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4. EXPERIMENTAL INVESTIGATIONS

The chemicals used throughout the research work were either A.R or G.R

grade and were purchased from different companies and are listed below:

Name of the Chemical Make

Methanol Sd-fine

Potassium dihydrogen phosphate ,,

Sodium carbonate ,,

Acetic acid ,,

Chloroform ,,

Starch ,,

Silica gel G ,,

Ammonium molybdate ,,

Phenolpthalein ,,

Disodium hydrogen phosphate ,,

Follin ciocalteau reagent ,,

Sodium thiosulphate Merck

Sodium hydroxide ,,

BHT, BHA ,,

Acetone ,,

Trichloroacetic acid ,,

Ethylacetate ,,

Liquid ammonia Sisco research lab

Lead acetate ,,

Propanol ,,

Ammonium ferric sulphate ,,

Butanol ,,

Ferric chloride ,,

Potasium iodide Finar reagent

Potassium ferricyanide Loba chemie

β- Carotene Himedia

Linoleic acid ,,

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2,2-Diphenyl-1- picryl hydrazyl Himedia

Ferulic acid ,,

Vanillic acid ,,

Tannic acid ,,

Kaempferol Sigma

Chlorogenic acid ,,

Caffeic acid ,,

Quercitin dihydrate ,,

Rutin hydrate ,,

Solutions

1. Phenolphthalein Indicator - One gram of phenolphthalein was dissolved in

100ml of ethyl alcohol.

2. Standard Aqueous Sodium Hydroxide - 0.1 N or 0.01N accurately

standardized.

3. Acetic Acid-Chloroform - Three parts of glacial acetic acid by volume was

mixed with 2 parts of chloroform by volume.

4. Saturated Potassium Iodide-Saturated solution of potassium iodide was

prepared by using recently boiled distilled water.

5. Standard sodium thiosulphate solution-0.1 N, 0.01N, accurately standardized.

6. Starch Solution-1 percent-1gm of starch was mixed in boiled and cooled

100ml water

7. 7.10-fold diluted Folin-Ciocalteu (F-C) reagent

8. 7.5% sodium carbonate solution.

9. 9. 6x10-5M methanolic solution of DPPH: Based upon the molecular weight of

DPPH; calculated amount was weighed and dissolved in methanol. Special

care was taken to store the solution. It was stored in amber coloured bottle.

119

The solution once prepared was used within two days and the absorbance

was checked every day.

10. 0.2M phosphate buffer (6.6pH) 133 ml of 0.5M KH2PO4

89.2 ml of 0.5M Na2HPO4

The pH of the solution was checked before using for analysis.

11. 1% potassium ferricyanide-1gm of potassium ferricyanide was dissolved in

100ml of water.

12. 10% trichloroacetic acid-10gms of trichloroacetic acid was dissolved in 100ml

of water

13. 1% ferric chloride -100mg of ferric chloride was dissolved in 100ml of water

14. Ferric reagent- Dissolve 3 gms of AR ammonium ferric sulphate, FeNH4

(SO4)2. 12H2O (iron alum), in 100 ml water and use immediately

15. Butanol/acetic acid/water-4:1:5

16. 2.51% linoleic acid-2.51 gm of linoleic acid was dissolved in 100ml

chloroform

17. 0.2M phosphate buffer (7pH)- 65.8 ml of 0.5 M KH2PO4

111ml of 0.5MNa2HPO4

18. 75% ethanol-75 ml of ethanol was made up with water upto 100ml.

19. 30% ammonium thiocyanate-30gms of ammonium thiocyanate was dissolved

in 100ml of water

20. 20mM Ferrous chloride-weighed amount of ferrous chloride was dissolved in

3.5% HCl.

21. 0.5 M KH2PO4 -17.011gm of KH2PO4 was dissolved in 250ml of water

22. 0.5 M Na2HPO4 -17.745gm of Na2HPO4 was dissolved in 250ml of water

23. 3.5% HCl- 3.5ml of concentrated HCl was made upto100ml

Made up to 1000ml

Made up to 1000ml

120

The raw materials selected for the study can be grouped as follows:

1) Peel of four different mango varieties baneesha, malgoa, totapuri, neelam and

Pomegranate peel.

2) Kernels of four different mango varieties baneesha, malgoa, totapuri, neelam

and broken pieces and powder waste of areca nut kernel.

3) Curry leaves, betel leaves and guava leaves.

Determination of AA of different plant extracts is carried out in the following

manner:

4.1 PREPARATION OF RAW MATERIALS

4.1.1 Peel

4.1.1a Mango Peel

Mangoes of four different varieties i.e., baneesha, malgoa, totapuri and

neelam were purchased. The peel was separated from pulp and cleaned by removing

extraneous matter with a knife. The cleaned peel was shade dried and then tray dried

at 500C. The dried peel was stored in airtight containers until use.

4.1.1b Pomegranate Peel

Ripe pomegranates were obtained from the local market. The peel was

separated manually. The fresh peel was collected and dried in sun and then in a tray

drier at 500C. The dried pomegranate peel was powdered and stored in air tight

containers until use.

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4.1.2. Kernels

4.1.2a Mango Kernels

Different varieties of mango namely, baneesha, malgoa, totapuri and neelam

fruits were purchased and the peels were separated for active compounds as discussed

in 4.1.1.a. The pulp was separated and the seeds were removed out. Seeds were

cracked manually; kernels were taken out and dried at 500C.The dried kernels were

reduced in size and stored in air tight containers.

4.1.2b Areca nut

Broken pieces and powder waste of areca nuts were procured from areca

processing unit, dried in a hot air oven at 500C, powdered and extracted for active

components.

4.1.3. Leaves

The beetle leaves (B.L), curry leaves (C.L) and guava leaves (G.L) were

collected from local area. They were dried in shade for 3-4 days and then oven dried

at 500C for 2-3 days. The dried leaves were powdered and stored in air tight

containers.

4.2. PREPARATION OF EXTRACTS

Dried peel, kernels and leaves were studied for extraction efficiency and total

phenolic content by using five different solvents namely methanol, ethanol, acetone,

ethyl acetate and chloroform.

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4.2.1. Peel extracts

4.2.1a Mango peel extracts

Extraction was based on the method of Yen and Duh1. Five grams of size

reduced different mango peels were weighed in different conical flasks. 50 ml

methanol was poured into one conical flask. The conical flask was placed in a shaking

incubator over night at room temperature. The extract was filtered through whatman

no.40 filter paper and the residue was re-extracted with fresh methanol and treated in

the similar manner. The procedure was repeated for three consecutive days. The

combined filtrate was evaporated in a Heidolph Rota evaporator at 400C. The

concentrated extract was stored under frozen conditions until use. The experiment was

carried out by using different solvents like ethanol, acetone, ethyl acetate and

chloroform for all the varieties of mango peels in the same manner.

4.2.1b Pomegranate Peel extracts

The dried and powdered pomegranate peel was extracted with different

solvents, like methanol, ethanol, acetone, ethylacetate and chloroform. The extraction

was carried out for pomegranate peel as discussed in 4.2.1a and the extracts of

different solvents were stored under frozen conditions in different amber coloured

bottles until further use.

4.2.2. Kernel extracts

4.2.2a Mango kernel extracts

Each variety of mango kernel was treated separately with different solvents

i.e., methanol, ethanol, acetone, chloroform and ethylacetate to know their extraction

efficiency. The extractions were carried out for different kernels using different

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solvents separately as discussed in 4.2.1a. The concentrated extracts of four kernels in

different solvents were stored under frozen conditions until use.

4.2.2b Areca nut extracts

Areca nut kernel powder was treated separately with different solvents i.e.,

methanol, ethanol, acetone, chloroform and ethylacetate to know their extraction

efficiency. The extraction was carried out using different solvents separately as

discussed in 4.2.1a. The concentrated extracts of different solvents were stored under

frozen conditions until use.

4.2.3. Leaf extracts

4.2.3a Betel leaf extracts (BLE)

BL was taken in different conical flasks and then treated with five different

solvents each. The extraction was carried out for three consecutive days as given in

4.2.1a. The filtrate was pooled up and was concentrated in Rota evaporator at 400C

under vacuum.

4.2.3b Curry leaf extracts (CLE)

Five different solvents were used to extract CL placed in different conical

flasks. The extraction was carried out for three consecutive days as explained in

4.2.1a.

4.2.3c Guava leaf extracts (GLE)

GL was treated in the same way as described in 4.2.1a to extract the active

components.

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4.3. EXTRACTION EFFICIENCY OF DIFFERENT SOLVENTS

The dried extracted sample was weighed for calculating the yield by the

following equation2;

Yield (% dry weight basis) = (W1 x100)/W2 --------------- 4.1

Where W1 is the weight of residue after extracting and drying and

W2 is the weight of dried samples

4.4. DETERMINATION OF TPC OF PEEL, KERNEL AND LEAF

EXTRACTS

The extracts of all raw materials obtained by using different solvents were

tested for TPC by using the following method2 which was a modification of Singleton

and Rossi3. One ml of each extract was diluted to 50ml as they were highly

concentrated.

Diluted extracts (0.2 ml) were mixed with 1.0 mL of tenfold diluted F-C

reagent and 0.8 mL 7.5% sodium carbonate solution. The absorbance of the mixtures

was measured at 765 nm in UV-Visible 160 A spectrophotometer (Shimadzu, Japan)

after incubating for 30 min at room temperature. The content of phenolics was

expressed as gallic acid equivalents (GAE) in mg/gm.

4.5. ASSESSMENT OF AA OF PEEL, KERNEL AND LEAF EXTRACTS

Based on the extraction efficiency (EE) and TPC, the methanol extracts of all

the raw materials were further used for different antioxidant assays.

125

4.5.1. DPPH activity

Free radical scavenging ability of different extracts was determined by DPPH

method proposed by Von Gadow4 (1997). Aliquot (50µL) of the different extract was

placed in a cuvette and 2 mL of 6x10-5M methanolic solution of DPPH radical was

added. Absorbance was measured immediately. The decrease in absorbance at 517nm

was determined after 16 min for all samples. Methanol was used as blank. The

absorbance of the DPPH radical without antioxidant, i.e., the control was measured

daily. Special care was taken to minimize the loss of free radical activity of the DPPH

radical stock solution by pouring it in amber coloured bottle. Methanolic solutions of

BHT and BHA were tested too at same concentrations as extracts. All determinations

were performed in triplicate. The percentage inhibition of the DPPH radical by the

samples was calculated according to the formula of Yen5 (1994)

%inhibition= [(AC(0)-AA(t))/AC(0)] x 100 --------------- 4.2

Where AC(0) is the absorbance of the control at t=0 min and

AA(t) is the absorbance of the antioxidant at t=16min6

4.5.2. RRP

The reducing power of different extracts was determined by a method

proposed by Oyaizu7. Different concentrations (50, 100, 250, 500, 1000 ppm) of

extracts were prepared by using methanol. 1 ml of this sample was mixed with 2.5ml

of phosphate buffer (0.2M, pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The

mixtures were incubated for 20 min at 500C. At the end of the incubation, 2.5ml of

10% trichloroacetic acid was added to the mixtures and centrifuged at 5000 rpm for

10 min. The upper layer (2.5 ml) was mixed with 2.5ml of distilled water and 0.5 ml

of 0.1% ferric chloride and the absorbance was measured at 700 nm. The reducing

126

power tests were done in triplicate. RRP of the extracts was calculated by the equation

given by Chang and Liu9.

RRP = (Sample absorption at 700nm/1.2) x 100 --------------- 4.3

4.5.3. FTC method

The AA of different extracts was determined by modified thiocyanate

method10. 100 ppm of extracts were calculated and added to a mixed solution of

linoleic acid (0.13ml) in 99.0% ethanol (10ml) and 0.2mM phosphate buffer (pH

7.0,10 ml) and the volume was made up to 25 ml with distilled water. The mixed

solution in a conical flask was kept in a constant temperature oven at 400C. The

absorbance of the solution was measured at regular intervals11. 0.1 ml of this solution

was added to 9.7 ml of 75% ethanol and 0.1ml of 30% ammonium thiocyanate.

Precisely after 3 minutes after the addition of 0.1 ml of 20mM ferrous chloride in

3.5% hydrochloric acid to the reaction mixture, the absorbance of the resultant red

colour was measured at 500nm with a spectrophotometer. Distilled water was used as

a control9.

4.5.4. β-carotene-linoleic acid bleaching assay

β- Carotene- linoleic acid bleaching assay of extracts was tested by the method

proposed by Taga12 (1984). β-carotene (2mg) was dissolved in chloroform (20ml). To

an aliquot (3ml) of the solution, linoleic acid (40mg) and Tween 40 (400mg) was

added. Chloroform was removed using a rotary evaporator at 500C, made up to 100

ml with oxygenated water and mixed well. A mixture prepared as above without β-

carotene emulsion served as blank. Aliquots of the β-carotene-linoleic acid emulsion

(2ml) were mixed with antioxidant extract (40µl) and incubated at 500C, for 120 min.

127

Absorbance was measured at 15 min intervals. All determinations were carried out in

triplicate. The AA of the extracts was calculated by measuring the absorbance of

emulsion at 470 nm in UV visible spectrophotometer. Distilled water was used as

control. The degradation rate of extracts was calculated as follows.

4.6. CHROMATOGRAPHIC SEPARATION

Chromatography can be defined as that technique for the separation of a

mixture of solutes in which separation is brought about by the differential movement

of the individual solutes through a porous medium under the influence of a moving

solvent.

4.6.1. Paper Chromatography

Paper chromatography is one of the simplest and most widely used of the

chromatographic techniques. Methanol extracts of different raw materials were first

tested for presence of phenolic compounds by paper chromatography. Later on the

same procedure was applied in carrying out the TLC.

The apparatus and procedure used for carrying out paper chromatography are

as follows:

Chromatographic chamber, Oven, Whatman No.1 paper (20cmx3cm),

Measuring cylinder, Beakers, Capillary tube, Solvent chamber (20cmx4cm), Sprayer,

Solvent system- Butanol: acetic acid: water (4:1:5)

Spraying reagent: FCR, Ferric chloride and ammonia solution.

Extracts of the plant materials were spotted on the paper with a capillary tube.

The solvent system i.e., Butanol: acetic acid: water (4:1:5) was prepared and poured

128

into the chromatographic chamber before 1 hr. of developing and lid was placed in

position.

The paper developed in the solvent chamber in ascending manner, was dried

and the spraying reagent was sprayed and it was exposed to ammonia vapours. The

spots were located and the Rf value was calculated for the spots13.

frontsolvent by the moved distance the

sample by the moved distance the=valueR f --------------- 4.4

4.6.2. Thin layer chromatography

It is a chromatographic technique in which an adsorbent layer is developed on

glass plates. Elution is carried out with solvents as in the case of paper

chromatography.

Chemicals required: Silica gel G, Butanol, Glacial acetic acid, Ammonium

ferric sulphate, Folin ciocalteau reagent, Ammonia

Apparatus required: Galss plate (20cmx19cm), Capillary tubes, Measuring

cylinder, Beakers, Solvent chamber (24cmx12cm).

Preparation of chromatoplates: Plates were washed with chromic acid and then

with water and dried in an oven. Chromatoplates were prepared by applying a uniform

layer of adsorbent (silica gel) in the form of aqueous slurry to the clean glass plates by

means of a special applicator. The thickness of the layer was maintained 25 mm. The

plate was allowed to stand for 15-20 minutes in the horizontal position and was dried

in a hot air oven at 100-1050C for about 2 hours. This is referred to as activation of

129

the layer. The polyphenols were identified by their colour reactions with specific

spray reagents, Rf values and spectral characteristics.

After development, the chromatograms were allowed to air dry and observed

visually. The chromatoplate was sprayed with FCR and was exposed to ferric chloride

by taking it in a petridish and holding the plate slightly above the dish and was

observed. Only those spots which give colour reactions with ferric chloride-

ferricyanide reagent were considered as polyphenols.

Phenolic compounds react with ferric reagent to produce yellow, orange, green

or blue colours, depending on the class of phenolic compound. Results were again

compared with external standards for tentative identification13.

4.6.3. Column Chromatography

CC is routinely carried out technique which is adaptable to all the major types

of chromatography. The columns used in the study was made up of glass and has a

diameter of 70mm and a length of 150 cm with a sintered glass disc at the bottom to

support the stationary phase. The column was packed with silica gel G. The solvent

was made to run through the column to make the silica gel set uniformly in the

column. The sample was allowed to just run into the column and the solvent was then

added to the column to a height of 5-10 cm. As column packing influences flow rate,

it was packed carefully. One ml of the sample was loaded and the sample was passed

through the column by using methanol as the mobile phase. The effluent was

collected from the column outlet and was analysed in LCMS and HPLC for

identification of phenolic compounds14.

130

4.6.4. HPLC and Identification of phenolic compounds

All the extracts were subjected to HPLC for identification of phenolic

components. HPLC is one among most useful tools, available for quantitative

analysis. HPLC analysis was performed by a slight modification of column conditions

proposed by Mi-Jung Kim15 using Shimadzu LC-20AD pumps, SPC-M20 with diode

array detector. Chromatographic separations were performed on Xtimate C-18 column

(4.6x 250 mm i.d., 5µm). The mobile phase was 0.2% glacial acetic acid in water as

solvent A and acetonitrile as solvent B. The gradient programme was 0 min 5% B in

A; 60 min 30% B in A with flow rate of 1ml/min.Operating conditions was as

follows:

Column temperature was 250C; injection vol. 20µl; photo diode array

detection at 275nm.The compounds were identified from their peak areas in relation

to respective reference standards.

4.6.5. LCMS

High Performance Liquid Chromatography-Mass Spectrophotometer (410

Prostar, Varian Inc, USA) fitted with electrospray ionization (ESI) source for –ve ion

mass spectra of column elute were recorded in the range m/z 100-2000. Nitrogen was

used as the drying gas, flow rate was 0.8ml/min and pressure was 150ps. The

temperature of nebulizer was 2500C and operated at 25 psi. Collision induced

dissociation spectra obtained with fragmentation amplitude of 1.0 V using helium as

collision gas (1.1x 10-5m bar) 16

131

4.7. ASSESSMENT OF AA OF DIFFERENT EXTRACTS IN PUFA RICH RBD

EDIBLE OILS

4.7.1. Stability of RBD SFO

RBD SFO, without any additives was purchased from sunflower oil industry

(M/S I.K. oil mills, Adoni). The oil was stored in amber coloured bottles. The extracts

were incorporated at 200 ppm concentration in RBD SFO which was stored in

different amber coloured bottles. The AA of extracts was compared individually with

BHT incorporated in RBD SFO at its permitted level i.e., 200 ppm. The control

sample of RBD SFO with out any additives was also used for comparison17.

AA evaluation10 & 18

To test the AA of the extracts in PUFA rich RBD edible oils, two methods

were employed, i.e., Rancimat method and hot air oven method in conjunction with

PV determination.

4.7.1a Rancimat method19

A 743 Rancimat (Metrohm, Herisau, Switzerland) was used. 5gm portions of

treated RBD SFO with different were loaded into different reaction vessels.

Measurements of eight different samples can be conducted in one batch. The air

supply was maintained at 20L/h and the heating temperature at 1300C throughout the

experiment. Induction time for the test samples was determined by measuring the

elapsed time from the beginning to the moment when a sudden change of conductivity

occurred. The induction time was recalculated to different temperatures from 40-

1300C.

132

4.7.1b Hot air oven method

15gm portions of treated RBD SFO with different extracts were placed in

different glass beakers and heated in a hot air oven to 40˚C for one hour. The samples

were tested for PV after heat treatment. Similar procedure was followed at different

temperatures (50-1300C) and PV was tested for the RBD SFO at each temperature.

Determination of PV20

The PV is a measure of the peroxides contained in a sample of fat, expressed

as milli-equivalents of peroxide per 1000 grams of the material. The material in an

acetic acid-chloroform medium is treated with an aqueous solution of potassium

iodide. The liberated iodine is titrated with standard sodium thiosulphate solution.

RBD SFO samples which were heated were weighed into a 250-ml glass

stoppered conical flask and then 30 ml of the acetic acid-chloroform solution was

added. The flask was swirled until the sample was dissolved. 0.5 ml of saturated

potassium iodide solution was added. The solution was allowed to stand exactly one

minute with occasional shaking and then 30 ml of distilled water was added. The

solution was titrated with 0.1 N sodium thiosulphate solution with constant and

vigorous shaking. Titration was continued until the yellow colour almost disappeared.

0.5 ml of starch solution was added and titration was continued till the blue colour just

disappeared.

The determination was repeated using 0.01 N sodium thiosulphate solution, if

the titer value was less than 0.5 ml.

133

A blank determination was conducted in the same way.

Calculation-

PV as milli-equivalents per 1000grams sample = g

NBS 1000)( ××− ---------- 4.5

Where

S = Volume in ml of sodium thiosulphate solution used up by the sample,

B = Volume in ml of the sodium thiosulphate solution used up in the blank

determination,

N = Normality of the sodium thiosulphate solution, and

g = weight in g of the sample.

4.7.2. STABILITY OF RBD SBO

RBD SBO with out any additives was procured from soybean oil industry

(M/s Cargill Foods, Pune). Different extracts were incorporated separately at 200

ppm concentration in RBD SBO which was stored in different amber coloured bottles.

The AA of extracts was compared with synthetic antioxidant, BHT incorporated in

RBD SBO at its permitted level; 200ppm. The control sample of RBD SBO with out

any additives was used for comparison.

AA of extracts in RBD SBO were tested by two methods i.e., Rancimat

method and hot air oven method in conjunction with PV determination.

4.7.2a Rancimat method

RBD SBO samples with different extracts were tested in Rancimat to know

the IP as mentioned in section 4.7.1a.

134

4.7.2b Hot air oven method

RBD SBO with different extracts, BHT and control was tested for PV after

heating for different temperatures as explained in section 4.7.1b.

4.8. STATISTICAL ANALYSIS

All determinations were carried out in triplicate. Statistical analyses were

performed by using Microsoft Excel Data Analysis, significant differences between

means were determined by Duncan’s multiple range tests21 and were considered to be

significant when Ρ<0.05.

135

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