International Journal of Pharmacy

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1 International Journal of Pharmacy Journal Homepage: Research Article CODEN: IJPNL6 Centratherum anthelminticum ameliorates antiatherogenic index in hyperlipidemic rabbits Tooba Lateef 1, 2 and Shamim A Qureshi 2 1 Department of Biochemistry, Jinnah University for Women, Karachi-74600, Pakistan 2 Department of Biochemistry, University of Karachi, Karachi-75270, Pakistan *Corresponding author qureshi29@live.com, t.lateef12@gmail.com ABSTRACT The present study first time scrutinized the lipid lowering effect of ethanolic seed extract (ESEt) of Centratherum anthelminticum in high-fat diet (HFD) induced hyperlipidemic rabbits. ESEt was used to determine acute toxicity in overnight fasted normal rabbits while its effect on biochemical parameters including serum total cholesterol (TC), triglycerides (TG), high (HDL-c), low (LDL-c), very low-density (VLDL-c) lipoproteins, antiatherogenic index (AAI), alanine aminotransferase (ALT), catalase (CAT), creatine kinase (CK), lipase, lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutase (SOD) and HMG-CoA/mevalonate ratio were estimated in HFD induced hyperlipidemic animal model. ESEt ( mg/kg) showed no sign of acute toxicity in rabbits. Similarly, three doses (200, 400, & 600 mg/kg) of same extract induced significant reduction in serum levels of TC, TG, LDLc, VLDL-c and an increase in HDL-c level with improvement in AAI found in test groups. Normal ALT and CK levels were also observed with positive progress found in antioxidant enzymes status, HMG-CoA / mevalonate ratio and lipase activity. Results conclude that ESEt exhibits antihyperlipidemic and antioxidant effects in HFD induced hyperlipidemic animal model. Keywords: Anti-atherogenic index, antioxidant, Centratherum anthelminticum, high-fat diet, hyperlipidemia. INTRODUCTION Increase consumption of high-fat diet (HFD) is the challenging issue of today for the development of hyperlipidemia, if it does not resolves timely, it brings obesity that opens the gate for serious outcomes like hypertension, diabetes, atherosclerosis and other cardiovascular problems. [1] It becomes more aggravates when it is associated with other risk factors like age, family history and sedentary lifestyle. [2] In this regard, 12 million deaths reported [3] each year with the same problem globally. Hyperlipidemia is characterized by elevated blood levels of triglycerides, total cholesterol, low-density and very low-density lipoproteins whereas low level of high-density lipoprotein in blood clearly tells that persons are at high risk. [4] The major negative aspect of hyperlipidemia is the hasty generation of reactive oxygen species (ROS) including oxygen and nonoxygen radicals which in turn involve in the oxidation of glucose, lipids and proteins thus become an initiator of chronic complications. [5] To reduce the risk of any accepted complication, researchers are focusing mainly on the treatment of hyperlipidemia and proving that decrease in blood lipids also reduces the chances of morbidity and mortality like a study reported that 20% decrease in blood cholesterol level can lowers approximately 31% of cardiovascular incidence and 33% of mortality rate. [6] Currently available drugs for the treatment of this health hazard include fibrate derivatives, 3-hydroxy-3- methylglutaryl-coa (HMG-CoA) reductase inhibitors (statins), bile acid binding agents, nicotinic acid, etc, which are not only providing gradual reduction in blood lipids but also associated with number of side effects that appeared on their prolong use. [7] Therefore, use of natural products for the treatment of hyperlipidemia is increasing day by day in all over the world due to their minimal side effects and cost effectiveness. [8] Centratherum anthelminticum Kuntze (syn. Vernonia anthelmintica Wild.) belongs to the family Asteraceae. [9] It is a 698

2 robust leafy annual plant distributed in Afghanistan, India, Malaysia, Pakistan, SriLanka and is wellreputed in folklore medicines. [10] Seeds of this plant have hot bitter taste and commonly known as kali ziri or zira dashti in Urdu. [9] These seeds are reported to contain variety of phyto-chemicals including alkaloids, carbohydrates, flavonoids, fatty acids, steroids, sterols, resin, etc. [11] Traditionally these seeds are not only used for anthelmintic and antiseptic purposes but also used to improve kidney troubles, asthma, hiccups, sores and white leprosy whereas seed powder is also reported to use externally to treat paralysis of legs. [10] Different organic solvent and aqueous extracts of these seeds were scientifically evaluated for analgesic, antipyretic, antibacterial, anticancer, anthelmintic, antidiabetic, hypotensive, spermicidal, laxative and smooth muscle stimulant activities. [12] Interestingly few herbal healers are also prescribed it for reducing blood lipids but no scientific evaluation has been found in literature. Therefore, the present work was designed to scrutinize the lipid lowering effect of ethanolic seed extract of C. anthelminticum in highfat diet induced hyperlipidemic animal model. MATERIALS AND METHODS Plant Material: Seeds of C. anthelminticum were bought from Hamdard dawakana, Saddar, Karachi, authenticated by taxonomist in Botany Department, University of Karachi, Karachi-75270, Pakistan and kept with a sample No. KU/BCH/SAQ/05. Preparation of ethanolic seed extract (ESEt): Forty grams of ground seed powder of C. anthelminticum was soaked in 95% ethanol (1 L) for overnight then filtered twice and concentrated at 40 o C by using rotary evaporator (Eylea-18) to get a dark brown residue which termed as ethanolic seed extract. [13] Dimethyl Sulphoxide (DMSO): 0.05% DMSO(Fisher Scientific, UK) was used as solvent for administering the doses of ESEt. [13] Simvastatin: Limitrol (simvastatin) of PharmaEvo (Pvt) Ltd, Pakistan and used as positive control (20 mg/kg) in present study. Induction of hyperlipidemia in experimental rabbits: Hyperlipidemia was induced in overnight fasted rabbits by oral administration of high-fat inducer (HFI) in a dose of 1ml/kg. The HFI consists of cholesterol (0.1 gm), bile salt (0.005 gm), butter (0.2 gm) in 1ml of peanut oil. [14] Determination of acute toxicity of ESEt: Overnight fasted normal rabbits were divided into control and test groups (n=4), each test group was administrated with different doses (10 to 2000 mg/kg) of ESEt orally while control group was given distilled water 1 ml/kg only through the same route. All groups after giving their respective treatments were kept under observation for 24 hours for signs like dullness, ruffled hair, depression, clumping together, itching, hyperactivity, sedation and mortality. [15] Experimental rabbits and determination of lipid lowering activity of ESEt: Healthy albino rabbits of both sexes weighing from kg were purchased from regular supplier of University of Karachi and kept in conventional animal house of Jinnah University for Women, Karachi-74600, Pakistan, according to the standard guidelines of animal handling. The rabbits were provided standard laboratory diet (SLD) with free access to water for 1 week before starting the experiment to acclimatize them to the environment. The procedure of present study was approved by Board of Advance Study and Research (BASR) of University of Karachi. Experiment was started by categorizing the overnight fasted rabbits into 7 different groups (n=6) according to the treatments (Figure I). Out of these, group I considered as normal control and kept them on SLD whereas rabbits in groups II-VII were given HFD (SLD plus HFI) with their assigned treatments. Each treatment was done orally once a day for 14 days consecutively. After completion of trial, rabbits were sacrificed, blood was collected to separate serum and livers were carefully dissected out to analyze biochemical parameters. Determination of biochemical parameters: Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c), alanine aminotransferase (ALT) and creatine kinase (CK) were estimated by using the commercially available kits of Randox, UK, while lipase enzyme was estimated by kit purchased from Roche diagnostic GmbH, Germany. Low-density lipoprotein (LDL-c) and very low-density lipoprotein (VLDL-c) were determined by below mentioned formulae [16] LDL-c = TC - (TG/5) - HDL-c (given in Randox reagent kit) VLDL-c = TG/5 Determination of anti-atherogenic index (AAI): The AAI was calculated by the following formula [17] AAI (%) = [HDL-c / (TC HDL-c)] x

3 Determination of antioxidant parameters: Catalase (CAT) activity: Liver homogenate (10%) in 0.01M phosphate buffer (ph 7.0) was prepared and filtered. Then 0.1 ml of filtrate was mixed with 1.4 ml of reaction mixture that contained 0.4 ml of 2 M hydrogen peroxide and 1 ml of same phosphate buffer. The reaction was terminated after 1 min by adding 2.0 ml of dichromate-acetic acid reagent. Blank contained distilled water in place of filtrate. The absorbance of both test and blank were measured at 620 nm to calculate percent inhibition of CAT. [18] Superoxide dismutase (SOD) activity: An aliquot of liver homogenate (10%) was treated with 0.75 ml of ethanol and 0.15 ml of ice chilled chloroform then centrifuged. Then 0.5 ml of EDTA (0.6 mm) and 1.0 ml of carbonate-bicarbonate (0.1M; ph 10.2) buffer was added in 0.5 ml of supernatant. The reaction was started by adding 0.5 ml of epinephrine (1.8 mm) and the absorbance was measured for 3 min at 480 nm. Blank contained all reagents except supernatant. Finally, percent inhibition of SOD was calculated. [19] Lipid peroxidation (LPO): 0.1 ml of liver homogenate (10%) was mixed with 2 ml of TBA- TCA-HCl reagent (thiobarbituric acid 0.37%, trichloroacetic acid 15% and 0.25N HCl), placed in a boiling water bath, cooled and centrifuged. The absorbance of clear supernatant was read at 535 nm which indicated the quantity of malondialdehyde produced. Percent inhibition of LPO was calculated against the blank. [20] Reduced glutathione (GSH): 0.5 ml of 10% liver homogenate and 2 ml of 2% TCA (trichloro-acetic acid) were mixed and centrifuged. Then 1 ml of supernatant allowed to reacts with 0.5 ml of Ellman s reagent (19.8 mg of 5, 5 -dithiobis-2-nitrobenzoic acid in 100 ml of 1% sodium citrate) and 3.0 ml of phosphate buffer (0.2M, ph 8.0). The absorbance was measured at 412 nm and percent inhibition was calculated. [21] Determination of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase activity: HMG-CoA reductase activity was determined in term of HMG-CoA / mevalonate ratio in liver homogenate prepared in sodium arsenate solution and filtered. Equal volumes of filtrate and dilute PCA (perchloric acid) were treated and centrifuged. Then 1.0 ml of supernatant reacted with 0.5 ml of 2M hydroxylamine reagent (alkaline ph in case of HMG- CoA) and 1.5 ml of ferric chloride, and incubated for 10 min. Absorbance was read at 540 nm. Finally, HMG-CoA / mevalonate ratio was calculated. [22] Statistical analysis: Outcomes of study are presented as mean SD (standard deviation) and analyzed by one way ANOVA (analysis of variance). The differences of means of test groups with hyperlipidemic controls were confirmed significant at p<0.05 through post hoc, least significant difference (LSD) test (SPSS, version 17.0). RESULTS AND DISCUSSION In vast literature of medicinal plants, the seeds of C.anthelminticum have been reported for many medicinal properties [12] but not for antihyperlipidemic activity which is the theme of present study. In this, interestingly, all three doses of ESEt exhibit strong antihyperlipidemic activity by inducing drastic decrease (p<0.05) in serum TC, TG, LDL-c and VLDL-c levels while increase (p<0.05) in serum HDL-c level in test groups as compared to HFD hyperlipidemic control groups (Figure 2). Conversely, simvastatin was found significantly effective in case of TC, LDL-c and VLDL-c (p<0.05) but not on TG and HDL-c when compared with same control groups. Studies have reported that consumption of HFD may leads to the production of increase amount of VLDL-c which in turn converts into LDL-c that actually transports cholesterol from liver to peripheral tissues including coronary arteries and form atherosclerotic plaques in their walls that cause blockage in normal blood flow. [23] Whereas HDL-c act as an antagonist of LDL-c and involves in efflux of cholesterol from peripheral tissues to liver to initiate its metabolism and excretion. [24] The decrease in serum TC may be due the decrease activity of HMG-CoA reductase, the rate-regulatory enzyme of cholesterol formation. [25] To prove this proposed mechanism, HMG-CoA/mevalonate ratio was calculated. [22] Amazingly, three of the doses of ESEt exponentially improved this ratio in all test groups which clearly indicates the inhibition (p<0.05) of HMG-CoA reductase activity whereas accelerated cholesterol production was observed in untreated groups including both hyperlipidemic controls that displayed low values of same ratio indicating the increase activity of HMG-CoA reductase (Table 1). Similarly, the same extract was also found an effective TG hydrolyzing agent by observing stimulated activity (p<0.05) of lipase enzyme in all three test groups (Table 1). The lipase enzyme hydrolyzes TG into its basic components including three free fatty acids and glycerol thus reduces the TG contents in adipose and non-adipose tissues. [26] Finally, the lipid lowering property of ESEt is more evident by observing improved AAI (Table 1) which is a powerful indicator for determining the risk of heart diseases and its low 700

4 value represents the high risk of atherosclerosis and vice versa. [27] It has been reported that hyperlipidemia induce the production of ROS which are powerfully involve in creating cellular stress and serve as the facilitator of secondary manifestations by forming oxidized-ldl (ox-ldl), affecting membrane bound enzymes and receptors, inhibiting antioxidant system of body especially by converting reduced glutathione (GSH) into oxidized, etc. [28] Therefore, the extent of lipid peroxidation of unsaturated fatty acids is an indicator of oxidative stress in body and it can enlighten the prognosis of many frequently prevalent diseases in population. [28] In the present study, marvelously, all three doses of ESEt not only provoke significant reduction in LPO in test groups but also elevate the status of antioxidant enzymes by showing the decrease in percent inhibition of CAT and SOD (Figure 3) and this represents the excellent antioxidant effect of seeds of C.anthelminticum through which it can scavenge the highly reactive oxygen and hydroxyl radicals. This protective effect of ESEt is more strengthen by restoring the percentage of GSH in all test groups (Figure 3). Glutathione is a protein which in its reduced form (GSH) plays an important role in antioxidant enzyme and non-enzyme (vitamin A, C and E) system. [29] Another beneficial effect of ESEt found was it did not disturb cellular integrity of liver and cardiac tissues by observing the normal serum levels of liver- and cardiac-specific enzymes viz., ALT and CK respectively in test groups as compared to hyperlipidemic control groups (Table 2). This finding is also compatible to results that showed that overnight fasted rabbits were easily ingested doses of ESEt from mg/kg without showing any sign of acute toxicity like dullness, ruffled hair, depression, clumping together, itching, hyperactivity, sedation and mortality (death). Therefore, ESEt could be targeted to isolate active principle that actually involved in improving the lipid profile of hyperlipidemic experimental animals. CONCLUSION. Table 1: Effect of ESEt on AAI, Lipase and HMG-CoA / Mevalonate ratio The results conclude that ESEt of C. anthelminticum is a potent antihyperlipidemic and antioxidant agent by significantly normalizing the lipid profile which was altered by HFD in experimental rabbits and elevating the status of antioxidant enzymes. ACKNOWLEDGEMENTS The authors are highly thankful to University of Karachi, Pakistan for providing facilities for conducting this experimental work. We are also grateful to Mr. Ali Zia, Application Specialist, Roche Diagnostic, Pakistan for providing the facility of estimating lipase enzyme. Groups AAI (%) Lipase (U/L) HMG-CoA / Mevalonate Ratio Group I ± 0.15 Group II ± 0.16 Group III ± ± ± 0.23 Group IV ± ± 0.11* Group V * ^ 1.31 ± 0.26* Group VI * ^ 1.50 ± 0.26* Group VII * ± 0.21* Each value is the mean SD (n=6). *=p<0.05, compared with group II and group III. ^=p<0.05, compared with group II. Table 2: Effect of ESEt on CK and ALT Groups CK(U/L) ALT(U/L) Group I Group II Group III ± ± 0.11 Group IV ± Group V Group VI Group VII Each value is the mean SD (n=6)

5 Figure 1: Animal grouping according to the treatments Experimental Rabbits (1 1.7 kg) Group I Normal (control) rabbits (Distilled water 1ml/kg) High-fat diet induced hyperlipidemic rabbits (HFD per kg body weight orally) Group II HFD Control (Distilled water 1ml/kg HFD Test Group III HFD Negative Control (0.05% DMSO 1 ml/kg) Group IV HFD Positive Control (Simvastatin 20 mg/kg) Group V (ESEt 200 mg/kg) Group VI (ESEt 400 mg/kg) Group VII (ESEt 600 mg/kg) Figure 2: Effect of ESEt on lipid profile Each value represents the mean SD (n=6). *=p<0.05, when compared with group II and III

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