Tea is one of the best refreshing drinks which have been using Worldwide since from hundreds of years. First discovered in China several years ago and later on it became popular in Japan, India and almost in all the parts of world. Different varieties of tea are available like green tea, black tea, oolong tea, white tea etc depending on the type of fermentation process. Tea is obtained from leaves, twigs, and leaf buds of Camellia sinensis species belonging to the family Theaceae. Camellia sinensis is commonly known as tea plant or tea shrub or tea tree and is mainly cultivated in tropical and sub-tropical regions.
Tea is the most popular beverage not only because of its good taste and pleasant aroma but also owing to its great beneficial effects. Consumption of tea reduces risk of cardiovascular diseases, reduces risk of cancer, decreases blood cholesterol levels, treatment of diabetes, anti-infective, produces stimulant effects etc. (Horzic D. et al, 2009). Besides this polyphenols present in tea might affect digestive enzymes like Î±- amylase, pepsin etc which are present in stomach thereby reduces digestibility of food. Moreover high levels of intake of tea may leads to sleeping disorders (Qiang et al, 2006).
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Fresh tea leaves are mainly composed of several components like polyphenols, methyl xanthines, amino acids and some trace elements.
LIST OF COMPONENTS PRESENT IN TEA :
Catechins like (-)- epicatechin (EC), (-)- epigallocatechin gallate (EGCG), (-)- epigallocatechin (EGC), (-)- epicatechin gallate (ECG), (+)- catechin.
Caffeine, theophylline & theobromine
Minerals & Trace elements
Calcium, potassium, magnesium, zinc, & nickel
Polyphenols are the major constituents of tea leaves. Among these catechins play a cruicial role. Catechins in presence of polyphenol oxidase undergoes oxidation thereby results in formation of theaflavins like theaflavin, theaflavin - 3 gallate, theaflavin - 3' gallate etc. which are golden yellow in colour. In order to obtain high quality products or constituents enzyme -substrate ratio plays a prominent role (Joseph et al, 2005).
A flavanol which is present in wide range in green tea, some of the fruits like apple, red wine,cocao products etc is (-) - epicatechin. It belongs to catechin family. Other derivatives of catechin involve (-)- epigallocatechin, (-) - epicatechin gallate, (-) - epigallocatechin gallate, (+) - catechin and so on. Among these catechins (-) - epigallocatechin gallate is present in high amounts in tea. Coming to structural activity relationship of epicatechin it possess two benzene rings A and B which are linked to each other by pyrone moiety C. Attachment of gallate group through an ester bond to the carbon present in the third position of ring C is observed in some of the compounds. Studies carried out on structure activity relationship of epicatechin shows that antioxidant activity pertains to ring B. Moreover existence of di-hydroxy or tri-hydroxy groups at ortho position in ring B is very essential for antioxidant activity. Enhance in activity of epictaechin was observed upon esterfication of carbon present at 3' position with gallic acid (Roy et al, 2010).
During diseased state or in severe chronic inflammation reactive oxygen species (ROS) were released in large amounts by metabolic process. Released ROS damages macromolecules like carbohydrates, lipids, proteins and DNA. Existence of ROS in large amounts destroys several large molecules which may further lead to the death of cells and moreover this may promote to occurrence of diseases like cancer. Generally ROS constitutes free radical oxygen molecules, peroxides, hydroxyl radical, peroxides and hypo-chlorus acid. They are highly reactive species because of presence unshared pair of electron in the outermost shell. Polyphenolic compounds like catechins which are present in human body suppress highly reactive free radicals thereby protecting from oxidative damage of cells which further leads to lethal effects. Owing to significant benefits of catechins present in tea they are used in treatment of cancer, obesity and diabetes. Besides this catechins present in green tea are also responsible for anti- aging effects in humans (Roy et al, 2010). Epicatechin also possess several physiological effects in related to its antioxidative ability.
Several in vivo studies were carried out in rats and humans to depict pharmacokinetic profile of catechins. (+) - catechin and (-)- epicatechin and in combination of them were administered orally to different groups. After absorption they enter liver through portal vein where metabolism takes place. Gastro intestinal tract also plays significant role in metabolism and conjugation of polyphenolic compounds prior to liver. Primary metabolites of (-) - epicatechin are glucornide and sulfo-glucornide exists as non-methylated forms and 3'- O - methylated sulphate form and metabolite observed in plasma for catechin was non- methylated glucornide. Urinary excretion of epicatechin metabolites were observed to be in high amounts when compared to catechin metabolites. In spite if this very low amounts of epicatechin and catechin levels were observed in urine among mixed groups. Moreover bioavailability of epicatechin is higher as it absorbs competitively in gastrointestinal tract when given along with catechin (Baba et al, 2001).
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Absorption of epicatechin and catechin was very fast in upper part of gastrointestinal tract and distribution takes place in plasma where they reach maximum peak concentration within 1-2 hrs after administration. Conjugation and methylation take place at phenolic hydrogen which is present on ring B thereby enhances antioxidant activity. O-methylated metabolites enters brain by crossing blood brain barrier (BBB) while glucornide metabolites cannot as they are polar in nature very low amounts of them crosses BBB. Intracisternal injection of epicatechin enhances memory impairement activated by intracisternal glucose oxidase (Mohsen et al, 2002).
A methyl xanthine derivative which is consumed worldwide extensively is caffeine. Chemical name of it is 1, 3, 7 - trimethylxanthine. It is an alkaloid present in leaves, fruits and beans of many plants like Camelia sinensis (tea leaves), Theobroma cacao (cacao beans) and so on. Concentration of caffeine varies among beverages mainly depends on type of plant, environmental conditions and method of brewing. In a 240ml cup of tea, concentration of caffeine accounts around 40 - 120 mg. Upon oral administration it absorbs completely and rapidly and eliminates within five hours. In vivo study carried out on healthy human volunteers shows that caffeine takes 1- 3.5h for gastric emptying. Half of the dose gets emptied within 60 minutes and it takes around 175 minutes for gastric emptying of 90% of dose. Metabolism of caffeine occurs in liver in presence of cytochrome P450. Major metabolites of caffeine include 1, 3 - dimethylxanthine, 3, 7 - dimethylxanthine, and 3, 7 - dimethylxanthine. At low concentration they form linear pharmacokinetics but at higher concentrations of caffeine they follow non-linear pharmacokinetics.
Caffeine enters brain rapidly by crossing BBB and exerts it action. Caffeine is mainly adenosine receptor antagonist. It has similar structure as that of adenosine hence it binds to adenosine receptors mainly to Al and A2a receptors present in the brain thus blocks the action of adenosine. This in turn promotes the activities of neurotransmitters like serotonin and dopamine. Thus caffeine improves mood and alertness by reversing the pharmacological effects of adenosine. Research conducted on caffeine shows that it is used in treatment of Parkinson's disease. Parkinson's disease is a neurodegenerative disorder which occurs due to loss of dopaminergic neurons present in brain. Significantly caffeine being adenosine antagonist blocks AA2 receptors in turn stimulates dopamine release. Consumption of tea containing caffeine is useful in management of weight. It induces oxidation of lipids, enhances metabolic rate & energy expenditure by activating ephedrine (sympathomimetic amine), thermogenic and lipolytic activities were reported. By inhibiting Cyclic AMP phosphodiesterase (PDE) caffeine exerts its thermogenic activity (Heckman et al, 2010).
Caffeinated tea has more beneficial effects than caffeinated coffee. Caffeinated tea when taken in lower doses at regular intervals elevates performance by making decisions quickly, lessens fatigue than coffee etc. Effect of these cognitive tasks at higher doses was observed to be low (Janet Bryan, 2007).
In spite of all these consumption of caffeine in high amounts has very low adverse effects which can be seen in rare cases. Adverse effects of it include nausea, drowsiness etc. Lethal effects of caffeine were observed only with medications but not with dietary intake of caffeine or through beverages like tea. Caffeine should be taken with caution in persons who are sensitive to it. For instance caffeine increases blood pressure if taken in high amounts in patients who are suffering with hypertension. Amount of caffeine to be taken by pregnant women should be less than 300 mg per day to reduce adverse effects as per FDA (Heckman et al, 2010).
An amino acid that is available extensively in tea is theanine. It is highly abundant amino acid present in leaves of Camellia sinensis. It was first identified in green tea and in Xerocomus badicus (mushroom). It constitutes around 1-2% of tea leaves. When injected in animals as caffeine it also readily reaches brain by partitioning through BBB through where it shows effects on central nervous system. Theanine enters brain within half an hour in dose dependent manner and attains maximum concentration around 5h. Theanine resembles to the structure of glutamic acid. It is present in the form of glutamate, a neurotransmitter present mainly in hippocampus region of brain. Theanine blocks the actions of glutamate by interacting with glutamic acid receptors. Other physiological responses of theanine include decreasing levels of norepinephrine which is released from nor-adrenergic neurons present in brain. Primarly norepinephrine acts by interacting with G-protein coupled receptors. Decrease of norephinephrine is importantly due to increase of Gamma- amino butyric acid (GABA). L-theanine antagonises actions of caffeine. As caffeine stimulates release of serotonin, theanine regulates its release thereby by decreases blood pressure. It plays a regulatory role by bringing down the tones of CNS. Theanine in turn plays a role in regulating anxiety.
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Studies were carried out on action of theanine on Î±-brainwave activity. Effects were analysed at 30 minutes after administration of 50mg - 200 mg of L-theanine. L-theanine mainly enhances Î±- brainwave activity in parietal and occipital areas of brain in turn reduces anxiety by lowering blood pressure without causing drowsiness. Î± - brainwave activity by theanine increases as time elapses when compared to placebo. Moreover L-theanine also improves concentration as caffeine (Janet Bryan, 2007).
Biosynthesis of theanine mainly takes place in roots than in leaves. In roots glutamic acid and ethylamine (acts as precursor) combines in presence of theanine synthetase enzyme by utilizing ATP (adenosine tri phosphate) to give theanine. Research carried out on distribution and biosyntheisis of theanine in 27 species of plants belonging to theaceae family reported that theanine is available in higher amounts in C.sinensis species than other species (Wei- Wei Deng et al, 2010). Theanine is also used to treat Alzheimer disease (AD), a neurodegenerative disease. Usually because of increased oxidative stress in brain AÎ² (a protein) accumulates in abnormal amounts in brain primarily in hippocampus and cortex regions. High amount of AÎ² protein directly induce mitochondrial damage which subsequently leads to death of neurons. L- theanine reduces oxidative stress thereby protects brain from neuronal cell death (Kim et al, 2009). Studies carried out on effect of theanine on brain serotonin levels reported that decline in serotonin concentration after administration of theanine (Yokogoshi et al, 1998).
INDIAN TEAS AND ITS ORIGIN:
India is one of the world's largest producer, consumer and exporter of tea. Assam state is famous for cultivation of teas of best quality because of its geographical conditions. Mostly plucking of tea leaves has been done by hands in order to avoid mechanical injury. Commercial production of tea in India started after establishment of East India Company by Britishers. Many varieties of teas are available in India. Major varieties include black tea, green tea, white tea, oolong tea etc. Assam, West Bengal, Kerala and Tamilnadu are the major tea cultivated places in India.
Assam tea is obtained from the leaves of Camellia sinensis var assamica. It is cultivated in low lands of Assam near the river Brahmaputra. Leaves are plucked two times in a year. Usually leaves are dark green in colour and wider. It is also known as black tea. It is mainly composed of caffeine. Leaves are allowed to oxidize during fermentation process hence it contains more amount of caffeine, theaflavins & theburgins and less amount of catechins (Balz Frei et al, 2003).
TWININGS LADY GREY:
Twinings Lady Grey is produced mainly from Assam tea leaves. It is pale gold in colour. It is a refreshing tea with good aroma as it is infused with flavours of lemon, orange and bergamot.
TULSI GREEN TEA:
Tulsi Green tea is also produced from leaves of Camellia sinensis. Leaves are plucked, withered, and steamed to inactivate the enzyme responsible for degradation of polyphenols. Green tea contains flavonoids like catchiness and they account for 10 % of dry weight of leaves. Caffeine is also present but in less amounts. Catechins present in green tea mainly contribute to its anti-oxidant activity. Additionally it has some medicinal properties due to presence of tulsi leaves in it.
YELLOW LABEL TEA:
Yellow label tea is a type of black tea with richest source of antioxidants. It is dark in colour and mainly constitutes flavanoids.
TETLEY LEMON TEA:
Tetley Lemon tea is a type of green tea with lemon fressness. It is a type of tea which retains its delicate flavours without any bitter taste.
AIM AND OBJECTIVE:
Main aim of the project is to carry out phytochemical analysis of different varieties of Indian teas and to analyse antioxidant properties of Indian tea samples at different time intervals.
To determine concentrations of caffeine, theanine and epicatechin by using HPLC.
To determine total polyphenolic content of Indian teas by using Folin - Ciocalteu phenol reagent.
To determine concentration of antioxidants by using ABTS+ radical cation decolourisation assay.
To compare total polyphenolic content and antioxidant activity.
Five types of Indian teas were used in the analysis:
Twinings Lady Grey tea
Tulsi Green tea
Yellow Label tea &
Tetley Lemon tea.
CHEMICALS USED: Caffeine, Theanine, Epicatechin (for preparation of standard solutions), Gallic acid, Trolox, Folin - Ciocalteu's phenol reagent, ABTS+ (2, 2' - azino -bis-3-ethylbenzthiazoline-6-sulphonic acid) were obtained from sigma.
Other chemicals like Sodium carbonate, potassium persulfate and potassium buffered saline were obtained from Fisher.
Beakers (500ml), Pipettes, volumetric flasks, thermometer, water bath, glass tubes, HPLC machine, and UV - Visible spectrophotometer.
Preparation of Tea samples:
0.5 g of each variety of tea powder was weighed twice and was used throughout the analysis. All teas of 0.5 g were exactly weighed into beaker. Water of about 100ml which was maintained at 70â° c by using thermometer was added to the beakers containing tea leaves. Stir the solution and leave it. 10ml of sample was collected from each beaker after 5 minutes, 60 minutes and 24 hours respectively. Collected tea samples were used for the analysis.
Table 1: Weight of Tea Samples
Weight in grams
Assam tea 1
Assam tea 2
Twinings Lady Grey 1
Twinings Lady Grey 2
Tulsi Green tea 1
Tulsi Green tea 2
Yellow Label tea 1
Yellow Label tea 2
Tetley Lemon tea 1
Tetley Lemon tea 2
Preparation of standard stock solution:
30.3 mg of caffeine was weighed and dissolved in 25ml of water in a volumetric flask to get concentration of 1.212 mg/ml.
28.6 mg of theanine was weighed and dissolved in 25 ml of water in a volumetric flask to get concentration of 1.144 mg/ml.
28.4 mg of epicatechin was weighed and dissolved in 25 ml of water in a volumetric flask to get concentration of 1.136 mg/ml.
Preparation of mixed standards:
Mixed standards were prepared in decreasing order of their concentrations by using above standard stock solutions. Therefore each mixed standard contains caffeine, theanine and epicatechin in equal volume.
Table 2: Concentrations of caffeine, theanine and epicatechin in mixed standards
Concentrations in mg/ml
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC):
Concentrations of caffeine, theanine and epicatechin in both standards and tea samples were analysed by using HPLC.
Model used: Dionex ICS 3000
Operating software: Chromeleon R
Column Oven: Thermostatted
Column used: SN-XF7
Dimension: 250 * 4.6 mm
Packing: Eurospher 100 - 5C18
Column temperature: 30â° c
Sample injection: Thermostatted auto sampler
Volume injected: 30 Âµl
Detector: PDA (Photo Diode Array Detector)
UV- Visible Wavelength: 210 nm
Mixture of 4 % acetonitrile (A) and 96 % of 0.1 % phosphoric acid (B) was used as mobile phase. Before starting entire process mobile phase was passed through column at a flow rate of 1 ml/min for 30 minutes at 68 bar pressure to clean the column and to obtain better separation of peaks. Standards and samples were injected with the help of auto sampler for the analysis of constituents and detected by PDA detector at UV- Visible wavelength of 210 nm.
TOTAL POLYPHENOLIC CONTENT:
50 Âµl of tea sample was taken in a test tube. To it 450 Âµl of filter water and Folin- Ciocalteau reagent of about 250 Âµl were added to sample. It should be kept for 5 minutes at room temperature before addition of 20 % sodium carbonate (Na2CO3) of about 1.5 ml. Then the solutions were allowed to stand for 20 mins at room temperature and absorbance was measured at 735 nm by using UV- Visible spectrophotometry by using water as blank. Gallic acid was used as standard and different concentrations of gallic acid like 10.4 Âµg/ml, 5.2 Âµg/ml, 2.6Âµg/ml and 1.3 Âµg/ml were prepared and analysed to obtain standard calibration curve.
ABTS+ RADICAL CATION DECOLOURISATION ASSAY:
2, 2'- azino- bis- 3- ethylbenzthiazoline- 6- sulphonic acid (ABTS) was dissolved in deionised water to get 7mM concentration. This was added to 2.5 mM concentration of potassium persulfate to generate ABTS radical cation. Mixture of 7 mM concentration of ABTS and 2.5 mM concentration of potassium persulfate was allowed to stand overnight at room temperature in dark thereby solution turns blue colour from green colour which indicates generation of ABTS radical cation. This ABTS radical cation solution was diluted with 5 mM potassium -buffered saline to get absorbance of 0.700 (Â± 0.020) at 734 nm. 10 Âµl of tea sample was added to 1 ml of diluted ABTS+ radical cation solution (Abs734 nm = 0.700 Â± 0.02) and incubated in water bath exactly at 300 c for one minute before measuring the absorbance at 734 nm by using water as blank. Trolox was used as standard and 10.4 Âµg/ml, 5.2 Âµg/ml, 2.6 Âµg/ml and 1.3 Âµg/ml of final concentrations were prepared to obtain standard calibration curve.
Peak areas of standards and samples obtained after injection through HPLC and their corresponding concentrations were listed below.
Table 3: Standard calibration curve of theanine, caffeine and epicatechin
Average Peak Area
Average peak area
Average peak area
Figure 1: Standard calibration curve of Theanine
Figure 2: Standard calibration curve of Caffeine
Figure 3: Standard calibration curve of epicatechin
Data obtained from the HPLC analysis of tea samples are used to calculate Concentrations of theanine, caffeine and epicatechin present in tea samples referencing to standard calibration curves of corresponding compounds.
Table 4: Concentration of Theanine found in different tea samples at different time intervals
Average peak area
Average peak area
Average peak area
Twinings Lady Grey
Table 5: Concentration of caffeine found in tea samples at different time intervals
Average peak area
Average peak area
Average peak area
Twinings Lady Grey
Table 6:Concentration of epicatechin found in tea samples at different time intervals
Average peak area
Average peak area
Average peak area
Twinings Lady Grey
TOTAL PHENOLIC CONTENT:
Gallic acid was used as standard to determine total phenolic content of tea samples.
Table 7: Absorbance of gallic at corresponding concentrations
Figure 4: Standard calibration curve of Gallic acid
Table 8: Total phenolic concentration of tea samples at different time intervals
Twinings Lady Grey
Improved high performance liquid chromatography (HPLC) is used for better analysis of the compounds present in tea samples. By using HPLC we can able to detect polyphenolic, alkaloids and methylxanthine compounds present in sample. Standards were injected in triplicate and samples in duplicate via auto sampler in order to avoid errors and to get better results. Mobile phase constitutes acetonitrile and acidified phosphoric acid. By using acidified phosphoric acid peak tailing is reduced hence more sharp peaks were obtained. Upon injection through auto sampler compounds were eluted at different retention times. Retention time plays a significant role in identification of analytes. Based on the retention time theanine, caffeine and epicatechin in standards were analysed. Standard retention time of theanine was found to be 3.47 min, caffeine at about 24.23 min, and epicatechin at about 25.68 min shown in figures 5, 6 & 7. After recognition of theanine, caffeine and epicatechin based on retention times in mixed standards their corresponding peak areas are noted which are helpful for plotting standard calibration curves. Standard calibration curve of three compounds observed to be linear with coefficient of determination 0.99 (shown in figures 1, 2 & 3).
Figure 5: Chromatogram obtained for single standard theanine by using HPLC
Figure 6: Chromatogram obtained for single standard Caffeine by using HPLC
Figure 7: Chromatogram obtained for single standard epicatechin by using HPLC
Figure 8: Chromatogram obtained for mixed standards showing the presence of theanine, caffeine and epicatechin by using HPLC.
Tea samples at different time intervals were injected in HPLC. Chromatograms thus obtained were analysed in reference to standards. Concentrations corresponding to peak areas were analysed by utilizing y = mx + c equation from standard calibration curve. Concentration of theanine, caffeine and epicatechin in different teas at different time intervals is noted in table 4, 5 & 6. On observing results concentration of theanine in Tetley lemon tea, Assam tea and Twinings lady grey increases from 5 min to 60 min and then from 60 min to 24 hours. Slight deviations are observed in case of yellow label tea and tulsi green tea. At 5 min time interval highest amount of theanine is found in Tulsi Green tea (0.0326 mg/ml), at 60 min it is observed in Assam tea (0.044 mg/ml) and at 24 hrs time interval it is observed in case of both Tulsi Green tea and Assam tea which is around 0.044 mg/ml. Similarly caffeine and epicatechin also shows increase of concentration as time elapses in all types of teas except in Assam tea. Caffeine concentrations are more in case of Assam tea (0.67 mg/ml) and Tetley lemon tea (0.622 mg/ml) at 5 min interval, yellow label tea possess high concentration of caffeine and epicatechin in both at 60 min and 24 hrs time interval. Epicatechin levels were not detected in case of Assam tea at both 60 min and 24 hrs time interval, only small amount of it observed at 5 min sample (0.012 mg/ml). Along with yellow label tea, tulsi tea also possesses high concentration of epicatechin in both 60 min and 24 hrs sample.
Figure 9: HPLC analysis of Assam tea
Figure 10: HPLC analysis of Tulsi Green tea
Figure 11: HPLC analysis of Yellow Label tea
On typical evaluation of data it is observed that concentration of theanine, caffeine and epicatechin increases as time elapses. Overall concentration of caffeine is more when compared to theanine and epicatechin in all teas at different timings. Among all teas Tulsi green tea constitutes high amount of theanine and epicatechin. Caffeine is also present but in less amounts. Existence of catechins in higher amounts is characteristic feature of Green teas (shown in figure 11). Assam tea constitutes more amounts of theanine and caffeine. Very less concentration of epicatechin is found in Assam tea. Concentration of epicatechin in all the teas observed to be very low when compared to caffeine and theanine and even negative values in concentration are reported. One reason might be because of instrumental errors. Other reasons like loss of catechins due to auto- oxidation. Extraction of tea sample also plays a prominent role in identification and quantitation of polyphenolic compounds like catechin. Water is potent solvent in case of alkaloids but it is less reactive/ effective solvent in case of polyphenolic compounds while methanol is best solvent for extraction of polyphenolic compounds like catechins. Other factors like temperature, time, and solvent to material ratio may also effect concentration of epicatechin present in sample. Factors like geographical origin of tea, difference in composition of chemical constituents in tea leaves, soil conditions, temperature, plucking of leaves in different seasons, extraction methods, solvent used for extraction etc effects concentration levels of Caffeine, theanine and epicatechin in different varieties of teas.
Total polyphenolic test was done by using Folin- Ciocalteu reagent to detect the concentration of polyphenolic compounds like catechins present in five varieties of teas. Folin- Ciocalteu reagent constitutes phosphotungstic acid (H3P[W3O10]4). When this reagent is added to sample in presence of alkali like sodium carbonate it gets reduced to phosphotungstic blue. Hence the final solution appears in blue colour. Moreover Folin- Ciocalteu reagent should be added to tea sample before addition of alkali in order to avoid oxidation of tea sample in presence of air. Absorbance thus measured is directly proportional to phenolic groups present in tea samples (Dunja Horzic et al, 2009). With reference to standard calibration curve of gallic acid concentration of polyphenolic groups present in tea samples are analysed and noted in table 8. On analysis of results as time increases absorbance of all tea samples increases thereby concentration as well since absorbance is directly proportional to concentration. Concentration of total polyphenolic content in Assam tea has been declined slightly at 24 hrs when compared to other teas which might be due to oxidation of catechins present in tea sample. Even though highest concentration of polyphenolic compounds reported in case of Assam tea at 5 min and 60 min time interval. Decreasing order of concentrations of polyphenolic compounds present at 60 min is as follows Assam tea > Yellow label tea > Tulsi Green tea > Tetley Lemon tea > Twinings Lady Grey. At 24 hrs Yellow label tea and Tulsi Green tea constitutes more concentration levels of polyphenols.