3. MATERIALS AND METHODS

Transcription

1 46 3. MATERIALS AND METHODS The present investigations Studies on flavan-3-ols and biological activity of Kangra tea [Camellia sinensis (L) O Kuntze] were carried out to explore fresh green tea shoots of Kangra local for their total polyphenols and flavan-3-ols profiles as affected by the variations in weather conditions during four harvesting flush seasons (first flush: April to mid May; second flush: mid May to June; rainy flush or main flush: July to mid September and winter flush: mid September to October) and to corroborate health claims associated with tea consumption. Samples of fresh tea shoots were collected from Wah Tea Estate, Rajpura, Palampur at seven day intervals throughout the plucking seasons (April to October/November) during three consecutive years (2007, 2008 and 2009). These samples were subjected to microwave heat treatment within twenty minutes of plucking, dried and analyzed for total polyphenols and total catechins (flavan- 3-ols), using standard techniques. (+)-Catechin (C), ( )-epicatechin (EC), ( )- epigallocatechin (EGC), ( )-epigallocatechin gallate (EGCG) and ( )-epicatechin gallate (ECG) were estimated in tea powders obtained by lyophilizing the aqueous extracts of fresh green tea shoots using chromatographic techniques. Antioxidant activity of aqueous extracts of green tea shoots and tea powders was evaluated in terms of IC 50 values using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical assay and metal ions chelating activity. Antibacterial activity of aqueous extracts was also evaluated against six selected bacterial pathogens. All analytical estimations were carried out in triplicate to minimize the source of error. The data on analytical estimations were pooled and analyzed statistically. 3.1 Sampling Samples of fresh green tea shoots (two leaves and a bud) were collected from Wah Tea Estate, Rajpura, Palampur at seven day intervals during four flush seasons (first flush: April to mid May; second flush: mid May to June; rainy flush or main flush: July to mid September and winter flush: mid September to October), for three consecutive years (2007 to 2009). Owing to low temperature during winters, the tea plant experience complete dormancy for about days. The schedule of sampling during three years along with sample codes is depicted in Table 3.1. The samples of fresh green tea shoots were always subjected to heat treatment in a microwave oven for three minutes at power

2 47 Table 3.1 Week number Week-number, date of collection and treatment of green tea shoots Date of collection Treatment W 1 April 03 April 03 April 06 The samples of fresh W 2 April 10 April 10 April 13 W 3 April 17 April 17 April 20 W 4 April 24 April 24 April 27 W 5 May 01 May 01 May 04 W 6 May 08 May 08 May 11 W 7 May 15 May 15 May 18 W 8 May 22 May 22 May 25 W 9 May 29 May 29 June 01 W 10 June 05 June 05 June 08 W 11 June 12 June 12 June 15 W 12 June 19 June 19 June 22 W 13 June 26 June 26 June 29 W 14 July 03 July 03 July 06 W 15 July 10 July 10 July 13 W 16 July 17 July 17 July 20 W 17 July 24 July 24 July 27 W 18 July 31 July 31 August 03 W 19 August 07 August 07 August 10 W 20 August 14 August 14 August 17 W 21 August 21 August 21 August 24 W 22 August 28 August 28 August 31 W 23 September 04 September 04 September 07 W 24 September 11 September 11 September 14 W 25 September 18 September 18 September 21 W 26 September 25 September 25 September 29 W 27 October 02 October 02 October 05 W 28 October 09 October 09 October 12 W 29 October 16 October 16 October 19 W 30 October 23 October 23 October 26 W 31 October 30 October 30 November 02 green tea shoots were subjected to heat treatment for 3 minutes in a microwave oven within 20 minutes of their plucking and finally dried in a hot air oven maintained at 45±5 C. During the year 2009 the samples after microwave heat treatment, were separated into four parts comprising whole leaves (two leaves and a bud), buds, first leaves and second leaves and dried separately. 100% (P-HI) in IFB convection microwave oven model: 30SC1 (30 L capacity, microwave 1.4 KW, frequency 2450 MHz), within twenty minutes of their plucking and finally dried in a hot air oven maintained at 45±5 C for 24 h. However, during the year 2009 the samples after microwave heat treatment were separated into four parts comprising whole leaves (two leaves and a bud), buds, first leaves and second leaves

3 48 and dried separately (Plate 3.1). The dried samples were ground using MAC Willey Grinder (Arthur H. Thomas Type) to pass through 60 mesh sieve and finally stored in air tight plastic containers. The containers were opened only at the time of analysis. 3.2 Moisture Content The stored samples were always dried completely prior to aqueous extractions for analytical estimations. Sample (1 g) of dried green tea shoots stored in air-tight container was taken in a pre-weighed Petri dish and placed in a hot air oven maintained at 60±5 0 C for 24 h. The Petri dish was taken out in a desiccator after regular intervals of 2 h till a constant weight is reached. The difference in initial and final weight was taken as the moisture content of the sample. 3.3 Preparation of tea extracts All quantitative estimations were always carried out in freshly prepared tea extracts. The method for aqueous extraction was standardized: Aqueous extractions were carried out at 60±5 C incorporating different weights of sample in various volumes of double distilled water for varying time periods in 250 ml Erlenmeyer conical flasks. After the stipulated time, the contents of the flasks were allowed to cool to room temperature and then filtered through Whatman Grade 1 filter paper. The final volume was made with the help of double distilled water. Total polyphenols and total catechins were estimated in each extract by standard procedures and given in Tables 3.2 and 3.3 (described in section 3.4).

4 49 49 a b c d Plate 3.1 Photographs of sample of green tea shoots: (a)two leaves and a Plate 3.1 Photographs of sample of green tea shoots: (a) two leaves and a bud, (b) before, (c) and after microwave heat treatment, (d) dried and grounded

5 50 Table 3.2 Variations in total polyphenols and total catechins as affected by the changes in weight of sample and volume of water S. no. Temperature ( o C) Duration of extraction (minutes) Sample weight (mg) Volume of water (ml) Total Polyphenols (%) Total catechins (%) Table 3.3 Variations in total polyphenols and total catechins as affected by duration of extraction period, weight of sample and volume of water S. no. Temperature ( o C) Duration of extraction (minutes) Sample weight (mg) Volume of water (ml) Total polyphenols (%) Total catechins (%) The content of total polyphenols and total catechins as affected by the number of extractions were also evaluated.

6 51 Table 3.4 Total polyphenols and total catechins as affected by number of extractions Sample no. Extraction no st 300 mg in 100 ml double distilled water 2 nd in 100 ml water 3 rd in 100 ml water 2. 1 st 300 mg in 100 ml double distilled water 2 nd in 100 ml water 3 rd in 100 ml water Temperature ( o C) Duration of extraction (minutes) Total polyphenols (%) Total catechins (%) The following procedure for the preparations of tea extracts was followed during the course of present investigations: Dried tea sample (300 mg) was taken in a 250 ml Erlenmeyer conical flask and 100 ml of pre-boiled hot double distilled water was poured into the flask. The flask was covered with aluminum foil and kept in a water bath shaker maintained at 60±5 C for 20 minutes. The contents of the flask were allowed to cool to room temperature and then filtered through Whatman Grade 1 filter paper in a 100 ml measuring flask. The final volume was made with the help of double distilled water. 3.4 Estimation of total polyphenols and total catechins Standard curves Standard curves for tannic acid and catechin were plotted employing standard techniques prior to quantitative estimation of total polyphenols and total catechins in aqueous tea extracts Tannic acid Standard curve was plotted employing protocol presented in Table 3.5. The reagents after mixing were allowed to incubate at 30 C for 40 minutes before recording

7 52 absorbance at 725 nm with the help of Merck Spectroquant Pharo 100 spectrophotometer. Reagents 1. Folin-Ciocalteu s phenol reagent (1 N): Folin-Ciocalteu s phenol reagent (2 N) (Merck, Mumbai) was diluted with double distilled water in the ratio of 1:1. The reagent was stored at 4 C. 2. Sodium carbonate solution (20%): 20 g anhydrous sodium carbonate (Qualigen Fine chemicals, Mumbai) was dissolved in 100 ml double distilled water and stored at room temperature. 3. Tannic acid (0.5 mg ml -1 ) solution: g Tannic acid (Sigma, USA) was dissolved in 10 ml double distilled water. Table 3.5 Protocol of standard curve for tannic acid Tube no. Vol. of tannic acid used (ml) Vol. of double distilled water (ml) Vol. of Folin- Ciocalteu s phenol reagent (ml) Vol. of sodium carbonate solution (ml) Conc. of tannic acid (µg) Blank T T T T T Standard curve along with equation of line, value of slope and y-intercept is presented in Figure 3.1.

8 Absorbance at 725 nm Concentration of tannic acid ( g) Equation of line: y = x Slope = ; y-intercept = ; r² = Figure 3.1 Standard curve for tannic acid Catechin Standard curve was plotted employing protocol presented in Table 3.6. The reagents after mixing were allowed to incubate at 30 C for 20 minutes in the dark before recording absorbance at 500 nm with the help of Merck Spectroquant Pharo 100 spectrophotometer. Reagents 1. Vanillin reagent (1%): 1 g Vanillin (s.d. FINE CHEM Ltd) was dissolved in 100 ml methanol (AR) (Merck, Mumbai) 2. Hydrochloric acid (9 M): To ml HCl was added ml methanol (AR) (Merck, Mumbai). 3. Catechin solution (1 mg ml -1 ): g Catechin (Sigma, USA) was dissolved in 5 ml methanol (AR) (Merck, Mumbai) Table 3.6 Protocol of standard curve for catechin Tube no. Vol. of catechin used Vol. of methanol Vol. of vanillin reagent Vol. of HCl Conc. of catechin

9 Absorbance at 500 nm 54 (ml) (ml) (ml) (ml) (µg) Blank T T T T T T Figure 3.2. Standard curve along with equation of line, slope and y-intercept is presented in Concentration of catechin ( g) Equation of line: y = x + ( ) Slope = ; y-intercept = ( ) ; r² = Figure 3.2 Standard curve for catechin Estimation of total polyphenols Total polyphenols were always estimated in freshly prepared tea extracts by the method of Makkar (2003).

10 55 To the freshly prepared tea extract (0.025 ml) was added double distilled water (0.975 ml), 1 N Folin-Ciocalteu s phenol reagent (0.500 ml) and 20% sodium carbonate solution (2.500 ml). The contents were mixed thoroughly with the help of vortex and incubated at 30 C for 40 minutes. Absorbance was recorded at 725 nm with the help of Merck Spectroquant Pharo 100 spectrophotometer. Total polyphenols were calculated using following equations: Absorbance at 725 nm ( ) Concentration (µg) = = X µg The concentration of total polyphenols (TP) was finally expressed in terms of g kg -1 of fresh green tea shoots on dry weight basis using following formula: X (µg) Concentration (g kg -1 ) = (Weight of sample Moisture content) Estimation of total catechins Total catechins were always estimated in freshly prepared tea extracts by the method of Sun et al. (1998). To the freshly prepared tea extract (0.250 ml) was added methanol (0.750 ml), 1% vanillin reagent (2.500 ml) and 9 M HCl (2.500 ml). The contents were mixed thoroughly with the help of vortex and allowed to incubate in dark at 30 C for 20 minutes. Finally absorbance was recorded at 500 nm with help of Merck Spectroquant Pharo 100 spectrophotometer. Total catechins were calculated using following equation: Absorbance at 500 nm + ( ) Concentration (µg) = = X µg The concentration of total catechins (TC) was finally expressed in terms of g kg -1 in fresh green tea shoots on dry weight basis using following formula: X Concentration (g kg -1 ) = (Weight of sample Moisture content) 3.5 Monthly representative samples and preparation of tea powders

11 56 Monthly samples of fresh green tea shoots were required to investigate monthly variations of phenolic constituents, preparation of tea powders and evaluation of flavan- 3-ols. Samples to represent a particular month were prepared by pooling and mixing thoroughly weekly samples of dried tea shoots/bud/first leaf/second leaf of the month in equal proportions by weight Preparation of tea powders Tea powders were prepared by lyophilizing aqueous extracts of monthly representative samples (Plate 3.2). A known weight (7 g) of the representative sample was taken in a 250 ml Erlenmeyer conical flask. To this was added 100 ml of pre-boiled hot double distilled water and the flask along with its contents was kept on water bath shaker (RSB-12 REMI) maintained at 50±5 C for 60 minutes. The contents of the flask were allowed to cool to room temperature; filtered through muslin cloth and then centrifuged at 764 x g for 15 minutes and finally filtered through Whatman Grade 1 filter paper to obtain absolutely transparent/clear aqueous extract. The aqueous extracts were finally dried to powder form with the help of Edwards EF 4 Modulyo freeze dryer and Heto Power Dry LL 3000 freeze dryer. The powders so obtained were immediately transferred into glass tubes fitted with air-tight stoppers which were stored in vacuum desiccator. The fractions from Sephadex G-25 (Sigma, USA) column ( ) containing significant amount of flavan-3-ols were pooled and freeze dried after removing ethanol with the help of rotary vacuum evaporator under reduced pressure.

12 57 57 a May b b b June August Plate 3.2 Photographs of (a) aqueous extracts of green tea shoots and (b) tea powders Plate 3.2 Photographs of (a) aqueous extracts of green tea shoots and (b) tea powders obtained by lyophilizing aqueous extracts of fresh green tea shoots during different months

13 Qualitative evaluation of flavan-3-ols Qualitative evaluations of flavan-3-ols in tea powders were carried out using chromatographic techniques. The tea powders were subjected to size-exclusion chromatographic separation using Sephadex G-25 (Sigma, USA) as stationary phase and 50% ethanol (AR) as mobile phase by the method of Cutler (2008). Various fractions collected were subjected to thin-layer chromatography along with standard flavan-3-ols by the method of Sherma and Fried (1996). i. Size-Exclusion chromatography Swollen Sephadex G-25 gel was degassed and packed in a glass column (48 x 2.5 cm) to a height of 30 cm (bed volume: cm 3 and void volume: cm 3 ). The column was equilibrated with 50% ethanol. Fresh solution prepared by dissolving 1 g tea powder into 6 ml of 50% ethanol and applied on the top of the equilibrated column. Elution at a flow rate of ml per minute was done with 50% ethanol. The eluted fractions, each of 5 ml, were collected in clean and dry test tubes. These fractions were further subjected to thin-layer chromatography and quantitative estimation of total polyphenols and total catechins by standard techniques. ii. Thin- layer chromatography Thin layer chromatography was done on glass plate coated with silica gel G by the method of Stahl (1969). Requirements: 1. Glass plate (20 x 20 cm) 2. Silica gel G (Merck, Mumbai) for thin-layer chromatography 3. Standard solutions (0.5 mg ml -1 ): g of each standard flavan-3-ols [(+)- catechin, ( )-epicatechin, ( )-epigallocatechin gallate, ( )-epicatechin gallate and ( )-epigallocatechin (Sigma, USA)] was dissolved in 2 ml methanol (AR). 4. Solvent system: Chloroform: Ethyl acetate: Acetic Acid:: 25:75:0.5 (By volume) 5. Detection reagent: Iodine vapours 6. Samples: fractions eluted from Sephadex G-25 column Procedure

14 59 Washed and dried glass plates were cleaned with acetone. Slurry was prepared by mixing 10 g of silica gel G (for each plate) in 20 ml double distilled water. Plates were coated with thin-layer (thickness 0.2 mm) of silica gel G slurry with the help of spreader. The plates were allowed to dry at room temperature and then activated at 110 C in hot air oven for 1 h. 5 µl of catechin, epicatechin, epigallocatechin, epigallocatechin gallate, epicatechin gallate solutions and fractions eluted from Sephadex G-25 column, were spotted on silica plate. The plate was developed in the solvent system. The developed plate was dried at room temperature. Detection was done by exposing the plate to iodine vapours in an iodine saturated chamber. 3.6 High performance liquid chromatography (HPLC) Samples of tea powder were characterized for flavan-3-ols profiles and their quantification by HPLC techniques by the method of Zhu and Chen (1999). Requirements: 1. WATERS HPLC system with 510 and 515 pumps, Rheodyne injector, Novapak C-18 (4 µm, 4.6x 250 mm) column, 490E Multiwavelength detector, Millennium 2010 data manager. 2. Whatman stainless steel syringe assembly with 0.22 µm Durapore (Millipore, USA) membrane filter. 3. Mobile phase constituents: acetonitrile (ACN), orthophosphoric acid (HPLC grade Merck, Mumbai) and double distilled water. 4. Standard solutions (0.5 mg ml -1 ): g of each standard flavan-3-ols [(+)- catechin, ( )-epicatechin, ( )-epigallocatechin gallate, ( )-epicatechin gallate and ( )-epigallocatechin] was dissolved in 2 ml 50% methanol (HPLC grade Merck, Mumbai). 5. Sample solutions (10 mg ml -1 ): g of tea powder was dissolved in 4 ml of double distilled water. Procedure Acetonitrile (ACN) and double distilled water both acidified with 0.025% H 3 PO 4 was filtered using Durapore (0.47 µm) membrane filter. The sample solutions and standard solutions were filtered through Whatman stainless steel syringe assembly using 0.22 µm Durapore membrane filter. 20 µl aliquot was used for HPLC injection.

15 60 The chromatograms were monitored at 220 nm. The flow rate was 1 ml min -1. The elution was done by the following gradient system: Time duration (minutes) ACN* (%) Water* (%) *acidified with 0.025% orthophosphoric acid (H 3 PO 4 ) Figure 3.3 represents the HPLC profile of standard catechins: catechin (C), epicatechin (EC), epigallocatechin (EGC), epigallocatechin gallate (EGCG) and epicatechin gallate (ECG). The retention times for EGC min, C min, EC min, EGCG min and ECG min (Table 3.7). Concentration of the standard solution injected = 0.1 mg ml -1 Volume of solution injected = 20 µl The qualitative and quantitative estimations of catechin and its derivatives in aqueous solutions of tea powders were carried out by comparing their retention time and spectrum with that of standards. The quantification was done with the help of following formula: Concentration of standard Concentration of unknown = Peak area of unknown Peak area of standard

16 61 61 EGC C EC EGCG ECG Figure 3.3 High performance liquid chromatogram of standards: catechin (C), epicatechin (EC), epigallocatechin (EGC), epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)

17 62 Table 3.7 HPLC profile of standard catechins monitored at 220 nm along with retention time, area and height of peak Standard Retention time (minutes) Area (uv*sec) Height (uv) EGC C EC EGCG ECG Evaluation of antioxidant property Antioxidant property of Kangra tea was evaluated on the basis of free radical scavenging potential of the aqueous extracts of fresh green tea shoots and aqueous solution of tea powders using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and metal ions chelating assays , 2-diphenyl-1-picrylhydrazyl assay The 2, 2-diphenyl-1-picrylhydrazyl free radical scavenging activity of aqueous extracts of green tea shoot samples and aqueous solutions of tea powders was evaluated by the methods of Kordali et al. (2005) and Sharma and Bhat (2009). Protocol for evaluating free radical scavenging activity in tea samples is presented in Table Reagents 1. DPPH solution (200 µm): g of 2, 2-diphenyl-1-picrylhydrazyl (Sigma, USA) was dissolved in 25 ml methanol (AR). 2. Acetate buffer (0.1 M, ph 5.5): 1.64 g of Sodium Acetate (Himedia, Mumbai) and 0.6 ml of Acetic Acid (Qualigen Fine chemicals, Mumbai) was dissolved in 50 ml double distilled water, ph was adjusted with ph meter CyberScan 510 to 5.5 and volume was made to 100 ml. 3. Buffered Methanol: 40 ml of 0.1 M acetate buffer (ph 5.5) was added to 60 ml methanol (AR).

18 , 2-diphenyl-1-picrylhydrazyl solution (200 µm): g of DPPH was dissolved in 25 ml buffered methanol (AR). 5. Samples solution: g tea powder was dissolved in 5 ml double distilled water to prepare a solution of 1 mg ml -1 concentration. Aqueous extracts of samples of fresh green tea shoots were prepared as described in section Ascorbic acid solution (1 mm): g of ascorbic acid (Himedia, Mumbai) was dissolved in 20 ml of methanol (AR). 7. Butylated hydroxytoluene (BHT) solution (1 mm): g of BHT (Merck, Mumbai) was dissolved in 20 ml methanol (AR). 8. Standard solutions (0.5 mg ml -1 ): g of each standard flavan-3-ols [(+)- catechin, ( )-epicatechin, ( )-epigallocatechin gallate, ( )-epicatechin gallate and ( )- epigallocatechin] was dissolved in 2 ml methanol (AR) (Merck, Mumbai). i. Standardization of technique To a known volume (20, 40, 60, 80, 100 µl) of 1 mm of ascorbic acid and 1 mm BHT solutions (each taken in two different sets of test tubes and each set comprising of five test tubes), was added methanol/ buffered methanol to make the final volume of 3 ml. 1 ml of 200 µm DPPH solution prepared in methanol/ buffered methanol were added to the test tubes containing methanolic/ buffered methanolic solutions of ascorbic acid and BHT. The contents were mixed thoroughly with the help of vortex mixer and allowed to incubate at 30 C for 30 minutes in the dark. Absorbance was measured at 517 nm with the help of Merck Spectroquant Pharo 100 spectrophotometer. Per cent of DPPH free radical scavenging activity (% inhibition) was calculated with the help of following equation: Absorbance control Absorbance sample % Inhibition = (i) Absorbance control Graphs were plotted between % inhibition and concentration of ascorbic acid/bht to evaluate the values of slope and y-intercepts. Figures 3.4 and 3.6 represent observations with respect to ascorbic acid and BHT in methanolic solutions and Figures 3.5 and 3.7 represent ascorbic acid and BHT in buffered methanolic solutions.

19 %Inhibition %Inhibition Concentration of ascorbic acid ( M) Slope = 4.127; y-intercept = ( ) 3.402; r² = Figure 3.4 Graph between ascorbic acid and per cent inhibition in methanol Concentration of ascorbic acid ( M) Slope = 4.145; y-intercept = ( ) 6.830; r² = Figure 3.5 Graph between ascorbic acid and per cent inhibition in buffered methanol

20 % Inhibition %Inhibition Concentration of BHT ( M) Slope = 2.679; y-intercept = 6.069; r² = Figure 3.6 Graph between BHT and per cent inhibition in methanol Concentration of BHT ( M) Slope = 3.120; y-intercept = 12.59; r² = Figure 3.7 Graph between BHT and per cent inhibition in buffered methanol IC 50 values (the amount of antioxidant necessary to decrease the initial DPPH concentration by 50%) for ascorbic acid and BHT in methanol/buffered methanol were evaluated using the following equation: IC 50 Value = (50 y-intercept)/ slope (ii) ii. Time course of scavenging of DPPH free radicals by ascorbic acid, BHT and tea powder sample

21 66 The kinetics of DPPH free radical scavenging activity by 1 mg ml -1 tea powder solution was evaluated and compared with that of 1mM of ascorbic acid and 1 mm BHT solutions reported in literature, to ascertain the period of incubation. Kinetics of DPPH free radical scavenging was calibrated incorporating following reaction: 1. DPPH and Ascorbic acid: 1 ml of DPPH solution (200 µm) was mixed with 60 µl of ascorbic acid solution (1 mm) in ml methanol/buffered methanol. 2. DPPH with BHT: 1 ml of DPPH solution (200 µm) was mixed with 60 µl of BHT solution (1 mm) in ml methanol/ buffered methanol. 3. DPPH with tea powder: 1 ml of DPPH solution (200 µm) was mixed with 12 µl of tea powder solution (1 mg ml -1 ) in ml methanol/ buffered methanol. The reactions were allowed to proceed at room temperature and absorbance at 517 nm was recorded immediately after mixing; followed by at regular intervals of five minutes each for 90 minutes duration with the help of Merck Spectroquant Pharo 100 spectrophotometer. The absorbance data recorded for different reactions is represented in Table 3.8 and 3.9. iii. Evaluation of standard catechin and its derivatives To a known volume (4, 8, 12, 16, 20, 24 µl) of 0.5 mg ml -1 of standard solutions (each taken in five different sets of test tubes and each set comprising of six test tubes), was added buffered methanol to make the final volume of 3 ml. 1 ml of 200 µm DPPH solution prepared in buffered methanol was added to the test tubes containing buffered methanolic standard solutions. The contents were mixed thoroughly with the help of vortex and allowed to incubate at 30 C for 30 minutes in the dark. Absorbance was measured at 517 nm with the help of Merck Spectroquant Pharo 100 spectrophotometer. Table 3.8 Time course of scavenging of DPPH free radicals in methanol Time Abs of Abs of DPPH + Abs of DPPH Abs of DPPH (minutes) DPPH ascorbic acid + BHT + tea sample

22

23 68 Table 3.9 Time course of scavenging of DPPH free radicals in buffered methanol Time Abs of Abs of DPPH+ Abs of DPPH Abs of DPPH (minutes) DPPH ascorbic acid + BHT +tea sample

24 69 Per cent of DPPH free radical scavenging activity and IC 50 values were calculated with the help of equations (i) and (ii). iv. Evaluation of tea samples Corroboration between the IC 50 values obtained in the present investigation and that of reported in literature for ascorbic acid and BHT confirmed the accuracy of the standardization of the technique, hence per cent of DPPH free radical scavenging activity and IC 50 values of aqueous extract of tea samples and aqueous solutions of tea powders were also evaluated adopting similar procedure and calculations incorporating the following protocol: Table 3.10 Protocol of DPPH free radicals scavenging activity in buffered methanol reaction medium Tube no. Vol. of sample solution Vol. of buffered methanol (ml) Vol. of DPPH solution in buffered methanol (ml) (ml) T 1 (Control) T T T T T T Metal ions chelation assay The binding potency of metal ions by tea catechins was evaluated using method of Tang et al. (2002). Protocol for evaluating metal chelating activity in tea powders is presented in Table Reagents

25 70 1. Ferrous chloride (FeCl 2.7H 2 O) solution (2 mm): g ferrous chloride (Himedia, Mumbai) was dissolved in 10 ml double distilled water. 2. Ferrozine solution (5 mm): g of ferrozine (Himedia, Mumbai) was dissolved in 10 ml of double distilled water. 3. Samples solution (5 mg ml -1 ): g of tea powder was dissolved in 10 ml double distilled water. 4. Ethylenediaminetetra-acetic acid (EDTA) solution (1 mm): g EDTA (Himedia, Mumbai) was dissolved in 10 ml double distilled water. 5. Citric acid solution (25 mm): 0.05 g citric acid (Himedia, Mumbai) was dissolved in 5 ml double distilled water. i. Standardization of technique The technique for the evaluation of metal ions chelation by the tea catechins a method was standardized using standard chelating agents namely EDTA and citric acid. a. EDTA-Metal ions chelation assay To a known volume (90, 105, 120, 135, 150, 165 µl) of 1 mm of EDTA solution taken in a set of six test tubes, was added double distilled water to make the final volume to 2.7 ml. To this was added 2 mm of FeCl 2.7H 2 O solution (0.1 ml) and 5 mm of ferrozine solution (0.2 ml). The contents were mixed thoroughly with the help of vortex and allowed to incubate at room temperature for 20 minutes in the dark. Absorbance was measured at 562 nm with the help of Spectronic GENESYS 5 spectrophotometer (Unicam, USA). Per cent of metal ions chelating activity of EDTA (% chelating activity) was calculated with the help of following equation: Absorbance control Absorbance sample % chelating activity = (iii) Absorbance control The value of slope and y-intercept were evaluated from a plot (Figure 3.8) between % chelating activity and concentration of EDTA.

26 %Chelating activity Concentration of EDTA ( M) Slope = 23.56; y-intercept = ( ) 50.62; r² = Figure 3.8 Graph between EDTA and per cent chelating activity 2+ IC 50(Fe ) value (the amount of metal chelator necessary to decrease the initial ferrous ions concentration by 50%) for EDTA was evaluated using the following equation: 2+ IC 50(Fe ) Value = (50 y-intercept)/ slope (iv) b. Citric acid-metal ions chelation assay To a known volume (150, 300, 450, 600, 750, 900, 1600 µl) of 25 mm citric acid solution, taken in a set of seven test tubes, was added double distilled water to make the final volume to 2.7 ml. To this was added 2 mm FeCl 2.7H 2 O solution (0.1 ml) and 5 mm ferrozine solution (0.2 ml). The contents were mixed thoroughly with the help of vortex mixer and allowed to incubate at room temperature for 20 minutes in the dark. Absorbance was measured at 562 nm with the help of Spectronic GENESYS 5 spectrophotometer (Unicam, USA). Per cent of metal ions chelating activity (% chelating activity) and IC 50 value of citric acid was calculated with the help of equations (iii) and (iv). The value of slope and y-intercept was evaluated from a plot (Figure 3.9) between % chelating activity and concentration of citric acid.

27 %Chelating activity concentration of citric acid (mm) Slope = ; y-intercept =16.66; r² = Figure 3.9 Graph between citric acid and per cent chelating activity ii. Evaluation of tea samples The metal ions chelation activity of tea catechins was evaluated in aqueous solutions of tea powders by adopting similar procedure and calculations as described above incorporating the following protocol: Table 3.11 Protocol of metal ions chelating activity Tube no. Vol. of stock sample solution (ml) Vol. of double distilled water (ml) Vol. of FeCl 2 (ml) Vol. of ferrozine (ml) T 1 (Control) T T T T T T The sample solution have pale yellow color and sample volumes used for evaluating metal ions chelating activity were quite large, it was noted that sample form blue color with ferrous ions. To correct this occurrence, sample blanks containing the

28 73 appropriate dilution of each volume of sample with 2 mm FeCl 2.7H 2 O solution (0.1 ml) were used. The absorbance of each sample blank was subtracted from the absorbance of sample, respectively. The final absorbance so obtained was used for further calculations. 3.8 Antibacterial activity Growth inhibitory activity of aqueous extracts of samples of fresh green tea shoots, bud, first leaf and second leaf and aqueous solutions of tea powders was evaluated against six selected pathogenic bacteria i.e. Escherichia coli (MTCC-443), Psuedomonas aeruginosa (MTCC-741), Listeria monocytogenes (MTCC-839), Bacillus cereus (MTCC-1272), Staphylococcus aureus (MTCC-96) and Streptococcus mutans (MTCC-890) procured from the Institute of Microbial Technology (IMTECH), Chandigarh. The bacterial cultures were maintained on freshly prepared nutrient agar (NA) medium and were stored at 4±0.5 C for further use. Requirements 1. Nutrient Agar: 28 g of nutrient agar (Himedia, Mumbai) was dissolved in 1 L double distilled water and sterilized in an autoclave at 121 C (15 lb inch -2 ) for 15 minutes. 2. Nutrient Broth: 13 g of nutrient broth (Himedia, Mumbai) was dissolved in 1 L double distilled water and sterilized in an autoclave at 121 C (15 lb inch -2 ) for 15 minutes. 3. Standard Solutions: g of each standard flavan-3-ols (catechin, epicatechin, epicatechin gallate, epigallocatechin gallate and epigallocatechin) was dissolved in 1 ml of double distilled water. The solutions were filter sterilized by 0.22 µm Durapore (Millipore, USA) membrane filter before evaluating for antibacterial activity. 4. Sample preparation: Aqueous extracts of dried green tea shoots were prepared using protocol as described in section except 10 g of sample was taken for extraction. The antibacterial activity was also evaluated in aqueous solution of tea powder (10 mg ml -1 ) obtained by lyophilizing column pooled fractions. The extracts/solutions were filter sterilized by 0.22 µm Durapore membrane filter before evaluating for antibacterial activity.

29 74 5. Ferrous sulphate (FeSO 4.7H 2 O) solution (0.1 M): g ferrous sulphate (Qualigen, Mumbai) was dissolved in 10 ml double distilled water. The solution was filter sterilized by 0.22 µm Durapore (Millipore, USA) membrane filter. 6. 2, 3, 5 Triphenyl tetrazolium chloride (0.1%): 0.1 g triphenyl tetrazolium chloride (Himedia, Mumbai) was dissolved in 100 ml double distilled water Antibiotic sensitivity Antibiotic sensitivity of six bacterial pathogens was tested against 18 standard antibiotics using paper disc method. 0.1 ml of 24 h old culture, grown in nutrient broth, was spread uniformly and aseptically on nutrient agar plate. Then antibiotic discs were placed aseptically on the agar plate. The plates were then incubated at 37 C for 24 h and the zone of clearance around the discs was observed and measured in mm Agar-well diffusion method for antibacterial activity Antibacterial activities of aqueous extracts of monthly pooled samples of green tea shoots, bud, first leaf and second leaf were evaluated by the method of Toda et al. (1989a). The antibacterial activity was always determined in freshly prepared extracts/solutions.

30 75 Table 3.12 List of the standard antibiotics used against six bacterial pathogens S. no. Antibiotic (Himedia, Mumbai) Code Concentration(µg/disc) 1 Amoxycillin Am 10 2 Carbenicillin Cb Cephadroxil Cq 30 5 Cepoxitin Cn 30 5 Chloramphenicol C 30 6 Ciprofloxacin Cf 5 7 Clindamycin Cd 2 8 Co-trimoxazole Co 25 9 Erythromycin E Fluconozole Fu Gentamycin G Metronidazole Mt 5 13 Nalidixic Acid Na Nitrofurantoin Nf Norfloxacin Nx Ofloxacin Of 5 17 Penicillin G P Tetracyclin T 30 To evaluate antibacterial activities of aqueous extracts of samples of green tea and aqueous solutions of tea powders, agar-well diffusion method was performed. 0.1 ml of 24 h old culture, grown in nutrient broth, was spread uniformly and aseptically on nutrient agar plate. The wells of 6.0 mm in diameter were aseptically punched with sterile stainless steel borer on each agar plate. In each well fixed volume 0.1 ml of extract was added and double distilled water was used as the control. Plates were kept at 4±0.5 C for 1 h to allow diffusion of the extract into the medium. The plates were then incubated at 37 C for 24 h. After 24 h, the diameter of zone of clearance around the wells was measured. For determining minimum Inhibitory concentration (the lowest concentration of the extract that did not permit turbidity or growth of the test organism) for pathogenic bacteria, increasing concentrations of aqueous solutions of standard flavan-3-ols, tea powders and aqueous extracts of samples of green tea were used.

31 Bioautography A TLC bioautographic method as described by Kline and Golab (1965) was used to detect active antibacterial components present in tea powder. For this 50 µl of aqueous solution of tea powder (1 mg ml -1 ) was applied on silica gel G TLC plate which was developed by using chloroform: ethyl acetate: acetic acid (25:75:0.5) as the solvent system and then dried for complete removal of solvent. Then TLC plate was placed in the Petri dish and 0.1 ml of inoculum of B. cereus grown in nutrient broth was added to agar medium and this medium was distributed over the entire TLC plate. The plate was incubated at 37 C for 24 h and the inhibition zone was visualized with 0.1% of triphenyl tetrazolium chloride solution. Inhibition zone was observed as clear area against a red colored background Determination of heat stability of tea catechins The aqueous solutions of standard EGCG and ECG (2 mg ml -1 ), and tea powder (10 mg ml -1 ) obtained by lyophilizing column pooled fractions were evaluated for heat stability of tea catechins. The heat stability of green tea catechins was measured by autoclaving these aqueous solutions for 15 minutes at 121 C. The antibacterial activity of these autoclaved solutions was evaluated by the agar-well diffusion method as described above Influence of metal ions on inhibitory properties of aqueous solution of tea powder Influence of metal ions on inhibitory properties of aqueous solution of tea powder was evaluated using agar-well diffusion method as described by West et al. (2001). The growth inhibition of aqueous solution of tea powder was determined against B. cereus and S. aureus using agar-well diffusion method as described above. Zones of inhibition were observed, then 0.1 ml of ferrous sulphate solution was added to one well whereas sterilized double distilled water was added to other well as a control. Plate was kept at 4 C for 1 h to allow diffusion of the solution into the medium. The plate was then incubated at 37 C for 24 h then appearance of growth was observed in inhibition zone. 3.9 Agro-climatic data Meteorological data on temperature, rainfall, relative humidity, bright sunshine hours and evaporation were collected from the Agro-meteorological Division,

32 77 Department of Agronomy, College of Agriculture, Himachal Pradesh Agricultural University Palampur, India Statistical analysis All analytical estimations were carried out in triplicate to minimize the experimental error. The data were pooled and analyzed statistically. The data analysis was carried out using WindoStat software version 8.0. Analysis of variance (ANOVA) followed by analysis of least significant difference (p<0.05) among means by the Duncan s Multiple Range Test (DMRT) was performed.

33 78 78 Fresh green tea shoots (weekly collected samples for three years 2007,2008 and 2009 from April to October/November) Enzyme inactivation(microwave heat treatment) Samples of green tea shoots divided in four parts (Two leaves and a bud, bud, first leaf and second leaf) during 2009 Dried at 45 5 C for 24 h, ground and stored TP and TC evaluation in weekly samples Pooled (monthly basis) TP and TC evaluation Aqueous extraction Lyophilization Biological activity (Antioxidant and Antibacterial) Tea powders TP and TC evaluation Fractionation Characterization and quantification of flavan-3-ols Qualitative and quantitative evaluation of flavan-3-ols Biological activity (Antioxidant and Antibacterial ) DPPH free radical scavenging Antioxidant Metal ion chelation Flow chart for flavan-3-ols profile and biological activities of fresh green tea shoots Flow chart for flavan-3-ols profile and biological activities of fresh green tea shoots of Kangra tea [Camellia sinensis (L) O Kuntze] (TP- total polyphenols; TC- total catechins)