CHAPTER 3 MATERIALS AND METHODS -...

59

CHAPTER 3

MATERIALS AND METHODS

Chemicals

Solvents like methanol, petroleum ether, and ethanol were purchased from

Sigma chem. Co, USA. Standard normal diets for rats are obtained from Tetra

chemi pvt, Ltd, Bangalore. All the biochemical kits were purchased from Spin

react, Spain and accurex Biomedical Pvt Ltd, Thane, India. All other reagents

were purchased from Merk India and all other from the authorized

dealers.Paracetamol obtained from Glaxo SmithKline Pharma and Silymarin from

Micro labs limited.

Plants collection and Identification

Hybanthus enneaspermus were collected from the campus of Tamil

University, Thanjavur and authenticated by Dr. G. V. S Murthy, Scientist and

Head, Botanical survey of India, Coimbatore.

Plant extraction

The plant material was pulverized with electric grinding machine into

minute pieces. The powdered and crushed materials were extracted using solvents

such as petroleum ether, 50% alcoholic (hydro-ethanolic and hydro-methanolic)

and water according to the methodology described in the Indian

Pharmacopoeia.1996. At the end of each respective extraction, the extract is

filtered using whatman No. 1 filter paper. The filtrate was concentrated under

reduced pressure in vacuum at 450°C using a rotary evaporator. The extracts were

stored at 4° C until use.

Print to PDF without this message by purchasing novaPDF (http://www.novapdf.com/)

http://www.novapdf.com/


60

Plate.3.1 Photograph of Hybanthus enneaspermus

Plate.3.2 Soxhlet apparatus




61

3.1. Standardization of plant material

The following physicochemical parameters were carried out in dried

Hybanthus enneaspermus powder (WHO, 2002; The ayurvedic pharmacopoeia of

India 2008) for the standardization of the plant material.

The % of yield was calculated using the formula,

Yield % (w/w) = amount of the extract / 100 g of dry material.

3.1.(a)Loss on drying

Eight grams of crude powder of Hybanthus enneaspermus was taken in an

evaporating dish and then dried in an oven at 105ºC till constant weight was

obtained. The weight after drying was noted and loss on drying was calculated.

The percentage was calculated on the basis of sample taken initially.

Loss on drying (%) = Loss in weight x 100/ W

Where W = Weight of the powder in gm.

3.1. (b).Determination of total ash value

2-3 gm of powder sample of Hybanthus enneaspermus was placed into a

previously dried crucible. The crucible was then kept in a muffle furnace at 100°C

for 30 minutes. Temperature was raised in 50°C increments up to 250°C at the

intervals of 30 minutes. After 30 minutes, the temperature was raised to 500°C

and the material was allowed to incinerate till it became white indicating the

absence of carbon. Then the process was stopped. Crucible was allowed to cool

completely in desiccators. Total ash was weighed and percentage of total ash was

calculated with reference to the powder sample taken initially.

Ash % = Loss in Weight x 100

W




62

3.1. ( c).Determination of acid insoluble ash value

About 25 ml of dilute hydrochloric acid was added to the crucible of total

ash, covered with a watch glass and boiled gently for 5 minutes. The watch-glass

was rinsed with 5 ml of hot water and this liquid was added to the crucible. The

acid solution was then filtered on an ash free filter paper. The paper along with the

precipitate was washed with hot water and was ignited in the crucible till constant

weight was obtained, later the residue was cooled in a suitable desiccators for 30

minutes and the weight was noted so as to get the acid-insoluble ash with

reference to powder sample taken initially.

3.1.(d).Water soluble ash

To the crucible containing the total ash, 25 ml of water was added and

boiled for 5minutes. The insoluble matter was collected on an ash less filter paper.

It was washed with hot water, dried and ignited in a crucible for 15 minutes. The

residue was allowed to cool and then weighed without delay. Weight of insoluble

matter was subtracted from the weight of total ash. The percentage of water

soluble ash was calculated on the basis of sample taken initially.

3.2.Qualitative phytochemical analysis

All the four successive extracts were subjected to qualitative

phytochemical analysis to identify the presence of constituents such as alkaloids,

carbohydrates, glycosides, flavonoids, phenolic compounds and tannins, proteins

and free amino acids, saponins, steroids and triterpenoids (The ayurvedic

pharmacopoeia of India 2008).




63

Alkaloids

a. -

presence of reddish brown precipitate indicates presence of alkaloids.

b. -

cream coloured precipitate indicates presence of alkaloids.

Amino acids

a. -To the extracts add about 2 ml of Millons reagent, white

precipitate indicates presence of amino acids.

b. Ninhydrine test: - To the extracts add ninhydrine solution and boil.Violet

colour indicates presence of amino acid.

Proteins

a. Biuret test: - To 2 ml of the extracts 2ml of Biuret reagent was added,

appearance of violet colour indicates presence of proteins.

b. Xanthoproteic test: -To 5ml of the extract, 1 ml of concentrated nitric acid

was added and boiled, presence of yellow precipitate is formed. On cooling

it, 40% sodium hydroxide solution was added and the formation of orange

colour indicates the presence of proteins.

Carbohydrates

a. est: -To the extracts -naphthol, and

few drops of concentrated sulphuric acid was added through the sides of

test tube . Appearance of purple to violet colour ring at the junction

indicates the presence of carbohydrates.




64

Flavonoids

a. Shinoda test: - To the extracts add few magnesium tunings and

concentrated hydrochloric acid added drop wise, appearance pink scarlet,

crimson red or occasionally green to blue colour appears after few minutes

indicates the presence of flavonoids.

b. Alkaline reagent test: - To the extracts add few drops of sodium

hydroxide solution, intense yellow colour is formed which turns to

colourless on addition of few drops of dilute acid indicate presence of

flavonoids.

Glycosides

a. - Boil the extracts separately with 1ml of sulphuric acid

in a test tube for 5minutes. Filter while hot. Cool the filtrate and shake with

equal volume of dichloromethane or chloroform. Separate the lower layer

of dichloromethane or chloroform and shake it with half of its volume of

dilute ammonia. Rose pink to red colour is produced in the ammonical

layer indicates the presence of glycosides.

Saponins

a. Froth formation test: - Place 2ml solution of all the extracts in water in a

test tube, shake well, stable froth (foam) is formed.

Phenolic compounds

a. Shinoda test: The presence of flavonoids was estimated by Shinoda test.

All the four extracts were treated with few drops of concentrated HCl and




65

magnesium ribbon. The appearance of pink or tomato red colour within

few minutes indicated the presence of flavonoids.

Steroids and tri-terpenoids

a. Libermann- Burchard test - Treat the extract with few drops of acetic

anhydride, boil and cool. Then add concentrated sulphuric acid from the

side of the test tube, brown ring is formed at the junction of two layers and

upper layer turns green which shows presence of steroids and formation of

deep red colour indicates presence of tri-terpenoids.

b. Salkowski test: -Treat the extract with few drops of concentrated sulphuric

acid red colour at lower layer indicates presence of steroids and formation

of yellow coloured lower layer indicates presence of tri-terpenoids.

3.3.Quantitative phytochemical analysis

3.3.1.Estimation of Carbohydrates

The total carbohydrates in each extract were estimated by Hedge and

Hofreieter (1962).

Reagents

1. 2.5 N Hydrochloric acid

2. Solid sodium (freshly prepared)

3. Anthrone reagent- 200 mg of anthrone was dissolved in 100ml of ice cold

95% H2SO4. This mixture was then neutralized using solid sodium

carbonate until the effervescence ceases, then it was made to 100 ml with

distilled water and used for estimation.




66

4. Stock Glucose solution: 10 mg of glucose was weighed and dissolved in

distilled water and made up to 10 ml in a standard flask (1mg/ml)

5. Working Standard: 1 ml of the stock was diluted to 10 ml with distilled

water in a standard flask.1 ml contains 100 µg glucose.

6. Preparation of extract: About 25 ml of the extract was hydrolyzed by

boiling it with 2.5 N HCl for 2 hours and then cooled to room temperature.

Procedure

To 0.5 ml of hydrolyzed extract 0.5 ml water was added to make the

volume 1 ml. 1 ml water serves as blank. 4 ml of anthrone reagent was added to

all the tubes. The mixture was heated in a boiling water bath for 8 minutes and

cooled. The green colour developed was read at 630 nm using UV/Vis

spectrometer. The experiments were repeated in triplicate and the results were

expressed as mg/g extract.

3.3.2.Estimation of protein

The total protein in each extract was determined by the method of Lowery

et al., 1951.

Reagents

1. Alkaline copper sulphate reagent: Reagent A: 2% sodium carbonate in

0.1N sodium hydroxide.Reagent B: 0.5% copper sulphate in 1% Potassium

sodium tartarate. Working Reagent: 50 ml of Reagent A and 1 ml of

Reagent B was mixed prior to use.




67

2.

use.

3. Stock protein solution: 50 mg of bovine serum albumin was weighed and

dissolved in 50 ml distilled water.

4. Working standard: 10 ml of stock was diluted to 50 ml with distilled water.

1ml contains 200 µg proteins.

5. Preparation of the extract: 10 mg of each extract was weighed separately

and dissolved in 1ml of their respective solvents and used for estimation.

Procedure

To 1 ml of prepared extract 5 ml of alkaline copper sulphate reagent

was added, mixed well and allowed to stand for 10 minutes and then 0.5 ml of

Folin- Ciocalteau reagent was added and mixed well. The mixture was allowed to

stand under dark for 30 minutes. The blue colour developed was read at 660 nm

using UV/Vis spectrophotometer. The experiments were in triplicates. The total

protein content of the extract was expressed as mg/g extract.

3.3.3.Estimation of Lipids

The lipid content of the extract was estimated by the method of Zak et al.,

(1953).

Reagents

1. Stock ferric chloride acetic acid reagent (0.5%)-50 mg of ferric chloride

Fecl3 was weighed and dissolved in 10 ml of glacial acetic acid.




68

2. Working ferric chloride acetic acid reagent (0.05%)-1 ml of the stock ferric

chloride acetic acid was diluted to 10 ml with glacial acetic acid.

3. 3:1 Ethanolic ether

4. 85% Concentrated sulphuric acid

5. Stock Cholesterol: 100 mg of Cholesterol dissolved in chloroform and

made to 100 ml in a standard flask (1mg/ml)

6. Working Standard: 1 ml of stock was diluted to 10 ml in a standard flask .1

ml contains 100 µg cholesterol.

7. Preparation of Extract : 100 mg of each extract was dissolved in 2 ml of

3:1 ethanolic ether mixture. The mixture was warmed, cooled and

centrifuged at 3000 rpm for 10 minutes. The supernatant was used for

estimation.

Procedure

0.1 ml of the supernatant in duplicates was pipetted out in to test tubes. The

volumes was made up to 5 ml with working FeCl3 acetic acid reagent (0.05%) and

kept at room temperature for 10 minutes. To this 3 ml of conc. sulphuric acid was

added. The tubes were kept ice- cold condition for 20 minutes. Pink colour

developed was read at 540nm. The total lipid content of the extract was expressed

as mg/g extract.

3.3.4.Estimation of Total Phenols

To analyse the total phenolic content (TPC), the method of Folin ciocalteu

assay was carried out (Marinova et al., 2005).




69

Reagents

1. Folin Ciocalteau reagents (1:2).

2. % Sodium Carbonate (Na2CO3)

3. Stock Gallic acid Solution: 10 mg of Gallic acid was dissolved in Methanol

and made up to 10 ml in Standard Flask (Concentration: 1mg/ml).

4. Standard Solution: 1 ml of the stock solution was diluted to 10 ml with

methanol in a standard flask. 1 ml of this contains 100 µg tannins.

5. Preparation of the extract: 1 ml of the extract was dissolved in 1 ml of

methanol: water (70:30). From this 1 ml is used for the estimation.

Procedure

1ml of standard or extract solution (20, 40, 60, 80,100 mg/l) was taken into

25ml volumetric flasks,each containing 9ml of HPLC grade distilled water. 1ml of

Folin-

of 7% Na2CO3 solution was added to the mixture. The solution was diluted up to

25ml with HPLC grade distilled water. Incubate the solution at room temperature

for 90 min. A reagent blank using HPLC Grade distilled water was prepared. The

absorbance was noted at 750nm using UV-Visible spectrophotometer. Values

were expressed as mg gallic acid (GAE) / g of the extract.

3.3.5. Estimation of total flavonoids

Colorimetric aluminum chloride method was used for flavonoids

determination (Marinova et al., 2005).




70

Reagents

1. Sodium acetate (1M)

2. Aluminium chloride (10%)

3. Stock quercetin: 10 mg of quercetin accurately weighed and dissolved in

10 ml of methanol (1 mg/ml).

4. Working Standard: 1 ml of the stock was diluted to 10 ml with methanol; 1

ml of this contains 100 µg quercetin.

5. Preparation of the Extract: 5 mg of the extract was weighed and dissolved

in methanol.

Procedure

1ml of standard or extract solution (20, 40, 60, 80,100 mg/l) was taken into

10ml volumetric flasks each containing 4ml of HPLC grade distilled water.

0.3ml of 5% NaNO2 added to the flask. After 5min, 0.3ml 10% AlCl3 was added

to the mixture. At the 6th min add 2ml of 1M NaOH was added and volume made

up to 10ml with HPLC grade distilled water. The absorbance was noted at 750nm

using UV-Visible spectrophotometer. A reagent blank using HPLC Grade distilled

water was prepared. The absorbance was noted at 415nm using UV-Visible

spectrophotometer. Values were expressed as mg of quercetin (QE) / g of extract.

3.3.6.Estimation of Vitamin C

Vitamin C content of the extract was estimated according to the method of

Oyaizu, 1986.




71

Reagents

1. DTC Reagent : 0.4 g of thio urea, 0.05 g of copper sulphate and 4 g of 2,

4-dinitro phenyl hydrazine weighed and dissolved in 100 ml of 9 N H2SO4.

2. 85% Sulphuric acid

3. Stock ascorbic acid: 10 mg of ascorbic acid was weighed and dissolved in

10 ml of 5% TCA (1mg/ml).

4. Working standard: 1 ml of the stock was diluted to 10 ml with 5% TCA

(100 µg/ ml).

5. Preparation of the extract: 10 ml of the extract was dissolved in 10 ml of

respective solvents and used for estimation.

Procedure

To 1 ml of the extract DTC was added and incubated at 37° C for 3 hours.

After incubation 1.25 ml of 85 % H2SO4 was added under ice-cold condition. The

mixture was kept at room temperature for 30 minutes. The absorbance was

measured at 540 nm against blank using UV/Vis spectrometer. The experiments

were repeated in triplicate. The results were expressed as mg of ascorbic acid /g of

extract.

3.3.7. Estimation of vitamin E

Estimation of vitamin E was performed according to the method of Prieto

et al., 1999.




72

Reagents

1. - Tocopherol reagent: 28mM sodium phosphate and 4 mN ammonium

molybdate were weighed and dissolved in 100 ml of 0.6 M sulphuric acid.

2. - tocophe - tocopherol was weighed and dissolved

in100 ml ethanol (1mg/ml).

3. Preparation of extract: 10 ml of the extract was weighed and dissolved in

10 ml of respective solvents and used for estimation.

Procedure

An aliquots of the prepared extract was mixed with - tocopherol reagent

- tocopherol. The respective solvents were used as blank. All the

tubes were capped and incubated in a boiling water bath at 95° C for 60-90

minutes. Samples were cooled to room temperature, the absorbance of each

samples were measured calorimetrically at 695 nm against blank in Perkin Elmer

UV/Vis spectrophotometer. The experiments were repeated in triplicates and

calculated using a standard graph and the values were expressed as

tocopherol / g of extract.

3.4. In-vivo study

Animals

Male wistar albino rats weighing between 150 220 gm were used for the

study. The animals were obtained from animal house, Perundurai IRT, Medical

College, Erode, Tamilnadu, India. On arrival the animals were placed at random

and allocated to treatment groups in polypropylene cages with paddy husk as

bedding. Animals were housed at a temperature of 24±2oC and relative humidity




73

of 30 70 %. The 12:12 (day: night) cycle was followed. All animals were

allowed free access to water and fed with standard commercial pellet rat chaw

(M/s. Hindustan Lever Ltd, Mumbai).All the experimental procedures and

protocols used in this study was conducted in Nandha college of

Pharmacy,Perundurai, Erode and were reviewed by the Institutional Animal

Ethical Committee (Regd no: 688/2/C-CPCSEA/ Proposal No:

NCP/IAEC/MTWU/Ph.D-01/2009-2010) and were in accordance with the

guidelines of the CPCSEA.

3.4.1.Acute toxicity study

Based on the qualitative and quantitative phytochemical analysis hydro-

ethanolic extract was chosen for the study. Acute toxicity studies were performed

according to OECD-423 guidelines (OECD, 2010). Male Swiss mice selected by

random sampling technique were employed in this study. The animals were fasted

for 4 h with free access to water only. Hydro- ethanolic extract of Hybanthus

enneaspermus plant was administered orally at a dose of 5 mg/kg initially and

mortality if any was observed for 3 days. If mortality was observed in two out of

three animals, then the dose administered was considered as toxic dose.

However, if the mortality was observed in only one animals out of three

animals then the same dose to be repeated again to confirm the toxic effect. If no

mortality was observed, then higher (50, 300, 2000 mg/kg) doses of hydro-

ethanolic extract of Hybanthus enneaspermus plant were administered orally for

further toxicity studies. From the maximum dose 1/10th and 1/5th of the values

were taken as the treatment dose for further studies (Paget and Barnes, 1983 and

Tanira et al., 1988).




74

Plate.3.3 Albino rats (150-220 g) used for the experiments

Plate3.4 Oral administration of drug




75

3.4.2. Methodology

The method of Chattopadhyay (2003) was used in the study. The animals

were divided in to 5 groups of 8 animals each.

Group- 1 : Receives normal saline (1ml/kg., p. o) for 7 days (control)

Group-2 : Serves as hepato-toxicant control received normal saline (1ml/kg

p.o) for 7 days. (Positive control)

Group - 3 : Receives sylimarin (100 mg/kg) for 7 days

Group- 4 : Receives 200 mg/kg of hydro-ethanolic extract of Hybanthus

enneaspermus once daily for 7 days.

Group-5 : Receives 400 mg/kg of hydro-ethanolic extract of Hybanthus

enneaspermus once daily for 7 days.

On the fifth day, of the administration of the respective treatments, the

animals of group II, III, IV and V were administered with paracetamol 2g/kg

orally using intragastric tubes.

3.4.2.1. Body and organ weights

Body weights of the animals were recorded at the initial and final stage of

the experiment. The liver and kidney weights were recorded in all the groups after

sacrifice on 7th day.

3.5. Blood collection and preparation of tissue homogenate for the analysis of

biochemical parameters

On 7th day, after 2 hours of respective treatments the blood was collected by

sinus puncture under light ether anesthesia and serum and plasma were separated




76

for haematological and biochemical estimations. Blood was collected in EDTA

(anticoagulant) for hematological experiments Liver and kidney tissues were

excised and washed thrice in phosphate buffered saline (PBS; 1:9, pH 7.4) blotted

dry and weighed. 10 % tissue homogenate was prepared in 0.1 M phosphate

buffer by a motor driven teflon glass homogenizer. The supernatant was

centrifuged at 15000 rpm for 1 hour and used for the determination of LPO,

enzymatic and non-enzymatic antioxidants and phospholipids (Navayath Sushma

and Thiagarajan Devasena , 2010).

3.5.1.Estimation of Hemoglobin

The hemoglobin was determined by Cyanmethemoglobin method

according to the procedure described by Drabkin and Austing (1932).

Principle

Hemoglobin is converted into Cyanmethemoglobin by the addition of

KCN and ferricyanide. The colour of Cyanmethemoglobin is read in a

photoelectric colorimeter at 540 nm against a standard solution.

Reagent

1. Drabkin reagent : Dissolve 200 mg of potassium ferricyanide, 50 mg

potassium cyanide and 1 g sodium carbonate in water and made up to 1

litre. The reagent had a pale yellow colour and a pH of 9.6.

2. Cyanomethemoglobin standard: 16g/100 ml.




77

Procedure

0.02 ml of blood was diluted with 5 ml of the reagent. The diluted blood

was mixed well and allowed to stand for 10 minutes, to ensure the completion of

the reagent. The solution was read at 540 nm together with the standard solution

of cyanomethemoglobin.

3.5.2. Determination of Prothrombine time

Prothrombine time was determined by the method of Mukerjee 1988.

Reagents

1. Sodium citrate 30%.

2. Liquiplastin.

Procedure

Nine parts of freshly collected blood was mixed with one part of sodium

citrate. Centrifuged immediately for 5 minutes at 7000 rpm and transferred the

plasma in to a clean test tube.0.1 ml of the plasma was taken in a test tube and

placed in the water bath for 3-5 minutes at 37°C. To the tube 0.2 ml of liquiplastin

reagent was added and simultaneously a stop watch was started and the contents

were mixed. The tube was tilted gently back and forth until a gel clot was formed.

The stop watch was stopped and the time in seconds was recorded. The

experiments were repeated in triplicate and the average time in seconds was

recorded as the prothrombine time.

3.5.3.Estimation of erythrocyte (RBC)

Erythrocyte count was estimated by the hemocytometer method of Ghai

(1993).




78

Reagents

1. e, 2.5 g of sodium sulphate

an0.25 g of mercuric chloride in 100 ml of distilled water.

Procedure

Blood was taken up to 0.5 ml mark in the Thoma red cell diluting pipette

of the blood sample. The diluted sample was filled in the counting chamber and

counted with the aid of the light microscope.Values are expressed as cells per

cubic millimetre of blood.

3.5.4.Estimation of total leukocytes (WBC)

Total leukocyte count was estimated by hemocytometer according to the

method of John (1972).

Reagents

1. 1.5% HCl.

Procedure

The WBC pipette was filled to 0.5 mark with whole blood and diluted to

the 11 mark with 1.5% HCl, resulting in a 1:20 dilution of the blood sample. The

hemocytometer was filled with the diluted blood and leukocytes present were

counted.Values are expressed as the number of leukocytes present per cubic

millimetre of the blood.




79

Table.3.1: Biochemical estimations performed in the study

S.

No Parameters Samples Methods

1. Total protein Serum Lowry et al.,1951

2. Albumin Serum Corcoran and Durnan (1977)

3. Blood glucose Serum Sasaki et al.,1972

4. Lactate Liver Barker and Summerson 1941

5. Pyruvate Liver Friedemann and Haugen 1943

6. Glycogen Liver Seifter et al., 1950

7. Urea Serum Kaplan, 1984

8. Creatinine Serum Murray, 1984

9. SGOT Serum Reitman and Franknel, 1957)

10. SGPT Serum Reitmanand Franknel,1957

11. ALP Serum King and King 1954

12. Total bilirubin Serum Malloy and Evelyn, 1937

13. GGT Serum Rosalki and Rau (1972).

14. LDH Serum King, 1965

15. Total cholesterol Serum Zlatkis et al., (1953

16. Total lipids Serum Fring and Dunn (1970)

17. Triglycerides Serum Trindar, 1969

18. LDL Serum Friedwald et al., 1972

19. VLDL Serum Friedwald et al., 1972

20. HDL Serum Wybenga et al., 1970

21. Total phospholipids Liver tissue Zilversmit and Davis (1950)

22. ACP Lysosomal Fraction Walter and Schutt, 1974




80

23. Cathepsin D Lysosomal Fraction Sapolsky et al (1973)

24. MPO Serum Bradley et al .,1982

25. C-Reactive protein Serum Handson and Lindquist 1997

26. TNF Alpha Serum Thomson, 1994

27. Isocitrate dehydrogenase Mitochondrial fraction Bell and Baron (1960)

28. Succinate dehydrogenase Mitochondrial fraction Slater and Bonner (1952)

29. Malate Dehydrogenase Mitochondrial fraction Mehler et al 1948

30. Hexose Liver homogenate Niebes, 1972

31. Hexosamine Liver homogenate Elson and Morgon 1933

32. Sialic acid Liver homogenate Welmer et al 1952

33. Fucose Liver homogenate Dische and Shettles 1948.

34. Ca 2+ ATPase Liver homogenate Bonting 1970

35. Mg 2+ ATPase Liver homogenate Hjerten and Pan 1983

36. Na+K+ ATPase Liver homogenate Ohnishi 1982

37. SOD Liver, Kidney Kakkar,1984.

38. CAT Liver, Kidney Sinha 1972

39. GPX Liver, Kidney Rotruck et al (1973)

40. GR Liver, Kidney Mannervik and Carlberg,1985

41. GSH Liver, Kidney Ellman (1959)

42. GST Liver, Kidney Habig et al (1974)

43. LPO Liver, Kidney Okhawa et al (1979).

44. Vitamin C. Plasma Roe and Keuther (1943)

45. Vitamin E Plasma Baker and Frank (1980)




81

3.5.5. Estimation of Serum Total protein

Total protein in serum was estimated according to the method of Lowry

et al., 1951.

Principle

The aromatic amino acids such as tyrosine and tryptophan present in

protein reacted with Folin-Ciocalteu reagent to give a dark blue color. The

intensity of the color obtained was directly proportional to the amount of protein

present in the sample.

Reagents

1. Reagent A 2% Sodium carbonate in 0.1 N NaOH.; Reagent B 0.5%

Copper Sulphate in 1% Sodium Potassium tartarate; Reagent C- 50 ml of

Reagent A was mixed with 0.5 ml of Reagent B just before use.

2. Folin Ciocalteau Reagent Dilute 1:2 dilution prepared with distilled

water.

3. Stock standard- 100 mg Bovine serum albumin in 100 ml distilled water.

4. Working standard 10ml of stock Standard was diluted to 100 ml to get

working standard containing 0.1 mg/100ml.

Procedure

0.5 ml of serum was mixed with 0.5 ml of 10% TCA and centrifuged for

10 minutes. The precipitate was dissolved in 1 ml of 0.1 N NaOH. From this an

aliquot was taken and 4.5 ml of alkaline copper sulphate was added and allowed

to stand at room temperature for 10 minutes. 0.5 ml of Folin Ciocalteau reagent




82

was added and the blue color developed was read after 20 minutes at 640 nm. A

standard curve was obtained with standard bovine serum albumin and was used to

assay the plasma protein level. Values are expressed as g/dl.

3.5.6. Estimation of serum albumin

The serum albumin was estimated by the method given by Corcoran and

Durnan (1977) using albumin test kit (Span Diagnostics Ltd.). Proteins form a

purple coloured complex with cupric ions in alkaline solution. The intensity of the

purple color is proportional to the amount of protein present in the sample.

Reagents

1. Reagent I: Albumin reagent (Succinic acid - 37 mM/L; bromocresol green

- 0.15M M/L; Sodium hydroxide - 1 mM/L; buffer pH - 3.68).

2. Reagent II: Albumin standard (BSA - 4 g/dl).

Procedure

3.0 ml of albumin reagent (Reagent I) was added to all the three test tubes.

Thereafter, 0.03ml serum was added to the test and 0.03 ml Reagent II was added

for the standard .To the blank 0.03 ml of purified water was added. They were

then mixed well and incubated at room temperature for 1 min. The absorbance

was read at 630 nm. The values were expressed in g/dl.

3.5.7.Estimation of blood glucose

Glucose was estimated by the method of Sasaki et al., (1972).




83

Principle

Glucose is oxidized by glucose oxidase to gluconic acid and hydrogen

peroxide. In a subsequent peroxidase catalyzed reaction, p-hydroxy benzoate

4-amino anti-pyrine react with hydrogen peroxide to form red colored quinone

complex. Absorbance data measured at 610nm using Spectrophotometer are

directly proportional to glucose concentrations.

Reagents

1. Orthotoluidine-boric acid reagent: This reagent consisted of 2.5 g of

thiourea and 2.4 g of boric acid in 100 ml of a mixture of water, acetic acid

(AR) and orthotoluidine (distilled) in the ratio of 10:75:15 and kept

overnight in the cold.

2. Standard: 100 mg of glucose in 0.1% benzoic acid. 10 ml of the above

Procedure

To 0.1 ml of serum, 4 ml of orthotoluidine reagent was added and heated in

a boiling water bath for 15 min along with standard solutions containing 25-100

3.5.8.Estimation of liver lactate

The lactate content of liver was estimated by the method of Baker and

Summerson (1941).




84

Reagents

1. Trichloro acetic acid: 10%

2. Calcium hydroxide powder: 2%

3. Copper Sulphate solution: 4%

4. Coppper sulphate solution 20%

5. P-hydroxy diphenyl reagent: 1.5%

6. Concentrated sulphuric acid.

7. Standard lactate solution : 21.3 mg of lithium lactate was dissolved in 100

ml of distilled water containing 0.1 ml of concentrated sulphuric acid.

Procedure

De-proteination of 0.5 ml of liver homogenate was done with 4.5 ml of

10% trichloroacetic acid. To 2.0 ml of supernatant obtained after centrifugation.1

ml of 20% copper sulphate was added and diluted to 10 ml with water. Aliquots of

standard lactate and blank were also treated in a similar manner. 1g of powdered

calcium hydroxide was added to each tube with stirring for half an hour .After

centrifugation, to 1 ml of the aliquot was added 0.05 ml of 4% copper sulphate

and 6 ml of sulphuric acid and heated for 5 minutes in a boiling water bath and

cooled. Then0.1 ml of p-hydroxy, diphenyl reagent was added. The tubes were left

at room temperature for 30 minutes. Finally the tubes were placed in a boiling

water bath for exactly 90 seconds, removed and cooled in cold water. The colour

developed was read at 540 nm in UV spectrophotometer.




85

3.5.9. Estimation of liver pyruvate

The pyruvate content of the liver as estimated by the method of Friedmann

and Haugen (1943).

Reagents

1. 2,4-Di-nittrophenyl Hydrazine (DNPH) : 1.0 mM in 20 ml HCl

2. Trichloroacetic acid :10%

3. Sodium hydroxide: 1.5 N

4. Standard pyruvate: 1.5 mg sodium pyruvate was dissolved in 100ml of

water.

Procedure

1 ml of liver homogenate was deproteinised with 5 ml of 10%

trichloroacetic acid. To 2ml of supernatant obtained after centrifugation,1 ml of

DNPH reagent was added. After 5 minutes, 5 ml of 1.5 N sodium hydroxide was

added. Aliquotes of standard pyruvate and blank containing 1 ml of water were

also treated in the same manner. The colour developed was read at 520nm in UV

spectrophotometer. The values were expressed as µg/mg protein.

3.5.10. Estimation of serum glutamate oxaloacetate trasaminase (SGOT)

AST catalyse the transfer of amino group from L- -

ketoglutarate with the formation of oxaloacetate and glutamate. The oxaloacetate

formed is reduced to malate by malate dehydrogenase and NADH, which is brown

colored in alkaline medium. The rate of decrease in NADH concentratioin was




86

measured using a commercial kit method according to the procedure of Reitman

and Franknel,1957.

Reagents

1. Phosphate Buffer 0.1 M (pH 7.4)Solution A: 14.2 g of disodium hydrogen

phosphate in 1 litre of distilled water. Solution B: 136 g of potassium di-

hydrogen phosphate in 1 litre of water.Mix 420 ml of solution A and 80

ml of solution B for Phosphate buffer.

2. - ketoglutarate

in 100 ml of phosphate buffer.

3. 2,4 -Dinitro phenyl hydrazine- 20 mg of 2,4 DNPH in 100 ml of 1N HCl.

4. 0.4 N NaOH.

5. Stock- 22 mg of sodium pyruvate in 100 ml of deionised water.

6. Working (0.2mM/100ml) -10 ml of stock made to 100 ml with water.

Procedure

The buffered substrate of volume 0.5 ml was incubated at 37º C for 5

minutes. 0.1 ml of serum was added to the substrate and stirred well and incubated

at 37 º C for 60 minutes. Subsequently 0.5 ml of colour reagent DNPH was added,

stirred well and placed at room temperature for 20 minutes to which 5 ml of 0.4 N

NaOH was added and stirred well and placed at room temperature for 10 minutes.

Finally, the optical density of the test was read with distilled water using green

filter. Values were expressed as IU/L.




87

3.5.11.Estimation of serum glutamyl pyruvate transaminase (SGPT)(Reitman

and Franknel ., 1957)

ALT catalyses the transfer of amino group from L-alanine to keto-

glutarate with the formation of pyruvate and glutamate.Alanine aminotransferase

-ketoglutarate

forming glutamate and pyruvate. The pyruvate formed is reduced to lactate by

lactate dehydrogenase and NADH. The rate of decrease in NADH concentration

was measured according to the procedure of Reitman and Franknel.

Reagents

1. Phosphate Buffer 0.1 M (pH 7.4):Solution A: 14.2 g of disodium hydrogen

phosphate in 1 litre of distilled water.Solution B: 13.6 g of potassium

dihydrogen phosphate in 1 litre water.Mix 420 ml of solution A and 80 ml

of Solution B for Phosphate buffer.

2. Buffered Substrate: 1.78 g of alanine and 29.2 mg of ketoglutartae in

100ml of phosphate buffer.

3. 2, 4 dinitrophenyl hydrazine 20 mg of 2, 4 DNPH in 100 ml of 1 N HCl.

4. 0.4 N NaOH

5. Stock 22mg of sodium pyruvate in 100ml of deionised water.

6. Working Standard : (0.2 nM/100 ml) 10 ml of stock was made to 100 ml

with deionised water.

Procedure

A test tube with 0.5 ml buffered substrate was incubated at 37 º C for 5

minutes. The serum of volume 0.1 ml was added to the substrate and stirred well

and incubated at 37º C for 30 minutes, to which 0.5 ml of DNPH and sodium




88

hydroxide was added, stirred and kept at room temperature for 10 minutes. Finally

the OD was read with distilled water using green filter. The values was calculated

and expressed as IU/L.

3.5.12.Estimation of serum alkaline phosphatase (ALP) (King and King 1954)

Phosphatases are enzymes which catalyses the splitting of phosphoric acid

from certain monosphoric esters in which the substrate is hydrolysed with the

liberation of phenol and disodium phosphate. The liberated phenol reacts with

phosphobolybdate in Folin Ciocalteau reagent in alkaline medium and produces

blue colored complex calorimetrically read at 620 nm using red filter.

Reagents

1. Carbonate bicarbonate buffer (pH 10)

2. Solution A (0.2 N anhydrous sodium carbonate:)-10.5 g of sodium

carbonate in 500 ml water.

3. Solution B 8.4 g of sodium bicarbonate in 500 ml water. Mix 38.5 ml

of solution A with 11.5 ml of solution B added and made to 100 ml with

distilled water for carbonate bicarbonate buffer.

4. Phenol Stock standard (1mg/mg) 100 mg of phenol weighed and 1 ml

HCl was added and the volume was made to 100 ml with water.

5. Phenol working standard 0.5 ml stock was made to 100 ml with water.

6. Buffered substrate 250 mg of sodium phenyl phosphate was weighed.

7. and dissolved in 0.01 M bicarbonate buffer and made to 100ml with buffer.

8. 15 % sodium carbonate.

9. Folin Ciocalteau reagent.




89

Procedure

In a series of dry test tubes marked as S1 - S5 0.5 ml of working standard

phenol was pipette out corresponding to a concentration range of 2.5 12.5 µg

phenol. 3.5 ml of distilled water serves as blank. The volume in all the tubes was

made to 3.5 ml with distilled water. 1 ml Folin Ciocalteau was added to all the

tubes. To estimate the enzyme activity 2 ml of buffered substrate added to the tube

marked as C and T. Incubated at room temperature for 5 minutes. 0.1 ml of serum

added to T only. They were centrifuged for 10 minutes at 2000 rpm. 2 ml of

supernatant was taken. 1.5 ml of distilled water and 1.5 ml of 15 % Na2CO3 were

added. The intensity of blue color developed was read at 640 nm and the values

were expressed as IU/L.

3.5.13.Estimation of serum lactate dehydrogenase

The LDH was estimated by the Method of King (1965).

Reagents

1. Stock sodium pyruvate (1mg/ml) 126 mg of sodium pyruvate was

dissolved in 100ml of distilled water.

2. Working sodium Pyruvate Buffered substrate (100 g/ml) 1.0 ml of the

stock pyruvate solution was made up to 100ml with distilled water.

3. Glycine buffer (0.1M) 7.5 g of glycine and 5.8 g of NaOH was weighed,

dissolved and made up to 100 ml with distilled water (pH-10).

4. Buffered lithium lactate substrate - 4 g of Lithium was weighed and

dissolved in 75ml of NaOH and the volume was made to 200 ml with




90

buffer.Nicotinamide adenine dinucleotide (NAD) 10 mg of NAD was

weighed and dissolved in 2 ml of Buffered substrate.

5. 2,4- Dinitro phenyl hydrazine 99 mg of DNPH was dissolved in 5 ml of

HCl and made to 100 ml with distilled water

6. 0.4 N NaOH- 16 g of NaOH was dissolved in 1 litre of distilled water.

Procedure

Three tubes marked as blank, control and test were taken. 1 ml of buffered

lactate substrate and 1.3 ml of glycine buffer were taken in test and control tubes.

The tubes were pre-incubated at 37° C for 15 minutes. This is followed by the

addition of 2.6ml distilled water in blank and 0.2 ml of NAD is added to the tube

and then the tubes were incubated at room temperature for 5 minutes. Then 0.1 ml

of enzyme added to test and control tubes and mixed well. The red colour

developed after addition of 7 ml of 0.4 N NaOH. The intensity of red colour

develop was read at 490 nm. The activity was expressed as IU/L.

3.5.14. Estimation of serum Gamma Glutamyl Transpeptidase

- -glutamyl -3-

carboxy- - -L-glutamyl glycine

and 2-nitro-5-aminobenzoic acid. The rate of 2-nitro-5 amino benzoic acid

formation was measured using commercial kit described by the method of Rosalki

and Rau (1972).




91

Reagents

1. 0.1 M Tris HCl, pH 8.5.

2. Substrate- 30.37 mg of L-gamma Glutamyl P- Nitroanilide was dissolved

in 10 ml distilled water by heating at 50-60° C.

3. Glycyl Glycine 13.2 mg/ml.

4. P- Nitroaniline 10 mg/ 100ml.

Procedure

The mixture under incubation contains 0.5 ml of substrate,1 ml of Tris

HCl, 2.2 ml of glycyl glycine, 0.2 ml of serum and the total volume was made up

to 4 ml with distilled water. After incubation for 30 minutes at 37° C the samples

heated at 100° C for 5 minutes and centrifuged. The amount of p-nitro aniline in

the supernatant was measured at 410nm. The values were expressed in IU/L.

3.5.15.Estimation of Serum bilirubin

Bilirubin and the diazo reagent form an azobilirubin complex, which can

be measured colorimetrically. The color of the azobilirubin varies with pH.

(Malloy and Evelyn, 1937).

Reagents

1. Absolute methanol

2. Hydrochloric acid, 1.5% v/v with water

3. Diazo-reagent - prepared by adding 0.3 ml of solution B to l0 ml of

solution A .Solution A : 1 g of sulphanilic acid was dissolved in 15 ml of

conc. HCI and made up to 1 liter with water.




92

4. Solution B : 0.5 g of sodium nitrite was dissolved in distilled water and

made up to 1 ml.

5. Standard solution of bilirubin. Solution containing 10 mg bilirubin per 100

ml chloroform was prepared.

Procedure

Two test tubes were taken and marked as blank and unknown. 0.2 ml of

serum and 1. 8 ml of distilled water were added to unknown and 2 ml distilled

water marked as blank. To the unknown 0.5 ml of diazo-reagent was added

and to the blank 0.5 ml of 1.5% HC1. Finally, 2.5 ml of methanol was added

to all the tubes. Allowed to stand for 30 min and the optical density was read

in a calorimeter set at 540 nm. The values were expressed as mg/dl.

3.5.16.Estimation of serum urea

Urea reacts with o-phthaldehyde in acid medium forming a colored

complex that can be measured spectrometrically using a commercial kit method

(Kaplan, 1984).

Reagents

1. O-phthaldehyde 4.8 mmol/L.

2. Borate- 87 mmol/L.

3. Standard Urea-50 mg/ dl.




93

Procedure

To 25 µl of serum and standard in a test tube, 1 ml of the o-pthaldehyde

and 1 ml borate reagent added. The contents were mixed well and incubated for

15 minutes at 37º C. The absorbance of the sample and standard was measured at

510 nm against distilled water as blank. The values were expressed as mg/dl.

3.5.17. Estimation of serum creatinine

The assay is based on reaction where creatinine reacts with sodium picrate

to form red colored complex using a commercial kit method (Murray, 1984).

Reagents

1. Picric acid - 17.5 m mol/L.

2. Alkaline reagent sodium hydroxide 0.29 m mol/L.

3. Standard Creatinine- 2 mg/dl.

Procedure

To 100 µl of serum and standard 1 ml of picric acid and alkaline sodium

hydroxide was added. The contents were mixed well and the absorbance was read

at 492 nm against distilled water as blank. The values were expressed as mg/dl.

3.5.18. Estimation of liver glycogen

The liver glycogen was estimated using the anthrone reagent method

(Seifter et al., 1950).




94

Principle

Glycogen in liver and skeletal muscle is liberated when heated with strong

alkali. Released glycogen precipitates by the addition of ethanol and sodium

sulphate (co-precipitant) to give a quantitative yield of glycogen. The

polysaccharide was then hydrolysed in acid and the released glucose was

estimated.

Reagents

1. 60% Potassium hydroxide

2. 30% KOH

3. Ethanol

4. Sodium hydroxide

5. Saturated sodium sulphate

6. Reagents for glucose estimation.

Procedure

1g of liver was weighed into a calibrated centrifuge tube that contained

2ml of KOH (30%) and heated in a boiling water bath for 20 minutes with

occasional shaking. The tubes were cooled by placing on ice and 0.2ml of

saturated Na2SO4 was added. Glycogen was precipitated by adding 5ml of ethanol

(95% v/v). The precipitate was separated by centrifugation and dissolved in 5ml

of water with gentle warming, and made to 10 ml with distilled water.

1ml sample of the glycogen solution was pipetted out into a test tube, 1ml

of HCl (1.2mol/L) was added and the test tube was heated in a boiling water bath




95

for 2hours. At the end of this period, 1 drop of phenol red indicator was added and

neutralized carefully with NaOH (0.5mol/L) till the indicator changes from pink

through orange to a yellow color. Finally, it was diluted to 5ml with distilled water

and preceded as for the estimation of glucose. Duplicates were maintained.The

values were expressed as mg/g tissue.

3.5.19.Estimation of serum Cholesterol

Serum Cholesterol was estimated according to the method of Zlatkis et al.,

(1953). Cholesterol is measured enzymatically in serum or plasma in a series of

coupled reactions that hydrolyze cholesteryl esters and oxidize the 3-OH group of

cholesterol. One of the reactions by product, H2O2 is measured quantitatively in a

peroxidase catalyzed reaction that produces a color. Absorbance is measured at

560 nm. The color intensity is proportional to cholesterol concentration.

Reagents

1. Ferric Chloride - 0.5 % solution of Fecl3.6 H20 in acetic acid.

2. Sulphuric acid.

3. StandardStock: Cholesterol (100 mg in 100 ml acetic acid).

4. Working Standard: The stock standard was diluted to 1: 25 with ferric

chloride- acetic acid reagent.

5. Acetone-ethanol reagent (1:1).

Procedure

0.1 ml of serum added to 10 ml of ferric chloride acetic acid reagent in a

stoppered centrifuge tube. It was mixed and allowed to stand for 10-15 minutes




96

for the protein to flocculate. 5 ml of clear supernatant fluid was transferred to a

stopper centrifuge. For standard 0.1 ml of physiological saline was mixed with 10

ml of cholesterol standard and 5 ml of ferric chloride-acetic acid reagent. 3 ml of

sulphuric acid was added to all the three tubes, stopper and mixed well. It was

then allowed to stand for 20-30 minutes. The unknown and the standard were read

against blank at 560nm in a spectrometer. The values were expressed as mg/dl.

3.5.20. Estimation of tissue Phospholipids

Tissue phospholipid was estimated by Zilversmit and Davis (1972).The

organic phosphorous is converted in to inorganic phosphorous which reacts with

ammonium molybdate to form phosphomolybdic acid which on reduction and

reaction with ANSA forms a stable blue colour. This colour can be measured at

660 nm.

Reagents

1. 5 N H2SO4

2. 5 % ammonium molybdate

3. Amino -2- napthol-4-Sulphonic acid (ANSA) 0.2 g ANSA was mixed

with 1.2 g of sodium bisulphate and 1.2 g sodium sulphate. 0.25 g was

taken from that and added with 10 ml distilled water. (Containing 8 g

phospholipid/ ml)

4. Standard phosphorous solution 35.1 mg of potassium dihydrogen

phosphate was dissolved in water. To this1 ml of 10 N H2SO4 added and

made to 100 ml with distilled water. 10 ml of this was diluted to 100 ml to

prepare working standard.




97

Procedure

An aliquot of the lipid extract was pipetted in to Kjeldhal flask and

evaporated to dryness. 1.0 ml of 5N H2SO4 was added and digested in a digestion

rack till it becomes light brown.To which 2-3 drops of concentrated nitric acid

was added and continued till it became colorless. The flask was cooled, 1ml

distilled water added and heated in boiling water bath for 5 minutes. Then 1 ml of

2.5 % ammonium molybdate and 0.1 ml ANSA added. The volume was made to

10 ml with distilled water and the absorbance was read at 660 nm within 10

minutes. The values were expressed as mg/ dl.

3.5.21.Estimation of serum Total Lipids

Total Lipids in serum was estimated according to the method of Fring and

Dunn (1970).

Reagents

1. Concentrated sulphuric acid

2. Vanillin (0.6 %)

3. Phospho-Vanillin reagent: 200 ml of 0.6 % vanillin in 800 ml of

concentrated ortho-phosphoric acid.

4. Standard Olive oil- Stock standard : 1 g in ethanol

5. Working Standard : 400 mg in ethanol.

Procedure

Three test tubes were labelled as test, blank and standard. 0.1 ml of serum,

0.1 ml of distilled water and 0.1 ml of working standard were taken respectively in




98

all the test tubes. 2 ml of concentrated sulphuric acid was added to each tube and

they were heated in boiling water bath for 10 min. It was cooled and then 0.1 ml

of the digested mixture was pipette from each tube and transferred them into

another tubes. 0.1 ml of phosphor -Vanillin reagent was added to all the tubes and

incubated at 37° C for 15 min. They are read at 540nm in colorimeter. The values

were expressed as mg/dl.

3.5.22.Estimation of serum HDL-cholesterol (Wybenga et al., 1970)

Principle

In the presence of precipitating reagent, all lipoproteins of the serum,

except HDL fraction, were precipitated. After centrifugation, the precipitate was

discarded and the HDL-cholesterol content of the supernatant was determined.

Reagents

1. Working reagent

2. Precipitating reagent

3. Standard (200mg/dl).

Procedure

In the assay of HDL-

precipitating reagent and kept at room temperature for 10min. Later, it was

centrifuged at 3000 rpm for 10 min. The clear supernatant was used for HDL-

incubated with 1ml of the reagent for 5 min at 37º C and the absorbance at 510nm

was measured against the reagent blank. For reagent blank, 10µl of distilled water




99

was added to 1ml of the reagent. The amount of HDL- cholesterol in the samples

was expressed as mg/dl.

3.5.23.Estimation of Serum VLDL- and LDL-cholesterol ( Friedwald et al.,

1972)

These are calculated using the formula

VLDL cholesterol = TG/5

LDL cholesterol = Total cholesterol (HDL cholesterol +VLDL

cholesterol). The values were expressed as mg/dl of plasma.

3.5.24. Estimation of Serum triglycerides (Trindar, 1969)

Triglycerides are measured enzymatically in serum or plasma using a

series of coupled reactions in which triglycerides are hydrolyzed to produce

glycerol. Glycerol is then oxidized using glycerol oxidase, and H2O2, one of the

reaction products, is measured at 500 nm

Reagents

1. Working reagent

2. Standard (200mg/dl).

Procedure

of serum and 1ml of working

standard were mixed and kept at room temperature for 15min and the absorbance

water was added to 1ml of working reagent. Concentration of triglycerides in

serum samples were expressed as mg/dl.




100

3.5.25.Estimation of acid phosphatase

The assay of acid phosphatase was done according to the method of Walter

and Schutt, 1974.

Reagents

1. Citrate buffer- 1M, pH 4.85

2. Tartarate -0.2 M

3. NaOH-0.1 N

4. Substrate- -nitrophenyl phosphate-0.4 %.

5. Standard- -nitrophenol 0.6 mM.

Procedure

Liver lysosomal enzymes were separated according to the method of

Wattiaux et al., (1978). Fresh liver is homogenized in 0.25 M sucrose solution.

The homogenate was filtered and centrifuged at 3000 rpm for 10 minutes in a

refrigerated high speed centrifuge. The pellets were removed and homogenized

again by centrifugation as done above. The supernatant was combined and

centrifuged at 12000 rpm for 20minutes. The lysosomal fraction was suspended in

1.15 % KCl. The homogenate was used for enzyme analysis.

-nitrophenyl phosphate substrate, 0.5 ml of 1 M citrate

buffer and 0.2 ml 0f 0.2 M tartarate were added to the tubes marked as test and

blank. The tubes were incubated at 37 º C for 5 minutes. The reaction is initiated

by the addition of 0.1 ml of the sample and distilled cwater to the test and blank

tubes and the time was noted. After 30 minutes of incubation at 37ºC the reaction




101

was arrested by the addition of 3.8 ml of 0.1 N NaOH. The values were measured

at 415 nm in spectrometer against blank and values were expressed as µmol of

phenol liberated/min/mg of protein.

3.5.26.Assay of cathepsin D

The cathepsin D level in lysosomal fraction was estimated according to the

method of Sapolsky et al (1973).

Reagents

1. Buffer : Sodium Formate-0.2M (pH 3.5)

2. Substrate: Hemoglobin 1 % in sodium formate buffer

3. TCA 10 %

4. Sodium Carbonate : 4 % in 0.1 N NaOH

5. Standard Tyrosine: 10 mg/ml in dilute HCl.

Procedure

The incubation mixture containing the following in a final volume of 2ml,

0.8 ml buffer 1 ml substrate and 0.2 ml enzyme homogenate. Tubes were

incubated at 37 C for 2 hrs. The enzyme reaction was arrested by the addition of

2 ml 10% TCA. The control tubes receive enzymes after arresting the reaction.

After 15 minutes the tubes were centrifuged at 1000 rpm for 15 minutes. To the

supernatant 2.5 ml Na2Co3 in NaOH added and the content were immediately

mixed well. Standards containings aliquots of tyrosine and blank containing water

were also treated in the same manner. The blue colour developed was read at 640

nm. The units were expressed as µmol of tyrosine liberated/min/mg of protein.




102

3.5.27.Estimation of Inflammatory markers

3.5.27.(i)Assay of Myeloperoxidase (MPO)

MPO oxidizes dianisidine hydrochloride and the rate of oxidation was

monitors as change in absorbance at 460 nm (Bradley et al., 1982).

Reagents

1. HTAB Buffer- 0.5 % (Hexa decyltrimethyl ammonium bromide).

2. Phosphate buffer (pH 6.0).

3. O-dianisidine hydrochloride (1.5mM in 0.0005 in H2O2).

Procedure

The liver tissues were homogenized in phosphate buffer 20 mmol/ l, pH

7.4, centrifuged at 10,000 rpm for 10 min at 4°C, and the resulting pellet

resuspended in phosphate buffer 50 mmol/L containing 0.5% hexa decyl trimethyl

ammonium bromide (Sigma). The sample was then centrifuged at 10,000 g for 5

min at 4°C, and the supernatant was used for the MPO assay (Szekanecz and

Koch, 2004).

To 1 ml of homogenate equal volume of HTAB added, mixed and

centrifuged at 4° C for 15 minutes at 3000 rpm. To 0.5 ml supernatant 3.5 ml

phosphate buffer was added followed by 1.5 ml of O-dianizidine hydrochloride

added. The absorbance was read at 450 nm in Thermo Scientific spectrometer at

The results were expressed in terms of

units (nmol of H2O2 liberated/ min/mg protein).




103

3.5.27.(ii).Estimation of C- reactive protein

Measurement of C-reactive protein was done using commercial kit

according to the method of Hanson and Lindquist (1997).

Principle

CRP turbilatex is a quantitative turbidimetric test where the latex particles

coated with specific anti-human. CRP is agglutinated when mixed with samples

containing CRP. The agglutination causes an absorbance change, depend upon the

CRP content of the sample that can be quantified from a calibrator of known CRP

concentration.

Reagents

1. Diluent (R1) - Tris buffer 20 mmol/L, pH 8.2; sodium azide 0.95 g/L.

2. Latex (R2) Latex particles coated with Ig G anti-human CRP, pH

10.sodium azide 0.95g/L.

3. CRP calibrator Reconstitute with 1 ml of distilled water (60mg/dl).

4. Working Reagent- Mix 1 ml latex reagent (R2) with 9 ml diluents (R1).

Procedure

To 50 µl of the working reagent add 5µl of the serum sample. Mix the

content well and read the absorbance in a semi-automated biochemical analyzer at

540 nm after 2 min against distilled water. The values were expressed as mg/dl.




104

3.5.27.(iii)Estimation of tumour necrosis factor alpha (TNF-

The TNF-

is specific for measuring TNF- , 1994).A monoclonal

antibody specific for TNF Alpha was coated onto the wells of the micro titer

strips provided. During the first incubation, TNF-

standard and a monoclonal anti TNF-á antibody conjugated to biotin were

simultaneously incubated. A coloured product was formed in proportion to the

amount of TNF- á present in the sample. The reaction was terminated addition of

acid and absorbance was measured at 450 nm.

Requirements

1. Capture antibody Pre-titrated, purified anti-rat TNF-

2. Sample-Serum.

3. Buffers - Wash Buffer: 1 x PBS, 0.05% Tween-20

4. 96 wells with pre-coated anti-rat TNF-alpha.

5. Elisa reader Measuring absorbance at 450-550 nm.

6. Stop solution- 2 N H2SO4.

All other reagents which are required for the experiments were prepared

according to the instruction.

Procedure

Initially 50µl of capture anti-body was added to all the 96 wells. To this 50

µl of standard and serum were added to the respective wells in duplicate. The

plate was then covered and incubated at room temperature for 2 hours. At the end

of incubation period, plates was washed three times with wash buffer, made it dry




105

and 100 µl of biotinylated anti-body reagent was added to each well. Again the

plate is covered and incubated at room temperature for 1 hour. After incubation

washing procedure was repeated for 3 times as before followed by the addition of

streptavidin-HRP reagent to all the wells covered and incubated the plates at room

temperature for 30 min. washing was carried out and added 100 µl of TMB

substrate to each wells. The blue colour was allowed to develop under the dark for

30 minutes. Then the reaction was arrested by adding the stop solution to each

well. The colour developed was measured on a plate reader at 450 nm. The results

were expressed as ng/ml.

3.5.28. Estimstion of TCA cycle enzymes

3.5.28(i) Assay of Isocitrate dehydrogenase

The enzyme activity was assayed according to the method of Bell and

Baron (1960).

Reagents

1. Tris HCl: 50mM, pH 7.4 containing 0.25 M Sucrose and 1mM EDTA.

2. Tris-HCl buffer: 0. 1 M, pH 7.5

3. Substrate : 0. 1M tri-sodium DL-Isocitrate in 0.9% saline

4. Manganous chloride: 0. 015 M in 0.9% saline

5. NADP : 0. 001 M in 0.9% saline

6. EDTA: 5%

7. Sodium hydroxide: 0. 4 N

8. 2, 4 dinitrophenyl hydrazine (DNPH) : 0. 001 M in 1N HCl .

9. -ketoglutarate in 50ml of buffer.




106

Procedure

Immediately after sacrifice, the liver was removed and all the blood vessels

and connective tissues were trimmed off. Wash the tissue free of blood in ice-cold

sucrose, lightly blot and place in a beaker to weigh. Cut the liver in to small

fragments and homogenize in buffer containing 0.25 M Sucrose and 1mM EDTA.

Centrifuge the suspension in a refrigerated centrifuge.

The homogenate was centrifuged at 1000g for 10 min; the supernatant was

transferred in to test tubes. The pellet was dissolved in sucrose buffer and

centrifuged for 10 min at 1000g. The supernatants were pooled and centrifuged

for 10 min at 10000g, the pellet collected represents mitochondrial fraction. Each

fraction was resuspended in sucrose and the washings combined with the

supernatants. This has the advantage of producing purer fractions. Carefully the

mitochondrial pellet was resuspended in about 2ml of sucrose and used as the

enzyme source and store on ice until required (Susin, 2000).

0.4ml buffer was taken in a test tube and 0.2ml of substrate, 0.3ml of

manganous chloride and 0.2ml of the mitochondrial suspension were added. A

control tube was also prepared simultaneously, 0.2ml of co-enzyme solution was

added to the test tube and 0.2ml of saline was added to the control tubes. After

mixing well, both the tubes were incubated for 60 min, 1.0 ml of DNPH was

added to both the tubes, followed by 0.5 ml of EDTA. The tubes were kept at

room temperature for 20 min and 10 ml of 0.4 N NaOH was added to the tubes. A

blank was run simultaneously. The colour intensity was measured at 390 nm in a

Shimadzu- -ketoglutarate.




107

-ketoglutarate

liberated per mg protein per hour.

3.5.28 (ii).Assay of succinate dehydrogenase

This enzyme activity was estimated accordingly to the method of Slater

and Bonner (1952). The rate of reduction of potassium ferric cyanide was

measured in the presence of sufficient potassium cyanide to inhibit cytochrome

oxidase by following the rate of decrease in the optical density at 420 nm.

Reagents

1. Phosphate buffer: 0.3 M pH 7.6

2. Sodium salt of EDTA : 0.03 M solution.

3. Potassium cyanide : 0.03 M solution.

4. Sodium Succinate : 0.4 m solution.

5. Bovine serum albumin : 3% solution.

6. Potassium ferric cyanide: 0.075 M solution.

Procedure

In a spectrophotometric cuvette, 1.0 ml of phosphate buffer, 0.1 ml of

EDTA, 0.1 ml of bovine serum albumin, 0.3 ml of sodium succinate, 0.2 ml of

potassium ferri cyanide and 0.1 ml of potassium cyanide were added and the total

volume was made up to 2.8 ml with double distilled water. The reaction was

started by the addition of 0.2 ml of mitochondrial suspension. Changes in optical

density at 420 nm were recorded in a Shimadzu- UV spectrophotometer at an




108

interval of 15 s for 5 min. The activity of succinate dehydrogenase is expressed as

per n moles of succinate oxidized/ minute /mg protein.

3.5.28.(iii)Assay of malate dehydrogenase

This enzyme activity was assayed by the method of Mehler et al 1948. The

activity determination is based on the measurement of the rate of oxidation of

NADH in the presence of the enzyme and excess of oxaloacetate.

Reagents

1. Tris HCl : 0.25M, pH 7.4

2. NADH: 0.015 M

3. Oxaloacetate : 0.0076 M, pH 7.4.

Procedure

To 0.3 ml of buffer 0.1 ml of NADH and 0.1 ml of oxaloacetate were

added and the total volume was made to 2.9 ml with water. The reaction was

started by adding 0.1 ml of mitochondrial suspension. The change in optical

density was measured at 340 nm in a Shimadzu- UV spectrophotometer at

intervals of 15 seconds for 5 min. The activity of malate dehydrogenase was

expressed as micromoles of NADH oxidized/minute/mg protein.

3.5.29.ESTIMATION OF GLYCOPROTEIN COMPONENTS

3.5.29 (i).Estimation of total hexoses

Total hexoses in tissues were estimated by the method of Niebes (1972).




109

Reagents

1. Orcinol sulphuric acid mixture: 1.6g of orcinol was dissolved in 100ml

of water. 1.0 ml of this was mixed with 7.5ml H2SO4: H2O mixture (3:2

v/v). This was prepared fresh before use.

2. 2. 5mg of galactose and 5.0mg of mannose were dissolved in 100ml of

water. This has a concentration of 100g/ml.

Procedure

0.2ml of the plasma or homogenate was mixed with 8.5ml of orcinol

H2SO4. The tubes were then heated at 80° C for 15 min, cooled and read at 540nm

after 20 min. Standard and blank containing 0.2ml of 0.2 N H2SO4 were also

processed similarly. Total hexose content was expressed as mg/dl of plasma or

mg/100g of tissue.

3.5.29 (ii). Estimation of hexosamine

Hexosamine in the tissues were determined by the method of Elson and

Morgon (1933).

Reagents

1. Ethanol: 95%

2. Hydrochloric acid: 3N

3. Sodium hydroxide: 3N

4. Acetyl acetone reagent: 1ml of acetyl acetone in 50ml of 0.5N sodium

carbonate, freshly prepared.




110

5. Ehrlich reagent: 0.8g of p-dimethyl amino benzaldehyde (recrystallized as

the hydrochloride) dissolved in 30ml of methanol and 30ml of con. HCl.

6. Glucosamine standard: 0.05mg/ml of free glucosamine in water.

Procedure

To 0.1ml of plasma or homogenate in a test tube 5ml of 95% ethanol was

added and mixed well, centrifuged for 15 min, decanted, and the precipitate was

suspended in 3ml of 95% ethanol, centrifuged and decanted. To the precipitated

protein, 2ml of 3N HCl was added and hydrolysed in a boiling water bath for 4

hours. The hydrolysate was neutralised with 3N NaOH. 1ml of the acetyl acetone

was added to 1ml of the aliquot, 1ml of the water (blank) and 1ml of standard. The

tubes were capped with marbles to prevent evaporation and placed in a boiling

water bath for 15 min. The tubes were cooled under tap water. 5ml of 95% ethanol

was added and mixed well. To these tubes 1ml of Ehrlich reagent was added and

mixed well. This was diluted to 10ml with 95% ethanol. Absorbance was

measured at 530nm after 30 min. Hexosamine content was expressed as mg/dl of

plasma or mg/100g of tissue.

3.5.29.(iii).Estimation of Sialic acid

Sialic acid in the tissues was estimated by the method of Welmer et al.

(1952).




111

Reagents

1. TCA: 5%

2. Acid mixture: 90ml of glacial acetic acid and 10ml of concentrated

sulphuric acid.

3. Diphenylamine reagent: 1g of diphenylamine recrystallized from ethanol

was dissolved in 100ml of mixture.

4. Sialic acid standard: 0.2mg/ml.

Procedure

4.8ml of 5% TCA was added slowly to 0.2ml of plasma or homogenate,

and 0.2ml of orosomucoid standard in a separate tube. The test tube was placed in

a boiling water bath for exactly 15 min with a glass marble to prevent evaporation;

the tubes were cooled by immersion in water and filtered. 2ml of clear filtrate in

each of tubes was pipette out and 4ml of DPA reagent was added into one of each

pair of tubes and 4ml of acid-mixture containing without DPA into another. The

reagent blank was prepared by adding 2ml of 5% TCA and 4ml of DPA reagent.

The tubes were mixed well, capped with a glass marble and immersed in a boiling

water bath for exactly 30 min. The tubes were cooled in water and the absorbance

was determined at 530 nm with a reagent blank set at zero. Sialic acid content

was expressed as mg/dl of plasma or mg/100g of tissue.

3.5.29.(iv).Estimation of Fucose

Fucose in the tissues was estimated by the method of Dische and Shettles

(1948).




112

Reagents

1. Sulphuric acid reagent: Con. H2SO4 and distilled H2O were mixed in the

ratio of 6:1.

2. Cysteine hydrochloride reagent: 3% cysteine hydrochloride in water 0.1N

NaOH.

Procedure

To 2.2ml of plasma or homogenate, 4.8ml of sulphuric acid reagent was

added and heated in a boiling water bath for 3 min. The sample was cooled and

0.1ml of cysteine hydrochloride reagent was added, 0.5ml of 0.1N NaOH was also

treated in the same way for blank, after 25 min the optical density was measured

at 393 and 430nm. Fucose content was expressed as mg/dL of plasma or mg/100g

of tissue.

3.5.30. Estimation of Membrane bound phosphatases on liver homogenate

3.5.30 (i). Assay of sodium potassium (na+-k+) ATPase

Na+-K + ATPase was assayed by the method of Bonting (1970). The

incubation mixture contained 1.0 ml of Tris-HCl buffer (90 mM, pH 7.5), 0.2 ml

each of 50 mM magnesium sulphate, 50 mM potassium chloride, 600 mM sodium

chloride, 1 mM EDTA, 40 mM ATP and the homogenate. The mixture was

incubated at 37oC for 15 minutes. The reaction was arrested by the addition of 1.0

ml of 10% TCA, mixed well and centrifuged. The phosphorus content of the

supernatant was estimated according to Fiske and Subbarow (1925) method. The

enzyme activity was expressed as µ moles of Pi liberated/min/mg protein.




113

3.5.30.(ii).Assay of calcium (ca2+) ATPase

The activity of Ca2+-ATPase was assayed according to the method of

Hjerten and Pan (1983). The incubation mixture contained 0.1 ml each of 125 mM

Tris-HCl buffer (pH8.0), 50 mM calcium chloride, 10 mM ATP and homogenate.

After incubation at 37oC for 15 minutes, the reaction was arrested by the addition

of 1.0 ml TCA. The amount of phosphorus liberated was estimated according to

the method of Fiske and Subbarow (1925). The enzyme activity was expressed as

µ moles of Pi liberated/min/mg protein.

3.5.30.(iii).Assay of Mg2+-ATPase

The activity of Mg2+-ATPase was assayed according to the method of

Ohnishi et al (1982). The incubation mixture contained 0.1 ml each of 375 mM

Tris-HCl buffer (pH 7.6), 25 mM magnesium chloride, 10 mM ATP and the

homogenate. The reaction mixture was incubated at 37oC for 15 minutes. The

reaction was arrested by the addition of 1.0 ml 10% TCA. The liberated

phosphorus was estimated according to the method of Fiske and Subbarow (1925).

The enzyme activity was expressed as µmoles of Pi liberated/minute/mg of

protein under incubation conditions.

3.5.30(iv).Estimation of phosphorus (Fiske and Subbarow, 1925)

Into a series of test tubes pipette out 1.0 5.0 ml of working standard

solution corresponding to µg values 8-40. 1.0 ml of the sample solution was taken

in separate test tubes. The volume in all the tubes was made up to 8.6 ml with

distilled water. Set up a blank with 8.6 ml of distilled water. Added 1.0 ml of

2.5% ammonium molybdate and 0.4 ml of ANSA to all the tubes. Mixed well and




114

allowed to stand for 10 minutes. The blue color developed was read at 660 nm in a

spectrophotometer.

3.5.31.Estimation of enzymatic, non-enzymatic antioxidants and lipid

peroxidation

3.5.31 (i) Estimation of SOD

The activity of SOD was estimated by Kakkar et al., (1984).

Principle

The assay is based on the inhibition of the formation of NADH- phenazine

methosulphate-nitroblue tetrazolium formazan. The reaction is initiated by the

addition of NADH. After incubation for 90 seconds, the reaction is stopped by

adding glacial acetic acid. The colour developed at the end of the reaction is

extracted into n-butanol layer and measured at 520 nm in spectrophotometer.

Reagents

1. 0.025 M sodium pyrophosphate Buffer (pH 8.3)

2. 186 m PMS

3. 300 m NBT

4. 780 m NADH

5. Glacial acetic acid

6. N-butanol

7. Chloroform

8. Ethanol.




115

Procedure

0.5 ml of liver homogenate was diluted to 1 ml with water. Then 2.5 ml of

ethanol and 1.5ml of chloroform (all reagents chilled) were added. This mixture

was shaken for 1 minute at 4oC and centrifuged. The enzyme activity in

supernatant was determined. The assay mixture contained 1.2 ml of 0.025 M

sodium pyrophosphate buffer, 0.1 ml of 186 M PMS, 0.3 ml of 300 M NBT and

0.3 ml of 780 m NADH, enzyme preparation and water in to total volume of 3.0

ml. Reaction was started by the addition of NADH. After incubation at 30° C for

90 seconds. The reaction was stopped by the addition of 1 ml of glacial acetic

acid. The reaction mixture stirred vigorously and shaken with 4.0 ml of n-butanol.

The Intensity of the chromogen in the butanol layer was measured at 560nm

against butanol blank. A system devoid of enzyme acts as blank control. One unit

of the enzyme activity is defined as the enzyme reaction which gave 50%

inhibition of NBY reduction in one minute under the assay condition. Values are

expressed as unit/mg protein for tissue and unit/ml for serum.

3.5.31.(ii)Estimation of Catalase

The catalase activity (CAT) was assayed by the method of Sinha (1972).

Principle

Dichromate in acetic acid was reduced to chromic acetate,

whenheated in presence of hydrogen peroxide with the formation of perchromic

acid as an unstable intermediate. The chromic acetate formed was measured at

590 nm. Catalase was allowed to split H2O2 for different periods of time. The

reaction was stopped at different time intervals by the addition of dichromate




116

acetic acid mixture and the remaining H2O2 was determined by measuring

chromic acetate colorimetrically after heating the reaction mixture.

Reagents

1. 0.01 M phosphate Buffer (pH 7).

2.H2O2 (2M)

3. 5% Potassium Dichromate

4. Dichromate- Acetic acid Reagent: 5% Potassium Dichromate and glacial

aceticacid were mixed together (1:3).

5. 2 M/ml Standard H2O2.

Procedure

To 1 ml of Phosphate Buffer 0.1 ml homogenate and 0.4 ml Hydrogen

peroxide were added. The reaction was stopped at 30 seconds by addition of 2 ml

dichromatic acetic acid reagent. The tubes were kept in boiling water bath for 10

minutes, cooled and read at 620 nm. A system devoid of enzyme serves as control.

Graded amounts of hydrogen peroxide ranging from 20-100 moles were used as

standard and processed along with a blank containing dichromatic reagent. Values

were expressed as nmoles of hydrogen peroxide decomposed/ min/ mg protein for

tissues as and n mole of H2O2 decomposed /min/ml of serum.

3.5.31 (iii).Estimation of GPx

Glutathione (GPx) was assayed by the method of Rotruck et al (1973). A

known amount of enzyme preparation was allowed to react with H2O2 in presence




117

of GSH for a specific time period. The GSH content remaining after the reaction

was measured at 530 nm.

Reagents

1. 0.4 M Phosphate Buffer (pH 7)

2. 10 mM Sodium Azide Solution

3. 10% TCA

4. 0.4 mM EDTA

5. 0.2 mM H2O2

6. 2mM Standard Glutathione

7. 0.11 mM DTNB.

Procedure

To 0.2 ml of buffer, 0.2ml of EDTA 0.1 ml sodium azide, 0.2 ml of liver

homogenate were added and mixed well. To the mixture 0.2 ml glutathione,

followed by 0.1 ml H2O2 were added, mixed and incubated at 37° C for 10

minutes along with control tube containing all reagents but no enzyme. After 10

minutes, the reaction was arrested by the addition of 0.4 ml of 10% TCA. The

tubes were centrifuged and the supernatant was estimated for Glutathione content

using Ellman reagent. The activity of GPx was expressed as n mol of GSH

consumed /min/mg protein for tissue and n mol GSH consumed/min/ml for serum.

3.5.31 (iv).Estimation of Glutathione reductase (GR)

The GR estimation was done according to the method of Mannervik and

Carlberg, 1985.




118

Reagents

1. Tris-HC1 : I M.

2. Ethyline diamine tetra acetic acid (EDTA ) : 5 mM, pH 8.

3. Glutathione oxidase (GSSG), 20.421 mg 1 ml.

4. Reduced nicotinamide adenine dinucleotide phosphate ( NADPH ) : 2 mM.

Procedure

50 µl of Tris - HCI - EDTA, 10µl of serum or 1ml tissue homogenate

and 790 µ1 of distilled water were mixed together, and incubated at 37ºC for 10

min. To this mixture 100 µl of GSSG added, and incubated for a further period of

10 min and then 50µl of NADPH was added and absorbance was read at 340 nm.

The values were expresses as U/mg of protein.

3.5.31.(v).Estimation of Glutathione- S-transferase (GST)

The activity of GST was assayed by the method Habig et al (1974) which

was measured by following the increase in absorbance at 340 nm using 1-chloro

2,4 dinitrobenzene (CDNB) as the substrate.

Reagents

1. 0.3 M phosphate Buffer (pH 6.5)

2. 30 mM GSH

3. 30 mM CDNB in 95% ethanol.




119

Procedure

The following solutions were taken in a quartz cuvette in the given

proportion. 1 ml phosphate buffer, 0.1 ml CDNB, 0.1 ml liver/ kidney

homogenate. The volume was adjusted to 2.9 ml with distilled water. The reaction

mixture was pre-incubated at 37 C for 5 minutes. The reaction was started by the

addition of 0.1ml 30mM Glutathione. The absorbance was at 340 nm after

5minutes. The reaction mixture without enzyme was blank. Values were

expressed as g of CDNB-GSH conjugate formed/min/mg protein.

3.5.31(vi).Estimation of Glutathione

Reduced glutathione (GSH) was determined by the method of Ellman

(1986).

Principle

This method was based on the -

dithio-bis-2-nitrobenzoic (DTNB) is added to compound containing sulphydryl

groups. The colour developed was readat 412 nm in spectrophotometer.

Reagents

1. 0.2M Phosphate buffer (pH 8).

2. 5% TCA.

3. Ellmans Reagent- 19.8 mg of DTNB in 100ml of 0.1 % sodium citrate.

4. Standard Glutathione- 10 mg of GSH/ 100ml distilled water.




120

Procedure

0.5 ml of liver homogenate was pipette out and precipitated with 2.0 ml of

5% TCA. 1.0 ml of supernatant was taken after centrifuge and 0.5 ml Ellman

Reagent and 3.0 ml Phosphate buffer were added to it. The yellow colour

developed was read at 412 nm. A series of standard were treated in simultaneous

manner along with blank continued by 3.5 ml buffer. Values were expressed as

mg/100 g of tissue.

3.5.31 (vii)Estimation of Vitamin C

Plasma ascorbic acid was estimated by the method of Roe and Keuther

(1943).

Principle

Ascorbic acid is oxidised by copper to form dehydroascorbic acid.The

product was treated with 2,4 dinitrophenyl hydrazine to form tris 2,4 dinitrophenyl

hydrazone which undergoes rearrangement to form a product with the absorption

maximum at 520 nm in spectrophotometer.

Reagents

1. 6% TCA

2. Acid washed Notril

3. 85% H2SO4

4. Stock L-Ascorbic acid - 100mg/100ml

5. 4% TCA




121

6. 2,4- Dinitro Phenyl Hydrazine (DNPH) The reagent was prepared by

dissolving 0.4 g of thio urea, 0.05 g of CuSO4 and 3 g of 2, 4- DNPH in

100 ml of 9 N H2SO4.

Procedure

To 0.5 ml of plasma, 1.5 ml of 6 % TCA was added and allowed to stand

for 5 minutes and centrifuged. The supernatant was removed and 0.3 g of acid

washed norit was added, shaken vigorously and filtered.2 ml of filtrate was taken

and 0.5 ml of DNPH added, stopper and placed in water bath for 37 C for 3

hours. After incubation the tubes were placed in ice bath, 2.5 ml of 85% H2SO4

added drop by drop. The contents were mixed and allowed to stand for 30

minutes. A set of standard containing 20-100 g of ascorbic acid acts as blank.

Color developed was read at 520 nm. Values were expressed in mg/dl.

3.5.31(viii).Estimation of Vitamin E

Plasma vitamin E was estimated by the method of Baker and Frank (1980).

Principle

-

tocopherol and the formation of red coloured complex with 2,2dipyridyl.

Absorbance of chromophore was measured at 520 nm in the spectrophotometer.

Reagents

1. Petroleum Ether (60-80° C)

2. Double distilled water.




122

3. - Di-pyridyl solution in ethanol.

4. 0.5 % Ferric Chloride solution in ethanol.

5. Stock Standard- 10 mg of Tocopherol/100ml distilled ethanol.

6. Working standard- Stock standard was diluted with distilled ethanol to get

a content of 10 g/ml.

Procedure

To 0.5 ml of plasma, 2 ml of petroleum ether and 1.5 ml ethanol added and

-dipyridyl solution

and 0.2 ml ferric chloride added, mixed well and kept and in dark for 5 minutes.

An intense red colour was developed. 4 ml of n-butanol was added to all the tubes,

-Tocopherol in the range of 10-100 g were taken and

treated. Similarly along with reagent blank it was read at 520nm. The values were

expressed as mg/ml.

3.5.32.Estimation of TBARS

LPO in tissue was estimated by the method of Okhawa et al (1979).

Serum and tissues was deproteinised with phosphotungstic acid and the precipitate

was treated with thiobarbituric acid at 90°C for 1 hour. The pink colour formed

gives the measure of thiobarbituric acid reactive substances (TBARS) which was

read at 530nm using spectrophotometer.

Reagents

1. 8.1 % SDS.

2. 20% Acetic acid.




123

3. 0.5 % TBA.

4. n-Butanol and Pyridine (15:1 v/v).

5. Stock Malondialdehyde solution (184

Procedure

To 0.2 ml of tissue homogenate, 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic

acid were added, mixed and 1.5 ml of 0.5% TBA was added. The mixture was

made up to 4.0 ml with distilled water and heated in water bath at 95 C for 60

minutes. After cooling with tap water, 1.0 ml distilled water, 5 ml of n-butanol:

pyridine was added and shaken vigorously. After centrifuge at 4000 rpm for

10minutes and the organic layer was removed and its absorbance was read at

535nm. Values were expressed as nano moles /100g tissue.

3.5.33. Histopathology of Liver (Humanson, 1962)

Processing of isolated liver

The animals were sacrificed and the liver of each animal was isolated and

was cut into small pieces, preserved and fixed in 10% formalin for two days. Then

the liver piece was washed in running water for about 12 hours to remove the

formalin and was followed by dehydration with isopropyl alcohol of increasing

strength (70%, 80% and 90%) for 12 hours each. Then finally dehydrations had

done using absolute alcohol with about three changes for 12 hours each.

Dehydration was performed to remove all traces of water. Further alcohol was

removed by using chloroform and chloroform removed by paraffin infiltration.

The clearing was done by using chloroform with two changes for 15 to 20 minutes




124

each. After paraffin infiltration the liver pieces were subjected to automatic tissue

processing unit.

Embedding in paraffin vaccum

Hard paraffin was melted and the hot paraffin was poured into L-shaped

blocks. The liver pieces were then dropped into the molten paraffin quickly and

allowed to cool.

Sectioning

The blocks were cut using microtome to get sections of thickness of

5micron .The sections were taken on a micro slide on which egg albumin i.e.,

sticking substance was applied. The sections were allowed to remain in an oven at

600°C for 1hour. Paraffin melts and egg albumin denatures, thereby fixing tissue

to slide.

Staining

Eosin is an acid stain, hence it stains all the cell constituents pink which

are basic in nature i.e., cytoplasm. Haematoxylin, a basic stain which stains all

the acidic cell components.

3.5.34.Statistical Analysis

using statistic software (Graph Pad ).The statistical analysis was carried out by

one way analysis of variance (ANOVA) followed by Tukeys-Kramer multiple

range test (TMRT). The analysis was performed using Graph Pad Prism software




125

version 5.0 (Saba et al., 2010). p values < 0.05 were considered statistically

significant.

3.6. Gas Chromatography Mass Spectrometry study for phytochemical

analysis

The GC-MS study was carried out at Indian Institute of Crop Processing

Technology, Thanjavur.

The GC-MS (ELAN 9000 ICP/MS) analysis was performed using Clarus

Perkin Elmer gas Chromatography equipped with a Elite 5 Capillary column (5 %

Diphenyl 95% dimethyl poly siloxane) and detector turbo mass gold. Helium was

the carrier gas at flow rate of 1 ml/min. The Injector was operated at 200oC and

the oven temperature was programmed as follows: - 60°C for 15 min, then

gradually increased to 280°C for 3 min. The Interpretation on Mass spectrum of

GC-MS conducted using the database of National Institute of Standard and

Technology (NIST) having more than 62,000 patterns. The spectrum of the

unknown components was compared with the spectrum of known components

stored in the NIST library (Paresh and Norman, 1998).

3.7. Hplc Analysis of Flavonoids (Shimadzu Corp., Kyoto HPLC Agilent 1200

G13653 MWD)

The HPLC analysis was carried out in Indian Institute of Crop Processing

Technology, Thanjavur.

Standard preparation: Standard stock solutions of five phenolic like gallic acid

(GA), caffeic acid (CA), rutin (RU), ferulic acid (FA) and quercetin (QU)




126

standard solutions were filtered through HPLC filter 0.45 mm membrane filter

(Millipore).

Sample preparation

The sample was prepared according to the procedure. The extraction was

carried out using 2 ml of fermented broth with 50 mL of 95% ethanol under 80

KHz, 45°C in ultrasonic extraction device for 30 min, repeated twice. The extract

was collected and filtered; the filtrate was dried at 50°C under reduced pressure in

a rotary evaporator. The dried crude extract was dissolved in the 100 ml mobile

phase. After filtering through a filter paper and a 0.45 mm membrane filter

(Millipore), the extract was injected into HPLC.

HPLC conditions

Flavonoids were analyzed using a RP-HPLC method, consisting of a LC-

10ATVp pump, SCL 10A system controller and a variable Shimadzu SPD-

1

area was calculated with CLASS VP software. Reverse phase chromatographic

analysis was carried out in isocratic conditions using a C-18 reverse phase column

(250×4.6 mm i.d., par -18; phenomenex, Torrance, CA,

USA) at 25°C. The gradient elution of solvent A (water-acetic acid (25:1 v/v) and

solvent B (methanol) had a significant effect on the resolution of compounds. As a

result, solvent gradients were formed, using dual pumping system, by varying the

proportion of solvent A (water-acetic acid (25:1, v/v) to solvent B (methanol).

Solvent B was increased to 50% in 4 min and subsequently increased to 80% in 10




127

min at a flow rate of 1.0 mL/min. Detection wavelength was 280 nm. Gallic acid

(GA), caffeic acid (CA), rutin (RU), ferulic acid (FA) and quercetin (QU) was

used as internal and external standards. Phenolic acids present in each sample

were identified by comparing chromatographic peaks with the retention time (Rt)

of individual standards and further confirmed by co-injection with isolated

standards (Weerasak Samee and Suwanna Vorara).




CHAPTER 3 MATERIALS AND METHODS -...

Documents

Transcript of CHAPTER 3 MATERIALS AND METHODS -...