materials and methods 76 4. materials and - shodhganga

Materials and Methods

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4. MATERIALS AND METHODS The following Chemicals are used for Experimental studies

Table: 4.1 List of chemicals used during experiments

1 Petroleum ether (Nice) 2 Methanol (Nice) 3 Chloroform (Nice) 4 Ethanol (Nice) 5 Potassium ferricyanide (SRL) 6 Trichloroacetic acid (TCA) –(SRL) 7 Ferric chloride (SRL) 8 Nitroblue tetrazolium (SRL) 9 NADH (SRL) 10 Phenazine methosulphate (SRL) 11 Phenyl hydrazine hydrochloride (sd fine chem ltd.) 12 Deoxy ribose (Merk) 13 Thiobarbituric acid (TBA) (Loba chemie) 14 Liquid paraffin (Nice) 15 Silymarin (Microlab) 16 Disodium hydrogen phosphate (Qualigen) 17 Dithiobisnitrobenzoate (DTNB) (Sigma Co.) 18 Paracetamol 19 Hydrochloric acid (HCl) (sd fine chem ltd.) 20 Formalin (Nice) 21 Anaesthetic ether (Sigma solvents and pharmaceuticals) 22 Sodium metabisulphate (sd fine chem ltd.) 23 Potassium dihydrogen phosphate (Merk) 24 KCl (Nice) 25 KOH (Nice) 26 Chemical Kits -SGOT, SGPT, HDL, Total Bilirubin, Direct

Bilirubin, Cholesterol, ALP (Span diagnostics). 27 Thioacetamide (sd fine chem ltd.) 28 Sodium nitroprusside (sd fine chem ltd.) 29 Sulphanilamide (sd fine chem ltd.) 30 o-phosphoric acid (sd fine chem ltd.) 31 Naphthyl ethylene diamine dihydrochloride (sd fine chem ltd.)


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All the chemicals used in our study were of analytical grade. 4.0. Plant materials

The plant materials such as whole plant of Commelina clavata

flowers of Kigelia AfricanaandSpathodea campanulatawere collected

from arid lands of Anantapur district of Andhra Pradesh state. The

plants materials were then identified and authentified by Dr.

Venkatapathi Raju,Professor, Department of Botany, Sri

Krishnadevaraya University, Anantapur. A voucher specimen of

Commelina clavata, Kigelia Africanaand Spathodea campanulatahas

been deposited in the Herbarium of the Department of botany for the

further reference.

4.1. Ash values

The powdered material of Commelina clavata, Kigelia

Africanaand Spathodea campanulatawere used for the determination

of total ash, acid insoluble ash, water soluble ash and sulphated ash

according to the procedure laid down in Indian pharmacopoeia.

Determination of total ash

About 3gm of dried powdered material was accurately weighed

and taken into previously ignited and tarred crucible. The power was

evenly spread as a fine layer and ignited gradually increasing the

temperature to 450o C until it is devoid of carbon particles. The

crucible was cooled in desiccators and weighed. The procedure was

repeated to get a constant weight. The percentage of total ash was

calculated with reference to air dried material.


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Determination of acid insoluble ash

The ash obtained as described in the above method was boiled

gently with 25ml of 2N HCl for five minutes. The insoluble ash was

collected on ash less filter paper and washed with hot water until the

filtrate was neutral. The filter paper containing the insoluble matter

was transferred into silica crucible and was ignited to a constant

weight. The percentage of acid insoluble ash was calculated with

respect to air dried drug.

Determination of water soluble ash

To the crucible containing the total ash, 25ml of distilled water

was added and boiled for 5min and filtered through ash less filter

paper. NO.41. The filter paper containing the insoluble matter was

transferred into a silica crucible and ignited for 15minutes at a

temperature not exceeding 450o C and the procedure was repeated to

get constant weight. Subtract the weight of the ash in milligrams from

weight of the total ash to get water soluble ash. The percentage of

water soluble ash was calculated with respect to air dried material.

Determination of Sulphated ash

Three grams of powdered drug was accurately weighed and

taken in a silica crucible, ignited until the substance was thoroughly

charred, cooled and moistened the residue with 1ml of sulphuric acid,

heat gently until white fumes are no longer evolved and ignited at

80±250C until all carbon particles have been disappeared. Cool the

crucible and add few drops of sulphuric acid. Then ignited as before


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and further allowed to cool and weighed. The procedure was repeated

until constant weight was obtained. The percentage of sulphated ash

value was calculated with respect to air dried drug.

4.2. Elemental analysis

The elements like sodium, potassium, magnesium, calcium,

arsenic, lead, palladium, and mercury were analysed by inductive

coupled plasma optical emission spectrophotometer (ICP-OES).

Digestion and preparation of sample

About 3 gms of dried powdered material was accurately

weighed and taken into crucible and ignited gradually increasing the

temperature to 500-600oc until colorless ash was obtained. 1 gm. of

above ash was treated with 10 ml HNO3 and ignited at 150oC until the

volume of nitric acid was reduced to half. To the above solution 10ml

of nitric acid was reduced to 5ml. The solution was cooled to room

temperature, then treated with 3ml per chloric acid and heated to

convert to dense fume, which was passed through inductively coupled

plasma optical emission spectrophotometer (ICP-OES).

Elemental analysis using inductive coupled plasma optical

emission spectrophotometer (ICP-OES-BERTY Series II VARIAN)

The elemental analysis of digested samples has been

determined by ICP-OES. The elements like sodium, potassium,

magnesium, calcium, arsenic, lead, palladium, and mercury have

been analyzed. In this method, the sample in the form of a

homogeneous liquid was introduced into plasma where the free atoms

capable of emitting characteristic wave length.


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The free atoms are excited to first exited state. Intensity of this

spontaneous emission is measured. The intensity of the radiated

emission can be measured by using

Icm = Aj×h×vj×N

Where

Aji = Transion probability for spontaneous emission

h = Planks constant

vji = Frequence of radiation

N = Number of atoms in the exited state.

4.3. Preparation of Extracts

The freshly collected plant materials were washed, shadow

dried and then dried in hot air oven at a temperature not more than

50°C. The dried materials were coarsely powdered using an electric

blender. Powdered materials (500g) were then packed in soxhlet

apparatus and successively extracted with Ethanol and methanol.

Each time before extraction with the next solvent, the powdered

materials were dried in hot air oven at below 50°C. Finally extracts

were concentrated in rotary evaporator at a temperature not more

than 50°C and then, dried under vacuum dessicator. The dried

extracts thus obtained were used for further experiments. In the

current research we have used the Ethanolic and methanolic extracts

of Commelina clavata, Kigelia AfricanaandSpathodea campanulata

plants.


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4.4. Phytochemical studies116, 117,118

Preliminary phytochemical screening was done using the

specified protocols for the qualitative analysis of Alkaloids,

carbohydrates, fixed oils, flavonoids, glycosides, phyto

sterol/terpenoids, saponins, and tannins/phenols. The screening

tests as follows:

1. Test for carbohydrates

About 100 mg of the extract was dissolved in 5 ml of distilled

water and filtered. The filtrate was subjected to the following tests.

i) Molisch’s test

To 2 ml of filtrate, two drops of alcoholic solution of α–naphthol

was added. The mixture was shaken well and 1 ml of concentrated

sulphuric acid was added slowly along the sides of the test tube. The

test tube was cooled in ice water and allowed to stand for few

minutes. A violet ring at the junction indicates the presence of

reducing sugars.

ii) Fehling’s test

1 ml of filtrate was boiled on a water bath with 1 ml each of

Fehling’s solution A and B. Formation of red precipitate indicates the

presence of sugars.


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iii) Barfoed’s test

To 1 ml of the filtrate, 1 ml of Barfoed’s reagent was added and

boiled on a boiling water bath for 2 minutes. Red precipitate indicates

the presence of sugars.

iv) Benedict’s test

To 0.5 ml of filtrate, 0.5 ml of Benedict’s reagent was added.

The mixture was heated on a water bath for 2 minutes. Red

precipitate indicates the presence of sugars.

2. Test for proteins and amino acids

About 100 mg of extract was dissolved in 10 ml of distilled

water and filtered through Whatmann’s No.1 filter paper and the

filtrate was subjected to tests for proteins and amino acids.

i) Millon’s test

To 2 ml of filtrate, few drops of Millon’s reagent were added. A

white precipitate indicates the presence of proteins.

ii) Biuret’s test

2 ml of filterate was treated with one drop of 2% copper

sulphate solution, 1 ml of 95% of ethanol, followed by excess of

potassium hydroxide pellets. Pink color in the ethanolic layer

indicates th0e presence of proteins.


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iii) Ninhydrin test

About 2 drops of ninhydrin solution was added to 2 ml of

aqueous filtrate. A characteristic purple colour indicates the presence

of amino acids.

3. Test for fats and oils

i) Spot test

A drop of the extract was placed on the filter paper and the

stain was observed. Stain remains, indicates the presence of fixed

oils.

ii) Saponification test

Added few drops of 0.5N alcoholic KOH to a small quantity of

various extracts along with a drop of phenopthalein separately and

heated on a water bath for 1-2 hours. The formation of soap or partial

neutralization of alkali indicates the presence of fixed oils and fats.

4. Test for Steroids

Libermann-Burchard Reaction

To few ml of the extract dissolved in few drops of chloroform,

3ml of acetic anhydride and 3ml of glacial acetic acid were added.

Warm and cooled under the tap. Drops of Conc. H2SO4 were added

along the sides of the test tube. Appearance of red orbluish green

colour indicates the presence of steroids.

5. Test for Glycosides


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i) The extract was mixed with a little anthrone on a watch glass.

One drop of Conc. H2SO4 was added, made into a paste and warmed

gently over a water bath. Dark green colour indicates the presence

glycosides.

A. Extract 200mg of drug with 5ml of dilute H2SO4 by warming

on a water bath. Filter, neutralized the acid extract with 5% solution

of NaOH. Add 0.1ml of Fehling’s solutions A&B until it becomes

alkaline and heat on a water bath for 2minutes. Note the quantity of

red precipitate formed and compared with that of test B.

B. Extract 200mg of drug with 5ml of H2O instead of H2SO4 by

warming on a water bath. After boiling add equal amount of water

assured for NaOH in the above test. Add 0.1ml Fehling’s solutions

A&B until alkaline. And heat it on water bath for 2minutes. Note the

quantity of red precipitate formed. Compare the quantity of

precipitate formed in test B with that of test A. The precipitate in test

A is greater than test B, indicates presence of Glycosides.

6. Test for Anthroquinones

i) Borntrager’s test

The extract was added with dil. H2SO4 boiled, and filtered,

added ether, filtered, aqueous ammonia or caustic soda was added.

Pink, red or violet colour in the aqueous layer after shaking indicates

the presence of anthroquinones.

If glycoside is present then the test should be modified by

hydrolysing with hydrochloric acid as the first step.

ii) Legal’s test


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About 50 mg of the extract was dissolved in pyridine. Sodium

nitroprusside solution was added and made alkaline using 10%

sodium hydroxide solution. Presence of glycoside is indicated by a

characteristic pink color.

7. Test for Coumarin Glycosides

Place a small amount of sample in test and cover the test tube

with a filter paper moistened with dilute NaOH. Keep it on a water

bath for several minutes, removed the paper and exposed to the UV

light. Green fluorescence indicates the presence of Coumarin

Glycosides.

8. Test for Saponins

i) Foam test

Few ml of the extract was shaken with water. Persistent foam

indicates the presence of saponons.

ii) Haemolytic test

One drop of the extract was placed on a glass slide with a drop

of blood. Haemolytic zone indicates the presence of saponins.

9. Test for flavonoids

i) Shinoda’s test

To the extract 5ml of 90% alcohol, 0.5gm of Magnesium

turnings and Conc. HCl was added, boiled for few minutes. Pink or

red colour indicates the presence of flavonoids.

ii) Alkaline test


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To the extract alcohol and 10% NaOH solution or ammonia

were added. Dark Yellow colour indicates the presence of flavonoids.

iii) Zinc Hydrochloric Acid test

To this test solution add a mixture of Zn dust and concentrated

HCl. Formation of red colour after few minutes indicates the presence

of flavonoids.

10. Test for Alkaloids

i) Dragendorff’s test

To the extract few drops of acetic acid was added followed by

Dragendroff’s reagent and shaken well. Orange red precipitate

indicates the presence of alkaloids.

ii) Mayer’s test

To the extract few drops of dilute hydrochloric acid and Mayer’s

reagent were added. White precipitate indicates the presence of

Alkaloids.

iii) Wagner’s test

To a few ml of filtrate, few drops of Wagner’s reagent were

added along the sides of the test tube. Formation of reddish brown

precipitate confirms the test as positive.

iv) Hager’s test

To a few ml of filtrate, 1 or 2 ml of Hager’s reagent was added.

A prominent yellow precipitate indicates positive test.


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11. Test for Tannins and Phenolic compounds

i) Ferric chloride test

About 50 mg of extract was dissolved in distilled water and to

this few drops of neutral 5% ferric chloride solution was added.

Formation of blue/green/violet color indicates the presence of

phenolic compounds.

ii) Gelatin test

A little quantity of extract was dissolved in distilled water and 2

ml of 1% solution of gelatin containing 10% sodium chloride was

added to it. Development of white precipitate indicates the presence of

phenolic compounds.

iii) Lead acetate test

A small quantity of extract was dissolved in distilled water and

to this, 3 ml of 10% lead acetate solution was added. A bulky white

precipitate indicates the presence of phenolic compounds.

12. Test for phytosterols and Triterpenoids

i) Libermann-Burchard’s test

The extract was dissolved in acetic anhydride, heated to boiling,

cooled and then 1ml of concentrated sulphuric acid was added along

the side of test tube. Red, pink or violet colour at the junction of the

liquids indicates the presence of steroids/triterpenoids and their

glycosides.

ii) Salkwoski test


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Few drops of concentrated sulphuric acid are added to the

chloroform extract, shaken on standing, red colour to the lower layer

indicates the presence of steroids and golden yellow colour indicates

the presence of triterpenoids.

iii) Noller’s test

The extract was warmed with tin and thionyl chloride. Pink

colour indicates the presence of terpenoids.

13. Test for Gums and Mucilages

i) The extract was slowly added into a test tube containing

alcohol with constant stirring. The formation of precipitate indicates

the presence of Gums and Mucilages.

14. Test for Resins

i) To the extract 5-10 ml of acetic anhydride was added. Gently

heated and cooled. To this 0.5ml H2SO4 was added. Bright purplish

red colour rapidly changed into violet indicates the presence of

Resins.

4.6. In-vitro models for evaluating antioxidant activities:

Herbal plants are known to contain a variety of antioxidants.

Numerous substances have been suggested to serve as antioxidants.

It has been revealed that various phenolic antioxidants, such as

flavonoids, tannins, coumarins, xanthones and more recently

procyanidins scavenge radicals dose-dependently, thus they are

viewed as promising therapeutic drugs for free radical pathologies125.

Reactive oxygen species (ROS) and free radicals such as superoxide


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anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH-) are

constantly formed in the human body by normal metabolic action and

have been implicated in the pathologies of certain human diseases,

including cancer, ageing, diabetes and atherosclerosis. Their action is

opposed by a balanced system of antioxidant defenses including

antioxidant compounds and enzymes. Upsetting this balance causes

oxidative stress, which can lead to cell injury and death. Current

research into free radicals has confirmed that foods rich in

antioxidants play an essential role in the prevention of cardiovascular

diseases, cancers and neurodegenerative diseases. Therefore, much

attention has been focused on the use of natural antioxidants to

inhibit lipid peroxidation, or to protect the damage of free radicals117.

Scavenging of hydrogen peroxide

A solution of H2O2 (20mm) was prepared in phosphate buffer

saline (PBS, PH 7.4). Various concentration (10µg-100µg) of standard

and extracts was prepared, 1ml of the extract and standard was

dissolved in methanol in a separate volumetric flask and to this

solution 2ml of H2O2 solution in PBS was added, the absorbance was

measured at 230nm, after 10min against blank solution.

Determination of Reducing Power

Method based on the principle of increase in the absorbance of

the reaction mixture. Increase in the absorbance indicates increase in

anti-oxidant activity. Different concentration of extracts (20µg-

100µg) in 1ml of distilled water were mixed with 2.5ml of phosphate

buffer (0.2m;PH6.6) & 2.5ml of potassium ferricyanide [K3Fe(CN)6]


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(1%), the resulting mixture was incubated at 50oC for half an hour.

Then, 2.5ml of trichloroacetic acid (10%) was added to the mixture,

which was then centrifuged at 3000rpm for 10min. Finally 2.5ml of

upper layer solution was mixed with 2.5ml of distilled water and

0.5ml of FeCl3 (0.1%) were added. The absorbance was measured at

700nm in UV-Vis spectrophotometer against blank. Increasing of the

reaction mixture indicates increasing reducing power.

Estimation of Phospho molybdenum

In this method quantitative determination of anti-oxidant

capacity, through the formation of phosphor molybdenum complex.

The assay is based on the reduction of Mo (VI) to Mo (V) by the

sample analyte and subsequent formation of a green phosphate Mo

(V) complex at acidic pH. An aliquot of 0.3ml of sample solution

containing a reducing species in DMSO was combined in a test tube

with 3ml of reagent solution (0.6m H2SO4, 28mm sodium phosphate

and 4mm ammonium molybdate) then the tubes were covered with

aluminium foil and kept in a water bath at 95Oc for 90min. Then the

samples were cooled to room temperature, absorbance of each

solution was measured at 695nm against blank. The total anti-

oxidant was expressed as mm equivalent to ascorbic acid. The results

are tabulated in -respectively.

4.6. Determination of acute toxicity:

The acute oral toxicity was performed according to OPPTS

following up and down procedure. Colony bred female albino rats

Wistar strain (150-200gm) was maintained under controlled animal


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house condition with access to food and water ad libitum. The limit

test carried out first at 3200 mg/kg. b.w. All animals were observed

for toxic symptoms and mortality for 72 h119.

An attempt is made to assess the influence of pre-treatment

with CCM and KAM on the levels of Glutathione in-vivo in

CCl4,paracetamol induced hepatotoxicity.

1. Gultathione (GSH) estimation in CCl4, paracetamol induced

heapatotoxicity in rats

Glutathione estimation:

Glutathione is present in all type of living cells. Tissues such

as mammalian liver normally contain high levels of reduced

Glutathione. It has been suggested that GSH protects thiol groups in

protein from oxidation, functions as an intracellular redox buffer and

serves as a reservoir of cysteine120.

The role of GSH in determining the extent of liver damage has

been demonstrated in experiments where the hepatic concentration of

GSH is altered by toxin treatments. Depletion of GSH contents has

been reported to potentiate hepatic necrosis and covalent bonding of

toxic metabolites to cellular macromolecules121.

4.7. GSH estimation in CCl4 induced hepatotoxicity:

In the dose response experiment, animals were randomly

assigned into 9 groups of 6 individuals each.

Group-I Animals (-ve Control) were administered 1ml distilled water p.o., for 5 days

Group-II Animals (+ve Control) were administered 1ml

distilled water p.o., for 5 days


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Group-III Animals were administered with silymarin 50mg/kg p.o., for 5days.

Group-IV Animals were administered with CCM 100 mg/kg

p.o., for 5 days. Group-V Animals were administered with CCM 200 mg/kg

p.o., for 5 days. Group-VI Animals were administered with KAM 100 mg/kg

p.o., for 5 days. Group-VII Animals were administered with KAM 200 mg/kg

p.o., for 5 days. Group-VIII Animals were administered with SCM 100 mg/kg

p.o., for 5 days. Group-IX Animals were administered with SCM 200 mg/kg

p.o., for 5 days.

Group-I received liquid paraffin (1ml/kg) s.c., on 2nd and 3rd

day. group-II, III, IV, V, VI, VII, VIII and IX received CCl4:liquid

paraffin (1:1) at a dose of 2ml/kg s.c., on 2nd and 3rd day, after 30

min of vehicle, 50mg/kg silymarin, 100, 200mg/kg of CCM, KAM and

SCM respectively. Animals were sacrificed on the 5th day under mild

ether anesthesia. Hepatic tissues were collected and assessed105.

Tissue samples were homogenized in ice cold Trichloroacetic

acid (1 gm tissue plus 10 ml 10% TCA) in a Ultra Turrax tissue

homogenizer. Glutathione measurements were performed using a

modification of the Ellamn procedure (Aykae, et.all.)122. Briefly, after

centrifugation at 3000 rpm for 10 minutes, 0.5 ml supernatant was

added to 2 ml of 0.3 M disodium hydrogen phosphate solution. A 0.2

ml solution of dithiobisnitrobenzoate (0.4 mg/ml in 1% sodium

citrate) was added and the absorbance at 412 nm was measured


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immediately after mixing. % increase in OD is directly proportional to

the increase in the levels of Glutathione. Hence, % increase in OD is

calculated.

The results are compiled in Table: 5.10 and graphically depicted

in Figure 5.6.

4.8. GSH estimation in Paracetamol induced hepatotoxicity:

The method of R.R. Chattopadhyay was followed21

In the dose response experiment, albino rats were randomly

assigned into 9 groups of 6 individuals each.

Group-I Animals (- ve Control) were administered normal saline 1ml/kg p.o., for 7 days

Group-II Animals (+ ve Control) were administered normal

saline 1ml/kg p.o., for 7 days

Group-III Animals were administered with silymarin 50mg/kg p.o., for 7 days.

Group-IV Animals were administered with CCM 100 mg/kg

p.o., for 7 days. Group-V Animals were administered with CCM 200 mg/kg

p.o., for 7 days. Group-VI Animals were administered with KAM 100 mg/kg

p.o., for 7 days. Group-VII Animals were administered with KAM 200 mg/kg

p.o., for 7 days. Group-VIII Animals were administered with SCM 100 mg/kg

p.o., for 7 days. Group-IX Animals were administered with SCM 200 mg/kg

p.o., for 7 days. On 7th day, 30 min after normal saline, 50 mg/kg silymarin,

and CCM and KAM 100, 200mg/kg administered to Group-II, III, VI,


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VII, VIII and IX respectively, received paracetamol 2g/kg p.o. After 48

hours of paracetamol feeding, rats were sacrificed under mild ether

anesthesia. Hepatic tissues were collected and assessed.

Tissue samples were homogenized in ice cold Trichloroacetic

acid (1 gm tissue plus 10 ml 10% TCA) in a Ultra Turrax tissue

homogenizer. Glutathione measurements were performed using a

modification of the Ellamn procedure (Aykae, et.all.,)122. Briefly, after

centrifugation at 3000 rpm for 10 minutes, 0.5 ml supernatant was

added to 2 ml of 0.3 M disodium hydrogen phosphate solution. A 0.2

ml solution of dithiobisnitrobenzoate (0.4 mg/ml in 1% sodium

citrate) was added and the absorbance at 412 nm was measured

immediately after mixing. % increase in OD is directly proportional to

the increase in the levels of Glutathione. Hence, % increase in OD is

calculated.

The results are compiled in Table:5.11 and graphically depicted in

Figure:5.8.

Estimation of biochemical markers to assess liver functions:

Parameter assess for the liver functions:

- Serum glutamate pyruvate transaminase (SGPT/ALT)

- Serum glutamate oxaloacetate transaminase (SGOT/AST)

- Serum alkaline phosphatase (ALP)

- Serum total bilirubin

Estimation of Serum SGPT:

Principle:


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Glutamic-pyruvic Transaminase (GPT - ALT) catalyses the

reaction between alpha-ketoglutaric acid and alanine giving L-

glutamic acid and pyruvic acid. Pyruvic acid, in the presence of

lactate dehydrogenase (LDH) reacts with NADH giving lactic acid and

NAD. The rate of NADH consumption is determined photometrically

and is directly proportional to the GPT activity in the sample123.

SGPT (ALT) Catalyses the following reaction:

α- ketoglutarate + L-alanine ↔ L-glutamate + pyruvate

Estimation of SGOT (AST):

Principle:

SGOT catalyses the transfer of the amino group of L-aspartate

(ASP) to α-ketoglutarate of the (α-KG) resulting in the formation of

oxaloacetate (OAA) and L-glutamate (L-Glu). The oxaloacetate so

formed, is allowed to react with 2,4-DNPH to form 2,4 dinitrophenyl

hydrazone derivative which is brown colored in alkaline medium. The

hydrazone derivate of oxaloacetate similar to pyruvate is considerable

more chromogenic than that of α-KG. The final color developed does

not obey Beer’s law124.

L-aspartate + alpha-ketoglutarate oxaloacetate L-glutamate+

SGOT, pH 7. 4

Estimation of Serum Alkaline Phosphatase (ALP):

Principle:

Under alkaline condition, colorless p-nitrophenol is converted

to 4-nitrophenoxide, which develops a very intense yellow color. Its


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intensity is proportional to the activity of alkaline phosphatase in the

sample125.

P-Nitro-phenyl phosphate H2 O ALP P - nitrophenol phosphate++

Estimation of Serum Bilirubin:

Principle:

Total bilirubin in the sample reacts with diazotised sulphanilic

acid in the presence of DMSO.

Direct bilirubin (conjugated) reacts in acid environment with

diazotised sulphanilic acid. The formed coloured azobilirubin is

measured photometrically at 546 nm126-128.

4.9. Histopathological studies:

Processing of isolated liver:

The animals were sacrificed and the liver of each animal was

isolated. The isolated liver was cut in to small pieces and preserved

and fixed in 10% formalin for two days. Following this was the

washing step where by the liver pieces were washed in running water

for about 12 hours. This was followed by dehydration with

isopropylalcohol of increasing strength (70%, 80% and 90%) for 12

hours each. Then the final dehydration is done using absolute alcohol

with about three changes for 12 hours each.

The clearing was done by using chloroform with two changes

for 15 to 20 minutes each. After clearing, the liver pieces were

subjected to paraffin infiltration in automatic tissue processing unit.


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The Liver pieces were washed with running water to remove

formalin completely. To remove the water, alcohol of increasing

strengths was used since it is a dehydrating agent. Further alcohol

was removed by using chloroform and chloroform removed by paraffin

infiltration.

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 allow cooling.

Sectioning:

The blocks were cut using microtone to get sections of

thickness of 5µ. The sections were taken on a micro slide on which a

egg albumin (sticking substance) was applied. The sections were

allowed to remain in an oven at 600C for 1 hour. Paraffin melts and

egg albumin denatures, thereby fixes tissues to slide.

Staining:

Eosin is an acid stain. Hence it stains all the cell constituents

pink which are basic in nature, eg: Cytoplasm. Hemotoxylin basic

stain which stains all the acidic cell components blue eg:DNA in the

nucleus.

Procedure:

1. Deparaffinized the sections by washing with chloroform for

about 15 minutes.

2. Hydrate the sections by washing in isopropylalcohol of

decreasing strength (100%, 90%, 80%, 70%).


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3. Finally washed with water.

4. Stained with haemotoxylin for 15 minutes.

5. Rinsed in tap water.

6. Differentiated in 1% acid alcohol by 10 quick dips. Checked the

differentiation with a microscope. Nuclei were distinct and the

back ground was very light (or colorless).

7. Washed in tap water.

8. Dipped in (Lithium carbonate) until sections become bright blue

(3-5 dips).

9. Washed in running tap water for 10 to 20 minutes, if washing

is inadequate eosin will not stain evenly.

10. Stained with eosin for 15 seconds – 2 minutes depending on the

age of the eosin and the depth of the counter stain desired. For

even staining results, dip slides several times before allowing

them to set in the eosin for the desired time.

11. Dehydrated in 95% isopropyl and absolute isopropyl alcohol

until excess eosin is removed, 2 changes of 2 minutes each

(check under microscope).

12. An absolute isopropyl alcohol 2 changes of 3 minutes each.

13. Chloroform 2 changes of 2 minutes each.

14. Mounted in DPX (Desterene dibutyl phthalate xylene).

Results:

Nuclei - Blue colour

Cytoplasm - Various shades of pink identifying different tissue

Components.


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All the sections of the tissues were examined under microscope

for the analyzing the altered architecture of the liver tissue due to

CCl4, paracetamol, thioacetamide and D-GalN/LPS challenge and

improved liver architecture due to pretreatment with test extracts and

standard drug129,130.

materials and methods 76 4. materials and - shodhganga

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