Orient Pharm Exp Med DOI 10.1007/s13596-013-0125-3

RESEARCH ARTICLE

Chemopreventive and antioxidant activity by Smilax zeylanica leaf extract against N-nitrosodiethylamine induced hepatocarcinogenesis in wistar albino rats

Venugopalan Rajesh & Perumal Perumal

Received: 8 April 2013 /Accepted: 28 May 2013 # Institute of Korean Medicine, Kyung Hee University 2013

Abstract The chemopreventive potential of methanol extract superoxide dismutase (SOD), catalase (CAT), glutathione per- of Smilax zeylanica leaves against N-nitrosodiethylamine oxidase (GSH-Px), glutathione reductase (GR), glutathione-S- (NDEA) induced hepatocarcinogenesis was investigated in transferase (GST), glutathione (GSH), vitamin- C and wistar albino male rats. The animals were divided into six vitamin-E in liver tissue. In contrast, the NDEA induced groups. Group I served as normal control group, group II and animals in group Vand group VI treated with Smilax zeylanica group III served as extract control received Smilax zeylanica leaf extract showed a significant improvement in body weight leaf extract 200 mg/kg and 400 mg/kg respectively once daily gain at the end of experimental period (16 weeks). A signif- orally. A single intraperitoneal injection of NDEA (200 mg/kg) icant decrease in relative liver weight with reduced levels of to group IV, group V and group VI animals followed by a liver injury and liver cancer markers in serum were observed single dose of carbon tetrachloride (CCl4, 2 ml/kg) 2 weeks in treatment groups. Moreover, treatment with Smilax later induced hepatocarcinogenesis in rats at the end of exper- zeylanica leaf extract decreased the extent of lipid peroxida- imental period (16 weeks). Starting 1 week prior to NDEA tion with concomitant increase in activities of enzymatic and administration, group Vand group VI animals received meth- non-enzymatic antioxidants when compared with NDEA in- anol extract of Smilax zeylanica 200 mg/kg and 400 mg/kg duced untreated group IV animals. The histological observa- respectively once daily orally for 16 weeks. At the end of the tions of liver tissue too correlated the biochemical observa- experimental period, the body weight, morphology of liver, tions. Furthermore, the extract did not produce any deleterious relative liver weight, the levels of liver injury and liver cancer effects in extract alone treated groups. These findings suggest markers, the levels of lipid peroxidation and the activities of that, the potential chemoprevention of methanol extract of enzymatic and non-enzymatic antioxidants in liver tissue were Smilax zeylanica leaves might be due to stabilization and assessed. Group IV NDEA induced hepatocarcinoma animals increase in all the components of antioxidant system attributed showed body weight loss with a significant increase in relative to antioxidant and free radical scavenging activity. liver weight. The levels of liver injury and liver cancer markers such as alanine transaminase (AST), aspartate trans- Keywords Smilax zeylanica leaves . N-nitrosodiethylamine . aminase (AST), alkaline phosphatase (ALP), gamma- Hepatocarcinogenesis . Tumour markers . Lipid glutamyl transferase (GGT), α-feto protein (AFT) and peroxidation . Antioxidant activity carcinoembryonic antigen (CEA) were substantially increased in group IV untreated animals. Moreover, with reference to lipid peroxidation and antioxidant system, group IV animals Introduction showed elevated levels of lipid peroxidation with a subse- quent decrease in activities of antioxidant enzymes viz., Liver cancer, especially hepatocellular carcinoma (HCC), is : the fifth most common cancer and the third foremost cause of V. Rajesh (*) P. Perumal cancer associated death globally (Befeler and Di Bisceglie Department of Pharmacology, JKK Nattraja College of Pharmacy, 2002; Bishayee and Dhir 2009). Hepatocellular carcinoma Natarajapuram, Komarapalayam, 638183, Namakkal District, Tamil Nadu, India can be secondary to hepatitis B or C, cirrhosis due to alcohol e-mail: [email protected] consumption, liver disease due to aflatoxin toxicity, hormon- P. Perumal al imbalance and certain metabolic diseases (Thorgeirsson e-mail: [email protected] and Grisham 2002). Hepatocarcinogenesis involves initial V. Rajesh, P. Perumal genotoxic insult (initiation), clonal expansion from prema- Several constituents present naturally in medicinal plants have lignant to malignant lesions (promotion) and finally tumor been shown to modify critical reactions that cause inhibition progression by further clonal expansion (Thorgeirsson and of chemically induced hepatocarcinogenesis (Soni et al. 1997; Grisham 2002; Halsted et al. 1996). Bhattracharya and Chatterjee 1998) and this worldwide effort N-Nitrosodiethylamine (NDEA) a potent hepatocarcinogen, continues to discover new anticancer drugs from plants. is known to cause perturbations in the nuclear enzymes in- Among the herbal resources is Smilax zeylanica Linn. belongs volved in DNA repair/replication (Bhosale et al. 2002)andis to family Smilacaceae. The genus Smilax Linn. consist of normally used as a carcinogen to induce liver cancer in animal about 350 species in the world (Shao et al. 2007), out of which models. Investigations have provided evidence that N- 24 species are found in India (Saldhana and Wicolson 1976; nitrosamines cause wide range of tumours in all animal species Ramaswamy et al. 2001; Santapau and Henry 1976). In south and these compounds are considered to be effective health India, the genus is represented by 4 species viz., Smilax hazards to man (Loeppky 1994). The main cause for concern zeylanica Linn., Smilax aspera Linn., Smilax perfoliata is that diethylnitrosamine is found in a wide variety of foods Roxb. and Smilax wightii A. DC (Gamble 2004). Many of like cheese, soybean, smoked, salted and dried fish, cured meat them have been long used as medicinal herbs, especially as and alcoholic beverages (Liao et al. 2001). Metabolism of traditional Chinese medicines in China (Abdala et al. 2008). certain therapeutic drugs is also reported to produce The extracts of Smilax china L. tubers are known to show diethylnitrosamine (Akintonwa 1985). It is also found in to- antitumor and antioxidant activities on mice and rats (Wang bacco smoke at a concentration ranging from 1 to 28- et al. 1996). Smilax zeylanica Linn. is distributed in tropical ng/cigarette and in baby bottle nipples at a level of 10 ppb and subtropical hills of Asia between an altitude of 500– (IARC 1972). It has been reported that, on metabolic activation, 1,800 m. Smilax zeylanica leaves were extensively used in it produces the pro-mutagenic products, O6-ethyl deoxy gua- traditional system of medicine against veneral diseases nosine and O4andO6- ethyl deoxy thymidine in liver which (Oommachan and Masih 1991), skin disorders, sores, swell- are responsible for its carcinogenic effects (Verna et al. ings, abscess (Ambasta 2006;Nadkarni1976) and also ap- 1996). It is also reported that the generation of reactive plied for rheumatism and pain in lower extremities (Kirtikar oxygen species (ROS) by NDEA causes carcinogenic and Basu 1991). Based on the widespread use of Smilax effects. Oxidative stress caused by ROS has been re- species in traditional medicine and the traditional importance ported in membrane lipid peroxidation, DNA damage of Smilax zeylanica Linn it was decided to select the plant for and mutagenesis associated with various stages of tumor investigation. As previous studies mainly focused on the plant formation process (Parola and Robino 2001). Human liver rhizomes, and there was little information about the Smilax appears to metabolize nitrosamines in a manner similar to that zeylanica leaves. In the present study, an effort was made to of rodent liver and also exhibits considerable similarities with ascertain the possible role of Smilax zeylanica Linn. leaves in regard to morphology, genomic alterations and gene expres- chemically induced hepatocarcinogenesis. sion, despite their different disease etiologies (Feo et al. 2000). Hence the model of NDEA-induced HCC is considered as one of the most accepted and widely used experimental models to Materials and methods study hepatocarcinogenesis (Hai et al. 2001). Chemoprevention offers a novel approach to control the Chemicals incidence of liver cancer (Wattenberg and Estensen 1996). A large number of chemotherapeutic agents have been identi- Chemicals used in this study including NDEA, carbon tetra- fied in epidemiological and experimental studies, preclinical chloride (CCl4), Oxidized and reduced glutathione (GSSG and and clinical observations (Takuji et al. 1993; Jagadeeswaran GSH), hydrogen peroxide (H2O2), dithionitrobenzene (DTNB), et al. 2000). However, the toxic effects produced by some of Sodium dodecyl sulphate (SDS), Thiobarbituric acid (TBA), 1- the agents have limited their extensive use. There is, there- chloro-2, 4-dinitrobenzene (CDNB), dinitrophenyl hydrazine fore, a need to identify synthetic or natural compound that (DNPH), nitro blue tetrazolium (NBT), reduced nicotinamide have significant chemopreventive potential to block initia- adenine dinucleotide (NADPH), ethylenediamine tetra acetic tion or arrest the progression in premalignant cells without acid (EDTA), phenyl methyl sulfonyl fluoride (PMSF) were undesirable side effects. procured from sigma chemicals, St. Louis, USA. Enzyme In recent years, there has been considerable interest in immunoassay kit for estimation of tumor markers α-feto natural products with antioxidant property to protect cellular protein (AFP) and carcinoembryonic antigen (CEA) were pur- components from oxidative damage and prevent diseases. chased from UBI MAGIWELL (USA) and all other chemicals Over 50 % of anticancer drugs approved by United States including petroleum ether (60 – 80 °C), methanol (HPLC) were Food and Drug Administration since 1960 were originated purchased from SD Fine Chemicals Ltd., Mumbai, India with from terrestrial plants (Kim and Park 2002;Mann2002). highest purity grade. Chemopreventive and antioxidant activity

Plant material procedure). Limit test was performed to determine the safety of extract. Five female wistar rats were used for the study. The fresh leaves of Smilax zeylanica Linn were collected in Animals were fasted prior to the dosing (food but not water the month of September from yercaud hills. Yercaud is a hill withheld for 3–4 h). Extract 2,000 mg/kg b.wt. suspended station in Salem District, in Tamil Nadu, India. It located in in 0.5 % w/v carboxy methyl cellulose was administered the Shevaroys range of hills in the Eastern Ghats. It is orally to one animal and observed for mortality. If the situated at an altitude of 1,515 m (4,970 ft) above sea level first animal survives, four additional animals were se- (Geoposition: Latitude- 11.781997 (11° 46′ 55.19″ N), quentially dosed orally so that a total of five animals Longitude- 78.209782 (78° 12′ 35.22″ E). The plant material are tested. Animals were observed individually at least was taxonomically indentified, confirmed and authenticated once during the first 30 min after dosing, periodically by Botanical Survery of India, Coimbatore, Tamilnadu duringthefirst24h(withspecialattentiongivenduring (BSI/SRC/5/23/2011-12/Tech-1256) and the voucher speci- the first 4 h), and daily thereafter for a total of 14 days. men was retained in our laboratory for further reference. The After the experimental period, the animals were weighed collected leaves were shade dried and the dried materials and humanely killed and their vital organs including were crushed to coarse powder with mechanical grinder. The heart, lungs, liver, kidneys, spleen, adrenals, sex organs powder was stored in an airtight container for extraction. and brain were grossly examined (OECD 2000).

Extraction Experimental design and induction of hepatocellular carcinoma (HCC) The dried coarse powder was defatted with petroleum ether (60 – 80 °C) in a soxhlet extractor in order to remove fatty The experimental protocol was approved by Institutional substances. The defatted marc was dried and it was subjected Animal Ethical Committee (887/ac/05/CPCSEA). Healthy to extraction with methanol using Soxhlet apparatus for 72 h. male albino wistar rats (6–8 weeks) weighing 130–150gwere After completion of extraction, methanol was removed by used for the study. Randomization in selection of animals for distillation. The residue obtained was air dried. The dried grouping was carried out to avoid statistical difference in the methanol extract was stored in air tight glass container for body weight of animals. The experimental animals were di- further investigation. The percentage yield obtained was vided into six groups, each group comprising of six animals. 22.03 % w/w. The experimental animals were divided into six groups of six animals each according to the following experimental regi- Phytochemical screening men. Group 1 animals served as normal control rats. Group 2 animals received Smilax zeylanica leaf extract 200 mg/kg/day The extract obtained was subjected to preliminary phyto- (p.o.) in 0.5 % carboxy methyl cellulose (CMC) for 16 weeks. chemical screening (Khandelwal and Kokate 1995). Group 3 animals received Smilax zeylanica leaf extract 400 mg/kg/day (p.o.) in 0.5 % CMC for 16 weeks. Group 4 Acute oral toxicity study of Smilax zeylanica leaf extract animals received vehicle 0.5 % CMC 2 ml/kg/day (p.o.) 1 week prior to NDEA 200 mg/kg single i.p. and 2 weeks Healthy, young adult albino wistar rats (150–200 g) of fe- later received a single dose of CCl4 (2 ml/kg) by gavage as 1:1 male sex were used for the study. The animals were obtained dilution in corn oil. The oral administration of vehicle was from Agricultural University, Mannuthy, Thrissur, kerala continued after NDEA injection throughout the experimental (328/99/CPCSEA) and were housed in polypropylene cages. period of 16 weeks. Group 5 animals were given Smilax The animals were maintained under standard laboratory con- zeylanica leaf extract 200 mg/kg/day (p.o.) in 0.5 % CMC ditions (25° ± 2 °C; 12 h light and dark cycle). The animals started 1 week prior to the injection of NDEA and 2 weeks were fed with standard diet and water ad libitum. Ethical later received a single dose of CCl4 2 ml/kg by gavage as 1:1 clearance (for handling of animals and the procedures used in dilution in corn oil. The oral administration of extract study) was obtained from the Institutional Animal Ethical was continued for 16 weeks after NDEA injection. Committee (887/ac/05/CPCSEA) before performing the Animalsingroup6weregivenSmilax zeylanica leaf study on animals. extract 400 mg/kg/day (p.o.) in 0.5 % CMC started 1 week prior to the injection of NDEA and 2 weeks

Experimental design later received a single dose of CCl4 2 ml/kg b.wt. by gavage as 1:1 dilution in corn oil. The oral administra- Acute oral toxicity study of Smilax zeylanica leaf extract was tion of extract was continued for 16 weeks after NDEA carried out as per Organization for Economic Cooperation injection. Body weight was recorded at the end of every week and Development (OECD) guideline 425 (Up and Down for 16 weeks. V. Rajesh, P. Perumal

Sample collection After boiled at 95o for 60 min, the reaction solution was extracted with 1.0 mL of distilled water and 5.0 mL of n- At the end of the experimental period (16 weeks), blood butanol and pyridine (15:1 v/v). The absorbance at 532 nm samples were collected from retro-orbital plexus under an- of the organic layer was determined after centrifugation. aesthesia in EDTA tubes and centrifuged at 2,200 g for 15 min at 4 °C. Plasma samples were stored at−20 °C for Estimation of superoxide dismutase (SOD) activity biochemical analysis. 0.5 ml of tissue homogenate was diluted with 1 ml of water. Biochemical analysis In this mixture, 2.5 ml of ethanol and 1.5 ml of chloroform (all reagents chilled) were added and shaken for 1 min at 4 °C Serum transaminases (AST and ALT) were determined by the then centrifuged. The enzyme activity in the supernatant was method of Reitman and Frankel (1957). The activity of serum determined. The assay mixture contained 1.2 ml of sodium alkaline phosphatase (ALP) was estimated by the method of pyrophosphate buffer (0.025 M, pH 8.3), 0.1 ml of 186 μM KindandKing(1954). Serum bilirubin (SB) and total protein phenyl methane sulphate (PMS), 0.3 ml of 30 μM nitro blue (TB) were estimated by the methods of Malloy and Evelyn tetrazolium (NBT), 0.2 ml of 780 μM nicotinamide adenine (1937) and Wooten (1964) respectively. Gamma glutamyl trans- dinucleotide reduced (NADH), appropriately diluted en- ferase (GGT) activity was determined according to the method zyme preparation and water in a total volume of 3 ml. of Szas (1976). Quantitative estimation of tumor markers α-feto Reaction was started by the addition of nicotinamide adenine protein (AFP) and carcinoembryonic antigen (CEA) were based dinucleotide reduced (NADH). After incubation at 30 °C for on solid phase enzyme linked immunosorbent assay (ELISA) 90 s the reaction was stopped by the addition of 1 ml glacial using the UBI MAGIWELL (USA) enzyme immunoassay kit acetic acid. The reaction mixture was stirred vigorously and (Sell and Beckar 1978;Macnabetal.1978). shaken with 4 ml of n-butanol. The intensity of the chromogen After blood collection animals were sacrificed by cervical in the butanol layer was measured at 560 nm against butanol decapitation and the liver was excised, washed in ice cold blank. A system devoid of enzyme served as control. One unit saline, blotted to dryness and examined for any deductable of the enzyme activity is defined as the enzyme reaction, which changes. A portion of liver tissue was then homogenized for gave 50 % inhibition of nitro blue tetrazolium (NBT) reduction biochemical assays. in 1 min under the assay conditions (Karkar et al. 1984)

Preparation of tissue homogenate Estimation of catalase activity

The tissues were weighed and 10 % tissue homogenate was The reaction mixture (1.5 ml) contained 1.0 ml of 0.01 M prepared with 0.025 M Tris–HCl buffer, pH 7.5. After cen- phosphate buffer (pH 7.0), 0.1 ml of tissue homogenate and trifugation at 10,000×g for 10 min, the clear supernatant was 0.4 ml of 2 M H2O2. The reaction was stopped by the used to measure thiobarbituric acid reactive substances addition of 2.0 ml of dichromate-acetic acid reagent (5 % (TBARS). For the determinations of vitamin E level, the potassium dichromate and glacial acetic acid were mixed in tissues were weighed and lipids were extracted from tissues 1:3 ratio). Then the absorbance was read at 620 nm; CAT by the method of Folch et al. (1957) using chloroform– activity was expressed as μMofH2O2 consumed/min/mg methanol mixture (CHCl3:CH3OH) (2:1; v/v). The lipid protein (Sinha 1972). fraction was used for the estimation of vitamin E. For the estimation of non-enzymatic and enzymatic antioxidants, Glutathione peroxidase (GSH-Px) tissues were minced and homogenized (10 % w/v) in 0.1 M phosphate buffer (pH 7.0) and centrifuged for 10 min and the Glutathione peroxidase activity was measured by the method resulting supernatant was used for enzyme assays. of Rotruck et al. (1973). To 0.2 ml of buffer, 0.2 ml of ethylenediaminetetraacetic acid (EDTA), 0.1 ml of sodium Estimation of lipid peroxidation (LPO) azide and 0.5 ml of tissue homogenate were added. To that mixture, 0.2 ml of glutathione solution and 0.1 ml of hydro- The levels of thiobarbituric acid reactive substances gen peroxide were added. The contents were mixed well and (TBARS) in the liver was measured by the method of incubated at 37 °C for 10 min along with the control tubes Ohkawa et al. (1979) as a marker for lipid peroxidation. An containing all the reagents but no enzyme. After 10 min, the aliquot of liver homogenates was mixed with 0.2 mL of reaction was arrested by the addition of 0.4 ml of 10 % TCA. 8.1 % Sodium dodecyl sulphate (SDS), 1.5 mL of 20 % 0.2 mL of tissue homogenate was added to the control tubes. acetic acid and 1.5 mL of 0.8 % Thiobarbituric acid (TBA), The tubes were centrifuged and supernatant was assayed for then the volume was adjusted to 4.0 mL with distilled water. glutathione content by adding Ellman’s reagent. Chemopreventive and antioxidant activity

Glutathione-S-transferase (GST) Histopathological examination

Glutathione-S-transferase activity was measured by the Representative sections of right, left and caudate lobes of method of Habig et al. (1974). The reaction mixture contain- liver were fixed in 10 % formalin and embedded in molten ing 1 ml of buffer, 0.1 ml of 1-chloro-2,4-dinitrobenzene paraffin wax and were ultra sectioned (5–6 μm thickness), (CDNB), 0.1 ml of homogenate and 1.7 ml of distilled water stained with hematoxylin and eosin and were examined was incubated at 37 °C for 5 min. The reaction was then under light microscope for histopathological changes. started by the addition of 1 ml of glutathione. The increase in absorbance was followed for 3 min at 340 nm. The reaction Statistical analysis mixture without the enzyme was used as blank. Results were expressed as mean±standard error of mean Glutathione reductase (GR) activity (SEM). The results were analysed for statistical significance by one way ANOVA followed by dunnett’s test (Graphpad Glutathione reductase that utilizes Nicotinamide adenine Software Inc,La Jolla, CA. Trial version). The criterion for dinucleotide phosphate (NADPH) to convert metabolized statistical significance was set at P<0.05. glutathione (GSSG) to the reduced form was assayed by the method of Horn and Burns (1978). Results Reduced glutathione (GSH) Percentage yield of extract Reduced glutathione was estimated by the method of Ellman (1959). 0.5 ml of tissue homogenate was precipitated with The percentage yield of extract obtained from extraction of 2 ml of 5 % TCA. After centrifugation, 1 ml of supernatant Smilax zeylanica leaves using methanol as solvent was found was taken and added 0.5 mL of Ellman’s reagent (19.8 mg of to be 22.03 % w/w. 5,5′ dithio (bis) nitrobenzoic acid in 100 ml of 1 % sodium The phytochemical examination of methanol extract of citrate) and 3 ml of phosphate buffer. Standards were treated in Smilax zeylanica leaves revealed the presence of carbohy- a similar way and the colour developed was read at 412 nm. drates, alkaloids, phenolics, tannins, flavonoids, glycosides and alkaloids. Estimation of ascorbic acid (vitamin C) Acute oral toxicity study 0.5 ml of tissue homogenate was mixed thoroughly with 1.5 ml of 6 % TCA and centrifuged for 10 min at 3,500 g. Acute oral toxicity study was carried out as per OECD After centrifugation, 0.5 ml of the supernatant was mixed with guideline 425. From the limit test results it was observed 0.5 ml of Dinitrophenylhydrazine (DNPH) reagent and that, the Smilax zeylanica leaf extract is safe upto a dose level allowed to stand at room temperature for an additional 3 h of 2,000 mg/kg. There was no mortality and the experimental then added 2.5 ml of 85 % sulphuric acid and allowed to stand animals did not show any toxic effect throughout the obser- for 30 min. Then the absorbance was read at 530 nm. A set of vation period of 14 days. standards containing 10–50 μg of ascorbic acid were taken and processed similarly along with a blank. Ascorbic acid Body weight values were expressed as μg/mg tissue (Omaye et al. 1979). Average body weights of different animal groups at various Estimation of vitamin E intervals are shown in Fig. 1. There was no significant differ- ence in final body weight between the control group (230.8± 0.1 ml of lipid extract, 1.5 ml of ethanol and 2 ml of 2.0 g) and extract alone treated groups (Extract 200 mg/kg− petroleum ether were added, mixed and centrifuged for 237.5±2.81 g, Extract 400 mg/kg− 231.7±2.1 g). The final 3,000 rpm for 10 min. The supernatant was evaporated to body weight of NDEA induced untreated animals (142.5± dryness at 80 °C then 0.2 ml of 2, 2-1-dipyridyl solution and 4.95 g) was significantly less (p<0.001) compared to control 0.2 ml of ferric chloride solution was added and mixed well. animals (230.8±2.0 g). The group V and group VI animals This was kept in dark for 5 min and added 2 ml of butanol. with NDEA induced and treated with extract 200 mg/kg and Then the absorbance was read at 520 nm. Standards of α- 400 mg/kg showed a significant increase (p<0.001) in final tocopherol in the range of 10–100 μg were taken and treated body weight (Extract 200 mg/kg− 194.2±9.61 g and Extract similarly along with blank containing only the reagent. The 400 mg/kg− 210±1.82 g) compared to untreated animals. It values were expressed as μg/mgtissue (Barker et al. 1951). shows the Smilax zeylanica leaf extract prevented the body V. Rajesh, P. Perumal

260 240 220 200 180 160 140 120 100 Body weight (g) weight Body 80 60 40 20 0 123456789101112131415161718 weeks

Normal E xtract 200 mg/kg Extract 400 mg/kg NDEA Control NDEA + Extract 200 mg/kg NDEA + Extract 400 mg/kg Fig. 1 Effect of Smilax zeylanica leaf extract on body weight gain during N- untreated animals (NDEA control) showed a significant decrease in body nitrosodiethylamine (NDEA) induced hepatocarcinogenesis in rats. Average weight (***p<0.001b) compared to control animals. The group V and group body weight of different animal groups at various intervals. Results are given VI animals with NDEA induced and treated with extract 200 mg/kg as mean±S.E.M. One -way ANOVA followed by Dunnett’s test was used to (NDEA+Extract 200 mg/kg) and 400 mg/kg (NDEA+Extract 400 mg/kg) compare experimental groups. There was no significant difference in final showed a significant increase (***p<0.001b) in final body weight compared body weight between the control (normal) group and extract alone treated to untreated animals (NDEA control) groups (Extract 200 mg/kg and Extract 400 mg/kg) (nsa). NDEA induced weight loss in NDEA induced animals and maintained the relative liver weight between control (2.15±0.09) and extract growth rate near normal. alone treated groups (Extract 200 mg/kg− 2.02±0.02, Extract 400 mg/kg− 2.03±0.02). The relative liver weight of group IV Gross morphology NDEA induced untreated animals (3.61±0.25) were signifi- cantly increased (p<0.001) compared to Group I control ani- As depicted in Fig. 2, the appearance of liver in control mals. A significant decrease (p<0.001) in relative liver weight group rats were normal and there were no macroscopi- was noted in NDEA induced group V and group VI animals cally detectable changes in liver (Fig. 2a). No obvious treated with extract 200 mg/kg (2.60±0.23) and 400 mg/kg changes in liver morphology were observed in extract (2.45±0.10). alone treated group which is indicative of nontoxic nature of Smilax zeylanica leaf extract (Fig. 2b and c). Effect of Smilax zeylanica leaf extract on serum marker The rats in NDEA group revealed enlarged liver and enzymes and hepatocarcinogenesis marker multiple lesions (whitish) with almost the entire surface of liver is occupied with abnormal growth in posterior The effect of Smilax zeylanica leaf extract on the activities of surface (Fig 2d Posterior surface of liver). In group of marker enzymes and serum protein levels in control and NDEA induced animals treated with Smilax zeylanica experimental groups are shown in Table 2. A significant leaf extract 200 mg/kg, compared to NDEA control increase (P<0.001) in AST (77.33±3.40), ALT (63.50± there was a marked reduction in damage caused by NDEA 2.06), ALP (107.5±4.78) and GGT (116.7±8.02) IU/L were on gross examination of anterior and posterior surface. There observed in NDEA induced untreated animals compared to were no detectable lesions or nodules (Fig 2e). The liver of control animals with AST (36.97±1.97), ALT (33.33±1.33), rats treated with Smilax zeylanica leaf extract 400 mg/kg ALP (46.83±1.47) and GGT (42.00±4.83) IU/L. The ani- appeared similar to normal rat liver. Both anterior and poste- mals treated with Smilax zeylanica leaf extract 200 mg/kg rior surface of liver appeared smooth and there were no and 400 mg/kg showed a significant decrease in AST, ALT, detectable lesions or nodules (Fig 1f). ALP and GGT levels from (38.17±4.28)–(42.00±1.71) (p< 0.001), (43.67±3.63)–(37.67±0.91) (p<0.001), (83.3± Relative liver weight 1.66)–(58±0.89) (p<0.001) and (58.00±8.18)–(42.67± 2.49) (p<0.001) respectively compared to NDEA untreated The relative liver weight of control and experimental group of animals . The total protein levels were decreased (P<0.001) animals are shown in Table 1 and the results were expressed as in NDEA untreated animals (6.28±0.16) compared to con- g liver/100 g body wt. There was no statistical difference in trol animals (8.43±0.51). However, upon treatment with Chemopreventive and antioxidant activity

Fig. 2 Gross morphology of liver. a Liver of control animal showing occupied with abnormal growth. e Liver of NDEA induced group V normal morphology. b and c Liver of animal dosed with extract alone animals treated with Smilax zeylanica leaf extract 200 mg/kg showing 200 mg/kg and 400 mg/kg respectively showing normal appearance marked reduction in damage caused by NDEA. f Liver of rats treated which is indicates nontoxic nature of extract. d Liver of group IV with Smilax zeylanica leaf extract 400 mg/kg showing normal morphol- NDEA induced untreated animals (NDEA control) showing multiple ogy similar to control animals. No detectable lesions or nodules were lesions (whitish) with almost the entire posterior surface of liver is noted in anterior and posterior surface of treatment groups

Table 1 Relative liver weight of various groups of rats at the end of the study (16 weeks)

Groups Relative liver weight (g liver/100 g body wt)

Group I (Control) (0.5 % CMC) 2 ml/kg p.o. 2.15±0.09 Group II (Extract 200 mg/kg p.o.) 2.02±0.02ns,a Group III (Extract 400 mg/kg p.o.) 2.03±0.02ns,a Group IV (NDEA alone) 3.61±0.25***,a Group V (NDEA+Extract 200 mg/kg p.o.) 2.60±0.23***,b Group VI (NDEA+Extract 400 mg/kg p.o.) 2.45±0.10***,b

All values are expressed as mean±S.E.M, n=6 in each group a Values are significantly different from control group; ns-non significant; *P<0.05; **P<0.01; ***P<0.001 b Values are significantly different from NDEA- induced group; ns-non significant; *P<0.05; **P<0.01; ***P<0.001 (one-way ANOVA followed by Dunnett’s test) V. Rajesh, P. Perumal

Table 2 Effect of Smilax zeylanica leaf extract on biochemical parameters in control and experimental groups of rats

Groups AST (IU/L) ALT (IU/L) ALP (IU/L) TB (mg/dl) TP (mg/dl) GGT (IU/L)

Group I (Control) (0.5 %CMC) 36.97±1.97 33.33±1.33 46.83±1.47 0.41±0.04 8.43±0.51 42.00±4.83 2 ml/kg p.o. Group II (Extract 200 mg/kg 46.17±1.42ns,a 35.00±1.41ns,a 53.50±1.92ns,a 0.33±0.04ns,a 8.23±0.21ns,a 37.83±2.41ns,a p.o.) Group III (Extract 400 mg/kg 46.67±1.90ns,a 39.17±0.74ns,a 57.17±0.90ns,a 0.33±0.03ns,a 8.41±0.15ns,a 32.17±1.32ns,a p.o.) Group IV (NDEA alone) 77.33±3.40***,a 63.50±2.06 ***,a 107.5±4.78 ***,a 0.41±0.03ns,a 6.28±0.16***,a 116.7±8.02***,a Group V (NDEA+Extract 38.17±4.28***,b 43.67±3.63***,b 83.3±1.66**,b 0.40±0.03ns,b 7.75±0.16*,b 58.00±8.18***,b 200 mg/kg p.o.) Group VI (NDEA+Extract 42.00±1.71***,b 37.67±0.91***,b 58±0.89***,b 0.36±0.02ns,b 8.76±0.56***,b 42.67±2.49***,b 400 mg/kg p.o.)

All values are expressed as mean±S.E.M, n=6 in each group. One -way ANOVA followed by Dunnett’s test was used to compare experimental groups a Values are significantly different from control group; ns-non significant; *P<0.05; **P<0.01; ***P<0.001 b Values are significantly different from NDEA- induced group; ns-non significant; *P<0.05; **P<0.01; ***P<0.001

Smilax zeylanica leaf extract 200 mg/kg and 400 mg/kg the zeylanica leaf extract 400 mg/kg. Non-significant alteration protein levels were significantly increased from (7.20± in serum marker enzymes and hepatocarcinogenesis marker 0.31)–(8.76±0.56 mg/dl) (p<0.01–p<0.001). in extract alone treated group II and group III animals when The effect of Smilax zeylanica leaf extract on AFP and compared with control group I animals indicates the non- CEA levels in serum of control and experimental groups are toxic nature of Smilax zeylanica leaf extract. shown in Table 3. A significant increase in AFP (23.67± 2.05) (p<0.001) and CEA (16.32±1.51) (p<0.05) were ob- Effect of Smilax zeylanica leaf extract on LPO and levels served in NDEA induced untreated animals compared to of antioxidant enzymes in liver control animals with AFP and CEA levels 12.55±1.24 and 11.55±1.23 ng/ml respectively. In group of animals treated The LPO levels in liver homogenate of control and experi- with Smilax zeylanica leaf extract 200 mg/kg and 400 mg/kg, mental animals are illustrated in Fig. 3a significant increase the AFP levels in serum were significantly reduced from (p<0.001) in the production of MDA (1.90±0.03) was ob- 16.43±0.85 – 13.87±0.30 (p<0.01–p<0.001) compared to served in the group IV NDEA intoxicated untreated animals NDEA untreated animals and there was no significant dif- compared to control animals (1.09±0.08). Administration of ference in CEA levels (14.75±0.21) in serum of animals Smilax zeylanica leaf extract showed a significant reduction treated with extract 200 mg/kg compared to NDEA untreated in LPO as evidenced by a significant fall in MDA levels to animals. However, CEA levels were significantly decreased 1.42±0.18 (p<0.01) and 1.10±0.05 (p<0.001) at doses 200- (11.60±0.40) (p<0.01) in animals treated with Smilax mg/kg and 400 mg/kg respectively.

Table 3 Effect of Smilax zeylanica leaf extract on α-feto protein levels (AFP) and carcino embryonic antigen (CEA) levels in the serum of control and experimental groups of rats

Groups α-feto protein (AFP) ng/ml Carcino embryonic antigen (CEA) ng/ml

Group I (Control) (0.5%CMC) 2 ml/kg p.o. 12.55±1.24 11.55±1.23 Group II (Extract 200 mg/kg p.o.) 11.45±0.38ns,a 10.02±0.31ns,a Group III (Extract 400 mg/kg p.o.) 11.82±0.32ns,a 9.90±0.15ns,a Group IV (NDEA alone) 23.67±2.05***,a 16.32±1.51*,a Group V (NDEA+Extract 200 mg/kg p.o.) 16.43±0.85**,b 14.75±0.21ns,b Group VI (NDEA+Extract 400 mg/kg p.o.) 13.87±0.30***,b 11.60±0.40**,b

All values are expressed as mean±S.E.M, n=6 in each group a Values are significantly different from control group; ns-non significant; *P<0.05; **P<0.01; ***P<0.001 b Values are significantly different from NDEA- induced group; ns-non significant; *P<0.05; **P<0.01; ***P<0.001 (one-way ANOVA followed by Dunnett’s test) Chemopreventive and antioxidant activity

Lipid peroxidation near normal levels. The SOD levels in liver of animals treated 2.5 with extract 200 mg/kg and 400 mg/kg were significantly – a increased (p<0.01 to p<0.001) 1.25±0.21 and 1.18±0.07 respectivelycomparedtoNDEA untreated animals. Extract *** 2.0 200 mg/kg and 400 mg/kg significantly increased the CAT, b GSH-Px, GR and GST levels from (1.41±0.08) – (1.44±0.03) ** (p<0.001), (1.53±0.04) – (1.73±0.08) (p<0.05 – p<0.01), (1.06±0.10) – (1.13±0.08) (p<0.01 – p<0.001) and (0.91± 1.5 b a 0.04) – (1.28±0.04) U/mg protein (p<0.01– p<0.001), respec- a *** ns tively compared to NDEA induced untreated animals. ns Similarly, administration of extract 200 mg/kg and 400 mg/kg 1.0 significantly increased the non-enzymatic antioxidants, GSH, Lipid peroxidation vitamin C and vitamin E levels from (0.86±0.04) – (0.95±

MDA nmole/hr/gm of tissue nmole/hr/gm MDA 0.02) (<0.05 – p<0.001), (10.45±0.39) – (10.63±0.37) (p<

0.5 0.01) and (0.75±0.02) – (0.90±0.02) (p<0.01 – p<0.001), respectively compared to NDEA induced untreated animals. The extract did not produce any deleterious effect on the antioxidant defense system in normal animals which is 0.0 evidenced from the non-significant alteration of the enzymatic g g/kg and non-enzymatic antioxidants along with the maintained rate Normal 0mg/k of lipid peroxidation in group II and group III animals when ct 200m NDEA Control compared with the normal control group I animals. The results Extract 200mg/kgExtract 400mg/kg indicate that, the level of lipid peroxidation which increased in liver of carcinogen administered animals was lowered in extract NDEA + ExtraNDEA + Extract 40 treated animals and in contrast the antioxidant status which was Fig. 3 Effect of Smilax zeylanica leaf extract on lipid peroxidation in found to be decreased in carcinogen administered animals was liver homogenate of experimental animals during NDEA induced liver improved to near normal upon Smilax zeylanica leaf extract carcinogenesis. Results are expressed as mean±S.E.M. a values are significantly different from control group; ns-non significant; *P< administration. This indicates that Smilax zeylanica leaf extract 0.05; **P<0.01; ***P<0.001. bvalues are significantly different from contributes to exert antioxidant defense mechanism. NDEA induced untreated group; ns-non significant; *P<0.05;**P< 0.01;***P<0.001. (ANOVA, followed by Dunnett’s test). Significant Histopathology increase (***P<0.001) in lipid peroxidation is noted in NDEA induced untreated animals (NDEA control) compared to normal animals. NDEA induced animals treated with extract (NDEA+Extract 200 mg/kg and Histopathological examination of liver sections from control NDEA+Extract 400 mg/kg) showed a significant decrease in lipid group animals revealed normal architecture (Fig. 5a). The peroxidation levels (200 mg/kg- **P<0.01, 400 mg/kg- (***P< liver sections of group II and group III animals given extract 0.001) compared to NDEA control animals alone for 16 weeks showed normal architecture. The portal tracts were normal. The hepatic parenchyma showed normal Figure 4a to h portrays the activities and the levels of hepatocytes. The central vein and sinusoids appeared normal. antioxidants in liver of control and experimental animals and There is no evidence of inflammation or malignancy or atyp- the results were expressed as U/mg of protein. A significant ical cells depicting the non-toxic nature of Smilax zeylanica decrease (p<0.001) in activities of SOD (0.93±0.02), cata- leaf extract (Fig. 5b and c). Section of liver from group IV lase (0.92±0.03), GSH-Px (0.98±0.13) GR (0.68±0.04), NDEA induced untreated animals revealed loss of architecture GST (0.68±0.04) were noted in group IV NDEA intoxicated with proliferation of tumour cells resembling bile ductular untreated animals compared to control animals with SOD (1.85 cells with suggestive adenocarcinoma (cholangio adenocarci- ±0.07), CAT (1.75±0.06), GSH-Px (2.40±0.24), GR (1.08± noma) (Fig. 5d). In contrast, NDEA induced animals treated 0.10), GST (1.50±0.03). The levels of non-enzymatic antiox- with Smilax zeylanica leaf extract 200 mg/kg showed normal idants GSH (0.59±0.09), vitamin-C (8.58±0.41) and vitamin- lobular architecture with mild inflammation in portal tracts E (0.63±0.01) were also significantly decreased (p<0.001) in (Fig. 5e), whereas the liver sections of NDEA induced animals liver of group IV untreated animals compared to control ani- treated with Smilax zeylanica leaf extract 400 mg/kg showed mals with GSH (1.08±0.07), vitamin-C (10.77±0.47) and normal hepatic architecture (Fig. 5f). The portal tracts were vitamin-E (0.78±0.01). However, administration of Smilax normal and the hepatic parenchyma showed normal hepato- zeylanica leaf extract at 200 mg/kg and 400 mg/kg significantly cytes with no evidence of neoplastically transformed cells increased the levels of SOD, CAT, GSH-Px, GR and GST to (40×, HE). V. Rajesh, P. Perumal

a Superoxide dismutase b Catalase 2.5 b 2.0 a a a ns ns ns ns b 2.0 b b *** 1.5 *** ** b ** 1.5 a a *** 1.0 SOD *** 1.0 Catalase U/mg of protein of U/mg cosumed/min/mgof protein 2 O

2 0.5 0.5

0.0 H of nmole 0.0

kg

Normal Normal 00mg/kg

act 400mg/kg NDEA Control NDEA Control Extract Extract200mg/kg 400mg/kg ExtractExtract 200mg/ 400mg/kg

NDEA +NDEA Extract + Extr200mg/kg NDEA +NDEA Extract + Extract 200mg/kg 4

c Glutathione peroxidase d Glutathione reductase 1.5 3 b b a a ** *** ns ns a a b ns ns 1.0 2 b ** a

x * *** a

GSH-P *** 1 0.5 Glutathione reductase Mmoles/min/mg of protein of Mmoles/min/mg mole of NADPH oxidized/min mg protein mg oxidized/min NADPH of mole 0 µ 0.0 g

Normal Normal mg/kg

NDEA Control DENA Control ExtractExtract 200mg/kg 400mg/kg ExtractExtract 200mg/kg 400mg/kg

NDEA +NDEA Extract + Extract 200mg/kg 400mg/kg DENA +DENA Extract + Extract 200mg/k 400 Fig. 4 Effect of Smilax zeylanica leaf extract on the status of enzymatic and animals (NDEA control) compared to normal animals. NDEA induced non-enzymatic antioxidants in liver homogenate of control and experimental animals treated with extract (NDEA+Extract 200 mg/kg and NDEA+Extract group of animals. Results are expressed as mean±S.E.M. a values are 400 mg/kg) showed a significant increase in activities of SOD (200 mg/kg- significantly different from control group; ns-non significant; *P<0.05; **P<0.01, 400 mg/kg P**<0.01), catalase (200 mg/kg- ***P<0.001, **P<0.01; ***P<0.001. b values are significantly different from NDEA 400 mg/kg- ***P<0.001), GSH-Px (200 mg/kg- *P<0.05, 400 mg/kg- induced untreated group; ns-non significant; *P<0.05;**P<0.01;***P< **P<0.01), GR (200 mg/kg- **P<0.01, 400 mg/kg ***P<0.001), GST 0.001. (ANOVA, followed by Dunnett’s test). Significant decrease in activites (200 mg/kg- **P<0.01, 400 mg/kg ***P<0.001), GSH (200 mg/kg- *P< of SOD (***P<0.001), catalase (***P<0.001), GSH-Px (***P<0.001),GR 0.05, 400 mg/kg- ***P<0.001), vitamin C (200 mg/kg- **P<0.01, 400 mg/ (***P<0.001), GST (***P<0.001), GSH (***P<0.001), vitamin C (***P< kg- **P<0.01) and vitamin E (200 mg/kg- **P<0.01, 400 mg/kg- ***P< 0.01) and vitamin E (***P<0.001) were noted in NDEA induced untreated 0.001) compared to NDEA control animals Chemopreventive and antioxidant activity

e Glutathione-S-transferase f Glutathione 2.0 1.5 a a a a ns ns ns b 1.5 ns b b *** *** 1.0 * b a ** 1.0 ***

a GSH *** 0.5

0.5 nmole/mg of protein units/min/mg of protein Glutathione-S-transferase

0.0 0.0

Normal Normal

NDEA Control DENA Control ExtractExtract 200mg/kg 400mg/kg ExtractExtract 200mg/kg 400mg/kg

NDEA +NDEA Extract + Extract 200mg/kg 400mg/kg DENA +DENA Extract + Extract 200mg/kg 400mg/kg

Vitamin E Vitamin C h g b 1.0 15 a a ***

a b b ns ns b ns ** a ** 0.8 a b ** ns *** 10 ** 0.6 Vitamin E Vitamin C 0.4 5

0.2

0 0.0

Normal Normal

NDEA Control NDEA Control ExtractExtract 200mg/kg 400mg/kg ExtractExtract 200mg/kg 400mg/kg

NDEA +NDEA Extract + Extract 200mg/kg 400mg/kg NDEA +NDEA Extract + Extract 200mg/kg 400mg/kg

Fig. 4 (continued)

Discussion N-nitrosodiethylamine (NDEA), one of the most important environmental carcinogen on metabolic transformation in liv- Liver cancer, one of the most common cancer worldwide, er produces ROS that act as promutagenic products is respon- accounts approximately 549,000 deaths each year (Liu et al. sible for carcinogenic effects (Bansal et al. 2005). Therefore, 2006). As liver, the major metabolic organ in our body is development of a compound with potent antioxidant activity responsible for production of ROS during metabolism of can prevent the formation of ROS and thus provide protection carcinogens might be responsible for carcinogenic effects. against the deteriorating outcome. Traditionally many herbal V. Rajesh, P. Perumal

Fig. 5 Histopathological image of liver tissues. a (40 ×) H and E tumour cells resembling bile ductular cells with suggestive adenocarci- stained section of liver tissue from group I normal animals showing noma. e (circle, 40 ×) H and E stained section of liver from NDEA normal architecture. b and c (40 ×) H and E stained section of liver from induced group V animals treated with Smilax zeylanica leaf extract group II and group III animals given extract alone for 16 weeks showing 200 mg/kg showing normal lobular architecture with mild inflammation normal hepatocytes in parenchyma. The portal traids appeared normal in portal tracts. f (arrows, 40 ×) H and E stained section of liver from with no evidence of inflammation or malignancy or atypical cells NDEA induced group VI animals treated with Smilax zeylanica leaf depicting the non-toxic nature of Smilax zeylanica leaf extract. d (40 extract 400 mg/kg showing normal hepatocytes and portal triads with ×) H and E stained section of liver from group IV NDEA induced no evidence of neoplastically transformed cells untreated animals showing loss of architecture with proliferation of medicines have revealed cancer chemopreventive potential glutathione, tissue proteins and enzymes viz. SOD, catalase through antioxidant activity. The present study was aimed to and peroxidase in CCl4 induced hepatotoxicity model in evaluate the chemopreventive and antioxidant activity of Wistar albino rats (Anita et al. 2010). methanol extract of Smilax zeylanica leaves against N- Decrease in body weight observed during the experimen- nitrosodiethylamine induced hepatocarcinogenesis. Dose tal period in NDEA induced untreated animals could be levels were selected based on the acute oral toxicity study largely due to deterioration of liver function and nutritional and our previous invivo studies, where the extract showed deprivation which might be due to reduced food intake. In protection against streptozotozin (STZ) induced oxidative addition, assessment of relative liver weight was used as stress (Rajesh et al. 2010) and substantial evidence from potential tool to diagnose change in liver size. NDEA in- studies on invitro and invivo antioxidant activities where the duced proliferation of cells in the liver was evident from methanol extract of Smilax zeylanica leaves at the dose levels increase in NDEA induced untreated animals. Treatment 200 mg/kg and 400 mg/kg body weight exhibited free radical with Smilax zeylanica leaf extract prevented body weight scavenging effect against the different free radicals tested loss and reduced the relative liver weight compared to (DPPH, hydrogen peroxide, ABTS, nitric oxide and superox- untreated animals, which signify the amelioration capacity ide free radicals) and significant increase in the levels of of extract upon carcinogen exposure. Significant decrease in Chemopreventive and antioxidant activity lesions in liver of treatment groups were supported by histo- CEA levels observed in NDEA induced untreated animals logical assessment of liver. The pathological changes in liver might have caused due to mutagenesis. The decrease in levels caused by NDEA were assessed by determining the levels of of AFP and CEA following treatment with Smilax zeylanica various biochemical hepatic markers (Kovalsky et al. 1996). leaf might be due to response to therapy. Marked elevation in AST and ALT in NDEA induced Increased ROS generation and decreased antioxidant de- untreated animals indicated the hepatocellular damage. fense in liver tissue has been reported in development of Because AST and ALT are cytoplasmic in location, they are hepatocarcinogenesis (Ramakrishnan et al. 2006; Kweon released into circulation after cellular damage and rupture of et al. 2003). ROS, through interaction with nucleic acids, plasma membrane (Wroblewski 1959; Sallie et al. 1991). proteins, lipids results in chromosomal instability, mutations, Hence AST and ALT are considered as sensitive markers loss of organelle function play an important role in develop- employed in diagnosis of hepatic damage. Treatment with ment of cancer (Waris and Ahsan 2006). Lipid peroxidation Smilax zeylanica leaf extract significantly reduced the levels is one of the major mechanisms of cell injury caused by free of AST and ALT in NDEA induced animals. This indicates radicals. Enormous amount of free radicals generated by that Smilax zeylanica leaf extract tends to prevent liver carcinogens reacts with lipids causing lipid peroxidation damage by maintaining integrity of plasma membrane, there- (Sikkim and Mulee 2000). The products of lipid peroxidation by suppressing the leakage of enzymes through membrane. include malondialdehyde that interacts with various mole- The elevation in ALP in NDEA induced untreated ani- cules leading to oxidative stress and has been reported to be mals might be due to proliferation of bile ductular cells and involved in the formation of tumours (Ramakrishnan et al. resulting blockade of bile ducts. Significant reduction in 2007). In the present study, significant increase in the levels ALP levels in NDEA induced extract treated animals indi- of lipid peroxidation observed in untreated group IV cancer cates the protective effect of Smilax zeylanica leaf extract bearing animals may be due to the excessive production of against NDEA induced hepatic injury. The reduction in free radicals and due to inhibition of antioxidant enzymes. serum protein levels in NDEA induced untreated animals Significantly reduced levels of lipid peroxidation were seen might be due to liver damage because hepatotoxicity impairs in Smilax zeylanica leaf extract treated group Vand group VI the synthetic function of liver (David 1999). Treatment with NDEA induced animals. This clearly shows that Smilax Smilax zeylanica leaf extract ameliorated the imbalance. zeylanica effectively controlled the rate of lipid peroxidation, GGT, an enzyme embedded in plasma membrane of he- which suggests the beneficial effect of the extract against patocytes, mainly in canalicular domain. Any damage in NDEA initiated free radical formation. The presence of plasma membrane causes liberation of GGT into serum flavonoids may contribute to this effect, because they are (Sivaramakrishnan et al. 2008). Hence, GGT is considered proved to be a potential inhibitor of lipid peroxidation as a best indicator of liver damage (Bulle et al. 1990). (Siegers and Younes 1981). Elevated levels of GGT observed in NDEA induced The enhanced formation of lipid peroxides is further untreated animals might be due to liver injury. Treatment evidenced by decrease in activities of antioxidant enzymes with Smilax zeylanica leaf extract significantly lowered the in liver of NDEA induced untreated animals as compared GGT level which indicates that Smilax zeylanica leaf extract with normal control animals. tends to prevent liver damage by maintaining integrity of SOD is said to act as the first line of defense against plasma membrane. superoxide radical generated as a by-product of oxidative phos- A tumour associated foetal protein, AFP has long been phorylation. SOD mediated dismutation of superoxide radical - employed as a serum tumour marker to monitor disease (O2 ) generates hydrogen peroxide (H2O2). Accumulation of progression (Abelev 1971). AFP is a 72 KDa α1 globulin excess of H2O2 causes toxic effects on cellular system. In this synthesized during embryonic life by foetal yolk sac, liver regard GSH-Px and catalase converts H2O2 into water (Li and intestinal tract with uncertain biological function. Since Shijun et al. 2000). GSH-Px catalyses the reduction of H2O2 AFP has high specificity for hepatocarcinoma (Liu et al. at the expense of reduced GSH, thereby protecting cells against

2006), its serum concentration can be used in diagnosis of oxidative damage. CAT detoxifies H2O2 into H2OandO2 hepatocarcinoma and for the diagnosis of tumour response to (Murray et al. 2003). Thus SOD, CAT and GSH-Px act mutu- therapy. ally and constitute the enzymatic defense mechanism against CEA is a glycoprotein found in different cells, but typically ROS (Bhattacharjee and Sil 2006). In the present study de- associated with certain tumours. Increase in CEA levels is crease in the activities of SOD, CAT and GSH-Px in untreated most frequent in cancer of colon and rectum. The conditions cancer bearing animals could be attributed to excessive utiliza- which can elevate CEA are smoking, pancreatitis, inflamma- tion of enzymes in detoxification of peroxides and hydroper- tory bowel disease, pancreatitis and cirrhosis of liver. CEA oxides generated during liver carcinogenesis. Restoration in the levels increase, with an increase in tumour size (Jahan et al. levels of lipid peroxidation upon treatment with Smilax 2011). In the present study, significant increase in AFP and zeylanica leaf extract might have resulted in the recoupment V. Rajesh, P. Perumal in the activities of the above antioxidant enzymes to normalcy. The histological observations clearly indicate the pres- SOD, catalase and GSH-Px require several secondary enzymes ence of neoplastic changes in NDEA induced untreated like GR and cofactors like GSH, NADPH, to function at high animals. In animals treated with Smilax zeylanica leaf extract efficacy. GR catalyzes the NADPH dependent reduction of the liver architecture was preserved which clearly supports glutathione disulphide to glutathione thus maintaining glutathi- the biochemical data. These suggest the Smilax zeylanica one levels (Katiyar et al. 1993). GST catalyzed GSH conjuga- leaves might have promising protective role against liver tion is an important mechanism for the detoxification process. carcinogenesis. In the present study the activities of GR and GST were signif- icantly reduced in untreated NDEA induced untreated animals. Upon treatment with Smilax zeylanica leaf extract, the GR and Conclusion GST levels were significantly increased. Increase in GR can protect the liver by maintaining the basal level of GSH, which Data from this study suggest that administration of is important for many other GSH dependent detoxification Smilax zeylanica leaf extract possesses significant pro- reactions. tection against NDEA induced liver carcinogenesis by a GSH, vitamin C and vitamin E are well known non- mechanism related, at least in part, to the ability of enzymatic antioxidant defense system of cells, act synergis- Smilax zeylanica leaves to decrease oxidative stress by tically to scavenge free radicals in biological system. GSH is stabilizing and increasing all the components of antioxi- found to be present in high concentration in cells, protects dant defense system which were disturbed system during cells against free radical attack (Farombi et al. 2000). GSH NDEA induced oxidative stress in rats. The combined acts directly as free radical scavenger by donating a activity of the phytomolecules in the extract might be hydrogen atom and thereby neutralizing hydroxyl radical. responsible for oxidative defense and chemoprevention. It reduces peroxides and maintains protein thiols in the reduced state (Nwanjo and Oze 2007). GSH-PX uses Acknowledgments The authors are thankful to the management GSH as a substrate to catalyze the reduction of hydro- of JKK Nattraja College of Pharmacy, Komarapalayam, Tamil Nadu, INDIA for providing necessary facilities to carry out the peroxide and H2O2 (Bebe and Panemangalore 2003). 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