INTRODUCTION:

Tea is grown and manufactured in numerous countries of the world and is blended or drunk in many more. Many phenolic compounds, particularly flavanoids, exhibit a wide range of biological effects. Reactive oxygen species and free radicals are constantly formed in the human body by normal metabolic action^[1]. Green tea is a leading beverage worldwide. Green tea is important source of polyphenol antioxidants.

A study found that the caffeine content of 1 g of green tea ranged from 11 to 20mg^[2]. The astringency in tea can be attributed to the presence of polyphenols including flavanoids, epigallocatechin gallate and other catechins^[3]. These are the most abundant compounds in tea leaves. Green tea protects against bacterial induced dental caries^[4]. Green tea reduces cholesterol HDL and LDL. Long term consumption of tea catechins can reduce the risk of coronary disease and could be beneficial against high fat diet induced obesity and typeII diabetes^[5]. The health promoting effect of antioxidants from plants is thought to arise mainly from their protective effects by counteracting reactive oxygen species, which are believed to play a significant role in the etiology and pathogenesis of various chronic diseases, premature ageing, and the oxidative deterioration of cosmetics, foods, and pharmaceutical preparations^[6]. Green tea has also shown effects on glucose uptake and utilization. It regulates glucose and lipid metabolism through activating phosphoinositide 3-kinaseand AMPK and their downstream signaling, so proven as potential anti-obesity and anti-diabetic agent^[7]. Green tea reduces iron induced lipid peroxidation in brain homogenates as well as in cultured C6 astrocytes and lung cells. It has also shown to reduce the formation of spin-adducts of hydroxyl radicals and hydroxyl radical to induced DNA strand breakage^[8]. Molecular oxygen is an essential component of all living organisms, but higher oxidative stress leads to generation of free radicals. These free radicals play a considerable role in the development of fibrosis and other diseases. Flavanoids act as anti-fibrotic agent by the free radical scavenging activity^[9]. Their action is opposed by a balanced system of antioxidant defense. Therefore, the evaluation of antioxidant activities of extracts and fractions is considered an important step^[10]. The objectives are: Determination of Total polyphenols as per International Standardization of an organisation - ISO 14502-1:2005(E): part of Quality control guidelines to Green/black Tea producers and industries to ensure the better grade product and Determination of Total polyphenolic content, Flavanoids and Antioxidant Potential on the basis of the method of tea preparation mentioned on product pack by optimizing the common method of analysis to ensure the quality in terms of better health benefits.

MATERIAL AND METHODS:

5% Ferric chloride, 10%Lead acetate, Ethanol, Chloroform water, Gallic acid, 7.5% Na₂CO₃, Folin-ciocatechu, 70% Methanol, Quercetin, 5%NaNO₂, 10%AlCl₃, 1M Na OH, Sodium Phosphate buffer, 10% Trichloro acetic acid, 1% Potassium ferric cyanide. UV-visible Spectrophotometer (Shimadzu-1800), Colorimeter (Systronics, 114), Hot air oven (Kemi, CRO405021144), Centrifuge (Rotek, RCH-6).

Statistical analysis:

Data were statistically evaluated by One- Way ANOVA, expressed as Mean±SD by Post Tukey- Kramer Multiple Comparison Test using Graph pad instat3 trial version free software, performed the post-test only if the P value <0.05 for the significance.

TOTAL POLYPHENOLS ESTIMATION:

Total Polyphenolic Content Estimation by as per ISO 14502-1 procedure:

Standard Preparation:

10mg of Gallic acid monohydrate was dissolved in 100 mL of methanol and water (70:30) mixture., Aliquots of 0.2, 0.4, 0.6, 0.8, 1, 1.2mL from above stock solution which corresponds to 2, 4, 6, 8, 10, 12µg/mL respectively were taken in separate 10mL volumetric flasks and added 5mL of Folin-Ciocalteu phenol reagent. After 3-8 minutes, 4mL of 7.5% of sodium carbonate solution added. Allowed to stand at room temperature for 60 minutes and measured the absorbance at 765nm to prepare the standard calibration curve of Gallic acid by plotting absorbance against concentration.

Test sample preparation:

Weigh (0.200±0.001)g of the test sample into an extraction tube. Methanol/water extraction mixture is placed in the water bath set at 70°C, allows 30 minutes to equilibrate. Place the extraction tube with sample at 70°C. Pipette out 1mL and made it to 10mL using methanol and water (70:30) mixture. Pipette out 1mL and add 5mL of FC reagent and stand it for 3-4 min and add 4mLof sodium carbonate. The absorbance is taken at 765nm^[11].

Total Polyphenolic Content Estimation in one cup of prepared tea as per Product Insert Procedure:

Standard Preparation:

10mg of Gallic acid monohydrate was dissolved in 100 mL water. Aliquots of 0.2, 0.4, 0.6, 0.8, 1, 1.2mL from above stock solution which corresponds to 2, 4, 6, 8, 10, 12µg/mL respectively were taken in separate 10mL volumetric flasks and added 5mL of Folin-Ciocalteu phenol reagent. After 3-8 minutes, 4mL of 7.5% of sodium carbonate solution added. Allowed to stand at room temperature for 60 minutes and measured the absorbance at 765nm to prepare the standard calibration curve of Gallic acid by plotting absorbance against concentration.

Test sample Preparation:

Tetley green tea taken 1 full teaspoon (approx. 3.006gm) Pour one cup (100mL) of freshly boiled water over and brew for 2 min. Stir and strain into a beaker (stock solution). Lipton green tea taken half teaspoon (approx. 1.15gm) Pour 100mL of freshly boiled water and brew3-4mins.Stir and strain into a beaker (stock solution). AVT green tea taken 1 full teaspoon (approx. 2.6332gm). Pour 100mL of freshly boiled water and brew for 2-3 min. Stir and strain into a beaker (stock solution) From the each stock solution, pipette out 1mL and make it to 10mL with water. From that pipette out 0.2mL add 5mL of FC reagent and wait for 3- 4 min. and add 4mL of sodium carbonate and the volume was made to10mL with distilled water and the absorbance is taken at 765nm.

TOTAL FLAVANOID ESTIMATION:

Total Flavanoid Content Estimation in one cup of prepared tea as per Product Insert Procedure:

Standard Preparation:

Standard Quercetin (10mg) were added to 10mL volumetric flask and made the volume to 10mL with ethanol and water mixture (70:30). From this 1mL, 2mL, 3mL, 4mL, 5mL, 6mL which corresponds to 10, 20, 30, 40, 50, 60µg/mL were taken into each 10mL volumetric flask and 0.3mL of 5% NaNO2 was added. After 5 minutes, 0.3mL of 10% AlCl3 was added. After 6 min, 2mL of 1 M NaOH was added and the volume was made to10mL with distilled water. The solution was mixed and the absorbance was measured against a freshly prepared blank at 502nm^[12].

Test sample preparation:

Pipette out each of 1mL of prepared green tea separately and make it up to 10mL and pipette out 0.4mL for Tetley brand and AVT brand but in case of Lipton 0.8mL to get to overcome the sensitivity of absorbance and get the signal of response and transferred to 10mL volumetric flask. To the above solutions add 0.3mL of 5% NaNO2 was added. After 5 minutes, 0.3mL of 10% AlCl3 was added. After 6 min, 2mL of 1 M NaOH was added and the total volume was made up to 10mL with distilled water. Then the solution was mixed and the absorbance was measured against a freshly prepared reagent blank at 502nm.

ANTIOXIDANT PROPERTY:

Estimation of Antioxidant Potential in one cup of prepared tea as per Product Insert Procedure:

It is investigated by using the potassium ferricyanide –ferric chloride method. Weigh accurately 0.1g of ascorbic acid standard and make it up to 100mL with water. For the test sample pipette out 10mL of prepared green tea separately and make it up to 100mL with water. Pipette out 1mL, 2mL, 3mL, 4mL, and 5mL to the respective test tubes and make up to 100 mL with water for both. From the above concentration pipette out each of 2.5mL and to it added 2.5mL of phosphate buffer (0.2M, pH 6.6) and 2.5mL potassium ferricyanide (1%), 2.5mL of 10% (w/v) trichloroacetic acid were mixed and kept for 20min (to reduce ferricyanide into ferrocyanide). The above solution is centrifuged for 10 min. From the above solution pipette out 2.5mL and simultaneously add 2.5mL of water. Finally added 0.5mL of FeCl3 and made up the 10 mL volume with distilled water and the absorbance was measured at 700 nm and % inhibition is calculated^[13].

% Inhibition = (Absorbance of test/Absorbance of control) - 1 *100

RESULTS:

Fig.1: Calibration curve of Standard Gallic acid using water ethanol and water (70:30)

Analyzed as per ISO 14502 procedure

Table 2: As per ISO 14502 (GAE gm/100gm)

	TETLEY	LIPTON	AVT
	20.2	29.8	17.8
	23.8	26.8	17.5
	21.6	28.3	17.70
Mean	21.9	28.3	17.67
SD	1.48	1.22	0.12
Mean ± SD	21.9±1.48	28.3±1.22	17.67±0.12

Fig.3: Calibration curve of Standard Gallic acid using water

AS PER PRODUCT INSERT PROCEDURE (GAE in gm/d L)

Table 4: As per product insert procedure (GAE in gm/d L)

	TETLEY	LIPTON	AVT
	0.261	0.074	0.230
	0.312	0.113	0.225
	0.227	0.094	0.228
Mean ± SD	0.266 ± 0.035	0.094 ± 0.016	0.227 ± 0.002

AS PER PRODUCT INSERT PROCEDURE (QE in gm/d L)

Table 5: As per product insert procedure (QE in gm/d L)

	TETLEY	LIPTON	AVT
	0.380	0.103	0.271
	0.333	0.117	0.323
	0.451	0.099	0.294
Mean ± SD	0.388 ± 0.049	0.107 ± 0.008	0.296 ± 0.021

Fig.6: IC 50 Value comparison

DISCUSSION:

As per ISO 14502 protocol, the quality of the Green tea is determined by estimating Total Polyphenols by UV- visible spectrophotometric method .The results show that the TETLEY brand contains GAE 21.9±1.48gm/100gm present in the tea leaf powder, in case of LIPTON brand contains GAE 28.3±1.22gm/100gm present in the tea leaf powder and AVT brand contains GAE 17.67±0.12 gm/100gm present in the tea leaf powder, statistically each value represents the mean value of Gallic acid equivalent ± SD for each tea brand by determining (n=3). The p value without * are considered as not significant (result are similar) when compared to ISO standards specified mean value. When comparison between ISO standards v/s TETLEY brand, p value is <0.05 show less significant, on comparison with LIPTON brand, p value is ** p <0.01 shows high significant and on comparison with AVT brand, p value is *** p <0.001 shows very high significant. From these only LIPTON tea brands possess and compliance with ISO standards (24.5±0.03gm./100gm).

As per the product insert procedure specified for estimating the Total Polyphenols by UV- visible spectrophotometric method, the results show that TETLEY brand contains GAE 0.266±0.035gm/dL present in the one teaspoon (2.961g.approx), in case of LIPTON brand contains GAE 0.094±0.016gm/dL present in the one teaspoon (3.01gm.approx) and AVT brand contains GAE0.227±0.002gm/dL present in the one teaspoon (3.01g.approx)weighed amount taken. When comparison between all the three brands, TETELY v/s LIPTON brand, p value is ***<0.0001, show significant difference in the mean value of results, on comparison with TETELY v/s AVT, p value is >0.05, shows significantly same in the mean value of results. LIPTON v/s AVT brand, p value is ***<0.001, show very high significant in the difference in mean value of result. Determining by gm/dL unit basis will gives the correct data to ensure the quality, so alternatively one may make use of this developed method.

As per the product insert procedure specified for estimating the Total Flavonoids by UV Visible spectrophotometric method, the results shows that TETLEY brand contains Quercetin equivalent 0.388± 0.049gm/dL present in the tea leaf powder, in case of LIPTON brand contains Quercetin equivalent 0.107± 0.008gm/dL present in the tea leaf powder and AVT brand contains Quercetin equivalent 0.296±0.021gm/dL present in the tea leaf powder. When comparison between all the three brands, TETELY v/s LIPTON brand, p value is *<0.05, show less significant different in the mean value of results, on comparison with TETELY v/s AVT, p and the LIPTON v/s AVT brand, p value is ***<0.001, shows very high significant in the difference in mean value of result. Determining by gm/dL unit basis will gives the correct data to ensure the quality, so alternatively one may make use of this developed method.

As per the product insert procedure specified for the method of tea preparation, the Antioxidant activity of the Green tea is determined by In vitro reducing power method by using UV- visible spectrophotometry. In case of antioxidant activity, the decrease in IC 50 value indicates that the potency of the product is greater, as a result: TETLEY shows 1.62 IC 50 value present in the one teaspoon (2.961g.approx), AVT shows 1.55 IC 50 value present in the one teaspoon (2.682g.approx) LIPTON shows 1.16 IC 50 value present in the one teaspoon (3.01g.approx) and shows 2.5 IC 50 value present in the half teaspoon (1.623 g. approx.).So in order to get the high antioxidant effect one teaspoon LIPTON is good.

CONCLUSION:

When both AVT and Tetley recommend for 1 teaspoon Lipton recommends for ½ teaspoon which means more number of servings can be provided as compared to the other two brands, the reason for the LIPTON may be like lower bitterness for better palatability, more number of servings shall be provided and last for more number of days and make the consumer to be affordable and economic and also and as per the quality concern, the testing is done by ISO 14502 protocol it shows that higher the content of Total Polyphenols when compared to other products, this may be the reason it withstand in top 10 in the market

While performing the experiment, the result obtained for total poly phenols and flavanoids and antioxidant effect was very poor in case of half teaspoon of Lipton. So considering from the results 1 teaspoon Lipton is best instead of ½ teaspoon specified by company insert procedure. So we suggest the results it is always better to take 1 teaspoon of top 10 Best-selling brand of LIPTON to obtain the maximum health benefits. In future this study can be extended to know the ground reality with subjective to human consumptions to know the actual amount of content is required to get maximum benefits for antioxidants potentiality.

ACKNOWLEDGEMENT:

Our heartful thanks to Administrator Rev. Fr. Jos Mathai Mailadiath, Nirmala College of Pharmacy, Muvattupuzha, for granting permission to utilize the facilities to carry out this work.

CONFLICT OF INTEREST:

None.

REFERENCES:

1. Bayarmaa Birasuren, Na Yeon Kim, Hye Lyun Jeon and Mee Ree Kim. Evaluation of the Antioxidant Capacity and Phenolic Content of Agriophyllum pungens Seed Extracts from Mongolia. PreventiveNutrition and food science, 2013;18(3):188-195.

2. Chatterjee A, Saluja M, Agarwal G, Alam, M. Greentea: A boon for periodontal and general health. Journal of Indian Periodontol. 2012; 16(2):161-167.

3. Harbow Matthew E. Tea Chemistry. Critical Reviews in Plant Science Society. 1997;16(5):415-485.

4. Amurdhavani. B.S. Benefits of Green Tea in Dentistry-A Review. Research J. Pharm. and Tech. 8(6): June, 2015: 772-774.

5. Rogith Kannan, Karpagam Krishnamoorthy. Evaluation of Lipid Profile Based on Consumption of Green Tea- A Systematic Review. Research J. Pharm. and Tech 2016;9(8):1277-1279.

6. Kaurc, Kapoor Hc. Antioxidants in fruits and vegetables –the millennium’s health. International Journal of Food science and Technology, 2008; 36 (7):703-725.

7. Sattar J, J AL-Shaeli, Ali M. Ethaeb. Decaffeinated green tea extract regulates glucose metabolism in insulin sensitive cell lines. Research J. Pharm. and Tech.2019; 12(6):2814-2823.

8. Leena Patil, Balaraman R. Protective effect of green tea extract on chemically induced testicular damage in rats. Research J.Pharm. and Tech.2(4): Oct-Dec, 2009: 837-841.

9. Merlin NJ, V Parthasarathy, R Manavalan. Role of Flavanoids in Free Radical Induced Hepatic Fibrosis. Research J. Pharm. and Tech. 2(1): Jan-Mar. 2009: 52-57.

10. Cook NC, Samman S. Flavonoids–Chemistry, metabolism, cardio protective effects and dietary sources. Journal of Nutrition Biochem. 1996; 7:66-76.

11. IS0 14502-1:2005(E) https:// www.iso. org/ obp/ui/# iso:std:iso:14502-1, First edition, 2005-03-01.

12. Lorenzo Bramati, Francesca Aquilano, and Piergiorgio Pietta. Unfermented rooibos tea-quantitative characterisation of flavanoids by HPLC and UV and determination of the total antioxidant activity. Journal of Agriculture and food chemistry, 2004; 51(25):7472- 7480.

13. R. Badmanaban, C N Patel, Vinay Patel. Determination of Polyphenolic Content and In-vitro Antioxidant Capacity of the Leaves of Lagenaria siceraria (mol.) standl.2010; 2(7): 162-169.

Received on 20.09.2019 Modified on 19.10.2019

Research J. Pharm. and Tech. 2020; 13(7): 3179-3183.

DOI: 10.5958/0974-360X.2020.00562.4