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J Pharm Pharmaceut Sci (www.cspsCanada.org) 8(3):507-515, 2005 Phytopreventative Anti-Hyperlipidemic INTRODUCTION Effects Of Gynostemma Pentaphyllum In Rats Gynostemma pentaphyllum (Thumb.) Makino (GP) (Jiaogulan-Chinese name) is an herbaceous vine Samer Megallia, Fugen Aktanb, Neal M. Daviesc, plant of the cucurbitaceous family and is distributed Basil D. Roufogalisa naturally in shaded and humid places (1). In traditional Chinese medicine it is indicated for heat aFaculty of Pharmacy and Herbal Medicines Research and clearing, detoxification, and as an anti-tussive and Education Centre University of Sydney, Sydney NSW, expectorant for relieving cough and chronic Australia b bronchitis. In Japan it is indicated as a diuretic, Faculty of Pharmacy, Ankara University, Tadogan, antipyretic, anti-inflammatory and tonic. GP contains Ankara, Turkey cCollege of Pharmacy, Department of Pharmaceutical saponins (triterpene glycosides or gypenosides); Sciences, Washington State University, Pullman, more than 100 dammarane-type glycosides, also Washington, U.S.A. called gypenosides, have been isolated and identified (2). A general structure of dammarane-type Received May 7, 2005; Revised September 12, 2005; Accepted September 13, 2005; Published September 16, 2005 gypenoside is illustrated in Figure 1. Some of these saponins are the same as those from Panax ginseng (ginsensides) (3-4). Flavonoids such as rutin are also Abstract Purpose: Gynostemma pentaphyllum is found. The medicinal properties of GP have been widely used in traditional Chinese medicine. mainly attributed to the saponins (2). Preliminary studies indicate Gynostemma isolated triterpine glycosides lower cholesterol. Our studies examine anti-hyperlipidemic effects of gypenosides. Methods: Gynostemma activity was examined in poloxamer P407 induced hyperlipidemia in rats. Results: 1 g/kg P407 induced plasma triglyceride (25 fold), total cholesterol (6 fold), low density lipoprotein cholesterol (LDL) (7 fold), high density lipoprotein cholesterol (HDL) (1.6 fold), and nitrite (8 fold). After acute (4 days) and chronic (12 days) oral administration the gypenoside extract (250 mg/kg) reduced triglyceride (53% and 85%, respectively) and total cholesterol levels (10% and Figure 1: General Structure of Dammarane-Type 44%, respectively). No significant effects on LDL or Gypenosides. Gypenoside consists of both the HDL cholesterol were observed. The gypenosides hydrophobic sapogenin part and the hydrophilic sugar part reduced nitrite ~80%. Similar results were obtained in the molecule (where R1 and R2 = glucose, rhamnose; with atorvastatin (75 mg/kg for 4 days); except that R3 = glucose, xylose). LDL cholesterol was reduced (17%) and HDL Poloxamer 407 (Pluronic RF-127) has been cholesterol increased. 50% of lipoprotein lipase used to induce hyperlipidemia in rats. P407 is a (LPL) plasma activity was inhibited by ~20 μM biocompatible, non-ionic surfactant (5) is considered P407. Gynostemma had no effect on LL, however, it non-toxic and safe in animal chronic administration reversed the P407 inhibition of LPL activity in a for long term studies (6). In the rat, once daily concentration-dependent manner, with a 2-fold intraperitoneal (ip) injections of 0.33 mg/kg P407 for increase at ~10 μg/ml. Conclusions: These studies four consecutive days resulted in the increase of demonstrate efficacy of Gynostemma pentaphyllum monocyte numbers with no other toxicological in lowering triglyceride, cholesterol and nitrite in complications (7). In another study, 0.5 g/kg of P407 acute hyperlipidemia. The results suggest further was given to C57BL/6 mice every third day for 200 investigations of Gynostemma gypenosides are days, without any significant weight loss or warranted to examine the mechanisms of this alterations in the liver enzymes aspartate activity. aminotransferase (AST) and alanine aminotransferase (ALT) (8). The increase in Corresponding Author: Dr. Sam Megalli PhD, Faculty of triglycerides (TG) seen following P407 injection is Pharmacy, Pharmacy building A15, The University of Sydney, NSW 2006, Australia. [email protected] mainly due to the inhibition of TG degradation, due 507 J Pharm Pharmaceut Sci (www.cspsCanada.org) 8(3):507-515, 2005 to a direct inhibitory effect on lipoprotein lipase Ethanol was obtained from Asia Pacific Specialty (LPL), the enzyme responsible for TG degradation. Chemicals Ltd. Sydney, Australia. Other materials However, the effect of P407 on hypercholesterolemia including ketamine hydrochloride and suggests that the elevations of cholesterol could be carboxymethylcellulose (CMC) were purchased from mediated through indirect activation on HMG-CoA Sigma-Aldrich (Australia). Total cholesterol (TC), reductase (6). total triglyceride, (TG) low density lipoprotein Herbal medicines and nutraeuticals are being cholesterol (LDL) and high density lipoprotein increasingly accepted and utilised in the Western cholesterol (HDL) measuring kits were obtained medicine to treat and/or prevent various diseases. In from Sigma-Aldrich (Australia) and Trace Scientific this present study, we investigate the effect of Ltd.(Australia) and used according to manufacturers gypenosides isolated from Gynostemma instructions after precipitation techniques and pentaphylumm on hyperlipidemia and nitrite levels in modified enzymatic procedures to plasma an acute model of hyperlipidemia. In addition, in samples.(9-11) All other reagents and chemicals vitro and in vivo studies have been utilised to further were of analytical grade. explore the possible modes of action of Gynostemma pentaphyllum. Animal Grouping and Treatments Experiments were conducted in accordance with the MATERIALS AND METHODS Animal Ethics Committee at The University of Sydney guidelines, Approval number L24/5- Purified gypenoside extract was provided by Ankang 2001/2/3369. Pharmaceutical Institution of Beijing Medical Sprague-Dawley (S-D) rats were purchased University, People’s Republic of China. The Ankang from Animal Services at the University of Sydney. extract in a capsule formulation was dissolved in The rats obtained were males, 4-8 weeks old and 90% ethanol and thoroughly mixed with a magnetic average weight 250-300 g. Rats were housed 3-4 per stirrer then filtered twice using filter paper and cage in a temperature controlled (22 ± 1)˚C room, evaporated down using a Buchi Rotavapor R-114. with a light/dark cycle of 12 hr. For a week The extract was then dried in a vacuum oven and following their receipt, the animals were allowed free kept in a bell jar with silica gel. A final purity of access to a standard rat chow diet and tap water approximately 90% gypenosides was obtained. while they were acclimating to the environment. Poloxamer 407, also called Pluronic RF-127, was During the experimentation all rats had free access to donated by BSAF Australia. The poloxamer 407 was standard rat chow and water at all times unless made at a final concentration of 30% (w/w) by otherwise stated in the methods section. Gynostemma dissolving the powder in distilled cool water; the pentaphyllum and atorvastatin samples were well solution was then kept refrigerated overnight to mixed in 1% carboxymethylcellulose. Treatments facilitate its dissolution. Needles and syringes used were administered to rats using oral gavage via a to administer P407 were cooled prior to curved feeding needle (Harvard Apparatus). At the administration to prevent P407 gelation within the start of the experiment animals were randomly syringe. Poloxamer was administered ip to the rats. distributed so that body weights, initial TG, TC and Atorvastatin was purchased in a tablet form at other parameters were similar in all the experimental strength 80 mg through Australian Pharmaceutical groups. Industries in Northmead, Sydney. After crushing the At the end of each study, animals were tablets, powder was then dissolved in 1% CMC for sacrificed and blood collected via cardiac puncture oral dosing via gavage. for analysis. Rats were sacrificed after the induction For the assay of lipoprotein lipase (LPL), of anaesthesia using 1 ml of ketamine injection (1 g Glycerol tri [9, 10(n)-3H] oleate ([3H] TO) was /10 ml) and sacrificed using a lethal injection of 0.5 obtained from Amersham Biosciences, Sydney ml concentrated solution of potassium chloride (Code TRA191). Lipoprotein enzyme from bovine (70%) solution directly into the heart. milk and cold glyceryl trioleate, also know as Triolein (TO), and lecithin stored in chloroform (100 Effect of Acute and Chronic Gynostemma mg/ml) were obtained from Sigma-Aldrich pentaphyllum (Australia). Glycerol and 0.2 M Tris-HCl (pH 8.0) Sprague-Dawley rats were divided into 3 groups containing 3% bovine serum albumin (BSA) were (Figure 2). A control (C) group (12 rats), did not also obtained from Sigma-Aldrich (Australia). receive any treatment apart from 1% CMC as an oral 508 J Pharm Pharmaceut Sci (www.cspsCanada.org) 8(3):507-515, 2005 gavage, a second group treated with P407 1 g/kg Each rat received an intravenous injection into the alone (P407) (12 rats) and a group receiving GP 250 tail vein of 1000 IU of heparin in a volume of 0.3 ml. mg/kg once daily as an oral gavage for 4 days for Two minutes following the heparin injection, blood acute experiments (12 rats) and 12 days for chronic was drawn using cardiac puncture. Blood was then experiments. In addition, atorvastatin was used as a pooled centrifuged and plasma frozen until further positive control. Rats were divided into 3 groups experimentation. (Figure