Quantitative Analysis Of Isoflavanoids Biology Essay

Tea has been stated to be since 5000 old ages ago, nevertheless was non discovered until 2737 B.C when Chinese Emperor Shen Nong, revered for his cognition of agribusiness and medical specialty, mandated, presumptively for wellness grounds, that his topics boil H2O before imbibing it. One twenty-four hours while the emperor was fixing his H2O, the tea leaves blew into the pot of boiling H2O. The olfactory property enticed Shen Nong to try the pot ‘s contents. At one time he found the spirit to his liking and his organic structure rejuvenated. That the emperor who had been enduring from strivings and achings, had been pain – free and his organic structure rejuvenated after imbibing the “ Brown H2O ” . This particular works is known as Camelia Sinensis ( L ) Kutze belongs to the household of Theaceae. At that clip, people did non prefer imbibing tea that much, but it ‘s curative consequence such as uncluttering sight, relieve concern, chase awaying thirst, detoxification, attracted more and, easy they start to lift in popularity..

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

Tea was non until the Tang and Song Dynasties, showed some marks in Chinese tradition. Tea production including cultivation, harvest home, and processing had developed quickly and a bookman named Lu Yu published the first unequivocal book, “ Cha Ching ” or “ The Tea Classic ” , on tea after he spent over twenty old ages analyzing the topic. This certification included his cognition of planting, processing, tasting, and brewing tea. His research helped to promote tea imbibing to a high position throughout China. This was when the art of tea imbibing was born.

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

Subsequently, a Song Dynasty emperor helped the spread of tea ingestion farther by indulging in this fantastic usage. He enjoyed imbibing tea so much, that he bestowed tea as gifts to merely those who were worthy. During the same clip, tea was really divine to many bookman to compose books, verse forms, vocals, and pictures. This non merely popularized tea, it besides elevated tea ‘s value which drew tea-growers to the capital.

Between the Yuan and Qing Dynasties, the engineering of tea production continuously advanced to go more simplified and to better the methods of heightening tea spirit. During this period, tea houses and other tea-drinking constitutions were opened up all over China. By 900 A.D. , tea imbibing spread from China to Japan where the Nipponese Tea Ceremony or Chanoyu, was created. In Japan, tea was elevated to an art signifier which requires old ages of dedicated survey. Unlike the Nipponese people, the Chinese people tend to see tea imbibing as a signifier of enjoyment after a repast or to function when invitees visit.

Introduction of tea in the West ne’er begin until the twelvemonth 1600, when Queen Elizabeth demanded the East India Company to catch the finest silk, spice, herbs and others richest which contain a high market value. However, the popularity of tea was non that high until about range 1700 throughout Europe and England. At that clip, the drink was enjoyed merely by the aristocracts because a lb of tea cost an norm. British laborer the equivalent of his nine months rewards. The British began to import tea in larger qualities to fulfill the quickly spread outing market. Tea became Britain ‘s most of import point of trade with China. All categories were able to imbibe tea, as the tea trade increased and became less of a luxury. Now, tea is low in monetary value and readily available.

( Citation: Tea ‘s Wonderful History, Chinese History and Culture Project hypertext transfer protocol: //www.chcp.org/tea.html/ )

Background of survey

There are a batch of assortments of tea are produced from the tea works, Camellia Sinensis ( L ) . Kuntze a perennial harvest. Tea works are grown in warm, humid clime with plentifulness of rainfall and besides spread visible radiation and weak acid, Good drainage system raise the cultivation. Tea can be divided into three chief groups based on their processing technique.

Fresh foliages can be processed to Black tea, Oolong tea and Green tea by different grades of agitation procedure. All three types of tea have different quality of word picture including olfactory property, visual aspect and gustatory sensation.

Green tea fundamentally retains about the same composing as the fresh tea foliages, the drying procedure and steam will kill the oxidising enzyme ( polyphenol oxidase ) nowadays in the foliages inactivated the procedure of agitation, . On the other manus, black tea requires a full oxidization of the foliages, and the enzyme is to the full utilized in tea processing to organize the pigment ( Theaflavin and thearubin ) . After the foliages are plucked, they are laid out to shrivel for approximately 8 to 24 hours. This lets most of the H2O evaporate. The foliages are rolled ( without steam ) in order to interrupt up the cell surface so that O will respond with the enzymes and get down the oxidization procedure. The foliages are left to wholly oxidise, therefore turning the foliages to a deep black colour. Black tea and Oolong tea are fermented, and leads to production of crisp tannic acids in the foliages.

Epidemiologic surveies suggest that the phenolic compounds present in tea foliages have the benefits of cut downing the hazard for assorted diseases such as malignant neoplastic disease and cardiovascular disease.

The chief hypothesis associated with the putative benefits of tea is linked to the strong free radical-scavenging and antioxidant belongingss of their polyphenols. Catechins and Gallic acid ( GA ) are considered to be the active constituents of tea polyphenols responsible for the good effects on human wellness

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

A comparing between different trade names of procedure black tea could be established by mensurating their antioxidant activity and separating the content of of import antioxidants present in the several teas. In this research, processed black teas are extracted hot H2O extraction and the quantitative analysis of the polyphenols presnt in the commercial black tea were measured utilizing Reversed Phase High Liquid Chromatography ( RP- HPLC ) , with UV sensor.

The entire phenolic content and flavonoid content were both steps utilizing spectrophotometric techniques based on Folin – Ciocalteu check and Aluminium Chloride severally.

Malaya is besides the biggest consumers of black tea, yet there is small information or research on the commercial black tea available in Malaysia. There are many arguments made in comparing between black tea and green tea, particularly in footings of its antioxidant contents. Green tea is normally claimed to hold the strongest antioxidant and leads other type of tea beneficially while black tea, which take up most of the tea market were said to hold less wellness consequence and therefore is non every bit good as green tea. Hence it is deserving to prove the content of antioxidant in different trade names of black tea..

Significance of survey

This information collected would supply a comparing on which trade name of the black tea bag provided most of the antioxidant activity between the 9 types ( 7 Brands ) of black teas selected. This survey will besides supply information on the consequence of tea processing on the flavonoids content. It is besides to set up tea leaf infusion as a safe and natural additives on nutrient, cosmetics and pharmaceutical merchandise. Research surveies result obtained can assist supply wellness information to the populaces, indirectly and promote younger coevals to imbibe tea.

The information obtained would be utile for farther application of tea leaf infusions as safe and natural additives to be used in nutrient, decorative and pharmaceutical.

Aim of survey:

The purpose of this research is to compare between 9 samples of black tea ( 7 trade names ) of pure black tea foliage from different parts in term of its antioxidants. The specific aims are as follows:

1. To measure the polyphenols and flavonoids contained in 9 processed black tea foliages.

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

3. To larn and understand the rule of Reversed- Phase HPLC with UV sensing

4. To quantify the entire phenolic content and flavonoids content utilizing calorimetric techniques based on Folin- Ciocaltue ‘s Phenol check and Aluminium chloride assay severally.

Scope And restriction:

Tea is comparatively sensitive to heat, air, and wet. Poor preserved methods will impact the quality of the tea even the finest merchandises. Tea easy absorbs wet, olfactory properties, and besides heat which will do debasement, which adversely affect the spirit of the tea. Commercialized tea might already been exposed to sunlight or any contaminant that affect the truth of the reading. Optimization is of import for keeping the truth of the reading


1.5 Future Surveies

The survey can foster enhance with it came – preventing belongingss as ability to scavenge for free extremist by tea phenols as curative drug against malignant neoplastic disease, CNS hurt, arthritis and besides Aquired Immune lack Syndrome ( AIDS ) .

Literature Review

2.1 Camellia sinensis ( L. ) Kuntze

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

There are two chief assortments of tea:

Camellia Sinensis ( Japan and China works with little foliages C. Sinensis form, and growing in the cool, high mountain part )

Camellia Assamica ( the Assam works with big foliages C. Sinensis volt-ampere, grows best in humid tropical Climate such as Northeast India and Szechuan, Yunnan Province of China )

Tea works was characterized as an evergreen bush. It can blossom yellowish-white flowers and long ( 10cm ) elliptic, serrate, leathered foliages. Flowers are alar, lone, or up to three in a bunch, the little small flowers have six to eight petals. Outer petals were spoiled and interior petals are obviate to loosely obovate. Young foliages have all right white hairs on their underside, terminal buds are silvern grey and downy. The immature foliages of C. Sickness by and large sprouts get downing from March thought October followed by blossoming of flowers till February and fruiting occurs from August. ( Chang and Bartolomew, 1984 ) .

The domestication procedure of C. Sinensis changed it Phytochemical and familial characteristic depending on the interaction between Artificial choice and environment factors. Some instances, cultivated tea workss have decreased heterozygosity comparison to their nearest relations ( Kaundun and Matsumoto, 2003 ) , whereas other cultivated C. Sinensis volt-ampere Assamica population has heterozygosity under choice on the footing of their increased foliage country, tweaking shoots weight, and caffeine and polyphenolic content compared to the remainder of the cultivated tea workss and their wild relations ( Chen et.al, 2005, Ahmed 2011 ) .

The soft seedling develops in a baby’s room for 10 months, which protect them from rough clime conditions. After they have strengthened, the tea works continues to develop in unfastened Fieldss that are sheltered by the broad tree shadiness.

Plucking of tea foliages is still conducted utilizing handpicked method, this is to guarantee that the quality of the tea collected is the best. Normally this tea picking season in Asia state starts with the beginning of spring and continues from May till August,

They are assorted types of tea foliages which are normally grouped into 4 groups such as Black tea, Green tea, Oolong tea, and White tea. All of the assortments were collected from the same tea works. In fact it was the tea processing process which differentiated them. The Fresh tea foliages are filled with 80 % of liquid and it dried boulder clay less than 10 % before it can be readily sold to the consumers. Hence drying is an of import phase in the tea procedure, because cut downing the concentration of the foliages and thereby beef uping and continuing the tea foliage.

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

2.1.1 Fresh tea flower

Young shoots of the tea works are by and large known as tea flower that consist if the terminal bud with next foliages. In the fresh tea flower it contains high values of non-volatile compounds such as polyphenols, flavanols, phenolic acid, aminic acids, chlorophyll, other pigments and food. The chemical composing of the tea leaves depends upon the foliage, the ringer that being examined, dirt and clime conditions are agronomic patterns.

The entire polyphenol in the tea flower is between 20-30 % , and divided chiefly 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 look intoing for their bioactivities and use.

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

Caffeine, theobromine, and Elixophyllin, the chief alkaloids, history for approximately 4 % of the dry weight. . A cup ( 200 milliliter ) of green tea ( Gun Powder, Hangzhou, China ) contains about 142 milligrams EGCG, 65 milligram EGC, 28 milligram ECG, 17 milligram EC, and 76 milligram caffeine.

The soluble portion of foliage includes about 30 polyphenolic organic structures, over 20 amino acid, a twelve sugar and half a twelve organic acids. The entire ash content of the tea foliage is about 5.5 per centums including both soluble and indissoluble parts. Polyphenol comprises about 30 per centums of solid affair in the tea set. Most of it belongs to a group named flavanols, colorless works pigments that readily turns brown

( Beginning: Belizt EL at, 2009 ) .

A Teas are a major beginning of flavan-3-ols in the diet. The flavan-3-ol subclasses are ranked by grade of polymerisation. 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 tannic acids. These compounds are theorized to be responsible for the positive wellness effects of tea. As degrees of agitation increased from green to oolong to black tea, the major flavan-3-ol profiles changed. Entire 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 tendency around the universe, Asia and Asean

Tea has rise in popularity in society particularly among younger coevals. Some of the most recent in tea tendencies is utilizing tea as topping or ingredient in day-to-day favourite nutrient and drinks. Assorted merchandise made up of tea has been seen widely from assorted drink, ice-cream, facial merchandise and decorative.

Another tea tendency is that of functioning loose leaf tea instead than the standard tea bags. Many eating house and person are looking into buying fring leaf tea for brewing. Many times, loose foliage tea will blossom one time submerged in wateradn produce a pleasant asthetic sight.

Tea drink in the market had widely developed, other than apparent tea, the debut of assorted flower, herbs and even fruit in tea made it go flavored, scented and herbal tea, greatly increases popularity among adolescents. Now immature coevals prefer imbibing more tea merchandise than sodium carbonate and sugar merchandise.

For many old ages, “ Teh Tarik ” was popular among Malayan and had been a symbolism drink for tourer milk was really popular. Now with all types of teas, Ice tea is another tea tendency that can be enjoyed in many spirits. This is and first-class manner for tea drinkers to bask a nice cool drink on a hot summer twenty-four hours. They possibly available in pulverization signifiers, every bit good as readily imbibe bottle version.

With the altering society of today ‘s universe, tea tendency are invariably altering, They are besides really different in different parts of the universe. However tea tendency comes about it is increasily going one of the hottest drink

Below was a statistical analysis conducted on the 7 states that are the screens about 80 % of the universe tea production. As can be seen below, the growing rate is rather impressive and about changeless.

The universe production averaged in these 47 old ages to about 2.2 1000000s of metric tons per twelvemonth with merely 983,825 dozenss being produced in 1961 against about 3,896 1000000s in 2008.

As illustrated, we can see that China remain the biggest tea bring forthing states from 3.3 % to 39 % . While india portions drop get downing from 2006 from 36 % to 20.7 % .

hypertext transfer protocol: //teaconomics.teatra.de/files/2011/05/003-World-production2.jpg


1.2 Black tea

Tea foliages are coming from the same works merely that the processing processs in tea processing were different. Black tea, normally known as to the full 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 green goods is named as Xiao Zhong Black tea.

Once the fresh tea leaves picked, they are left to shrivel for several hours under the Sun or drying the foliages is by blowing currents of warm air over them. This measure is indispensable for the terminal quality of the tea H2O is released to let the agitation and oxidization of the foliage to get down. Rolling measure comes next when the wet content is reduced to about 60 % . There are few types of turn overing techniques such as Orthodox roller or rotor vane, or CTC ( oppressing, rupturing, and curving ) machine. This measure is important to interrupt the cell and let go of the enzyme, included polyphenol oxidase and peroxidase, and originate the agitation procedure with the debut of O. The smaller the tea foliage is crushed the wider the surface exposed. The four cathechins are enzymatically oxidize and undefgoes complex chemical alterations.

After the foliages are rolled by manus or machine a alone compound is formed between the enzymes and the polyphenols. The agitation procedure can so take place.A Fermentation conditions ( clip, temperature, and wet ) determine the concluding spirit and olfactory property of black teas. The last measure consists of drying of the tea leaves on the drying beds with forced hot air between 200F-250F ( 93.3-121 Celsius ) fluxing around it. When the foliages are about 80 per centum dried, theyA complete their drying over wood fires.

The resulting merchandise is red-brown in colour and is sorted harmonizing to size. The sorted largerA tea leaveA is considered theA ” foliage class ” and the smaller 1 asA ” broken class ” . These two gradesA are sold as loose-leaf teas. The “ fanning ” or “ dust ” class isA normally used in tea bags.A The concluding merchandise will hold a wet content of approximately 4 % .A

When the agitation occurs, the monomeric flavan-3-ols undergo polyphenol oxidase-dependent oxidative polymerisation taking to the formation of bi-flavanols, thea-flavins, thearubigins, and other oligomers in a procedure normally known as “ oxidization. ” Theaflavins ( about 1 % -2 % of the entire dry affair 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 permutation systems, which give the characteristic colour, and gustatory sensation of black tea. About 10 % -20 % of the dry weight of black tea is due to thearubigens, which are even more extended oxidized and polymerized, have a broad scope of molecular weights, and are less good characterized. ( Chung S. Yang, Zhi-Yuan Wang )

An approximate of the black tea drink average per centums of constituents of solid infusions are: catechins ( 10-12 % ) , theaflavins ( 3-6 % ) , thearubigins ( 12-18 % ) , flavonols ( 6-8 % ) , phenolic acids and depsides ( 10-12 % ) , aminic acids ( 13- 15 % ) , methylxanthines ( 8-11 % ) , carbohydrates ( 15 % ) , proteins ( 1 % ) , mineral affair ( 10 % ) , and volatiles ( & lt ; 0.1 % ) ( Graham, 1992 )

Figure 2.1.2 Oxidation of single catechin

2.2 Antioxidant activity

2.2.1 Antioxidant belongingss and mechanism of the tea polyphenol

Antioxidants act as protection against the detrimental effects of free extremist green goodss of course in organic structure. The globalisation of the universe, development of scientific discipline had bring benefit to us, yet there are besides adversed effects such as environemt pollution, smoke or physiological upset which overhelmed the map of the antioxidants, interrupt the It ‘s system in our body.Free extremist production cause harm to proteins and DNA within the cells.

Recent surveies on tea suggest that the comsupmtion of tea can assist to forestall tumour formation in worlds, This is because tea foliage contains abundant group ot polyphenols. Polyphenols besides suggested to hold antimutagenic and anticarcinigenic properties.It besides have beneficiall protective belongingss against cholesterin ( Maron and other,2003 )

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

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


hypertext transfer protocol: //www.caymanchem.com/images/catalog/70940.png

Catechin is a polyphenolic flavonoid which has been isolated from a assortment of natural beginnings including tea foliages, cocoas, apples, and the wood and bark of trees such as acacia. It was constructing blocks for procyanidins or known as condensed tannic acids. 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’- place of the C- ring, Each of the isomers can besides be as two optical isomers ( + ) or ( – ) catechins. ( Friedman,2005 ) . Cathechin from enzymatic oxidization will organize theaflavin and which farther oxidized to thearubigins There are many sorts of catechins in this survey we merely study the undermentioned compound:


Epicatechin-3-gallate, CG

Epicatechin, EC

Epigallocatechin, ECG

EGCG is the most copiousness catechin, which account for 65 % of the entire catechins in green tea. However black tea catechins was undergoes that undergoes agitation procedure, had convert catechins into polymer such as theaflavin and thearubigins. The degree of catechins decrease from 35 % -50 % in fresh tea foliage to 10 % in black tea. Below was the comparision of the entire catechins in green tea and black tea.

Type of tea


( mg/100g )


( mg/100g )


( mg/100g )

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 powerful antioxidant than ascorbate or I±-tocopherol in certainA in vitroA checks of lipid peroxidation.A Catechin inhibits the free radical-induced oxidization of stray LDL by AAPH.A It has besides been reported to move as an inhibitor of COX-1 with an IC50A of approximately 80 AµM.3A Catechin and other related procyanidin compounds have antitumor activity when tested in a two-stage mouse cuticular carcinoma theoretical account using topical application.


Theaflavin is a chemical made up ofA polyphenols. Normally found inA black tea, the plant-produced chemical has been clinically proven to forestall the accretion of free groups in the organic structure. As a consequence, theaflavin is a popular compound in malignant neoplastic disease research, every bit good as in other disease surveies and interventions

WhenA green teaA is fermented to go black tea, three different theaflavins are created. CalledA theaflavin-3-gallate, theaflavin-3′-gallant and theaflavin-3,3′-digallate, they can besides be found inA Oolong tea. The presence of these antioxidants alters both the chromaticity every bit good as the spirit of the tea.

Theaflavins have been utile in decelerating the growing of human immunodeficiency virus, or HIV, cells, which could assist scientists detect a remedy for the disease. Some surveies suggest that regular ingestion of theaflavin may assist lower one ‘s degrees of low-density lipoprotein, or LDL, A cholesterin. Peoples who on a regular basis drank theaflavins for 90 yearss in one survey were able to take down their cholesterin by about 12 per centum. Levels of LDL cholesterin specifically were reduced by 16 per centum. Participants in the survey were besides able to raise their degrees of high-density lipoprotein, orA HDLA cholesterin that the organic structure needs, somewhat.

Several other wellness benefits may ensue from the tea antioxidants. An antiviral agent, theaflavin may assist lower blood sugar degrees. One survey shows that it may assist heighten the maps of the liver, every bit good as cut down the sum of lipoids and organic structure weight of an being. Most of these possible benefits remain under reappraisal by the scientific community. Many doctors besides point out that black tea consumed in concurrence with a low-fat diet outputs the most wellness benefits.

To imbibe the benefits of theaflavins, five cups of black tea are by and large recommended daily. While this redress is considered safe for most people, adult females who are pregnant or nursing should avoid it.

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


Medical Researched on Tea

Harmonizing to research, black tea was found to take down blood force per unit area, thereby cut downing the hazard of bosom disease. It may besides cut degrees of “ bad ” LDL cholesterin and blood sugar. Again, given that bosom disease is one of the major causes of decease in industrialised states, the regular cupper is get downing to look like an progressively promising and unnoticeable solution to maintain your wellness in cheque.

From a concern point of position, tea makers operate under two long-held beliefs: that the 30-50 twelvemonth old female is the current primary client and that suppressing the immature and hep coevals of teens and twentysomethings is the lone manner to spread out the class in the old ages to come. Both points are surely valid, but they slightly fail to reflect the economic and demographic worlds of today. That is because the hereafter, at least for the following twosome of decennaries at least, really belongs an full population of baby-boomers that is presently come ining retirement.A

With huge sums of wealth and legitimate concerns about ageing and wellness, this mark should non be forgotten by tea concerns. Yes, that population may be non as gastronomically sophisticated or as creatively minded as others, yet no maker or retail merchant can afford to pretermit its impact on the bottom line, particularly given that tea is so relevant to its wellness concerns. Indeed, most of the outstanding wellness benefits of tea, such as cut downing the incidence of cardiovascular diseases, malignant neoplastic disease, Alzheimer ‘s and fleshiness, to call but a few, are evidently more relevant to a babe boomer than a college pupil. Add to that increased wellness and medical consciousness, more free clip to bask repasts ( and brew proper tea ) , every bit good as the disbursement power that the aged possess and you ‘ve got an offer hard to decline.

Viewed in this visible radiation, these medical surveies are more than merely about probationary scientific findings, because they may indicate to a slightly new way for the full tea industry. Despite restrictions on wellness claims in selling, they offer new ways of believing about the relevancy and potency of a population that goes unnoticed far excessively frequently.

2.2.2 Test on antioxidant activity Test of Total Polyphenol Content

Folin – Ciocalteu Assay


Folin & A ; Ciocalteu ‘s phenol or can be called theA Gallic AcidA Equivalence methodA ( GAE ) , is a mixture ofA phosphomolybdateA andA phosphotungstateA used for the colorimetricA assayA of phenolic andA polyphenolic antioxidants. [ 1 ] A It is named afterA Otto Folin, A VintilA? Ciocalteu, andA Willey Glover Denis.

The mixture of the phosphomolydate and phosphotungstate with acid and reflux will bring forth composites with construction in the solution.



Folin & A ; Ciocalteu ‘s phenol reagent does non incorporate phenol. Rather, the reagent will respond with phenols and non – phenolic cut downing substances to organize chromogens that can be detected spectrophotometric technique. The colour development is due to the transportation of negatrons on the basic pH to cut down the phosphomolybdic /phosphotungstic acid composites to organize chromogens in which the metals have lower valency.

The most common use of this reagent is in the Lowry method for finding protein concentration.

In this method, protein is pre – treated with Cu ( II ) in a modified biuret reagent ( alkalic Cu solution stabilized with Na K tartrate ) . Folin & A ; Ciocalteu ‘s phenol reagent generates chromogens that give an increasing optical density between 550 NM and 750 NM. Normally, optical density at the extremum ( 750 nanometer ) or shoulder ( 660 nanometer ) are used to quantify protein concentrations between 1-100 Aµg/ml while the optical density at 550 nmis used to quantitate higher protein concentrations. In the absence of Cu, colour strength

would be determined chiefly by the tyrosine and tryptophan content of the protein, and to a lesser extent by cysteine, and histidine. Copper ( II ) enhances colour formation by chelation with the peptide anchor, therefore easing the transportation of negatrons to the chromogens. Copper ( II ) has no consequence on colour formation by tyrosine, tryptophan, or histidine, but reduces that due to cysteine.

To command for the consequence of these compounds on colour development and, therefore, on the deliberate protein concentration of the sample, it is indispensable that the space and criterions be made up in the same medium as the samples.

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

Reversed Phase HPLC

Reversed stage chromatography has found both analytical and preparatory applications in the country of biochemical separation and purification. Molecules that possess some grade of hydrophobic character, such as proteins, peptides and nucleic acids, can be separated by reversed stage chromatography with first-class recovery and declaration. Separation mechanism in rearward stage chromatography depends on the hydrophobic adhering interaction between the solute molecules in the nomadic stage and was immobilized hydrophobic ligand, ( stationary stage ) .

Materials and Methods

Tea Sample

Samples of different trade names of tea bag were collected from the local market. The tea is blended and boxing from Sri Lanka ( Ceylon Grandeur ) , Cameron ( Cameron, Boh ) , London ( Twinning, Ahmad Tea ) and France ( St Dalfour Tea ) . The samples had been manufactured in commercial mills utilizing standard fabrication conditions. Each tea bag weighted about 2-2.5g. Tea brewed utilizing H2O at temperature at 800 C, and the tea bag was soaked for 10 proceedingss before it is removed. The tea was so filtered utilizing Filter paper ( Whatman no. 1 ) to take the staying residues.


The chemicals and reagents were of AR or HPLC class. Folin-Ciocalteu, Sodium carbonate, Acetic acid were of analytical class and supplied from Kemika ( Zagreb, Croatia ) . and methyl alcohol ( HPLC class ) 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,

Germany ) .

Standard Stock Solutions

A standard solution of EGCG was prepared by fade outing 4.72 milligram of EGCG in 50 milliliter methyl alcohol. Standard secret plan for HPLC analysis was prepared by shooting in triplicate a changeless volume of 5 I?l of serially diluted concentrations incorporating 9.85, 19.70, 39.35, 78.50, 157.50 and 283.20 mg/5 I?l of EGCG and observing AUC matching to each concentration.

The standard solution of ECG was prepared by fade outing 3.02 milligram of ECG in 10 milliliter methyl alcohol. The stock solution was diluted to do 9.40, 18.85, 37.50, 75.50, 151.0 and 302 ng/5 I?l dilutions of ECG. A changeless volume of 5 I?l of each concentration was injected in triplicate. The criterion secret plan was prepared as described for EGCG and same conditions of analysis were used for the two catechins.

Appraisal of polyphenols in tea samples

The appraisal of polyphenol was done utilizing calorimetric techniques harmonizing to modified describes by the Lachman, Hosnedl, Pives and Orsak ( 1998 ) Folin- Ciocalteu check. The sample infusions were diluted 10-fold utilizing H2O, doing its volume to 40Aµl.. The diluted samples are so added with 160 Aµl of H2O and 300 Aµl of 10 % Folin- Ciocalteu check and incubate about 30 Sec up to 8 proceedingss. A 20 % of Sodium Carbonate solution measured 400 Aµl was so added to the mixture. After 2 hours at room temperature sample optical density is measured at 765nm utilizing a UV-Visible Spectrophotometer. The phenoplast were quantified by the standardization curve of a known concentration of Gallic acid criterion ( 0.5mm – 2.0 millimeter ) . The concentration was expressed as g of Gallic acid tantamount per g of dry weight. The reading was done triplicate for each tea sample and the consequence is shown in the tabular array aˆ¦

Appraisal of entire flavanoids Content

The flavonoid besides detected utilizing e Aluminium Chloride calorimetric check and aliquot ( 0.25ml ) of pull outing or standard Quercetin ( 0.1mM, 0.12mM, 0.14mM, 0.16mM, 0.18mM, 0.20mM ) as the standard curves. The standard / tea infusion is added into 1.5ml of the extractor tubing incorporating 0.25 milliliter of H2O. To the tubing was added to 0.5 milliliters 2 % AlCl3.

The optical density was read utilizing UV -Visible spectrometer against prepared reagent space at 420nm. The entire flavonoid content was expressed as milligram Quacetin tantamount /g dry mass. The measurings were conducted 3 times for each tea sample and the consequence is shown on tabular array.

HPLC analysis of the Polyphenols and Flavanoids

A Shimadzu LC 20 AT fitted with a SIL 20 A car sampling station and the SPD -20 UV- seeable sensor with a category LC 10 chromatography workstation was used for the analysis of the prepare sample. The chromatographic separation was performed utilizing a reversed-phase Zorbax Eclipse XDB – C18 ( Agilent Technologies ) , ( 4.6 x 150mm 5 – micrometer ) . The samples and criterions were filtered through a 0.45Aµm membrane filter ( nylon membrane, Supelco, USA ) , and 20Aµl of each samples was injected for HPLC analysis. Elution was carried out at ambient temperature of 380C. The binary gradient manner system was carried out utilizing the undermentioned dissolver system. Mobile stage 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 rhythm takes about 30 proceedingss. With initial pump force per unit area of 49atm..


Mobile Phase Composition ( % ) Solvent Angstrom

Mobile Phase Composition ( % ) Solvent B

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