MATERIALS AND METHODS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/8943/13/13_chapter...

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MATERIALS AND METHODS

4.1. Plant materials

Bark of Tecomella undulate and roots of Plumbago zeylanica were procured from

Khambadia village near to Jamnagar, Gujarat. Fruits of Ziziphus jujube and dried

rhizomes of Zingiber officinale were purchased from the market, Khari Bavli, New Delhi.

All the plant materials were authentified by Prof. A.K. Rawat, National Botanical

Research Institute (NBRI), Lucknow, India and the voucher specimens were deposited

into Institutional Herbarium (NBRI-SOP-202).

4.2. Extraction of plant material

The dried plant materials were pulverized and coarsely powdered. Each powdered batch

(500g) was defatted using petroleum ether and extracted with 95% methanol using a

Soxhlet extractor. The bark of T.undulate was extracted using 90 % ethanol, fractionated

using solvents of graded polarities like the butanolic and water fractions. After exhaustive

extraction, the methanolic extract was dried using rota-evap apparatus.

4.3. Preliminary phytochemical screening

Qualitative phytochemical screenings of each plant extract as made to investigate the

presence of major chemical classes of components e.g. alkaloids, flavonoids, glycosides,

tannins, phenols and steroids (Cannell, 1998).

4.4. Animals

Wistar albino rats (180-240 g) of either sex, procured from Animal House, I.S.F. College

of Pharmacy, Moga, Punjab, were employed in the present study. They were maintained

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under standard conditions of temperature (25±2 oC), humidity (45-55 %) and 12/12 h

light and dark cycles. Animals were fed on standard chow diet purchased from

Aashirwad Industries Ltd, Ropar, Punjab and water ad libitium. All the experiments were

conducted after approval of study protocol by the Institutional Animal Ethics Committee

(IAEC) and in accordance with the guidelines of the Committee for the purpose of

control and supervision on experiments on animals (CPCSEA) for the use and care of

experimental animals.

4.5. Drugs and chemicals

The drugs used were purchased as Paracetamol from Glaxo SmithKline Pharma,

Bangalore; Silymarin from Micro Labs Limited., Baddi; Carbon tetrachloride from

Merck Specialities Pvt. Ltd., Mumbai; and ethanol (99.9%) from Rankem Ltd., Mumbai.

All other chemicals and biochemical reagents of analytical grade were used as freshly

prepared solutions. Biochemical enzymatic kits were purchased from ERBA, diagnostics

Mannheim Gmbh, Germany.

4.6. Acute toxicity study

Wistar albino rats (n=3), selected by random sampling technique, were used in the study.

Acute oral toxicity was performed as per OECD-423 guidelines (Acute toxic class

method) (Botham, 2004). The animals were fasted overnight, provided only water after

which test drugs were administered to groups orally at the dose level of 5 mg/kg and

observed for 14 days. If mortality was not observed, the procedure was repeated for

further higher doses such as 50, 300 and 2,000 mg/kg body weight. The animals were

observed for toxic symptoms such as behavioral changes, locomotion and mortality.

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4.7. High performance liquid chromatographic (HPLC) study of plant extracts:

The plant extracts were studied for the presence of plumbagin in P.zeylanica, and

gingerol in Z.officinale using Reverse Phase HPLC. Moreover, the presence of gallic acid

(GA) (a tannin part) was also studied in each plant extract. Waters 515 HPLC equipped

with 20 µl Rheodyne manual injector and W2998 PDA detector was used for HPLC

analysis. The separation was achieved using Thermo Hypersil C18 column

(150mmx4.6mm ID, 3um) with isocratic flow rate of 1ml/min (for plumbagin and gallic

acid), 1.3 ml/min (for gingerol), and the UV detection technique was used. The weighed

amount of standard and test samples were diluted in mobile phases: methanol: water

(70:30) (for plumbagin at 265 nm wave length); acetonitrile : water (55:45) (for gingerol

at 280 nm wavelenght); and water/acetic acid/methanol (isocratic, 80: 5: 15 v/v/v) for GA

respectively, filtered through 0.22 µ syringe filters and injected into chromatograph. The

injection volume was 20 �l and the column temperature was maintained at 30 ºC.

4.8. Paracetamol (PCM)-induced hepatotoxicity:

Animals were divided into 15 groups as mentioned below (n=8). Test drugs and standard,

dissolved in distilled water, were administered orally for 7 days. Paracetamol (3g/kg,

p.o.) was given on 3rd and 5th days to induce hepatic injury (Porchezhiana and Ansari,

2005).

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Table. 1. Experimental protocol for paracetamol induced hepatotoxicity

S.No. Group Treatments

1. Control Normal saline treated

2. Paracetamol control (PC) Paracetamol 3g/kg, p.o.

3. T.undulate control 100

(TU100)

Tecomella undulate extract 100mg/kg, p.o. +

Paracetamol 3g/kg


(TU200)


Paracetamol 3g/kg


(TU400)


Paracetamol 3g/kg

6. P.zeylanica extract 100

(PZ100)

Plumbago zeylanica extract 100mg/kg, p.o. +

Paracetamol 3g/kg


(PZ200)


Paracetamol 3g/kg


(PZ400)


Paracetamol 3g/kg

9. Z.officinale extract 100

(ZO100)

Zingiber officinale extract 100mg/kg, p.o. +

Paracetamol 3g/kg


(ZO200)


Paracetamol 3g/kg


(ZO400)


Paracetamol 3g/kg

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12. Z.jujube extract 100 (ZE100) Ziziphus jujube extract 100mg/kg, p.o. +

Paracetamol 3g/kg


Paracetamol 3g/kg


Paracetamol 3g/kg

15. Silymarin (SILY50) Silymarin 50mg/kg, p.o. + Paracetamol 3g/kg

4.9. Carbon tetrachloride (CCl4) induced hepatotoxicity:

Animals were divided into 15 groups as mentioned below (n=8). Test drugs and standard

dissolved in distilled water, were administered orally for 7 days. CCl4 (1ml/kg, s.c.)

diluted in olive oil (1:1) was given on 4th and 5th days, 2 hrs after drug administration, to

induce hepatic injury (Rosa and Solis, 2009).

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Table. 2. Experimental protocol for CCl4 induced hepatotoxicity



2. CCl4 control (CCl4C) CCl4 1ml/kg, s.c., (1:1) in Olive oil


(TU100)


CCl4 1ml/kg


(TU200)


CCl4 1ml/kg


(TU400)


CCl4 1ml/kg


(PZ100)


CCl4 1ml/kg


(PZ200)


CCl4 1ml/kg


(PZ400)


CCl4 1ml/kg


(ZO100)


CCl4 1ml/kg


(ZO200)


CCl4 1ml/kg

11. Z.officinale extract 400 Zingiber officinale extract 400mg/kg, p.o. +

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(ZO400) CCl4 1ml/kg

12. Z.jujube extract 100

(ZE100)

Ziziphus jujube extract 100mg/kg, p.o. + CCl4

1ml/kg


(ZE200)


1ml/kg


(ZE400)


1ml/kg

15. Silymarin (SILY50) Silymarin 50mg/kg, p.o. + CCl4 1ml/kg

4.10. Alcohol-induced hepatotoxicity:

Animals were divided into 15 groups as mentioned below (n=8). Alcohol (ethanol 28%)

15g/kg, p.o. was given daily for 4 weeks to induce chronic hepatic injury (Tsukamoto,

1998). Test drugs and standard were given orally for 4 weeks, two hr before the ethanol

administration .

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Table. 3. Experimental protocol for alcohol induced hepatotoxicity



2. Alcohol control (AC) Ethanol (30%), 15 g/kg, p.o.


(TU100)


Ethanol 15g/kg


(TU200)


Ethanol 15g/kg


(TU400)


Ethanol 15g/kg


(PZ100)


Ethanol 15g/kg


(PZ200)


Ethanol 15g/kg


(PZ400)


Ethanol 15g/kg


(ZO100)


Ethanol 15g/kg


(ZO200)


Ethanol 15g/kg

11. Z.officinale extract 400 Zingiber officinale extract 400mg/kg, p.o. +

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(ZO400) Ethanol 15g/kg


(ZE100)

Ziziphus jujube extract 100mg/kg, p.o. +

Ethanol 15g/kg


(ZE200)


Ethanol 15g/kg


(ZE400)


Ethanol 15g/kg

15. Silymarin (SILY50) Silymarin 50mg/kg, p.o. + Ethanol 15g/kg

Animals were anaesthesized with di-ethyl ether, blood was collected and centrifuged at

3000 rpm to obtain serum for biochemical estimations. The animals were sacrificed and

liver was dissected out. The isolated liver was washed with cold saline, blotted dry and

weighed. A 10 % liver homogenate, of each liver, was prepared in 0.15 M Tris HCl

buffer (pH 7.4) for tissue biochemical estimations. A part of homogenate after

precipitating proteins with trichloro-acetic acid (TCA) was used for estimation of

glutathione using phosphate buffer (pH 7.4). A major lobe of the each liver was fixed in

10% formalin solution for histological examination.

4.11. Pharmacological assessments:

4.11.1. Ponderal changes:

4.11.1.1. Body weight: Change in body weight was expressed as % body weight and

calculated using following formula:

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% Body weight = Final b.wt. - Initial b.wt. × 100

Final b. wt.

4.11.1.2. Liver weight (Relative weight):

Change in liver weight was expressed as liver weight/100 g body weight referred as

relative wt. of liver.

4.11.2. Biochemical Estimations:

4.11.2.1. Serum Biochemical Estimations: The serum separated from the blood, was

subjected to the following biochemical estimations using commercial biochemical

enzymatic diagnostic kits:

4.11.2.1.1. Alanine transaminase (ALT) (Bradley et al., 1972):

Principle:

ALT catalyzes the transfer of amino group between L-Alanine and �-Ketoglutarate to

form pyruvate and glutamate. The pyruvate formed reacts with NADH in the presence of

lactate dehydrogenase to form NAD. The rate of oxidation of NADH to NAD is

measured as a decrease in absorbance which is proportional to the ALT.

L-Alanine +�-ketoglutarate Pruvate + L-Glutamate

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Pyruvate + NADH + H+ Lactate + NAD+

Procedure:

Wavelength/ filter : 340nm

Temprature : 37• C/ 30• C/ 25• C

Light path : 1cm

Calculations:

ALT activity in U/L 25•C/30•C = A/min. × 952

ALT activity in U/L 37•C = A/min. × 1768

4.11.2.1.2. Aspartate transaminase (AST) (Bradley et al., 1972):

Principle:

AST catalyzes the transfer of amino group between L-Alanine and �-Ketoglutarate to

form oxaloacetate and glutamate. The oxaloacetate formed reacts with NADH in the

presence of Lactate Dehydrogenase to form NAD. The rate of oxidation of NADH to

NAD is measured as a decrease in absorbance which is proportional to the AST.

L-Alanine + �-ketoglutarate Oxaloacetate + L-Glutamate

Pyruvate + NADH + H+ Malate + NAD+

Procedure:

Wavelength/ filter : 340 nm

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Temprature : 37• C/ 30• C/ 25• C

Light path : 1cm

Calculations:

AST activity in U/L 25•C/30•C = A/min. × 952

AST activity in U/L 37•C = A/min. × 1768

4.11.2.1.3. Alkaline Phosphatase (ALP) (Lieberman and Phillips, 1990):

Principle:

ALP at an alkaline pH hydrolysis p-nitrophenylphosphate to form p-nitrophenol and

phosphate. The rate of formation of p-nitrophenol is measured as an increase in

absorbance which is proportional to the ALP activity in the sample.

p-Nitrophenylphosphate p-Nitrophenol + Phosphate

Procedure:

Wavelength/ Filter : 405 nm

Temprature : 37 •C / 30• C / 25• C

Light Path : 1cm

Calculation:

ALP activity in U/L = A/min. × 2754

4.11.2.1.4. Bilirubin level (Jendrassik and Grof, 1938):

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Principle:

Bilirubin reacts with diazotised sulphanilic acid to form coloured azobilirubin compound.

The unconjugate bilirubin couples with the sulphanilic acid in the presence of a caffeine-

benzoate accelerato. The intensity of the colour formed is directly proportional to the

amount of bilirubin present in the sample.

Bilirubin + Diazotised Sulphanilic acid Azobilirubin Compound

Procedure:

Wavelength : 546 nm

Temperature : Room temperature

Light Path : 1cm

Calculations:

Serum bilirubin in mg/dl = (Abs.of test – Abs. of blank) × 26.312

4.11.2.2. Tissue Biochemical Estimations:

4.11.2.2.1. Lipid Peroxidation (TBARS)

The tissue lipid peroxidation reaction was assessed by estimating thiobarbituric acid

reactive substances (TBARS) by the method of Wills et al. (1966) with some

modifications. Briefly, 500 µl homogenate was added to equal amount of the same buffer,

and incubated for 2 hrs at 37oC. After incubation, 1 ml of 10 % trichloroacetic acid

(TCA) was added to the mixture and centrifuged at 3000 rpm for 10 min. The supernant

(1 ml) was added to 1 ml of 0.67 % TBA solution and boiled for 15 minutes on water

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bath. The samples were cooled in tap water and 1 ml of distilled water was added. The

color intensity was determined at 532 nm. TBA reacting compound was expressed as nM

of malonaldehyde (MDA) / g of liver. Tubes containing known levels of MDA were

treated similarly and used as standards.

4.11.2.2.2. Reduced Glutathione (GSH)

The reduced glutathione (GSH) level was assessed by the method of Ellman, 1959.

Briefly, the tissue homogenate in 0.1 M phosphate buffer pH 7.4 was mixed with equal

volume of 20% trichloroacetic acid (TCA) containing 1 mM EDTA to precipitate the

tissue proteins. The mixture was allowed to stand for 5 min prior to centrifugation for 10

min at 200 rpm. The supernatant (200 �l) was then transferred to a new set of test tubes

and added 1.8 ml of the Ellman's reagent (DTNB: 5, 5'-dithio bis-2-nitrobenzoic acid)

(0.1 mM) was prepared in 0.3 M phosphate buffer with 1% of sodium citrate solution.

Then, all the test tubes make up to 2 ml volume. The absorbance of the solution was

measured at 412 nm against blank and the amount of reduced GSH was expressed as

µM/mg of protein.

4.11.2.2.3. Superoxide dismutase (SOD)

The tissue superoxide dismutase (SOD) activity was assessed by the method of Kakkar et

al (1984). The assay mixture contained 0.1 ml of sample, 1.2 ml of sodium

pyrophosphate buffer (pH 8.3, 0.052 M), 0.1 ml phenazine methosulphate (186 �M), 0.3

ml of 300 �M nitroblue tetrazolium, and 0.2 ml NADH (750 �M). Reaction was started

by addition of NADH. After incubation for 90 s at 30°C, the reaction was stopped by the

addition of 0.1 ml glacial acetic acid. Reaction mixture was stirred vigorously with 4.0 ml

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of n-butanol. Mixture was allowed to stand for 10 min, centrifuged and butanol layer was

separated. Color intensity of the chromogen in the butanol layer was measured at 560 nm

spectrophotometrically.�

4.11.2.2.4. Tissue Nitrite/Nitrate level

The amount of stable nitrite (nitrite and nitrate), the end product of NO generation was

determined by a colorimetric assay as described by Green et al (1982). Briefly, 500µl of

supernatant obtained from homogenate was mixed with an equal volume of Griess

reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2.5%

H3PO4), and incubated at room temperature for 10 min. The absorbance was read at 540

nm. The amount of nitrite was calculated from a sodium nitrite (NaNO2) standard curve

and was expressed as µM/mg of protein.

4.11.2.2.5. Na+K

+ATPase activity

Na+K+ATPase activity was determined by measuring ATP hydrolysis in a reaction

medium containing 100 mM NaCl, 30 mM KCl, 10 mM MgCl2 and 0.5 mM EGTA in 20

mM imidazole buffer (pH 7.4) by the method of Bonting (1970). An aliquot of the sample

was added to the reaction mixture and pre-incubated for 5 min at 30 °C. The reaction was

initiated by the addition of ATP (final concentration 5 mM). Incubation was carried out at

30 °C for 15 min. The reaction was stopped by addition of 2 ml of cooled Bonting's

reagent (560 mM sulphuric acid, 8.1 mM ammonium molybdate and 176 mM ferrous

sulphate). After 20 min at room temperature, the amount of released Pi was determined

by reading the absorbance at 700 nm of the reduced phosphomolybdate complex.

4.11.2.2.6. Tissue Collagen level:

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Tissue collagen level was assessed by estimating hydroxyproline content colorimetrically

by the modified method of Jamall et al (1981). Briefly, the frozen tissue was

homogenized in 4 ml of 6N HCl and hydrolyzed at 110oC for 16 h. The hydrolysate was

filtered, and then 30 �l aliquot of the sample was evaporated under vacuum. The

sediment was dissolved in 1.2 ml of isopropanol and incubated with 0.2 ml of 0.84%

chloramines-T in acetate citrate buffer (pH 6.0) for 10 min at room temperature. Then,

1.0 ml of Ehrlich’s reagent was added and the mixture was incubated at 60oC for 25 min.

The absorbance of the sample solution was measured at 560 nm. The hydroxyproline

content in 100 mg of liver was calculated from the standard curve of 4-hydroxy-L-proline

and expressed as �g/100 mg liver weight.

Figure. 9. Standard curve for tissue TBARS level using Malondialdehyde (TBARS)

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Figure. 10. Standard curve for tissue reduced glutahione using Glutathione (GSH):

Figure. 11. Standard curve for tissue nitrite/nitrate level using Sodium nitrite:

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Figure. 12. Standard curve for tissue Na+K

+ATPase level using Potassium

dihydrogen phosphate:

Figure. 13. Standard curve for tissue collagen level using Hydroxyproline:

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4.11.3. Histopathological studies:

The major lobe of liver was transferred to formalin solution for fixation and later on

processed for histopathological studies following the standard procedure. The microtome

sections were cut processed and stained with haematoxylin and eosin.

4.11.3.1. Fixation

The tissues were excised out immediately after sacrifice, extraneous tissues were cleaned

off, cut into pieces of such appropriate thickness that the fixative readily penetrated

throughout the tissue to be fixed. Tissues transferred to the 10% formaldehyde solution

(37-40% formaldehyde) and allowed to remain in it till they were taken up for processing.

4.11.3.2. Tissue processing

Tissue processing involves dehydration, cleaning and infiltration of the tissue with

paraffin. The dehydrating agent used was ethyl alcohol; acetone and isoporopyl alcohol

can also be used. Following dehydration the tissue was transferred to a paraffin solvent.

Which is miscible with the dehydrating agent as well? These are known as clearing

agents such as chloroform and xylene.

Tissues were thoroughly washed by placing them under running tap water and

then conveyed through a series of following solvents as per schedule for dehydration,

clearing and paraffin infiltration.

Alcohol 70% 20 min

Alcohol 80% 20 min

Alcohol 90% 20 min

Alcohol 95% (2 changes) 20 min each

Isopropyl alcohol 20 min

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Acetone (2 changes) 20 min each

Chloroform (3 changes) 20 min each

Melted Paraffin Wax (60°C) 40 min

Next the tissues were embedded in paraffin wax to prepare tissue blocks, which

are oriented so that the sections are cut in desired plane of the tissue. Tissues are fixed to

metal object holder after trimming them to suitable size.

4.11.3.3. Section Cutting:

A smear of 5% Mayer's egg albumin was prepared and smeared on to the slide and dried.

The tissue sections of the 5 �m thickness were cut with the help of spencer type rotating

microtome. The tissue sections were put on slide and drops of water and then section

were floated in water on slide between 55-600C water drained off and slide dried on hot

plate at about 500C for 30 min. This section is ready for staining.

4.11.3.4. Staining procedure:

4.11.3.4.1. Mayer's haematoxylin stain:

Dissolve 50 g of ammonium or potassium alum in one liter of water with out heating.

Then dissolve haematoxylin 1.0 g in this solution; further add 0.2 g sodium iodate, 1.0 g

citric acid and 50 g chloral hydrate. Shake until colour solution is formed. The final

colour of stain is reddish violet

4.11.3.4.2. Eosin stain 2% w/v in alcohol

After fixing the section on slide the section were stained by serially placing them in the

following reagents

Xylol (2 changes) 3 min each

Acetone 3 min

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Alcohol 95% 3 min

Haematoxyline stain 20 min

Running water 20 min

Eosin Stain 5 min

Alcohol 95% (3 changes) 3 min each

Acetone (2 changes) 3 min each

Xylol (2 changes) 3 min each

After passing through, all the above reagents and stains the slides were mounted with

DPX (Diphenyl Phthalein Xylene) and cover slips were placed. Care was taken for

avoiding the air bubbles during mounting the slide. The slides were viewed under

Trinocular research Motik microscope at 10x magnification to note down the changes in

the microscopic features of the tissues studied.

4.12. Statistical Analysis

Results were expressed as mean ± Standard deviation (SD), analyzed by one-way

ANOVA followed by Bonferroni’s multiple comparison analysis as post-hoc test. p value

<0.05 was considered to be statistically significant. A Graphpad Prism Instate software

was used as statistical tool.

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