Smilax zeylanica: A Promising Antilipidemix and Antiobesity Agent

Background: It is well established fact that obesity has now said to be a disease status.The phytochemicals have always been looked for their enormous stored potential. Similax zeylanica is a naturally occurring flavonoid known for its pharmacological potential. In this study it was aimed to determine the antiobesity and antilipidemic effect of various Similax zeylanica extract. Materials and Methods: The pharmacological screening of Cyclohexane extract of Smilax zeylanica (SZH), ethyl acetate extract Smilax zeylanica (SZE) and methanolic extract of Smilax zeylanica (SZM) was performed. The acute toxicity studies were performed on various animal groups. Obesity was diet induced.The plasma samples were investigated for various biochemical parameters such as glucose, lipid profile (Cholesterol, TG, HDL, LDL and VLDL), Liver function test (SGPT, SGOT). Results and Conclusion: The various parameters studied showed that the methanolic extracts of Similax zeylanica showed enormous antiobesity potential and concluded for the supplementation for antilipidemic effect.


INTRODUCTION
Heftiness is a constant metabolic problem brought about by awkwardness between energy admission and use. Overweight and heftiness are characterized as strange or exorbitant fat collection that presents a danger to wellbeing [1]. Numerous mainstream researchers have gotten progressively keen on the sub-atomic guideline of fatty oil combination and in drug ways to deal with lessen fat assimilation and capacity because of phytochemicals, introducing an energizing open door for the revelation of more up to date hostile to weight specialists. The guideline of unsaturated fat and fatty substance accessibility in natural reactions relies upon the movement of lipolytic chemicals present in unsaturated fat digestion in fat tissue. The portrayal and ID of a few qualities associated with lipid digestion have yielded a rich pool of possible focuses for medications to treat weight and other metabolic conditions [2]. Pancreatic lipase, the principle lipid processing chemical, eliminates unsaturated fats from the α and α′ places of dietary fatty oils, which yield the lipolytic item β-monoglyceride and long chain soaked and polyunsaturated unsaturated fats. Restraint of pancreatic lipase is an alluring focused on methodology for the revelation of powerful enemy of stoutness specialists for weight treatment [3]. One of the screening techniques utilized in the disclosure of against heftiness drugs is to look for powerful lipase inhibitors from plant removes. Plants have been utilized as customary regular meds for mending numerous infections. Specifically, different oriental therapeutic plants are accounted for to have natural movement.Smilax zeylanica Linn. (Smilacaceae) regularly known as Jangliashbha (Hindi) is broadly disseminated in Indian woods. It is a brambled, woody plant that grows up to 50 m long [4]. It creates little blossoms and dark, blue or red berry-like natural products which are eaten regularly by bird. Plants bloom in May and June with white/green grouped blossoms. On the off chance that fertilization happens, the plant will deliver a splendid red to blue-dark circular berry organic product around 5-10 mm in width that develops in the fall [5]. The writing overview uncovers that different pieces of Smilax zeylanica have been utilized as a fables medication for relieving Investigation on Phytochemicals, antiobesity and antilipidemic activities of Smilax zeylanica Linn. Verdant Extracts [6,7]. The current examination includes the starter examination of phytochemical constituents, intense harmfulness study and the antiobesity effect of various concentrates of leaves of S. zeylanica utilizing oil ether, diethyl ether, chloroform and methanol as solvents.

Collection, Identification and Authentication of Plant Material
The plant smilax zeylanica was collected in June from Yamuna biodiversity park, New Delhi.
The formal authentication and identification was done by Dr. Bhawana.S .Bajpai Department of Botany Ch.Charan Singh University, Meerut voucher specimen number B01-PB565/1/2/2017. The roots were allowed to air dry, away from sunlight. The dried material was grounded coarsely to a powder and transferred to labelled brown bottles until required.
The dried, powdered plant material (500 g) was defatted with petroleum ether and was then extracted consecutively with solvents of increasing polarity Cyclohexane, Ethyl acetate and methanol for 72 hrs each with intermittent stirring. The collected mass was subjected to drying to evaporate the excess of solvent in a rotary evaporator; this procedure was as per protocol. The collected dark coloured material were stored at 4 o C and were termed as Smilax zeylanica cyclohexane extract(SZH), Smilax zeylanica(SZE), Smilax zeylanica (SZM).

Acute Toxicity Study
The acute toxicity was performed according to OECD guidelines (OECD 423, 2001). The selected Wistar rats were used for toxicity studies. The animals were divided into three groups of three in each. The animals were fasted overnight prior to the experimental procedure. The acute toxicity study was performed for deciding safe doses for further pharmacological studies along with this any behavioural or physiological changes due to extract administration was also observed. Extracts were given orally to rats at the graded dose of 1000, 2000, 4000 mg/kg body wt. Immediately, after dosing, the animals were observed continuously for first four hours for behavioural changes and for mortality at the end of 24 h and daily up to 14 days for any behavioural change or mortality. Since no mortality was reported even after 14 days. This indicated that the extracts are safe up to a single dose of 4000 mg/kg body weight. Hence the selected doses for the administration in experimental animals were considered 1/10th and 1/5th of maximum safe dose (Table 1). Since no mortality was observed the animals were then kept in a washing period for 20 days, the same animals were then used for the anti-obesity activity [8].

Anti-obesity Activity
In order to screen the phytoextracts for antiobesity activity Wistar albino rats were used. Obtained the approval from CPCSEA Committee at ITS College of Pharmacy, Ghaziabad. Registration number 1044/PO/Re/S/07/CPCSE. Anti-obesity study was carried out on 36 animals which were divided into 6 groups with six animals in each group. Except the normal diet rats all other groups were fed with Cafeteria diet for a period of 8 weeks [9], pre-weighed food pellet/s was presented to rats in the home cage every day. Food intake was measured daily as the in the weight of left over feed from the weight of total feed presented to each animal daily. Orlistat drug (10 mg/kg) was used as standard drug, during the course of the study each group was then dosed accordingly. Table 2 gives the summary of treatment groups, dosage and duration of activity.

Diet Induced Obesity
The obesity in rats was induced by feeding them cafeteria diet for a period of 8 weeks. Three different cafeteria diets were given every day.
The diets were prepared and calculated according to per kg b w and given to all the groups of rats (except ND) any three of this snack food were given daily along with chow diet [10].
Preparation of Cafeteria diet (CD): The CD pellets were prepared fresh daily in combination of 70% Cafeteria diet with 30% of normal rat pellet.
Anthropometric measurements of each rat were monitored every week, during the course of the 8 weeks experiment (Table 3).
Animals were euthanized at the end of 8 th week and the weight of the liver of each Rat and white adipose tissues in all the nine groups were measured at the end of the experiment. Each tissue was then rinsed with phosphate-buffered saline, and stored at −40⁰C until histopathological analysis.

Statistical Analysis
The factual investigation of the considerable number of results was completed utilizing oneway ANOVA took after by Dunnet's numerous correlations test and Bonferroni post-Hoc test. Blood collection and biochemical assays: A 12-h fasting blood difference samples was drawn at the end of 8 weeks from the retro-orbital plexus into an anti-coagulant containing tube. Plasma was obtained by centrifuging the blood at 3000 rpm for 15 min at 4°C.
Biochemical parameters viz., Plasma glucose, TC, TG and Plasma SGOT, SGPT were estimated using commercially available kits as per manufacturer's instructions.

Anthropometric Measurements
The various Anthropometric measurements like weight, and thenasal length were Documented for each rat according to standard techniques [11]. Weightwas measured using scales.
Body Weight: Weight was recorded every week using weighing scale.
Body Mass Index: BMI was calculated as weight (g) divided by height squared (cm 2 ).
Lee Index: Lee index is given by was calculated before and after the treatment as an index of obesity.Body weight for all groups was recorded weekly. The plasma samples were investigated for various biochemical parameters such as glucose, lipidprofile (Cholesterol, TG, HDL, LDL and VLDL), Liver function test (SGPT, SGOT).

Plasma Glucose Levels
The glucose levels in plasma were measured using a commercially available kit (AutoZyme) Plasma Glucose, Accurex Biomedical Pvt. Ltd. The set of tests were performed in duplicate as per the manufacturer'sInstructions using a UV spectrophotometer and absorbance were measuredat 505 nm.

Plasma Cholesterol Levels
Plasma cholesterol levels were measured using commercially available kit by Sigma code no. 120194.Tests were performed induplicate as per the manufacturer's instructions using a UV spectrophotometerand absorbance was measured at 510 nm upon scanning from 505 -530 nmrange.

Plasma Triglyceride Levels
Plasma triglyceride levels were measured using commercially available kitby Sigma code: 120211). This kit contains areagent set for determination of triglycerides, based on enzymatic method usingLipoprotein lipase, Glycerol kinase, Glycerol phosphate oxidase and Peroxidase.Tests were performed in duplicate as per the manufacturer's instructions using UV spectrophotometer and absorbance was measured at 510 nm uponscanning from 505-530 nm range.

Plasma High Density Lipoprotein Cholesterol (HDL Chol.) Levels
Plasma HDL-Cholesterol levels were measured using commercially available kit.HDL-Cholesterol precipitating reagent method (PTA) is intended for theseparation and quantitative determination of HDL cholesterol and utilizes the well-established precipitating properties of phosphotungstic acid to precipitate non HDL-Chol. The remaining cholesterol in the supernatant, HDL-Chol was then measured using commercially available kit for the estimation of Cholesterol by Erba code no. 120194.

Plasma Low Density Lipoprotein (LDL) and Very Low Density
Lipoprotein (VLDL) levels. The plasma LDL and VLDL levels were calculated from the Friedewald equation [12].

VLDL Level
Serum VLDL levels (mg/dl) = Triglyceride level/ 5 Plasma SGPT (ALT) levels Serum SGPT levels (ALT) were estimated using commercially available kit ALT Activity Assay kit:SIGMA (MAK052)SGPT (ALT), catalyses the transfer of amino group from L-alanine to 2-Oxoglutarate to form pyruvate and L-Glutamate. The pyruvate thus formed reacts with 2, 4dinitrophenyl hydrazine to form a corresponding hydrazone, a brownish red colored complex in an alkaline medium. The absorbance was measured at 505 nm using a UV spectrophotometer.

Plasma SGOT (AST) Levels
Serum SGOT levels were estimated using commercially available kit (AST Activity Assay Kit: SIGMA (MAK055),SGOT(AST), catalyses the transfer of amino group from aspartic acid to 2-Oxoglutarate toform oxaloacetate and L-Glutamate. The oxaloacetate thus formed reacts with 2, 4-dinitrophenylhydrazine to form a corresponding hydrazone, a brownish red colored complex in an alkaline medium. The absorbance was measured at 505 nm using a semi-auto biochemistry analyzer.

Histological Analysis
At the end of the experimental period, animals were sacrificed. Adipose tissue (fat pads) from each rat was removed, weighed andstored at-40°C. For histological examination, adipose tissue was fixed in 10% formalin solution and embedded inparaffin. Standard sections of 5μm thickness were cut, stained using haematoxylin and eosin, viewed under an optical microscope (40X).Histological analysis was carried out at the oral pathology department, I.T.S Dental college Muradnagar.

Effects of High-Fat Diet and Treatment on Body Weight
There was no momentous difference in all of the treatment groups at the commencement of the study. However, animals feed with high-fat diet showed significant increase in body weight compared to those fed with normal pellet diet (NPD). There was no significant difference in body weight of Positive Control group and normal but there was a significant difference in the body weight of Normal group and other treatmentgroups(p<0.05). The group treated with SZM Which is GP III showed no significant difference with POSITIVE CONTROL (Figs.1, 2).

. Effects of high-fat diet on food intake
The average daily feed intake of all the groups was the same at the start of the study; however, 8 weeks treatment with CD resulted in an increase in food intake (Fig. 3). The intake of food was calculated as gm/day/animal. Using the weighing scale the food intake was calculated every day.
CDgroup fed with high fat diet showed significant (p<0.001) increase in daily food intake when compared with Normal group and POSITIVE CONTROL group Treatment with TOM (400 mg/kg/p.o) and SZM showed significant (p<0.001) decrease in daily food intake as compared with CD group animals. Results are shown in (Fig. 3). Fig. 4 shows weekly comparison of Food intake ND group and other treatment groups.

Weekly Food Intake
There was a significant increase in the food intake in rats fed with CD but after the treatment of 3-4 weeksthere was a decrease in food intake.

BMI and Lee index
Body weight for all groups was recorded every week. The mean BMI and Lee Index were calculated at the end of the entire experimental duration.
Body Mass Index of Rats Fed on the Experimental Diets for 8weeks. Values are mean + SEM; n=6. Means are statistically significantly different (p < 0.05) (Fig. 5).

Locomotor Activity
Locomotion or physical activity is found reduced in obese animals hence this parameter can be used for determining the extent of obesity. This experiment was carried out using anactophotometer (Fig.  7),Dunnets multiple comparison test between Negative control CD and Other groups.

Adiposity Index Adipose tissue weight
Adipose tissue (fat pad) weight was taken at the end of the experiment after euthanisation of animals from all the groups. The fat pads were separated, washed in saline and weighed for all the rats (Figs. 8,9).
It was observed that there was a significant increase in adipose tissue weight in rats fed with Cafeteria diet (p<0.05). Whereas the POSITIVE CONTROL group and the group treated with SZM did not show any significant increase in weight of fat pads when compared to the normal group, there was significant increase in weight of fat pads in all other treatment groups (p<0.001) in comparison with normal group.

Plasma Glucose
The plasma Glucose levels were measured in each group. There was no significant difference between the normal group and POSITIVE CONTROL group, and similarly there was no significant difference between the SZM treated group i.e. GP III and Normal group.Whereas other treatment groups showed significant difference from the normal group (p<0.001) (Fig.  10).

Total Cholesterol, Triglycerides, LDL, HDL,VLDL
Rats fed with CD showed increased levels of serum Triglycerides, Total cholesterol, Fig. 11 LDL, VLDL Fig. 12, Fig. 13 and decreased HDL levels Fig. 14, However oral administration of SZM extract significantly suppressed the rise of Lipid profile and led to raise in the HDL levels.
When compared to Normal group and POSITIVE CONTROL group. Below given are the charts representing the same.

Faecal Lipids
Faecal matter was collected from the experimental rats at the end of 9th week and 15th week, dried and powdered. Faecal lipids were extracted with chloroform and methanol (2:1), then dissolved in 1% triton × 100 and estimated by standard kit method.
Increased excretion of faecal tri glycerides was observed in Orlistat and TOM treated CD groups, indicating that TOM might reduce lipid digestion and absorption. Similarly slightly significant TG in the faeces was observed in rats treated with SZM.

Liver Biochemical Parameters
Liver function tests are important indicators to reveal the functional status of liver since it is the vital organ involved in detoxification of compounds and in general metabolism. During diet induced obesity, the liver of obese rats displayed characteristic features of hepatic steatosis such as fat accumulation and swelling of roughendoplasmic reticulum and mitochondria in hepatocytes. Increased levels of SGOT (Fig.  19), SGPT (Tables 2, 3, Fig. 20), ALP in CD group, as shown in the present study indicate alterations in liver metabolic function. SZM extract administration has effectively lowered the CD-induced elevated levels of these hepatic enzymes and lipid profiles demonstrating its protective activity.

Nmol/Gm
Our results showed a statistically significant increase in the hepatic tissues levels of MDA in obese rats when compared to the POSITIVE CONTROL group, (p<0.001) (Fig.  22) Concerning the antioxidant enzymes activities in obese animals, the present study showed a statistical significant decrease in the activities of hepatic GSH level in obese rats compared to positive control (Fig. 23).

3.11Abdominal Fat Depots and Fatty Liver: Dissected Figures of ND andCD Fed Rats
The increase in accumulation of abdominal fat on feeding rats with high fat diet became further evident on dissecting the animals (Fig. 23). The increase in the fat deposit was evident even through the naked eyes. The differentiation between the fatty liver and normal liver could also be made through naked eyes. The liver in all animals of the CD group were enlarged and yellowish in colour, indicative of liver steatosis. On administration of treatment Orlistat, and SZM the liver remained reddish brown in colour and appeared normal in texture and colour with no abdominal or extra-hepatic fat deposition.

DISCUSSION
The deadly dose(LD50) value of the Smilax zeylanica indicated that the concentrate (methanol) was protected and nontoxic up to 5 g/kg [13].

Treated Rat
Smilax zeylanica in high fat diet-induced obesity. Weight is a significant danger factor for increased bleakness and mortality and is associated with various medical ailments [14]. High fat eating regimen actuated stoutness has been considered as the most mainstream model among scientists because of its high closeness of imitating the typical course of heftiness scenes in human thus why it is considered as a dependable instrument for examining corpulence as they will promptly put on weight when feed high-fat weight control plans. Human examinations have uncovered that expanded fat admission is related with body weight acquire, which can prompt corpulence and other related metabolic illnesses. This examination hence demonstrated that rodents presented to highfat diet for about fourteen days cause a critical increment of creatures' body weight, along these lines confirming the stout status [15]. Despite the fact that there was asignificant difference in the body weights between the high-fat and normal diet groups, nosignificant difference was seen in the everyday food admission of creatures. This perception furnishes us with the way that an expansion in body weight is independent of the amount of food consumed by the creatures. Treatment of HFD rodents with Smilax zeylanica at 200 mg/kg and 400 mg/kg p.o. conversely causes a remarkable decrease of body loads when contrasted with the high-fat diet administered rats.The result also suggests that Smilax zeylanica supplementation at 200 mg/kg and 400 mg/kg are fit for forestalling body weight acquire, correspondingly helping in keeping up the current body weight. This outcome was in accordance with the results reported from the previous study where a portion subordinate diminishing in the body weight was observed. Further, treatment with Smilax zeylanicare markably decreases the organ weight of rats feed on high-fat diet. Thus it proved the weight reducingpot ential of Smilax zeylanica. Further, dyslipidemia is another significant trademark in the pathogenesis of heftiness described by hypertriglyceridemia with diminished degree of LDL and VLDL [16]. Chronic dys lipid emia has been characterized asa major risk factor for cardiovascular danger, including atherosclerosis. In the current examination separated from decrease in weight, supplementation with Smilax zeylanicawas seen to lessen altogether the degrees of complete cholesterol and LDL and expanded the degree of HDL level in rodents feed with HFD. The expansion in the degree of HDL was discovered to be in a portion subordinate way; that is, supplementation with Smilax zeylanica at a portion of 400 mg/kg shows a superior impact in contrast with 200 mg/kg. where treatment with crudeextract of Smilax zeylanica ledto an expanded serum HDL level and diminished degrees of complete cholesterol, LDL, and triglyceride. Thus, it canbe concluded that leaves of Smilax zeylanicahave cardioprotective potential [17]. Further, atherogenic is viewed as a marker for different cardiovascular problems; the higher the worth, the higher the risk of developing cardiovascular disease and the other way around. High-fat eating regimen openness brought about the increased atherogenic index. Treatment with 200 mg/kg and 400 mg/kg significantly attenuated the atherogenic index and thus gives cardioprotection. The diminished atherogenic index by Smilaxzeylanica thus supports the cardioprotectant nature of Smilax zeylanica. To enhance the outcomes, the histopathological considers were likewise performed. The writing audit revealed that high fat diet-induced obesity and abnormal lipid metabolic small collectively are associated with inflammation, clog, and non alcoholic greasy liver sickness (NAFLD) prompting hepatic disappointment causing a lift in SGOT, SGPT, and complete bilirubin level in the serum [18]. Our outcomes showed that consumption of high-fat diet may play acrucial part in the pathogenesis of greasy liver or hepatic steatosis associated with obesity depicted via ballooning degeneration. Raised degrees of liver chemicals are a screen of hepatocellular harm and connect with expanded liver weight [19]. The results acquired in the current examination set up that high-fat diet causes hepatocellular damage, as clearly seen by the stamped height of serum proteins (SGOT, SGPT, and total bilirubin) activities and histopathological studies of liver misrepresented with hepatic steatosis. Nonetheless, treatment with Smilax zeylanica causes amomentary reduction in thee nzyme levels, meaning the job of Smilax zeylanicain forestalling liver harm causedbyhigh -fatdiet. Insulin resistance is associated with a number of metabolic problems, for example, corpulence, hyperlipidemia, and hypertension. HFD admissions were appeared to add to conditions, for example, hyperlipidemia, glucose narrow mindedness, hypertension, and atherosclerosis . Various confirmations showed that in exploratory creatures, high-fat eating regimens brought about unsettling influence in glucose digestion and disabled glucose resilience, and the current investigation likewise exhibit the decrease in blood glucose level those treated with similax (200 and 400 mg/kg).The hypolipedemic potential is related with the presence of -sitosterol20in unrefined concentrate of Smilax zeylanica.. Accordingly, further investigation should be done for distinguishing proof of explicit constituents present in Smilax zeylanica for its observed effects. The current examination consequently reasons that the concentrate of leaves of Smilax zeylanica have hypolipedemic and antiobesity potential that secures the body against unfriendly impacts of high fat dietinduced obesity. Further, we demonstrated that the day by day supplementation of Smilax zeylanicaleaves concentrate may switch the arrangement of hepatic steatosis and nonalcoholic fatty liver disorder. The results in the present study established that high-fat diet causes elevation in body weight and reduces lipid digestion as obviously seen by the checked rise of liver proteins and lipid level. Nonetheless, supplementation with smilax reversal the parameters thus suggesting its weight-reducing potential.

CONCLUSION
Hence, from the current examination it tends to be inferred that the methanolic concentrate of Smilax zeylanicais valuable to the weight the board, which underpins its customary case. Further, contemplates are done to decide the dynamic guideline of this plant, trailed by the distinguishing proof of the unthinking methodology of Smilax zeylanicathat helps in weight the executives.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
Animal Ethic committee approval has been taken to carry out this study.