MATERIALS AND METHODS

2.1. MATERIALS

2.1.1. Plant material

Authenticated plants of Ophiorrhiza rugosa var. decurnbens was collected from

Kozhikode, a northern district of Kerala and Hibiscus furcatus plants were collected

locally from Trichur, Kerala. Gossypin was generously supplied by Dr. G.A.Nair,

Research Scientist, Regional Research Institute, Thiruvananthapuram, Kerala, India.

For further biological activity studies gossypin was purchased from Sigma Chemical

Co. St. Luis, MO, USA.

2.1.2. Chemicals

1. Murashige and Skoog (MS) medium

2. Gamborg (B5) medium

3. Whites medium

4. Woody plant medium (WPM)

5. Minimal Essential Medium (MEM)

6. Rosewell Park Memorial Institute

medium (RPMI-1640)

7. 2,4-Dichloro phenoxy acetic acid

(214-D)

8. Naphthalene acetic acid (NAA)

9. Indole acetic acid (IAA)

10. Benzyl adenine (BA)

11. Indole 3-buhic acid (IBA)

12. Gibberrllic acid ( G h )

13. Kinetin (KN)

14. Carnptothecin (CPT)

Himedia Laboratories Pvt Ltd. India

Himedia Laboratories Pvt Ltd. India

Himedia Laboratories Pvt Ltd. India

Himedia Laboratories Pvt Ltd. India

Himedia Laboratories Pvt Ltd. India

Himedia Laboratories Pvt Ltd. India

Sigma Chemical Co. St Luis, USA

Sigma Chemical Co. St Luis, USA

Sigma Chemical Co. St Luis, USA

Sigma Chemical Co. St Luis, USA

Sigma Chemical Co. St Luis, USA

Sigma Chemical Co. St Luis, USA

Sigma Chemical Co. St Luis, USA

Sigma Chemical Co. St Luis, USA

15. Silica gel G Qualigens India

16. Silica gel for column chromatography Qualigens India

(60-120 mesh)

17. Trypsin E.Merck India Ltd

18. Streptomycin

19. Cefotaxim

E.Merck India Ltd

Alkem Laboratories Pvt Ltd, India

20. Agar agar Qualigens India '

21. Nitro blue tetrazolium (NBT) Sigma Chemical Co. St Luis, USA

22. MTT Sigma Chemical Co. St Luis, USA

23. Deoxyribose E.Merck India Ltd

24.7,12-Dimethyl benz (a)anthrazene Sigma Chemical Co. St Luis, USA (DMBA)

25. Cyclophosphamide

27. Cisplatin

28. Dimethyl sulphoxide (DMSO)

29.5,5'-dithio-bis 2-nitrobenzoic acid

30. Thiobarbituric acid

31. Gossypin

32. Aspirin

33. Ranitidin

2.1.3. Instruments

1. Laminar Flow chamber

2. Refrigerated centrifuge

3. Freeze drier

4. Microscope

5. Inverted microscope

6. UV chamber

7. UV spectrophotometer

8. Infra red spectrophotometer

Dabur India Ltd, India

Khandelwal Laboratories, India

E.Merck India Ltd

Sisco Research Laboratories, India

Sisco Research Laboratories, India

Sigma Chemical Co. St Luis, USA

German remedies Lid.

Torrent pharmaceuticals, India

Kemi Pvt Ltd, India

Remi Pvt Itd, India

Labconco, USA

Meiji, Japan

Willvert Will

Commag

Elico India Ltd.

Perkin Elmer

9. High performance liquid chromatography (HPLC)

Jasco, Japan

10. Mass Spectrophotometer (MS) Micromass Quattropole Mass spectrophotometer

11. Nuclear magnetic resonance (NMR) FT NMR 300MHz

12. Elisa Plate reader Awareness Technology Inc. USA

13. Melting point apparatus Kofler hot stage

2.1.4. Cell lines

Dalton's Lymphoma Ascites (DLA) cells Ada y ar Cancer Institute

Ehrlich ascites tumour (EAC) cells Adayar Cancer Institute

Mouse lung fibroblast (L929) cells National facility for animal cell and tissue culture, Pune, India.

Human myelogenous leukemia cells Indiana state University, USA (K562)

Human colon carcinoma cells (HT 29) Indiana state University, USA

2.1.5. Animals

Swiss albino mice

Wistar Rats

National Institute of Nutrition,

Hyderabad, India

College of Veterinary and Animal

Sciences, Thrissur, India

2.2. METHODS

2.2.1. Explants and surface sterilization

Varoius parts of the plant viz. internode, leaf disc and petiole served as the

explants. To minimize the rate of contamination in in vitro cultures prophylactic

sprays were given to source plants with bavistin 0.1% at weekly intervals. The

explants excised from the parent plant in early morning were soaked in tween 20 for

5 min and thoroughly washed with running tapwater for 30 min. The explants were

transferred to sterile laminar airflow hood (Kemi, India) for processing under

aseptic conditions. Surface sterilization was done by immersing the explants into

0.01-0.1 % MgClz for 1-4 min. The plant materials was then rinsed five times with

sterilized water, once quickly and 4 times for 10 rnin and then the explants were

transferred to appropriate nutrient medium in culture tubes/ flasks.

2.2.2. Preparation of Nutrient media

All cultures or the present study were inoculated either in Murashige and

Skoog medium (Murashige and Skoog, 1962), White's (White, 1943), and Gamborg

(B5) medium (Gamborg et al., 1977). For preparing the medium, all stock soIutions

were taken in appropriate portions and final volume was made up by the addition

of double distilled water and stock of plant growth regulators.

The prepared medium was supplemented with 10, 20, 30 and 40 % (w/v)

sucrose experimentally. After adding the growth regulators, the pH was adjusted to

5.6-5.8. Agar 0.8% (w/v) dissolved by heating, was used as the gelling agent for

semisolid medium and without agar for liquid medium.

The medium containing agar and all other ingredients was dispensed into

culture tubes and conical flasks, which were presterilized by autoclaving. The tubes

and flasks were plugged with sterile cotton plugs. The tubes containing medium

were finally sterilized by autoclaving at 121 OC and 15 lbs pressure for 20 min. The

cultures were placed in a culture room on a 16h/8h photoperiod with light intensity

2000-3000 lux by cool incandescent tubes maintained at 25k1° C and relative

humidity 60-80%.

2.2.3. Culture environment

Primary explants as well as pieces of calli were cultured in media and

incubated in a culture room maintained at 25OC. Callus induction started under dark

and calli were subcultured every 4 weeks to the same medium or other

combinations of the media. All cultures were alternatively exposed to 16 h

photoperiod at a photon flux density 30-50~ Em-2s-1 light fluorescent lamp.

2.2.4. Effect of auxins on callusing

Leaf, stem and petiole explants were cultured in 1/2 MS basal medium

supplemented with auxins such as 2,4-dichlorophenoxy acetic acid (2,4-D),

naphthalene acetic acid (NAA) and indole 3 acetic acid (IAA), each at 0.5,I.O and 2.0

mgl-I to study their relative effects on callus induction and growth. Observations

were recorded on percentage of cultures initiating calli, callus growth rate and

period taken for the calli to initiate. Pieces of calli (200 mg fw) were transferred to

fresh nutrient media and incubated for various periods of time. To determine the

growth kinetics of the callus cells, calli grown on different media were collected and

fresh weight (fw) and dry weight (dw) of calli were determined.

2.2.5. Effect of cytokinins on callusing

Major cytokinins at variable concentrations, benzyl adenine (BA), Kinetin

(KN) each were incorporated singly to the basel medium suplemented with auxins,

(2,4-D or NAA or IAA), Response of leaf, stem and petiole explants to varying

concentrations of auxins and cytokinins were evaluated with respect to percentage

of cultures initiating calli and callus growth rate.

2.2.6. Estimation of relative growth rates of calli

Fresh weight of initiated calli in promising culture media were recorded at

the time of initial subculture (four weeks after callus initiation). All the experiments

were replicated three times. Growth of established calli was estimated as relative

growth rate (RGR) over 28 days subculture period.

Wherc Wi = Initial callus mass at t~ (g)

W2 = Final callus mass at t2 (g)

t = Period (days), tz-ti = 28 days

2.2.7. Establishing suspension cultures

Standardization of subculturing intervals and assessment of critical cell

density in MS medium at half strength was supplemented with BA and NAA, was

distributed to Erlenmayer flasks (250ml), 50 rnl in each. The flasks with media were

plugged with non-absorbent cotton and autoclaved without agar. pH of the medium

was adjusted to 5.6-5.8 before autoclaving.

Suspension cultures were established from leaf callus of varying fresh weight

(1.0-2.0) in 50 ml of each callus growth medium. Flasks were incubated at 110 rpm at

26&l0 C with a 16h photoperiod. Subculturing intervals and critical cell density were

established after observing the cell count at specific intervals for each of the varying

fresh weight employing a haemocytorneter.

2.2.8. Subculture of suspensions

Subculture was performed at 20 day interval. The supernatant liquid in batch

cultures retaining small cell clumps was poured into sterilized flasks using an

inoculurn ratio (1:4v/v), and cultured in an orbital shaker.

2.2.9. Determination of packed cell volume (PCV)

Suspension cultures were shaken thoroughly. 5ml of the suspension was

pipetted out to a graduated centrifuge tube. The samples were centrifuged at 2000

rpm for 5 min. and sedimented mass expressed as ml pellet per ml culture.

2.2.10. Screening suspensions for camptothecin content

Apparently valuable callus lines of experimental species with respect to

expression of camptothecin were made into suspensions, extracted in chloroform

and screened for the presence of camptothecin by HPLC.

2.2.11. Transformation and establishment of hairy root cultures

Agrobacten'urn rhizogenes strains ATCC A4, 15834, RlOOO were used for the

study. Leaves, stem and calli of 4-8 weeks old plants were infected by these strains.

The experiment was conducted in three steps.

2.2.12. Assessing the virulence of experimental strains of bacteria

Streak plating was conducted to assess the comparative virulence of strains

employed. Yeast extract mannitol (YEM) medium incorporated with 1.8% agar was

poured nto sterilized petri plates and dried in the laminar hood for 20 min. One

loopful of liquid bacterial culture obtained from the stab cultures was streaked on to

the media in petri plates and incubated at 30°C to obtain single colonies. The extent

of growth of the srains in petri plates was observed.

2.2.13. Revival of cultures

One single colony was inoculated, each from the three bacterial strains into

YEM broth for overnight shaking at 25OC. After 24h of incubation the optical density

of these cultures were noted in order to adjust the OD between 0.5-0.7. This one-day-

old culture was employed to infect plant parts and in vitro cultures of Ophiorvhiza

rugosn var decumbens.

2.2.14. Inoculation of in vitro cultures with bacterial strains

The experimental calli and plantlets were inoculated with strains ATCC A4,

15834 and RlOOO by wounding the tissue with a scalpel and pouring 2 0 ~ 1 bacterial

suspensions per 0.5 g calli. The inoculated calli were cultured in half MS medium

devoid of growth hormones and incubated at 250°C for a day. After 24h, the

inoculated calli were transplanted to half strength MS medium incorporated with

500mgl-I cefotaxime, a broad-spectrum antibiotic. Cultures observed for induction of

hairy roots with respect to days to root initiation and percentage of cultures

initiating roots. When medium is found to be exhausted, roots were harvested and

CPT content was estimated using HPLC.

2.2.15. Preparation of the plant extract

Air-dried plant powder (100g) is extracted with 70% methanol in soxhlet

apparatus. Extract is concentrated, evaporated to dryness under vacuum using rotary

evaporator. The dried extract is suspended in Phosphate buffered saline (PBS) and

used for the further studies. Preparation of the callus extract by freeze drying the calli

and powdered using a mortar and pestle and extracted with methanol.

2.2.16. Preparation and application of spray reagents

Liebermnann- Burchar d reagent for terpenes: 5 ml acetic anhydride were mixed

with 5ml conc. HS04 at 4O C. This mixture was used to spray on TLC plates (Stahl,

1969).

Aluminium chloride for flavanoids: The TLC plates were sprayed with 1%

alcoholic ammonium chloride and observed under UV illumination for fluorescent

spot (Stahl, 1969).

Lead Acetate for flavanoids: The TLC plates were sprayed with 25% aq. solution of

basic lead acetate and observed under UV illumination for fluorescent spot

(Harborne, 1976).

Dragondorff reagent: The TLC plates were sprayed with dragondorff reagent,

which gives orange red colour for alkaloids (Stahl, 1969).

2.2.17. Extraction of crude CPT

Different parts of the plant were dried and powdered, defatted with light

petrol (60-80°C bp). The residual part was extracted 3 times with 70% methanol in a

soxhlet apparatus. The filtrate was concentrated under low vacuum, suspended in

distilled water and extracted with chloroform. Chloroform layer was evaporated to

dryness and subjected to column chromatography using silica gel (60-120 mesh)

packed in a column (50 cm x20mm diameter) and eluted with varying proportions

of chloroform and methanol (v/v). Each fraction was screened for CPT by TLC

using chloroform: acetone (70:30v/v) as the moving solvents and fluorescent spots

were detected under a UV chamber (350 nm).

2.2.18. Isolation and crystallization of CPT Fractions containing CPT were pooled and further purified by preparative

TLC using CHC13: acetone (70:30 v/v) as soIvent system (Roja and Heble, 1994).

Fluorescent band corresponding to standard CPT were scraped from the TLC plates

and extracted with chloroform. The CHCb extract evaporated and small volume

MeOH was added and kept at 4O C overnight. Crystallized CPT (pale yellow) was

isolated by centrifugatin (Tafur et al., 1976).

2.2.19. TLC analysis

Standard CPT and isolated CPT were chromatographed on silica gel HFzs4

plates using different solvent systems and the relative front (Rj) was calculated.

Distance travelled by the spot Rfvalue =

Distance travelled by the solvent front

2.2.20. Melting Point

Meltingpoint of the crystallized CPT was determined on Kofler hot stage and

was uncorrected.

2.2.21. UV-Visible absorption spectra

The purified fraction was dissolved in chloroform and absorption spectrum

was recorded between 200-400 nm using Elico spectrophotometer.

2.2.22. IR- Spectra

The functional groups present in CPT were determined by IR- spectra. The

spectra were measured using Perkin Elmer PF2 800 in KBr.

2.2.23. High performance liquid chromatography (HPLC)

The purified samples were subjected to HPLC analysis. The HPLC consists of

an HPLC pump (LC8A, reverse phase column, 4 mm diameter, 250rnm length C18),

UV absorbance detector set at 256 nm for detection of CPT. The mobile phase used

was acetonitrile: water (30:70v/v) with a flow rate 1 ml/min. The quantity of CIT

was calculated by standard calibration with authentic samples.

2.2.24. Electron spray mass spectrometry

The mass of isolated CPT was recorded on micro mass Quattroll triple

quadrupole mass spectrophotometer. The samples were dissolved suitable solvent

such as CHCI3, MeOH, acetonitrile, H20 were introduced into the ESI source

through a syringe pump at 0.4 ml/hr. The ESI sourse capillary was 3.5 KV and the

cone voltage 2530V, and the spectra were collected in 4 sec.

2.2.25.1H-Nuclear magnetic resonance

The number of protons present in the purified samples was recorded by

Bruker DRX-300 Mz FT NMR in CdC13 solution.

2.3. Cytotoxicity assays

2.3.1. Brine shrimp cytotoxicity (BST) assay

2.3.l.l.Hatching the Shrimp

Brine shrimp eggs were hatched in a shallow rectangular dish (22x32 cm)

filled with artificial sea water prepared with a commercial salt mixture and double

distilled water. A steel divider with several 2 mm holes was clamped in the dish to

make two unequal compartments. The eggs (20 mg) were sprinkled into the larger

compartment which was darkened, while the smaller compartment was illuminated.

After 48 h photohopic nauplii were collected by pipette from the lighted side,

having been seperated by the divider from their shells.

2.3.1.2. Sample preparation:

Samples were prepared by dissolving the compound in DMSO (0.2 %) and

different concentrations were transferred to 1.25 cm discs of filter paper (Whatman

No.1) . The discs were dried in hot air oven for 1 h, placed in dram vials. Control

discs were prepared using only DMSO. Five replicates were prepared for each dose

level.

2.3.1.3.Bioassay

Ten shrimps were transferred to each sample vial using pipette, and artificial

sea water was added to make 5 ml. The nauplii can be counted macroscopically in

the stem of the pipette against the light background. A drop of yeast suspension (3

mg/5d) was added as food to each vial. The vials were maintained under

illumination, after 6 and 24h the percent death at each dose and the control were

determined, using the formula

[(Test - Control) / Control] xlOO (Mayer et al., 1982).

2.3.2. Determinatiom of 3 h - time cytotoxicity of the extract

Cytotoxicity studies were carried out using Dalton's Lymphoma Ascites

(DLA) and Ehrlich Ascites Carcinoma (EAC) tumour cells. Different concentrations

of the extract (10-1000pg/ml) were incubated with tumour cells (lo6) suspended in

PBS (pH 7.2), and cytotoxicity was determined after 3h using the trypan blue

exclusion method (Babu et al., 2002)

2.3.3. Determination of 72 h-time cytotoxicity of the extract

Cytotoxicity of the extract in culture was determined using L-929 cells (Anis

and Kuttan, 1999). Cells (lo4) were plated in a 96 well flat-bottomed titre plates and

maintained with MEM (Minimum Essential Medium) containing 10% goat serum.

Various concentrations of the extract (10-100 pg/rnl) were added into the wells and

incubated for 72h. MTT [3-(4,5-Dime thy l thiazol-2-y1)-2,5-diphenyltetrazolium

bromide] (5%, 20 pl) was added to the wells 4h before the end of incubation.

Medium and reagents were aspirated, 98% DMSO was added and after shaking for

15 min, the absorbance was measured at 545 nm with a reference wavelength of 630

nm using an Elisa plate reader (Awarness Technology Inc.). CPT was treated as the

positive control. The percentage cytotoxicity was calculated and compared with

untreated controls.

2.4. Topoisomerase I and I1 inhibition assay

Saccharomyces cervisiae mutant cultures JN 394, JN 394t-1 and JN 3%-2-5 used

for the topoisomerase assays. JN 394 is hypersensitive to topoisomerase I poisons

while JN 394t-I lack top0 I gene and therefore shows lack of response to

topoisomerase I poisons. JN 3%-2-5 carry top I1 gene, which is resistant to

topoisomerase I1 poisons but respond to topoisomerse I poisons. The organisms

were supplied by Dr. John Nistiss of St. Jude Children's Hospital, Memphis,

Tennesee, USA and were cultured in petridishes containing Yeast Potato Dextrose

Agar (YPDA) medium (20 ml). The cells from a fully grown plate of each organisms

were suspended in saline solution (10 ml) and then diluted to obtain 5x106 CFU/ rnl.

50pl of this suspension was then used to inoculate petridishes containing YPDA

media and allowed to air dry in the laminar flow hood for 20 rnin. The test material

was dissolved in DMSO and added to the inoculated plates (20pl) to give a final

concentration of 250 pg/ ml. These plates were inoculated at 27 OC for 72-96h. At the

end of incubation period, the zones of inhibition were recorded for each test

organism. Control was prepared by adding DMSO (20 pl) to inoculated plate

(Chang et al., 1995; Roth et al., 1998).

2.5. Propagation of tumour cell lines

DLA and EAC tumour cell lines obtained from Adayar Cancer Institute,

Chennai, India and were propagated as transplantable acites tumours in Swiss

albino mice. For this, tumour cells were aspirated from the peritoneal cavity of

mouse was washed thrice with saline and one million cells were inoculated to fresh

animals every two weeks to propagate the cell lines. '

2.6. Determination of tumour reducing activity of plant products

2.6.1. Solid tumour model

One million tumour cells (DLA/EAC) were injected into the right hind limb

of mice (6 mice/ group). Drug administration was started as prophylactically (10

days before tumour challenge), simultanoiusly (24h after tumour challenge) and 10

days after tumour challenge. Mode of administration of the drug was given

intraleision, intraperitoneall y and orally. Diameter of the tumour was measured on

every fifth day using vernier calipers and volume was calculated using the formula,

Volume = n/6 x DI x D2 x D3 where Dl is the length, D2 is the breadth and Ds is the

height of the tumour (Maity et al., 2000).

2.6.2. Ascites tumour model

One million tumour cells (DLA/EAC) were injected in to the peritoneal

cavity of the mice (6 mice/ group). Drug administration (p.o and i.p) was started

24h sfter tumour inoculation and continued daily for 10 days. Animals were

observed for the development of ascites turnour and death due to tumour burden

was recorded. The increase in life span (percentage ILS) of treated group was

calculated using the formula, percentage ILS= (T-C)/Cx100, where 'T and 'C are

mean survival of treated and control mice, respectively (Babu et al., 2002).

2.7. Determination of in vitro antioxidant activity

2.7.1. Superoxide scavenging activity

It was determined by the light induced superoxide generation by riboflavin

and subsequent reduction of NBT as described by Mc Cord and Fridovich (1969).

The reaction mixture contained EDTA (6pM) containing 3 pg NaCN, riboflavin

(2pM), NBT (50pM), various concentrations of the test material and phosphate

buffer in a final volume of 3 ml. The tubes containing the reaction mixture were

uniformly illuminated with an incandescent lamp for 15rnin and the optical density

was measured at 530nm before after illumination. The percentage inhibition was

evaluated by comparing the absorbency value of the control tubes and experimental

tubes.

2.7.2. Hydroxyl radical scavenging activity

Hydroxyl radical scavenging activity of the test material was measured by

studying the competition between deoxyribose and test compounds for hy droxyl

radicals generated from the Few/ ascorbate/ EDTA/H202 system (Fenton reaction).

The hydroxyl radicals attack deoxyribose, which eventually results in the formation

of thiobarbituric acid reacting substances (Elizabeth and Rao, 1990). The reaction

mixture containing deoxyribose (2.8 mM), ferric chloride (O.lpM), EDTA (0.1 mM),

H202 (ImM), ascorbate (0.1 mM) KH2P04 -KOH (20 mM, pH 7.4) and various

concentrations of the sample in a volume of 1 ml was incubated for l h at 370 C.

Deoxyribose degradation was measured as thiobarbituric acid reactive substances

by the method of Ohkawa et a1 (1979). The inhibition produced by different

concentrations of the sample as well as the concentration required for 50% inhibition

was calcuIated.

2.7.3. Lipid Peroxidation assay

Lipid peroxidation was induced in rat liver homogenate by the method

described Bishayee and Balasubramonian (1971) in the presence of different

concentrations of the test material and estimated by thiobarbituric acid reactive

substances by the method Ohkawa et a1 (1979). Different concentrations of the test

material was incubated with 0.1 d of rat liver homogenate (25%) containing

30rnM KCI, Tris- HCl buffer (0.04 M, pH 7.0), ascorbic acid (0.06 mM) and ferrous

ion (0.16 mM) in a total volume 0.5 ml for lh. At the end of the incubation period,

0.4 ml of the reaction mixture was treated with 0.2 ml SDS ($.I%), 1.5 ml

thiobarbituric acid (0.8%) and 1.5 ml acetic acid (20%) pH 3.5). The total volume was

made up to 4ml by adding distilled water and kept in water bath at 95O C for lh.

After cooling, iml distilled water and 5 rnl butanol-pyridine mixture (15:1, v/v) was

added. After vigorous shaking, the tubes were centrifuged and the upper layer

containing the chromophore was read at 532 nm. The percentage inhibition was

calculated and the concentration required for 50 % inhibition was calculated.

2.7.4. Nitric oxide radical inhibition activity

Nitric oxide, generated from sodium nitroprusside in aqueous solution at

physiological pH, interacts with oxygen to produce nitrite ions, which were

measured by Griess reaction (Green et al., 1982; Marcocci et ai., 1994a). The reaction

mixture (3 ml) containing sodium nitroprusside (10 mM) in phosphate buffered

saline (PBS) and the compound (from 1 to 100 pg/ml) was incubated at 25' C for 150

min. After incubation, 0.5 ml of the reaction mixture was removed and 0.5 ml of

Griess reagent (1 % sulphanilamide, 2% and 0.1% naphthylethylene diamine

dihydrochloride) was added. The absorbance of the chromophore formed was

reviewuated at 546 nm.

2.8. Estimation of protein

Procedure described by Lowry et a1 (1951) was used for protein estimation.

The method based on the formation a protein-copper complex and reduction of

phosphomol ybdate-phosphotungstate reagent (Folin-Ciocalteuphenol reagent) by

tyrosine and tryptophan residues of protein to form a coloured product.

Reagents: Solution A consists of lml CuS04 5H20 (1 %) + Irnl sodium potassium

tartrate (2%) + 98 ml 2% Na2C03 in I N NaOH. Solution B contains Folin

Ciocalteuphenol reagent and distilled water mixed in 1:l ratio before use.

Procedure: 0.1 ml of tissue homogenate (2.5%) was diluted to 1.2 ml and mixed with 6

ml of the solution A. The mixture was incubated at room temperature for 10 min and

add 0.3 ml of solution B, mix immediately, kept at room temperature for 30 min.

Optical density was taken at 750 nm. The amount of protein was calculated from the

54

standard curve of bovine serum albumin (BSA).

2.9. Estimation of tissue glutathione (GSH)

The method described by Moron et al (1979) was used. Glutathione in the

tissue homogenate was determined by using 5'5'-dithio-bis-2-nitrobenzoic acid

(DTNB) to give a yellow coloured complex with absorption maximum at 412 nm.

hwedure: 2 5 ~ 1 of 25% trichloroacetic acid (TCA) were added to 0 . 5 d of homogenate

to precipitate the protein. The tubes were cooled in ice for 5 rnin and the mixture was

further diluted with 0.6 ml of 5% TCA and centrifuged at 1000 rpm for 10 min. From

the supernatant, 0.3 ml was taken for estimation. The volume of the aliquot was made

up to I d with 0.2 M sodium phosphate buffer (pH 8.0) and 2nd of the freshly

prepared DTNB solution (0.6 mM in 0.2 M phosphate buffer, pH 8.0) were added to

the tubes. The intensity of the yellow colour formed was read at 412 nm in a

spectrophotometer after 10 min. Standard curve GSH was prepared using reduced

glutathione. Values were expressed in nmol/ mg protein.

2.10. Estimation of serum glutamate -pyruvate transaminase (GPT)

activity

The method of Bergmayer and Burnt (1980') was used to assay GPT. The

enzyme GFT catalyses the reaction between Zoxo-glutamate and L-alanine forming

L-glutamate and pyruvate. The pyruvate produced thus react with Zdinitrophenyl

hydrazene giving a product, with absorption maximum at 520 nm.

Reagents: Phosphate buffer pH 7.4, substrate 1.78 g of DL alanine and 30 mg of

ketoglutarate dissolved in 20 rnl buffer containing 1.25 ml of 0.4 N NaOH. The

solution was made up to 100 ml with buffer, pH 7.4 and kept at 4OC. Dinitrophenyl

hydrazene (DNPH, 20 mg % in I N HCl), NaOH (0.4N), pyruvate standard 1 %.

Procedure: 0.5 ml of the substrate was incubated for 3 min at 37 OC. Serum (100p1)

was added after incubation. Mixed well and was incubated for 3 min at 37 OC. 0.5 ml

of DNPH was added to this mixture and kept at room temperature for20 min. The

reaction was stopped by adding 5 ml of 0.4 N NaOH, vortexed and kept at room

temperature for 5min. The absorbence was measured at 520 nm. The enzyme activity

was expressed as a measure of pyruvate formed, which was calculated form the

standard curve of pyruvate.

2.11. Estimation of Alkaline phosphatase (ALP) activity

The method of King and Amstrong (1980) was used for this assay. 4-amino

antipyrene reacts with compounds containing phenolic groups. In presence of an

alkaline oxidising agent, to give purple colour which can be measured at 520 nm.

Reagents: Alkaline phosphate buffer, pH 10 (6.36 g of anhydrous sodium carbonate

and 3.36 g of sodium bicarbonate in 1 liter of distilled water). Disodium phenyl

phosphate (IOOmM), NaOH (0.5 N), 4-aminoantip yrene (0.6 %), potassium

ferricynide (2.4%), phenol standard 1 mg/ml.

Procedure: lml of substrate was incubated with lml bicarbonate buffer and

incubated for 3 min at 37O C. After incubation 100~11 serum was added, vortexed well

and incubated for 15 min at 37O C. After incubation 0.8 mI of 0.5 N NaOH, 1.21111

NaHC03, lml of amino antipyrene and 1 ml potassium ferricyanide were added,

mixed well and absorbance was measured at 520 nm. The enzyme activity was

expressed as the amount of phenol produced and was calculated from the standard

graph of phenol.

2.12. Histopathology

A small portion of the tissue was fixed in 10% buffered formalin immediately

after sacrifice. Passing through ascending grades of alcohol, cleared in x y lene,

impregnated and embedded in paraffin dehydrated specimens. 3-5pm sections were

56

made using a microtome and stained with Hematoxylin and Eosin. The sections

were mounted in DPX and observed under light microscopy.

2.13. Statistical analysis

Student's 't' test was used for the statistical evaluation of the data. To

determine the statistical significance between two values in the control (X) and

treated (Y) group, 't' value was calculated using the equation.

Where X and Y are the means of the two samples X and Y; nx and ny are the sample

size and S was found out using the equation,

Where 'Sx' and 'Sy' are the standard deviation of the iwo samples. By knowing the ' t

'value and degree of freedom (nx+ny-2), statistical significance was deduced from 't'

distribution table.