ijapr - international journal of advances in pharmaceutical research

M.S Latha et al. / International Journal of Advances in Pharmaceutical Research 

IJAPR 

Available Online through 

www.ijapronline.org 

CHEMOPREVENTIVE EFFECT OF WOODFORDIA FRUTICOSA KURZ FLOWERS 

ON N-NITROSODIETHYLAMINE INDUCED HEPATOCELLULAR 

CARCINOMA IN RATS 

M.S Latha * , A. Nitha, P.N Ansil, S.P Prabha, 

Biochemistry and Pharmacognosy Research Laboratory, School of Biosciences, 

Mahatma Gandhi University, P.D. Hills P.O, Kottayam, Kerala-686560, India. 

Received on 21 – 10 - 2012 Revised on 22 – 11- 2012 Accepted on 02– 12 – 2012 

ABSTRACT 

To evaluate the chemopreventive effect of methanolic extract of Woodfordia fruticosa dried flowers on Nnitrosodiethylamine 

induced hepatocellular carcinoma in experimental rats. Two different doses of methanolic 

extract of Woodfordia fruticosa (MEWF 100mg/kg and 200mg/kg) were used to study the chemopreventive effect in 

experimental rats against N-nitrosodiethylamine induced hepatocellular carcinoma. NDEA (0.02%, 2ml, 

5days/week) was administered to the rats in all groups except normal and drug control. Various serum parameters 

like aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyl transferase (GGT) 

were studied. The antioxidant status of liver was evaluated by using the following parameters: - glutathione-Stransferase 

(GST), glutathione reductase (GR) and glutathione peroxidase (GPx). Immunohistochemical 

localizations of liver tissue for two important cancer markers: - Proliferating cell nuclear antigen (PCNA) and 

Cyclin D1 were evaluated. MEWF significantly (p ≤ 0.05) prevented the elevation of serum AST, ALT and GGT 

levels. Hepatic GST, GR and GPx levels were remarkably increased by the treatment with the extract. 

Immunohistochemical analysis revealed the overexpression of proliferating cell nuclear antigen (PCNA) and cyclin 

D1 in the liver tissue of rat intoxicated with NDEA. The overexpression was effectively reduced by the treatment 

with MEWF. This study demonstrates the chemopreventive effect of MEWF, and thus scientifically supports the 

use of this plant in traditional medicine for the treatment of liver cancer. 

Keywords: Chemoprevention, Cyclin D1, N- nitrosodiethylamine (NDEA), Proliferating cell nuclear antigen 

(PCNA), Silymarin, Woodfordia fruticosa. 

1. INTRODUCTION 

Hepatocellular carcinoma (HCC) is the fifth most 

common cancer worldwide and the third most 

common cause of mortality with a continuously 

increasing incidence annually (1). Liver is an organ 

of paramount importance and it plays an essential 

role in drug and xenobiotic metabolism. Chronic 

infection of hepatitis B and/ C, toxic industrial 

chemicals, aflatoxin exposure in diets, cigarette 

smoking, alcohol consumption, air and water 

pollutants etc are the major risk factors of liver 

diseases. Intake of acetaminophen like drugs and 

certain chemicals may also lead to hepatocellular 

carcinoma .Moreover, due to the high tolerance of 

liver, HCC is seldom detected at the early stage and 

once detected treatment faces a poor prognosis in 

most cases (2). 

Research Paper 

ISSN: 2230 – 7583 

N-nitrosodiethyalamine (NDEA) is a potent 

hepatocarcinogen in rats producing reproducible 

tumor after repeated administration and is the most 

important environmental carcinogen among N-niroso 

compounds. Administration of NDEA to animals 

causes cancer in liver and at low incidence in other 

organs also. It has been shown that the mechanism of 

action is due to metabolism of NDEA to alkylating 

agents and reactive oxygen species and further 

interaction with DNA molecule , forming various 

DNA adducts that can lead to mutations (3). The O4 – 

ethyl deoxythimidine adduct (O4- Etdt) accumulates 

in hepatocyte DNA following NDEA administration 

which is thought to be important in tumor initiation 

(4). 

Hepatic chemical carcinogenesis is a multistep 

process in experimental animals. Carcinogens initiate 

IJAPR / Dec. 2012/ Vol. 3 /Issue. 12 / 1322 – 1330 1322


the process, which is followed by regeneration, 

growth and clonal proliferation, eventually leading to 

cancer (5). There is extensive evidence that the free 

radicals participate in NDEA induced 

hepatocarcinogenesis (6). Enzymes such as GST, GR 

and GPx reduce oxidative stress in the normal tissues. 

Extensive liver damage causes decrease in levels of 

these enzymes suggesting a high oxidative stress in 

the tissues, which may be one of the key factors in 

the etiology of cancer (7). They have been proved to 

cause numerous cellular anomalies, including but not 

limited to protein damage, deactivation of enzymatic 

activity, alteration of DNA and lipid peroxidation of 

membranes (8). Several herbal drugs like Abrus 

precatorius, Scutia myrtina, Funaria indica etc. have 

been evaluated for its potential as liver protectant 

against NDEA induced hepatocellular carcinoma in 

rats (9- 11). 

Chemoprevention, which is referred to as the use of 

nontoxic natural or synthetic chemicals to intervene 

in multistage carcinogenesis, has emerged as a 

promising and pragmatic medical approach to reduce 

the risk of cancer. Numerous components of plants, 

collectively termed “phytochemicals” have been 

reported to possess substantial chemopreventive 

properties. For many years cancer chemotherapy has 

been dominated by potent drugs that either interrupt 

the synthesis of DNA or destroy its structure once it 

has formed. Unfortunately, their toxicity is not 

limited to cancer cells and normal cells are also 

harmed (12). 

Use of plants and plant derivatives considered as 

alternative medicine for many ailments are on the 

increase on a global perspective. Woodfordia 

fruticosa is a traditional medicinal plant belongs to 

the family Lythraceae is used against a wide variety 

of diseases. All parts of the plant possess valuable 

medicinal properties such as anti inflammatory, 

antitumor, hepatoprotective and free radical 

scavenging activity but flowers are of maximum 

demand. Dried flowers are used as tonic in disorders 

of mucous membrane, hemorrhoids and in 

derangement of liver (13). Phenolics, particularly 

hydrolysable tannins and flavonoids were identified 

as major components in Woodfordia fruticosa 

flowers. In view of these the present study was 

undertaken to evaluate the chemopreventive effect of 

Woodfordia fruticosa flower extract on NDEA 

induced hepatocellular carcinoma in experimental 

rats. 

2. MATERIALS AND METHODS 

2.1 Chemicals 

N-nitrosodiethylamine (NDEA), silymarin, 

proliferating cell nuclear antigen (PCNA), cyclin D1, 

anti-mouse IgG horse radish peroxidase, streptavidin 

horse radish peroxidase conjugate and 

diaminobenzidine were purchased from Sigma 

Chemical Co., St. Louis, MO, USA. Assay kits for 

serum aspartate aminotransferase (AST), alanine 

aminotransferase (ALT) and gamma glutamyl 

transferase (GGT) were purchased from Agappe 

Diagnostics, India. All other chemicals were of 

analytical grade. 

2.2 Collection of plant material and preparation of 

plant extracts 

Woodfordia fruticosa flowers were collected from 

natural habitat (Kollam, Kerala, India) during 

November - January. Plant material was identified by 

Dr. V.T Antony, S.B College. A voucher specimen 

(Acc. No. 7566) is deposited at the herbarium of the 

Department of Botany, S.B College, Changanassery, 

Kottayam, Kerala. Flowers were shade-dried and 

powdered and 50 g of dried powder was soxhlet 

extracted with 400 mL of methanol for 48 h. The 

extract was concentrated under reduced pressure 

using a rotary evaporator and was kept under 

refrigeration. The yield of methanolic extract of 

Woodfordia fruticosa (MEWF) was 12.5 % (w/w). 

The concentrate was suspended in 5% Tween 80 for 

the present studies. 

2.3 Animals and diets 

Male Wistar rats weighing 150-160 gm were used in 

this study. The animals were housed in 

polypropylene cages and had free access to standard 

pellet diet (Sai Durga Feeds, Bangalore, India) and 

drinking water. The animals were maintained at a 

controlled condition of temperature of 26–28 o C with 

a 12 h light: 12 h dark cycle. Animal studies were 

followed according to Institute Animal Ethics 

Committee regulations approved by Committee for 

the Purpose of Control and Supervision of 

Experiments on Animals (Reg. No. B 2442009/4) and 

conducted humanely. 

2.4 Induction of hepatocellular carcinoma 

Hepatocellular carcinoma (HCC) was induced by oral 

administration of 0.02% NDEA (2ml, 5 days/week 

for 20 weeks (12). Silymarin at an oral dose of 100 

mg/kg body weight was used as standard control in 

the experiment (14-15). Two different doses of 

MEWF (100mg/kg and 200 mg/kg) suspended in 5% 

Tween 80 were prepared for oral administration to 

the animals. It is reported that the extract of W. 

fruticosa flowers are safe up to the dose of 

2000mg/kg, p.o (13). 

2.5 Experimental design 



Thirty six rats were divided into six groups, 

Group I - Normal control 

Group II - NDEA control (0.02% NDEA, 2 

ml, 5days/week, p.o) 

Group III - Silymarin (100mg/kg b.w) + 

NDEA 

Group IV - MEWF (100mg/kg, b.w ) + 

NDEA 

Group V - MEWF (200mg/kg, b.w) + NDEA 

Group VI - MEWF (200 mg/kg) alone 

Daily doses of silymarin and MEWF treatments were 

started in groups III to V animals 1 week before the 

onset of NDEA administration and continued up to 

20 weeks. Group VI served as drug control received 

MEWF alone for the entire period. The rats were 

sacrificed 48 h after the last dose of NDEA 

administration. 

2.6 Serum enzyme analysis 

Blood was collected from neck blood vessels and 

kept for 30 min at 4 0 C. Serum was separated by 

centrifugation at 2500rpm at 4 0 C for 15 min. 

Quantifying the serum levels of aspartate 

aminotransferase (AST), alanine aminotransferase 

(ALT) and gamma glutamyl transferase (GGT) by 

using a standard diagnostic kit (Agappe Diagnostic 

Ltd., India). Activities of these serum enzymes were 

measured by using semi autoanalyzer (RMS, India). 

2.7 Tissue analysis 

Liver tissue was excised, washed thoroughly in icecold 

saline to remove the blood. Then the dissected 

livers were cut into separate portions for biochemical 

assays and immunohistochemical examination. 

2.7.1 Biochemical assays 

Ten percent of homogenate was prepared in 0.1M 

Tris HCl buffer (pH – 7.4). The homogenate was 

centrifuged at 3000 rpm for 20 min at 4 o C and the 

supernatant was used for the estimation of 

glutathione-S-transferase (GST), glutathione 

reductase (GR), glutathione peroxidase (GPx) and 

total protein. 

GST (EC 2.5.1.18) activity was determined from the 

rate of increase in conjugate formation between 

reduced glutathione and CDNB (16). GR (EC 

1.6.4.2) activity was assayed at 37 o C and 340 nm by 

following the oxidation of NADPH by GSSG (17). 

GPx (EC 1.11.1.9) activity was determined by 

measuring the decrease in GSH content after 

incubating the sample in the presence of H2O2 and 

NaN3 (18). Protein content in the tissue was 

determined using bovine serum albumin (BSA) as the 

standard (19). 

2.7.2 Immunohistochemical analysis 

Immunohistochemical analysis of the two cancer 

markers, namely proliferating cell nuclear antigen 

(PCNA) and Cyclin D1 were conducted. 

Tissue sections were deparaffinised in three changes 

of xylene at 60 o C for 10min each and hydrated 

through a graded series of alcohol. For antigen 

retrieval evaluation, slides were placed in citrate 

buffer (pH 6.0) for three cycles of 5 min each in a 

microwave oven. The sections were then allowed to 

cool to room temperature and then rinsed with 1x tris 

buffered saline (TBS), and treated with 0.3% H2O2 in 

water for 10 min to block endogenous peroxidase 

activity. Non specific binding was blocked with 3% 

BSA in room temperature for 1 h. Subsequently the 

primary antibody was applied at predetermined 

dilutions (PCNA antibody diluted 1:500 / Cyclin 

D1antibody diluted 1:80) with 1% BSA in PBS for 

overnight at 4 o C. After this incubation washed thrice 

in PBS and incubated with anti-mouse horseradish 

peroxidase for 45 min. After triplicate washing with 

PBS, sections were incubated for 30 min with 

streptavidin–HRP complex. Then washed with PBS 

and incubated for 5–10 min in a solution of 

diaminobenzidine (6 mg/10mL 50mM Tris–HCl, pH 

7.6) containing 0.01% H2O2. Counterstaining was 

performed with hematoxylin. Images were taken at 

original magnification of 100× ( Motic AE 21, 

Germany and Moticam 1000 camera). 

2.8 Statistical analysis 

Results were expressed as mean ± S.D and all 

statistical comparisons were made by means of oneway 

ANOVA test followed by Tukey’s post hoc 

analysis and p-values less than or equal to 0.05 were 

considered significant. 

3. RESULTS 

3.1 Serum enzyme analysis 

The serum levels of AST, ALT, and GGT in group II 

were significantly (p ≤ 0.05) elevated by the 

administration of NDEA, when compared to normal 

control. The treatment of MEWF at a dose of 100 and 

200 mg/kg showed a significant decrease (p ≤ 0.05) 

in AST, ALT, and GGT (Fig.1). Standard control 

drug, Silymarin at a dose of 100 mg/kg also 

prevented the elevation of serum enzymes. Treatment 

with MEWF at 200 mg/kg and Silymarin exhibited a 

protection of 98% and 80.7% in AST levels, 97% and 

88% in ALT levels and 98.2% and 84% in GGT 

levels, respectively. 

3.2 Liver morphology 

Fig.2 shows the morphological variations of rat livers 

in different experimental groups. NDEA treated rat 



liver (Fig.2B) is morphologically different from 

normal liver. In NDEA treated groups rat liver 

become very large in size and a large number of 

hepatic nodules were observed in the liver. In MEWF 

200mg/kg treated group and in MEWF alone treated 

groups liver morphology is very similar to normal 

rats. 

3.3 Tissue biochemical analysis 

3.3.1 Estimation of Glutathione - S - transferase 

(GST) 

The GST activity of liver tissues were significantly (p 

≤ 0.05) reduced in NDEA intoxicated rats of pretreatment 

groups compared to normal control. The 

MEWF dose dependently increased (p ≤ 0.05) the 

activity of GST in hepatic tissues (Table 1). 

Treatment with 200 mg/kg methanolic extract 

exhibited prominently increased i.e., 89.9% GST 

levels. In addition, Silymarin treated rats also 

prevented the NDEA induced decrease in GST 

activity by 69.9% in hepatic tissue. 

3.3.2 Estimation of Glutathione Reductase (GR) 

GR activity was significantly decreased (p ≤ 0.05) in 

NDEA treated animals when compared to control. A 

significant increase (p ≤ 0.05) in the level of GR was 

observed in MEWF (100 and 200 mg/kg) and 

Silymarin (100 mg/kg) treated rats intoxicated with 

NDEA (Table 1). The percentage of protection in liver 

tissue was 83.8% for 200 mg/kg of MEWF. Silymarin 

restored the GR activity up to 62.6% in rat liver. 

3.3.3 Estimation of Glutathione Peroxidase (GPx) 

Activities of hepatic GPx was significantly (p ≤ 0.05) 

lowered in NDEA treated rats (Table 1). MEWF dose 

dependently prevented the lowering of GPx 

compared to NDEA alone treated groups. In liver, 

200 mg/kg of methanolic extract showed a protection 

of 94.5%. Silymarin-treated rats also prevented the 

lowering of GPx by 73.3% in hepatic tissues. 

3.4 Immunohistochemical analysis 

Fig. 3 and 4 shows the immunohistochemical 

analysis of PCNA and Cyclin D1 respectively. Here 

normal rat tissue showed regularly stained nucleus. 

NDEA administered rat liver tissue showed 

overexpression of Proliferating Cell Nuclear Antigen 

(PCNA) and Cyclin D1. Administration of MEWF 

showed a reduction in the expression of PCNA and 

Cyclin D1. PCNA is a common index for 

proliferation of hepatocytes at late G1 stage and early 

S stage. Fig. 3B and 4B (HCC bearing) groups 

stained positively for PCNA and Cyclin D1 

indicating active cell proliferation when compared 

with normal control. The plant extract given at a 

concentration of 200mg/kg showed a remarkable 

lowering of the expression of PCNA and cyclin D1 

and was comparable to the effect rendered by 

silymarin at a concentration of 100mg/kg. 

4. DISCUSSION 

Abnormal proliferation of cells is the main feature of 

carcinogenesis, and there for exploration of drugs 

that can affect malignant proliferation of liver cells is 

of primary importance in chemical prevention of liver 

cancer. PCNA present in cell nucleus is directly 

involved in DNA replication. It has been found that 

the positive expression of PCNA were a common 

index for proliferation of hepatocytes at late G1 stage 

and early S stage (20). The positive expression of 

proteins was mainly found in the precancerous 

proliferation focus and cancerous liver tissue during 

NDEA induced hepatocarcinogenesis. The high 

expression of PCNA in the present studies suggested 

that the ability of cell proliferation become stronger, 

and this was closely related to malignant cell 

proliferation and carcinogenesis (21). The results 

indicated that the administration of MEWF could 

remarkably decrease the PCNA in HCC bearing 

tissue, suggesting that MEWF had the ability to 

suppress malignant proliferation of hepatocytes in 

experimental liver cancer and inhibited tumor growth 

through inhibition of DNA synthesis. 

Cyclin D1 is another important cancer marker has 

been associated with a variety of cancers including 

HCC. Cyclin D1 is the regulatory subunit of a 

holoenzyme that promotes progression through the 

G1-S phase of cell cycle. Amplification or 

overexpression of Cyclin D1 plays important roles in 

the development of a subset of human tumorigenesis 

and cellular metastasis (22). 

Amino transferases (AST, ALT) and GGT are the 

important cytoplasmic enzymes present in the liver. 

During chemical intoxication the membrane integrity 

is lost due to necrosis and these enzymes move into 

the circulatory system followed by elevated levels of 

enzymes in the serum (23). Increased levels of serum 

AST, ALT and GGT was detected in NDEA treated 

rats, effectively down- regulated by the treatment 

with MEWF indicating the protective role of 

Woodfordia fruticosa in NDEA induced HCC. 

The intra cellular antioxidant system comprises of 

different free radical scavenging antioxidant enzymes 

such as GST, GR and GPx constitute the first line of 

cellular antioxidant defense system. When excess 

free radicals are produced, the equilibrium is lost and 

consequently oxidative insult is established (24). 

GST offers protection against lipid peroxidation by 

promoting the conjugation of toxic electrophiles with 

GSH (25). GR is also essential for the maintenance of 



GSH levels in vivo. The significant (p ≤ 0.05) 

restoration of GPx activity in MEWF and silymarin 

might be due to the antioxidant activity by 

detoxifying the endogenous metabolic peroxides 

generated after NDEA intoxication in hepatic tissues. 

These biochemical restorations may be due to the 

inhibitory effects of MEWF in the process of cellular 

damage in the liver. Free radical scavenging activity 

of Woodfordia fruticosa methanolic extract 

is well established and that effectively protects the liver against carbon tetrachloride [13], diclofenac (26) 

thioacetamide (27) and phenytoin (28) induced hepatotoxicity in rats. 

Preliminary phytochemical analysis identified the major class of chemical components present in methanolic extract 

are saponins (steroids and terpenes), phenolics, alkaloids, flavanoids, tannins etc. The phytochemicals identified are 

more potent in inhibiting free radicals and this could be protective against the progression of NDEA induced HCC. 

The compounds reported from the dried flowers of Woodfordia fruticosa are β-sitosterol, kaempferol, ellagic acid, 

octacosanol, meso-inositol, quercetin, woodfordins A, B, C, oenothein B, woodfordin D and oenothein A (29). 

Ellagic acid is a compound reported to have chemopreventive effect and is useful in inflammatory disorders like 

colorectal cancer (30). Quercetin is a plant derived Flavanoid, used in cancer prevention and treatment. Antiinflammatory 

effect of quercetin in hepatoma cell lines (Hep G2) was reported (31). Woodfordin C and oenothein B, 

a class of macrocyclic hydrolysable tannins exhibited potent host-mediated antitumor activity against sarcoma 180 

in mice (32-33). Woodfordin C shows remarkable inhibition of DNA topoisomerase II (34). Woodfordin D and 

Oenothein A, trimeric hydrolysable tannins also have antitumor activity (35). Thus protection yielded by this extract 

may be due to the combined effects of these compounds or fractions rather than any single component. 

AND FIGURES 

Table 1: Effect of MEWF on GST, GR and GPx in the liver tissue of different experimental groups 

Treatment groups 

Normal control 

NDEA control (0.02%) 

Silymarin(100mg/kg)+ 

NDEA 

MEWF(100mg/kg)+ NDEA 

MEWF(200mg/kg)+ NDEA 

MEWF alone 

GST 1 

72.5±0.70 

33.1±0.43 * 

58.3±0.52 ** 

53.9±0.78 ** 

68.5±0.51 ** 

70.2±0.53 ** 

1: μmol CDNB-GSH conjugate formed/min/mg protein ; 2 : nmol of GSSG utilized/min/mg protein; 3: nmol of GSH 

oxidized/min/mg protein 

Values are mean± S.D., n=6 

* p≤ 0.05 versus normal control 

** p≤ 0.05 versus NDEA control 

GR 2 

21.4±0.77 

7.2±0.38 * 

16.16±0.43 ** 

12.3±0.45 ** 

19.15±0.83 ** 

21.7±0.61 ** 

GPx 3 

283.1±2.85 

162.4±3.09 * 

250.8±3.05 ** 

217.5±2.51 ** 

276.4±3.81 ** 

281.8±3.0 ** 



Fig.1. Effects of MEWF on changes in serum enzyme levels of rats treated with NDEA. 

Fig. (A) Aspartate aminotransferase, (B) Alanine aminotransferase and (C) Gamma glutamyl transferase. (I) Normal 

control, (II) NDEA control (III) Silymarin + NDEA, (IV) MEWF (100 mg/kg) + NDEA (V) MEWF (200 mg/kg) + 

NDEA, (VI) MEWF alone. Values are mean ± S.D, error bar indicating the standard deviation, n = 6 animals. * p ≤ 

0.05 vs. normal control. **p ≤ 0.05 vs. NDEA control. 

Fig.2. Morphological variations of liver in control and treated rats 

Fig.2 (A) Normal control; (B) NDEA control (0.02%); (C) Silymarin (100 mg/kg) + NDEA; (D) MEWF (100 

mg/kg) + NDEA; (E) MEWF (200 mg/kg) + NDEA and (F) MEWF (200mg/kg) alone. 



Fig. 3. Immunohistochemical localizations of PCNA in control and treated groups. 

Fig. 3. Liver tissue was immunostained for PCNA followed by staining with hemetoxilin (100x). (A) Normal 

control; (B) NDEA control (0.02%); (C) Silymarin (100 mg/kg) + NDEA; (D) MEWF (100 mg/kg) + NDEA; 

(E) MEWF (200 mg/kg) + NDEA and (F) MEWF (200mg/kg) alone. 

Fig. 4. Immunohistochemical localizations of Cyclin D1 in control and treated groups 

Fig. 4. Liver tissue was immunostained for Cyclin D1 (indicated by brown stained nuclei) followed by staining with 

hemetoxilin (100x). (A) Normal control; (B) NDEA control (0.02%); (C) Silymarin (100 mg/kg) + NDEA; (D) 

MEWF (100 mg/kg) + NDEA; (E) MEWF (200 mg/kg) + NDEA and (F) MEWF (200mg/kg) alone. 



CONCLUSION 

The results presented in this study indicate that the 

HCC induced by N-nitrosodiethylamine was 

effectively inhibited by the treatment with MEWF at 

a dose of 200 mg/kg, b.w. When compared to 

standard drug silymarin treated group, the MEWF 

treated group showed better efficacy towards NDEA 

induced hepatocellular carcinoma. In rats treated with 

MEWF alone no significant change was observed. 

The pretreatment with MEWF on pathological 

conditions in chronic hepatic disorder such as HCC 

showed remarkable protection; provide a new 

approach to study the active principle responsible for 

chemoprevention in tumor models. However further 

work is required for the fractionation of MEWF and 

identification of the active compound which is 

underway. 

Conflict of interest statement 

We declare that we have no conflict of interest. 

Acknowledgments 

Financial assistance from Mahatma Gandhi 

University as Junior Research Fellowship to Nitha 

Anand is thankfully acknowledged. Authors also 

thankful to Dr. P.J Wills, Scientist, MIMS research 

foundation, Calicut., Ms. Jyothi Lekshmi M, 

Research scholar, SBS, M. G. University, Kottayam 

and Ms. Reshmi Radhakrishnan, project staff, SBS, 

M.G University, Kottayam for their help in this work. 

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