CHAPTER II

MATERIALS AND METHODS

CHEMICALS

Diethyl phthalate was purchased from Sisco Research Laboratories Pvt. Ltd.,

Mumbai, India and was of analytical grade. All the other chemicals used in the

present study were of analytical grade and purchased from Hi Media Laboratories Pvt.

Ltd., Mumbai, India, Sisco Research Laboratories Pvt. Ltd., Mumbai, India and

Sigma Aldrich, St. Louis, MO, USA. Olive oil was obtained from Figaro, Madrid,

Spain.

HOUSING AND CARE OF ANIMALS

All animal studies were sanctioned by Institutional Animal Ethics Committee

of Gujarat University, Ahmedabad and approved by the Committee for the Purpose of

Control and Supervision of Experiments on Animals, New Delhi, India. Healthy

young female albino mice of Swiss strain weighing 30-35 gm were obtained from

Zydus Research Centre, Ahmedabad, India. The animals were kept in the Animal

House of Zoology Department of Gujarat University, Ahmedabad, India under

controlled conditions (Temperature 25±2˚C, relative humidity 50-55% and 12h

light/dark cycle). They were fed with certified pelleted rodent feed supplied by Amrut

Feeds, Pranav Agro Industries Ltd., Pune, India and potable water ad libitum.

Animals were handled according to the guidelines published by the Indian National

Science Academy, New Delhi, India (1991).

27
PLAN OF STUDY

The present study was carried out in three parts:

PART-I: To study the toxic effects of diethyl phthalate both in vitro and in vivo

conditions

PART- II : Phytochemical analysis of the Nigella sativa seed extract

PART-III: To study the ameliorative effect of Nigella sativa seed extract on

toxicity induced by diethyl phthalate

28
 PART-I

TO STUDY THE TOXIC EFFECTS OF DIETHYL PHTHALATE

BOTH IN VITRO AND IN VIVO CONDITIONS

A. In vitro study

To evaluate the toxicity of diethyl phthalate in vitro conditions lipid peroxidation and

protein content was studied.

Healthy adult Swiss strain female albino mice were humanly sacrificed by cervical

dislocation. Liver and kidney were isolated, blotted free of blood and used in the

study.

Lipid peroxidation assay:

Liver and kidney homogenates prepared in phosphate buffered saline were

used for various treatments followed by estimation of lipid peroxidation.

Following sets of tubes were prepared:

1. Control tubes: These tubes contained 0.2 mL of tissue homogenates and

phosphate buffered saline.

2. DMSO control tubes: These tubes contained 0.2 mL of tissue homogenates

and DMSO.

3. Antidote control tubes: These tubes contained 0.2 mL of tissue homogenates

and Nigella sativa seed extract (200 µg/mL).

4. DEP - treated tubes: These tubes contained 0.2 mL of tissue homogenates with

different concentrations of DEP (10-40 µg/mL) in DMSO.

29
 5. DEP and Nigella sativa seed extract tubes: These tubes contained 0.2 mL of

tissue homogenates with different concentrations of Nigella sativa seed extract

(25 – 200 µg/ml) along with DEP (40 µg/mL).

The final volume of each tube was made up to 1 mL with phosphate buffered

saline. Each tube was added with 0.1 mL H2O2 to induce lipid peroxidation.

All the tubes were subjected to incubation for 30 min at 37˚C .

The lipid peroxidation in control, DMSO control, toxin and toxin plus antidote-

treated tubes were measured by quantification of thiobarbituric acid reactive

substance (TBARS) by the method of Ohkawa et al with slight modifications. This

method is based on the formation of a red chromophore that absorbs light at 532 nm

following the reaction of thiobarbituric acid (TBA) with products of lipid

peroxidation like malondialdehyde (MDA) and others collectively called as

thiobarbituric acid reactive substances (TBARS). The results were expressed as n

moles MDA formed/mg protein/60 min.

Protein assay:

Liver and kidney were homogenized in chilled distilled water and used for

various treatment followed by estimation of protein content.

Following sets of tubes were prepared.

1. Control tubes: These tubes contained 0.2 mL of tissue homogenates and

distilled water.

2. DMSO control tubes: These tubes contained 0.2 mL of tissue homogenates

and DMSO.

30
 3. Antidote control tubes: These tubes contained 0.2 mL of tissue homogenates

and Nigella sativa seed extract (200 µg/mL).

4. DEP - treated tubes: These tubes contained 0.2 mL of tissue homogenates with

different concentrations of DEP (10-40 µg/mL).

5. DEP plus Nigella sativa seed extract – treated tubes: These tubes contained

0.2 mL of tissue homogenates with different concentrations of Nigella sativa

seed extract (25 – 200 µg/mL) along with DEP (40 µg/mL).

The final volume of each tube was made up to 1 mL with distilled water. All

the tubes were subjected to incubation for 30 min at 37˚C.

The protein content in control, DMSO control, toxin and toxin plus antidote-

treated tubes were estimated by the method of Lowry et al with slight modification.

Bovine serum albumin was used as a standard. Reaction of protein with Folin

Ciocalteau reagent results in blue colour which is due to two reactions occurring

simultaneously i.e., the reaction of alkaline copper sulphate solution with peptide

bonds and reduction of phosphomolybdic and phosphotungstic acids by aromatic

amino acids present in the protein. Resulting blue colour was measured at 540 nm.

The protein contents were expressed as mg/100 mg tissue weight.

B. In vivo study

To evaluate the toxicity of diethyl phthalate (DEP) in vivo conditions following

parameters were studied.

STUDY DESIGN

Fifty animals were randomly divided into five different groups and caged

separately. Animals of Group 1 (untreated control) were maintained without any

31
treatment. Animals of Group 2 (vehicle control) received olive oil (0.2

mL/animal/day) for 30 days as olive oil was used as vehicle to dissolve DEP. Animals

of Group 3, 4 and 5 were treated with low (310 mg/kg body weight/day), mid (620

mg/kg body weight/day) and high (1240 mg/kg body weight/day) dose of DEP

respectively. All treatments were given using a feeding tube attached to a hypodermal

syringe for 30 days. Doses of DEP were based on LD50 value i.e. 6200 mg/kg body

weight (NTP, 2006) (Table 2.1).

Behavioral and clinical changes throughout the experiments were recorded. The

body weight of control and all treated groups of mice were recorded to the nearest gm

on an animals weighing balance. At the end of treatment, the animals were weighed

and humanely sacrificed by cervical dislocation. Blood samples collected by cardiac

puncture in non - anticoagulant added tubes were allowed to clot and centrifuged at

1000 x g for 10 min at 4˚C. Obtained non – haemolysed serum samples were stored at

-4˚C and used for biochemical analysis. The liver and kidneys were dissected out,

blotted free of blood and weighed to the nearest mg on a balance and used for

histopathological and biochemical analysis.

BIOCHEMICAL ANALYSIS

Protein, lipid, carbohydrate and nucleic acid contents

Protein content:

Methodology used for protein estimation was as described in part I in vitro study

section. The protein content was expressed as mg/100 mg tissue weight.

Glycogen content:

The glycogen content in the liver was estimated by the method of Seifter et al.

(1950). The glycogen present in tissue is converted to glucose, which reacts with

32
anthrone reagent to give a green coloured product which was read at 620 nm. The

glycogen content was expressed as mg/100 mg tissue weight.

Total lipid content:

Total lipid content in the liver and kidney was estimated according to the method

of Fringes et al. (1972) using olive oil as a standard. Lipid on being heated with

sulphuric acid followed by addition of vanillin and phosphoric acid produces a pink

colour whose optical density is measured at 530 nm. The total lipid content was

expressed as mg/100 mg of tissue weight.

Total cholesterol content:

The concentration of cholesterol was estimated in the liver and kidney by the

method of Zlatki et al. (1953). Cholesterol forms a coloured complex with FeCl3 in

the presence of concentrated sulphuric acid and glacial acetic acid which can be

measured at 540 nm. The cholesterol content was expressed as mg/100 mg tissue

weight in liver and mg/dL.

Estimation of nucleic acid:

Extraction:

A known weight of fresh tissue was homogenized in 5 mL of cold 5% TCA and

the homogenate was kept at 0-4ºC for 30 min. The precipitate obtained after

centrifugation (10 min at 1000 x g) was dissolved in 5 mL of cold 5% TCA and left

for 30 min at 0-4°C. Thereafter, centrifugation (10 min at 1000 x g) was carried out

and the precipitate obtained was dissolved in alcohol: ether (1:3, v/v) mixture and left

for 30 min at 50°C. This process was repeated once again. The tubes were centrifuged

33
at 1000 x g for 10 min and the supernatant was discarded. The lipid free pellet

obtained was dissolved in 5 mL of 0.1 N KOH and incubated at 37°C for 16-18 h.

Then 0.17 mL of 6 N HCl and 5 mL of 10% TCA were added to the incubated

suspension and precipitate was allowed to be formed at 4°C for 30 min. After

centrifugation at 1000 x g for 10 min the supernatant was separated and used for

estimation of RNA. The pellet containing DNA and protein was heated at 90°C for 15

min after adding 5 mL of 5% TCA. The supernatant was then separated by

centrifugation (10 min at 1000 x g) after cooling at 4 °C for 30 min and used for

estimation of DNA.

(i) Deoxyribonucleic acid (DNA) :

The estimation of DNA in the liver and kidney was carried out by the method of

Giles and Meyer (1965). The DNA in the supernatant reacts with diphenylamine to

give a blue coloured complex whose optical density was read at 620 nm. The DNA

content was expressed as µ moles/100 mg tissue weight.

(ii) Ribonucleic acid (RNA):

The estimation of RNA in liver and kidney was carried out by the method of

Schneider (1939). The RNA in the supernatant reacts with the orcinol reagent to give

a greenish colour, whose absorbance was read at 670 nm. The concentration of RNA

was expressed as µ moles/100 mg tissue weight.

Enzymatic assays

Alanine transaminase (EC [Link]) activity:

34
 The alanine transaminase (ALT) activity in liver and serum was assayed by the

method of Reitman and Frankel (1957). A buffered solution of α-ketoglutarate and L-

alanine were made to react with the liver homogenate for 30 min. The pyruvate

formed from L-alanine by the enzymatic reaction reacts with 2, 4-dinitrophenyl

hydrazine (DNPH) in alkaline medium and formazon formed was measured at 540

nm. The enzyme activity was expressed as mU/mg protein/30 min in case of liver and

mU/mL in case of serum.

Aspartate transaminase (EC [Link]) activity:

The aspartate transaminase (AST) activity was assayed by the method of Reitman

and Frankel (1957). Assay method is similar as described in ALT activity assay,

except buffered solution which contained L- aspartate instead of L-alanine and

allowed to react for 1 hr. The enzyme activity was expressed as mU/mg protein/60

min in case of liver and mU/mL in case of serum.

Alkaline phosphatase (E.C.[Link]) activity:

The alkaline phosphatase (ALP) activity in liver and serum was determined by the

method of Bessey et al. (1946). Alkaline phosphatase at optimum pH 10.5 catalyzes

the hydrolysis of p-nitrophenyl phosphate (disodium salt) to p-nitrophenol and

inorganic phosphate. The liberated p-nitrophenol reacts with sodium hydroxide to

form yellow coloured complex which was measured at 410 nm. The ALP activities in

liver were expressed as µmoles p-nitrophenol released/mg protein/30 min and IU/mL

in serum.

Acid phosphatase (E.C.[Link]) activity:

35
 The acid phosphatase (ACP) activity was assayed in the liver, kidney and serum

by the method as described in Sigma Technical Bulletin (Sigma Technical Bulletin,

MO, USA). Acid phosphatase at optimum pH 4.8 catalyzes the hydrolysis of p-

nitrophenyl phosphate (disodium salt) to p-nitrophenol and inorganic phosphate. The

liberated p-nitrophenol reacts with sodium hydroxide to form a yellow coloured

complex which was measured at 420 nm. The enzyme activity was expressed as

µmoles p-nitrophenol released/mg protein/30 min in liver and kidney and IU/mL in

serum.

Adenosine triphosphatase (EC.[Link]) activity:

The adenosine triphosphatase (ATPase) activity in the liver and kidney was

assayed by the method of Quinn and White (1968). ATPase causes hydrolysis of

adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and inorganic

phosphate (i.p.). The liberated inorganic phosphate was estimated by the method of

Fiske and Subbarow (1925). The optical density was read at 660 nm. The enzyme

activity was expressed as µmoles inorganic phosphate released/mg protein/30 min.

Succinic dehydrogenase (EC.[Link]) activity:

The succinic dehydrogenase (SDH) activity in the liver and kidney was assayed

by the method of Beatty et al. (1966) using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-

phenyl- 2H – tetrazolium chloride (INT) as an electron acceptor. The electrons

released by the enzyme SDH from the substrate are taken up by INT, which was

reduced to a red coloured formazon. This was extracted in ethyl acetate and the

absorbance was read at 420 nm. The enzyme activity was expressed as µg formazon

formed/mg protein/15 min.

36
Phosphorylase (EC [Link]) activity:

The liver phosphorylase activity was assayed by the method of Cori et al. (1943).

The inorganic phosphate (i.p.) formed at the end of the reactions was estimated by the

method of Fiske and Subbarow (1925). The enzyme phosphorylase hydrolyses the

substrate glucose -1-phosphate. The inorganic phosphate formed at the end was

treated with an acidic molybdate solution; it forms phosphomolybdic acid which on

addition of 1-amino-2-nephthol-4-sulphonic acid (ANSA) is quantitatively reduced to

a blue coloured complex which is measured spectrophotometrically at 660 nm. The

enzyme activity is expressed as µg phosphorus released/100 mg fresh tissue/15 min.

LIPID PEROXIDATION AND ANTIOXIDATIVE DEFENSE MECHANISM

Lipid peroxidation:

The level of lipid peroxidation in the liver and kidney was measured by the

method as described by Ohkawa et al. (1979). This method is based on the formation

of a red chromophore that absorbs light at 532 nm following the reaction of

thiobarbituric acid (TBA) with products of lipid peroxidation like malondialdehyde

(MDA) and others collectively called as thiobarbituric acid reactive substances

(TBARS). The results were expressed as n moles MDA formed/mg protein/60 min.

Non-enzymatic antioxidants:

Glutathione content:

The glutathione (GSH) content in the liver and kidney was measured by the

method of Grunert and Philips (1951). In saturated alkaline medium, the GSH present

in the tissues reactes with sodium nitroprusside to give a red coloured complex which

37
was measured at 520 nm. The glutathione content was expressed as µg/100 mg tissue

weight.

Total ascorbic acid content:

Total ascorbic acid (TAA) content was estimated in the liver and kidney by the

method of Roe and Kuether (1943). TAA is oxidized to dehydroascorbic acid (DHA)

by Norit reagent in the presence of TCA. This couples with 2, 4-dinitrophenyl

hydrazine in the presence of thiourea and sulphuric acid to yield a red coloured

complex which was read at 540 nm. TAA content was expressed as mg/gm tissue

weight.

Enzymatic antioxidants:

Catalase (EC.[Link]) activity:

The catalase (CAT) activity was assayed in the liver and kidney by the method of

Luck (1939). The assay mixture consisted of 50 mM phosphate buffer (pH 7.0),

aliquot and 10 mM H2O2 which was added to initiate the reaction. The decrease in

absorbance was noted every 5 seconds at 240 nm. The enzyme activity was expressed

as µ moles H2O2 consumed/mg protein/min.

Superoxide dismutase (EC.[Link]) activity:

The superoxide dismutase (SOD) activity in the liver and kidney was assayed by

the method of Kakkar el al. (1984) with slight modification. This method is based on

the NADH-phenazine methosulfate-nitroblue tetrazolium formazon inhibition. The

formazon formed at the end of the reaction was extracted into butanol layer, upon

inactivation of the reaction with acetic acid. The enzyme activity was expressed as

38
units/mg protein. One unit of enzyme activity is defined as the enzyme concentration

required to inhibit the optical density of chromogen production at 560 nm by 50% in 1

min under the assay condition. The enzyme activity was expressed as units/mg

protein.

Glutathione reductase (E.C.[Link]) activity:

The glutathione reductase (GR) activity in liver and kidney was assayed by the

method of Mavis and Stellwagen (1968). The enzyme catalyzes the conversion of

oxidized glutathione (GSSG) to reduced glutathione (GSH). The decrease in

absorbance was recorded for 5 min at 340 nm. The enzyme activity was calculated as

nmoles NADPH consumed/mg protein/min.

Glutathione-S-transferase (EC [Link]) activity:

The glutathione-S-transferase (GST) activity was assayed by the method of Habig

et al. (1974). The increase in absorbance was noted at 340 nm using 1-chloro-2,4-

dinitrobenzene (CDNB). The enzyme activity was calculated as µmoles CDNB

conjugates formed/mg protein/min.

Glutathione peroxidase (EC.[Link]) activity:

The glutathione peroxidase (GSH-Px) activity in the liver and kidney was assayed

by the modified method of Pagila and Valentine (1967). The enzyme activity was

expressed as units/mg protein/min, where 1 unit of GSH-Px equals to nmoles of

NADPH consumed/mg protein/min.

Serum parameters

39
 Methods for measurement of alanine transaminase (ALT), aspartate

transaminase (AST), alkaline phosphatase (ALP) and acid phosphatase (ACP), as

well as protein and cholesterol contents were as described earlier in part I section.

Serum creatinine content:

Creatinine present in the serum reacts with picric acid in alkaline medium to

form an orange red colour which can be measured colourimetrically (Jaffe reaction).

Estimation of creatinine was performed by the alkaline picrate method of Bonsnes and

Taussky (1945) as described by Varley (1988). The optical density of the orange

colour developed was read at 520 nm. The creatinine content was expressed as mg/dL

serum.

HISTOPATHOLOGICAL STUDIES:

Histopathological studies were carried out using the standard technique of

hematoxylin and eosin (H & E) staining. Liver and kidney tissues of all control and

treated animals were preserved in 10% neutral buffered formalin immediately after

the autopsy. The tissues were dehydrated by passing through ascending grades of

alcohol, cleared in xylene and embedded in paraffin wax (58 to 60˚C mp). 5 µm thick

sections were cut on a rotary microtome and stained with H & E, dehydrated in

alcohol, cleared in xylene, mounted in DPX and examined under a light microscope.

40
 PART II

PHYTOCHEMICAL ANALYSIS OF THE NIGELLA SATIVA

SEED EXTRACT

Extract preparation

Seeds of Nigella sativa was purchased from LVG, Ahmedabad and extract

was prepared according to the method of Bhargava and Singh (1981) with slight

modification. The finely ground seed powder was mixed with 50% methanol in water

and allowed to stand overnight for maximum extraction of polyphenols. Percolation

of the extract was performed at room temperature. Collected filtrate was evaporated

below 50˚C to obtain a residue which was stored under refrigerated conditions.

Percent yield of the extract was calculated. Polyphenolic contents of the extract was

qualitatively and quantitatively analyzed using standardized methods. The

antioxidative potency of both the extracts was estimated using various chemical assay

systems as described below:

Qualitative and quantitative analysis of polyphenolic content:

Qualitative analysis

Qualitative analysis for determining the presence of tannins, saponins,

flavonoids and alkaloids in the plant extracts were carried out using standard methods

as described by Harborne (1973), Trease and Evans (1989) and Sofowara (1993).

Test for tannins:

41
 0.5 gm of extract was dissolved in 20 mL distilled water in a test tube and then

filtered. A few drops of 0.1% FeCl3 was added and observed for brownish green or

blue black colour.

Test for saponins:

2 gm of the extract was boiled in 20 mL of distilled water in a water bath and

filtered. 10 mL of filtrate was mixed with 5 mL of distilled water and shaken

vigorously. The forth was mixed with 3 drops of olive oil and shaken vigorously and

then observed for formation of emulsion.

Test for flavonoids:

5 mL of dilute ammonia solution was added to plant extract, followed by

addition of concentrated H2SO4. A yellow colouration indicated presence of

flavonoids.

Test for alkaloids:

The test was performed with Mayer’s, Wagner’s and Dragendorff’s reagents.

Observation of white, brown, orange colouration indicated the presence of alkaloids.

Quantitative analysis:

The quantitative analysis of the plant extract was performed using standard

protocols as mentioned below:

Total phenolic content (TPC):

Total phenolic content of hydro-alcoholic extract was estimated by the method

as described by Singleton et al. (1999). Briefly extract react with Folin-Ciocalteu

42
reagent in the presence of sodium carbonate to form a blue coloured complex which

was read at 760 nm. Total phenolic content of the extract was expressed as mg gallic

acid equivalents/gm dry weight of extract.

Flavonoid content:

The flavonoid content in the extract was estimated by the method of Lamaison

and Carnat (1990). Briefly 1 ml of plant extract was mixed with 1 ml of aluminium

chloride reagent and resultant colour was read at 430 nm. The flavonoid content of the

extract was expressed as mg quercetin equivalents/gm dry weight of extract.

Tannin content:

The tannin content of the extract was estimated by the method as described by

Price and Butler (1977). Plant extract was allowed to react with K3Fe(CN)6-FeCl3

reagent for five min and the intensity of colour developed was measured

spectrophotometrically at 720 nm. The tannin content of the extract was expressed as

mg rutin equivalents/gm dry wt. of extract.

Ascorbic acid content:

Ascorbic acid, also known as vitamin C, is one of the most abundant

antioxidant present in plant, was quantified by the method of Jagota and Dani (1982).

The ascorbic content of plant extract was expressed as µg/gm dry weight of extract.

Antioxidative potency:

Superoxide radical scavenging assay:

Superoxide radical scavenging activity was assessed by the method of Liu et

al. (1997). In the PMS/NADH-NBT system, superoxide anion derived from dissolved

43
O2 by PMS/NADH coupling reaction reduces NBT. The addition of various

concentrations of hydro-alcoholic extract resulted in decreased colour intensity which

was read at 560 nm against blank to determine the quantity of the formazon generated.

IC50 value of the extract (concentration required to scavenge 50% of the radicals) was

calculated.

Hydroxyl radical scavenging assay:

The hydroxyl radical scavenging activity of the extract was estimated by the

method of Halliwell et al. (1987), where radicals were generated from

Fe+3/ascorbate/EDTA/H2O2 from Fenton’s reaction. Briefly different concentrations of

plant extract was made to react with 2-deoxy-2-ribose, H2O2, FeCl3 and EDTA. The

reaction was initiated by the addition of ascorbic acid. After incubation of 90 min the

reaction was estimated by addition of thiobarbituric acid (TBA) and resulting colour

was red at 590 nm. Percent inhibition by various concentrations of plant extract and

IC50 was calculated.

Nitrous oxide radical scavenging assay:

Nitrous oxide radical scavenging acivity was measured using method of

Sreejayan and Rao (1997). Various concentrations of plant extract was incubated

with 10 mM sodium nitroprusside and incubated for 150 min. After incubation, Griess

reagent was added to the tubes and absorbance of chromophore formed was read at

590 nm. IC50 value and percent inhibition by various concentrations of plant extract

was calculated comparing the absorbance of control and test compounds against

blank.

44
DPPH radical scavenging assays:

Ability of plant extract to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH)

radical was measured by the method of Gymfi et al. (1999). DPPH is a purple colour

radical compound which changes to stable compounds having yellow colour by

reacting with antioxidant compounds. Addition of 0.1 mM DPPH solution in various

concentrations of extract in presence of tris-HCl buffer (50 mM, pH-7.4) resulted in

decreased absorbance which was measured at 517 nm. Percent inhibition was

calculated by measuring the absorbance of plant extract treated samples against blank.

IC50 value for the extract was calculated.

45
 PART III

TO STUDY THE AMELIORATIVE EFFECT OF NIGELLA SATIVA

SEED EXTRACT ON TOXICITY INDUCED BY DIETHYL

PHTHALATE

STUDY DESIGN

Based on the results of part – I, the high dose (HD) of DEP was chosen further

to evaluate the hepatoprotective effect of Nigella sativa seed extract. Fifty animals

were divided into five groups. Animals of Group 1 received 0.2 mL olive

oil/animal/day for 30 days and marked as vehicle control. Antidote control group

(Group 2) animals were orally administered with Nigella sativa seed extract (300

mg/kg body weight/day) for 30 days. Animals of Group 3 received HD (1240 mg/kg

body weight/day) of DEP for 30 days. Animals of Group 4 and 5 were treated with

HD of DEP along with 150 and 300 mg/kg body weight/day of Nigella sativa seed

extract for 30 days.

Behavioural and clinical changes throughout the treatment were observed in the

animals of all groups. On the completion of the treatment (30 days), animals were

humanely sacrificed on 31st day by cervical dislocation. The liver and kidney were

dissected out carefully, blotted free of blood, weighed to the nearest mg and utilized

for the study. For studying serum parameters, the blood collected by cardiac puncture

was allowed to clot and centrifuged at 1000 x g for 10 min to obtain serum. The

obtained serum samples were stored under refrigerated conditions and used within 24

h.

46
BIOCHEMICAL ANALYSIS

Protein, lipid, carbohydrate and nucleic acid contents

The protein, glycogen, total protein, cholesterol, DNA and RNA contents were

estimated using standard protocols as described in part I materials and methods

section.

Enzymatic assays

The activities of ALT, AST, ACP and ALP in liver and kidney were done using

standard methods as described in part I materials and methods section.

Lipid peroxidation and antioxidative defense mechanism:

Lipid peroxidation:

The lipid peroxidation was estimated using standard methods as described in part I

materials and method section.

Non- enzymatic antioxidants:

Methods used for measurement of glutathione and total ascorbic acid

contents were described in part I material method section.

Enzymatic antioxidants:

Methods used for assessment of superoxide dismutase, catalase, glutathione

reductase, glutathione transferase and glutathione peroxidase were described in

part I materials and method section.

Histopathological studies:

Tissues of all control and treated animals were preserved in 10% neutral

buffered formalin for histopathological examination after autopsy. The standard

technique of H & E was followed as described in part I material methods section.

47
Hepato/reno-protective index:

The liver/kidney protecting activity of the Nigella sativa seed extract was

expressed as hepatoprotective/renoprotective percentage (H/R) (Prakash et al. 2008)

which was calculated using the formula:

Where T is the mean value of plant extracts along with the DEP, C is the mean value

of DEP alone, and V is the mean value of vehicle control animals.

Statistical analysis:

All the data are expressed as the means ± standard error mean (SEM).

Statistical analysis, linear regression analysis and Pearson correlation analysis of

relationship between experimental parameters were performed using Graphpad Instat

software version 5.03. The data were statistically analyzed using One - way Analysis

of Variance (ANOVA) followed by Tukey’s test. The level of significance was

accepted with p < 0.05. The IC50 values were calculated by probit analysis.

48