Journal of Renewable Natural Resources Bhutan ISSN: 1608-4330

Effect of Peganum Harmala L. on lipid Metabolism and Changes Cyp7a1 gene Expression in male Wistar Rat

Nazila Vahidi-eyrisofla1, Sepide Jafarzade Rastin2, Fateme Taghvaei2, Mehdi Ahmadifar3,4, Ali Mohammad Eini5*

1Department of Biology, Damghan Branch, Islamic Azad University, Semnan, Iran 2International campus, shahid Sadoughi University of medical science, Yazd, Iran

3Department of Biology, College of Science, University of Science and Culture, ACECR, Tehran branch, Iran

4Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

corresponding: 5Young Researchers and Elites club, science and research branch, Islamic Azad

University, Tehran, Iran* Academic center for education culture and research, Qom branch, Qom, Iran

ABSTRACT Aims: Hypercholesterolemia is a problem faced by many societies and is a cause of concern for health professionals, since it organizes one of the main risk factors for the increscent of cardiovascular diseases. Dietary factors such as continuous ingestion of high amounts of saturated fats and cholesterol are believed to be directly related to hypercholesterolemia and susceptibility to atherosclerosis hypercholesterolemia animals are useful models for studies on cholesterol homeostasis, and drug trials to better understand the relationship between disorders in cholesterol metabolism, atherosclerosis as well as possible treatments for reduction of circulatory cholesterol levels. The present study was aimed at investigating the effect of Peganum harmala (P.harmala) on lipid metabolism and changes Cyp7a1 gene expression in the diet on hypercholesterolemia in male Wistar Rats. Methods: In the present study the effects of P.harmala on lipid metabolism were investigated in a 28-days feeding trial. Total of 64 Rats (200±15) were divided into six groups: (G1 control, G2) control plus 100 mg/kg, G3) control plus 200 mg/kg, G4) control plus 400 mg/kg, experimental was 8 weeks period. The blood and liver tissue samples for serum lipid profile were collected and Cyp7a1 Gene expression determined. Results: In this study, we further investigated the protective effect of P.harmala on examination parameters in Rat significant differences (P<0.05). Effect of P.harmala with 200, 400 mg/kg dosage on average body weight (g) in treatment group and control group significant differences (P<0.05). Plasma glucose was changed by dietary treatments. P. harmala decreased low density lipoprotein LDL in 200, 400 mg/kg of the extract groups and increased high density lipoprotein cholesterol in 100 mg/kg of the extract groups significant difference (P<0.05), but decreased plasma triglycerides only in 100 mg/kg of the diet group (P<0.05) and on serum VLDL, cholesterol was not significant differences in Rat. The results show that with increasing extract dose, CYP7A1 gene expression is increased significant difference (P<0.05). Conclusions It is concluded that methanolic extract of P. harmala could be effectively used in Rat to optimize serum lipid profile. These results demonstrate that P.harmala increases the effect of excretion cholesterol and Cyp7a1 gene.

Keywords: Peganum harmala L, Lipid metabolism, cholesterol, lipoprotein, Cyp7a1 gene

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INTRODUCTION The hepatocytes are major complex in cholesterol catabolism and metabolism, biliary cholesterol spatter, and transport. Cholesterol homeostasis in the hepatocytes are maintained by modification anew cholesterol metabolism and catabolism (1). 7a-hydroxylase cytochrome P4507A1 (CYP7A1) is the regulator enzyme in the bile acid biosynthetic pathway in the hepatocytes and such regulate cholesterol and bile acid homeostasis. The CYP7A1 in humans is associated with hypercholesterolemia and premature atherosclerosis (1-3). The cholesterol-lowering effect of stimulation of bile acid synthesis has been attributed to increased conversion of cholesterol into bile acids and stimulation of low-density lipoprotein (LDL) receptor–mediated cholesterol uptake into the liver (5). Hypercholesterolemia is a problem faced by many societies and is a cause of concern for health professionals, since it organizes one of the main risk factors for the increscent of cardiovascular diseases (6, 7). Medicinal plants have been used for centuries as remedies for human and animal ailments. They have many pharmacologically active chemical compounds (8), antibacterial (9) and antifungal (10) agents P. harmala belong is to the family of Zygophylaceae and have been shown to possess a diverse range of medicinal properties. Numerous beta Carboline alkaloids like Harmaline, Harmine, Harmalol and Harmol were present in P. harmala extract (11). P.harmala extract exhibited great variety of pharmacological and biological activities such as antibacterial and antifungal agents as well as mono amine oxidase (MAO) inhibition and hypothermia. Similarly analgesic, anti-inflammatory (12), disinfectant (13), growth promoting (14), cholesterol lowering and hepato protective effects (15) have also been reported. Present study was designed to examine the potential benefits of methanolic extract of P. harmala research article in terms of reducing protective effects of P.harmala L. On lipid metabolism extract Harmine and Harmaline against human low density lipoprotein oxidation and also its analgesic effect were studied (16). The principal compounds of this plant are Carboline alkaloids such as Harmine, Harmaline, Harmalol, Harman, Vasicine and Vasicinon (12). However, it has been reported that P. harmala contains some flavonoids (16). P. harmala alkaloids have a wide spectrum of pharmacological action let including plate aggregation inhibition (17), mono amine oxidase inhibition, anxiolytic and behavioral effects, and immune modulator influences (18). There were some reports concerning the cardio vascular actions of alkaloids and antioxidants that reduced systemic arterial blood pressure (19). So effect of antioxidant compounds of this plant (Harmine and Harmaline) has not been investigated thoroughly.

Figure 1. P.harmala L. early May- Month (in flowering time)

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MATERIALS AND METHODS Animals and experimental design. Animals and experimental design. The experiment used 64 male Wistar Rats each weighing approximately 200±15 g that was obtained from Academic Center for Education, Culture and Research (ACECR), Qom, Iran. Animals were divided into eight groups in a controlled environment with 12 h light and dark cycles at 22-25C˚ and humidity 45-50% humidity. The room was lighted from 06:00 to 18:00 hours. Experiments were conducted. In the first experiment: G1) control, G2) control plus 100 mg/kg, G3) control plus 200 mg/kg, G4) control plus 400 mg/kg, experimental was 8 week period, diets and water were provided ad libitum. Each dietary treatment group was composed of eight Rats. Methanolic extraction of P. harmala. P. harmala were collected in the month of May 2013 from desert of Qom city (Figure 1). To obtain methanolic extract 2 kg powder P. harmala of ground was immersed in 10 liters 80 % (v/v) aqueous methanol at room temperature for five days and filtered through Wattmann Filter Paper (No.42). Extraction by Barij esans Co. (Golkaran Ltd.), Kashan City. The extract was then transferred to a glass bottle and stored in refrigerator before use. Sample collection At the end of experiments, animals were fasted for 16 hours and weakly anesthetized with phenobarbital. Then, blood samples were taken by cardiac puncture into Vacutainer tubes (Becton-Dickinson Co., Rutherford, NJ). Immediately, the liver was excised, two washed in saline solution and was kept in at -165C° Assay kits and Biochemical determinations. Plasma glucose concentrations were determined using the Clinical Chemistry system (Pars Azmoon Co, IRAN). Plasma triglyceride was analyzed enzymatically using Tri-Es® (Pars Azmoon Co, IRAN). The separation of plasma lipoproteins and analysis of total plasma cholesterol and each lipoprotein cholesterol were performed using a lipoprotein profiling system (Beckman Instruments, Inc., Fullerton). RNA preparation and Real Time PCR (RT –PCR) Total RNA was prepared from frozen liver extraction of RNA and reverse transcription of RNA to cDNA was performed using RNX-Plus (Cinna gen, Karaj, Iran) and 2-steps RT-PCR kit (Vivantis, UK), respectively, due to manufacturer instructions. Real time-PCR performance using SYBR Green PCR Master Mix (Amplicon) and Rotor-Gene 6000 Series software version 1.7.65 (Corbett Life Science), and primers of each gene were designed as follows utilizing primer 3 program. The heating to 95°C for 15 min., and 40 cycles of elongament at 58°C for 30 sec and denaturation at 95°C for 15 sec. Statistical analysis. Data were showed as mean ± standard error of the mean (SEM). That using one-way ANOVA, followed by post hoc Tukey tests. A significance level was less 0.05 (p<0.05). RESULTS and Discussion

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In this study, we further investigated the protective effect of P.harmala on examination parameters in Rat significant differences (P<0.05) that shown in (Table 1). Effect of P.harmala with 200, 400 mg/kg dosage on average body weight (g) in treatment group and control group Significant differences (P<0.05) (figure 2). Low density lipoprotein (LDL) and High density lipoprotein (HDL) cholesterol significant differences (P<0.05). To our knowledge, no such data has been reported regarding effects of P. harmala on LDL cholesterol, however, effect of other medicinal plants has been reported for LDL decreasing potentials (M Eini, et al, 2014). That were recorded between the control group and treated groups as well as among the treated groups at all recorded stages. There was gradual increase in the HDL level with increasing level of P. harmala at all recorded stages in all groups. However, other medicinal plant shave been explored for their HDL increasing potential (Figure 3). The effect of different levels of P. harmala methanolic extract on serum VLDL, cholesterol was not significant differences in Rat is presented in (Table 1) (Figure 3). Lowering effect of P.harmala L 200,400 mg/kg dosage on triglyceride in treatment group compare by control group significant differences (P<0.05) (figure 3). The results show that with increasing extract dose, CYP7A1 gene expression is increased significant difference (P<0.05) (Figure 4).

Table1. Effect of P.harmala on treatment and control Groups Rat

Treatment groups for 8 weeks, diets and water were provided ad libitum. Each dietary treatment group was composed of eight Rats a) The significant difference (P<0.05).

Figure 2.Effect of P.harmala L on average body weight (g) in treatment and control groups

PARAMETER/GROUP CONTROL CONTROL PLUS

100MG/KG

CONTROL PLUS

200MG/KG

CONTROL PLUS

400MG/KG

Plasma triglyceride 173±17 190±13 164±16a 165.5±8

Plasma glucose 121±14 146±19a 144±8a 137±2a

Plasma cholesterol 119±35 128±13 131±6 126±5

VLDL cholesterol 35.75±4 33±6 33±3 38.37±2

HDL cholesterol 46±3 55±6a 45±2 22±5

LDL cholesterol 32.25±2 31.62±2 26.12±6a 25.12±2a

BODY WEIGHT AFTER 8 WEEKS (G)

272±8 258±6 253±14A

296±7

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G1) control, G2) control plus 100mg/kg, G3) control plus 200mg/kg, G4) control plus 400mg/kg.

Figure3. Effect of P.harmala L on parameters lipid and glucose in treatment and control groups.

G1) control, G2) treatment plus 100mg/kg, G3) treatment plus 200mg/kg, G4) treatment plus 400mg/kg *) significant difference (P<0.05)

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Conclusion The effects of different levels of P. harmala methanolic extract on total cholesterol were presented in (Table 1). Decrease in total cholesterol by P. harmala might be due to the increasing CYP7A1 by different alkaloids Harmine, Harmaline, and Harmol present in P. harmala. These alkaloids have also been reported to have hypoglycemic properties (4), (20). P. harmala has antioxidant property (21) which reduces LDL oxidation and thereby reducing total cholesterol content (22) reported 26-31% reduction of serum cholesterol when supplemented with Trigonella foenumgraecum on mice (23). Results shown a new mechanism that increased CYP7A1 activity may stimulate cholesterol synthesis and cholesterol absorption. It is well known that serum cholesterol in mice consists of mainly HDL. Thus, the LDL receptor–mediated cholesterol uptake, as previously suggested (24). The mechanism and efficacy of diverse medicinal herbs to reduce serum cholesterol level might be due to the presence of different levels and types of alkaloid. It is concluded from the results of the study that total cholesterol, triglycerides and LDL cholesterol showed gradual significant decrease with the increasing dose level of P. harmala up to 250 mg/ml in drinking water, while HDL cholesterol showed significant increase with increasing level of P. harmala Symptoms of P. harmala toxicity experienced by cases were similar to what had been reported for animals (25) and in French patient (26) However that the cholesterol transport in Rats is very different from humans. Rats do not express the CETP enzyme and carry most of their cholesterol in HDL particles (27, 28). This major consist of neurosensory symptoms, hallucination, slight elevation of body temperature (29) and cardio-vascular disorder such as bradycardia and low blood pressure (30, 31, 32). In summary, we showed effect of P.harmala on hypercholesteromia and level of CYP7A1 gene and dilation of a hydrophobic bile acid pool stimulate cholesterol exchange into bile acids, anew cholesterol synthesis, and biliary free cholesterol discharge, without increasing intestinal cholesterol sorption. This study affirmation the importance of bile acid signaling in holding cholesterol preventing hypercholesterolemia and homeostasis. Acknowledgment In addition, a thank you to Reza Tabatabaei qomi, who introduced us to the Methodology of work, and whose passion for the “underlying structures” had lasting effect. We also thank the ACECR Qom branch for consent to include copyrighted pictures as a part of our paper.

Figure 4. The relative expression of CYP7A1 gene affected by the different concentration of P.harmala extract. The effects of treatment with P.harmala extract on the expression of CYP7A1 gene is G1) control, G2) control plus 100mg/kg, G3) control plus 200mg/kg, G4) control plus 400mg/kg. The results show that with increasing extract dose, CYP7A1 gene expression is increased

*) significant difference (P<0.05)

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References (1) Chiang JY. Bile acids: regulation of synthesis. J Lipid Res 2009 Aug 50; 1955-66. (2) Pullinger CR, Eng C, Salen G, Shefer S, Batta AK, Erickson SK. Human cholesterol 7a-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype. J Clin Invest 2002 Jul 110(1);109-117. PMID: 12093894 (3) Hylemon PB, Zhou H, Pandak WM, Ren S, Gil G, Dent P. Bile acids as regulatory molecules. J Lipid Res 2009 Aug 50(8);1509-1520. PMID: 19346331 (4) Miyake JH, Duong-Polk XT, Taylor JM, Du EZ, Castellani LW, Lusis AJ. Transgenic expression of cholesterol 7a-hydroxylase prevents atherosclerosis in C57BL/6J mice. Arterioscler Thromb Vasc Biol 2002 Jan 22(1);121-126. PMID: 11788471 (5) Li T, Owsley E, Matozel M, Hsu P, Novak CM, Chiang JY. Transgenic expression of cholesterol 7a-hydroxylase in the liver prevents high-fat diet-induced obesity and insulin resistance in mice. HEPATOLOGY 2010 Aug 52(2);678-690. PMID: 20623580 (6) Gomes MR, Tomaso H, Nazarian, GK, Bjarnason H, Dietz Jr, Hunter DW. Upper extremity deep vein thrombosis and chronic pulmonary embolism resulting in pulmonary artery hypertension. Roentgenol. 1998 Jun 170(6);1532-34. PMID: 9609168 (7) Mohammad Eini A. Effect of Peganum harmala L. on Lipid parameters in hypercholesterolemiainduced male Wistar Rat. Academia Journal of Medicinal Plants. 2014 Nov 2(5);074-078. (8) Chaturvedi M, Dwivedi S, Dwivedi A, Barpete PK, Sachan R. Formulation and evaluation of polyherbal anthelmintic prepaRation. Ethnobotanical Leaflets. 2009 Aug 13;329-31. (9) Shah A, Rasapalli S, Mello C, Singh B R, Cai S. Antibacterial activity of commonly used traditional Chinese medicines as cold and flu remedies. J Med Plants Res. 2012 Jan 6;234-42. (10) Rosina K, Islam B, Akram M, Shakil S, Ahmad A, Ali SM, Siddiqui M, et al. Antimicrobial activity of five herbal extracts against multi drug resistant (MDR) strains of bacteria and fungus of clinical origin. Molecules. 2009 Fev 14(2);586-97. PMID: 19214149 (11) Herraiz T, González D, Ancín-Azpilicueta C, Arán VJ, Guillén H. Beta-Carboline and Alkaloids in Pegannumharmala and inhibition of human monoamine oxidase (MAO). Food Chem Toxicol. 2010 Mar 48(3);839-45. PMID: 20036304 (12) Monsef HR, Ghobadi A, Iranshahi M, Abdollahi M. Antinociceptive effects of Peganumharmala L. alkaloid extract on mouse formalin test. J Pharm Sci. 2004 Feb 19,7(1);65-69. PMID: 15144736 (13) Shahverdi AR, Monsef-Esfahani HR, Nickavar B, Bitarafan L, Khodaee S, Khoshakhlagh N. Antimicrobial activity and mainchemical composition of two smoke condensates from Peganumharmala seeds. Z Naturforsch C. 2005 Sep-Oct;60(9-10). PMID: 16320612 (14) Qazan WS. The effect of low levels of dietary Peganumharmala L. and Ballotaundulata or their mixture on chicks. J Anim Vet Adv. 2009 Jun 8;1535-38, PMID:24054692. (15) Hamdan K, Masmoudi H, Ellouz F, Feki AE, Carreau S. Protective effects of Peganumharmala extracts on thiourea induced diseases in adult male Rat. J Env Biol. 2008 Jan;29(1):73-7. PMID: 18831335 (16) Sharaf M, El-Ansari MA, Matlin SA, Saleh NA. Four flavonoid glycosides from Peganumharmala. Phytochem. 1997 Feb 44(3);533-36. PMID: 9014375 (17) Saeed SA, Farnaz S, Simjee RU, Malik AT. Riterpenes and B-sitosterol from piper betel;isolation, antiplatelet and anti-inflammatory effect. Biochem Soc Trans. 1993 Nov;21(4):462S. PMID: 8132030 (18) Wang X, Wang H, He A. Study on the antitumor effect of total harmala. J Chin Med Univ. 1996 Jul-Dec; 7(14): 199–212. PMCID: 3841998

Bhu.J.RNR. Vol 3.1, 213-220: 2015 Nazila Vahidi-eyrisofla et al

220

(19) Wang Y, Ohtani K, Kasai R, Yamasaki K. Steroidal saponins from fruits of Tribulus terrestris. Phytochemistry. 1997 Jul;42(5):1417-22. PMID: 9397208 (20) Singh AB, Chaturvedi JP, Narender T, Srivastava AK. Preliminary study on the hypoglycemic effect of Peganumharmalaseeds ethanol extract on normal and streptozocine induced diabetic Rats. Indian J Clin Biochem. 2008 Oct;23(4):391-3. PMID: 23105794 (21) Jinous A, Fereshteh R. Chemistry, pharmacology and medicinal properties of Peganumharmala L. Afr J Pharm Pharmaco. 2012 Jan 6;1573-80. PMCID: 3841998 (22) Tse SYH, Mak IT, Dickens BF. Antioxidative properties of harmane and b-carboline alkaloids. Biochem Pharmacol. 2001 Jul 15;42(3):459-64. PMID: 1859459 (23) Anuja P, Sunil N, Shashikant P, Vijay T, Manoj T. Anti diabetic effect of polyherbal combinations in STZ induced diabetes involve inhibition of α-amylase and α-glucosidase with amelioRation of lipid profile. Phytopharmacology. 2012 Aug 2;46-57. PMCID: 3988503 (24) Miyake JH, Doung XD, Strauss W, Moore GL, Castellani LW, Curtiss LK. Increased production of Apo B100-containing lipoproteins in the absence of hyperlipidemia in transgenic mice expressing cholesterol 7a- hydroxylase. J Biol Chem (2001) Jun 29;276(26):23304-11. PMID: 11323427 (25) El-Bahri L, Chemli R. Peganumharmala L;a poisonous plant of North Africa. Vet Hum Toxico. 1991 Jun;33(3):276-7. PMID: 1858311 (26) Al-Allaf TA, Khuzaie RF, Rashan LJ, Halaseh WF. Cytotoxic activity of a series of tumor cell lines with various tumor ligands. Boll. Chem. Pharm. 1999 Jun;138(6):267-71. PMID: 10464976 (27) Shapira Z, Terkel J, Egozi Y, Nyska A, Fiedman J. Abortifacient potential for the epigeal parts of Peganumharmala. J Ethno-pharmacol. 1989 Dec;27(3):319-25. PMID: 2615437 (28) Aarons DH, Rossi GV, Orzechowski RF. Cardiovascular actions of three Harmala alkaloids;harmine, haramaline, and harmalol. J Pharm Sci. 1977 Fev 66;1244-4, PMID: 903860. (29) Abdel-Fattah A, Matsumoto K, Murakami Y, El-Hady KA, Mohamed MF, Watanabe H. Facilitato. Inhibitory effects of Harmaline on the tryptophan induced 5-hvdroxyt syndrome and body tempeRature changes in pargyline-pretreated Rats. Jpn J Pharmacol. 2006 Jun 72;39-47, PMID:9068981. (30) Berrougui H, Lopez-Lazaro M, Martin-Cordero C, Hmamouchi M, Ettai A. Totoxic activity of methanolic extract and two alkaloidsextracted from seeds of Peganumharmala L. J Nat Remedies. 2005 Sep 5;41-45. (31) Manan A, Chand N, Khan S, Qureshi MS, Rehman AU, Jan B. Effect of periodic supplementation of herbal infusion on the liver function and lipid profile of broiler chickens. Sarhad J Agric. 2012 Nov 28;75-82. (32) Monsef HR, Ghobadi A, Iranshahi M. Antinociceptive effects of Peganumharmala L.alkalid extract on mouse formalin test. J Pharm Pharmaceut Sci. 2004 Feb 19;7(1):65-9. PMID:15144736