chapter-ii experimental 2. 1. tea samples and...

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CHAPTER-II

EXPERIMENTAL

2. 1. Tea samples and chemicals

Commercial green tea (United Nilgiris Tea Estate Co. Ltd, Coimbatore) and

black tea (Tata Kannan Devan tea-Kolkatta) were purchased from local departmental

store, Erode, Tamil Nadu, India. The purchased tea samples were consequently

subjected to analysis for the present study. All the chemicals used in this work were

of analytical grade. The chemicals used in this work are listed in Table 2.1. All the

solutions and reagents were prepared from triply distilled water.

Table 2.1. List of Chemicals

Name of the chemicals

Grade/purity

Make

Acetonitrile

HPLC grade

S.D. Fine Chemicals, Mumbai

Acetyl acetone

AR


Agar

AR


Ammonia

AR


Ammonium acetate

AR


Ammonium hydroxide

AR


Ammonium molybdate

AR


Ammonium sulphate

AR


Anthrone reagent

AR


Ascorbic acid

AR


2,2’-azino-bis (3-

ethylbenzothiazoline-6-

sulfonic acid) [ABTS]

AR


58

Acetic acid

AR


Bromophenol blue

AR


Caffeine

AR


Butanol

AR


(+) Catechin

AR

Sigma-Aldrich, Bengaluru

Chloro auric acid 3H2O

AR

Loba Chemie

Citrate buffer

AR


Chloroform

AR


2,2-diphenyl-1-picryl hydrazyl

radical [DPPH]

AR


(+)- Dextrose

AR


Distilled water

HPLC grade


Dimethyl sulphoxide

AR


Dipotassium hydrogen

phosphate

AR


Disodium hydrogen phosphate

AR


Ethanol

AR


Ethedium bromide

AR


Ethyelenediamine tetra acetic

acid (EDTA)

AR


Ethyl acetate

AR


Ferrous ammonium sulphate

AR


Ferrous sulphate

AR


Ferric chloride

AR


Flucanazole

AR


59

Folin-Ciocalteu’s reagent

AR


Gentamycin

AR


Gallic acid

AR


Glacial acetic acid

AR


Hydrogen peroxide

AR


Hydrochloric acid

AR


Isobutyl methyl ketone

AR


Magnesium sulphate

AR


Methanol

AR


Nitric acid

AR


Perchloric acid

AR


Phosphate buffer

AR


Potassium chlorate

AR


Potassium chloride

AR


Potassium persulphate

AR


Peptone

AR


Potassium hydroxide

AR


Potassium ferricyanide

AR


Potassium hexacyanoferrate

AR


Pyrocatechol

AR


Pyrogallol

AR


Salicylic acid

AR


60

Silver nitrate

AR


Sodium hydroxide

AR


Sodium chloride

AR


Sodium carbonate

AR


Sodium sulphate (anhydrous)

AR


Sodium nitrate

AR


Sucrose

AR


Sulphuric acid

AR


Trichloro acetic acid

AR


Tris-Hcl buffer

AR


Vanillin

AR


Yeast

AR


2. 2. Extract preparation

2.2.1. Extract preparation for estimation of polyphenol and catechins

About 70% (v/v) ethanol was prepared using distilled water and was kept in a

water bath for 30 min at 70°C. 2 g of powdered black and green tea samples were taken

in a centrifuge tube and 5 ml of hot 70% (v/v) ethanol was added to it. The mixture was

kept in a water bath maintained at 70°C with intermittent stirring. It was then

centrifuged at 5000 rpm for 10 min. The supernatants were transferred to 10 ml standard

measuring flask. The residual waste was subsequently extracted again with 5 ml of 70%

(v/v) hot ethanol for 10 min. The contents were pooled and made up to the volume with

cold 70% (v/v) ethanol. 1 ml of the extract was diluted to 100 ml with distilled water

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and directly used for the estimation of total polyphenols and total catechins. For

reducing sugar, 1 ml of the extract was diluted with 10 ml of distilled water.

2. 2.2. Extract preparation for antioxidant assay

Tea extracts for antioxidant assay was prepared by solvent extraction method

[1]. 50 g of powdered green and black tea samples were added to 200 ml of ethanol

and water separately. The mixture was kept for 24 h in tightly sealed vessel at room

temperature, protected from sunlight and stirred several times with a sterile glass

rod. This mixture was filtered through Whatman No.1 filter paper and the residue

was adjusted to the required concentration (50 ml of solvent for the residue of 50 g

of tea) with the extraction fluid of further extraction and it was repeated thrice and a

clear colourless supernatant extraction liquid was finally obtained. The extracted

liquid was subjected to rotary evaporation in order to remove the solvents. Then the

extract was stored in an airtight container at 4•C in refrigerator for further use.

2.3. Biochemical Analysis

2. 3.1. Estimation of total polyphenols

Polyphenols in both teas were estimated according to ISO procedure [2].

Briefly, 1 ml of diluted extract was mixed with 5 ml of 10 % (v/v) Folin- Ciocalteu’s

reagent and 4 ml of 7.5 % (w/v) sodium carbonate solution. The mixture was shaken

vigorously and kept aside for completion of the reaction. After 1 h, the blue colour

developed was measured at 765 nm against reagent blank in Perkin Elmer Lambda

35 UV-Visible spectrophotometer. The total polyphenols present in tea extracts were

calculated from the calibration curve obtained for gallic acid of known

concentrations and the results were expressed in gallic acid equivalent as mg/g.

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2. 3.2. Determination of total catechins

Total catechins were measured by following the method of Swain and Hills

[3]. 2 ml of the diluted extract was kept under ice cold condition and 6.5 ml of ice

cold acidified vanillin reagent was added to it and the contents were diluted to 10 ml

with distilled water. It was shaken well and allowed to stand for 15 min for

completion of the reaction. Absorbance of the orange colour developed was

measured at 500 nm against the reagent blank using a UV-Visible

spectrophotometer. The amount of catechins present in tea leaves extracts were

calculated using the calibration curve of known concentrations of (+) catechin and

the results were expressed in (+) catechin equivalents as mg/g.

2. 3.3. Measurement of total reducing sugars

Hedge and Hofreiter [4] method was adopted for determination of total

reducing sugars in both teas. Briefly, to 2 ml of the diluted extract kept under ice

cold condition, 4.0 ml of ice cold anthrone reagent was added. The test tube with

content was heated in a boiling water bath for 8 min and cooled under running water.

Absorbance of the green colour developed was measured at 630 nm against the

reagent blank using a UV-Vis spectrophotometer. The amount of reducing sugar

present in tea leaves extracts was calculated using the calibration curve of known

concentrations of (+) dextrose and the results were expressed in (+) dextrose

equivalent as mg/g.

2.3.4. Estimation of caffeine

Caffeine content of tea was determined according to Ronald and Ronald

method [5]. About 1 g of the powdered tea sample was taken in a 125 ml separating

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funnel and to which 5 ml of 40 % (v/v) aqueous ammonia was added and extracted

with three 25 ml portions of chloroform. The pooled chloroform layer was shaken

well with 10 ml of 1 % (w/v) potassium hydroxide solution. The chloroform layer

was filtered through anhydrous sodium sulphate and made up to 100 ml with

chloroform. Absorbance of the solution was measured at 274 nm in Perkin Elmer

Lambda-35 UV-Vis spectrophotometer against blank. The amount of caffeine was

calculated from the calibration curve obtained with 5 to 25 ppm of standard caffeine

and the results were expressed in mg/g.

2. 3.5. Determination of theaflavins and thearubigins

Theaflavins and thearubigins were estimated according to Thanaraj and

Seshadri method [6]. About 2 g of tea sample was taken in a 250 ml conical flask, to

which, 100 ml of freshly boiled distilled water was added and the contents were

infused over the boiling water bath for 10 min with intermittent shaking. It was then

filtered through cotton wool and the analysis was carried out as per the scheme in

Flow chart 2.1. The solvent extraction of tea extract was carried out in separating

funnels with adequate shaking at every stage. Contents of theaflavin (TF),

thearubigin (TR), highly polymerised substances (HPS) and total liquor colour

(TLC) were calculated from the absorbance values (A to E as given in Flow chart

2.1) as follows.

TF % = 4.313 x C

TR % = 13.643 x (B+D-C)

HPS % = 13.643 x E

TLC % = 10 x A

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where A, B, C, D, E are the absorbance values from the Flow chart 2.1. DMC- Dry

matter content. Multiplication factors for the calculation of TF and TR were derived

from molar extinction coefficient of pure compounds and dilution fraction. HPS was

represented as TR. In the case of TLC, factor 10 was the dilution factor.

Flow chart 2.1. Estimation of TF, TR, HPS and TLC in green & black tea

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2.3.6. Measurement of total proteins and lipids

The amount of nitrogen was measured using well known Kjeldahl method

[7]. The protein was calculated by multiplying the amount of nitrogen by a

factor 6.25. Total lipid content was measured according to the literature [8]. 1 g of

the dried leaf powder was added to 25 ml of chloroform and methanol mixture

(2:1) in a separating funnel, to which 5 ml of 0.9 % (w/v) sodium chloride was

added and the mixture was shaken well, allowed to stand till the separation of

layers and the chloroform layer was carefully transferred to a pre weighed

china dish. The entire extraction procedure was repeated twice with two more

25 ml portions of chloroform and methanol mixture. The pooled chloroform

extract was evaporated to dryness on a boiling water bath. The china dish with

lipids were dried and weighed again. From the difference in weights, the

percentage of lipids present in the leaf material was calculated gravimetrically and

expressed in %.

2. 3.7. Estimation of moisture content

Moisture content was estimated according to Indian Standard [9]. A known

weight of tea sample was taken in a weighing bottle and it was kept in hot air oven

maintained at 103oC for 16 h. The dried tea sample was weighed and dry matter

content was computed. From the dry matter content, moisture content was calculated

according to the formula.

Moisture content (%) = (100 – Dry matter content)

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2. 3.8. Estimation of individual catechins

Individual catechins were measured according to ISO procedure [10], a

simple method developed for estimation of ECG, C, EC, EGCG and EGC. About 0.2

g of tea powder was extracted using 10 ml of 70 % (v /v) ethanol maintained at 70

°C. 1 ml of the extract was diluted to 5 ml with stabilizing solution. The stabilizing

solution was prepared by mixing 0.025 % (w/v) each of EDTA and ascorbic acid and

10 % (v/v) acetonitrile in HPLC grade water. It was prepared as fresh on the day of

use and was filtered through 0.45 µ nylon membrane filter, and used for HPLC

analysis.

Mobile phase A – 2 % (v/v) acetic acid in 9 % (v/v) acetonitrile in water,

mobile phase B – 80 % (v/v) acetonitrile in water. The gradient programme is as

follows.

- Flow rate of mobile phase: 1.0 ml /min.

- Binary gradient conditions: 100 % mobile phase A for 10 min, then over 15 min a

linear gradient to 68 % mobile phase A, 32 % mobile phase B and hold at this

composition for 10 min. Then, reset to 100 % mobile phase A and allowed to

equilibrate for 10 min before next injection.

A Phenomenex Luna 5 µm phenyl – hexyl column (250 mm length x 4.6 mm

internal diameter) fitted with a Phenomenex Security Guard 4 mm x 3 mm phenyl

hexyl bonded cartridge was used for the analysis. The column was maintained at 35

± 0.5 °C. The absorbance of compounds was measured at the wave length of 278 nm

using UV – detector. Integration and calculations were carried out using HP

Chemstation software.

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2.4. Antioxidant activities

2.4.1. DPPH radical scavenging activity

Free radical scavenging effect was estimated according to the method of Zhu

et al. [11]. Briefly, a 1mM DPPH radical -solution in ethanol was prepared, and 1 ml

of this solution was mixed with 3 ml (20-100 µg) each of ethanol and water extract

of tea. The mixture was then vortexed vigorously and left for 30 min at room

temperature in the dark and the absorbance was measured for various concentrations

at 517 nm with Perkin Elmer Lambda UV-Visible spectrophotometer. A control

sample containing the same amount of water and DPPH radical was prepared and

measured at the same wavelength. All determinations were carried out in triplicates.

The DPPH radical scavenging activity was calculated as follows,

2.4.2. Ferric reducing power

The method of Yildirin et al. [12] was adopted for the estimation of reducing

power. Exactly 1 ml (20-100 µg) each of ethanol and water extract of tea was mixed

with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1% (w/v) potassium

ferricyanide. The mixture was incubated at 50o

C for 30 min, afterwards, 2.5 ml of

10 % (v/v) trichloroacetic acid was added to the mixture and centrifuged at 3000

rpm for 10 min. Finally, 2.5 ml of the upper layer was pipetted out and mixed with

2.5 ml of distilled water and 0.5 ml of 0.1% (w/v) ferric chloride was added. The

absorbance of the extracts was measured for various concentrations at 700 nm using

a Perkin Elmer Lambda 35 UV-Visible Spectrophotometer. The increased

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absorbance of the reaction mixture indicated increased reducing power. The

experiments were carried out in triplicates.

2.4.3. ABTS radical scavenging activity

To determine ABTS radical scavenging assay, the method of Re et al. [13]

was adopted. The stock solutions included 7mM ABTS and 2.4 mM potassium

persulphate. The working solution was then prepared by mixing the two stock

solutions in equal quantities and allowing them to react for overnight at room

temperature in the dark. The solution was then diluted by mixing 1 ml of ABTS

solution with 60 ml ethanol to obtain an absorbance of 0.706 ± 0.001 units at 734

nm. Fresh ABTS solution was prepared for each assay. The tea extract (1 ml) (20-

100 µg ) was allowed to react with 1 ml of the ABTS solution and the absorbance

was measured for various concentrations at 734 nm after 7 min. Triplicate

determinations were made and the percentage inhibition was calculated as,

Ethanol without tea extract was used as control.

2.4.4. Hydrogen peroxide scavenging activity

The ability of the extracts to scavenge hydrogen peroxide was determined

according to the method of Ruch et al. [14]. Shortly, hydrogen peroxide solution

(2mM/l) was prepared with 50 mM phosphate buffer (pH 7.4). 1 ml each of various

concentrations (20-100 µg) of ethanol and water extracts of teas were allowed to

react with 0.6 ml of hydrogen peroxide solution. Absorbance was measured for

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various concentrations at 230 nm after 10 min against a blank solution containing

phosphate buffer without hydrogen peroxide. The experiments were carried out in

triplicate.

where the control is ethanol instead of tea extract.

2.4.5. Hydroxyl radical scavenging activity

Hydroxyl radical scavenging activity was determined by following the

method of Halliwell and Gutteridge (15). 0.2 ml each of various concentrations (20-

100 µg) of tea extracts were added with 1.0 ml of EDTA (Ethylene diammine tetra

acetic acid) solution, (0.13 g of ferrous ammonium sulphate and 0.26 g of EDTA

were dissolved in 100 ml of water) and mixed with 1.0 ml of 0.85 % (v/v) DMSO in

0.1M phosphate buffer (pH 7.4) to initiate the reaction followed by the addition of

0.5 ml of 0.22 % (w/v) ascorbic acid. The reaction mixture was kept in water bath at

90ºC for 15 min and the reaction was terminated by adding 1.0 ml of ice-cold 17.5 %

(w/v) of trichloroacetic acid. Further 3.0 ml of Nash reagent (75 g of ammonium

acetate, 3.0 ml of glacial acetic acid and 2.0 ml of acetyl acetone in 1.0 lit of

water) was added to all the test tubes and incubated for 15 min for colour

development. Absorbance was measured at 412 nm using a Perkin Elmer Lambda

35 UV-Visible Spectrophotometer. Reaction mixture without ascorbic acid served as

control. Triplicate determinations were made and the percentage inhibition was

calculated as,

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2. 4.6. Polyphenol oxidase activity

Polyphenol oxidase activity was determined according to Molla method [16].

Shortly, 1 g of the powdered sample was ground with 10 ml of the prechilled 10 mM

citrate buffer (pH 5.6) and the mixture was centrifuged at maximum speed for 10

min, and the PPO activity was assayed with oxygen electrode (Model- Inolab oxi-

730). PPO enzyme activity was estimated by adding 2 ml of crude enzyme in the

reaction chamber containing 10 ml of 10 mM citrate buffer and 25 ml of 100 mM

pyrocatechol which was saturated for few min and the decrease in oxygen

concentration was observed at periodic intervals of 1 min up to 5 min using oxygen

electrode. 1 unit of enzyme activity is defined as µM of oxygen utilized per min per

g dry weight.

2. 4.7. Peroxidase enzyme activity.

POD enzyme activity was measured according to Takeo and Kato method

[17]. Briefly 1 g of the powdered tea sample was homogenized in 1 ml of prechilled

10 mM citrate buffer (pH 5.6) and the activity was assayed using oxygen electrode

(model- inolab oxi-730). POD activity was estimated by adding 1.4 ml of crude

enzyme in the reaction chamber containing 15 ml of 0.025 M phosphate buffer and

10 ml of 0.01 M pyrogallol which was saturated for few min and the increase in

oxygen concentration was observed by adding 7 ml of 300 mM hydrogen peroxide

up to 5 min at periodic intervals of one min. One unit of enzyme activity was defined

as µM of oxygen formed per min per g dry weight.

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2.4.8. Superoxide dismutase activity

SOD activity was assayed according to the method of Marklund and

Marklund [18]. The reaction mixture was prepared with 0.2 ml of the ethanolic

extract, 2.6 ml of tris-HCl buffer (pH 8.5) and 0.2 ml of 7.2 mM pyrogallol

and was subsequently incubated at 25°C for 15 min. The reaction was stopped by

adding 0.1 ml of 1N HCl. The oxidized pyrogallol was measured at 420 nm using

UV-Visible spectrophotometer. The results were expressed as percentage of

inhibition.

2.5. Determination of heavy metals and minerals

2.5.1. Cleaning procedure for glasswares

The glassware and polyethylene containers used for analysis were washed

with tap water, then soaked overnight in 6N HNO3 solution and rinsed several times

with ultra pure water to eliminate absorbance due to detergent [19].

2.5.2. Extraction procedure for mineral nutrients

About 1.0 g of homogenized sample of tea extract was taken in 150 ml

conical flask to which 10 ml of 9:4 mixture of nitric acid and perchloric acid was

added and kept overnight for predigestion to avoid the danger of explosion with

perchloric acid [20] . The samples were digested on an electric hot plate in the

temperature range of 70-80ºC till the content become colourless. The digested

samples were transferred to 100 ml standard measuring flasks and made up with

distilled water and used as a stock solution.

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2.5.3. Estimation of Fe, Zn, Mn and Mg

The wet digested extract from the stock solutions was directly fed into the

Atomic Absorption Spectrophotometer (Perkin Elmer AAS) and the readings were

taken using the respective hollow cathode lamps after preparing a standard curve by

feeding the certified standard solutions (make Merck; certified by NIST) for each

nutrient [21]. Operation parameters for the elements are given in Table 2.2.

Table 2.2. Operation parameters for determination of elements

Elements Wavelength (nm) Fuel gas Oxidant gas

Zn

213.9

Acetylene (2.0 ml/min)

Air (17 ml/min)

Fe

248.3


Air (17 ml/min)

Mn

280.1


Air (17 ml/min)

Ca

422.7


Air (17 ml/min)

As

193.7


Air (17 ml/min)

Hg

253.7


Air (17 ml/min)

Cr

357.9


Air (17 ml/min)

Cu

324.7


Air (17 ml/min)

Ni

232.0


Air (17 ml/min)

Pb

283.3

Inert gas-Argon

Mafrs’s recom.

Cd

228.8

Argon

Mafrs’s recom.

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2.5.4. Estimation of phosphorus

5 ml of wet digested extract from stock solution was taken in a 25 ml

standard measuring flask, to which 10 ml of distilled water, 5 ml of freshly prepared

ascorbic acid and 4 ml of ammonium molybdate solutions were added. The contents

were diluted to 25 ml with distilled water, shaken well and allowed to stand for 30

min for completion of the reaction. Absorbance of the developed blue colour was

measured at 882 nm against the reagent blank solution using UV-Visible

Spectrophotometer [22]. The amount of phosphorus present in the sample was

calculated using the calibration curve of known concentrations of phosphorus and

the results were expressed in mg/kg.

2.5.5. Estimation of nitrogen

About 0.5-1g of ground tea sample was taken in a Kjeldahl tube and the

analysis was carried out using Kjeldahl method [7]. The principle involves the

conversion of all forms of nitrogen into ammonia form by digesting with 3.33%

salicylic acid in sulphuric acid at 450° C. Distillation was carried out in Gerhardt or

Kjeldahl instrument. The distillate was collected in a conical flask, which contains a

known quantity of 0.05 N hydrochloric acid. The excess hydrochloric acid was then

titrated against standard sodium hydroxide solution using digital burette III (Make,

GmbH). The result was expressed in mg/kg.

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2.5.6. Estimation of potassium

The wet digested extract from the stock solution was directly fed into the

flame photometer and set the potassium filter. The potassium content was estimated

using direct reading of Flame Photometer [21].

2.5.7. Estimation of sodium

The digested sample was directly fed into the flame photometer and set the

sodium filter. The sodium content was estimated using direct reading of Flame

Photometer (Sherwood 410) [21].

2.5.8. Estimation of arsenic and mercury

In a pre-acid washed and cleaned dry beaker, 0.5 g of accurately weighed tea

sample and 5 ml of HNO3 were added and placed on a hot plate. The solution was

evaporated to near dryness and cooled. Additionally, 1 ml of HNO3 and 0.5 ml

H2SO4 were added. The above mixture was evaporated to dryness and 1.0-2.0 ml of

H2O2 was added drop wise and the mixture was heated to destroy the remaining

nitrogen oxides that might interfere in the measurement set up. The residue was

dissolved in 8 ml HCl and the volume was brought to 25 ml in calibrated flask using

double distilled water. Reagent blank was prepared containing same amounts of

acids taken from the lots evaporated as above. After sample preparation, the samples

were analyzed in AAS (Perkin Elmer AAS-Flame (AAnalyst 800) as per the

conditions given in Journal of AOAC international, 1996 [23].

2.5.9. Estimation of chromium

In a pre-acid washed and cleaned dry beaker, 1.0 g of accurately weighed tea

sample and 100 mL of HNO3-HCl acid mixture (3:1 v/v) were added and placed on a

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hot plate. The solution was evaporated to near dryness and cooled. Additionally 50

ml of above acid mixture was added and evaporated to obtain clear solution. The

solution was transferred to 25 ml volumetric flask and diluted with double distilled

water. Reagents blank was prepared containing same amounts of acids taken from

lots evaporated as above. After sample preparation, the samples were analyzed in

AAS (Perkin Elmer AAS-Flame (AAnalyst 800)) as per conditions given in Bull

Environ.Contam.Toxicol. 2004 [24].

2.5.10. Estimation of copper and nickel

In a pre-acid washed, cleaned and dried 500 ml beaker, 3.0 g of accurately

weighed tea sample and 100 ml HNO3 were added and swirled. The reaction

mixture was covered, set aside for 10 min and then heated on a hot plate.

Then the mixture was evaporated to near dryness and cooled. Additionally, 50 ml

of HNO3 and 10 ml of KClO4 were added. The evaporation was continued to

obtain clear solution. The solution was transferred to 50 ml volumetric flaks and

diluted to the volume with water (Insoluble KClO4 settles down at the bottom).

Reagent blank was prepared containing same amounts of acids taken from lots

evaporated as above. After sample preparation, the samples were analyzed in AAS

(Perkin Elmer AAS-Flame (Analyst 800) as per conditions given in AOAC -999.11,

2005 [25].

2.5.11. Estimation of lead and cadmium

About 0.5 g of tea sample was placed in a pre-acid washed and cleaned dry

crucible, kept in a muffle furnace at 450˚C for 3 h. After cooling the crucible, 5 ml

of 6M HCl was added, ensuring that all ash comes into contact with acid. The acid

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solution was evaporated using hot plate. The contents were dissolved using

0.1 M HNO3 and filtered into a 50 ml standard measuring flask. The crucible

was washed completely with care. Blanks were treated in the same way as that of

sample. After sample preparation, the samples were analyzed in AAS (Perkin

Elmer AAS-Flame (AAnalyst 800) as per the conditions given in AOAC - 999.11,

2005 [25].

The concentration, C of the metal (As, Hg, Cr, Cu, Ni, Pb, Cd, Zn and Mn) in

the test sample was calculated according to the formula,

where, C is the concentration of the metal (µg/ml), a is the concentration of test

sample (µg/ml), b is the concentration in the blank solution (µg/ml), W is the weight

of the sample taken and V is the volume of test solution (ml).

2.6. Antimicrobial activities

2.6.1. Preparation of extracts

The extraction of tea samples by solvents like water, ethanol and ethyl

acetate was carried out as described by Moon et al. [26]. In this method, 20 g of

black and green tea samples were extracted using soxhlet extractor with 250 ml

water, ethanol and ethyl acetate as solvents. The extraction lasted for 6 h for ethanol

and ethyl acetate and 8 h for water. The extract was concentrated to dryness by using

rotary vacuum evaporator to yield a dense residue and stored in bottles and

refrigerated at 4ºC prior to use.

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2.6.2. Test organisms

Six bacterial species namely, Escherichia coli (MTCC 723), Vibrio cholerae

(MTCC 3906), Bacillus polymyxa (ATCC 842), Staphylococcus aureus (MTCC

3160), Bacillus subtilis (MTCC 736), Pseudomonas aeruginosa (MTCC 7837) and

three fungal species namely, Aspergillus niger (MTCC 1881), Candida albicans

(MTCC 3958), Aspergillus flavus (MTCC 1883) were procured from culture

collection of S.D.M. Medical college, Sattur, Dharwad, Karnataka. The stock

cultures of bacteria were revived by inoculating in nutrient broth and grown at 37ºC

for 18 h, while the stock cultures of fungi were revived by inoculating in czapek-dox

agar media and grown at 27ºC for 48 h.

2.6.3. Preparation of medium for antibacterial and antifungal activity

The antibacterial activity was studied using nutrient broth medium, which

was prepared by mixing 10 g of peptone, 10 g of NaCl, 5 g of yeast extract and 20 g

of agar in 1000 ml of distilled water. The antifungal activity was determined using

czapek-dox agar medium which was prepared by mixing 30 g of sucrose, 2 g of

sodium nitrate, 1 g of K2HPO4, 0.5 g of MgSO4. 7H2O, 0.5 g of KCl, 0.01 g of

FeSO4 and 20 g of agar and the volume was made up to 1000 ml by distilled water.

2.6.4. Antibacterial and antifungal activity

The antibacterial and antifungal activities of the tea extracts were tested on

the test stains using the agar well diffusion method [27]. In this method, 18 h old

culture (100 µl, 10-4

CFU) of bacteria and 48 h old culture (100 µl, 10-4

CFU) of

fungi were introduced and evenly spread on the surface of sterile nutrient agar

medium plates and czapex-dox agar medium plates respectively. Six wells of about 5

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mm diameter were aseptically cut on agar plates using a gel puncture. The wells

were filled with 100 µl of black and green tea extracts (10 mg/ml and 20 mg/ml).

The control well was filled with antibiotic gentamycin for bacteria and flucanazole

for fungi (100 µl from 10 mg/ml stock) and the respective solvents (100 µl) used for

extraction of samples were used as negative control. The plates were incubated at 37

ºC for 24 h for bacteria and 27 ºC for 48 h for fungal spores. Sensitivity was

recorded by measuring the clean zone of inhibition on the agar surface around the

disc. All experiments were carried out in triplicates.

2.7. Biosynthesis of nanoparticles from aqueous tea extracts

2.7.1. Preparation of the tea extract for the synthesis of silver nanoparticles

5 g of tea samples (black tea and green tea) was separately boiled in 100 ml

of double distilled water for 10 min and filtered through suction pump and Whatman

No.1 filter paper.

2.7.2. Preparation of the tea extract for the synthesis of gold nanoparticles

1 g of the tea samples (black and green tea) was separately boiled in 100 ml

of double distilled water for 10 min and filtered through Whatman No.1 filter paper.

2.7.3. Synthesis of silver nanoparticles

0.1M aqueous solution of silver nitrate was prepared in double distilled water

and used for the synthesis. For the synthesis of silver nanoparticles, various

concentrations of the black and green tea extracts (2, 5 and 7 ml) were taken, to

which added 2, 5 and 7 ml of distilled water followed by the addition of 1 ml of

0.1M silver nitrate. The solutions were then shaken well for thorough mixing to get

silver nanoparticles. Then, the solution was kept in freeze drying flask under the

79

vacuum below 100 Pascals at 50°C. Freeze drying (lyophilization) is a process

typically used to preserve perishable material.

2.7.4 Synthesis of gold nanoparticles

1 mM solution of chloro auric acid was prepared in double distilled water and

used for the synthesis. For the synthesis of gold nanoparticles, various

concentrations of the black and green tea extracts (0.2, 0.6, 1.2 ml) were added to 10

ml of 1 mM chloro auric acid and stirring was continued for 2 min to get gold

nanoparticles.

2.7.5. Characterization of silver and gold nanoparticles

2.7.5.1. UV-Visible spectral analysis

The colour change in reaction mixture (metal ion solution + tea extracts) was

recorded through visual observation. The reaction mixture was monitored as a

function of time using Elico-UV-Visible spectrophotometer (Model S3-159)

operated at a resolution of 1 nm in the range of 200-800 nm after 5 fold dilution of

sample with double distilled water. Distilled water was used as blank.

2.7.5.2. Fourier transform Infra-Red spectroscopy analysis

To remove any residue or compound that is not the capping ligand of the

nanoparticles, the residual solution of 100 ml, after reaction was centrifuged at 5000

rpm for 10 min and the resulting suspension was redispersed in 10 ml of double

distilled water. The centrifuging and redispersing processes were repeated three

times, thereafter the purified suspension was fire dried to obtain dried powder. FTIR

spectra of the samples were recorded using Bruker-Tensor 27 FTIR spectrometer in

80

attenuated total reflection mode (Pike technologies, Gladia ATR for FTIR with

diamond crystal) in the range of 4000-400 cm-1

.

2.7.5.3. Scanning electron microscope analysis

Thin films of samples were prepared by dropping a very small amount of the

solution containing nanoparticles on a carbon coated copper grid. Extra solution was

removed using a blotting paper and then allowed to dry prior to the measurements.

The SEM analysis was performed on a JEOL, model JSM-6390 instrument operated

at an accelerating voltage of 20 KeV and counting time of 100 s.

2.7.5.4. Energy dispersive X-ray spectroscopy

Energy dispersive X-ray (EDX) analysis of the synthesized silver and gold

nanoparticles was performed on a JEOL JSM-6390 microscope fitted with Oxford-

EDX analyzer.

2.7.5.5. Transmission electron microscope analysis

The morphology of the synthesized silver and gold nanoparticles was

determined by Transmission electron microscopy (TEM). For TEM studies, the

solution containing nanoparticles was diluted and a drop of it was placed on copper

grid and allowed to dry in vacuum. The transmission electron micrographs of silver

nanoparticles were taken using Technai-G-10, an 80 KV TEM with a tungsten

source and an ultra high resolution pole piece. Similarly, the morphology of the gold

nanoparticles was studied using high resolution Technai F 20 TEM.

2.7.5.6. X-ray diffraction measurements

The silver and gold nanoparticle solutions obtained were purified by repeated

centrifugation at 5000 rpm for 20 min, followed by redispersion of the pellet of

81

silver and gold nanoparticles into 10 ml of double distilled water. After

freeze drying, the structure and composition of the purified nanoparticles were

determined by using X-ray diffactomoter, (Shimadzu model Lab-X-XRD-6000)

operated at a voltage of 40 kV and a current of 30 mA with Cu K α radiation with a

wavelength of 1.5406 A°. The diffracted intensities were recorded form 10° to 90°

of 2θ angles.

The crystallite domain size was calculated from the width of the XRD

peaks, assuming that they are free from non-uniform strains, using the Scherer

formula,

D= 0.94 λ / β cos θ ………………….. (1)

where, D is the average crystallite domain size perpendicular to the reflecting planes,

λ is the x-ray wavelength, β is the full width at half maximum (FWHM), and θ is the

diffraction angle. To eliminate additional instrumental broadening, the FWHM was

corrected using the FWHM from a large grained Si sample.

β corrected = (FWHM2

sample-------FWHM2

standard) 1/2

-------- (2)

This modified formula is valid only when the crystallite size is less than 100 nm

2.7.5.7. Particle size analysis

The particle size range of the silver nanoparticles along with its

polydispersity was determined using a particle size analyzer (Sympatec GMBH,

Nanophox particle size analyzer). Particle size was arrived based on measuring the

time dependent fluctuation of scattering of laser light by the nanoparticles

undergoing Brownian motion.

82

2.7.5.8. Thermo gravimetric Analysis

Thermogravimetric analysis of the powder of the biologically synthesized

silver nanoparticles was carried out using TGA Q500 V20.10 build 36 instrument at

heating rate of 10 C/min under nitrogen atmosphere.

2.7.5.9. Photoluminescence spectral analysis

Photoluminescent spectra of the synthesized silver and gold nanoparticles

were recorded using Flurolog III spectroflurophotometer.

2.7.5.10. Antibacterial activity of silver and gold nanoparticles

Silver and gold nanoparticles synthesized using aqueous extracts of black

and green teas were tested for their potent antibacterial activity against six

pathogenic bacteria as given in 2.6.2. The antibacterial activities of the gold and

silver nanoparticles were determined by agar well diffusion technique [27]. Nutrient

agar medium was prepared and poured into sterilized Petri plates and wells of 5 mm

diameter were made in the plate using gel puncture. The agar plates were inoculated

with 18 h old culture (100 µl, 10-4

CFU) and spread evenly on the plate. After 20

min, the wells were filled with 100 µl of silver and gold nanoparticles. The control

wells were filled with gentamycin (100 µl from 10mg/ml stock) and with 100 µl of

1mM AgNO3 or chloroauric acid. All the plates were incubated at 37ºC for 24 h and

the diameter of inhibition zones were noted. All experiments were carried out in

triplicate.

2.7.5.11. Antifungal activity of silver and gold nanoparticles

Silver and gold nanoparticles synthesized using aqueous extracts of black

and green teas were tested for their potent antifungal activity against two pathogenic

83

fungi as given in 2.6.2 by Agar well diffusion technique [27]. Czapek-Dox Agar

medium was prepared, media was poured into sterilized Petri plates and wells of 5

mm diameter were made in the plate using gel puncture. Each plate was inoculated

with 48 h old culture (100 µl 10-4

CFU) and spread evenly on the plate. After 20

min, the wells were filled with 100 µl of silver and gold nanoparticles. The control

wells were filled with flucanazole (100 µl from 10mg/ml stock) and with 100 µl of

1mM AgNO3 or chloroauric acid. All the plates were incubated at 270C for 48 h and

the diameter of inhibition zone were noted. All experiments were carried out in

triplicates.

2.8. Statistical analysis

All the experiments were carried out in triplicate. The results were expressed

as mean ± SD.

84

References

1 M. Chessbrough, Medical laboratory manual for tropical countries, Linacre

House, Jordan Hill, Oxford. pp.260 (2000).

2 ISO 14502-1:2005 (E) Determination of substances characteristics of green

and black tea. Part-I: Content of total polyphenols in tea- Colorimetric method

using Folin Ciocalteu’s reagent.

3 T. Swain and W.E. Hills, J. Sci. Food Agric. 10 (1959) 63-68.

4 J.E. Hedge and B.T. Hofreiter, Carbohydrate Chemistry. Academic press, New

York, USA. pp.17-22 (1962).

5 S.K. Ronald and S. Ronald, Beverages and chocolate. In Pearson’s

Composition and analysis of foods, 9th

ed., Longman scientific and Technical

Publishers, England, pp.356-362 (1991).

6 S.N.S. Thanraj and R. Seshadri, J. Sci. Food Agric. 51 (1990) 57-69.

7 J.P.E. Anderson, Soil respiration: in A.L. Page, R.H. Miller and D. Keeney,

editors, Agronomy monograph 9: Methods of soil analysis, Part 2. Chemical

and biological properties, second edition.American Society of Agronomy and

Soil Science Society of America, Madison, Winconsin, USA. pp.831-871

(1982).

8 R. Ravichandran and R. Parthiban, Food Chem. 68 (2000) 7-13.

9 IS. 13853: Indian Standard Procedure. 1994.

10 ISO 14502-2: 2005 Tea method for determination of substances characteristic

of green and black tea, Part-2. Content of catechins in green tea-method using

high performance liquid chromatography.

85

11 Q.Y. Zhu, R.M. Hackman, J.L. Ensunsa, R.R. Holt and C.L. Keen J. Agric.

Food Chem. 50 (2002) 6929-6934.

12 A. Yildrin, A. Mavi and A.A. Kara, J. Agric. Food Chem. 49 (2001) 4083-

4089.

13 R.Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang and C. Rice Evans,

Free Radic. Biol. Med. 26 (1999) 1231-1237.

14 R.J. Ruch, S.J. Cheng and J.E. Klaunig, Carcinogenesis 10 (1989) 1003-1008.

15 B. Halliwell and J. Gutteridge, FEBS Lett. 128 (1981) 347-352.

16 M..M. Molla, Sri Lanka J. Tea Sci. 61 (1992) 15-19.

17 T. Takeo and Y. Kato, Plant Cell Physiol. 12 (1971) 217-223.

18 S. Marklund and G. Marklund, Eur. J. Biochem. 47 (1974) 468-474.

19 C. Cabrera, F.Lloris, R. Gimenez, M. Olalla and M.C. Lopez, Sci. Total

Environ. 308 (2003) 1-14.

20 B.A. Haunter, M.S. Johnson, and D.J Thompson. J. Appl. Ecol. 24 (1987) 573-

586.

21 B.S.Bhargava and H.B.Raghupathi, Analysis of plant materials for macro and

micronutrients. In: Methods of analysis of soils, plants, waters and

fertilizers.(Ed. Tandon, H. L. S.) Fertiliser Association and Consultation

Organization, New Delhi, pp. 49-82 (2001).

22 R.H. Bray, and L.T.Kurtz, Soil Sci. 59 (1945) 39-45.

23 O. Jimenez De Blas and N. Rodriguez-Mateos, J. AOAC Internat. 79 (1996)

764-768.

24 I. Narin, H. Colak, O. Turkoglu, M. Soylak and M. Dogan. Bull Environ.

Contam. Toxicol. 72 (2004) 844-849.

86

25 AOAC official methods of analysis 999.11. Metals and other contaminants.

Chapter 9, 18th

edition, pp.19-22 (2005).

26 H.P. Moon, W.K. Gyoung, H.K. Joung and J.S. You, Korean Chem. Soc. 26

(2005) 747-754.

27 C. Perez, M. Pauli and P. Bazerque, Acta Biol. Med. Exp. 15 (1990) 113-115.

chapter-ii experimental 2. 1. tea samples and...

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