbance versus concentration graph was plotted and equation was formed. The catechin hydrate equation was used as the standard curve equation for the calculation of total catechins in 54
different samples. Catechin standard curve equation: Y=3.25x+0.002; (R2=1.000)……………………………………………………….…….(4.2) In the standard curve equation, y denotes the absorbance and x denotes the concentration of the compound. These equations were used to estimate the concentration of total catechins in the crude extracts and purified compounds.
4.2 HPLC Standard curves of Polyphenols, Catechins & Caffeine All the standards were made in mg/ml concentration and were run on HPLC by the method developed by Sharma et al. (2005) using a C-18 reverse phase 250x4.0mmx5µm column fitted with a C-18 guard column with a quaternary solvent manager using a gradient elution employing acetonitrile (A) and 0.1% orthophosphoric acid (B) as the mobile phase. The areas of all the standards were noted with respect to retention time and concentration versus area graph was plotted table (Table A.3). The equations of each standard were calculated according to the trend line formed and the value of R2 was kept more than 0.9. Gallic acid,
y = 2.5704x - 0.2015…………………………………………..(4.3)
Catechin Hyd
y= 2.4251x - 0.0053……………………………………………..(4.4)
Cafffeine,
y = 7.8478x - 0.1475……………………………………………..(4.5)
EC,
y = 1.6503x - 0.0005………………………………………………(4.6)
EGCG,
y = 5.2837x - 0.2462……………………………………………….(4.7)
CG,
y = 1.01x + 0.0113……………………………………………….(4.8)
ECG,
y = 4.6375x + 0.0962……………………………………………..(4.9)
55
4.3 Total polyphenols of tea leaves: The extracted tea leaves were analysed for total polyphenol of Bray, and Thrope (1954). 100µl of the sample was mixed with 500 µl of FR reagent, 100 µl 35% sodium carbonate and was filled upto the mark of 25ml with water. After half hour of incubation the absorbance was taken at 730nm and the readings were put into the standard equation (Eq 4.1). The results revealed that the total polyphenol content was 26.15%.
4.4 Total polyphenols of different parts of leaf parts: Different parts of the leaf were separately dried, curshed and extracted for the analysis. 100µl of the different sample were put in place of Gallic acid and total polyphenols were estimated. Reading was taken at 730 nm against the blank after 30 minutes of incubation. The absorbance was found to increase as we go down from bud to 3rd leaf. The value of absorbance was put in the gallic acid standard equation 4.1 along with the dilution factor. The total polyphenol concentration varied from 27.17 % to 14.6%. The concentration was maximum in 1st stem(27.17%) followed by the 2nd stem , Bud, 1st leaf,2nd leaf , 3rd stem and and with the least concentration at 3rd leaf(14.69%)(Table 4.1). These results suggested that the total polyphenols are high in the upper stem parts than the leafs. Also the fig 4.1 suggests that the trend tends to decrease as we go down from the bud to the 3rd leaf.
56
Table 4.1: % Concentration of polyphenols in different leaf parts Leaf parts
Concentration(%)
Bud 1st Stem 1st Leaf 2nd Stem 2nd Leaf 3rd Stem 3rd Leaf
23.4 27.17 21.25 27.04 18.5 17.92 14.69
30
Concentration(%)
25 20 15 10 5 0 Bud 1st Stem 1st Leaf
2nd 2nd 3rd 3rd Leaf Stem Leaf Stem Different parts of tea shoot
Fig 4.1.comparison of total polyphenols in different leaf parts
4.5 Total catechins of tea leaves: 100mg of dried tea leaves were crushed and extracted with 60% methanol. 100µl of extracted samples were mixed with 1ml of catechin reagent and 1ml of 30% HCl followed by 1 hr of 57
incubation. The absorbance was measured at 425nm and the values were put into the standard curve equation number 4.2. Te results suggested that the tea leaves were 17.2% pure. HPLC analysis revealed that the total purified catechins were 15.64% including the major catechins i.e. EC, EGCg, CG and ECg making a total of 15.64% (Table 4.2) Table 4.2: major catechins in tea by HPLC
Catechins
Area
%
GA CAT CAFF EC EGCG CG ECG
0.3545077 1.1007547 0.194835 1.57338 0.25166 1.169026
1.279 nd 3.181 2.367 5.887 3.759 3.627 15.64114
Total Catechins
4.6 Total catechins in different parts of leaf: Different parts of the tea leaf were separately dried, crushed and extracted for the analysis. 100µl of each extracted part was added in place of catechin standard in the process of total catechin estimation and absorbance was recorded at 425 nm after 1 hour of incubation. The absorbance measured was put in the standard catechin hydrate equation 4.2 and the concentration was calculated. Concentration of the total catechins ranged from 16.25% to 14.53%. Concentration was found to be maximum in the bud (16.25%) followed by the 1st stem, 1stleaf, 2nd stem, 2nd leaf, 3rd leaf and 3rd stem (14.63%) (Table 4.3). The results in the fig 4.2 suggested that the Catechins are found to be maximum in top most part of the bush 58
i.e the bud and it decreases as we go down the stem. Also the total catechin concentration was found to be more in case of leaves as compared with the stems. Table 4.3:Total catechins of different leaf parts
Leaf part Bud 1st Stem 1st Leaf 2nd Stem 2nd Leaf 3rd Stem 3rd Leaf
Concentration (%) 16.25 16.14 15.55 15.01 14.81 14.54 14.63
16.5
Concentration(%)
16 15.5 15 14.5 14 13.5 Bud
1st 1st Leaf 2nd 2nd 3rd 3rd Leaf Stem Stem Leaf Stem Different parts of tea leaf
Fig 4.2: Comparison of total catechins in different shoot components
59
4.7 Comparative estimation of Catechins of purified Catechins from XAD-4, XAD-7 and XAD-16: 8
Purified catechins from different resins were
100µl of each sample prepared in mg/ml concentration was added in place of catechin
7.02 Yield (%)
analyzed and total catechins were calculated.
7.5 7
6.815 6.55
6.5 6 5.5
hydrate in the process of total catechins
5 XAD-4
estimation. The absorbance was noted at
XAD-7
XAD-16
Resins
425nm after 1 hour of incubation and was put in the catechin standard curve equation 4.2 to
Fig 4.3: % Yield comparison of different resins
calculate concentration of total Catechins. The yield was found to be almost same in all the resins but XAD-4 was found to be on the higher side than other resins (Fig 4.3).Purity of catechins was found to be maximum with XAD-16(77.2%) followed by XAD-7(68.6%) and XAD-4(59.3%)(Table 4.4) Table 4.4: % Purity of catechins from different resins Resin
Purity(%)
XAD-4
59.3
XAD-7
68.6
XAD-16
77.2
60
100 80
possesses the highest percentage purity of Catechins and is a better resin than
Purity(%)
Fig 4.4 clearly shows that XAD-16
XAD-4 and XAD-7 for the purification
60 40 20 0 XAD-4
of Catechins and can be used for bulk
XAD-7 Resins
XAD-16
purification. Fig 4.4: % Purity comparison of different resins
4.8 Total purified Catechins by XAD-16: Bulk purified catechins along with the crude sample kept before the purification process by XAD-16 were made in mg/ml concentration and 100µl of the sample was added in place of catechin hydrate in the process for analyses of total catechins. The absorbance was measured at 425nm after incubation of 1 hr and was put in the catechin standard curve equation number 4.2 to calculate the purity concentration. The purity of the catechins was found to be 43.5% as compared with the purity of the crude that is 7.5%. This clearly shows that XAD-16 has increased the purity of the catechins. Catechin profile of purified catechins (Fig 4.6) was compared with the crude tea extract (Fig 4.5). Table (4.6) shows the major purified catechins (C,EC, EGC, ECg and EGCg) with a total purity of 41.613%.
61
Fig 4.5: Catechin profile of crude using HPLC
Fig 4.6:Catechin profile of purified catechins using HPLC.
62
Table 4.6:Major catechins purified by XAD-16 Catechins GA CAT EC EGCG EGC ECG
Area 2.60839 0.96197 0.867942 5.589009 1.147506 2.417922
Total Catechins(%)
% 5.346 3.34 6.876 14.75 7.56 9.087 41.613
4.9 DPPH radical –scavenging activity: DPPH antioxidant assay is frequently used to determine a radical scavenging activity of natural compounds. DPPH radical involves a hydrogen atom transfer process. In the presence of a hydrogen-donating antioxidant, unstable DPPH radical is reduced to a stable yellow- colored diphenyl picryl hydrazine (Sun et al,. 2011). Fig 4.7 illustrates the concentration dependent curves of DPPH radical scavenging activity of catechins. These catechins are found to be capable of scavenging DPPH in concentration dependent manner. As the concentration was increased the antioxidant activity of catechins also increased. The activity was expressed as IC50 value (µg/ml), i.e. , the concentration necessary to decrease the DPPH concentration by 50%. The IC50 value of purified catechins was found to be 2.12 µg/ml.
63
120
Inhibition(%)
100 80 60 40 20 0 2
4 6 Concentration (µg/ml)
8
Fig 4.7: Scavenging effect of catechins on DPPH radical and each value is expressed as mean of triplicate.
4.10 Isolation of individual catechins by column chromatogpraphy Individual catechins were isolated by column chromatography using C-18 silica as the stationary phase. Simple and easy method was developed after several trials and errors. The column was run in normal phase where C-18 was brought to the polar conditions and non polar solvent system of chloroform-methanol was used. Polarity of the solvent system was increased gradually by increasing methanol. About 25 fractions were collected, 5ml each and were analyzed on TLC and HPLC. The TLC was performed on aluminum coated silica gel with the mobile phase: chloroform: methanol: acetic acid (9:3:3). The catechins were identified by a yellow spot when they were put in the iodine chamber.
64
Standards (EC, EGCg, CG, Catechin hydrate) in mg/ml concentration.
Crude extract
Fig 4.8: TLC plate of standard and crude extract
The Fig 4.8 clearly marks the presence of different catechins separated at different polarity and marked by yellow spots in crude extract. Different catechin standards can also be seen which are separated at different heights.
65
Different fractions collected were spotted on the TLC plate, against the standards to identify each of the isolated catechins (Fig 4.9)
Fractions showing positive result for one of the Catechins
Standards(EC, EGCG, CG, Catechin Hydrate) in mg/ml concentration.
Fig 4.9: TLC plate of different fractions against standard catechins. The identified catechins were later analyzed on HPLC with a run time of 28 minutes and gradient elution. The HPLC profile of the partially purified extract showed a number of peaks indicating the presence of several other constituents. In order to achieve complete purification the polarity of the mobile phase was adjusted and active fractions were pooled 66
and concentrated on rotary evaporator and re-analyzed by HPLC for the estimation of purity of catechins. The five major catechins i.e Catechin Hydrate, ECG, EC, EGC and EGCg were confirmed by the HPLC chromatograms. Fig 4.10 represents the HPLC chromatogram of purified and isolated ECG, Fig 4.11 purified EGCG, fig 4.12 purified and isolated EGC, fig 4.13 purified EC and fig 4.14 represents isolated catechin hydrate. ECG
Fig 4.10: HPLC profile of purified ECG EGCG
Fig 4.11:HPLC profile of purified EGCG. 67
EGC
Fig 4.12:HPLC profile of purified EGC EC
Fig 4.13:HPLC profile of purified EC
68
CAT
Fig4.14 :HPLC profile of purified Catechin Hydrate
The HPLC chromatograms of catechins before isolation (Fig 4.6) were compared with the respected isolated catechins . The results suggests that the concentration of Catechin Hydrate has increased from 3.34% to 31.43%, EGC has increased from 7.56% to 44.70%, EC from 6.87% to 35.66%, ECG from 9.08% to 40.38% EGCG content which was 14.75% before purification has increased to 42.08% after isolation and purification (Table 4.7) Table 4.7: Comparison of catechins before and after purification. Catechins EGCG ECG CAT EC EGC
Before purification 14.75% 9.087 3.34 6.87% 7.56 69
After purification 49.08% 43.38% 31.43% 35.66% 40.705%
5. CONCLUSIONS AND FUTURE SCOPE OF TRAINING 5.1 Conclusion The present process of isolation of catechins was different and simple because the catechins are very difficult to separate. The catechins were purified before the isolation by different resins which as a new concept.The results suggested that catechins purified by XAD-16 had the maximum purity(77.8%) as compared with XAD-7 and XAD-4. Also among the isolated catechins EGCG was isolated to the max(49.08%) followed by ECG,EGC,EC and also CAT(31.43%)(table 4.7). The chemical properties of the components to be extracted plays a very important role in deciding the solvents to be used for partitioning. Caffeine for example is non polar so if we choose a solvent that is less polar than water, then caffeine will go into that solvent layer. But at the same time extractability of the solvent to be used is also equally important. We should ensure that only caffeine and no other constituent of interest should go in that layer. Similarly catechins are polar, so we choose a solvent having polarity greater than that of water, all the catechins will go into that solvent layer. Again extractability should be considered. That solvent is preferred which is capable of extracting only the components of interest. We should go by using the solvents with increasing polarity and accordingly choose the appropriate one.
70
5.2 Future perspective:
Catechins have been found to possess antioxidant properties which have increased its use in the Nutraceutical Industry.
Catechins can also be mixed with various other drugs in a formulation to increase the effect of a particular drug in the body.
Catechins possess anti cancer properties thus they can be used in the manufacturing of medicines for treatment of cancer.
Being a natural source, it can be consumed directly or with some food products but in small doses.
Health drinks can also be made using catechins as an ingredient.
Catechins have an antibiotic as well as anti diabetic effect, thus they can be used for the treatment of diabetes.
71
Appendix Standard curves for Spectrophotometer: Gallic acid: Table: A.1 Absorbance of Gallic acid as standard
Gallic Acid Concentration
Absorbance at
(mg/ml)
730nm
0.33
0.2
0.308
0.32
0.3
0.319
0.31
0.4
0.326
0.5
0.338
0.34
y = 0.097x + 0.2888 R² = 0.9909
0.3 0
0.2
0.4
0.6
Fig:A.1 Gallic acid standard curve Catechin Hydrate: Table: A.2 Absorbance of Catechin hydrate
Catechin Hydrate
as standard
2
Concentration
Absorbance at
(mg/ml)
425nm
1.5 1
0.1
0.321
0.5
0.3
0.977
0
y = 3.25x - 0.002 R² = 1 0
0.5
0.2
0.4
1.621 Fig:A.2 Catechin Hydrate standard curve 72
0.6
Standard curves of HPLC: Table A.3: Areas of different standards with respect to concentration GA
CAT
CAFF
Concentration (mg/ml)
EC
EGCG
CG
ECG
EGC
Area
0.05 0.28217 0.11932 0.27553
0.076 0.08758 0.05348 0.28488
0.1 0.50316 0.24862 0.64929 0.15818 0.20591 0.10869 0.57296 0.2 0.73248 0.45662 1.35056 0.34942
0.8032 0.23329 1.07987
0.4 1.21609 0.97296 3.02059 0.65195 1.88134 0.40725
Gallic acid:
1.9253
Gallic Acid 1.5
Standard Curve of Gallic Acid: y = 2.5704x - 0.2015
1 0.5
R² = 0.9901
0 0
0.1
0.2
0.3
0.4
0.5
Fig:A.3 Gallic acid standard curve by HPLC
Catechin Hydrate: Standard Curve of Catechin Hydrate y = 2.4251x - 0.0053
Catechin Hydrate
1.5 1 0.5
R² = 0.9982 0
0
0.1
0.2
0.3
0.4
0.5
Fig:A.4 Catechin Hydrate standard curve by HPLC 73
Caffeine 4
Caffeine:
3 2
Standard Curve of Caffeine , y = 7.8478x - 0.1475
R² = 0.9984
1
0 0
0.1
0.2
0.3
0.4
0.5
Fig:A.5 Caffeine standard curve by HPLC
EC EC:
0.8 0.6
Standard Curve of EC y = 1.6503x - 0.0005
0.4 0.2
R² = 0.9973
0 0
0.1
0.2
0.3
0.4
0.5
Fig:A.6 EC standard curve by HPLC
ECG 2.5
ECG: Standard Curve of EGCG y = 5.2837x - 0.2462
2 1.5 1
R² = 0.9962
0.5 0 0
0.1
0.2
0.3
0.4
Fig:A.7 ECG standard curve by HPLC 74
0.5
EGCG EGCG: Standard Curve of CG, y = 1.01x + 0.0113
2 1.5 1 R² = 0.9946
0.5 0 0
0.1
0.2
0.3
0.4
0.5
Fig:A.8 ECGG standard curve by HPLC
CG: Standard curve of ECG
CG 0.5 0.4
y = 4.6375x + 0.0962
0.3 0.2
R² = 0.9926
0.1 0 0
0.1
0.2
0.3
0.4
Fig:A.9 CG standard curve by HPLC
75
0.5
0.60
ECG
EGC
0.50
AU
0.40
0.30
GA CG
0.20
CAFF CAT
0.10
EC
EGCG
0.00 2.00
4.00
6.00
8.00
10.00
12.00 Minutes
14.00
16.00
18.00
20.00
22.00
Fig A.10: Catechin profile of standard mix. In these HPLC standard curve equations y denotes the area covered by peak and x denotes the concentration of the compound. These equations could be used to estimate the concentration of respective compound in the crude extract and purified compounds.
76
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