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Research Article
 

Inhibitory Activity of Some Plant Methanol Extracts on 3-Hydroxy-3-Methylglutaryl Coenzyme a Reductase



A. Gholamhoseinian, B. Shahouzehi and F. Sharifi-Far
 
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ABSTRACT

Beta-hydroxy-beta-methylglutaryl coenzyme a reductase (HMG CoA reductase) is the key enzyme in cholesterol biosynthesis. Inhibition of this enzyme reduces the synthesis of cholesterol and could be used in the management of coronary artery disease. Drugs used for the management of cholesterol biosynthesis have showed some side effects that are cause of new trends in the nature. This study was designed to find new HMG CoA reductase inhibitors from natural resources. One hundred plants were botanically identified and their methanol extracts were prepared. Anti HMG-CoA reductase activity of the extracts were determined spectrophotometrically by NADPH oxidation, using HMG-CoA as the substrate. Quercus infectoria, Rosa damascena and Myrtus communis extracts showed more than 50% inhibitory effect on the enzyme activity and 21 extracts showed an inhibitory effect between 30-50 percent on activity of HMG-CoA reductase. Kinetic study of the enzyme was performed in the presence of two concentrations of the effective extracts (0.05 and 0.15 mg mL-1). These active extracts showed non-competitive inhibition by Lineweaver-Burk plot analysis. Under the standard condition, Km value for enzyme was 0.6 mM and Vmax value was 0.011 mM min-1. When 0.15 mg mL-1 of extracts of Quercus infectoria, Rosa damascena and Myrtus communis were used the Vmax values of 0.0041, 0.0031 and 0.0028 mM min-1 were obtained, respectively. Therefore, purification and characterization of their active constituents and in vivo examination of these active extracts, is necessary in order to be used as safer therapeutic agents in the future.

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  How to cite this article:

A. Gholamhoseinian, B. Shahouzehi and F. Sharifi-Far, 2010. Inhibitory Activity of Some Plant Methanol Extracts on 3-Hydroxy-3-Methylglutaryl Coenzyme a Reductase. International Journal of Pharmacology, 6: 705-711.

DOI: 10.3923/ijp.2010.705.711

URL: https://scialert.net/abstract/?doi=ijp.2010.705.711
 
Received: May 02, 2010; Accepted: July 06, 2010; Published: August 13, 2010



INTRODUCTION

Cholesterol is a sterol and a constituent of all eukaryotic plasma membrane. It is essential for the growth and viability of higher organisms. Maintenance of cholesterol homeostasis is very important for healthy statue and is accomplished by a regulatory complex network (Avci et al., 2006; Deng, 2009). However, hypercholesterolemia is one of the known risk factors in the development of Coronary Artery Disease (CAD) and enhances free radical generation which plays an important role in the pathogenesis of other diseases besides cardiovascular diseases such as cancer and inflammatory disorders. Epidemiological studies have shown a continuous relationship between total cholesterol and Coronary Heart Disease (CHD) (Avci et al., 2006; Parab and Mengi, 2002). CHD is the leading cause of death in developing countries, where it is responsible for more than 50% of all mortalities (Faergeman, 2000). Elevated plasma cholesterol level also promotes other debilitating diseases including certain forms of cancer, diabetes and obesity. Therefore, hypercholesterolemia and its associated cardiovascular diseases represent one of the greatest worldwide economic, social and medical challenges that we are facing now (Hui and Howles, 2005; Deng, 2009).

HMG-CoA reductase reaction is the rate limiting step in the biosynthesis of cholesterol and isopernoides and is a unique molecular target in anti cancer therapy (Wong et al., 2002; Bochar et al., 1997). Drugs that lower cholesterol level have been used for decades, but the high prevalence of their adverse effects such as myopathy, liver damages and potential drug-drug interactions has been reported too (Deng, 2009). Therefore, finding and development of other therapeutic agents for controlling cholesterol level especially safer agents are is warranted. Plants are the best resources of new drug agent and their use for medicinal purposes has a long history (Deng, 2009).

The existence of cholesterol lowering agents and HMG-CoA reductase inhibitors have been demonstrated in different plant species including Garlic (Chetty et al., 2003), Morus alba, Melissa officinalis, Artemisia capillaries (Lee et al., 2008), Vitis vinifera (Koo et al., 2008), Ananas comosus (Xie et al., 2007), Kiwifruit (Jung et al., 2005) and Gynostemma pentaphyllum (Megalli et al., 2005). However, more searches for finding the most effective HMG-CoA reductase inhibitors from natural sources are needed. In the present study, we have screened methanol extracts of various plants for their anti- HMG-CoA reductase activity to find safer and cheaper agents which may be used as medications in future for prevention and control of hypercholesterolemia and related diseases.

MATERIALS AND METHODS

Reagents: HMG-CoA reductase and HMG-CoA were obtained from SIGMA and NADPH purchased from Fluka, USA. All other reagents were analytical grade.

Plants: Different parts of plants were collected from various regions of Kerman province. Plants were botanically identified by Dr. Mirtajaldini, Department of Botany, Bahonar University, Kerman, Iran during 2008-2009 (Table 1). A voucher specimen of each plant was deposited at the herbarium of the Herbal Medicines Research Center, Faculty of Pharmacy, Kerman University of Medical Sciences. Plant materials were air dried in dark place and grounded into fine powder. The powdered material (20 g) was extracted with 200 mL of absolute methanol for 24 h. The suspensions were filtered and air-dried; these air-dried samples were stored at -20°C in dark vials until use (Gholamhoseinian et al., 2009; Sharma et al., 2005).

Enzyme assay: HMG-CoA reductase activity was measured spectrophotometrically. The HMG-CoA dependent oxidation of NADPH was measured in 340 nm and pH 7.5; 10 μL of each preparation containing 50 μg crude extract was added to the reaction mixture including HMG-CoA (1.2 mmol L-1), NADPH (1.2 mmol L-1), HMG-CoA reductase (1 unit), 0.1 mol L-1 potassium phosphate buffer containing (400 mM KCl, 0.1 mg mL-1 of bovine serum albumin and 3.5 mmol L-1 EDTA) (final volume of 1 mL). Absorbance reduction at 340 nm was measured in 6 min interval (Xie et al., 2007). HMG-CoA reductase inhibitory activity was calculated by using the following formula (Jung et al., 2005):

Image for - Inhibitory Activity of Some Plant Methanol Extracts on 3-Hydroxy-3-Methylglutaryl Coenzyme a Reductase

Kinetic study: In order to determine the kinetic properties of the HMG-CoA reductase after addition of methanol extracts of Quercus infectoria, Rosa damascena and Myrtus communis, the activity was assayed by using various concentrations of HMG-CoA (0.3, 0. 6, 0. 9 and 1.2 mmol L-1) in the absence and presence of two different concentrations of the extracts (0.05 and 0.15 mg mL-1). Inhibition mode was determined by double-reciprocal Lineweaver-Burk plot analysis according to the Michaelis-Menten kinetics (Zhao and Kim, 2004; Carbonell and Freire, 2005; Gholamhoseinian et al., 2008b).

RESULTS

Plants with HMG-CoA reductase inhibitory effect: Among one hundred extracts; Quercus infectoria, Rosa damascena and Myrtus communis showed 84, 70 and 62% inhibitory effect on HMG-CoA reductase activity, respectively.

Twenty one extracts showed an inhibitory effect between 30-50% on HMG-CoA reductase activity. The rest of plant extracts showed less than 30% or no inhibition on the activity of the enzyme in this study (Table 1).

Kinetic analysis of HMG-CoA reductase inhibition: The inhibition mode of Quercus infectoria, Rosa damascena and Myrtus communis extracts was analyzed by double-reciprocal Lineweaver-Burk plot analysis. The enzyme kinetics demonstrated non-competitive inhibition on HMG-CoA reductase activity by Quercus infectoria (Fig. 1), Rosa damascena (Fig. 2) and Myrtus communis (Fig. 3). The Km value of HMG-CoA for HMG-CoA reductase in the absent of the extracts was 0.6 mM and Vmax value was 0.011 mM min-1. When the extracts were added to the enzyme mixture the Vmax value in the presence of Quercus infectoria extract was 0.0041, Rosa damascena extract was 0.0031 and for Myrtus communis extract was 0.0028 mM min-1.

Table 1: Anti HMG-Co A reductase activity of plants extract. Reaction mixture was contained 1.2 mmol L-1 NADPH, 1.2 mmol L-1 HMG-Co A, 1U enzyme, 0.1 mol L-1 potassium phosphate buffer pH 7.5, 400 mM KCl, 0.1 mg mL-1 BSA and 3.5 mmol L-1 EDTA; Reduction in absorbance was assayed at 340 nm
Image for - Inhibitory Activity of Some Plant Methanol Extracts on 3-Hydroxy-3-Methylglutaryl Coenzyme a Reductase
Image for - Inhibitory Activity of Some Plant Methanol Extracts on 3-Hydroxy-3-Methylglutaryl Coenzyme a Reductase

Image for - Inhibitory Activity of Some Plant Methanol Extracts on 3-Hydroxy-3-Methylglutaryl Coenzyme a Reductase
Fig. 1: The Lineweaver-Burk plot analysis for HMG-CoA reductase in presence of different concentrations of HMG-CoA (0.3, 0.6, 0.9 and 1.2 mmol L-1) and two different concentrations (0.05 and 0.15 mg L-1) of Quercus infectoria methanolic extract at 340 nm

Image for - Inhibitory Activity of Some Plant Methanol Extracts on 3-Hydroxy-3-Methylglutaryl Coenzyme a Reductase
Fig. 2: The Lineweaver-Burk plot analysis for HMG-CoA reductase in presence of different concentrations of HMG-CoA (0.3, 0.6, 0.9 and 1.2 mmol L-1) and two concentrations (0.05 and 0.15 mg L-1) of Rosa damascena methanolic extract at 340 nm

Image for - Inhibitory Activity of Some Plant Methanol Extracts on 3-Hydroxy-3-Methylglutaryl Coenzyme a Reductase
Fig. 3: The Lineweaver-Burk plot analysis for HMG-CoA reductase in presence of different concentrations of HMG-CoA (0.3, 0.6, 0.9 and 1.2 mmol L-1) and two concentrations (0.05 and 0.15 mg L-1) of Myrtus communis methanolic extract at 340 nm

DISCUSSION

The importance of plasma cholesterol level in the pathogenesis of atherosclerotic plaques has been represented by numerous studies (Yoshie et al., 2004). Because of various side effects of some cholesterol lowering drugs, herbal remedies have gained special attention to find the new safer compounds (Deng, 2009). Our results showed that Quercus infectoria, Rosa damascena and Myrtus communis are potent inhibitors of HMG-CoA reductase. Effectiveness and kinetic properties of these extracts were determined against HMG-CoA reductase which has not been showed so far. No such properties have been found for other plant extracts used in this study. The presence of HMG-CoA reductase inhibitors has been reported in some natural resources especially Kiwifruit extracts and Vitis vinifera (Koo et al., 2008; Jung et al., 2005). Inhibitory activities of Kiwifruit on HMG-CoA reductase activity were 14% at 10 mg mL-1 and 20-30% at 50 mg mL-1 (Jung et al., 2005) but our effective extracts were used at 0.15 mg mL-1 and showed more than 60% inhibitory effect on activity of HMG-CoA reductase which showed our plants are more potent inhibitor of HMG CoA reductase.

Yazdanparast and Bahramikia (2008) showed that Anethum graveolens L. crude extract has a strong hypocholesterolemic effects in rats and they showed that Anethum graveolens exert it’s effect through inhibition of HMG CoA reductase.

It has been previously showed that Quercus infectoria and Rosa damascena have anti-porcine pancreatic lipase, anti-alpha mannosidase and anti-alpha glucosidase activities (Gholamhoseinian et al., 2010; 2008a, b). These are target enzymes with therapeutic potential in the treatment of hyperlipidaemia, diabetes, metastatic cancer and lysosomal storage disease. It has been also showed that Eucaliptus galbie and Levisticum officinale are potent inhibitors of lipase whereas no strong inhibitory effect was found on HMG-CoA reductase by them (Gholamhoseinian et al., 2010). The existence of cholesterol lowering agents have been demonstrated in different plant species including garlic (Chetty et al., 2003), Morus alba, Melissa officinalis, Artemisia capillaries (Lee et al., 2008), Ananas comosus (Xie et al., 2007) and Gynostemma pentaphyllum (Megalli et al., 2005) but their mechanism of cholesterol lowering effects have not been established. Probably the active ingredient (s) of these plants affects the absorption of cholesterol in the intestine. The structural similarities to cholesterol might cause the mal-absorption and excretion or affects the biosynthesis stage of cholesterol in the liver and lead to cholesterol lowering effect in vivo (Ros, 2000).

Quercus infectoria have great medicinal values. In Asia the galls of this plant have been used for long time in treating inflammatory diseases and possess many therapeutic activities such as antidiabetic, anti-parkinsonian, antiviral, anti- microbial, antioxidant and larvicidal properties (Aroonrerk and Kamkaen, 2009). By antioxidant and anti HMG CoA reductase activity, Quercus infectoria can be considered as a potent cardio-protective plant. Rosa damascena showed anti-diabetic effect by reducing carbohydrate absorption from the intestine (Gholamhoseinian et al., 2009). Other therapeutic effects of Rosa damascena include cardiac strengthening and anti inflammatory effect. In addition, Rosa damascena extract can act on central nervous system including brain and showed hypnotic effect (Rakhshandeh et al., 2004). Myrtus communis also has been employed as an antiseptic and anti inflammatory agent and in the treatment of diabetes mellitus (Yoshimura et al., 2008).

HMG-CoA reduction is the target enzyme for anti-cancer therapy (Wong et al., 2002). With this regard, plant extracts that inhibits HMG-CoA reductase may be used as cancer therapeutic agents. Statins are a group of drugs that inhibit HMG-CoA reductase in a competitive manner (Carbonell and Freire, 2005). In contrast, we showed that Quercus infectoria, Rosa damascena and Myrtus communis inhibit HMG-CoA reductase in a non-competitive manner. This type of inhibition probably is dependent on especial components which bind to enzyme or enzyme-substrate complex. The anti HMG-CoA reductase activity of these plants encourages us to design an in vivo study on animal models to confirm their effects. It would be interesting to purify and characterize the active constituents of these extracts, to establish the composition and mechanism of action and to confirm their pharmacological potentials.

ACKNOWLEDGMENT

This study was supported by research funds No. 87/163, provided by the Vice Chancellor for research and the Kerman Physiology Research Center, Kerman University of Medical Sciences, Kerman, Iran.

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