Chapter-5 - Information and Library Network Centreshodhganga.inflibnet.ac.in/bitstream/10603/17901/5/14_chapter 5.pdf · the efficacy of any drug or the accomplishment of Kaya Karpam

Chapter-5

Materials& Methods

CHAPTER 5 METHODOLOGY

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Materials and Methods

5.1 List of materials and equipments used during experiment

Table 5.1: List of materials and equipments used during experiment

Sl. No Name of the materials and equipments

1. Chang liver cells (Human normal liver cells)

2. Trypsin-phosphate-versene-glucose (TPVG) solution

3. DMEM (Dulbecco’s modified eagles medium)

4. Fetal Calf Serum (FCS)

5. Propanol

6. Haemocytometer, Pasteur Pipettes, Sterile Pipettes, Cell Counter

7. Microplate reader (ELISA Reader, Bio tek)

8. SRB dye (0.4% prepared in 1% acetic acid) (Sigma Chemicals)

9. 10mM Tris base

10. MTT (prepared in Hank’s Balanced Salt Solution (HBSS) without phenol

red, 2mg/ml) (Sigma Chemicals)

11. 50% trichloro acetic acid

12. Anaesthetic ether (Sigma solvents and Pharmaceuticals- Mumbai).

13. 95% Ethanol

14. Formalin (Nice–Cochin, Fischer Scientific. Lot No:- 91026906-5., Pdt

No.24005).

15. Chloroform (S.D. Fine – Chem Ltd. Mumbai. Mole. Wt-119.5.

16. Heparin (HEP-5, Gland Pharma Ltd, Hyderabad. Batch – No. UJ918)

17. Kcl (S.D. Fine – Chem Ltd.)

18. Nacl (S.D. Fine – Chem Ltd.)

19. EDTA(S.D. Fine – Chem Ltd.)


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20. KH2PO4 (S.D. Fine – Chem Ltd.)

21. H2O2 (SDFCL-38694L05,Batch No- G09A/2209/0807/13)

22. Centrifuge- (REMI)

23. Digital balance (Essae-teraoka Ltd)

24. Deep freezer (Whirlpool-23classic)

25. Rotary flash evaporator (SUPERFIT, Rotary “Vaccum Digital Bath”)

26. Albino rats (Wistar strain)

27. Methanol (SD Fine Chemical Ltd. Mumbai)

28. Anaesthetic ether (Sigma)

29. Carbon tetrachloride (SD Fine Chemical Ltd. Mumbai)

30. Paracetamol (Strides Arcolabs Ltd. Bangalore)

31. Thioacetamide (SD Fine Chemical Ltd. Mumbai)

32. Silymarin (Micro Labs Bangalore)

33. Liquid paraffin (CDH, Mumbai)

34. Nitroblue tetrazolium NBT (Loba Chemical, Mumbai)

35. EDTA (Nice Chemicals, Cochin)

36. Hydroxylamine hydrochloride (Nice Chemicals, Cochin)

37. Sodium carbonate (SD Fine Chemical Ltd. Mumbai)

38. Folin Cioalteau Reagent (Loba Chemical, Mumbai)

39. Copper sulphate (SD Fine Chemical Ltd. Mumbai)

40. Sodium hydroxide (SD Fine Chemical Ltd. Mumbai)

41. 90% Alcohol (SD Fine Chemical Ltd. Mumbai)

42. Standard bovine albumin (SD Fine Chemical Ltd. Mumbai)

43. Pottasium dihydrogen orthophosphate (SD Fine Chemical Ltd. Mumbai)

44. Disodium hydrogen orthophosphate (SD Fine Chemical Ltd. Mumbai)


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45. Hydrogen peroxide (SD Fine Chemical Ltd. Mumbai)

46. ASAT kit (Crest Biosystems, Coral Clinical Systems, Goa)

47. ALAT kit (Crest Biosystems, Coral Clinical Systems, Goa)

48. ALP kit9 (Crest Biosystems, Coral Clinical Systems, Goa)

49. Bilirubin kit (Crest Biosystems, Coral Clinical Systems, Goa)

50. Analytical balance (Schimadzu/Japan)

51. Semi analyzer (Qualigen, AR 601 Mumbai)

5.2. Collection of plant material

The used plant materials for the present studies were collected from herb

collectors from Bidadi, Ramanagaram District, Karnataka. The plant was identified,

confirmed and authenticated by Botanist Prof. T. Nagendra department of botany,

Bharathi college, Bharathi nagara, Mandya. The respective parts of the dried

materials were then pulverized separately into coarse powder by a mechanical

grinder. The resulting powder was then used for extraction.

5.3 Preparation of Extracts89

The study design comprises of Total extract, chloroform fractionation of

extract and formulation of total extract (Amuri).

Total extract is prepared by using 70% ethyl alcohol. The powdered drug was

dried and packed well in Soxhlet apparatus and extracted with 1500 ml of ethanol for

72 hours. The extract was concentrated and dried using Rotary vacuum evaporator.

It was kept in a desiccator until used.

Fractionation is done by using chloroform. Formulation is done by dissolving

total extract in ‘Amuri.’


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5.3.1. Preparation of Amuri90

Amuri is a primordial liquid elixir obtained from plantain tree named Musa

paradicica through a special process.

The process is described in unpublished siddha palm leaf manuscript named

‘kandhar nadi vakkiyam’. As per the procedure plantain trees before attaining

flowering stage have to be selected. The trees are prepared in the following manner

on Ashtami that is 8th day after New moon [Ammavasai]. The roots are treated with a

siddha preparation named ‘Shangu bashpam’ and a two feet pit made around the

root to irrigate with water. A window of approximately 3-5 inches is made on the

upper portion of the stem about ½-1 foot from the top and guru chooranam

preparation is placed inside the window and sealed with dried banana leaf and tied

tightly. The medicines are allowed to soak till chathurdasi [6th day after Ashtami]. On

fullmoon day [pournami] the tree is cut about one to two feet above the ground. A

tube is inserted on the smoothened top of the plant and drained out. It is collected in

a clean container. The collected liquid is distilled and the distillate obtained is ‘Amuri’.

Amuri, Muppu and Guru are highly acclaimed preparations in siddha tradition.

It is mentioned in the literature that without these, the preparation of any medicine,

the efficacy of any drug or the accomplishment of Kaya Karpam is incomplete. Even

amongst these three, Amuri is primordial in the preparation of Muppu itself. It is

considered as a very basic drug. Because of its importance, siddhars in their

literature have used various synonyms in cryptic poetic language. In Kandar Nadi

Vakiyam, an unpublished nadi literature, a reference is found that a specially

prepared plantain juice is Amuri.

Amuri is used i) during treatment (Vaidya Amuri) ii) as a base for processing

metals/minerals (Vada Amuri) iii) to facilitate salvation (Gnana Amuri) iv) to

rejuvenate and prolong life (Karpa Amuri).


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5.4 Qualitative phytochemical screening 91

Standard phytochemical tests were used to screen the extract for the

presence of different constituents. The 3 plant extracts taken for the study were

subjected to qualitative analysis for the phytoconstituents like alkaloids,

carbohydrates, glycosides, steroids, tannins, proteins, amino acids and flavonoids as

per the standard procedures before carrying out pharmacological studies. The results

are tabulated as shown in.

A. Tests for Alkaloids:

Hager’s test: - Extract treated with a few drops of Hager’s reagent (saturated

solution of picric acid) – yellow precipitates indicate presence of alkaloids.

Mayer’s test: - Extract treated with Mayer’s reagent (potassium mercuric iodide

solution) – formation of cream precipitates indicates presence of alkaloids.

Dragendorff’s test: - Extract treated with Dragendorff’s reagent (potassium bismuth

iodide solution) - orange precipitates.

Wagner’s test: - Extract treated with Wagner’s reagent (iodine, potassium–iodine

solution) –reddish brown precipitates.

B. Tests for carbohydrates:

Molish’s test: -

Extract treated with Molish’s reagent (alpha naphthol in 95% ethanol) and a

few drops of concentrated sulphuric acid were added by the sides of the test tubes. A

violet ring at the junction is formed.

Fehling’s test: -

Extract treated with Fehling’s reagent (Fehling’s reagent A-copper sulphate in

water and Fehling’s reagent B-sodium potassium tartarate) followed by heating. Red

colored precipitates indicate presence of reducing sugars.

Barford’s test: -

Extract treated with Barford’s reagent (copper acetate in water and glacial

acetic acid) - red coloration.


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Benedict’s test: -

Extract treated with Benedict’s reagent (copper sulphate, sodium citrate and

sodium carbonate in water)- red colored precipitates.

C. Tests for Steroids, Terpenoids and Cardiac Glycosides:

Liebermann-Burchard test: -

10 g of extract was dissolved in 1 ml of chloroform, 1 ml of acetic anhydride

was added followed by the addition of 2 ml of concentrated sulphuric acid from the

sides of the test tube. Formation of reddish brown colour at the junction indicates the

presence of steroids/terpenoids/cardiac glycosides.

Salkowski test: -

1 ml of concentrated sulphuric acid was added to 10 g of the extract dissolved

in 1 ml of chloroform. A reddish brown layer exhibited by the chloroform layer and

green fluorescence by the acid layer suggests the presence of steroids.

Noller’s test: -

5 g of the extract was dissolved in 2 ml of 0.01% anhydrous stannic chloride

in pure thionyl chloride .Yellow colour indicates the presence of triterpenoids.

Legal’s test: -

The extract was treated with sodium nitroprusside in pyridine and methanolic

alkali. The formation of pink color indicates the presence of cardiac glycosides.

Balget’s test: -

1 ml of the extract solution was treated with a few drops of sodium picrate

reagent, yellowish orange color indicates the presence of cardiac glycosides.

Killer Killani’s test: -

5 g of the extract was treated with 1 ml of glacial acetic acid and a few drops

of ferric chloride solution. 2 ml of concentrated sulphuric acid were carefully added

from the sides of the test tube. Formation of a reddish brown color at the junction of


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the two layers and formation of bluish green color in the upper layer indicates the

presence of deoxy sugar in the carbohydrates.

D. Tests for Saponins:

Diluted 1 ml of the extract with distilled water to 10 ml and shaken in a

graduated cylinder for 15 minutes. Formation of 1cm layer of foam indicates the

presence of saponins.

Haemolysis test: -

2 ml of 1.8% sodium chloride solution was taken in two test tubes. To one test

tube 2 ml of distilled water were added and to another 2 ml of 1% extract were

added. Blood is obtained by pricking the thumb and 5 drops of blood were added to

each tube, the contents were gently mixed and observed under the microscope.

Haemolysis indicates the presence of saponins.

E. Tests for Tannins:

Ferric chloride test: - Extract treated with ferric chloride solution–blue color is

formed.

Gelatin test: - Extract treated with gelatin solution–white precipitates appear.

Lead acetate test: - Extract treated with lead acetate solution–yellow precipitates

appear.

F. Tests for Proteins and Amino Acids:

Millon’s test: - Extract treated with Millon’s reagent (mercuric nitrate in nitric acid)-

red color is formed.

Biuret test: - Extract treated with sodium hydroxide and copper sulphate solution

drop wise –violet color.

Ninhydrin test: - Extract treated with ninhydrin reagent and ammonia and heated –

violet color is formed.

G. Tests for Flavonoids:

Ferric chloride test: - To the extract add a few drops of neutral ferric chloride

solution- blackish red color is formed.


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Lead acetate test: - To the extract add lead acetate solution-yellow precipitates.

Magnesium ribbon test: - To the extract add few fragments of magnesium ribbon

and concentrated hydrochloric acid along the sides of the test tube-magenta color is

formed is formed.

Zinc–hydrochloride test: - To the extract, a pinch of zinc dust was added followed

by addition of concentrated hydrochloric acid along the sides of the test tubes-

magenta color is formed.

5.5 Experimental Animals

Male Swiss albino mice (18-22g) were used for the study. Animals were

housed in polycarbonate colony cages (6/cage) in a well ventilated room (air cycle:

15/min; 70:30) under an ambient temperature of 23±2C and 40–65% relative

humidity, with artificial photoperiod 12-h light/12-h dark cycle. They were provided

with standard rodent pellet diet (Provimi, India) and purified water ad libitum (RIOS,

USA). Experimental animals were acclimatized for 7 days to the laboratory conditions

prior to experimentation. Guidelines of “Guide for the Care and Use of Laboratory

Animals” (Institute of Laboratory Animal Resources, National Academic Press 1996;

NIH publication number #85-23, revised 1996) were strictly followed throughout the

study. Institutional Animal Ethical Committee (IAEC-XIV/SRU/100/2008), Sri

Ramachandra University, Chennai, India, approved the study protocol.

5.6 Acute oral toxicity study (OECD Guide line 423)92

The acute oral toxicity study was carried out as per the guidelines set by

Organization for Economic co-operation of Development [OECD],received drafts

guidelines 423,revised from committee for the purpose of control and supervision of

experiment on animals[CPCSEA]. Female Sprague Dawley rats (160-180g) were

divided into two groups of 3 animals / group. The test drug was administered once

orally via gastric intubation at a dose level of 2000 mg/kg b.wt. Lethality, abnormal

clinical signs and body weight changes were observed on the day of dosing and


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thereafter for 13 days. Gross pathological changes were also observed on the

completion of the experiment.

5.7 Cell lines and growth media93

Chang liver cells (Human normal liver cells) were cultured in DMEM

(Dulbecco’s modified eagles medium) medium. The medium also contains 10% fetal

calf serum, penicillin (100 U) and streptomycin (100 µg).

5.8 Method for Passaging the Cells

Procedure

All the reagents were brought to 37oC before use.

Sufficient amount of TPVG solution was added to cover the monolayer, rinsed

and discarded.

Fresh TPVG solution was added and allowed to stand at room temperature

for 2-3 minutes.

TPVG solution was discarded and the flask containing the monolayer was

incubated at 37oC for 3-5 minutes and slightly tapped to free the cells from the

surface.

10ml of DMEM containing 10% serum was added to the flask and pipetted to

breakdown the clumps of cells.

Total cell count was taken using a haemocytometer.

Calculated the total number of cells. The medium was added according to

the cell population needed. Required amount of medium containing the

required number of cells (0.5-1.0x105 cells/ml) was transferred into bottles

according to the cell count and the volume was made up with medium and


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required amount of serum (10% growth medium and 2% maintenance

medium) was added.

The flasks were incubated at 37oC and the cells were periodically checked for

any morphological changes and contamination. After the formation of

monolayer, the cells were further utilized.

5.9 Cytotoxicity Screening94, 95

5.9.1 Determination of Mitochondrial Synthesis by Micro culture Tetrazolium

(MTT) Assay

The ability of the cells to survive a toxic insult has been the basis of most

cytotoxicity assays. This assay is based on the assumption that dead cells or their

products do not reduce tetrazolium. The assay depends both on the number of cells

present and on the mitochondrial activity per cell. The cleavage of MTT to a blue

formazan derivative by living cells in clearly a very effective principle on which the

assay is based.

The principle involved is the cleavage of tetrazolium salt 3-(4,5 dimethyl

thiazole-2 yl) - 2,5-diphenyl tetrazolium bromide (MTT) into a blue colored product

(formazan) by mitochondrial enzyme succinate dehydrogenase. The number of cells

was found to be proportional to the extent of formazan production by the cells used.

Procedure

1. The monolayer cell culture was trypsinized and the cell count was adjusted to

1.0x105 cells/ml using medium containing 10% new born calf serum.

2. To each well of the 96 well microtitre plate, 0.1ml of the diluted cell

suspension (approximately 10,000 cells) was added.


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3. After 24 hours, when a partial monolayer was formed, the supernatant was

flicked off, washed the monolayer once and 100l of different drug

concentrations was added to the cells in microtitre plates. The plates were

then incubated at 37oC for 3 days in 5% CO2 atmosphere, and microscopic

examination was carried out and observations recorded every 24 hours.

4. After 72 hours, the drug solutions in the wells were discarded and 50l of

MTT in DMEM was added to each well.

5. The plates were gently shaken and incubated for 3 hours at 37oC in 5% CO2

atmosphere.

6. The supernatant was removed and 50l of propanol was added and the

plates were gently shaken to solubilize the formed formazan.

7. The absorbance was measured using a micro plate reader at a wavelength of

540nm.

The percentage growth inhibition was calculated using the formula below:

Mean OD of Individual Test Group

Mean OD of Control Group

5.9.2 Determination of Total Cell Protein Content by Sulphorhodamine B (SRB)

assay

SRB is a bright pink aminoxanthene dye with two sulfonic groups. Under mild acidic

conditions, SRB binds to protein basic amino acid residues in TCA (Trichloro acetic

acid) fixed cells to provide a sensitive index of cellular protein content that is linear

over a cell density range of at least two orders of magnitude.

Colour development in SRB assay is rapid, stable and visible. The developed colour

can be measured over a broad range of visible wavelength in either a

spectrophotometer or a 96 well plate reader. When TCA-fixed and SRB stained

samples are air-dried, they can be stored indefinitely without deterioration.

% Growth Inhibition = 100 – X 100


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Procedure

Same as that of MTT assay (Sl. No. 1. to 3.)

After 72 hours, 25l of 50% trichloro acetic acid was added to the wells gently

such that it forms a thin layer over the drug dilutions to form a overall

concentration of 10%.

e) The plates were incubated at 4oC for one hour.

f) The plates were flicked and washed five times with tap water to remove

traces of medium, drug and serum, and were then air-dried.

g) The air-dried plates were stained with SRB for 30 minutes. The unbound

dye was then removed by rapidly washing four times with 1% acetic acid.

The plates were then air-dried.

100l of 10mM tris base was then added to the wells to solubilise the dye.

The plates were shaken vigorously for 5 minutes.

The absorbance was measured using micro plate reader at a wavelength of

540nm.


Mean OD of Individual Test Group


5.10 In vitro hepatoprotective activity against CCl4 induced toxicity96

Methodology:

Below the CTC50 value two dose levels were selected for each extract and used for

further studies.

The monolayer cell culture of Chang liver cells was trypsinized and the cell

count was adjusted to 1.0x105 cells/mL using medium containing 10% new

born calf serum.



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To each well of the 96 well microtitre plate, 0.1mL of the diluted cell

suspension (approximately 10,000 cells) was added.

After 24 hours, when a partial monolayer was formed, the supernatant was

flicked off, the monolayer was washed once and treated with 100l of different

drug concentrations for 24 hrs.

After 24 hrs of pretreatment with the extracts, the cells were challenged with

CCl4 (15 mM) where 100l of different drug concentration and 100l of CCl4

was added. The plates were then incubated at 37oC for further 24 hours in 5%

CO2 atmosphere. Microscopic examination was carried out and observations

were recorded every 24 hours.

After 72 hours, the drug solutions in the wells were discarded and 50l of

MTT in MEM - PR was added to each well.

The plates were gently shaken and incubated for 3 hours at 37oC in 5% CO2

atmosphere.

The supernatant was removed and 50l of n-propanol was added and the

plates were gently shaken to solubilize the formed formazan.

The absorbance was measured using a microplate reader at a wavelength of

540nm.


Mean OD of Individual Test group


Cells which are exposed only with toxicant CCl4 showed a percentage viability of

42%. Cells which are pretreated with extracts showed an increase in percentage

viability and the results were highly significant (P < 0.001, when compared to CCl4

intoxicated cells). The percentage viability ranged from 72 to 84 % cell viability, when

pretreated with the extracts.



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The above study indicates the hepatoprotective activity of all the three extracts in

vitro against CCl4 induced toxicity.

5.11 ADAPTOGENIC ACTIVITY

5.11.1 Swim Endurance Test97, 98

Force Swim test

Forced swimming test (FST) is a screening model for antidepressants/

adaptogens. Mice were placed individually in a Plexiglas cylinder (height 40 cm,

diameter 10 cm) filled with tap water (27± 2C) to a height of 18 cm. Two swimming

sessions were conducted: a 15 min pre-test followed 24 h later by a 6 min test. After

the pre-test, the animals were removed from the water, dried before a room heater

and returned back to their home cages. The total duration of immobility behaviour

was recorded during the second 6 min test. Mouse was judged immobile, when it

remained floating in water, in an upright position making only small movements to

keep the head above water.

Treatment and Groups98

To test the adaptogenic effect of drugs in FST, animals were pretreated with

drugs at 100 mg/kg, p.o daily for a

period 14 days. Test drugs were prepared in 0.5% CMC.

There were nine groups:

Group I: vehicle – received no CMC or FST;

Group II: vehicle received 0.5% CMC (at dose volume of 10ml/kg, p.o) +

FST;

Group III: Fluoxetine (15 mg/kg, p.o) + FST;

Group IV: TE + FST (100mg/kg, p.o);

Group V: TE + FST (200mg/kg, p.o);

Group VI: FE + FST (100mg/kg, p.o);


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Group VII: FE + FST (200mg/kg, p.o);

Group VIII: Formulation + FST (100mg/kg, p.o) and

Group IX: Formulation + FST (200mg/kg, p.o).

Mice from groups I and II, injected with saline solution, served as controls.

Fluoxetine was used as a classical antidepressant. All groups were treated daily for

14 days. FST was performed 1hour after the last dose. The experimental animals

were euthanized and their brains were removed immediately, and the prefrontal

cortexes (PFC) were dissected out on ice for biochemical analysis.

5.12 Biochemical estimation

5.12.1 Superoxide dismutase99

Principle

The enzyme is necessary for survival in all oxygen metabolizing cells. It is

found in the cytosol and intermembrane space of mitochondria of eukaryotic cells. It

contains copper and zinc. In normal cells, this radical alone is the precursor of

hydrogen peroxide.

Superoxide dismutase scavenges the super oxide (O-2) and thus provides a first line

defense against free radical damage. SOD’S are a family of metalloenzyme that

catalyze the dismutation of super oxide anion (O2) to hydrogen peroxide and

molecular oxygen in the following manner.

2H2O2 + 2O- → 2H2O + O2

In the erythrocytes, the super oxide anion (O-2) interacts with peroxides to form

hydroxyl radicals (-OH), which causes heamolyses in the absence of SOD activity.

SOD measurement was carried out on the ability of SOD to inhibit spontaneous

oxidation of epinephrine to adrenochrome.


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Superoxide dismutase (SOD) was assayed by taking 0.05 ml of brain

homogenate followed by the addition of 0.3 ml of sodium pyrophosphate buffer

(0.025 M, pH 8.3), 0.025 ml of PMS (186M) and 0.075 ml of NBT (300M in buffer,

pH 8.3). Reaction was started by addition of 0.075 ml of NADH (780M in buffer of

pH 8.3). After incubation of the reaction mixture at 30 ◦C for 90 s, the reaction was

stopped by addition of glacial acetic acid (0.25 ml). Then, the reaction mixture was

stirred vigorously and shaken with 2.0 ml of n-butanol. The mixture was allowed to

stand for 10 min and centrifuged. 1.5 ml of n-butanol alone served as blank. The

colour intensity was read at 560nm using spectrophotometer (Perkin Elmer, 25,

USA). Enzyme activity was expressed as 1 Unit = 50% inhibition/minute/mg of

protein (Kakkar et al., 1984).

5.12.2 Reduced glutathione100

Glutathione (GSH) content was estimated by following Jollow et al. (1974)

method. 0.25 ml of tissue homogenate was added to an equal volume of ice cold 5%

TCA. The precipitate was removed by centrifugation at 4000rpm for 10 min. To 1ml

aliquot of supernatant, 0.25 ml of 0.2Mphosphate buffer (pH 8.0) and 0.5 ml of DTNB

(0.6mM in 0.2M phosphate buffer, pH 8.0) were added and mixed well. The

absorbance was read at 412nm using spectrophotometer (Perkin Elmer, 25, USA).

The values were expressed in nanomoles/g tissue.

5.12.3 Thiobarbituric acid reactive substances (TBARS)101

Lipid peroxidation was evaluated by measuring the TBARS content according

to the TBA test described by with slight modifications. 0.2 ml of the brain tissue

homogenate was taken and to this 0.8 ml saline, 0.5 ml of BHT and 3.5 ml TBA

reagent (0.8%) were added and incubated at 60 ◦C in a boiling water bath. After

cooling, the solution was centrifuged at 2000rpmfor 10 min. The absorbance of the


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supernatant was determined at 532nm using spectrophotometer (Perkin Elmer, 25,

USA) against the blank. TBARS content were expressed in nanomoles/mg tissue.

Standard calibration was plotted using 1, 1,3,3-tetra ethoxy propane in the

concentration range of 0.50–4.00 g.

5.13 ANTIOXIDANT ACTIVITY

5.13.1 Evaluation of Total Antioxidant Capacity102

The total antioxidant capacity was determined by phosphomolybdenum

method which is based on the reduction of Mo (VI) to Mo (V) by the antioxidant

compounds and the formation of a green Mo (V) complex which has the maximal

absorption at 695 nm

.

Preparation of test and standard solutions

The extracts and the standards, ascorbic acid and α-tocopherol (55 mg each)

were separately dissolved in 5 ml of freshly distilled DMSO. The lower dilutions were

made serially with freshly distilled DMSO.

Procedure

An aliquot of 0.1 ml of sample solution containing a reducing species in

freshly distilled DMSO was combined in an Eppendorff tube with 1 ml of

reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate, and 4 mM

ammonium molybdate).

The tubes were capped and incubated in water bath at 95 °C for 90 min.

The samples were cooled to room temperature, and the absorbance was

measured at 695 nm.

The total antioxidant capacity was expressed as mM equivalent of ascorbic

acid.

Documents

Chapter-5 - Information and Library Network Centreshodhganga.inflibnet.ac.in/bitstream/10603/17901/5/14_chapter 5.pdf · the efficacy of any drug or the accomplishment of Kaya Karpam