4. EXPERIMENTAL INVESTIGATIONS Name of the Chemical...
Transcript of 4. EXPERIMENTAL INVESTIGATIONS Name of the Chemical...
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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.
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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
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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.
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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
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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.
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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
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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
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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.
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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
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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.
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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.
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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.
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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.
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