Antihyperlipidemic Effects of Methanol Extract of Seeds of Citrullus lanatus in Hyperlipidemic Rats

It has been considered that natural drugs are free from adverse effects and are less toxic than the synthetic ones. The practitioners of traditional medicine prescribe these drugs even though active compounds of many herbs are not known. These drugs have been widely prescribed due to their low cost and less adverse effects (Valiathan, 1998). The synthetic drugs are known to cause several side effects, i.e., elevated level of liver enzymes, dry skin, flushing, gastric irritation, myositis, nausea, diarrhoea and hyperuricemia (Kumar et al., 2008). Medicinal plants play a significant role in lowering the lipid levels by inhibiting the biosynthesis of cholesterol and the reduction of absorption of lipids in the intestine (Gramza and Korezak, 2005). Many individuals who suffer from hyperlipidaemia use plants as folk medicines for the treatment of hyperlipidaemia in developing countries. A vast number of medicinal plants are being evaluated for anti-hyperlipidemicactivity (Calixto, 2005).Hyperlipidaemia, a condition shown by an increase in the quantity of atherogenic lipoproteins i.e., total cholesterol (TC), triglycerides (TG), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) with a decrease in the quantity of anti-atherogenic lipoprotein i.e., high density lipoprotein (HDL). The main predisposing aspect is hyperlipidaemia which is responsible for developing atherosclerosis (Saravanan et al., 2003). Worldwide, about 17 million deaths are caused due to hyperlipidaemia every year (Mendis et al., 2011). Atherosclerosis is a condition characterized by chronic inflammatory disease of arterial network. It is prompted by numerous factors with strong involvement of lipid peroxidation related to endothelial damage. Invasion of LDL by intima layer increases due to endothelial damage, leading to oxidation of LDL and formation of plaques (Li et al., 2014).

Citrulus lanata is a well-known medicinal plant. Seeds of this plant have shown anti-laxative (Sharma et al., 2011), anti-inflammatory, anti-giardia (Hassan et al., 2011), analgesic (Hassan et al., 2011), anti-oxidant (Singh et al., 2011), anti-microbial (Hassan et al., 2011) and anti-ulcer (Lucky et al., 2012) activities. Traditionally this plant is being used as vermifuge, demulcent, diuretic, hypotensive, tonic, burns and to cure bed wetting (Hassan et al., 2011). Phytochemistry showed the presence of different phytochemicals in the methanol extract of seeds of this plant e.g., alkaloids, flavonoids, saponins, glycosides and tannins. The analytical study revealed the occurrence of calcium, sodium, magnesium and zinc. Proximate analytical study has shown high concentration of fat, carbohydrates and protein (Lucky et al., 2012).

No previous investigation has been carried out to assess anti-hyperlipidemic effect of the seeds of this plant in Triton X-100 induced hyperlipidaemia rat model. So, the objective of present investigation was to evaluate antihyperlipidemic effects of the seeds of this plant in hyperlipidemic albino rats.

Seeds of Citrullus lanatus were purchased from Gulberg, Lahore. Identification and authentication of the plant was done by Prof. Dr. Zaheer-ud-Din Khan, Botany Department, G.C University, Lahore. A voucher specimen with number (GC. Herb. Bot. 3383) was deposited in the herbarium of University.

Seeds of the plant were washed, air dried, chopped and powdered with herbal grinder. The plant material was placed in well closed container and stored in the refrigerator at 4°C before use. 500 g of the powdered seeds was added to 2100 ml of methanol (95%) and the extraction was processed in hot Soxhlet apparatus at 70°C for 72 h. The extract was dried by a rotary evaporator (Heidolph, Laborata 4002, Germany) under reduced pressure at 60˚C. Methanol extract was stored in sample bottles at 0 - 4 ˚C for further studies (Sathya and Shoba, 2014).

All the animal experimental methods were approved by the Animal Ethical Committee (AEC) of The University of Lahore (Approval number: IREC-2019-79, 28/03/2019). The animals were purchased from Post Graduate Medical Institute and were placed in the animal house, Faculty of Pharmacy, The University of Lahore. The rats were placed in polyacrylic cages and were maintained at the temperature 25±1°C under the standard laboratory conditions for 20 days. The rats were permitted to free access of water and the standard dry pellet diet ad libitum and acclimatized at the laboratory conditions before the start of experiment.

Toxicological studies of methanol extract (5000 mg/kg) of the plant were done as per OECD (Organization for Economic Co-operation Development) guidelines No. 425 (OECD, 2008).

The standard pellet diet was mixed with egg yolks and butter and given to the groups (B, C and D) daily for the development of hyperlipidaemia. Group A was kept on normal standard diet. The solution of Triton X-100 was freshly prepared in the normal saline solution and was given through i.p. injection at the dose 100 mg/kg to groups B, C and D after fasting for 18 h.

The study was done using 20 male Wistar albino rats weighing 180-250 g. After 72 h of i.p. injection of Triton X-100 to all groups except group A. The rats were randomly distributed into four groups (n=5) labelled as Group A, B, C and D. Group A was kept as normal control while group B was kept without treatment, group C was treated with the standard simvastatin (10 mg/kg O.D) and group D was treated with the methanol extract of seeds of the plant (800 mg/kg) orally for 15 days. The amount of seed extract for every rat was calculated based on body weight. The suspension of the plant extract was administered by oral route to every rat by the use of a stomach tube with disposable syringe. Detail of the groups was as follow: Group A, normal control (NC) administered with dry pellet diet, normal saline and fresh water by oral route during treatment. Group B, hyperlipidaemia control (HC) in which Hyperlipidaemia was induced by high cholesterol diet with 100 mg/kg intraperitoneal injection of Triton X-100 (Sudha et al., 2011). This groups was administered with the normal saline by oral route during the treatment. Group C, hyperlipidaemia standard (H-S): This group was administered with Simvastatin (10 mg/kg) O.D. after 72 h of induction of hyperlipidaemia for 7 days (Shinde et al., 2013) and Group D, hyperlipidaemia C. lanatus (H-CL): This group was administered with methanol extract of seeds of the plant (800 mg/kg) orally for 7 days (Lucky et al., 2012).

Finally, the samples of blood were taken by the cardiac puncture and were taken into the gel clot activator tubes to separate serum. The liver and heart tissues were placed into 100 mL beakers containing formalin (10%) and stored at temperature of -20°C for further histopathological studies.

TC, TG, HDL-C, ALT, AST levels were determined using respective assay kits of Chemistry Analyzer Cobas C111 (Roche Diagnostics).

The sample was pipetted into a prepared Cobas sample tube (RD standard false bottom tube) and placed in Cobas C111 analyzer. The desired test was selected in the user menu and the measurement was started. The measurement was carried out automatically. Cholesterol level was measured using CHOD-PAP method. Applying Friedewald’s formula LDL and VLDL levels were calculated as follows:

LDL= TC-(HDL-VLDL) and VLDL= TG/5 respectively (Friedewald et al., 1972) while Non-HDL was determined using the following formula (Non HDL-C = TC-HDL) (Ridker et al., 2005).

Tissue samples were collected for histopathological examination. The tissues were passed through various standard techniques like fixation, washing, dehydration, clearing, placed, sectioning and staining (Carleton et al., 1980; Winsor, 1998). Fixation of tissues was done by chemical fixation procedure using aqueous solution of formaldehyde (10%) at normal pH. Dehydration and clearing process was done using xylene. The tissues were placed in paraffin. Using microtome tissue sections of 5-8 µm thickness were cut and floated on water.

At the end, the slides with various sections were placed in the incubator at temperature 37°C for about 3 h for further adhesion. The histological sections were stained with eosin and haematoxylin (Winsor, 1998) and then examined under light microscope (Leitz, Wetzle, Germany).

The results were presented as mean±SEM. The statistical analysis of data was done by one way analysis of variance (ANOVA) which is followed by Turkey’s Multiple Comparison as post hoc test using Graph Pad Prism version 5. The value at P < 0.05 was considered significant statistically.

Intraperitoneal (i.p.) administration of methanol extract (5000 mg/kg b.w.) of the plant seeds caused no death in 2 stages of the test. Thus i.p. LD50 of methanol extract was estimated to be equal to or greater than 5000 mg/kg b. w. (Omoboyowa and Ajayi, 2016).

The effect of plant seed extract, administered for 15 days, on serum cholesterol, triglycerides, LDL-C, HDL-C and VLDL-C (mg/dl ± SEM) in hyperlipidaemia induced rats is shown in Table I. The levels of total cholesterol, total glycerides, LDL-C, non HDL-C, VLDL-C and ALT in serum of group D treated with methanol extract were found to be significantly (P < 0.05) decreased as compared to group A. The serum levels of HDL and AST in group D not significantly (P > 0.05) different from those of group A.

Normal configuration of liver in control rats is shown in Figure 1A. Severe congestion of blood vessels, necrotic foci along with fatty changes of hepatocytes resulted in shrunken sinusoids in Triton X-100 treated rats (Fig. 1B). Hepatocytes of zone 1 are normal but of zone 3 had undergone mild fatty changes due to the accumulation of fat droplets in rats treated with simvastatin (Fig. 1C). The seed extract administered rats depicted mild congestion, accumulation of some inflammatory cells in portal areas but no hyperlipidaemia signs are seen in Figure 1D.

Table I. Effect of C. lanatus seeds on serum cholesterol, triglyceride, HDL-C, Non-HDL-C, LDL-C, VLDL-C, ALT and AST levels in triton X-100 induced rats after 15 days of methanol extract administration.

The values are expressed as mean±SEM (n=3). Both significance (P<0.05) and non-significance (P>0.05) results were obtained when compared to normal control. NC, normal control; HC, hyperlipidaemia control; H-S, hyperlipidaemia standard; H-CL, hyperlipidaemia C. lanatus; TC, total cholesterol; TG, triglyceride; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; VLDL-C, very low density lipoprotein cholesterol; ALT, alanine aminotransferase; AST, aspartate transaminase.

Severe hyperlipidaemia condition e.g., accumulation of lipid particles and severe congestion of blood vessels are seen in the hearts of Group B rats (Fig. 2B) as compared to group A control rats (Fig. 2A). Group C treated with simvastatin shows severe congestion of blood vessels with mild hyperlipidaemia condition. Histological structure of myocardium shows lipids droplets in cardiac muscle fibres (Fig. 2C). The group D treated with the plant had mild congestion of blood vessels and no hyperlipidaemia condition. No evidence of the accumulation of lipid droplet is found (Fig. 2D).

In this study, seeds extract of C. lanatus caused a significant improvement in lipid profile, ALT and AST levels as well as histological features of liver and heart in hyperlipidaemia induced rats. Hyperlipidaemia is the most vital risk factor of atherosclerosis, manifested by elevated plasma levels of lipid profile (TC, TG, non-HDL, LDL and VLDL) accompanied with a decline in levels of HDL (Koba and Hirano, 2011). Lipid profile was kept to be high in hyperlipidaemia untreated group while improvements were seen in the simvastatin and the seed extract treated groups. The abnormal lipid profile is the indicator of hyperlipidaemia where liver and small intestine could be the hypothesized as sites of the effect of plant extract on lipid profile. The improvement of lipids would be compensated by upsurge excretion of total cholesterol. The intestinal antihyperlipidemic effect might be facilitated by obstructing absorption of cholesterol in intestine and eliminating reverse movement of cholesterol, as explained with the standard therapeutic agents like Ezetimibe (Sudhop et al., 2002; Catapano et al., 2014) and bile acid sequestrant, cholestyramine (Davidson et al., 2013; Terunuma et al., 2013).

The administration of methanol extract of the plant led to low concentration of TC which could be due to lower levels of LDL. It might be due to the anti-oxidant effects of the plant (Singh et al., 2011). Our findings are related with the previous studies showing that the fruit juice of this plant decreased LDL and TC levels with significant (P < 0.05) results in Sprague-Dawley rats linked with anti-oxidant effect by the changes in gene expression by lipid metabolism (Hong et al., 2015). The plant extract increased HDL-C levels which indicates its anti-atherogenic activity in hyperlipidaemia condition. Our results are concordant with the earlier research in which the anti-atherosclerotic activity of extract prepared from the plant on LDL-deficient mice model was evaluated in which decreased levels of cholesterol by the plant exhibited its antihyperlipidaemia activity at significant level (Poduri et al., 2013). Initially by treating with the statins was a cause of an increased level of AST and ALT. Treatment with statins was a cause of obesity that is related with fatty liver that caused the elevated levels of ALT (Athyros et al., 2010). The extract of plant led to decreased level of ALT and AST in comparison to the standard simvastatin showing its hepatoprotective activity.

The plant C. lanatus has shown to exert significant antihyperlipideamia effects in hyperlipidaemia rats induced by Triton X-100. This shows the presence of active phytochemicals which are responsible for hyperlipidaemia effect. This plant besides having antihyperlipidaemic effect has fewer side effects of abnormal liver enzymes and weight gain. However, further studies are recommended to investigate the exact mechanism(s) by which the plant shows antihyperlipidaemia effect. There is need for establishing the efficacy and safety for further clinical use. Also, there is the requirement for activity–oriented fractionation to isolate and identify the active constituent(s) of this well-known medicinal plant.

Authors are grateful to Professor Dr. Muhammad Shahzad, Department of Pharmacology, UHS, Lahore for helpful guidance and support.

The authors have declared no conflict of interest.