Quantitative Analysis Of Isoflavanoids Biology Essay

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Tea has been stated to exist since 5000 years ago, however was not discovered until 2737 B.C when Chinese Emperor Shen Nong, revered for his knowledge of agriculture and medicine, mandated, presumably for health reasons, that his subjects boil water before drinking it. One day while the emperor was preparing his water, the tea leaves blew into the pot of boiling water. The aroma enticed Shen Nong to sample the pot's contents. At once he found the flavor to his liking and his body rejuvenated. That the emperor who had been suffering from pains and aches, had been pain - free and his body rejuvenated after drinking the "Brownish water". This special plant is known as Camelia Sinensis (L) Kutze belongs to the family of Theaceae. At that time, people did not prefer drinking tea that much, but it's therapeutic effect such as clearing sight, relieve headache, dispelling thirst, detoxification, attracted more and, slowly they start to rise in popularity..

(Citation: , Edited by Chi- Tang Ho, Jen-Kun Lin, Feriedoon Shahidi, 2009,Tea and Tea Products, London , UK )

Tea was not until the Tang and Song Dynasties, showed some signs in Chinese tradition. Tea production including cultivation, harvesting, and processing had developed rapidly and a scholar named Lu Yu published the first definitive book, "Cha Ching" or "The Tea Classic", on tea after he spent over twenty years studying the subject. This documentation included his knowledge of planting, processing, tasting, and brewing tea. His research helped to elevate tea drinking to a high status throughout China. This was when the art of tea drinking was born.

(Citation Kit Cow & Ione Kramer 1990: All the Tea in China, China.)

Later, a Song Dynasty emperor helped the spread of tea consumption further by indulging in this wonderful custom. He enjoyed drinking tea so much, that he bestowed tea as gifts to only those who were worthy. During the same time, tea was very inspired to many scholar to write books, poems, songs, and paintings. This not only popularized tea, it also elevated tea's value which drew tea-growers to the capital.

Between the Yuan and Qing Dynasties, the technology of tea production continuously advanced to become more simplified and to improve the methods of enhancing tea flavor. During this period, tea houses and other tea-drinking establishments were opened up all over China. By 900 A.D., tea drinking spread from China to Japan where the Japanese Tea Ceremony or Chanoyu, was created. In Japan, tea was elevated to an art form which requires years of dedicated study. Unlike the Japanese people, the Chinese people tend to view tea drinking as a form of enjoyment after a meal or to serve when guests visit.

Introduction of tea in the west never begin until the year 1600, when Queen Elizabeth demanded the East India Company to grab the finest silk, spice, herbs and others richest which contain a high market value. However, the popularity of tea was not that high until nearly reach 1700 throughout Europe and England . At that time, the drink was enjoyed only by the aristocracts because a pound of tea cost an average. British laborer the equivalent of his nine months wages. The British began to import tea in larger qualities to satisfy the rapidly expanding market. Tea became Britain's most important item of trade with China. All classes were able to drink tea, as the tea trade increased and became less of a luxury. Now, tea is low in price and readily available.

( Citation : Tea's Wonderful History , Chinese History and Culture Project http://www.chcp.org/tea.html/ )

Background of study

There are a lot of varieties of tea are produced from the tea plant, Camellia Sinensis ( L). Kuntze a perennial crop. Tea plant are grown in warm , humid climate with plenty of rainfall and also diffuse light and weak acid, Good drainage system raise the cultivation. Tea can be divided into three main groups based on their processing technique.

Fresh leaves can be processed to Black tea , Oolong tea and Green tea by different degrees of fermentation process. All three types of tea have different quality of characterization including aroma , appearance and taste.

Green tea basically retains almost the same composition as the fresh tea leaves, the drying process and steam will kill the oxidizing enzyme (polyphenol oxidase) present in the leaves inactivated the process of fermentation, . On the other hand, black tea requires a full oxidation of the leaves, and the enzyme is fully utilized in tea processing to form the pigment ( Theaflavin and thearubin). After the leaves are plucked, they are laid out to wither for about 8 to 24 hours. This lets most of the water evaporate. The leaves are rolled (without steam ) in order to break up the cell surface so that oxygen will react with the enzymes and begin the oxidation process. The leaves are left to completely oxidize, thus turning the leaves to a deep black color. Black tea and Oolong tea are fermented, and leads to production of sharp tannins in the leaves.

Epidemiological studies suggest that the phenolic compounds present in tea leaves have the benefits of reducing the risk for various diseases such as cancer and cardiovascular disease.

The principal hypothesis associated with the putative benefits of tea is linked to the strong free radical-scavenging and antioxidant properties of their polyphenols. Catechins and Gallic acid (GA) are considered to be the active components of tea polyphenols responsible for the beneficial effects on human health

All tea are rich in polyphenol content which, includes flavonoids and phenolic acids which constitutes almost 30 % of the dry weight (Chung et a.l, 1993). However the total antioxidant content will change due to process involved in tea production. The difference is significant when compared to green tea and black tea.

A comparison between different brands of process black tea could be established by measuring their antioxidant activity and distinguishing the content of important antioxidants present in the respective teas. In this research, processed black teas are extracted hot water extraction and the quantitative analysis of the polyphenols presnt in the commercial black tea were measured using Reversed Phase High Liquid Chromatography (RP- HPLC), with UV detector.

The total phenolic content and flavonoid content were both measures using spectrophotometric techniques based on Folin - Ciocalteu assay and Aluminium Chloride respectively.

Malaysia is also the biggest consumers of black tea, yet there is little information or research on the commercial black tea available in Malaysia. There are many debates made in comparison between black tea and green tea, especially in terms of its antioxidant contents. Green tea is usually claimed to have the strongest antioxidant and leads other type of tea beneficially while black tea, which take up most of the tea market were said to have less health effect and therefore is not as good as green tea. Hence it is worth to test the content of antioxidant in different brands of black tea..

Significance of study

This data collected would provided a comparison on which brand of the black tea bag provided most of the antioxidant activity between the 9 types (7 Brands) of black teas selected. This study will also provide information on the effect of tea processing on the flavonoids content. It is also to establish tea leaf extract as a safe and natural additives on food, cosmetics and pharmaceutical product. Research studies result obtained can help provide health information to the publics, indirectly and encourage younger generation to drink tea.

The data obtained would be useful for further application of tea leaf extracts as safe and natural additives to be used in food, cosmetic and pharmaceutical.

Objective of study :

The intention of this research is to compare between 9 samples of black tea (7 brands) of pure black tea leaf from different regions in term of its antioxidants. The specific objectives are as follows:

1. To evaluate the polyphenols and flavonoids contained in 9 processed black tea leaves.

2. To quantify the amount of the specific Isoflavonoid in 9 types of commercial black tea using High Performance Liquid Chromatography.

3. To learn and understand the principle of Reversed- Phase HPLC with UV detection

4. To quantify the total phenolic content and flavonoids content using calorimetric techniques based on Folin- Ciocaltue's Phenol assay and Aluminium chloride assay respectively.

Scope And limitation :

Tea is relatively sensitive to heat, air, and moisture. Poor preserved methods will affect the quality of the tea even the finest products. Tea easily absorbs moisture, odors, and also heat which will cause degradation, which adversely affect the flavor of the tea. Commercialized tea might already been exposed to sunlight or any contaminant that affect the accuracy of the reading. Optimization is important for maintaining the accuracy of the reading


1.5 Future Studies

The study can further enhance with it came - preventing properties as ability to scavenge for free radical by tea phenols as therapeutic drug against cancer, CNS injury , arthritis and also Aquired Immune deficiency Syndrome (AIDS).

Literature Review

2.1 Camellia sinensis (L.) Kuntze

Camelia Sinensis , commonly known as the tea plant belongs to the family of Theaceae. It was formerly named Thea Japonenense. Later Linnaeus renamed it Thea sinensis. In 1959, the generic name was changed to Camellia. The plant is a diploid with 2n = 2x = 30.

There are two main varieties of tea:

Camellia Sinensis ( Japan and China plant with small leaves C. Sinensis var., and growth in the cool, high mountain region )

Camellia Assamica (the Assam plant with large leaves C. Sinensis var, grows best in humid tropical Climate such as Northeast India and Szechuan, Yunnan Province of China)

Tea plant was characterized as an evergreen shrub. It can bloom yellow-white flowers and long (10cm) elliptic, serrate, coriaceous leaves. Flowers are axillary , solitary , or up to three in a cluster, the small little flowers have six to eight petals. Outer petals were spoiled and inner petals are obviate to broadly obovate. Young leaves have fine white hairs on their underside, terminal buds are silvery gray and sericeous. The young leaves of C. Sickness generally sprouts starting from March thought October followed by blooming of flowers till February and fruiting occurs from August. (Chang and Bartolomew, 1984) .

The domestication process of C. Sinensis changed it Phytochemical and genetic characteristic depending on the interaction between Artificial selection and environment factors. Some cases , cultivated tea plants have decreased heterozygosity compare to their nearest relatives ( Kaundun and Matsumoto , 2003), whereas other cultivated C. Sinensis var Assamica population has heterozygosity under selection on the basis of their increased leaf area , plucking shoots weight, and caffeine and polyphenolic content compared to the rest of the cultivated tea plants and their wild relatives ( Chen et.al, 2005, Ahmed 2011).

The soft seedling develops in a nursery for ten months, which protect them from harsh climate conditions. After they have strengthened, the tea plant continues to develop in open fields that are sheltered by the wide tree shade.

Plucking of tea leaves is still conducted using handpicked method, this is to ensure that the quality of the tea collected is the best. Usually this tea picking season in Asia country starts with the beginning of spring and continues from May till August,

They are various types of tea leaves which are commonly grouped into 4 groups such as Black tea, Green tea , Oolong tea , and White tea. All of the varieties were collected from the same tea plant. In fact it was the tea processing procedure which differentiated them. The Fresh tea leaves are filled with 80% of liquid and it dried till less than 10% before it can be readily sold to the consumers. Hence drying is an important stage in the tea process, because reducing the concentration of the leaves and thereby strengthening and preserving the tea leaf.

( Citation : http://www.wtea.com/about-tea_growth.aspx , Wissotzky Tea 2007.)

2.1.1 Fresh tea flush

Young shoots of the tea plant are generally known as tea flush that consist if the terminal bud with adjacent leaves. In the fresh tea flush it contains high values of non-volatile compounds such as polyphenols, flavanols, phenolic acid , amino acids, chlorophyll, other pigments and nutrient. The chemical composition of the tea leaves depends upon the leafage, the clone that being examined, soil and climate conditions are agronomic practices.

The total polyphenol in the tea flush is between 20-30 %, and divided mainly into six groups of compounds. Among them catechins have the highest content, there are 4 types of catechins such as (-) -epigallocatechin-3-gallate (EGCG), (-) -epigallocatechin (EGC), (-) -epicatechin-3-gallate (ECG), (-) -epicatechin (EC), (+) -gallocatechin (GC), and (+) -catechin (C). It is widely investigating for their bioactivities and utilization.

(Citation : Yong -Su Zhen(edi), 2002, Tea, Bioactivity and its therapeutic Potential , )

Caffeine, theobromine, and theophylline, the principal alkaloids, account for about 4% of the dry weight. . A cup (200 ml) of green tea (Gun Powder, Hangzhou, China) contains about 142 mg EGCG, 65 mg EGC, 28 mg ECG, 17 mg EC, and 76 mg caffeine.

The soluble part of leaf includes about thirty polyphenolic bodies, over twenty amino acid, a dozen sugar and half a dozen organic acids. The total ash content of the tea leaf is about 5.5 percents including both soluble and insoluble portions. Polyphenol comprises about 30 percents of solid matter in the tea set. Most of it belongs to a group named flavanols, colorless plant pigments that readily turns brown

( Source : Belizt el at , 2009 ).

 Teas are a major source of flavan-3-ols in the diet. The flavan-3-ol subclasses are ranked by degree of polymerization. The catechins are monomers (catechin, epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate), the theaflavins are dimers (theaflavin, theaflavin 3-gallate, theaflavin 3'-gallate, theaflavin 3,3'digallate), and the derived tannins. These compounds are theorized to be responsible for the positive health effects of tea. As levels of fermentation increased from green to oolong to black tea, the major flavan-3-ol profiles changed. Total catechins were 13.6 g/100 g in dry weight green tea and 4.2 g/100 g dry weight in black tea.

(Citation : , Major Flavonoids in Dry Tea, USDA , Researcher from TURF University )

Tea trend around the world , Asia and Asean

Tea has rise in popularity in society especially among younger generation. Some of the most recent in tea trends is using tea as topping or ingredient in daily favorite food and drinks. Various product made up of tea has been seen widely from mixed drink, ice-cream, facial product and cosmetic.

Another tea trend is that of serving loose leaf tea rather than the standard tea bags. Many restaurant and individual are looking into purchasing loosing leaf tea for brewing. Many times, loose leaf tea will unfold once submerged in wateradn produce a pleasant asthetic sight.

Tea drink in the market had widely developed, other than plain tea, the introduction of various flower , herbs and even fruit in tea made it become flavored , scented and herbal tea , greatly increases popularity among teenagers. Now young generation prefer drinking more tea product than soda and sugar product.

For many years, " Teh Tarik" was popular among Malaysian and had been a symbolism drink for tourist milk was very popular. Now with all types of teas,Ice tea is another tea trend that can be enjoyed in many flavors. This is and excellent way for tea drinkers to enjoy a nice cool drink on a hot summer day. They maybe available in powder forms , as well as readily drink bottle version.

With the changing society of today's world, tea trend are constantly changing, They are also very different in different parts of the world. However tea trend comes about it is increasily becoming one of the hottest drink

Below was a statistical analysis conducted on the 7 countries that are the covers almost 80% of the world tea production. As can be seen below, the growth rate is quite impressive and almost constant.

The world production averaged in these 47 years to nearly 2.2 millions of tonnes per year with only 983,825 tons being produced in 1961 against nearly 3,896 millions in 2008.

As illustrated, we can see that China remain the biggest tea producing countries from 3.3 % to 39%. While india shares drop starting from 2006 from 36% to 20.7 %.



1.2 Black tea

Tea leaves are coming from the same plant just that the processing procedures in tea processing were different. Black tea , commonly known as fully fermented tea had covered about 80% of the tea markets. It was developed in the mid-17th century in Chongan Country, Fujian Province of China. The first black tea produce is named as Xiao Zhong Black tea.

Once the fresh tea leaves picked, they are left to wither for several hours under the sun or drying the leaves is by blowing currents of warm air over them. This step is essential for the end quality of the tea water is released to allow the fermentation and oxidation of the leaf to begin. Rolling step comes next when the moisture content is reduced to about 60 %. There are few types of rolling techniques such as orthodox roller or rotor vane, or CTC ( crushing, tearing, and curling) machine. This step is crucial to break the cell and release the enzyme , included polyphenol oxidase and peroxidase, and initiate the fermentation process with the introduction of oxygen. The smaller the tea leaf is crushed the wider the surface exposed. The four cathechins are enzymatically oxidize and undefgoes complex chemical changes.

After the leaves are rolled by hand or machine a unique compound is formed between the enzymes and the polyphenols. The fermentation process can then take place. Fermentation conditions (time, temperature, and moisture) determine the final flavor and aromas of black teas. The last step consists of drying of the tea leaves on the drying beds with forced hot air between 200F-250F (93.3-121 Celsius) flowing around it. When the leaves are about 80 percent dried, they complete their drying over wood fires.

The resulting product is reddish-brown in color and is sorted according to size. The sorted larger tea leave is considered the "leaf grade" and the smaller one as "broken grade". These two grades are sold as loose-leaf teas. The "fanning" or "dust" grade is usually used in tea bags. The final product will have a moisture content of about 4%. 

When the fermentation occurs, the monomeric flavan-3-ols undergo polyphenol oxidase-dependent oxidative polymerization leading to the formation of bi-flavanols, thea-flavins, thearubigins, and other oligomers in a process commonly known as "oxidation." Theaflavins (about 1%-2% of the total dry matter of black tea), including theaflavin, theaflavin-3-O-gallate, theaflavin-3'-O-gallate, and theaflavin-3,3'-O-digallate, possess benzotropolone rings with dihydroxy or trihydroxy substitution systems, which give the characteristic color, and taste of black tea. About 10%-20% of the dry weight of black tea is due to thearubigens, which are even more extensive oxidized and polymerized, have a wide range of molecular weights, and are less well characterized. (Chung S. Yang, Zhi-Yuan Wang )

An approximate of the black tea beverage mean percentages of components of solid extracts are: catechins (10-12%), theaflavins (3-6%), thearubigins (12-18%), flavonols (6-8%), phenolic acids and depsides (10-12%), amino acids (13- 15%), methylxanthines (8-11%), carbohydrates (15%), proteins (1%), mineral matter (10%), and volatiles (<0.1%) (Graham, 1992)

Figure 2.1.2 Oxidation of individual catechin

2.2 Antioxidant activity

2.2.1 Antioxidant properties and mechanism of the tea polyphenol

Antioxidants act as protection against the damaging effects of free radical produces naturally in body. The globalization of the world, development of science had bring benefit to us, yet there are also adversed effects such as environemt pollution, smoking or physiological disorder which overhelmed the function of the antioxidants, interrupt the It's system in our body.Free radical production cause damage to proteins and DNA within the cells.

Recent studies on tea suggest that the comsupmtion of tea can help to prevent tumor formation in humans, This is because tea leaf contains abundant group ot polyphenols. Polyphenols also suggested to have antimutagenic and anticarcinigenic properties.It also have beneficiall protective properties against cholesterol (Maron and other,2003)

In fresh tea, major matabolites in the tea leaf includes polyphenoloc catechins and theaflavins, and alkaloids caffeine and theobromine catechins cover 80% of the total polyphenols in the tea leaf Inactivation of phenol oxidase in green tea orevent oxidations of the catechins, whereas enzyme phenolase-catalyzed the formation of dimeric theaflavins and polymeric thearubigin, which differentiate the black color to black tea ( Schwimmer 1981: Shahidi and Narczk 2004).

In this experiment there are 7 types of standard are comparable, which belongs to Catechins ( Catechin , Epicatechin-3-gallate, Epicatechin , Epigallocatechin, ) and Theaflavin , Theobromine, Gallic acid,and Caffeine.



Catechin is a polyphenolic flavonoid which has been isolated from a variety of natural sources including tea leaves, chocolates, apples, and the wood and bark of trees such as acacia. It was building blocks for procyanidins or known as condensed tannins. Tea catechins exist as two geometrical isomers: Trans-catechins and cis- catechins. Depending on the stereochemicla confriguration of the 3', 4' - dihydroxyphenyl and hydroxyl group at the 2'- and 3'- position of the C- ring, Each of the isomers can also exist as two optical isomers (+) or (-) catechins.(Friedman,2005). Cathechin from enzymatic oxidation will forms theaflavin and which further oxidized to thearubigins There are many kinds of catechins in this study we only study the following compound :


Epicatechin-3-gallate, CG

Epicatechin , EC

Epigallocatechin, ECG

EGCG is the most abundance catechin, which account for 65%of the total catechins in green tea. However black tea catechins was undergoes that undergoes fermentation process, had convert catechins into polymer such as theaflavin and thearubigins. The level of catechins decrease from 35%-50% in fresh tea leaf to 10% in black tea. Below was the comparision of the total catechins in green tea and black tea.

Type of tea







Brewed black tea




Brewed Green tea




(Citaion : Karrie Heneman, PhD, Sheri Zidenberg-Cherr,Nutrition and Health info sheet, PhD UC Cooperative Extension Center for Health andNutrition Research )

Function of catechins

Catechinsis a more potent antioxidant than ascorbate or α-tocopherol in certain in vitro assays of lipid peroxidation. Catechin inhibits the free radical-induced oxidation of isolated LDL by AAPH. It has also been reported to act as an inhibitor of COX-1 with an IC50 of about 80 µM.3 Catechin and other related procyanidin compounds have antitumor activity when tested in a two-stage mouse epidermal carcinoma model employing topical application.


Theaflavin is a chemical made up of polyphenols. Commonly found in black tea, the plant-produced chemical has been clinically proven to prevent the accumulation of free radicals in the body. As a result, theaflavin is a popular compound in cancer research, as well as in other disease studies and treatments

When green tea is fermented to become black tea, three different theaflavins are created. Called theaflavin-3-gallate, theaflavin-3'-gallant and theaflavin-3,3'-digallate, they can also be found in Oolong tea. The presence of these antioxidants alters both the hue as well as the flavor of the tea.

Theaflavins have been useful in slowing the growth of human immunodeficiency virus, or HIV, cells, which could help scientists discover a cure for the disease. Some studies suggest that regular consumption of theaflavin may help lower one's levels of low-density lipoprotein, or LDL, cholesterol. People who regularly drank theaflavins for 90 days in one study were able to lower their cholesterol by nearly 12 percent. Levels of LDL cholesterol specifically were reduced by 16 percent. Participants in the study were also able to raise their levels of high-density lipoprotein, or HDL cholesterol that the body needs, slightly.

Several other health benefits may result from the tea antioxidants. An antiviral agent, theaflavin may help lower blood sugar levels. One study shows that it may help enhance the functions of the liver, as well as reduce the amount of lipids and body weight of an organism. Most of these possible benefits remain under review by the scientific community. Many physicians also point out that black tea consumed in conjunction with a low-fat diet yields the most health benefits.

To drink the benefits of theaflavins, five cups of black tea are generally recommended daily. While this remedy is considered safe for most people, women who are pregnant or nursing should avoid it.

( Citation : Ness AR, Powles JW. Fruit and vegetables, and cardiovascular disease: a review. Int J Epidemiol1997; 26:1-3. )


Medical Researched on Tea

According to research, black tea was found to lower blood pressure, thereby reducing the risk of heart disease. It may also cut levels of "bad" LDL cholesterol and blood sugar. Again, given that heart disease is one of the major causes of death in industrialized countries, the regular cuppa is starting to look like an increasingly promising and unobtrusive solution to keep your health in check.

From a business point of view, tea manufacturers operate under two long-held beliefs: that the 30-50 year old female is the current primary customer and that conquering the young and hip generation of teens and twentysomethings is the only way to expand the category in the years to come. Both points are certainly valid, but they somewhat fail to reflect the economic and demographic realities of today. That is because the future, at least for the next couple of decades at least, actually belongs an entire population of baby-boomers that is currently entering retirement. 

With vast amounts of wealth and legitimate concerns about ageing and health, this target should not be forgotten by tea businesses. Yes, that population may be not as gastronomically sophisticated or as creatively minded as others, yet no manufacturer or retailer can afford to neglect its impact on the bottom line, especially given that tea is so relevant to its health concerns. Indeed, most of the prominent health benefits of tea, such as reducing the incidence of cardiovascular diseases, cancer, Alzheimer's and obesity, to name but a few, are obviously more relevant to a baby boomer than a college student. Add to that increased health and medical awareness, more free time to enjoy meals (and brew proper tea), as well as the spending power that the elderly possess and you've got an offer difficult to refuse.

Viewed in this light, these medical studies are more than just about tentative scientific findings, because they may point to a somewhat new direction for the entire tea industry. Despite limitations on health claims in marketing, they offer new ways of thinking about the relevance and potential of a population that goes unnoticed far too often.

2.2.2 Test on antioxidant activity Test of Total Polyphenol Content

Folin - Ciocalteu Assay


Folin & Ciocalteu's phenol or can be called the Gallic Acid Equivalence method (GAE), is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric assay of phenolic and polyphenolic antioxidants. [1] It is named after Otto Folin, Vintilă Ciocâlteu, and Willey Glover Denis.

The mixture of the phosphomolydate and phosphotungstate with acid and reflux will produce complexes with structure in the solution.



Folin & Ciocalteu's phenol reagent does not contain phenol. Rather, the reagent will react with phenols and non - phenolic reducing substances to form chromogens that can be detected spectrophotometric technique. The color development is due to the transfer of electrons on the basic pH to reduce the phosphomolybdic /phosphotungstic acid complexes to form chromogens in which the metals have lower valence.

The most common usage of this reagent is in the Lowry method for determining protein concentration.

In this method, protein is pre - treated with copper (II) in a modified biuret reagent (alkaline copper solution stabilized with sodium potassium tartrate). Folin & Ciocalteu's phenol reagent generates chromogens that give an increasing absorbance between 550 NM and 750 NM. Normally, absorbance at the peak (750 nm) or shoulder (660 nm) are used to quantify protein concentrations between 1-100 µg/ml while the absorbance at 550 nmis used to quantitate higher protein concentrations. In the absence of copper, color intensity

would be determined primarily by the tyrosine and tryptophan content of the protein, and to a lesser extent by cysteine, and histidine. Copper (II) enhances color formation by chelation with the peptide backbone, thus facilitating the transfer of electrons to the chromogens. Copper (II) has no effect on color formation by tyrosine, tryptophan, or histidine, but reduces that due to cysteine.

To control for the effect of these compounds on color development and, thus, on the calculated protein concentration of the sample, it is essential that the blank and standards be made up in the same medium as the samples.

(Citation : Fluka Analytical ,Sigma-Aldrich Chemie GmbH ·)

Reversed Phase HPLC

Reversed phase chromatography has found both analytical and preparative applications in the area of biochemical separation and purification. Molecules that possess some degree of hydrophobic character, such as proteins, peptides and nucleic acids, can be separated by reversed phase chromatography with excellent recovery and resolution. Separation mechanism in reverse phase chromatography depends on the hydrophobic binding interaction between the solute molecules in the mobile phase and was immobilized hydrophobic ligand, (stationary phase ).

Materials and Methods

Tea Sample

Samples of different brands of tea bag were collected from the local market. The tea is blended and packaging from Sri Lanka (Ceylon Grandeur), Cameron ( Cameron , Boh ), London ( Twinning, Ahmad Tea) and France ( St Dalfour Tea). The samples had been manufactured in commercial factories using standard manufacturing conditions. Each tea bag weighted about 2-2.5g. Tea brewed using water at temperature at 800 C, and the tea bag was soaked for 10 minutes before it is removed. The tea was then filtered using Filter paper ( Whatman no. 1 ) to remove the remaining residues.


The chemicals and reagents were of AR or HPLC grade. Folin-Ciocalteu, Sodium carbonate, Acetic acid were of analytical grade and supplied from Kemika (Zagreb, Croatia). and methanol (HPLC grade) was purchased from J.T. Caffeine (CF), Theobromine (TB), (-) epicatechin (EC), (-) -epicatechin gallate (ECG), (+) -gallocatechin (GC),), (+) -catechin (C), Gallic acid (GA), were obtained from Aldrich (Sigma-Aldrich Chemie, Steinheim,


Standard Stock Solutions

A standard solution of EGCG was prepared by dissolving 4.72 mg of EGCG in 50 ml methanol. Standard plot for HPLC analysis was prepared by injecting in triplicate a constant volume of 5 μl of serially diluted concentrations containing 9.85, 19.70, 39.35, 78.50, 157.50 and 283.20 mg/5 μl of EGCG and noting AUC corresponding to each concentration.

The standard solution of ECG was prepared by dissolving 3.02 mg of ECG in 10 ml methanol. The stock solution was diluted to make 9.40, 18.85, 37.50, 75.50, 151.0 and 302 ng/5 μl dilutions of ECG. A constant volume of 5 μl of each concentration was injected in triplicate. The standard plot was prepared as described for EGCG and same conditions of analysis were used for the two catechins.

Estimation of polyphenols in tea samples

The estimation of polyphenol was done using calorimetric techniques according to modified describes by the Lachman , Hosnedl, Pives and Orsak(1998) Folin- Ciocalteu assay. The sample extracts were diluted 10-fold using water, making its volume to 40µl.. The diluted samples are then added with 160 µl of water and 300 µl of 10% Folin- Ciocalteu assay and incubate about 30 Sec up to 8 minutes. A 20 % of Sodium Carbonate solution measured 400 µl was then added to the mixture. After 2 hours at room temperature sample absorbance is measured at 765nm using a UV-Visible Spectrophotometer. The phenolic were quantified by the calibration curve of a known concentration of Gallic acid standard (0.5mm - 2.0 mm). The concentration was expressed as g of Gallic acid equivalent per g of dry weight. The reading was done triplicate for each tea sample and the result is shown in the table …

Estimation of total flavanoids Content

The flavonoid also detected using e Aluminium Chloride calorimetric assay and aliquot (0.25ml) of extracting or standard Quercetin ( 0.1mM, 0.12mM, 0.14mM, 0.16mM, 0.18mM , 0.20mM) as the standard curves. The standard / tea extract is added into 1.5ml of the centrifuge tube containing 0.25 ml of water. To the tube was added to 0.5 ml 2% AlCl3.

The absorbance was read using UV -Visible spectrometer against prepared reagent blank at 420nm. The total flavonoid content was expressed as mg Quacetin equivalent /g dry mass. The measurements were conducted 3 times for each tea sample and the result is shown on table.

HPLC analysis of the Polyphenols and Flavanoids

A Shimadzu LC 20 AT fitted with a SIL 20 A auto sampler and the SPD -20 UV- visible detector with a class LC 10 chromatography workstation was used for the analysis of the prepare sample. The chromatographic separation was performed using a reversed-phase Zorbax Eclipse XDB - C18 ( Agilent Technologies), (4.6 x 150mm 5 - micron ). The samples and standards were filtered through a 0.45µm membrane filter (nylon membrane, Supelco , USA) , and 20µl of each samples was injected for HPLC analysis. Elution was carried out at ambient temperature of 380C. The binary gradient mode system was carried out using the following solvent system. Mobile phase A ( Water : acetic acid - 98:2 v:v, maintain at pH 2.65 ) , Mobile Phase B ( Acetonitrile ).The flow rate for the elution is 0.8ml/min . The whole cycle takes about 30 minutes. With initial pump pressure of 49atm..


Mobile Phase Composition (% ) Solvent A

Mobile Phase Composition (% ) Solvent B

0 -0.01