Orient Pharm Exp Med (2013) 13:267–277 DOI 10.1007/s13596-013-0114-6

RESEARCH ARTICLE

Cytoprotective effect of zeylanica Linn. leaves against Benzo[a]pyrene induced lung cancer with reference to lipid peroxidation and antioxidant system in Swiss albino mice

Venugopalan Rajesh & Perumal Perumal

Received: 29 January 2013 /Accepted: 1 April 2013 /Published online: 25 April 2013 # Institute of Korean Medicine, Kyung Hee University 2013

Abstract The cytoprotective effect of methanol extract of and increase in all the components of antioxidant system Smilax zeylanica leaves on benzo(a)pyrene induced experi- attributed to antioxidant and free radical scavenging activity. mental lung cancer was investigated in swiss albino mice. Lung cancer was induced by 50 mg/kg body weight of Keywords Smilax zeylanica leaves . Benzo(a)pyrene . Lung benzo(a)pyrene, given orally twice a week for four succes- carcinogenesis . Lipid peroxidation . Antioxidant activity sive weeks to male swiss albino mice. Benzo(a)pyrene in- duced mice were treated with methanol extract of Smilax zeylanica leaves once daily orally started 1 week before Introduction benzo(a)pyrene induction and continued for 16 weeks. At the end of the experiment various parameters were analyzed. Lung cancer remains a major cause of death in men and It was observed that, the treatment with 200 mg/kg and women accounting 29 % of all deaths (Perin and Notani 400 mg/kg methanol extract of Smilax zeylanica leaves effec- 2001). Tobacco smoking is the most important risk factor for tively suppressed the lung cancer as revealed by significant lung cancer (Stefani et al. 1999). Benzo[a]pyrene, a polycyclic decrease in number of nodules in lung with a significant hydrocarbon present in tobacco smoke play a major role in the weight gain during experimental period. Moreover, with ref- aetiology of lung cancer (Hecht et al. 2002). Benzo[a]pyrene erence to lipid peroxidation and antioxidant system, Smilax is metabolically activated into benzo[a]pyrene 7, 8-diol-9, 10- zeylanica leaf extract decreased the extent of lipid peroxida- epoxide, which reacts with DNA predominantly to form DNA tion with concomitant increase in the activities of enzymatic adduct and progression of disease (Osborn et al. 1976). The antioxidants (superoxide dismutase, catalase, glutathione per- pathogenesis of carcinogenesis involves both free radical and oxidase, glutathione reductase and glutathione-S-transferase) peroxidation products, which damage many cellular macro- and non-enzymatic antioxidants (reduced glutathione, vitamin molecules (Kim et al. 2000). Oxidative stress resulting tobac- C and vitamin E) levels compared to lung cancer bearing co plays a critical role in lung carcinogenesis (Sun 1990). untreated animals. Histopathological examination of lung tis- Chemoprevention offers a novel approach to control the sue too correlated with morphological and biochemical obser- incidence of lung cancer (Wattenberg and Estensen 1996). A vations. Furthermore, the extract did not produce any large number of chemotherapeutic agents have been identi- deleterious effects in extract alone treated groups. Our data fied in epidemiological and experimental studies, preclinical suggest that, the potential chemoprevention of methanol ex- and clinical observations (Takuji et al. 1993; Jagadeeswaran tract of Smilax zeylanica leaves might be due to stabilization et al. 2000). However, the toxic effects produced by some of the agents have limited their extensive use. There is, there- : fore, a need to identify synthetic or natural compound that V. Rajesh (*) P. Perumal have significant chemopreventive potential to block initia- Department of Pharmacology, JKK Nattraja College of Pharmacy, tion or arrest the progression in premalignant cells without Natarajapuram, Komarapalayam, 638183, Namakkal District, Tamil Nadu, undesirable side effects. e-mail: [email protected] In recent years, there has been considerable interest in P. Perumal natural products with antioxidant property to protect cellular e-mail: [email protected] components from oxidative damage and prevent diseases. 268 V. Rajesh, P. Perumal

Among the herbal resources belonging to , the Extraction genus Smilax Linn. consist of about 350 species in the world (Shao et al. 2007), out of which 24 species are found in India The powdered leaves of Smilax zeylanica were extracted (Saldhana and Wicolson 1976; Ramaswamy et al. 2001; with methanol using Soxhlet apparatus for 72 h. After Santapau and Henry 1976). In south India, the genus is rep- completion of extraction, methanol was removed by distil- resented by four species viz., Smilax zeylanica Linn., Smilax lation. The residue obtained was air dried. The percentage aspera Linn., Smilax perfoliata Roxb. and Smilax wightii A. yield of methanol extract was 22.03 % w/w. The dried DC (Gamble 2004). Many of them have been long used as methanol extract was stored in air tight glass container for medicinal herbs, especially as traditional Chinese medicines further investigation. in China (Abdala et al. 2008). The extracts of Smilax china L. tubers are known to show antitumor and antioxidant activities Phytochemical screening on mice and rats (Wang et al. 1996). Smilax zeylanica Linn. is distributed in tropical and subtropical hills of Asia between an The extract obtained was subjected to preliminary phyto- altitude of 500–1,800 m. This perennial climbing shrub is chemical screening (Khandelwal and Kokate 1995). extensively used in traditional system of medicine against veneral diseases (Oommachan and Masih 1991), skin disor- Quantitative estimation ders, sores, swellings, abscess (Ambasta 2006; Nadkarni 1976) and also applied for rheumatism and pain in lower Estimation of total phenolic content extremities (Kirtikar and Basu 1991). Species of Smilax Linn. contain dioscin (spirostanol triglycoside), smilagenin The total phenolic content was determined spectrophotometri- and sarasapogenin (1–3%)(Evans2002). The root of cally using the Folin–Ciocalteu method. This test is based on the Smilax zeylanica Linn. contain diosgenin, a steroidal sa- oxidation of phenolic groups by phosphomolybdic and ponin glycoside (Kar and Sen 1984). Based on the wide- phosphotungstic acids (FC reagent). This reagent, based on the spread use of Smilax species in traditional medicine and Slinkard and Singleton method (1977),andtheearlyworkof the traditional importance of Smilax zeylanica Linn it was Singleton and Rossi (1965) is a colorimetric oxidation/reduction decided to select the for investigation. As previous method for phenolic compounds. The products of the metal studies mainly focused on the plant rhizomes, and there oxide reduction have a blue color that exhibits a broad light was little information about the Smilax zeylanica leaves. In absorption with a maximum at 764 nm. The intensity of light the present study, an effort was made to ascertain the absorption at that wavelength is proportional to the concentra- possible role of Smilax zeylanica Linn. leaves in chemi- tion of phenols. Briefly, a 20 μL of the diluted sample was cally induced lung carcinogenesis. added to 100 μL of Folin–Ciocalteu reagent. After 8 min, 300 μL of saturated sodium carbonate solution (25 %) was added. The absorbance was measured at 764 nm. The calibra- Materials and methods tion curve was prepared with gallic acid solutions ranging from 10 to 1,000 μg/ml, and the results are given as gallic acid Chemicals equivalents (GAE).

Benzo[a]pyrene was purchased from Sigma Chemical Determination of total flavonoids Company St. Louis, MO, USA. All other chemicals used were of analytical grade. Flavonoid content was estimated using aluminium chloride colorimetric method (Chang et al. 2002). Various concen- Plant material trations of extract in methanol were mixed with 0.1 ml of 10 % aluminium chloride (w/v), 0.1 ml of 1 M potassium The fresh leaves of Smilax zeylanica Linn were collected acetate and 2.8 ml distilled water. The mixture was allowed in the month of September from yercaud hills, to stand at room temperature for 30 min. The absorbance of Tamilnadu, India. The plant material was Taxonomically reaction mixture was read at 415 nm. Results are expressed indentified, confirmed and authenticated by Botaniocal as mg/g quercetin equivalent. Survery of India, Coimbatore, Tamilnadu and the voucher specimen was retained in our laboratory for further ref- Animals erence. The collected leaves were shade dried and the dried materials were crushed to coarse powder with me- Healthy Swiss albino mice (6–8 weeks old) weighing 20– chanical grinder. The powder was stored in an airtight 25 g were used for the study. The animals were obtained container for extraction. from Agricultural University, Mannuthy, Thrissur, kerala Cytoprotective effect of Smilax zeylanica Linn. leaves 269

(328/99/CPCSEA) and were housed in polypropylene was carried out for any deductable changes. A portion of lung cages. The animals were acclimated to laboratory conditions tissue was then homogenized for biochemical assays. and were maintained under standard conditions of tempera- ture and light (25±2°C, 12 h light and dark cycle). They Preparation of tissue homogenate were fed with standard pellet diet and had free access to water. Ethical clearance (for handling of animals and the pro- The tissues were weighed and 10 % tissue homogenate was cedures used in study) was obtained from the Institutional prepared with 0.025 M Tris–HCl buffer, pH 7.5. After centri- Animal Ethical Committee (887/ac/05/CPCSEA) before fugation at 10,000 × g for 10 min, the clear supernatant was performing the study on animals. used to measure TBARS. For the determinations of vitamin E level, the tissues were weighed and lipids were extracted from Acute oral toxicity study tissues by the method of Folch et al. (1957) using chloroform–

methanol mixture (CHCl3:CH3OH) (2:1; v/v). The extract Healthy Swiss albino mice of female sex (20–25 g) were used for the estimation of vitamin E. For the estimation of used for the study. Acute oral toxicity study for methanol non-enzymatic and enzymatic antioxidants, tissues were extract of Smilax zeylanica leaves was carried out as per minced and homogenized (10 % w/v) in 0.1 M phosphate Organization for Economic Cooperation and Development buffer (pH 7.0) and centrifuged for 10 min and the resulting (OECD) guideline 425 (Up and Down procedure). Limit test supernatant was used for enzyme assays. was performed to determine the safety of extract. Animals were observed individually at least once during the first Enzyme assays 30 min after dosing, periodically during the first 24 h (with special attention given during the first 4 h), and daily there- The levels of thiobarbituric acid reactive substances (TBARS) after for a total of 14 days. After the experimental period, in the tissue was measured by the method of Ohkawa et al. the animals were weighed and humanely killed and their (1979) as a marker for lipid peroxidation. Superoxide vital organs including heart, lungs, liver, kidneys, spleen, dismutase was determined by the method of Kakkar et al. adrenals, sex organs and brain were grossly examined (1984). Catalase (CAT) activity was assayed by the method (OECD 2000). method of Sinha (1972). The activity of glutathione peroxi- dase (GSH-Px) was determined in lung tissue homogenate In-vivo study on benzo[a]pyrene induced lung carcinoma according to the method of Rotruck et al. (1973). The activity in Swiss albino mice of glutathione-S-transferase (GST) was estimated by the method of Habig et al. (1974). The activity of glutathione Healthy Swiss albino mice of male sex (20–25 g) were used reductase (GR) was estimated by the method of Horn and for the study randomization in selection of animals for Burns (1978). Reduced glutathione (GSH) was determined grouping was carried out to avoid statistical difference in according to the methods of Ellman (1959). The vitamin C the body weight of animals. The experimental animals were (ascorbic acid) content in the lung tissue was determined divided into six groups, each group comprising of six ani- according to the method of Omaye et al. 1979. The vitamin mals. Group 1 served as normal control. Group 2 animals E(α-tocopherol) content in the lung tissue was estimated as received extract 200 mg/kg suspended in 0.5 % carboxy detailed in Barker et al. 1951. methyl cellulose (CMC), once daily, orally for 16 weeks. Group 3 animals received extract 400 mg/kg suspended in Histopathological examination 0.5 % CMC, once daily, orally for 16 weeks. Group 4 animals received Benzo[a]pyrene 50 mg/kg body weight dissolved in A portion of lung tissue was fixed in 10 % formalin and corn oil, twice a week orally for four successive weeks to embedded in molten paraffin wax. Sections were cut at induce lung cancer by 16th week. Group 5 animals were 4 μm thickness, stained with hematoxylin and eosin and received extract 200 mg/kg, suspended in 0.5 % CMC, once were examined under light microscope for histopathological dailyorallystarted1weekbeforethefirstdoseof changes. benzo[a]pyrene induction and continued for 16 weeks. Group 6 animals were received extract 400 mg/kg (as in Statistical analysis Group 5) 1 week before the first dose of benzo[a]pyrene induction and continued for 16 weeks (Magesh et al. 2007). Results were expressed as mean ± standard error of mean At the end of the experimental period, the animals were (SEM). The results were analysed for statistical significance sacrificed by cervical decapitation, lung tissue were immedi- by one way ANOVA followed by dunnett’s test (Graphpad ately excised, washed in ice cold saline, blotted dry and Software Inc,La Jolla, CA. Trial version). The criterion for weighed. Gross morphological examination of lung tissue statistical significance was set at P<0.05. 270 V. Rajesh, P. Perumal

Results

Phytochemical studies

The percentage yield of extract obtained from extraction of Smilax zeylanica leaves using methanol as solvent was found to be 22.03 % w/w. The phytochemical examination of methanol extract of Smilax zeylanica leaves revealed the presence of carbohy- drates, alkaloids, phenolics, tannins, flavonoids, glycosides and alkaloids. The total phenolic content in methanol extract was found to be 620±2.36 mg GAE/g of dry extract. The concentration of flavonoids was found to be 1,137± 12.45 mg quercetin equivalent/g of extract (Table 1).

Acute oral toxicity study

Acute oral toxicity study was carried out as per OECD guideline 425. From the limit test results it was observed that, the Smilax zeylanica leaf extract is safe upto a dose level of 2,000 mg/kg. There was no mortality and the experimental animals did not show any toxic effect through- out the observation period of 14 days. Fig. 1 Gross morphology of lung. a Lung of control animal showing normal morphology. b and c Lung of animal dosed with methanol Gross morphology extract of Smilax zeylanica leaves 200 mg/kg and 400 mg/kg respec- tively showing normal appearance which is indicates nontoxic nature of extract. d Lung of benzo[a]pyrene induced untreated animals show- As depicted in Fig. 1, the appearance of lung in control ing macroscopically visible nodules. e Lung of benzo[a]pyrene in- group mice were normal and there were no macroscopically duced animals treated with Smilax zeylanica leaf extract 200 mg/kg detectable changes in lung (Fig. 1a). No obvious changes in showing reduced number of nodules. f Lung of benzo[a]pyrene in- duced animals treated with Smilax zeylanica leaf extract 400 mg/kg lung morphology were observed in extract alone treated showing absence of nodules group which is indicative of nontoxic nature of Smilax zeylanica leaf extract (Fig. 1b and c). There were no visible nodules in lung of control and extract alone treated groups. In average number of nodules in lung of animals treated with benzo[a]pyrene induced untreated animals, macroscopically extract 200 mg/kg was 3.33±0.33 and animals treated with visible nodules (12.50±1.23) were noted in lung with en- extract 400 mg/kg was 1.33±0.22. The reduced incidence of larged appearance of lobes (Fig. 1d). In group of nodule in treated groups made the appearance of lung near benzo[a]pyrene induced animals treated with Smilax zeylanica normal without any enlargement/inflammation. leaf extract the nodule incidence were reduced and the average number of nodules/nodule bearing lung were significantly less Body weight (p<0.001) compared to untreated animals (Fig. 1e and f). The Average body weights of different animal groups at various intervals are shown in Fig. 2. There was no significant Table 1 Total phenolic content (TPC) and total flavonoid content difference in final body weight between the control group (TFC) in methanol extract of Smilax zeylanica leaves (31.5±0.28) and extract alone treated groups (Extract Extract TPC (mg of TFC (mg of 200 mg/kg- 32.1±0.65, Extract 400 mg/kg- 34.8±0.37). GAE/g of extract) quercetin/g of The final body weight of benzo[a]pyrene induced untreated extract) animals (24.12±2.04) was significantly less (p<0.001) Methanol extract of 620±2.36 1,137.5±12.45 compared to control animals (31.5±0.28). The group V and Smilax zeylanica leaves group VI animals with benzo[a]pyrene induced and treated with extract 200 mg/kg and 400 mg/kg showed a significant Values are means ± SD from three determinations. Total phenolic increase (p<0.001) in final body weight (Extract 200 mg/kg- content (TPC) is expressed as milligram of gallic acid equivalent per gram of extract. Total flavonoid content (TFC) is expressed as milli- 30.6±1.10 and Extract 400 mg/kg- 28.9±0.59) compared to grams of quercetin equivalents per gram of dry extract untreated animals. It shows the Smilax zeylanica leaf extract Cytoprotective effect of Smilax zeylanica Linn. leaves 271

40

35

30

25 Normal Extract 200 mg/kg 20 Extract 400 mg/kg Benzo(a)pyrene

Body weight (g) 15 B(a)P + Extract 200 mg/kg 10 B(a)P + Extract 400 mg/kg

5

0 1 2 3 4 5 6 7 8 9 101112131415161718 weeks

Fig. 2 Effect of Smilax zeylanica leaf extract on body weight of Benzo[a]pyrene induced untreated animals showed a significant de- control and experimental group of animals during benzo[a]pyrene crease in body weight (p<0.001) compared to control animals. The [B(a)P] induced lung carcinogenesis. Average body weight of different group V and group VI animals with benzo[a]pyrene induced and animal groups at various intervals. Results are given as mean ± S.E.M. treated with extract 200 mg/kg and 400 mg/kg showed a significant There was no significant difference in final body weight between the increase (p<0.001) in final body weight compared to untreated animals control (normal) group and extract alone treated groups (ns).

prevented the body weight loss in benzo[a]pyrene induced GST with a significant decrease (p<0.01) in activity of GSH- animals and maintained the growth rate near normal. Px was noted in group IV benzo[a]pyrene intoxicated untreated animals compared to control animals. The levels Relative lung weight of non-enzymatic antioxidants GSH, vitamin-C and vitamin-

There was no statistical difference in relative lung weight between control (0.667±0.019) and extract alone treated Table 2 Effect of Smilax zeylanica Linn. leaf extract on relative lung weight and nodule incidence in benzo[a]pyrene [B(a)P] induced lung groups (Extract 200 mg/kg- 0.601±0.024, Extract 400 mg/kg- carcinogenesis 0.616±0.014). The relative lung weight of group IV benzo[a]pyrene induced untreated animals (1.270±0.32) were Groups Parameters significantly increased (p<0.05) compared to Group I control Relative lung weight Average number of animals. A significant decrease (p<0.01) in relative lung (g lung/100 g body wt) nodules/nodule weight was noted in benzo[a]pyrene induced group V and bearing lung group VI animals treated with extract 200 mg/kg and – 400 mg/kg (Table 2). Group I (control) 0.667±0.01 Group II Extract 0.601±0.02ns, a – (200 mg/kg) Lipid peroxidation and antioxidant status Group III Extract 0.616±0.01ns, a – (400 mg/kg) The levels of lipid peroxidation in lung tissue of control and Group IV B(a)P 1.270±0.32*, a 12.50±1.23 experimental animals are illustrated in Fig. 3. A significant (untreated) Group V B(a)P + 0.621±0.01**, b 3.33±0.33***, b (p<0.001) enhancement in the production of malondialdehyde Extract 200 mg/kg (MDA) was observed in group IV benzo[a]pyrene intoxicated Group VI B(a)P + 0.614±0.02**,b 1.33±0.22***,b untreated animals compared to control animals. Treatment with Extract 400 mg/kg extract offered significant protection against benzo[a]pyrene All values are expressed as mean ± S.E.M, n=6 in each group induced elevation in lipid peroxidation as evidenced by a a Values are significantly different from control group; ns-non signifi- significant fall in MDA levels. * ** *** cant; P<0.05; P<0.01; P<0.001 Figure 4a–h portrays the activities and the levels of antiox- b Values are significantly different from benzo[a]pyrene untreated idants in lung of control and experimental animals. A signif- group; ns non significant; * P<0.05;** P<0.01;*** P<0.001 (ANOVA, icant decrease (p<0.001) in activities of SOD, Catalase, GR, followed by Dunnett’s test) 272 V. Rajesh, P. Perumal

2.0 a interstitium (Fig. 5A1 and A2). Extract alone treated group II *** b b (Fig. 5B1 and B2) and group III animals (Fig. 5C1 and C2) 1.5 * showed normal architecture with no evidence of inflam a *** a mation/malignancy/atypical cells depicting the non-toxic 1.0 ns ns nature of Smilax zeylanica leaf extract. Section of lung from group IV benzo[a]pyrene induced untreated animals re- 0.5 vealed loss of architecture with tumour cells growing in

Lipid peroxidation nests and thick cords that are separated from one another 0.0

MDA nmole/hr/gmMDA tissue of by thin walled sinusoids. Histological appearance of solid areas with adenocarcinoma is also extremely var- Normal iegated. Numerous mitotic cells with surrounding region showing dense lymphocytic inflammation were noted

Extract 200Extract mg/kg 400 mg/kg (Fig. 5D1 and D2). Section of lung from benzo[a]pyrene Benzo(a)pyrene [B(a)P] intoxicated animals treated with Smilax zeylanica leaf extract [B(a)P] + Extract[B(a)P]+ 200 Extract mg/kg 400 mg/kg 200 mg/kg showed atypical cells with hyperchromatic nucleus Fig. 3 Effect of Smilax zeylanica leaf extract on the level of lipid in parenchyma and alveolar spaces. The adjacent alveoli were peroxidation in lung of control and experimental group of ani- normal with no evidence of inflammation (Fig. 5E1 and E2). mals. Effect of Smilax zeylanica leaf extract on lipid peroxidation Group IV benzo[a]pyrene intoxicated animals treated with in lung tissue of experimental animals during benzo[a]pyrene induced lung carcinogenesis. Results are expressed as mean ± S.E.M.a values are Smilax zeylanica leaf extract 400 mg/kg showed foamy mac- significantly different from control group; ns-non significant; *P<0.05; rophages in alveolar spaces with reduced alveolar damage and **P<0.01; ***P<0.001. bvalues are significantly different from near normal architecture. There were no atypical cells or benzo[a]pyrene untreated group; ns-non significant; *P<0.05;**P< adenocarcinoma and the cut sections was unremarkable 0.01;***P<0.001. (ANOVA, followed by Dunnett’stest) (Fig. 5F1 and F2).

E were also decreased in lung tissue of group IV untreated animals. Administration of extract to group V and group Discussion VI benzo[a]pyrene intoxicated animals significantly in- creased the activities of SOD, Catalase, GR, GST to near Reactive oxygen species (ROS) and organic free radical normal levels. GSH-Px activity was to be moderate in intermediates formed from many carcinogens are suggested treated animals compared to group IV untreated animals. to be involved in the initiation and progression of carcino- The non-enzymatic antioxidants, GSH, vitamin C and genic transformation (Barker et al. 1951). The lung is par- vitamin E levels were significantly increased in group V ticularly at risk to the toxic effects of ROS because it and group VI animals treated with extract compared to interfaces with various oxidants, such as environmental untreated group IV animals. pollutants and cigarette smoke. Benzo[a]pyrene, a potent The extract did not produce any deleterious effect on the tobacco carcinogen belonging to polycyclic aromatic antioxidant defense system in normal animals which is group of compounds is generated by complete combus- evidenced from the non-significant alteration of the enzy- tion of tobacco during smoking. Progressive cellular matic and non-enzymatic antioxidants along with the architectural changes due to oxidative stress, lipid per- maintained rate of lipid peroxidation in group II and group oxidation and modulation of various cellular or molec- III animals when compared with the normal control group I ular pathways by reactive free radicals (reactive oxygen animals. species and reactive nitrogen species) generated during

The results indicate that, the level of lipid peroxidation the cytochrome P450 (CYP1A1) dependent metabolism which increased in lung of carcinogen administered ani- of benzo[a]pyrene has been implicated during the path- mals was lowered in extract treated animals and in con- ogenesis of lung carcinogenesis (Banerjee and Das 2004). trast the antioxidant status which was found to be One of the promising strategies for chemoprevention is to decreased in carcinogen administered animals was im- alleviate oxidative stress which is implicated in all stages of proved to near normal upon Smilax zeylanica leaf extract carcinogenesis. administration. Herbs are recognized as sources of natural antioxidants and thus play an important role in the chemoprevention of diseases Histopathology by combating the Reactive Oxygen Species (ROS) induced oxidative damage (Li Shijun et al. 2000). Traditionally many Histological examination of lung sections from control an- herbal have revealed their cancer chemopreven- imals revealed normal architecture with normal alveoli and tive potential. Smilax plants, (about 350 species) are Cytoprotective effect of Smilax zeylanica Linn. leaves 273

ab

b a a 1.5 aa b b 1.5 *** *** ns ns b ns ns *** a a 1.0 1.0 *** ns *** SOD

0.5 Catalase 0.5 cosumed/min/mg of protein U/mg of protein U/mg of 2 O 2 0.0 0.0

g g g g )P] k k P] a ) (a mg/ mg/k Normal Normal 0 [B 0

t 400 mg/kg nmole H of ct 20 trac xtra Extract 200Extract mg/kg 400 mg/kg Ex Extract 200Extract mg/ 40o(a)pyrene Extract 400 mg/k ]+ E P] + Benzo(a)pyrene [B( )P] + Benz (a [B(a)P]+ Extract[B 200 mg/kg [B(a)P [B(a) cd

a a b a a ns b 1.5 ns b 1.5 ns *** ns b ** a ns a ns *** 1.0 ** 1.0

GSH-Px 0.5 0.5 Glutathione reductase Mmoles/min/mg of protein 0.0 0.0

g /k al (a)P] g/kg Normal [B 0 mg mg/kg e 00 m Norm

4 mole oxidized/min of NADPH mg protein

act µ tract 20 r ct 200 mg/kg Extract 200 Extractmg/kg 400 mg/kg )pyrene [B(a)P] + Ext Extract 200Extract mg/kg 400 mg/kg ] Benzo(a)pyren Benzo(a [B(a)P] + Ex[B(a)P (a)P] + Extract 400 [B(a)P] + Extra[B

Fig. 4 Effect of Smilax zeylanica leaf extract on the status of enzymatic expressed as mean ± S.E.M. avalues are significantly different from and non-enzymatic antioxidants in lung of control and experimental control group; ns-non significant; *P<0.05; **P<0.01; ***P<0.001. group of animals. Effect of Smilax zeylanica leaf extract on the status of bvalues are significantly different from B(a)P untreated group; ns-non enzymatic and non-enzymatic antioxidants in lung tissue of experimental significant; *P<0.05;**P<0.01;***P<0.001. (ANOVA, followed by animals during benzo[a]pyrene induced lung carcinogenesis. Results are Dunnett’stest) distributed widely in tropical and temperate regions through- 2010). Hence, based on the extensive use of Smilax zeylanica out the world. Many of them have long been used as medicinal Linn in traditional medicine and very limited and scattered herbsandtheyareknowntoberichinsteroidalsapo- record of Smilax zeylanica leaves, a study was undertaken to nins. Recent pharmacological investigations showed that investigate the effect of Smilax zeylanica leaf extract against some steroidal saponins isolated from Smilax china benzo[a]pyrene induced lung cancer with reference to lipid exhibited significant cytotoxicity against several tumour cell peroxidation and antioxidant status in swiss albino mice. From lines (Hu and Yao 2002) and diosgenin, a steroidal sapogenin the study, it was observed that the methanol extract of Smilax isolated from Smilax zeylanica leaves and roots are used as zeylanica leaves showed a significant protection against precursors of many pharmacological active steroids (Kar and benzo[a]pyrene induced lung carcinoma. Dose levels were Sen 1984). Also Smilax zeylanica leaves were reported to selected based on the acute oral toxicity study and our previ- exert a potent free radical scavenging effect (Anita et al. ous invivo studies, where the extract showed protection 274 V. Rajesh, P. Perumal

e f b a a a 1.5 a b b 2.0 a ns ns b *** ns ns * ** 1.5 a 1.0 *** ** *** 1.0 GSH 0.5 0.5 nmole/mg protein of units/min/mg of protein of units/min/mg

Glutathione-S-transferase 0.0 0.0

g g g g )P] k a /k mal g (a)P] Normal Nor mg/kg 00 400 m act 400 mg/k tract 200 mg/ktract tract 200 mg/kg tract 2 tract 400 mg/ Extract 200Extract mg/kg 400 mg/kg Ex Extr Ex Ex + Ex enzo(a)pyrene [B Benzo(a)pyrene [B( B (a)P] a)P] + a)P] + [B(a)P] + Ex[B [B( [B(

g h b b b a a a *** 8 a 6 a *** b *** ns * ns ns ns a 6 * *** 4

4

2 Vitamin C Vitamin Vitamin E Vitamin 2

0 0

kg rmal g/kg mg/kg B(a)P] mg/kg Normal 0 mg/kg No e [B(a)P] 00 n 200 mg/ 2 yrene [ ract 40 ract 200 p ract xtract 400 a)m Extract 200Extract mg/kg 400 mg/kg Ext Ext E ( + Extract 400 mg/kg Benzo(a)pyre Benzo [B(a)P] + Ext[B(a)P] [B(a)P] + Extract[B(a)P] +

Fig. 4 (continued) against streptozotozin (STZ) induced oxidative stress (Rajesh number of nodules in treatment groups were supported by et al. 2010) and substantial evidence from studies on histological assessment of lung that presented a near normal invitro and invivo antioxidant activities where the meth- architecture compared to increased nodules in benzo[a]pyrene anol extract of Smilax zeylanica leaves at the dose induced untreated animals. levels 200 mg/kg and 400 mg/kg body weight exhibited Lipid peroxidation is one of the major mechanisms of cell strong antioxidant and free radical scavenging effect injury caused by free radicals. Enormous amount of free (Anita et al. 2010). radicals generated by carcinogens reacts with lipids causing Reduction in body weight gain observed in benzo[a]pyrene lipid peroxidation (Sikkim and Mulee 2000). The products induced untreated animals, could be largely due to deteriora- of lipid peroxidation include malondialdehyde, that in- tion of lung function and nutritional deprivation. In addition, teracts with various molecules leading to oxidative stress assessment of the relative lung weight was used to investigate and has been reported to be involved in the formation of potential changes in the lung size. A significant increase in tumours (Ramakrishnan et al. 2007). In the present study, relative lung weight was noted in benzo[a]pyrene induced significant increase in the levels of lipid peroxidation untreated animals, which could be due to increased incidence observed in untreated group IV cancer bearing animals of lung nodules and inflammation. Treatment with Smilax may be due to the excessive production of free radicals zeylanica leaf extract prevented the body weight loss and and due to inhibition of free radical scavenging enzymes. reduced the nodule incidence. Significant decrease in average Significantly reduced levels of lipid peroxidation was seen in Cytoprotective effect of Smilax zeylanica Linn. leaves 275

Fig. 5 Histopathological image of lung tissues. Sections of lung or malignancy (C1 &C2). Sections of lung tissue of animal intox- tissue of control animal showing normal parenchyma. External icated with benzo[a]pyrene and left untreated showing solid areas surface and cut sections were unremarkable. The bronchi, alveoli with adenocarcinoma. The surrounding region showing dense lym- and the interstitium were normal. There was no inflammation, phocytic inflammation (D1 &D2). Sections of lung tissue of animal atypical cells or malignancy (A1 &A2). Sections of lung tissue intoxicated with benzo[a]pyrene and treated with extract 200 mg/kg of animal given extract 200 mg/kg alone for 16 weeks showing showing atypical cells with hyperchromatic nucleus in parenchyma normal parenchyma. External surface and cut sections were and alveolar spaces. The adjacent alveoli were normal. There was unremarkable. The bronchi, alveoli and the interstitium were nor- no inflammation (E1 &E2). Sections of lung tissue of animal mal. There was no inflammation, atypical cells or malignancy (B1 intoxicated with benzo[a]pyrene and treated with extract 400 mg/ &B2). Sections of lung tissue of animal given extract 400 mg/kg kg showing foamy macrophages in alveolar spaces. There were no alone for 16 weeks showing normal parenchyma. External surface atypical cells or adenocarcinoma. The bronchi, alveoli and the and cut sections were unremarkable. The bronchi, alveoli and the interstitium were normal. External surface and cut sections were interstitium were normal. There was no inflammation, atypical cells unremarkable (F1 &F2)

Smilax zeylanica leaf extract treated group V and group VI reduced GSH, thereby protecting cells against oxidative dam- benzo[a]pyrene intoxicated animals. This clearly shows that age. Catalase detoxifies H2O2 into H2OandO2 (Murray et al. Smilax zeylanica effectively controlled the rate of lipid perox- 2003). Thus SOD, catalase and glutathione peroxidase (GPx) idation, which suggest the beneficial effect of the extract act mutually and constitute the enzymatic defense mechanism against benzo[a]pyrene initiated free radical formation. The against ROS (Bhattacharjee and Sil 2006). In the present study presence of flavonoids may contribute to this effect, because decrease in the activities of SOD, catalase and GPx in they are proved to be a potential inhibitor of lipid peroxidation untreated cancer bearing animals could be attributed to exces- (Siegers and Younes 1981). sive utilization of enzymes in detoxification of peroxides and The enhanced formation of lipid peroxides is further hydroperoxides generated during lung carcinogenesis. evidenced by decrease in activities of antioxidant enzymes Restoration in the levels of lipid peroxidation upon treatment in lung of benzo[a]pyrene intoxicated animals as compared with Smilax zeylanica leaf extract might have resulted in the with normal control animals. recoupment in the activities of the above antioxidant enzymes SOD is said to act as the first line of defense against to normalcy. SOD, catalase and GPx require several superoxide radical generated as a by-product of oxidative secondary enzymes like glutathione reductase and co- phosphorylation. SOD mediated dismutation of superox- factors like GSH, NADPH, to function at high efficacy. − ide radical (O2 ) generates hydrogen peroxide (H2O2). Glutathione reductase catalyzes the NADPH dependent re- Accumulation of excess of H2O2 causes toxic effects on duction of glutathione disulphide to glutathione thus cellular system. In this regard GPx and catalase converts maintaining glutathione levels (Katiyar et al. 1993). GST

H2O2 into water (Li et al. 2000). Glutathione peroxidase catalyzed GSH conjugation is an important mechanism for (GPx) catalyses the reduction of H2O2 at the expense of the detoxification process. In the present study the activities of 276 V. Rajesh, P. Perumal glutathione reductase and Glutathione-S-transferase were sig- Conclusion nificantly reduced in untreated benzo[a]pyrene intoxicated animals. Upon treatment with Smilax zeylanica leaf ex- In conclusion, our present data shows that administration of tract, the glutathione reductase and GST levels were Smilax zeylanica leaf extract possesses significant protec- significantly increased. Increase in glutathione reductase tion against benzo[a]pyrene induced lung carcinogenesis by can protect the lung by maintaining the basal level of stabilizing and increasing all the components of antioxidant GSH, which is important for many other GSH depen- defense system which were disturbed during dent detoxification reactions. benzo[a]pyrene induced oxidative stress in mice. The po- GSH, vitamin C and vitamin E are well known non- tential chemoprevention might be due to antioxidant prop- enzymatic antioxidant defense system of cells, act synergisti- erty of the methanol extract of Smilax zeylanica leaves. cally to scavenge free radicals in biological system. GSH is Preliminary phytochemical examination revealed the pres- found to be present in high concentration in cells, protects ence of flavonoids, phenolics and tannins in the methanol cells against free radical attack (Farombi et al. 2000). GSH extract of Smilax zeylanica leaves and the quantitative anal- acts directly as free radical scavenger by donating a hydrogen ysis revealed the presence of phenolics and flavonoids, atom and thereby neutralizing hydroxyl radical. It reduces which are powerful antioxidants might be responsible for peroxides and maintains protein thiols in the reduced state defense and potential chemoprevention. (Nwanjo and Oze 2007). Glutathione peroxidase uses GSH as a substrate to catalyze the reduction of hydroperoxide and Acknowledgments The authors are thankful to the management of H2O2 (Bebe and Panemangalore 2003). Reduced glutathione JKK Nattraja College of Pharmacy, Komarapalayam, Tamil Nadu, (GSH) in tissues maintains maintains the cellular levels of INDIA for providing necessary facilities to carry out the research work. vitamin C and Vitamin E in active form. Vitamin C and vitamin E act synergistically in scavenging wide variety of ROS. 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