Analysis of Phenolic acids in some samples of Indian and Nepal Tea by High Performance Liquid Chromatography
R Srivastava, A Singh, S Misra, U Singh, A Tiwari
R Srivastava, A Singh, S Misra, U Singh, A Tiwari. Analysis of Phenolic acids in some samples of Indian and Nepal Tea by High Performance Liquid Chromatography. The Internet Journal of Alternative Medicine. 2008 Volume 6 Number 2.
The beneficial effects of tea (
), Brooke bond Taaza™ (BBTZ), Lipton Darjeeling Tea™ (LDT), Tea city™ (TCT), Taj Mahal Tea™ (TAJM), Tetley Tea™ (TET), Assam dana (DASM), Assam dana 1 (ASMD-1), Assam dana 2 (ASMD-2), Assam Tea (ASM), North Bengal tea (NB), North Bengal dana-1 (DNB-1), North Bengal-2 (DNB-2), North Bengal Upper (NBUP), Darjeeling tea-FOP (DARJ-FOP), Darjeeling tea-FOP1 (DARJ-FOP1), Darjeeling tea- brake (DARJ-br), Indian tea garden (Darjeeling-ITGA (DARJ-ITGA), Darjeeling-ITGB (DARJ-ITGB), Darjeeling-ITGC (DARJ-ITGC) and Nepal tea garden (KP-33A, KP-16A, KP-9A, KP-C6 and KP-C2).
Tea, made from the leaves of
Recently, phenolic acid metabolites as biomarkers for tea and coffee-derived polyphenol exposure in human subjects have been used 9,10 . Good amount of phenolic acids have recently been observed in tea by some other worker also 12 .
In this report, we analyzed several samples of tea available in market and tea garden of India as well as Nepal for the presence and amount of phenolic acids and correlated to health. The results are presented here.
Materials and Methods
Preparation of Samples for High Performance Liquid Chromatography (HPLC)
The tea samples of the market and tea garden, Indian and Nepal were macerated in a pestle-mortar and extracted by two methods, viz., hot and cold extractions.
Ethanol: water (80:20) was added in the samples, which were heated up to 60-70º C for 30 min. The samples were cooled to room temperature and then centrifuged at 7,500 rpm for 15 min. The supernatants were evaporated at room temperature. Finally dried materials were re-suspended in 1.0 ml HPLC grade methanol by vortexing. Again the solution was filtered through membrane filter (Millipore, pore size 0.45 µm). The prepared samples were stored at 4 ° C for HPLC analysis.
Tea samples were macerated in a pestle-mortar and extracted twice with ethanol-water (80:20,v/v, 10 ml). The extracts were kept for overnight in the solvent and then centrifuged at 7,500 rpm for 15 min. The supernatants were evaporated at room temperature. Finally dried materials were re-suspended in 1.0 ml HPLC grade methanol by vortexing. Again the solution was filtered through membrane filter (Millipore, pore size 0.45 µm). The prepared samples were stored at 4 ° C for HPLC analysis.
High Performance Liquid Chromatographic (HPLC) analysis
High performance liquid chromatography (HPLC) of the samples was performed in binary conditions with HPLC system (Shimadzu Corporation, Kyoto, Japan) comprising Shimadzu LC-20 ATVP reciprocating pumps, a variable SPD-20, AVP UV VIS detector, C-18 reverse phase HPLC column (250x4.6 mm) i.d., particle size 5 µm Luna 5µ C-18 (2), (Phenomenex, USA) at 36 ° C. Operating conditions were injection volume (10 µl), mobile phase methanol: water containing 0.4% acetic acid (66:34, v/v) flow rate 1 ml/min and detection at 290 nm and 254 nm. Tannic, gallic, caffeic, ferulic, vanillic, benzoic, cinnamic and salicylic acids were used as internal and external standards. Phenolic acids present in the samples were identified through comparing retention time (Rt. in min.) of standards. E.g. TA (Rt. 2.600), CT (Rt. 2.758), GA (Rt. 2.900), Caf-A (Rt. 3.358), FA (Rt. 3.825), BA (Rt. 5.650), CA (Rt. 6.792) and SA (Rt. 11.400). These phenolic acids were identified by co-injection of internal and external standards for their confirmation. Amount of individual compound was calculated by comparing peak areas of reference compounds with those in the samples run under same elution conditions.
Indian Tea Garden and Nepal Tea Garden
In the cold extracted samples, catechin, caffeic and ferulic acids were present in all the samples. Catechin was maximum in KP-C6 (2070.6290 µg/g) and minimum in DARJ-ITGC (227.2576 µg/g). Caffeic and ferulic acids were also maximum in KP-C6 (784.1483 µg/g CfA) (227.7835 µg/g FA) and minimum in DARJ-ITGC (258.2662 µg/g CfA) (35.4526 µg/g FA), respectively. Benzoic acid was present only in KP-9A (18248.0204 µg/g). The amount of catechin was high amount in cold extraction while in hot extraction the amount reduced and caffeic acid also showed this variation in hot and cold extraction in Nepal tea samples (Fig. 1).
In the hot extracted samples, the amount of catechin was reduced in comparison with cold extracted samples. This may be due to degradation of those compounds during hot extraction. In hot extraction, catechin, caffeic and ferulic acids were present in all the samples. The amount of catechin was maximum in KP-16A (1430.1092 µg/g) minimum in DARJ-ITGC (622.1591 µg/g) and tannic acid was present only in DARJ-ITGC (1050.6685 µg/g). Ferulic acid was also present in all the samples being maximum in KP-C6 (199.9919 µg/g) and minimum in KP-16A (47.7598 µg/g) (Fig.2).
Tea samples of Indian Tea Market
HPLC analysis of other 18 tea samples collected from Indian market indicated that the number and quantity of phenolic acids varied in different tea samples. In cold extraction, catechin was present all the samples being maximum in Darjeeling tea-brake (1090.20 µg/g). Caffeic acid, which was also present in all tea samples was maximum in DNB2 (1270.2038µg/g) and minimum in JGT (22.3209 µg/g), Tannic acid was present in ASM, DASM, DNB2, ASMD-1 ASMD-2 and DARJ-FOP1 being maximum in DASM (1833.9346µg/g). Gallic acid was absent in all the tea samples. Ferulic acid was present in Lipton Darjeeling ten, Tea city, Tetley tea and Darjeeling tea-brake and DARJ-FOP1 and maximum in Tetley tea (148.92 µ/g). Benzoic acid was present only in Janhit green tea, Tea city and Taj Mahal and maximum in Taj Mahal tea (11087.80 µg/g), while Cinnamic acid was present only in Tetley tea, Assam dana-I and Darjeeling tea-brake. Salicylic acid was present in Assam dana-I and Darjeeling tea-brake (98.79 µg/g) (Fig. 3).
In hot extraction, catechin and caffeic acid were present in all the samples. Catechin was maximum in ASMD-1 (2282.5348 µg/g) and minimum in NB (313.4076 µg/g) while caffeic acid was maximum in ASM (1069.4067µg/g) and minimum in NB (246.0799µg/g). Tannic acid was present in TT(Pre), ASM-1, ASM-2 and DARJ-FOP maximum in TT(Pre) (1430.3377 µg/g) and minimum in DARJ-FOP (34.5491µg/g). FA was present all the branded samples and DARJ-Br and DARJ-FOP and maximum in BBTZ (246.5178µg/g) and minimum in DARJ-Br (81.4652µg/g). Benzoic acid was present in TET (154.86.5346µg/g) while cinnamic acid in DARJ-br (10.2654µg/g) and salicylic acid in DARJ-br (40.6413 µg/g) (Fig. 4).
Phenolic acids constitute an important group of natural products contributing significantly to the marked pharmacological value of a number of plants, including fruits, vegetables and spices 12 . Tea is particularly rich in polyphenols, including catechin, flavone, flavonol, theaflavin, anthocyanidin, thearubigin and miniphenol formaldehyde, which are thought in contribute to the health benefits of tea 7 . Tea polyphenols act as antioxidants
Tea consumption has been shown to be associated with reduced risk of both cancer and cardiovascular disease in human population studies, but clinical data demonstrating bioavailability of the individual catechins and other polyphenolic components of tea are limited 20 . Caffeic acid besides being antioxidant also shows anti-inflammatory and anti cancer properties. It also reduces aflatoxin production 17 catachin a major constituent decreased the frequencies of sister chromatid exchanges (SCE) induced by paraquat (PQ) which is a generator of reactive oxygen species 16 . 21 reported that green tea and food containing polyphenol might be beneficial to human health by protecting against reactive oxygen species induced genotoxicity. Since both these compounds (caffeic acid and catechin) are in maximum amount in tea the positive response for human health can be attributed to these compounds. The phenolic acids together with several other compounds are responsible for global gravitation towards tea consumption and recommendation for its use since time immemorial.
According to the present results, caffeic acid and catechin were present in Darjeeling tea-brake and Assam dana-I and several other phenolic acids were also present in Darjeeling tea-brake and Assam dana-I, so these are recommended as better tea than other tea samples for human health.
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