Indian Journal of Traditional Knowledge Vol. 16 (2), April 2017, pp. 216-220

Effect of crude extract of asiatica L. on lipid profile and coagulation parameters of rats and mice

Alamgeer*, Raheela Qayyum & Hira Asif Department of Pharmacology, Faculty of Pharmacy, University of Sargodha, Pakistan E-mails: [email protected]; [email protected]

Received 23 June 2016, revised 17 October 2017

The present study was aimed to investigate the effect of aqueous methanol extract of Grewia asiatica L. (500 mg/kg; p.o for 28 days) on lipid profile and coagulation parameters of Sprague Dawley rats and Swiss Albino mice. Lipid profile (triglyceride, cholesterol, high density lipoprotein, low density lipoprotein and very low density lipoprotein in mg/dL) and coagulation parameters (thrombin, prothrombin, activated partial thromboplastin and fibrinogen time in s) were studied on 29th day at the completion of treatment period using standard kits. Aqueous methanol extract at the dose of 500 mg/kg showed significant decrease in serum cholesterol, triglyceride and low density lipoprotein level in both rats and mice as compared to control and hyperlipidemic group. Moreover, there was a significant increase in high density lipoprotein, thrombin, prothrombin, activated partial thromboplastin and fibrinogen time both in rats as well as mice. There was no mortality and sign of toxicity observed in the extract treated animals during the study.

Keywords: Grewia asiatica L., Lipid profile, Coagulation parameters. IPC Int.Cl.8: A61K 36/00, G01N 33/48, A01D 14/00, A61B 5/15

The recent few decades have witnessed a renewed diseases. Hypercholesterolemia leads to endothelial interest in the search of new therapeutic agents derived dysfunction, which is an early stage of atherogenesis3. from natural source due to their low cost and minimum There is a strong associations between serum side effects1. Indigenous have been extensively cholesterol and risks of Coronary heart disease (CHD). used to treat various cardiovascular disorders for over a Large epidemiologic studies have demonstrated that millennium in developing countries, the reason of this subjects with hypertension have a marked increase could be attributed to lack of modern health facilities in the prevalence of hypercholesterolemia, diabetes, and rising cost of synthetic agents. Despite the great hypomagnesemia, hypertriglyceridemia5. Lipids are major effectiveness of medicinal plants, there are few reports cell membrane components essential for various describing pharmacological effects and their underlying biological functions including cell growth and division of mechanism of action. Therefore, pharmacological normal and malignant tissues. They are homogeneous validation of medicinal plants could greatly benefit group of compounds related more by physical than 6 in the development of new therapeutic agents. chemical properties . Lipoprotein disorders or Grewia asiatica L. is a of plain areas bearing in hyperlipidemia may result from a primary abnormality in summer. Locally the bark of this is used to treat lipid metabolism or is a secondary manifestation of 5 trouble and burning in vagina and its fruit is used for its some other condition . Saturated fats have a profound cooling and digestible effect. It cures inflammation, hypercholesterolemic effect, being a major risk factor for heart and blood disorders and trouble of throat2. Its atherosclerosis and related occlusive vascular diseases. are reported to have good antibacterial, antiviral, The normal level of blood cholesterol in human beings is and antifungal activity3 and fruit has radio protective 150 - 250 mg/dL but when its level rises to more than effect on Swiss albino mice against radiation induced 250 mg/dL, it may lead to dangerous consequences hematological and biochemical alterations4. like cardiovascular diseases. Increased serum cholesterol levels can be induced when animal fats (saturated fats) Many studies have confirmed the relation between 7 hypertension, hypercholesterolemia and ischemic heart and food high in cholesterol are consumed . —————— Coagulation system has been intended as a possible *Corresponding author mechanism of thrombogenesis and atherosclerosis in ALAMGEER et al.: EFFECT OF GREWIA ASIATICA ON LIPID PROFILE AND COAGULATION PARAMETERS 217

patients with hyperlipidemia. While hyperlipidemia is administered normal saline through same route a risk factor for ischemic heart disease, suggesting equivalent to the volume of respective doses that hypercoagulability may perform a role in patients according to their body weight. Body weights of the with hyperlipidemia. animals were measured weekly. The present investigation was undertaken to Estimation of lipid profile examine the effect of aqueous methanol extract of After 28 days of treatment blood sample of about Grewia asiatica on lipid profile and blood coagulation 5 mL were collected in gel tube. Serum was parameters of rats and mice. immediately separated out by centrifugation at 3000 Material and methods rpm for 15 min in 14K Humax centrifuge. Lipid profile were analyzed on Humalyzer 3000 (semi- Animal selection automatic chemistry analyzer, Model # 16700) The present study was carried out on male (Human Germany) using standard kits supplied by Sprague-Dawley rats weighing 200 – 250 gm and Human. Cholesterol and low density lipoprotein male Swiss albino mice weighing 30 – 35 gm. The cholesterol (LDL-C) was estimated by CHOD-PAP animals were housed in stainless cages under standard method; triglyceride by GPO-PAP methods9, and high laboratory conditions (light period: 8:00 am to 8:00 density lipoprotein cholesterol (HDL-C) following the pm, 21 ± 2 °C, relative humidity 55 %). Animals were method of Friedewald et al.10. provided with balanced diet and water ad libitum. The study protocol was approved by the Institutional Estimation of coagulation parameters Animal Ethics Committee, University of Sargodha. In Blood samples of about 3 mL were collected in addition, experiments comply with the declarations of coagulation tubes containing 3.2 % sodium citrate. National Research Council 8. Plasma was separated by centrifugation at 3000 rpm for 15 min in 14 K Humax centrifuge. Thrombin time Plant material used (TT), prothrombin time (PT), activated partial The of Grewia asiatica were collected from thromboplastin time (APTT) and fibrinogen time (FT) DI Khan district of Khyber Pakhtoon khwa Pakistan were measured by Humaclot duo, using standard during the month of June, identified and authenticated reagent kits supplied by Human11. by Botanist Dr Sarfaraz Khan Marwat, Assistant Gross toxicities professor Botany at WENSUM College DI Khan. The gross toxicities were observed on weekly basis A voucher specimen has been deposited in the during administration of aqueous methanol extract herbarium, Faculty of Pharmacy, University of obtained from fruits of Grewia asiatica for a period of Sargodha, Sargodha, Pakistan for future reference, 28 days. catalogued under number GA-200E5. Mortality rate Preparation of extract Mortality rates were observed in animals treated The fresh fruit of Grewia asiatica was macerated in with crude extract of Grewia asiatica during the total aqueous methanol solution (30:70) and agitated daily period of experiment. The number of animals died for 72 hrs. The mash was filtered through muslin was also noted. cloth and Whatman filter paper and concentrated in a rotating evaporator under reduced pressure and Results temperature. Estimation of lipid profile Experimental design To evaluate hypolipidemic potential, aqueous Rats and mice were divided into three groups of methanol extract obtained from fruits of Grewia seven animals in each group. Group I served as asiatica was administered in the dose of 500 mg/kg to control group while animals in group II considered as rats and mice for 28 days. Results showed that hyperlipidemic control and received 25 % of fructose crude extract significantly reduced the triglyceride, in drinking water for 28 days. Group III (treatment cholesterol and low density lipoprotein, while level group) received the crude extract of Grewia asiatica of very low density lipoprotein was not changed in dose of 500 mg/kg; p.o of body weight for a period significantly. In addition, significant increase in of 28 days, while animals of control group were level of high density lipoprotein as compare to control 218 INDIAN J TRADIT KNOWLE, VOL. 16, NO. 2, APRIL 2017

group and hyperlipidemic group was also observed. high density lipoprotein was increased significantly As shown in Figs. 1 & 2 Grewia asiatica reduced both in rats and mice that is 41.6 ± 3.1, 38 ± 0.6 as level of triglyceride by 51.7 ± 6.6 as compare to compare to control group that is 21 ± 0.6 and control group that is 96.5 ± 1.4 in rats and also in 36 ± 1.7. However minor decrease in level of VLDL mice that is 86.5 ± 0.3 as compare to control that is was observed that was insignificant statistically. 95 ± 1.2. Cholesterol level and low density lipoprotein is also profoundly reduced that is 88.6 ± Coagulation profile 14.9, 90.5 ± 0.9, as compare to control group that is The effect of aqueous methanol extract of 151 ± 1.7, 90.5 ± 0.9, in rats and also in mice that is Grewia asiatica in the dose of 500 mg/kg on 143 ± 1.2, 86.5 ± 0.3 as compare to control that is coagulation parameters of rats and mice after 28 days 152 ± 1.7,92.5 ± 0.3, 18.5 ± 0.3, while the level of showed that crude extract significantly increases the thrombin (TT), prothrombin (PT), activated partial thromboplastin (aPTT) and fibrinogen (FT) time as compare to control group (Figs. 3 & 4).

Fig.1—Effect of Grewia asiatica on lipid profile of rats after 28 days. where, HDL = Hgh density lipoprotein, LDL = low density lipoprotein and VLDL = very low density lipoprotein, n = 7; *** Fig. 3—Effect of Grewia asiatica on coagulation profile Mean ± S.E.M; = P ˂ 0.001 as compared to control. of rats after 28 days. Where TT = thrombin time, PT = prothrombin time, aPTT = activated partial thromboplastin time and FT = fibrinogen time; n = 7; Mean ± SEM; *** = P˂ 0.001 as compare to control.

Fig. 2—Effect of Grewia asiatica on lipid profile of mice after 28 Fig. 4—Effect of Grewia asiatica on coagulation profile of mice days. where, HDL = Hgh density lipoprotein, LDL = low density after 28 days. Where TT = thrombin time, PT = prothrombin time, lipoprotein and VLDL = very low density lipoprotein, n = 7; a PTT = activated partial thromboplastin time and FT = fibrinogen Mean ± S.E.M; *** = P ˂ 0.001 as compared to control. time; n = 7; Mean ± SEM; *** = P˂ 0.001 as compare to control. ALAMGEER et al.: EFFECT OF GREWIA ASIATICA ON LIPID PROFILE AND COAGULATION PARAMETERS 219

Gross toxicities prevention and control of coronary heart disease. No gross toxicities were observed in any group of Alterations in the lipid profile have also been animals during the total period of experiment. associated with age17. The lowering of LDL-C of the rats and mice may be due to the presence of Mortality rate flavonoids in the extract which prevent the oxidation Result of present study revealed that no death was of LDL-C which is atherogenic. It may be possible observed in the animals of control group and treated that lipid lowering activity of the crude extract of the groups. fresh fruit may be due to the presence of alkaloids, Discussion which are said to be responsible for lowering In recent years, an increasing percentage of people hyperlipidemia. Furthermore the cholesterol lowering from industrialized countries have been using activity of the crude extract may be due to the complementary and alternative medicines (CAM). presence of saponins, which may be present in fresh Herbal medicine is used by up to 80 % of the fruit of plant whichis a cholesterol lowering agent12. population in the developing countries12. Total lipid The HDL was also increased which is one of the most profile of an individual is a contributive principle important criteria of an antihypercholesterolemic resulting from blood cholesterol along with its agent. Previous studies show that high level of HDL associated varieties of lipoproteins, i.e., high-density is associated with a lower incidence of cardiovascular lipoproteins (HDL, or α-lipoproteins), low-density disease. The increase in HDL level might be due to lipoproteins (LDL, or β-lipoproteins), very-low stimulation of pre HDL and reverse cholesterol density lipoproteins (VLDL, or pre-β-lipoproteins) transport. So, it is suggested that lipid lowering effect and triglycerides. Disposition of blood pressure and of crude extract may be due to combination of the coronary heart disease has been found to be in strong enriched phenolic contents in the extract and correlation with lipid profile particularly with blood enhanced bio synthesis of hepatic HDL resulting in cholesterol level and also with coagulation increased plasma HDL concentration18. The process parameters. Previously, it has been reported that of blood coagulation is an important chemical fructose fed hyperlipidemia has been associated with reaction in the body in response to vascular injuries increased availability of precursor molecule glycerol- thrombosis and CVD. 3-phosphate and excess free fatty acids while the The result shows the effect of crude extract of diminished activity of LPL results in increased Grewia asiatica on TT, PT, aPTT, and fibrinogen on content and impaired clearance from circulation. rats and mice. This study indicated the increase in TT Fructose also causes increase in cholesterol synthesis which might be linked with the deficiency of that is in line with a previous findings, addition of fibrinogen or inhibition of thrombin. This may fructose to cultured rat hepatocytes increase HMG- be due to reduced activity of coagulation factors CoA reductase activity 3 folds13,14 . Natural product like IX, X, XI, and XII which are essential medicines from plant sources of wide diversity have for thrombin generation14. Present study also long been used effectively in the treatment of blood showed that the crude extract of Grewia asiatica pressure and hyperlipidemia15. There is a strong significantly increased the fibrinogen time, it means correlation between lipid profile and coagulation that there was decrease in fibrinogen level, which is parameters, so the alterations in lipid levels influence increased in vascular diseases; thus crude extract thrombosis. This modification is due to changing the could be helpful in combating the risk of vascular activity of coagulation proteins, platelets and diseases by decreasing the fibrinogen level. There was fibrinolytic factors16. Results showed the effect of also decrease in PT, so it is possible that this decrease aqueous methanol extract at the dose of 500 mg/kg on in PT may be due to increase production of lipid profile of rats and mice. The result showed that coagulation factors like, V, VII & X11. The the extract significantly reduced serum cholesterol, results of present investigation were in line with triglycerides and LDL-C. High circulating serum the treatment of lipid lowering drugs like statins. cholesterol, low density lipoprotein- cholesterol Which stabilizes atheromatous plaque and has (LDL-C) and serum triglycerides are major risk antithrombotic effects14, so it is possible that crude factors of cardiovascular diseases. The modification extract of Grewia asiatica L. also produce these of lipid profile may be important both in the effects in the similar manner. 220 INDIAN J TRADIT KNOWLE, VOL. 16, NO. 2, APRIL 2017

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