Antihyperlipidemic effect of Pistacia khinjuk

Antihyperlipidemic effect of Pistacia khinjuk

Biomedicine & Pharmacotherapy 96 (2017) 695–699 Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsevi...

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Biomedicine & Pharmacotherapy 96 (2017) 695–699

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Original article

Antihyperlipidemic effect of Pistacia khinjuk a

b,⁎

MARK

c

a

Faiza Kamal , Muhammad Shahzad , Tanveer Ahmad , Zaheer Ahmed , Rasool Bakhash Tareend, Rabiya Naza, Ali Ahmade a

Department of Home and Health Sciences, Allama Iqbal Open University Islamabad, Pakistan Department of Pharmacology, University of Health Sciences Lahore, Pakistan Arid Agriculture University, Rawalpindi, Pakistan d Department of Botany, University of Baluchistan, Quetta, Pakistan e Experimental Animal Laboratory, University of Health Sciences Lahore, Pakistan b c

A R T I C L E I N F O

A B S T R A C T

Keywords: Antihyperlipidemic effect Pistacia khinjuk Body weight and proximate analysis

Background: Hyperlipidemia is a metabolic disorder that is caused by high levels of triglycerides and cholesterol in blood. Using of plants for the treatment of various ailments is a traditional practice in developing countries. The main objective of the study is to analyse the nutritional composition of Pistacia khinjuk and to assess its antihyperlipidemic activity. Materials and methods: In the present study, the ethanolic extract of Pistacia khinjuk leaves was evaluated for antihyperlipidemic activity. 40 adult albino rats were divided into five groups. Hyperlipidemia was induced using high fat diet for 60 days. Pistacia khinjuk extract of 150 mg/kg body weight and 300 mg/kg of body weight was administered to the rats for 28 days. Proximate analysis of Pistacia khinjuk was also performed. Results: Proximate analysis of Pistacia khinjuk leaves showed 6.8% ash content, 12.8% crude fiber and only 6.85% fat content. Findings of present study revealed that both doses of Pistacia khinjuk extract improved the serum lipid profile in albino rats by reducing total lipids, total cholesterol, triglycerides and low density lipoprotein, and increasing high density lipoprotein levels in Pistacia khinjuk treated groups. Body weight findings showed significant increase in body weight of all groups from zero day to 60th day. But before sacrifice, there was a decrease in hyperlipidemic and high dose Pistacia khinjuk while increase in control and low dose Pistacia khinjuk group respectively. Conclusion: From the results of the present study, it is concluded that Pistacia khinjuk extract has curative effect against hyperlipidemia.

1. Introduction Obesity and dyslipidemia is considered as one of the contributing risk factors in the prevalence of coronary heart diseases. It is considered as the main cause of morbidity and mortality nationwide as well as worldwide. It is estimated that increased levels of cholesterol may cause 29.7 million disability adjusted life years and 2.6 million deaths. Ratio of hyperlipidemia in women and men is 40:37 with increased risk of vascular diseases like myocardial infarction and cerebrovascular accident. Prevalence of hyperlipidemia in developed countries, developing countries and worldwide is 51%, 26% and 39% respectively. The principal target of treatment is to lessen the rate of cerebrovascular and cardiovascular diseases [1]. The elevated serum level of triglycerides, cholesterol and LDL are major risk factors for the premature development of cardiovascular diseases like arthrosclerosis, hypertension, coronary heart disease etc. ⁎

Increased plasma lipid levels mainly total cholesterol; triglycerides and LDL along with decrease in HDL are known to cause hyperlipidemia which is the reason for initiation and progression of atherosclerosis process [2]. Dyslipidemia could be primary or secondary. Primary dyslipidemia could be treated with antilipidemic drugs and secondary dyslipidemia that is originating from diabetes, renal diseases and hypothyroidism needs the treatment of the original disease rather than treating hyperlipidemia. Now a days, already existing hypolipidemic drugs have been associated with a huge number of side effects like hyperuricemia, diarrhea, nausea, myositis, gastric irritation, flushing, dry skin and abnormal liver function [3]. Extracts of different plants have been widely used as an effective therapy to treat many health ailments. In the field of natural sciences, medicinal plants have been used as therapeutic agents due to their wider use as in pharmacological and natural products and as herbal

Corresponding author. E-mail address: [email protected] (M. Shahzad).

http://dx.doi.org/10.1016/j.biopha.2017.10.061 Received 4 July 2017; Received in revised form 27 September 2017; Accepted 11 October 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.

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Group IV (Test group B). Rats were administered with high fat diet and treated with ethanolic extract of 300 mg/kg body weight orally for 28days. Group V (Standard drug). Rats were administered with Simvastatin 1 mg/kg body weight intraperitoneally for 28 days [8].

remedies. With time increasing concern of health and nutrition encourages the use of medicinal plants [4]. These plants gained popularity among researchers, academician and clinicians for the management of chronic diseases. Using of medicinal plants as antihyperlipidemic drugs gained much more importance these days as an alternative therapy. Medicinal plants lipid lowering activities are mediated by inhibiting hepatic cholesterol biosynthesis and reducing lipid absorption in the intestine [3]. Pistacia khinjuk is a classic plant; it has been used in managing problems like indigestion and toothache. Leaves of Pistacia khinjuk were found to be rich in essential oil, consisting terpene hydrocarbons [5]. Traditionally the species of Pistacia showed anti-atherogenic, anti-inflammatory, gastrointestinal, antioxidant, antimicrobial and hypoglycemic effects [6]. Other studies stated that some species have diuretic properties and as an antiseptic in treating urinary and respiratory infections [7]. Antioxidant activity (flavonoids and phenolic compounds) of Pistacia species has shown protective effect against cancers by delaying tumor growth and blocking cell proliferation. In cardiovascular diseases same effect helps to regress aortic surface lesions [6]. The main aim of the study is to assess antihyperlipidemic effect of pistacia khinjuk in albino rats.

2.5. Composition of high fat diet The composition of hypercholesterolemia diet was cholesterol (1%), cholic acid (0.5%), casein (20%), choline (0.25%), d-L-methionine (0.4%), coconut oil (25%), multi vitamin mix (3.5%) and sucrose (48.4%) [9]. 2.6. Proximate analysis In the first step, proximate analysis was performed to find out the nutritional composition of the Pistacia khinjuk. This included the assessment of ash, crude lipid and crude fiber [10]. 2.7. Acute toxicity of Pistacia khinjuk Acute toxicity of Pistacia khinjuk was measured by giving different doses of extract to observe mortality, behavior changes, eating habits and weight loss. Adult healthy male rats were divided into four groups (n = 4). The plant extract at doses of 500, 1000, 2000 and 5000 mg/kg body weight was administered orally by gavage to the animals of all the groups respectively. The rats were observed closely for the first 4 h and then after every 4 h for next 48 h of extract administration to see any behavioral changes, toxicity symptoms and mortality.

2. Materials and methods 2.1. Study design and setting of the study It was an experimental study to assess the antihyperlipidemic effect of Pistacia khinjuk. Research was carried out in an experimental research laboratory and Department of Pharmacology, University of Health Sciences, Lahore. It was approved by the institutional ethical review committee of UHS, Lahore and the approved number is UHS/ Pharm/9423. It was performed according to the international guidelines of performing studies on animals.

2.8. Lipid profile test Serum total cholesterol, triglycerides, HDL, and LDL levels were estimated by using chemistry analyzer (Rx Monza made in UK), with commercial kits of Randox. On the 90th day of the experiment blood samples were collected by intracardiac puncture and all the animals were sacrificed. Blood sample were collected separately in sterilized tubes and allowed to stand for 30 min at 20–25 °C, and centrifuged to separate serum at 4000 rpm for 20 min using centrifuge [11].

2.2. Collection of plant material and extraction method The leaves of Pistacia khinjuk were shade-dried and ground with an electric grinder into coarse powder. The powdered material was then extracted with 95% ethanol at room temperature for 7 days with occasional shaking and stirring. The extract was filtered through cotton filter and then the filtrate was concentrated with a rotary evaporator under reduced temperature and pressure to get brownish mass.

2.9. Statistical analysis Data were presented as Means ± SD and analyzed using analysis of variance technique (ANOVA) followed by Post hoc Tukey’s test through SPSS (Statistical Package for Social Sciences) version 18 and GraphPad version 6.

2.3. Animal selection, housing and experimental layout Total 40 healthy adult male albino rats of 8-week age were selected and kept in the animal house of the University of Health Sciences Lahore. The animals were housed under standard conditions (12 h light and dark cycles, at 25 °C temperature and 35–60% humidity) and fed with high cholesterol diet for 90 days. To confirm the induction of hyperlipidemia in rats, blood samples were collected by intracardiac puncture. The rats then were randomly divided into five groups and treatments were administered orally, once daily for 28 days.

3. Results 3.1. Proximate analysis Proximate analysis of Pistacia khinjuk leaves was done along with diet given to induce hyperlipidemia. Results showed that normal diet that is given to control group includes 10.6% fat and high fat diet contains 19% fat. Hence it showed 9% difference in fat content. Crude fiber and ash content showed same findings in both feeds as 2.2% and 2.3% crude fiber and 3.3% and 3.0% ash content in normal and high fat diet respectively. Pistacia khinjuk leaves showed 6.8% ash content, 12.8% crude fiber and only 6.85% fat content.

2.4. Drugs and dosage

• Ethanolic extract of Pistacia khinjuk, 150 mg/ kg body weight and 300 mg/kg body b.w. • Simvastatin 1 mg/kg b.w. [8]. Group I (Control group). The rats were fed with standard rat chow. Group II (Hyperlipidemic control group). The rats were administered with high fat diet for 90 days. Group III (Test group A). Animals were administered with high fat diet and treated with ethanolic extract of 150 mg/kg body weight orally for 28 days.

3.2. Acute toxicity Acute toxicity was assessed by giving four different doses of extract (500, 1000, 2000 and 5000 mg/kg body weight). Extract was given orally by gavage and weights were measured before administering extract and after 48 h. Observations were made at different intervals like 696

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Table 1 Change in body weight with different doses of Pistacia Khinjuk extract at day zero and after 48 h. Mean ± SD of weight (gm) in acute toxicity (n = 4). Pistacia khinjuk extract doses

Day zero

After 48 h

5000 mg/kg b.w. 2000 md/kg b.w. 1000 mg/ kg b.w. 500 mg/ kg b.w. Control

360.0 ± 33.04 351.33 ± 34.19 300.66 ± 31.89 297.33 ± 20.52 195.66 ± 4.04

350.66 347.33 279.33 274.00 201.66

± ± ± ± ±

P-value 26.56 37.54 25.16 11.13 3.51

0.401 0.321 0.040* 0.051 0.009*

* P value ≤0.05.

30 min, 2, 4, 12, 18, 24 and 48 h. Changes in their skin or fur, eating, sleeping pattern, diarrhea, convulsion and tremors along with mortality was observed. No significant findings were observed in all four groups. Hence it was found that the dose of 5000 mg/kg of body weight extract was considered to be safe. The mean weight of rats before administering 5000 mg/kg b.w. and 2000 mg/ kg b.w. extracts of Pistacia khinjuk was 360.0 ± 33.04 g and 351.33 ± 34.19 g; after extract was administered the mean weight was 350.66 ± 26.56 g and 347.33 ± 37.54 g respectively, which is statistically not significant. The mean weight of rats before administering 1000 mg/kg b.w. extract of Pistacia khinjuk was 300.66 ± 31.89 g, after extract was administered the mean weight was 279.33 ± 25.16 g which is statistically significant. The mean weight of rats before administering 500 mg/kg b.w. extract of Pistacia khinjuk was 297.33 ± 20.52 g; after extract was administered the mean weight was 274.00 ± 11.13 g, which is numerically significant but statistically not significant. The mean weight of control group before and after 48 h was 195.66 ± 4.04 g and 201.66 ± 3.51 g respectively, which is statistically significant only with 1000 mg/kg b.w. extract of Pistacia khinjuk (Table 1).

Fig. 1. Effect of Low Dose, High Dose Pistacia Khinjuk extract on total cholesterol in comparison with high fat diet and standard drug group. ^ shows P value < 0.001 indicating significant difference compared to control group. * shows P value < 0.05, ** P value < 0.01 and *** P value < 0.001 indicating significant difference compared to high fat diet group.

3.3. Effect of low dose, high dose Pistacia Khinjuk extract on lipid profile of hyperlipidemic rats (n = 6)

Fig. 2. Effect of low dose, high dose Pistacia Khinjuk extract on triglycerides in comparison with high fat diet and standard drug group. ^^^^ shows P value < 0.001 indicating significant difference compared to control group. *** indicates P value < 0.001 showing significant difference compared to high fat diet group.

3.3.1. Effect of Pistacia khinjuk on total cholesterol Our findings showed a significant increase in the levels of total cholesterol in hyperlipidemic group as compared to control group (194.3 ± 23.36 vs 88.83 ± 26.01 P < 0.001). Both low dose and high dose treatment of Pistacia khinjuk showed a significant decrease in total cholesterol levels as compared to hyperlipidemic group (134.7 ± 32.22 vs 194.3 ± 23.36 P < 0.01) and (115.7 ± 19.81vs 194.3 ± 23.36 P < 0.001) respectively. There was no significant difference found in the cholesterol lowering effect of low dose and high dose treatment of Pistacia khinjuk when compared with each other (134.7 ± 32.22 vs 115.7 ± 19.81 P > 0.05). Simvastatin showed significant difference in total cholesterol when compared with hyperlipidemic group (58.33 ± 3.98 vs 194.3 ± 23.36 P > 0.05) (Fig. 1).

3.3.3. Effect of Pistacia khinjuk on HDL levels Our findings showed a significant decrease in the HDL levels in hyperlipidemic group as compared to control group (38.50 ± 10.82 vs 96.33 ± 18.42 P < 0.001). Both low dose and high dose treatment of Pistacia khinjuk showed a significant increase in HDL levels as compared to hyperlipidemic group (69.17 ± 8.704 vs 38.50 ± 10.82 P < 0.01) and (77.33 ± 20.94 vs 38.50 ± 10.82 P < 0.001) respectively. There was no significant difference in the HDL levels, lowering effect of low dose and high dose treatment of Pistacia khinjuk when compared with each other (69.17 ± 8.704 vs 77.33 ± 20.94 P > 0.05). Simvastatin showed significant difference in HDL level when compared with hyperlipidemic group (50.00 ± 8.944 vs 38.50 ± 10.82 P > 0.05) (Fig. 3).

3.3.2. Effect of Pistacia khinjuk on triglycerides Our results showed a significant increase in the levels of triglycerides in hyperlipidemic group as compared to control group (433.2 ± 60.22 vs 192.7 ± 42.88 P < 0.001). Both low dose and high dose treatment of Pistacia khinjuk showed a significant decrease in triglycerides levels as compared to hyperlipidemic group (273.5 ± 56.42 vs 433.2 ± 60.22 P < 0.01) and (249.8 ± 53.92 vs 433.2 ± 60.22 P < 0.001) respectively. There was no significant difference found in the triglycerides lowering effect of low dose and high dose treatment of Pistacia khinjuk when compared with each other (273.5 ± 56.42 vs 249.8 ± 53.92 P > 0.05). Simvastatin showed significant difference in triglycerides when compared with hyperlipidemic group (166.3 ± 23.52 vs 433.2 ± 60.22 P > 0.05) (Fig. 2).

3.3.4. Effect of Pistacia khinjuk on LDL levels Our findings showed a significant increase in the LDL levels in hyperlipidemic group as compared to control group (159.5 ± 30.08 vs 66.03 ± 18.36 P < 0.001). Both low dose and high dose treatment of Pistacia khinjuk showed a significant decrease in LDL levels as compared to hyperlipidemic group (78.53 ± 23.75 vs 159.5 ± 30.08 P < 0.01) and (67.70 ± 8.880 vs 159.5 ± 30.08 P < 0.001) respectively. There was no significant difference found in the cholesterol lowering effect of low dose and high dose treatment of Pistacia khinjuk when compared with each other (78.53 ± 23.75 vs 67.70 ± 8.880 P > 0.05). Simvastatin showed significant difference in LDL level 697

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Fig. 3. Effect of low dose, high dose Pistacia Khinjuk extract on HDL levels in comparison with high fat diet and standard drug group. ^^^ shows P value < 0.001 indicating significant difference compared to control group. ** indicates P value < 0.01 showing significant difference compared to high fat diet group.

Fig. 5. Effect of body weight change at day 60 in control, hyperlipidemic, low dose Pistacia Khinjuk and standard drug group, high dose Pistacia Khinjuk and standard drug group. *** indicates P value < 0.001 showing significant difference as compared to control group at day 60.

Fig. 6. Effect of body weight change before sacrifice in control, hyperlipidemic, low dose Pistacia Khinjuk and standard drug group, high dose Pistacia Khinjuk and standard drug group. Body weight at sacrifice day shows no significant difference in all groups.

Fig. 4. Effect of low dose, high dose Pistacia Khinjuk extract on LDL levels in comparison with high fat diet and standard drug group. ^^^ shows P value < 0.001 indicating significant difference compared to control group. *** indicates P value < 0.001 showing significant difference compared to high fat diet group.

when compared with hyperlipidemic 159.5 ± 30.08 P > 0.05) (Fig. 4).

group

4. Discussion In last two decades, worldwide, number of patients with morbid obesity has been doubled. Out of total diabetic population 44% belongs to the obese and overweight group. On the other hand, majority of CVD patients are far away from normal BMI limits; more than 23% fall in overweight to obese category. If we consider obesity alone, it is responsible for 2.3 million deaths in adults. Situation is equally worse in case of childhood obesity where in 2010, 43 million children under 5 years of age were found overweight. Two third of the world’s population is inhabitant of those areas where obesity is the major cause of morbidity and mortality [12]. In spite of the remarkable progress achieved through prevention, cardiovascular diseases still remain a major cause of mortality in men and women. Hyperlipidemia needs interventions as it is considered one of the major contributing causes of premature mortality and morbidity with an increase in health expenditure. Large epidemiological studies and clinical trials showed reduction in cardiovascular disease by using dietary intervention and herbal medicines [13]. In this study, we determined the lipid lowering effect of Pistacia khinjuk in albino rats and results indicated there was a noteworthy decrease in LDL levels as compared to hyperlipidemic group. On the other hand, both low dose and high dose treatment of Pistacia khinjuk resulted in lowering the cholesterol level. Similarly, a significant decrease in triglycerides levels and HDL levels was observed. No

(44.17 ± 10.11vs

3.4. Body weight change in control, hyperlipidemic, low dose Pistacia khinjuk and standard drug group, high dose Pistacia khinjuk and standard drug group (n = 6) 3.4.1. Body weight change in all groups At day zero, the mean body weight of control, hyperlipidemic, low dose Pistacia khinjuk, high dose Pistacia khinjuk and standard drug was 181.2 ± 12.92, 183.9 ± 13.78, 221.7 ± 20.72, 218.2 ± 25.11 and 208 ± 20.06 respectively. At day 60, there was significant increase in body weight of all control, hyperlipidemic, low dose Pistacia khinjuk and high dose Pistacia khinjuk groups as 288.3 ± 26.8, 312.6 ± 33.26, 332.5 ± 35.42, 351.0 ± 18.96. Before sacrifice, there was a decrease in hyperlipidemic and high dose Pistacia khinjuk and 310.6 ± 27.90, 345.0 ± 46.47 while there was increase in control and low dose Pistacia khinjuk group 317.3 ± 23.44, 345.0 ± 46.47 respectively (Figs. 5 and 6).

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References

substantial transformations were found in the effect of low and high doses of Pistacia khinjuk. These results are similar to the study conducted on rabbits, using the Pistacia terebinthus L. to analyse its possible pharmacological effects and showed that Pistacia terebinthus L. had a hypolipidemic effect in hypercholesterolemic rabbits, also restraining the progression of the atherosclerotic lesions in the thoracic artery [14]. In another study, Pistacia lentiscus fatty oil and simvastatin were given to hyperlipidemic rabbits and results showed significant decrease in total cholesterol, LDL and triglycerides; however decrease in HDL levels was also observed [15]. Yet another recent study, conducted on 32 normo-lipidemic healthy young men, fed with a pistachio diet showed encouraging lipid lowering effects along with improving the blood glucose level [16]. A study published in 2002 showed the lipid lowering effect of simvastatin, as simvastatins are widely used in treating hypercholesterolemia and atherosclerosis diseases. Results of the study indicate that continuous dose of lipid lowering agents are associated with significant reversal of atherosclerotic lesions in humans. Our findings showed same significance, as total cholesterol, triglycerides and LDL levels were significantly abrogated [17]. Body weight results of our study showed increase in the body weight after 60 days but a decrease was observed before sacrifice, which is in contrast with the study conducted by Katerina et al., percent change in body weight of control group was 9.71%–8.99%, in methanolic group 5.05%–9.47% and in cyclohexane group 7.74%–10.76% [14]. Nutritive value of Pistacia khinjuk seeds from three different climates was determined by using 3 samples. Gross chemical composition includes dry matter, ash, crude fiber, crude fat, crude protein, nitrogenfree extract and starch, macro & micro – element content, essential and non-essential amino acids, fatty acid composition. Mean value of Pistacia khinjuk dry matter was 95.9 ± 0.28%, 9.15 ± 0.64% crude protein, 39.1 ± 1.56% crude fat content, 22.85 ± 0.21% crude fiber content, 2.50% crude ash content, 22.3 ± 0.85% nitrogen free extract and 4.4% starch. Whereas, in our study Pistacia khinjuk leaves showed 6.8% ash content, 12.8% crude fiber and only 6.85% fat content [10].

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5. Conclusion Our study reported that Pistacia khinjuk extract possesses lipid lowering activity but it requires further studies to identify and isolate the active compound/s of Pistcia khinjuk. Competing interests All authors declare that they have no competing interests Funding We received no grant for this study

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