Chapter -3 Materials Methodsshodhganga.inflibnet.ac.in/bitstream/10603/105697/8... · V Vitamin C+...

Chapter -3 Materials

& Methods

TTTThe present investigation has been undertaken to evaluate the role of

Tribulus terrestris root and fruit extract in modifying the lead acetate induced

toxicity in the liver and blood of Swiss albino mice.

The materials and methodology employed in the present investigation are as

under-

1. Animals selection

Swiss albino mice (Mus musculus) procured from IVRI - Izatnagar, India,

were bred in an animal house under controlled conditions of temperature (25±3oC)

and light (12 hrs. light and 12 hrs. darkness). These animals were given pelleted

standard mice feed (obtained from Hindustan Lever Ltd., Delhi) and water ad

libitum. A total check on cleanliness of the cages and animals were observed

throughout the experimental period. Occasionally soaked black gram and wheat

were given as supplementary diet. Tetracycline water once a fortnight was given to

these animals as one of the preventive measures against any infection.

A close-bred colony of these animals has been maintained by random

breeding. For this purpose, adult female and male mice were kept in the ratio of 3:1

polypropylene cages (15" × 10" × 6 size) having a wire gauge top. Sawdust was

used as breeding material in the cages.

Healthy young mice 6-8 weeks old with an average body weight (22±2 gm)

selected from the above colony were used for the present study.

2. Source of heavy metal toxicant

Heavy metal lead in the form of lead acetate was used for the present study,

it was obtained from Central Drug House (India). Lead acetate was dissolved in

vehicle Double Distilled Water (DDW) and administered subcutaneously.

3. Source of plant extract

The plant Tribulus terrestris (T. terrestris) was collected locally in the month

of July and August from the University of Rajasthan campus and was identified in

the Herbarium of Botany Department, University of Rajasthan, Jaipur as an RUBL-

Tribulus terrestris (RUBL 20824)

fruit

root

Plate A

Material and Methods 33

20824 variety (Plate A). The roots and fruits of the plant were air dried in shade and

powdered. Both the powders were distilled separately in soxhlet apparatus (for 36

hours using DDW) at 40°C. The remaining material was dried in oven at 36°C and

was used for the study. The dose was administrated through oral gavage to the mice.

4. Evaluation of antioxidant variables from Tribulus terrestris root and

fruit extract

DPPH radical scavenging assay

DPPH radical scavenging activity was determined by the method of Blois

(1958).

Principle: DPPH is a stable radical of purple color which is reduced to

yellow colored diphenylpicrylhydrazine, when it reacts with an antioxidant

compound which can donate hydrogen. The free radical scavenging activity of T.

terrestris root and fruit extract was measured in terms of hydrogen donating or

radical scavenging ability using the stable radical DPPH.

Procedure

i. 0.1 mM solution of DPPH in ethanol was prepared and 1.0 ml of this

solution was added to 3.0 ml of extract solution in water at different

concentrations (10-60 µg/ml).

ii. Thirty minutes later the absorbance was measured at 517 nm.

Lower absorbance of the reaction mixture indicates higher free radical

scavenging activity. Ascorbic acid was used as the standard antioxidant compound.

The capability to scavenge the DPPH radical was calculated using the following

equation:

DPPH Scavenged (%) = Acont - Atest x 100

Acont Where Acont is the absorbance in the presence of the control reaction and Atest is the

absorbance in the presence of the sample of the extracts.


5. Drug tolerance study & optimum dose selection of T. terrestris root and

fruit extract

Tribulus terrestris root and fruit extracts at the dose level of 800 mg/kg b.wt.

were given orally for 30 days. This dose was selected on the basis of previous

studies done in our lab. Mice for drug tolerance study were divided into various

groups of 5 animals each, out of which one is control and remaining are treatment

groups. Treatment groups were given 100, 400 and 800 mg/kg body weight/day T.

terrestris root and fruit extracts in double distilled water by oral gavage for 7

consecutive days. All these animals were observed regularly till 30 days for any sign

of morbidity, mortality and behavioural toxicity. Maximum tolerance doses of T.

terrestris root and T. terrestris fruit extracts were determined accordingly by

evaluating LPO (Lipid peroxidation) level and GSH (reduced glutathione) level in

mouse liver.

6. Dose selection of lead acetate

Lead was given subcutaneously in the form of lead acetate at different dose

levels (0, 5, 10, 20 and 40 mg/kg body weight) only once.

The dose was decided on the basis of body weight changes, and GSH and

LPO levels in the liver. After administration of 5 mg/kg b.wt. lead acetate

subcutaneously in Swiss albino mice, no significant changes were found in body

weight, GSH and LPO levels of liver tissue and the values were very similar to the

normal control values after 7 days. On the dose level of 10 mg/kg body weight,

slight changes were observed with decreased body weight and GSH level and

increased LPO level as compared to normal control. Symptoms of sickness, ruffled

hair and loss of appetite were also observed after 7 days but no mortality was

observed up to 30 days of experiment. While on dose levels 20 and 40 mg/kg b.wt.,

body weight and GSH level was decreased more and LPO level was increased

highly as compared to normal control. Symptoms of sickness were more severe and

localized infection of skin (skin lesions) after lead-intoxication subcutaneously was

also observed on these dose levels but no skin lesions were observed on 5 and 10

Normal Swiss albino mice

Ruffled hair Swiss albino mice

Swiss albino mice with skin lesion

Plate B


mg/kg b.wt. doses (Plate B). On the basis of above study for dose determination of

lead acetate, we selected a dose of 10 mg/kg body weight for our experiment.

Table A: Showing body weight changes of animals during 7 days of experiment

Groups Lead acetate (mg/kg b. wt.)

Body weight after lead acetate treatment (gm)

1st day 3rd day 5th day 7th day

I Normal Control (DDW orally)

30±1.80 30±1.20 30.5±1.60 30.5±0.90

II 5 mg/kg 30.5±0.90 30±1.40 30±1.20 29.5±1.80

III 10 mg/kg 31±2.40 30±1.70 28.5±0.70 27±1.90

IV 20 mg/kg 30±1.30 28.5±2.10 27±1.80 25±1.60

V 40 mg/kg 29.5±1.70 27±1.40 25.5±0.90 24±1.20

Table B: Showing GSH and LPO changes on different lead acetate doses in

liver of animals during 7 day experiment

Groups Lead acetate (mg/kg b. wt.)

No. of Animals

GSH level in liver (mg GSH/gm

tissue)

LPO level in liver (µMTBARS/mg

protein)

I Normal Control (DDW orally)

5 8.03±0.41 3.79±0.15

II 5 mg/kg 5 7.59±0.11 4.11±0.10

III 10 mg/kg 5 6.67±0.23 4.88±0.13

IV 20 mg/kg 5 6.51±0.31 5.61±0.23

V 40 mg/kg 5 5.99±0.33 6.63±0.11

7. Normal Studies

The animals did not receive lead acetate or root and fruit extract (Tribulus

terrestris). They were autopsied to study the normal structure of liver.

Experimental Protocol

Group I (Control group): Normal control, untreated (mice received DDW as

vehicle).

Group IIa and IIb ( T. terrestris group): Selected dose of T. terrestris root (IIa) and

fruit extract (IIb) dissolved in DDW was administered orally upto 30 days.


Group III (Heavy metal treated group): Freshly dissolved lead acetate in 0.1 ml

double distilled water was given subcutaneously only once at a dose of 10mg/kg

body weight. This day was considered as day zero and the experiment was continued

for 30 days.

Group IVa and IVb ( T. terrestris + Lead acetate + T. terrestris): T. terrestris root

and fruit extracts were given at a selected dose level (800mg/kg body weight) for 7

days and on the 7th day just after 30 minutes of T. terrestris root and fruit extract

administration lead acetate was given only once, this day was considered as day

zero. Then from the next day T. terrestris was continued for 30 days. The total

experimental period was of 37 days.

Group V [Vitamin C + Lead acetate + Vitamin C (positive control group)]:

Vitamin C was administered at a dose level of 100 mg/kg body weight for 7 days

and on the 7th day just after 30 minutes of Vitamin C administration lead acetate

was given only once, this day was considered as day zero. Then from the next day

Vitamin C was continued for 30 days. The total experimental period was of 37 days.

Group VI [Lead Acetate + DMSA (for 7 days)]: DMSA was administered at a

dose level of 50 mg/kg body weight for 7 days after 30 minutes of lead acetate

treatment.

Group VIIa and VIIb ( T. terrestris + Lead acetate + DMSA + T. terrestris

group): T. terrestris was administered orally for 7 days and on the7th day just after

30 minutes of T. terrestris root and fruit extract administration lead acetate was

given only once, this day was considered as day zero. Then from the next day

DMSA was given for 7 days only and T. terrestris was continued for 30 days. The

total experimental period was of 37 days.

Autopsy Intervals

At least 5 animals from all the above groups were sacrificed under light

chloroform anesthesia on 1, 3, 7, 15 and 30 days. Liver, blood and serum were taken

and processed for various histopathological and biochemical parameters.


Table C: Design of Experiment

Groups Experimental Groups

No. of Animals

Dose Days of Treatment

I Normal control 25 DDW (orally) Upto 30 days

IIa T. terrestris (root) treated

25 T. terrestris root extract (800mg/kg b.

wt.)

Upto 30 days

IIb T. terrestris (fruit) treated

25 T. terrestris fruit extract (800mg/kg b.

wt.)

Upto 30 days

III Lead acetate treated 25 10 mg/kg b.wt. (in DDW, subcutaneously

only once)

30 day experiment

IVa T. terrestris + Lead acetate + T. terrestris

(root extract)

25 800 mg/kg b.wt. T. terrestris + 10 mg/kg

b. wt. lead acetate + T. terrestris

7 days T. terrestris + lead acetate on

7th day + T. terrestris for 30

days

IVb T. terrestris + Lead acetate + T. terrestris

(fruit extract)

25 800 mg/kg b.wt. T. terrestris+ 10 mg/kg

b. wt. lead acetate + T. terrestris

7days T. terrestris +lead acetate on

7th day+ T. terrestris for 30

days

V Vitamin C+ Lead acetate + Vitamin C

25 100 mg/kg b.wt. Vitamin C+ 10 mg/kg b. wt. lead acetate +

Vitamin C

7days Vitamin C + lead acetate on 7th day + Vitamin C

for 30 days

VI Lead acetate + DMSA

25 10 mg/kg b. wt. lead acetate + 50 mg/kg b.

wt. DMSA

1st day Lead acetate + up to 7 days DMSA (30 day experiment)

VIIa T. terrestris +Lead acetate+ DMSA and

T. terrestris(root extract)

25 800 mg/kg b.wt. T. terrestris + 10 mg/kg b. wt. lead acetate +

DMSA &T. terrestris

7days T. terrestris + lead acetate on 7th day + DMSA for 7 days and T. terrestris for 30

days

VIIb T. terrestris +Lead acetate+ DMSA and

T. terrestris (fruit extract)

25 800 mg/kg b.wt. T. terrestris+ 10 mg/kg b. wt. lead acetate +

DMSA &T. terrestris

7days T. terrestris + lead acetate on 7th day + DMSA for 7 days and T. terrestris for 30

days


Parameters studied

To study the toxic effects of lead and the modulatory effects of T. terrestris

against lead induced alterations, following parameters were assessed:

A. General Parameters

1. Mortality

2. Sickness

3. Body weight

4. Liver weight

B. General Toxicity:

To assess the toxicity different histopathological, biochemical and

haematological parameters were done.

I. Histological Studies (Normal and Histopatholgoical)

Liver from autopsied animals were excised out and fixed in Bouin's fixative

for 24 hrs. The fixed tissue were further processed by standard method and sections

were cut at 5µ and stained with Haematoxylin and Eosine.

II. Biochemical Parameters

For all biochemical estimation, liver was taken out from animals and

homogenates were prepared. The parameters selected for biochemical study were as

follows -

i. Lipid Peroxidation (LPO) (Ohkawa et al.,1979)

ii. Reduced Glutathione (GSH) (Moron et al.,1979)

iii. Total Protein (Lowry et al., 1951)

iv. Glycogen (Montgomery, 1957)

v. Superoxide Dismutase (SOD) (Marklund and Marklund, 1974)

vi. Catalase (CAT) (Aebi, 1984)

vii. Glutathione Peroxidase (GPx) (Paglia and Valentine, 1967)

viii. Glutathione-S-Transferase (GST) (Habig et al., 1974)


Liver Function Tests

i. Serum Alkaline Phosphatase (Kind and King, 1954)

ii. Serum Glutamate Oxaloacetate Transaminase (Reitman and Frankel, 1957)

iii. Serum Glutamate Pyruvate Transaminase (Reitman and Frankel, 1957)

Haematological Study

i. Haemoglobin concentration (Wintrobe et al., 1981)

Homogenate Preparation for GSH and LPO:

1 gm of tissue and 9 ml of Tris KCl were mixed with the help of pestle

mortar and sea sand. 10% homogenate w/v of tissue in Tris KCl was prepared.

i. Lipid Peroxidation (LPO) assay

Lipid peroxidation level in liver was estimated by the method of Ohkawa et

al. (1979) as thiobarbituric acid reactive substances. The concentration of TBARS

was expressed as n moles of malondialdehyde per mg of tissue using 1, 1, 3, 3 -

tetromethoxy propane (TMP) as the standard.

Principle : Malondialdehyde formed from the breakdown of polyunsaturated fatty

acids serves as a convenient index for determining the extent of the peroxidation

reaction. Peroxidation of lipids generates MDA, which reacts with thiobarbituric

acid gives a red species absorbing 532 nm.

Procedure:

i. 0.8 ml of tissue sample was taken in test tubes and to the 0.2 ml of 8.1% of

SDS, 1.5 ml of 20% acetic acid was added and the pH was adjusted to 3.5

with NaOH.

ii. To the above solution 1.5 ml of 0.6% aqueous solution of TBA was added.

iii. The mixture was made upto 4ml with distilled water (0.6 ml) and then heated

at 95°C for 60 min.

iv. After cooling in tap water, 1 ml of distilled water and 5 ml of n-butanol and

pyridine (15% µ/v) were added and shaken vigorously.


v. The solution was centrifuged at 4000 rpm for 10 minutes. The organic layer

was taken and its absorbance was measured at 532 nm using UV-VIS

systronic spectrophotometer.

Standard Curve: A standard curve was prepared by using tetromethoxy-propane

(TMP, mol. wt. = 164.20) purchased from Lancaster, England). 1 to 12 n mol of

TMP was taken in corresponding no. of test tubes with all the reagents (minus

sample) were added. Standard curve was plotted by taking the absorbance against

TMP concentration on the abscissa.

ii. Reduced Glutathione (GSH) assay

The GSH level in liver was determined by the method of Moron et al.

(1979).

Principle: The reduced glutathione reacts with DTNB and forms a yellow coloured

complex with DTNB that absorbs at 412 nm.

Reduced glutathione + DTNB → 5-thio-2-nitrobenzoate (yellow)

The tissue homogenates (10% w/v) were immediately precipitated with 0.1

ml of 25% trichloroacetic acid (TCA), and the precipitate was removed after

centrifugation.

Free endogenous -SH was assayed in a total 3 ml volume by the addition of 2

ml of 0.5m M DTNB prepared in 0.2 M phosphate buffer (pH = 8.0) to 0.1 ml of the

supernatant, and absorbance was read at 412 nm using a UV-VIS systronic

spectrophotometer. GSH was used as an standard to calculate µ mole GSH /gm

tissue.

Homogenate Preparation for Total Protein, SOD, CAT, GPx and GST:

1 mg (or 0.001 gm) of tissue and 1 ml of double distilled water were mixed

with the help of pestle mortar and sea sand.

iii. Total Protein assay

Total protein content in liver was estimated by Lowry et al.(1951) method.


Principle: Here, protein is precipitated with trichloroacetic acid. When Folin

Ciocatteu reagent (Phosphatungstic phosphomolybdic acid) is added, a complex

results, the intensity of which accounts for the amount of proteins present in tissue

Folin Ciocatteu reagent after 50 percent dilution in double distilled water.

Procedure

i. 0.5 ml of 10% TCA was added was added to 0.5 ml of homogenate

ii. It was centrifuged for 15 minutes and the supernatant was discarded.

iii. Precipitate was then washed with 1 ml of 5% TCA.

iv. It was again centrifuged and the supernatant was discarded.

v. Precipitate was washed twice with 2 ml of GDW each time.

vi. It was further centrifuged and the supernatant discarded.

vii. Precipitate was dissolved in 4 ml of 1 N NaOH.

viii. Test tubes were placed in a water bath/oven at 60-70° for 10 minutes.

ix. After cooling and centrifuging, 0.5 ml of the above liquid was added to 4.5

ml of Lowry's solution.

x. Finally 0.5 ml of 1 N Folin-Phenol reagent was added and mixed well.

xi. Reading was taken at 640 nm against blank solution.

Blank solution: 0.5 ml of 1 N NaOH + 4.5 ml of Lowry's solution + 0.5 ml of

Folin-Phenol reagent.

Calculation: Amount of Total Protein was calculated by the following formula:

Reading of unknown ×

Concentration of standard × 1000 ×

4

Reading of standard Tissue taken 0.05

Where:

4/0.05 is volume taken.

Protein content was expressed as mg of protein/gm of tissue.

iv. Glycogen assay:

The Glycogen content in liver was estimated by Montgomery(1957)


Procedure:

i. 0.1 gm tissue was digested in 2 ml of 30% KOH and kept in boiling water

for 2-3 minutes.

ii. The digested tissue was brought to room temperature by cooling and 2.4 ml

absolute alcohol was added and left for 10 minutes.

iii. It was centrifuged was 30 minutes and supernatant was discarded and again

2.4 ml absolute alcohol was added.

iv. It was again centrifuged for 10-15 minutes and supernatant was discarded.

v. Tubes were kept in dessicator for overnight.

vi. On next day, the precipitate was dissolved in 4 ml GDW and mixed well.

vii. 2 ml of the above was taken and to it 0.1 ml of 80% phenol and 5 ml of

H2SO4 were added.

viii. It was mixed and cool.

ix. The absorbance was read at 624 nm spectrophotometrically against blank

solution.

Blank solution: 2 ml DDW +0.1 ml phenol reagent+ 5 ml of conc. H2SO4

Calculation: Amount of Glycogen was calculated by the following formula:

Reading of unknown

×

Concentration of standard × 1000 ×

4

Reading of standard Tissue taken 1

Where:

4/1 is volume taken.

Glycogen content was expressed as mg of glycogen/gm of tissue.

v. Superoxide Dismutase (SOD) assay:

The superoxide dismutase activity in liver was estimated by the method of

Marklund and Marklund (1974)

Principle: Pyrogallol is used to generate a reproducible O2. The estimation of

superoxide dismutase is based on inhibition of pyrogallol auto-oxidation at pH 8.0.


Procedure:

i. To 0.1 ml tissue homogenate, 1.5 ml Tris HCl buffer was added.

ii. Then 0.5 ml EDTA was added to above.

iii. 1 ml Pyrogallol was added (during readings only)

iv. Absorbance was taken at 420 nm in spectrophotometer every minute after a

lag of 30 seconds upto 4 minutes.

Calculation: SOD activity was calculated by the following formula:

SOD = ∆ EGR / minute × 3 × 60

100× 10 µl

Where,

100= SOD enzyme activity

SOD activity was expressed as µmol/ mg protein

vi. Catalase (CAT) assay:

Catalase activity in liver was estimated by the method of Aebi (1984).

Principle: Estimation of catalase activity is based on the decomposition of H2O2.

The decomposition of H2O2 followed directly by the decrease in absorbance at 240

nm.

Procedure:

i. 2 ml of 50 mM (pH=7) phosphate buffer was added to 0.1 ml tissue

homogenate

ii. 1 ml of H2O2 (30 mM) was added to the above (during readings only).

iii. Absorbance was taken at 240 nm in spectrophotometer where readings were

taken at every 10 second interval for 30 seconds.

Calculation: CAT activity was calculated by the following formula:

CAT = ∆ EGR / minute × 3 × 60

71 × 10 µl

Where,

71= CAT enzymatic activity

CAT activity was expressed as µmol H2O2 / minute/ mg protein


vii. Glutathione Peroxidase (GPx) assay:

Glutathione Peroxidase activity in liver was estimated by the method of

Paglia and Valentine (1967).

Principle: GPx activity was measured by the rate of GSSG formation by following

the decrease in absorbance of the reaction mixture at 340 nm as NADPH is

converted to NADP+.

Procedure:

i. 2.58 ml of 0.05 M (pH=7.0) phosphate buffer was pipette out in a quartz

cuvette.

ii. 0.01 ml tissue homogenate, 0.1 ml of 8.4 mM NADPH, 0.01 ml GR, 0.01 ml

of 1.125 M sodium azide and 0.1 ml of 0.15 M GSH was added to the above

cuvette.

iii. The reaction mixture was allowed to equilibrate at 20°C.

iv. The reaction was initiated by addition of 0.1 ml of 2.2 mM H2O2.

v. The decrease in absorbance was recorded at 340 nm between 2 to 4 minutes,

after initiation of reaction employing a cuvette with 1 cm light path in

spectrophotometer.

Calculation: GPx activity was calculated by the following formula:

GPx activity = ∆A340 × DF

6.22 × V

Where,

∆A340= A340/ min(blank)- A340/ min(sample)

6.22= mM extinction coefficient for NADPH

DF= dilution factor of sample before adding to reaction

V= sample volume in ml

GPx activity was expressed as nm of NADPH oxidized / minute/ mg protein

viii. Glutathione-S-Transferase (GST) assay:

Glutathione-S-Transferase activity in liver was estimated by the method of

Habig et al.(1974).


Procedure:

i. 2.8 ml of 0.1 M phosphate buffer and 0.1 ml of 30 mM GSH solution was

added to 3 ml cuvette.

ii. 10-50 µl of tissue homogenate was added to the cuvette and mixed well.

iii. 0.1 ml of 30 mM CDNB solution was added to the above cuvette to initiate

the reaction.

iv. Increase in O.D. at 340 nm in a spectrophotometer was recorded every

minute for 3 minutes after a lag of 30 seconds

Calculation: GST activity was calculated by the following formula:

GST = A340(t2) – A340(t1)

(t2-t1) × (Volume (in ml) of sample added) Where,

A340(t2) = Absorbance at 340 nm at time t2 in minutes

A340(t1) = Absorbance at 340 nm at time t1 in minutes

GST activity was expressed as µ mol GSH-CDNB conjugate formed/ min/ mg

protein

Collection of blood:

Swiss albino mice were carefully dissected. The blood samples were

collected directly from the ventricle of the heart with the help of the sterilized

disposable syringe (5.0 ml) fitted with hypodermic needle. The blood samples drawn

in the syringes were immediately transferred to respective vials dipped in EDTA

solution. Sterilized plain glass centrifuge tubes were used for the separation of the

serum.

Separation of serum :

The centrifuge tubes were allowed to stand in slanting position for clotting.

After the clots have started retracting, these tubes were then centrifuged at 2,800

rpm for 30 minutes. The supernatant serum was then carefully transferred to

sterilized plain glass vials with the help of the glass dropper.


Liver Function Tests

To assess the liver function following biochemical parameters in serum were

also done.

(i) Serum alkaline phosphatase

Serum alkaline phosphatase was measured by Kind and King's method

(1954) using commercially accessible kits (Span Diagnostics Ltd., Surat, India).

Optical density was measured at 510 nm.

Principle: Alkaline phosphatase from serum converts phenyl phosphate to inorganic

phosphate and phenol, at pH 10.0, phenol so formed reacts in alkaline medium with

4-amino antipyrine in the presence of an oxidizing agent, potassium ferricyanide,

and forms an orange-red coloured complex, which can be measured colorimetrically.

The colour intensity is proportional to the enzyme activity in the presence of an

oxidizing agent potassium ferricyanide, and forms an orange-red coloured complex,

which is measured colorimetrically.

The reaction can be represented as :

Phenyl phosphate Alkaline phosphatse Phenyl + Pi (inorganic phosphate)

pH 10.0

Phenol + 4- aminoantipyrine Potassium Phenyl + Pi (inorganic phosphate)

Ferricyanide OH_

Calculation:

Serum alkaline phosphatase activity (in KAU units)

O.D. Test - O.D. Control ×10

O.D. Std. - O.D. Blank (ii) SGOT (Serum Glutamate Oxaloacetate Transaminase) or AST

(Aspartate Transaminase)

The SGOT activity was estimated by method of Reitman and Frankel (1957)

using commercially accessible kits (Span Diagnostics Ltd., Surat, India). Optical

density was measured at 505 nm.

Principle: SGOT (AST) catalyse the following reaction:

α Ketoglutarate + L- Aspartate L – Glutamate + Oxaloacetate


Oxaloacetate so formed is coupled with 2,4-dinitrophenyl hydrazine (2,4-

DNPH) to give the corresponding hydrazone, which gives brown color in alkaline

medium and this is measured colorimetrically.

Calculation:

AST (SGOT) activity (in IU/L) = O.D. of Test - O.D. of Control

× Conc. of Std. O.D. of Std. – O.D. of Blank

(iii) SGPT (Serum Glutamate Pyruvate Transaminase) or ALT (Alanine

Transaminase)

The SGPT activity was measured by the method of Reitman and Frankel

(1957) using DNPH as colour agent by using commercially accessible kits (Span

Diagnostics Ltd., Surat, India). Optical density was measured at 505 nm.

Principle : SGPT (ALT) catalyse the following reaction :

α Ketoglutarate + L- Alanine L – Glutamate + Pyruvate

Pyruvate so formed is coupled with 2,4-dinitrophenyl hydrazine (2,4-DNPH)

to give the corresponding hydrazone, which gives brown color in alkaline medium

and this is measured colorimetrically.

Calculation:

AST (SGPT) activity(in IU/L) = O.D. of Test - O.D. of Control

× Conc. of Std. O.D. of Std. – O.D. of Blank

Haematological Study

The blood after collection in disodium EDTA vials was used for the

estimation of the haemoglobin:

Haemoglobin concentration (Hb)

The haemoglobin concentration was estimated by the standard Sahlis method

(Wintrobe et al., 1981). The method is based on the principle of making acid

haematin solution of blood in graduated haemoglobin tube and then comparing it


with the sealed comparison tubes containing the standard acid haematin solution.

The reading was finally recorded in gm/100 ml of blood.

Statistical Analysis

Analysis of Variance (ANOVA) Test:

The statistical significance in the different enzyme activities between the

control and experimental was assessed by ANOVA. The purpose of analysis of

variance (ANOVA) is to test for significant differences between means. One way

ANOVA carried out between two groups. This test is carried by Analystsoft

Statplus 2009 software.

The significance level was obtained from the table of significance provided

for the ANOVA’s distribution. The values of P as 0.05, 0.01 and 0.001 were

considered to be almost significant, significant and highly significant, respectively.

The data generated from the above observations are expressed as the mean ± SEM.

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