899 Baggiya Selvi D and Raja S. / International Journal of Biological & Pharmaceutical Research. 2015; 6(11): 899-908.

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HEPATOPROTECTIVE AND ANTIOXIDANT EFFECTS OF BEDDOMEI IN RATS

Baggiya Selvi David1 and Raja Sundararajan2*

1Department of Pharmacognosy, Karnataka College of Pharmacy, Bangalore-560064, Karnataka-State, . 2GITAM Institute of Pharmacy, GITAM University, Visakhapatnam-500045, , India.

ABSTRACT The aspire of the present study was to investigate the antioxidant and hepatotective effects of Cycas beddomei methanol extract on carbon tetrachloride and olive oil (1:1) induced liver damage in wistar albino rats. In vivo antioxidant activity of methanol extract of Cycas beddomei was evaluated by varied assays together with superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GRD), reduced glutathione, glutathione peroxidase (GPx) and malondialdehyde (MDA) levels in liver tissues. Serum glutamic oxaloacetate transaminases (SGOT), Serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin and total protein (TP) were estimated from serum. The serum levels of above biochemical parameters (SGOT, SGPT, ALP, LDH, bilirubin and total protein) were significantly increased; however, a significant decrease in the levels of total protein (TP) was observed in carbon tetrachloride and olive oil treated groups compared to normal control. Treatment with methanol extract of Cycas beddomei and silymarin prior to carbon tetrachloride and olive oil intoxication, afforded protection by lowering of the above serum markers as well as by increasing the total protein content. The extract showed potent activities on SOD, CAT, GRD, GPx, reduced glutathione and MDA levels. The outcome indicated that Cycas beddomei methanol extract has strong antioxidant activities and significant protective consequence against hepatotoxicity induced by carbon tetrachloride and olive oil (1:1) and have been supported by the assessment of liver histopathology in rats.

Key Words: Cycas beddomei, Serum transaminases, Carbon tetrachloride, Hepatoprotective, Histopathology.

INTRODUCTION The liver demonstrates a big role in metabolism completely different noxious chemicals at the side of (Law of xenobiotics by management the synthesis, secretion and and Burnt, 2010; Stickel and Schuppan, 2007) metabolism of xenobiotics. Varied physiochemical environmental pollutants area unit thought to be liable for functions of the body beside reaction, reduction, wholly completely different sort of liver injury and hepatic hydroxylation, hydrolysis, conjugation, sulfation, chemical parenchyma injury. Recent analysis in free radical biology change etc area unit well balanced by the liver alone. in addition prompts the pathophysiological role of free Injury to liver and harm to the hepatic parenchyma area radicals and aerobic stress in liver damage and injury. unit perpetually evidenced to be related to distortion of Revealing the mechanism of actions of potent poisonous various metabolic functions of liver. Etiologically varied substance like CCl4, paracetamol etc in addition indicated infectious agents together with viruses and wholly the role of aerobic stress and free radicals at intervals the pathophysiology of internal organ injury. The free radicals Corresponding Author sometimes generated throughout the conventional body metabolic pathways and in addition they're going to be S. Raja nontransmissible from the environment additionally. Email: [email protected] 900 Baggiya Selvi D and Raja S. / International Journal of Biological & Pharmaceutical Research. 2015; 6(11): 899-908.

Free radicals primarily act by attacking the and joint swellings (Latheef et al., 2008). The seeds are unsaturated fatty acids within the biomembranes that ground to a paste with coconut oil and are used as a causes membrane macromolecule peroxidation (a poultice to treat skin complaints such as wounds, sores and hallmark sign of hepatotoxicity), decrease in membrane boils (Jain & Sastry, 1980). Cycas beddomei showed fluidness and reduction of catalyst and receptor activity significant pharmacological activities such as anti- and injury to membrane super molecule that finally inflammatory (Alekhya et al., 2014), antioxidant triggers the cell inactivation and death (Klaunig et al., (Mahendra Nath et al., 2014), antidiabetic (Alekhya et al., 2011; Radak et al., 2011). Free radicals contain odd 2013), anthelmintic (Alekhya et al., 2013) and (Feijóo et al., 2010; Chandan et al., 2008) electrons. The antibacterial (Alekhya et al., 2013) activities. Different oxygen radicals, like superoxide (O2-), radical (•OH) and active constituents such as alkaloids, flavonoids, phenols, non free radical species, such as hydrogen peroxide (H2O2) terpenoids, saponins, tannins, glycosides, lignins, fatty and singlet O (•O2), are generated in several redox acids are a unit to date according in Cycas beddomei processes of traditional physiochemical pathways. (Prance and Mori, 1986). The objective of the present Antioxidant weaponry comprising wholly completely study was to investigate the hepatoprotective and different enzymes (Kataki et al., 2012) like SOD, catalase antioxidant activities of the methanol extract of Cycas and glutathione peroxidise etc lure and destroy these free beddomei. radicals. Sustenance deficiency alongside production of free radicals and a reduced level of on prime of mentioned MATERIALS AND METHODS enzymes is taken under consideration as a result of the material most offender for producing aerobic stress analysis on The whole plant of Cycas beddomei was oxidants and antioxidants over the past few years has collected from Hills of chittoor district, Andhra shown a link between most diseases like vas diseases, Pradesh, India and the plant material was taxonomically cancer, pathology, chronic diseases etc and production of identified and authenticated by the Dr. Madhava chetty reactive oxygen species (ROS) at the aspect of aerobic (Research Officer) botany, Andhra Pradesh. Voucher stress (Kataki et al., 2012). Therefore, antioxidants are specimen (KCP-114/2012) of this plant has been retained also accustomed reverse the harmful and pathological in the Karnataka College of Pharmacy, Bangalore, action of free radicals. These antioxidants usually restore Karnataka, India. the standard physiological system by scavenging the free radicals. The antioxidants in use are either derived Chemicals naturally from or synthetically. Owing to malignant All chemicals used in the study were of analytical neoplastic disease probability artificial antioxidants are not grade. CCl4 was procured from Krishna Chemicals Pvt. the popular sort of antioxidants. Current analysis within Ltd., Gujarat and Silymarin from GVK Bio Sci, the field of free radical biology thus accentuates the use of Hyderabad. antioxidants from natural origin and visual of this plenty of antioxidants of natural origin unit of measurement Preparation of plant material being investigated. CCl4 is one in each of the foremost The whole plant of Cycas beddomei was washed common poisonous substance used for experimental with tap water. They were cut in to small pieces and air- induction of liver injury in animal studies. Numerous dried thoroughly under shade (at room temperature) for 2 healthful plants are unit being researched for a decent months to avoid direct loss of phytoconstituents from hepatoprotective remedy. In view of this several healthful sunlight. The shade dried materials were powdered using preparations and sort of healthful plants mentioned in the pulverizer and sieved up to 60 meshes. It was then piece of writing for treatment of liver disorders area unit homogenized to fine powder and stored in air‐tight being investigated. Furthermore ancient folklore} and container for furthers analysis. autochthonal knowledge of medicative uses of plants also are (Kshirsagar et al., 2011; Shaik et al., 2012) currently Preparation of Cycas beddomei extract being explored and documented for potential bioactive The whole plant of Cycas beddomei was refluxed molecules to be future medicine. with methanol in a soxhlet extractor for 72 hrs. The excess Cycas beddomei Dyer (Cycadaceae) is an solvent was removed from the extract by vacuum rotary endemic and critically endangered gymnosperm confined flash evaporator and concentrated over the hot water bath. to Seshachalam Biosphere Reserve, Eastern Ghats, India Finally dried extract was stored in desiccators for (Rao et al., 2010). This plant initially considered as a rare hepatoprotective and antioxidant studies. species (Jain & Sastry, 1980) and vulnerable in Indian Red Data (Nayar and Sastry, 1987). Cycas circinalis var. Preliminary phytochemical screening beddomei (Dyer) J. Schust. is a synonym of Cycas The methanolic extract was subjected to various beddomei (Selvam, 2012). Traditionally male cones of this phytochemical studies to identify the presence of various plant were used to cure rheumatoid arthritis, muscle pains phytoconstituents like alkaloids, tannins, glycosides, 901 Baggiya Selvi D and Raja S. / International Journal of Biological & Pharmaceutical Research. 2015; 6(11): 899-908. carbohydrates, aminoacids, anthocyanidins, steroids, normal saline followed by 0.15M Tris-HCl (pH-7.4) flavonoids, reducing sugars, saponins, gums and oils blotted dry and weighed. 10% w/v of homogenate was (Kokate, 1997). prepared in 0.15M Tris-HCl buffer and processed for the estimation of lipid peroxidation (TBARS). The remaining Toxicity study homogenate was centrifuged at 1500 rpm for 15 min at The toxicity study was carried out using OECD 40C. The supernatant thus obtained was used for the guide lines No. 423. Three female rats of the same age estimation of superoxide dismutase, GPx, GSH and group and weight were taken in a single dose up to the catalase. highest dose of 2000 mg/kg B/W orally. The animals were observed for 1 hr continuously and then hourly for 4 hr, Estimation of Biochemical parameters and finally after every 24 hr up to 15 days for any SGOT & SGPT mortality or gross behavioral changes (Chandan et al., Serum transaminases (GOT and GPT) were 2007). determined by the method of Reitman and Frankel (1957). Each substrate (0.5mL) (either α -L-alanine (200mM) or In vivo hepatoprotective activity L-aspartate (200mM) with 2mM α - ketoglutarate) was Experimental animals incubated for 5 min at 37°C. A 0.1mL of serum was Wistar albino rats (150-200gm) of either sex were added and the volume was adjusted to 1.0mL with selected for hepatoprotective and antioxidant studies. sodium phosphate buffer (pH 7.4; 0.1M). The reaction Animals were housed in polypropylene cages in room mixture was incubated for 30 and 60 min for GPT and under standard condition like controlled light / dark GOT, respectively. A 0.5mL of 2, 4-dinitrophenyl cycle‟s temperature (22 ±2°C), 60 to 70% relative hydrazine (1mM) was added to the reaction mixture and humidity. The study protocol was approved by the left for 30 min at room temperature. Finally, the color Institutional Animal Ethics Committee of our Institute as was developed by the addition of 5mL NaOH (0.4 N) and per the requirements of Committee for the Purpose of the product formed was read at 505nm. Data were Control and Supervision on Animals (CPCSEA), New expressed as IUL-1. Delhi. Alkaline phosphatase Experimental design Alkaline phosphatase (ALP) was assayed by the Rats were divided into five groups of five strategy of Kind and King (1954). The reaction mixture of animals. 3.0 ml containing 1.5 ml of buffer (carbonate-bicarbonate Group I served as vehicle control and received normal buffer, 0.1M, pH 10.0), one ml of substrate and requisite saline (5ml/kg). amount of the enzyme sources was incubated at 37oC for 5 Group II was administered with CCl4/ olive oil (1:1, minutes. The reaction was inactive by the addition of 1.0 0.7 ml/kg i.p. for seven days). ml of Folins phenol chemical agent. The management Group III and IV received methanol extract (250 tubes were received the enzyme once stunning the mg/kg and 500 mg/kg p.o respectively daily for seven reaction. The contents was centrifuged and to the days) simultaneously with toxicant CCl4 and olive oil. supernatant, 1.0 ml of 15% sodium carbonate solution Group V was administered with reference drug, 1945 sodium carbonate solution, 1.0ml of substrate and silymarin (Majumdar et al., 1998) (25mg/kg p.o.) 0.1ml of metal chloride (0.1M), was intercalary and simultaneously with toxicant (Singh et al., 1999). All rats mixture was incubated for ten minutes at 370C. The color were sacrificed by cervical dislocation 24hrs after the last was scan out 640 nm against the blank. treatment. Bilirubin Biochemical parameters Bilirubin content was calculable by technique of The blood samples were allowed to clot for Malloy and Evelyn (1937). The two test tubes were taken 45minutes at room temperature. Serum was separated by and every into was added 0.2ml of blood serum sample and centrifugation at 3500 rpm at 370C for 15 min and 1.8 ml of water. To the unknown, 0.5 ml of diazo chemical analysed for various biochemical parameters such as agent and to the blank, 0.5 ml of 1.5% hydrochoric acid serum glutamic pyruvate transaminases (SGPT), serum was added. Finally, to every tube, 2.5 metric capacity unit glutamic oxaloacetate transaminases (SGOT), alkaline of alcohol was added so allowed to stand for half-hour in phosphatase (ALP), bilirubin, lactate dehydrogenase ice and absorbance was scan at 540nm. For a regular curve, (LDH) and total protein (TP). the higher than customary was diluted 1in 5ml methanol. The number of direct reacting bilirubin resolves equally by Antioxidant parameters work 2.5ml of water for 2.5ml of methanol. The values For estimating antioxidant activity, animals were were expressed as mg/dl. sacrificed and liver was excised, rinsed in ice –cold 902 Baggiya Selvi D and Raja S. / International Journal of Biological & Pharmaceutical Research. 2015; 6(11): 899-908.

Lactate dehydrogenase activity the addition of 0.1mL of glacialacetic acid. Reaction Lactate dehydrogenase (LDH) activity was mixture was stirred vigorously with 4.0mL of n-butanol. calculable in blood serum by the standard technique Color intensity of the compound within the alcohol was (Kornberg, 1955; Raja et al., 2007). The reaction mixture measured spectrophotometrically at 560nm and therefore consisted of 0.1mL of nicotinamide A dinucleotide the concentration of SOD was expressed as Umg-1 of (NADH)-reduced disodium salt (0.02 M), 0.1mL of metal protein. pyruvate (0.01 M), 0.1mL of blood serum and created up to 3mL with sodium orthophosphate buffer (0.1M; pH 7.4). Glutathione peroxidase assay The changes within the absorbance was recorded at 340nm Glutathione peroxidase (GPx) activity decided by at 30s interval each for 3 min and also the enzyme activity the method delineated by Wendel (1981). The reaction was calculated using a molar extinction constant of 6.220M- mixture accommodates 400μL of 0.25M potassium 1 cm-1 and it had been expressed as nanomoles NADH phosphate buffer (pH- 7.0), 200µl supernatant, 100 μL oxidised min-1 mg-1 protein. GSH (10 mM), a hundred μL NADPH (2.5mM) and 100μL GRD (6Uml-1). Reaction was started by adding Total protein 100μL hydrogen peroxide (12mM) and absorbance was Protein content in the tissue was determined by measured at 366nm at 1min intervals for five min earlier method reported (Lowry et al., 1951), using bovine employing a molar extinction constant of 6.22X 103 M- serum albumin (BSA) as the standard. 1cm-1. Information was expressed as mU mg-1 of protein.

Estimation of antioxidant parameters Glutathione reductase assay Catalase assay Glutathione reductase (GRD) activity was Catalase activity (CAT) was measured by the assayed by the method of Mohandas et al. (1984). The method of Aebi (1974). A 0.1mL of supernatant was assay system accommodates 1.65mL sodium added to cuvette containing 1.9mL of 50mM phosphate orthophosphate buffer (0.1M; pH 7.4), 0.1mL EDTA (0.5 buffer (pH 7.0). Reaction was started by the addition of mM), 0.05mL oxidised glutathione (1mM), 0.1mL 1.0mL of freshly prepared 30mM H2O2. The speed of the NADPH (0.1 mM), and 0.05mL supernatant in a total decomposition of H2O2 was measured mixture of 2mL. The accelerator activity was quantified spectrophotometrically at 240 nm. Activity of CAT was by measure the disappearance of NADPH at 340nm at 30s expressed as Umg-1 of protein. intervals for 3min. The activity was calculated employing a molar extinction constant of 6.22 X 103M-1cm-1 and was Lipid peroxidation assay expressed as nanomoles of NADPH oxidised min-1 mg-1 Lipid peroxidation (LPO) was measured by the protein. method of Liu et al (1990). Acetic acid 1.5mL (20%; pH scale 3.5), 1.5 of TBA (0.8%) and 0.2mL of sodium Reduced glutathione assay dodecyl sulphate (8.1%) was added to 0.1ml of Reduced glutathione (GSH) was measured supernatant and heated at 100oC for cooled and sixty min. according to the method of Ellman (1959). The equal To the present mixture add 5mL of n-butanol: NaoH amount of homogenate was mixed with 10% (15:1) mixture, 1mL of H2O and vortexed smartly. When trichloroacetic acid and centrifuged to separate the activity at 1200g for 10min, the organic layer was proteins. To 0.01 ml of this supernatant, 2ml of phosphate separated and therefore the absorbance was measured at buffer (pH 8.4), 0.5 ml of 5‟5-dithio, bis-(2-nitrobenzoic 532nm employing a spectrophotometer. acid) and 0.4ml double distilled water was added. Mixture Malonyldialdehyde (MDA) was an end product of LPO, was vortexed and the absorbance scan at 412nm among 15 that reacts with TBA to make pink chromogen–TBA min. The concentration of glutathione was expressed as reactive substance. It had been calculated employing a µg/mg of protein. molar extinction constant of 1.56 X 105M-1 cm-1 and it had been expressed as nanomoles of TBARS mg-1 of protein. Histopathological studies The method for histological studies was as Superoxide dismutase assay described by (Garg et al., 1996). Briefly the procedure SOD activity was analyzed by the method used included fixation of the tissue with formalin, delineated by Rai et al (2006). Assay mixture contain embedding in paraffin blocks, sectioning with microtome 0.1mL of supernatant, 1.2mL of tetrasodium and finally staining by Haemotoxylin and Eosin stain pyrophosphate buffer (pH 8.3; 0.052M), 0.1mL of technique. Haemotoxylin stains nucleus light blue, which phenazine methosulfate (186 mM), 0.3mL of nitroblue turns red in presence of acid. The cell differentiation was tetrazolium (300 mM) and 0.2mL of NADH (750 mM). achieved by treating the tissue with acid solution the Reaction was started by the addition of NADH, when counter staining was performed by using Eosin, which incubation at 30oC for 90s, the reaction was stopped by 903 Baggiya Selvi D and Raja S. / International Journal of Biological & Pharmaceutical Research. 2015; 6(11): 899-908. imparts pink colour to cytoplasm staining. Tissues were protein content (Figures 1–3). The activities of these stained using Haemotoxylin and Eosin (H & E) stain. enzymes were normalized after therapy with different doses of the extract of Cycas beddomei (250 and 500 mg Statistical analysis kg-1, p.o.) in dose-dependent manner, which indicated an Results are presented as mean ± S.E.M. activation of the regeneration process. Silymarin-treated Statistical differences between the mean of the various animals also showed that these enzymes were normalized. groups were evaluated using one-way analysis of variance The extract at a dose of 500 mg kg-1 exhibited significant (ANOVA) followed by Tukeys test for preventive effect (p<0.001) hepatoprotective activity almost equivalent to studies. Data were considered statistical significant at P silymarin-treated animals. value < 0.05. In vivo antioxidant assays RESULTS As shown in the Figure 4 and 5, carbon Percentage yield tetrachloride + olive oil (1:1) intoxication produced The percentage yield (Table No 1) of the significant (P<0.001) reduction in GSH,SOD,CAT, GPx, methanol extract of Cycas beddomei was found to be and GRD activities along with significantly increased 34.2% given. lipid peroxidation level (expressed as MDA) when compared to normal control. Treatment with methanol leaf Preliminary phytochemical screening of Cycas beddomei extract of Cycas beddomei at doses 250 and 500 mg/kg It was observed that the preliminary b.w for 7 days showed significant higher levels of GSH, phytochemical screening of Cycas beddomei showed the SOD,CAT, GPx, and GRD in addition to significant presence of alkaloids, flavonoids, steroids, carbohydrates, (P<0.001) lower levels of hepatic MDA as compared to phenolics, proteins, lignans, saponins and anthocyanidins, carbon tetrachloride + olive oil (1:1) intoxicated rats. where as absence of reducing sugars and gums. The preliminary phytochemical screening for various Histopathological observations functional groups was tabulated as Table No. 2. Histology of liver (Figure No: 6) of normal saline treated group showed normal architecture. The hepatic Toxicity study cords and the sinusoids were well visible (I). The Rats when fed with methanol extract of Cycas histopathological studies of the liver showed fatty beddomei up to 2000 mg kg-1,p.o. exhibited no mortality changes, swelling, necrosis, cell vacuolization, or any sign of gross behavioral changes when observed degenerated nuclei and infiltration with loss of initially for 24 h, and finally up to 15 days. hepatocytes in CCl4: olive oil (1:1 ratio) intoxicated rats in comparison with normal rats (II). The liver sections of rats Carbon tetrachloride + olive oil (1:1) induced treated with the lower (250mg/kg) and higher (500mg/kg) hepatotoxicity doses of the extract showed reduced degeneration of In this experiment, the extent of liver injury after hepatocytes, normalization of fatty changes, decrease in 7 days of CCl4 administration was assessed by the vacuolization and necrosis of the liver (III & IV). significant (p<0.001) increase of SGOT, SGPT, LDH, Silymarin treated group showed considerable reduction in ALP and bilirubin and significant (p <0.001) decrease in necrosis and damage of liver cells (V).

Figure 1. Effect of methanol extract of Cycas beddomei and silymarin (standard drug) on serum levels of SGOT (IU -1 -1 -1 L ) and LDH (nanomoles NADH oxidized min mg protein) during CCl4-induced oxidative stress in rats.

Note: Values are mean-SE (n=6). Group 2 (CCl4-induced rats) compared with Group 1(control rats). Groups 3–5 (CCl4 individually treated with methanol extract of CB (Cycas beddomei) and silymarin rats) compared with Group 2 (CCl4-induced rats). **p<0.001;* p<0.01. 904 Baggiya Selvi D and Raja S. / International Journal of Biological & Pharmaceutical Research. 2015; 6(11): 899-908.

Figure 2. Effect of methanol extract of Cycas beddomei and silymarin (standard drug) on serum levels of total protein (mg/dl) and bilirubin (mg/dl) during CCl4-induced oxidative stress in rats.

Note: Values are mean-SE (n=6). Group 4 (CCl4-induced rats) compared with Group 5 (control rats). Groups 3–5 (CCl4 individually treated with silymarin and methanol extract of CB (Cycas beddomei) rats) compared with Group 4 (CCl4-induced rats). **p<0.001;*p<0.01. Figure 3. Effect of methanol extract of Cycas beddomei and silymarin (standard drug) on serum levels of SGPT (IU -1 L ) and ALP (KA Units) during CCl4-induced oxidative stress in rats.

Note: Values are mean-SE (n=6). Group 2 (CCl4-induced rats) compared with Group 1 (control rats). Groups 3–5 (CCl4 individually treated with methanol extract of CB (Cycas beddomei) and silymarin rats) compared with Group 2 (CCl4-induced rats). **p<0.001; *p<0.01. Figure 4. Effect of methanol extract of Cycas beddomei and silymarin (standard drug) on hepatic levels of GSH (µg/mg of protein), GRD (nanomoles of NADPH oxidized min-1 mg-1 protein) and MDA (nanomoles of TBARS mg-1 of protein) during CCl4-induced oxidative stress in rats.

Note: Values are mean-SE (n=6). Group 2 (CCl4-induced rats) compared with Group 1 (control rats). Groups 3–5 (CCl4 individually treated with methanol extract of CB (Cycas beddomei) and silymarin rats) compared with Group 2 (CCl4-induced rats). **p<0.001; *p<0.01. 905 Baggiya Selvi D and Raja S. / International Journal of Biological & Pharmaceutical Research. 2015; 6(11): 899-908.

Figure 5. Effect of methanol extract of Cycas beddomei and silymarin (standard drug) on hepatic levels of GPx -1 (mu mg of protein), CAT (µ/mg of protein) and SOD (µ/mg of protein) during CCl4-induced oxidative stress in rats.

Note: Values are mean-SE (n=6). Group 4 (CCl4-induced rats) compared with Group 5 (control rats). Groups 3–5 (CCl4 individually treated with silymarin and methanol extract of CB (Cycas beddomei) rats) compared with Group 4 (CCl4-induced rats). **p<0.001; *p<0.01. Figure 6. Hepatoprotective action of methanol extract of Cycas beddomei against CCl4: olive oil (1:1) induced hepatotoxicity [I] Normal control; [II] CCl4-olive oil (1:1, 1ml/kg); [III] Cycas beddomei (250 mg/kg) + CCl4 + olive oil (1:1) [IV] Cycas beddomei (500 mg/kg) + CCl4 + olive oil (1:1); [V] Standard Silymarin (25 mg/kg).

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Table 1. Percentage yield of methanol extract of Cycas beddomei Extract name % Yield (w/w) methanol extract of Cycas beddomei 34.2

Table 2. Qualitative analysis (group tests) of methanol extract of Cycas beddomei Phytochemical test Observation Alkaloids + Flavonoids + Steroids + Carbohydrates + Phenolics + Proteins + Lignans + Saponins + Anthocyanidins + Gums - Reducing sugars - “+” indicates positive; “-” indicates negative

DISCUSSION deregulates the cellular functions leading to hepatic Acute toxicity studies disclosed the non-toxic gangrene (Naik and Panda, 2007). nature of the methanolic extract of Cycas beddomei up to a The reduced activities of SOD, GRD, GPx and dose level of 2000mg/kg weight in rats. There was no catalase observed suggests the hepatic damage inside the morbidity or unwholesome reaction found at any of the rats administered with carbon tetrachloride + olive oil doses selected throughout the study. Inside the current (1:1) but they treated with, 250 and 500mg/kg of Cycas investigation, Cycas beddomei methanol extract was beddomei methanol extract groups showed vital increase evaluated for the elicited hepatotoxicity in rats. The inside the amount of these enzymes, that indicates the hepatotoxicity elicited by carbon tetrachloride + olive oil antioxidant activity of the Cycas beddomei. Regarding non (1:1), hepatotoxicity is as a results of its matter CCl3•, a enzymic antioxidants, GSH can be an important radical that alkylates cellular proteins and totally different determinant of tissue condition to aerobic damage and so macromolecules with a concurrent attack on unsaturated the depletion of hepatic GSH has been shown to be fatty acids, inside the presence of oxygen, to produce associated with degree exaggerated toxicity to chemicals, macromolecule peroxides, leading to liver also as carbon tetrachloride + olive oil (1:1). Moreover, a damage(Clawson, 1989). Hepatocellular gangrene ends up decrease in hepatic tissue GSH level decided inside the in elevation of the serum marker enzymes like SGOT, carbon tetrachloride + olive oil (1:1) (Hewawasam et al., SGPT, ALP and bilirubin which are free from the liver 2003) treated groups. The increase in hepatic GSH level into blood (Zeashan et al., 2008). This study disclosed a inside the rats treated with, 250 and five hundred mg/kg of significant increase inside the activities of SGOT, SGPT, Cycas beddomei methanol extract is additionally as a ALP and serum bilirubin levels on, indicating sizeable result of de novo GSH synthesis or GSH regeneration. The hepatocellular injury (Ashok Shenoy et al., 2001; Wang et amount of macromolecule peroxide can be alive of al., 2011). Administration of Cycas beddomei methanol membrane harm and alterations in structure and extract at completely totally different dose level (250 and performance of cellular membranes. Inside the current five hundred mg/kg) attenuated the increased levels of the study, elevation of macromolecule peroxidation inside the serum enzymes, made by carbon tetrachloride + olive oil liver of rats treated with carbon tetrachloride + olive oil (1:1) and caused an ensuing recovery towards (1:1) was determined. The increase in LPO levels in liver normalization kind of like the control teams animals. The suggests exaggerated macromolecule peroxidation leading hepatoprotective results of the Cycas beddomei methanol to tissue damage and failure of antioxidant defense extract was further accomplished by the histopathological mechanisms to forestall the formation of excessive free examinations. Cycas beddomei methanol extract at radicals (Becker et al., 1987). Treatment with Cycas completely different dose levels offers hepatoprotection, beddomei methanol extract significantly reversed all the but 500mg/kg is much better than the lower dose. In changes. Hence, it's possible that the mechanism of carbon tetrachloride + olive oil (1:1) elicited hepatoprotection of Cycas beddomei is additionally as hepatotoxicity, the balance between ROS production and results of its antioxidant activity. these inhibitor defenses is additionally lost, „oxidative stress‟ results, which through a series of events

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CONCLUSION was compared with silymarin showing a better activity on Taken together, our promising results indicate some hepatic markers with respect to this reference that Cycas beddomei methanol extract, which does not compound. The CBME has shown dose dependent activity show any toxicity and mortality up to 2000 mg/kg dose, among that at the dose level of 500 mg/kg, p.o. shows may serve as a candidate with strong hepatoprotective better activity that is comparable with the control and effect and antioxidant activity in a rat model of carbon customary groups. tetrachloride and olive oil (1:1) induced liver damage. This study showed that the Cycas beddomei methanol CONFLICT OF INTREST extract has hepatoprotective effects that were evidenced The authors declare that they have no conflict of by biochemical, antioxidant and histopathological interest. analysis. The potency of Cycas beddomei methanol extract

REFERENCES Aebi H. Catalase. In Methods in enzymatic analysis. H.V. Bergmeyer. New York, Cheime, Weinheim, FRG: Academic press. 1974; 2: 674–684. Alekhya C, Yasodamma N and Chaithra D. Antibacterial and physico-chemical studies of Cycas beddomei dyer, male and female cones. International Journal of Pharma and Bio Sciences. 2013; 4(2): 647-656. Alekhya C, Yasodamma Nand Chaithra D. Anti-inflammatory activity of Cycas beddomei dyer. male cone extracts. Indo American Journal of Pharmaceutical research. 2014; 4(01): 132-137. Ashok Shenoy K, Somayaji SN, Bairy KL. Hepatoprotective effects of Ginkgo biloba against carbon tetrachloride induced hepatic injury in rats. Indian Journal of Pharmacology. 2001; 33:260-266. Becker E, Messner B, Berndt J. Two mechanisms of CCl4-induced fatty liver: lipid peroxidation or covalent binding studied in cultured rat hepatocytes. Free Radical Research and Communication. 1987, 3(1-5):299-308. Chandan BK, Saxena AK, Shukla S, Sharma N, Gupta DK, Singh K. Hepatoprotective activity of Wood fordia fruticosa Kurz flowers against carbon tetrachloride induced hepatotoxicity. Journal of Ethnopharmacology. 2008; 119 (2): 218-224. Chandan BK, Saxena AK, Shukla S, Sharma N, Gupta DK, Suri KA, Suri J, Bhadauria M, and Singh B. Hepatoprotective potential of Aloe barbadensis mill against carbon tetrachloride induced hepatotoxicity. Journal of Ethnopharmacology. 2007; 111: 560–566. Clawson GA. Mechanism of carbon tetrachloride hepatotoxicity. Pathology and Immunopahtological Research. 1989, 8: 104– 112. Ellman GL. Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics.1959; 82, 70–77. Feijóo M, Túnez I, Ruiz A, Tasset I, Muñoz E, Collantes E. Oxidative stress biomarkers as indicator of chronic inflammatory joint diseases stage. Reumatología Clínica. 2010; 6(2): 91-94. Garg K, Bhal I, Kaul ML. A Text Book of Histology. 2nd ed. CBS Publishers and Distributors, New Delhi.1996; 152-154. Hewawasam RP, Jayatilaka KAPW, Pathirana C, Mudduwa LKB. Protective effect of Asteracantha longifolia extracts mouse liver injury induced by carbon tetrachloride and paracetamol. Journal of Pharmacy and Pharmacology. 2003; 55: 1413-1418. Jain SK and Sastry ARK. Threatened Plants of India. A State-of-the Art Report. 1980; 40. Kataki MS, Ahmed MZ, Awasthi D, Tomar B, Mehra P, Yadav RS. In vitro antioxidant profile of Wedelia calandulaceae . Pharmacologia. 2012; 3(3): 75-83. Kind PR and King EJ. Estimation of plasma phosphatase by determination of hydrolysed phenol with antipyrine. Journal of Clinical Pathology. 1954; 7, 322-326. Klaunig JE, Wang Z, Pu X, Zhou S. Oxidative stress and oxidative damage in chemical carcinogenesis. Toxicology and Applied Pharmacology. 2011; 254 (2): 86-99. Kornberg A, Colowick SP, Kaplan NO. Lactic dehydrgenase of muscle. Academic press, New York. Methods in Enzymology. 1955; 1: 441-443. Kshirsagar AD, Mohite R, Aggrawal AS, Suralkar UR. Hepatoprotective medicinal plants of Ayurveda-A review. Asian Journal of Pharmacy and Clinical Research. 2011; 4(3):1-8. Latheef SA, Prasad B, Bavaji M, Subramanyam G. A Database on endemic plants at Tirumala hills in India. Bioinformation. 2008; 2(6): 260-262. Law K, Brunt EM. Nonalcoholic fatty liver disease. Clinics in Liver Disease. 2010; 14 (4): 591-604. Liu JR. Edamatsu H, Kabuto and Mori A. Antioxidant action of Guilingji in the brain of rats with FeCl3 induced epilepsy. Free Radical Biology and Medicine. 1990; 9: 451–54. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry. 1951; 193: 265–275. 908 Baggiya Selvi D and Raja S. / International Journal of Biological & Pharmaceutical Research. 2015; 6(11): 899-908.

Mahendra Nath M, Sankara Rao M, Ramesh L, Madhava Chetty K. Phyto-chemical evaluation and anti-oxidant potentiality of Cycas beddomei Dyer male cone aqueous extract. International Journal of Drug Delivery and Research. 2014; 6 (2): 220-227. Majumdar, A.M., Anuradha, S., Upadhye, Pradhan, A.M.: Effect of Azadirachta indica leaf extract on carbontetrachloride induced hepatic damage in rats. Indian Journal of Pharmaceutical Sciences.1988; 60(6): 363. Malloy HT, Evelyn KA. The determination of bilirubin with the photometric colorimeter. Journal of Biological Chemistry. 1937; 119: 481-490. Mohandas J, Marshall JJ, Duggin GG, Horvath JS, Tiller D. Differential distribution of glutathione and glutathione related enzymes in rabbit kidney: possible interactions in analgesic neuropathy. Cancer Research. 1984; 44: 5086–5091. Naik SR, Panda VS. Antioxidant and hepatoprotective effects of Ginkgo biloba phytosomes in carbon tetrachloride-induced liver injury in rodents. Liver International. 2007, 27(3):393-399. Nayar MP, Sastry ARK. Red Data Book of Indian Plants, Botanical Survey of India, Calcutta. 1987; 359. Prance GT and Mori SA. “Annals of the Missouri Botanical Garden”. 1986; 73: 99-101. Radak Z, Zhao Z, Goto S, Koltai E. Age-associated neurodegeneration and oxidative damage to lipids, proteins and DNA. Molecular Aspects of Medicine. 2011; 32 (4-6): 305-315. Rai S, Wahile A, Mukherjee K, Saha BP, Mukherjee PK. Antioxidant activity of Nelumbo nucifera (sacred lotus) seeds. Journal of Ethno pharmacology. 2006; 104:322-327. Raja S, Nazeer Ahamed KFH, Kumar V, Kakali M, Bandyopadhyay A, Pulok K. Mukherjee. Antioxidant effect of Cytisus scoparius against carbon tetrachloride treated liver injury in rats. Journal of Ethnopharmacology. 2007; 109: 41–47. Rao BRP, Babu MVS, Donaldson J. A Reassessment of the conservation status of Cycas beddomei Dyer (Cycadaceae), an Endemic of the – Kadapa hills, Andhra Pradesh, India, and comments on its CITES status. Encephalartos. 2010; 102:19-24. Reitman S, Frankel SA. Colourimetric method for the determination of serum oxaloacetatic and glutamic pyruvic transaminases. American Journal of Clinical Pathology. 1957; 28: 56–63. Selvam ABD. Cycas beddomei Dyer. Pharmacognosy of Negative Listed Plants. Botanical Survey of India. Ministry of Environment and Forest. 2012; 49. Shaik A, Elumalai A, Eswaraiah MC, Usha. An updated review on hepatoprotective medicinal plants. Journal of Drug Delivery and Therapeutics. 2012; 2 (2): 1-3. Singh K, Khanna AK, Chander R. Hepatoprotective activity of ellagic acid against carbon tetrachloride induced hepatotoxicity in rats. Indian Journal of Experimental Biology. 1999; 37: 1025. Stickel F, Schuppan D. Herbal medicine in the treatment of liver diseases. Digestive Liver Disease. 2007; 39(4): 293-304. Wang L, Ran Q, Li DH, Yao HQ, Zhang YH, Yuan ST. Synthesis and anti-tumor activity of 14-o-derivatives of natural oridonin. Chinese Journal of Natural Medicine. 2011; 9 (3): 194-198. Wendel A. 1981. Glutathione peroxidase. Methods in Enzymology 77: 325–33. Zeashan H, Amresh G, Singh S, Rao ChV. Hepatoprotective activity of Amaranthus spinosus in experimental animals. Food and Chemical Toxicology. 2008; 46: 3417-3421.