The Phytochemicals In Tea Biology Essay

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From centuries, tea has been the second most widely consumed beverage next to water and has become an indispensable part of life due to its immaculate health benefits (Chan, 2010). Tea is majorly brewed from Camellia sinensis in various parts of the world like China, Japan, India, Srilanka, Indonesia and African countries like Mauritius.(Luximon, 2005).

Tea comprises various beneficial phytochemicals which contribute to putative therapeutic benefits as an anticancer, anti inflammatory, reduces risk of cardiovascular diseases coronary heart disease, atherosclerosis and stroke, promotes insulin sensitivity and guards against neurodegenerative disorders, inhibits mutagenecity(Luximon,2005). Studies reveal that the above mentioned benefits can be attributed to phytochemical content of tea which is flavanoids, phenolic acids, catechins and flavanoid derivatives ( Luximon,2005). In addition ,tea is also a good source of methylxanthines like caffeine,theophylline,theobromine amino acids theanine and essential trace elements such as potassium, magnesium,calcium,nickel and zinc, minerals in adequate amounts which augment its potential medicinal properties along with antioxidant effects of polyphenols(flavanoids)(Horzic,2009).Consideration of all these benefits together with favourable effects of polyphenols on human health in dietary sources like food and beverages(tea, coffee) the phytochemical composition of tea drew interest of researchers towards its analysis

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Phytochemical composition of tea relies on species, climate, season, brewing techniques, fermentation process and agricultural practices and thus results in varied content in different types of tea which are of different origin(Luximon,2005). Green tea, Black tea, Oolong tea (semi fermented) and white tea (unfermented) are the common major forms of tea with differences in their chemical constituents and taste influenced by production process. Green tea is produced from young tea leaves (flush) followed by withering and steaming allowing for minimal or no fermentation. For black tea, tea leaves are subjected to fermentation for 11/2 -2 hrs in contrast to green tea before drying where the polyphenol oxidase in the tea leaves gets activated and oxidises monomeric flavon 3-ols resulting in the formation of theaflavins, thearubigins which are responsible for bright orange-red colour of black tea,bisflavanols and other polymers. High molecular mass polymeric products also results from polyphenol oxidase dependent oxidation of catechins. Fermentation plays a key role in the chemical composition of the tea as it triggers natural enzymatic oxidation of tea components leading to the altered phytochemical content of tea(Luximon,2005).

Identification and analysis of polyphenolic content and their antioxidant capacity in different tea brands of commonly used Indian teas would give a better understanding of bioactive components of tea and open up new avenues for the therapeutic pathways for averting various pathological disorders. Moreover, on account of promising health benefits obtained with tea drinking it is appealing to explore the healthy drink of high antioxidant capacity which would pave the way to gain better means for healthy life style in the world of aging population and modern medicine limitations.

Furthermore, as extraction process affects the tea composition scrutinizing and comparing the polyphenol content, methylxanthines with respective antioxidant capacity in different tea samples helps to identify optimal extraction process by determining the influence of extraction process on the

bioactive constituents of tea. In this study, polyphenolic content of different Indian teas were determined and simultaneously assessed by Gallic acid assay.Correspondingly,ABTS assay was used to find the antioxidant potential of tea samples.

PHYTOCHEMICALS IN TEA

Phytochemicals are the defensive products of plants which have the property to protect and prevent diseases in plants and have been finding numerous applications for ages in human pathological disorders. The major phytochemical constituents of tea are polyphenols which exert antioxidant, anti-inflammatory and anticancer properties. These are ubiquitously distributed in wide variety of dietary sources and structurally contain atleast one aromatic ring with hydroxyl groups as general functional group together with other substituents(Ferrazzano,2009). Flavanoids, phenolic acids, phenolic alcohols,stilbenes and lignans are major groups of polyphenols.Of these, flavanoids are the largest and well diverse family of polyphenols and constitute the major phytochemical composition of tea(Ferrazzano,2009). Flavanoids consist of two aromatic carbon rings, benzopyran ring and the other benzene ring linked by three carbon atoms often resulting in a heterocyclic ring(Spencer,2009). Catechins are monomeric flavanoids predominantly present in fresh tea leaves which include epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3 gallate (ECG) and epicatechin (EC) (Ferrazzano,2009). Gallic acid is the polyphenol comes under the category of phenolic acids and accounts for 4.5g/kg of fresh weight tea (Gupta,2007).Methylxanthines constitute the second major proportion of tea components include caffeine, theobromine and theophylline of which former is present in bulk than the later two. Tea also contains several amino acids , but major contribution is made by theanine 50 percent of total aminoacids.Apart from this, it contains more than 600 volatile fractions carbohydrates,tannins,gallotannins,adenine,quercetin glycosides,caratenoids,tocopherols,vitamins(A,K,B,C),aminophylline and yellow volatile oil which exists as solid at 25°C(De Mejia,2009).

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Of these wide range of tea components , epicatechin ,caffeine and theanine are of particular interest in analysis of phytochemical content of tea as these are present in adequate quantities suitable for analysis, gets extracted easily in short time with hot water.

FLAVANOID: EPICATECHIN

Flavanoids, significant chemical constituents of tea imparting plethora of benefits to tea as an antioxidant, anti-inflammatory and anticancer .Green tea is abundant source of flavanoids making up 30-40% of dry weight of fresh green tea leaves with about 70% of catechins(monomeric flavanoids),10% minor flavanols and 20% polymeric flavanoids. In contrast to green tea and oolong tea, black tea contains lower content of EGCG where the former comprises 50-80% EGCG of total catechins and EGC(De Mejia,2009). Catechins constitute 8-15% of dry weight of tea leaf and are non-volatile components which impart taste and colour to tea. These effectively scavenge reactive oxygen species (ROS), free radicals and even chelate metals for which they are subsequently regarded as major antioxidant contributors of tea. The biological benefits of flavanoids may be attributed to their antioxidant potential. Besides this, structural features of flavanoids identified recently disclosed that they are in compliance with required features of antioxidants to exhibit antioxidant activity which are ortho-dihydroxy catechol (3, 4, O-H) arrangement on B-ring of flavan-3ols and C2-C3 double bond in C ring along with carbonyl group at C4, hydroxypyranone structure induces the formation of stable phenoxyl radical due to electron delocalization. The catechol group on B-ring also serves for metal chelating property of flavanoids and assists in the reduction of ROS species catalyzed by metals(Wiseman.1997).

Interest of investigating the valuable pharmacological and physiological effects of flavanoids have been heightened in recent years unveiling the factors and mechanisms responsible for its multiple actions. Recent studies have shown that flavanoids play a crucial role in neuro-cognitive performance via interaction with memory associated components of brain on both cellular and molecular levels with enhancement in neurogenesis and decline in neurodegenerative processes resulting in overturn of age related declines(Spencer,2009). Epicatechin exists in conjugated form as it undergoes different metabolic processes sulfonidation and glucuronidation with two thirds in the sulphate form and one third in glucuronide form. Epicatechin is a monomeric polyphenol neutralize NF-Kb activation induced by IFN-gamma which has been evident by the reduction of IFN dependent nitric oxide production in pretreated macrophage cells in comparision to IFN treated controls.

Epicatechin coupled with exercise has been shown to boost retention of rat spatial memory in water maze tasks which was linked with improved angiogenesis and neuronal spine density in the dentate gyrus of hippocampus(Spencer,2009).

Epicatechin also serves as an anticancer agent in suppressing the factors related to growth and development of tumour cells a process called metastasis which has been reported in human colon cancer cells by inhibition of hepatocyte growth factor receptor (MET kinase) activity, a protein involved in metastasis of various tumours through molecular docking and kinetic studies(Larsen,2009).

METHYLXANTHINE: CAFFEINE

Methylxanthines are a class of compounds, show stimulant effects such as mood elevation, fight against fatigue and enhance the competency to work, probably caffeine being the most popular one.

The briskness and cream formation in tea may be attributed to caffeine content in tea. Caffeine accounts for 2.5 -4% of dry tea leaf(Balentine,1997). Black, Oolong and green tea contains same amount of caffeine when extracted from same amount of tea leaves and depends on the age of leaf, brewing techniques, temperature, size of leaf(Balentine,1997). These compounds are widely used in treatment of asthma by relaxation of bronchial smooth muscle but limited due to low potency.

Caffeine, is a trimethylxanthine, effective stimulant which acts indirectly on catecholamine neurons unlike direct effect by inhibition of enzyme phosphodiesterase which metabolizes cyclic adenosine monophosphate (AMP).On the other hand, it acts as an adenosine receptor antagonist by binding to adenosine receptors which blocks receptor stimulation and subsequently depresses the release of adenosine, a purine nucleotide which behave either as a transmitter or alter other transmitters. Unlike caffeine, adenosine is a depressant and thus inhibition of this neurotransmitter results in stimulant effects. Besides this, dopamine release also increases via adenoreceptor blockage which adds to arousal function of caffeine. Caffeine, an antagonist of adenosine blocks the effects of adenosine, inhibits vasodialtion & stimulates catecholamine levels in plasma, renin release & lipolysis, effects of platelet aggregation leading to increased blood pressure and free fatty acid on acute administration of caffeine(Rebec,2005).

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Caffeine also inhibits cyctokinesis without affecting nuclear division revealed on treatment of mitotic plant cells with caffeine. The proposed mechanism of action is disruption of cell plate assembly process inhibiting the transformation of the membrane tubes into tubulovesicular network following reduced callose content of caffeine treated plates specifying that tubulovesicular network formation is required for the deposition of callose(Cyr,J.R,2001).Some studies suggests that caffeine has chemopreventive effect which was found in black tea preparation, inhibited lung tumerogenesis in F344 rats induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).In addition, comparing with caffeinated teas, the antitumour effect of caffeine was appraised by reduced inhibition of UVB light stimulated skin tumours in SKH-1 mice when treated with decaffeinated teas. Furthermore, another observation in pregrant and nursing B6129SF1 female mice treated with dibenzopyrene after administered with caffeine offered cancer chemoprevention to her progeny which further supports and concludes caffeine as an effective chempreventive(Ju,2007).On the other hand, significant amounts of caffeine attenuates the risk of ovarian cancer via induction of CYP1A2, a enzyme involved in the activation of some heterocyclic compounds which might be carcinogenic. Caffeine content in green tea leaves acts as one of the biomarkers in assessing the quality of black tea.

AMINOACID: THEANINE

Theanine is a unique amino acid with tea plant as the only existing natural source which was discovered by Sakato in tea leaves with chemical structure as gamma-ethylamino-L-glutamic acid. It is one of the tea components which determine taste of tea especially green tea. It constitutes about 1-2% of dry weight of tea. It is well known for its potential therapeutic benefits as relaxant, blood pressure inhibitor and better enhancer of one's learning ability. In addition, underlying molecular mechanisms further supports its relaxant effect which is due to generation of α waves, generally 30-60 minutes after theanine administration in dose dependant manner in the parietal and occipital regions of brain without drowsiness as 0 waves are not affected which are enhancers of drowsiness.

Similarly, it lowers blood pressure as it inhibits the levels of serotonin probably the rationale behind its calming effect. Furthermore, inhibition of serotonin together with dopamine by theanine enables it to improve learning ability. It has also been evident that theanine is involved in suppression of tumours by increasing the concentration of antitumor agent like doxorubicin in mice. Theanine is also involved in immune functions providing resistance against microbial infections as it's a precursor of the non-peptide antigen ethylamine. Additionally, it possess strong antiischemic property which has been identified in SH-SY5Y,a cultured human dopaminergic cell line by the protective effects of theanine on neurotoxic inducers like rotenone, dieldrin and PD-related neurotoxicants.

L-Theanine is a glutamic acid analogue abundant of total amino acid content present in green tea sharing similar structural features with L-glutamic acid which prevent the binding of later to glutamate receptors in the brain. It possess cognition and mood elevating properties synergistic to caffeine due to its characteristic features which affect physiological and psychological stress responses experimentally observed in a laboratory setting using acute stressor. Significant anti stress effects were observed by oral administration of L-Theanine in twelve participants after four trials probably due to inhibition of excited cortical neurons. One more study reported that it has the potential to affect neurotransmitters in terms of secretion and function even half an hour after its administration in brain. It has also been postulated that L-theanine has greater efficacy in exerting neuroprotective action at a moderate degree despite its lower affinity than glutamic acid to glutamate receptors subtypes which emphasizes the functional role of L-Theanine in down regulation of cerebral functions. Previous studies in animals suggested that it counteracts the excitatory effects of caffeine and lowers blood pressure. Furthermore, it could be logically stated that L-theanine reduce physiological or psychological stress as a function of neurotransmitters since the later controls the emotional behaviours in all states and L-theanine has influence on it(Kimura,2007).

L-Theanine is at its maximum in tea leaves at the period of germination after 45 days which implies that it works as nitrogen source and precursor for building the carbon skeleton compounds of tea plant.Theanine is distributed all over the tea plant but abundantly exists in young and active tissues, young plants which signify its role in the metabolic processes in the tea plant. The presence of theanine in shoots shows that the major constituents of the tea "polyphenols" are derived from theanine with the first leaf as main site of synthesis.(mail).

It is deemed as one of the significant therapeutic agents and used as additive in many products owing to its cardiovascular, oncological and neurological effects. It has been consumed extensively in the form of green tea due to its relaxing and calming effect(book).

MATERIALS AND METHODS:

Chemicals:

Folin- Ciocalteau, Sodium carbonate,ABTS((2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)diammonium salt),Trolox((6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid),Phosphate buffer saline tablets, Potassium persulphate powder as well as caffeine, theanine, epicatechine of analytical grade were obtained from Sigma Aldrich,UK.HPLC grade acetonitrile and phosphoric acid were also obtained from Aldrich.

Equipment:

Six 500ml beakers, Brita water filter, five 10 ml volumetric flasks, Pipettes in the range of 200-1000µl, 20-40µl, 10µl, medium size glass tubes, glass pipettes, glass pipette bulbs, measuring cylinders of 25ml, 100ml,400ml.

Tea Samples:

Five different types of Indian teas in either powder form/sachets were purchased from local supermarkets namely-Natco spiced tea (Masala tea), Organic Tulsi tea, Twinings English breakfast, Twinings Blueberry and Apple, Twinings Camomile and Spearmint.

Preparation of Tea-water extract:

Aqueous extraction procedure was preferred to simulate the general brewing conditions used in household for a cup of tea. Approximately 0.5g of all tea samples were weighed in two trials which had been transferred to respective 500ml beakers and 100ml of hot water was added to tea samples under 85°C temperature control followed by stirring with a glass rod. Hot water was obtained from boiling of tap water in Brita water filter for purification. With the aim of monitoring the effect of multiple extractions on phytochemical composition of tea mainly polyphenols, methylxanthine and aminoacid each sample of two different weights were extracted three times under same conditions sequentially after 5min, 60min and 24 hrs which made up six extractions for each sample and thirty in total.

Preparation of Standards:

Standards of caffeine, epicatechin and theanine were prepared by weighing equivalent amounts required for standardisation and analysis of unknown concentrations in tea samples.25 ml volumetric flasks were used for preparation of stock solutions of caffeiene,epicatechin and theanine.30.7mg of caffeine, 31.3mg of epicatechin, 27.1mg of theanine were made to 25ml solutions with water in respective volumetric flasks which resulted in the concentrations of 1.228mg/ml for caffeine,1.084mg/ml for theanine and 1.252mg/ml of epicatechin.

Preparation of mixture standards;

Mixture standards containing caffeine, theanine and epicatechin were prepared by mixing equal amounts of standard solutions of caffeine, epicatechin and theanine, 1ml of each standard solution was taken from stock solution with 1ml micropipette making the final volume of mixture standard to 3ml for the initial one. Then different concentrations of mixture standards were prepared by diluting with water with the final volume as 3ml, sequentially decreasing the amount of standards with the increasing amounts of water. Five mixture standards were prepared by mixing 0.8ml, 0.6ml, 0.4ml, 0.2ml of each standard stock solution with respective amounts of water which give five different concentrations for each standard.

HPLC Analysis of Tea extracts:

Dionex Summit HPLC system comprising of UVD 340 pump for solvent delivery and UPDA UV/Visible photodiode array detector regulated by Chromeleon version 6.87 software was used for analysis of tea samples. Seperations of tea samples were achieved using a Eurospher 100-5 C18 reverse phase column (250mmÃ-4.6mm).10µl of each sample was injected for HPLC analysis. The temperature of the column oven was fixed at 30°C. Mobile phase or the solvent compositions used were 4% of Acetonitrile(solvent A) and 96% of 0.1% phosphoric acid(solvent B) were used for eluting the tea amples at a flow rate of 1ml/min. The elution consisted of linear gradient program starting at 96% B to reach 83%B at 25min, maintained at 83%B for 0.5min and then returned to 96%B at 30min following a ramp gradient flow. Chromatograms were recorded at a wavelength of 210nm.Absorbance of elutes were determined by photodiode array detector which was set between 210 and 595nm .Unknown concentrations of caffeine, theanine and epicatechine in tea samples were determined by comparing the peak area, retention times and spectral data with those of the valid standards. The analysis of all samples was done in two injections.

Total phenolic content:

Phenolic content of tea was estimated by well known Folin-Ciocalteu method which is generally used for determining polyphenolic content in various foods and beverages.250µl of Folin- Ciocalteu was added to a solution containing 50µl of tea extract and 450µl of deionised water. The whole mixture was left to stand for 5min and then 1.5ml of 20% Sodium carbonate was added. The solution was allowed to stand for 20min at room temperature and the absorbance was recorded at 735nm in UV spectrophotometer with water as blank. The process was done with all thirty samples of tea in two trials.

Method Validation

The unknown concentrations of total polyphenols in tea samples were determined by validating the obtained data with Gallic acid as reference compound. Calibration curve was plotted with different concentrations of Gallic acid by concentration versus absorbance. Standard stock solution of Gallic acid was prepared by dissolving 10.02mg of Gallic acid in water with final volume as 100ml to produce concentration of 10.02µg/ml. With the dilutions of stock solution by water, various concentrations were generated as 5.01, 2.5 and 1.25 µg/ml respectively. For effective validation of the data, two more controls were used to shun any deviations and study the effects caused by other chemicals used in Folin-C method. One of these was without tea sample and 500µl water, 250µl Folin-C reagent, 1.5ml of Sodium carbonate. The other one was done by addition of 700µl water, 1.5ml of Sodium carbonate to 50µl of sample.

Measurement of antioxidant activity:

ABTS radical cation decolourization assay:

The radical cation of ABTS(2,2'-azinobis-(3-ethylbenzthiazoline)-6-sulfonic acid was used to evaluate the antioxidant capacity of antioxidants in the tea samples.7mM concentrated solution of ABTS was taken as stock and the radical cation solution of ABTS+ with the same concentration was prepared by reacting 0.36gms of ABTS with 0.06gms of potassium persulphate in 100ml water. Before use, this solution was allowed to stand for 12-16hr in dark at room temperature to facilitate complete oxidation of ABTS with potassium persulphate as these two react stochiometrically in the ratio 1:0.5.Although, the oxidation of ABTS set off immediately in the solution mixture sufficient time should be allowed to get maximal and stable absorbance. The ABTS+ radical cation solution was then diluted with 5mM phosphate buffer saline (PBS) or water until an absorbance of 0.07(±0.02) at 734 nm. For the antioxidant assay, 1ml of diluted ABTS+ radical cation solution was added to 10µl aliquot of each tea sample and absorbance was recorded exactly 1min after the addition of diluted ABTS+ radical cation solution in PBS or water

RESULTS:

HPLC Separation of Authentic Catechins, Methylxanthines and Amino acids:

A mixture of standards containing epicatechin, caffeine and theanine of five different concentrations were separated by the HPLC procedure described above. A baseline resolution was achieved by gradient HPLC which was completed in 30min for one injection of each mixture standard as shown in Figure 1.Each mixture standard was allowed for three injections in a volume of 10µl.A reequilibriate time of 0.5min was set to make the column get back to its initial conditions for next sample injection.

The molar absorbency of epicatechin is rather high when compared to the peak heights of caffeine and theanine in all standards. The peak areas of all the three constituents in mixture standard 2 are higher than the mixture standard 1 despite its low concentration than the later (Figure 2). The peak areas of the succeeding mixture standards with decreasing concentrations are in accordance with their respective concentrations (Figure 3 and Figure 4).

Figure .First Standard Mixture of Theanine,Caffeine,Epicatechin

Figure : Chromatogram of Second Standard Mixture of Theanine,Caffeine,Epicatechin

Figure : Chromatogram of third standard mixture of Theanine,Caffeine,Epicatechin

Figure .Chromatogram of fourth standard mixture of Theanine,Caffeine,Epicatechin

HPLC Separation of epicatechin, caffeine and theanine in Indian Spiced tea:

The typical HPLC patterns of Indian spiced tea are illustrated in Figures 5, 6 and 7 suggests that high amount of caffeine is present in Spiced tea even after multiple extractions than theanine and epicatechin with a high average peak area between (60.23 -84.51). It was also observed that there is increase in all these three constituents when extracted after 60min than 5min.However, almost similar peak areas are observed for these constituents even after 24hrs extraction when compared with those of 60mins extract with only slight variations.

Figure .Chromatogram of fve minutes extract of Spiced tea

Figure 6: Chromatogram of sixty minutes extract of Spiced tea

Figure 7:Chromatogram of 24hrs extract of Spiced tea

HPLC Separation of epicatechin, caffeine and theanine in Indian Organic Tulsi tea:

Tulsi tea is a type of green tea mostly consumed in India for its attractive aroma and taste. The representative chromatograms of Tulsi tea are given in Figures 8,9,10 which depicts that caffeine is present in higher quantities in all three extracts (5min, 60min, and 20hrs) with least amount of theanine. There is increase in all the three constituents in the tea extract after 1hr but 1.12 times decrease in average peak area was observed for theanine and caffeine in 24 hrs extract in contrast to Epicatechin.

Figure 8.Chomatogram of five minute extract of Tulsi tea

Figure 9.Chromatogram of sixty minutes extract of Tulsi tea

Figure 10.Chromatogram of 24hrs extract of Tulsi tea

HPLC Separation of epicatechin, caffeine and theanine in Twinings Camomile and Spearmint tea:

A representative HPLC pattern of Twinings Camomile and Spearmint tea (Figure 11) demonstrates that significant amounts of caffeine and epicatechin are present in 60min tea extract. Average peak area of theanine was increased from 0.4415-1.746 correspondingly with tea extracts of long time period. Although, there is steep decrease in peak areas of Caffeine and Epicatechin in 24hrs extract.

Among these, Caffeine is present in high amounts than the other two in all extracts.

Figure 11.Chromatogram of sixty minutes extract of Camomile and Spearmint

HPLC Separation of epicatechin, caffeine and theanine in Twinings English breakfast:

Extremely high levels of caffeine are observed with respect to its high peak area which is approximately 100 times greater than that of theanine and 10 times of epicatechin in all three extracts.

Excluding epicatechin in 24hrs tea extract, three constituents showed elevated peak areas in ascending order of extraction time from 5min -24hrs extract although with only slight variations. It contains low levels of Theanine.

HPLC Separation of epicatechin, caffeine and theanine in Twinings Blueberry and Apple:

Unlike other tea samples, distinct feature of Twinings Blueberry and Apple lies in existence of exceptionally high levels of Theanine and least amount of caffeine with moderate levels of Epicatechin which is postulated in the characteristic HPLC pattern of these constituents in Figure 12.

Moreover, observation of peak areas of Caffeine indicated that its content decreased with increase in extraction time i.e. maximum in 5min tea extract and the opposite in 24hr extract.

Figure 12.Chromatogram of five minutes extract of Blueberry and apple

Standard calibration curves of Theanine, Caffeine and Epicatechin.

HPLC peak areas for corresponding concentrations of Theanine

Theanine (mg/ml)

HPLC peak area

1.084

15.21833

0.8672

13.11033

0.6504

11.45333

0.4336

9.091667

0.2168

6.288667

HPLC peak areas for corresponding concentrations of Caffeine:

Caffeine(mg/ml)

Hplc peak area

1.228

230.3066667

0.9824

208.1496667

0.7368

171.7206667

0.4912

142.4836667

0.2456

108.0356667

HPLC peak areas for corresponding concentrations of Epicatechin

Epicatechine(mg/ml)

HPLC peak area

1.252

387.0293333

1.0016

340.6693333

0.7512

263.98

0.5008

199.5573333

0.2504

129.8106667

Total phenolic content:

Total phenolic content of 5 brands of India tea are shown in Table 1.The data presented illustrates that total phenolic content was obtained in the range of 4.166-6.515 mg of Gallic acid/gm of tea. On an average of all extractions in different tea samples Spiced tea has high proportion of phenolic content(6.515mg/g of tea) .Significant values were observed in Camomile and Spearmint, Blueberry and Apple of 4.636 and 4.166 mg/gm respectively. Total phenolic content of English Breakfast(6.462mg/gm) is close to that of Spiced tea. Tulsi tea is intermediate between Spiced tea and Blueberry & apple with a total phenolic content of 5.458mg/g.

Calibration curve of Standard Gallic Acid in decreasing concentrations

Concentration of Gallic acid (µg/ml)

Absorbance

10.02

4.419

5.01

1.962

2.5

0.679

1.25

0.235

Table 1: Total Phenolic Content (TPC) of different brands of Indian tea.

Gallic Acid equivalent(GAE mg)/g

Code

5min

60min

24hrs

Total average

GA eq(mg)/g tea

Spiced tea

2.011

4.094

3.6677

3.257566667

6.515

Tulsi tea

1.792

1.7829

4.615

2.729966667

5.458

Camomile and Spearmint

1.56

2.6006

2.795

2.318533333

4.636

English breakfast

2.1011

3.164

4.428

3.231033333

6.462

Blueberry and apple

1.393

1.981

2.876

2.083333333

4.166

Estimation of antioxidant activity:

Polyphenols are a rich source of antioxidants in tea and the measurement of antioxidant capacity by ABTS radical cation decolourisation helps in finding optimal extraction techniques and their therapeutic utilisation in the field of medicine.

Observed absorbance values of tea samples in ABTS Assay with water as diluent:

All tea samples of three extractions were subjected to ABTS assay according to the procedure mentioned above. To acquire absorbance of diluted ABTS radical cation solution as 0.07, 1ml of ABTS radical cation solution was diluted in 75ml of water and the absorbance readings of the tea samples were recorded which is shown in Table 2.

The results demonstrates that three extractions of Blueberry and Apple tea has highest absorbance values of 0.407(5 min), 0.268(60 min) and 0.321 (24hrs) when compared to other teas whereas first (5min) and second extractions (60 min) of Camomile and Spearmint has second highest absorbance of 0.178 and 0.1907.Least absorbance was observed in 24hr extract of English Breakfast followed by Tulsi tea with negative values of -0.0008 and -0.002 respectively. Spectral data of Spiced tea depicts that there is significant raise in absorbance in 24 hrs extract of 0.07 which is nearly 10 times greater than 0.006 of 5min extract and 0.0055 of 60 min extract. Comparable absorbance value of English Breakfast in first extraction(5min) with that of Spiced tea(5min) shows that the former(0.06) is 10 times more than the later(0.006) unlike slight difference in second extraction with Spiced tea(0.0055) and English Breakfast(0.001).Analysis of absorbance data of all tea samples in view of increasing extraction time period illustrates that considerable decrease in absorbance was observed in Tulsi tea and English breakfast from +0.0036 to -0.0026 and +0.0607 to -0.00088 respectively. Conversely, small increase of absorbance has resulted in Camomile and Spearmint tea from +0.178-+0.263.

Table 2:Observed absorbance values of different tea samples by ABTS assay with water as diluent

a. Absorbance readings of five minutes extract of two different weights of each tea.

Code

105

Average

205

Average

Average 5min

Spiced tea

-0.0088

0.003

-0.0029

0.0152

0.0175

0.01635

0.006725

Tulsi tea

-0.006

0.008

0.001

0.0015

0.011

0.00625

0.003625

Camomile and Spearmint

0.278

0.1399

0.20895

0.0413

0.2537

0.1475

0.178225

English Breakfast

0.0013

0.0215

0.0114

0.189

0.031

0.11

0.0607

Blueberry and Apple

0.443

0.49

0.4665

0.3233

0.3752

0.34925

0.407875

b. Absorbance readings of sixty minutes extract of two different weights of each tea.

Code

160

Average

260

Average

Average 60 min

Spiced tea

0.085

0.024

0.0545

0.0682

0.0482

0.0582

0.05635

Tulsi tea

0.031

0.029

0.03

0.2025

0.2023

0.2024

0.1162

Camomile and Spearmint

0.054

0.032

0.043

0.029

0.011

0.02

0.0315

English Breakfast

0.0231

0.0234

0.02325

0.0232

0.0201

0.02165

0.02245

Blueberry and Apple

0.025

0.034

0.0295

0.023

0.041

0.032

0.03075

c. Absorbance readings of twenty hours extract of two different weights of each tea.

Code

124

Average

224

Average

Average 24hrs

Spiced tea

0.0122

0.294

0.1531

0.006

-0.002

0.002

0.07755

Tulsi tea

0

-0.01

-0.005

-0.0005

0

-0.00025

-0.002625

Camomile and Spearmint

0.247

0.239

0.243

0.243

0.323

0.283

0.263

English Breakfast

-0.001

-0.001

-0.001

-0.0015

0

-0.00075

-0.000875

Blueberry and Apple

0.4215

0.012

0.21675

0.3537

0.4932

0.42345

0.3201

Observed absorbance of tea samples in ABTS Assay with PBS as diluent:

Similarly, ABTS assay was done using PBS as a diluent for ABTS radical cation solution as the results with the above method turned very low where water was used as diluent. Moreover, this time ABTS was used in double concentration to have high absorbance(1.4) than standard(0.7) in order to have significant results for which 2ml of ABTS radical cation solution was made up to final volume of 82ml with water. The spectral data of all the tea samples was shown in Table 3.

Observations of all the three extractions in all tea samples elucidates that no tea sample absorbance was high in all three extractions but showing high molar absorbency in at least one of the extractions.

For instance, first extraction of Spiced tea, second extraction of Tulsi tea and third extraction of English Breakfast has high absorbance of 0.0945, 0.1162, and 0.07 respectively in relation to their extractions and of all samples. Very similar results in absorbance were observed in 60min extract of Camomile and Spearmint (0.0315) and Blueberry & Apple (0.03075) but minor difference in their 24 hrs extracts which are 0.022 and 0.069 respectively. Least absorbance was observed in first extract of English Breakfast (0.02) which is nearly 4.5 times less than that of Spiced tea. 0.0729 and 0.069 absorbance values of English Breakfast and Blueberry & Apple are closely related in their 24hrs extract.

Table 3.Observed absorbance values of different tea samples by ABTS assay with PBS as diluents

a. Absorbance readings of five minutes extract of two different weights of each tea.

Code

105

Average

205

Average

Average 5min

Spiced tea

0.1647

0.0783

0.1215

0.082

0.0548

0.0684

0.09495

Tulsi tea

0.0465

0.0396

0.04305

0.0275

0.0198

0.02365

0.03335

Camomile and Spearmint

0.0813

0.0672

0.07425

0.038

0.015

0.0265

0.050375

English Breakfast

0.023

0.0211

0.02205

0.0261

0.024

0.02505

0.02355

Blueberry and Apple

0.033

0.045

0.039

0.024

0.025

0.0245

0.03175

b. Absorbance readings of sixty minutes extract of two different weights of each tea.

Code

160

Average

260

Average

Average 60 min

Spiced tea

0.085

0.024

0.0545

0.0682

0.0482

0.0582

0.05635

Tulsi tea

0.031

0.029

0.03

0.2025

0.2023

0.2024

0.1162

Camomile and Spearmint

0.054

0.032

0.043

0.029

0.011

0.02

0.0315

English Breakfast

0.0231

0.0234

0.02325

0.0232

0.0201

0.02165

0.02245

Blueberry and Apple

0.025

0.034

0.0295

0.023

0.041

0.032

0.03075

c. Absorbance readings of twenty hours extract of two different weights of each tea

Code

124

Average

224

Average

Average 24hrs

Spiced tea

0.0232

0.0205

0.02185

0.0232

0.0238

0.0235

0.022675

Tulsi tea

0.023

0.019

0.021

0.023

0.021

0.022

0.0215

Camomile and Spearmint

0.0235

0.0233

0.0234

0.0232

0.0192

0.0212

0.0223

English Breakfast

0.0232

0.221

0.1221

0.0235

0.024

0.02375

0.072925

Blueberry and Apple

0.0235

0.02

0.02175

0.0232

0.211

0.1171

0.069425

DISCUSSION:

The aim of this study was to explore whether any correlation existed between total phenolic content and antioxidant activity in five different types of Indian tea and to investigate the influence of tea extraction in different time frames on the phytochemical composition of tea.

The main findings in this study were identification of different phytochemical contents present in Indian tea with particular attention towards methylxanthines, flavanoids, aminoacids, total phenolic content and their antioxidant efficiency. To assist with the identification of methylxanthines, flavanoids, aminoacids HPLC analysis of all tea samples was carried out in reference with authentic theanine, caffeine and epicatechin and the relative unknown concentrations were worked out. As per the HPLC analysis, highest amount of caffeine was observed in Twinings English Breakfast with the least in Twinings Blueberry and apple. On the contrary, BA contained theanine in peak levels with the least in Tulsi tea. With respect to Epicatechin, scarce amount was present in Camomile and Spearmint and elevated levels in Tulsi tea. Significant levels of caffeine and theanine were observed in Spiced tea with low levels of Epicatechin. These results were deduced on comparison of three extractions of all tea samples on an average.

The total phenolic content(TPC) which includes flavanoids, flavanols, phenolic acids and other phenolic group containing compounds was determined by Folin-Ciocalteu method and observed as Gallic acid equivalent(mg) per gm of tea. It is based on the ability of phenolic groups in the tea samples to reduce phosphomolybdate ion of Folin-Ciocalteu. The calculated TPC suggests that TPC in a tea sample depends on type of tea ,extraction time period which was clearly observed in Camomile & Spearmint, Blueberry & Apple and English Breakfast with steady increase in their TPC from 5min-24hrs although slightly.

On the other hand, no specific pattern was observed in TPC of Spiced tea and Tulsi tea. For example,

TPC of Tulsi tea remained nearly same in first and second extractions but steeply rose in its third extraction. Likely, TPC of Spiced tea has elevated from 5min-60 min and slightly decreased in 24hr extract.

ABTS radical cation solution serves as an effective estimator of antioxidant capacity of polyphenols

in tea samples due to the significant hydrogen donating ability of the later to stable free radical cation of ABTS. This method is based on measurement of scavenging ability of tea antioxidants of stable-blue ABTS radical cation in relation to the decrease in the absorbance of scavenged ABTS radical cation which is subsequently compared to that of a reference compound normally Trolox ,a water soluble vitamin E analogue of known amount.

Antioxidant capacity could also be monitored in the colour change of ABTS radical cation solution when added to tea sample on the basis of relation between colour intensity and absorbance. As can see from Table 2(a, b, c), Blueberry and Apple tea has higher absorbance and Tulsi tea with least absorbance.

This implies that, the former has the least antioxidant activity and the later has high antioxidant activity which is further supported by the immediate colour change of ABTS radical cation solution to colourless in Tulsi Tea whereas the opposite in Blueberry and Apple with no colour change.

These results were deviated when PBS was used as diluent showing high antioxidant activity in Camomile and Spearmint & low in Spiced tea in terms of absorbance and colour change.