Evaluation of hypolipidemic Marrubium vulgare effect in Triton WR-1339-induced hyperlipidemia in mice

Abeer Y Ibrahim, Saber F Hendawy, Ahmed A A Elsayed, Elsayed A OmerMedicinal and Aromatic Plants Researches Dept., Pharmaceutical and Drug Industries Division, National Research Centre, 12622 Egypt

ABSTRACT

Objective: To evaluate the hypocholesterolemic and hypotriglyceridemic activities of four Marrbium vulgare (M. vulgare) herb extracts using Triton WR-1339-induced hyperlipidemia in mice. Methods: Hyperlipidemia was developed by intraperitoneal injection of Triton (200 mg/kg body weight ). The animals were divided into main four groups of eight mice each: normal control group, hyperlipidemic control group, hyperlipidemic plus tween-40 control and treated group. The fourth one was divided into four subgroups, petroleum ether extract group, chloroform extract group, ethyl acetate extract group and methanol extract treated group each of them contains two sub-sub group for treating animals with two doses at 0.1 and 0.25 LD50. Results: After 7 h and 24 h of treatment, the intragastric administration of all extracts caused a signifi cant decrease of plasma total cholesterol. Triglyceride levels were also signifi cantly lowered by all extracts while petroleum ether produced the lowest decreasing level. Similar results were observed for LDL-cholesterol concentrations. Furthermore, more polar extracts (methanol and ethyl acetate)-soluble fractions showed a signifi cant ameliorative action on elevated atherogenic index (AI) and LDL/HDL-C ratios, while these atherogenic markers were not statistically suppressed by the chloroform and petroleum ether-soluble extract. Conclusion: The fi ndings indicated that Marrubium may contain polar products able to lower plasma lipid concentrations and might be benefi cial in treatment of hyperlipidemia and atherosclerosis.

ARTICLE INFO

Article history:

Received 15 January 2016

Received in revised form 16 February 2016

Accepted 15 March 2016

Available online 20 May 2016



Evaluation of hypolipidemic Marrubium vulgare effect in Triton WR-1339-induced hyperlipidemia in mice

Abeer Y Ibrahim*, Saber F Hendawy, Ahmed A A Elsayed, Elsayed A Omer
Medicinal and Aromatic Plants Researches Dept., Pharmaceutical and Drug Industries Division, National Research Centre, 12622 Egypt

ABSTRACT

Objective: To evaluate the hypocholesterolemic and hypotriglyceridemic activities of four Marrbium vulgare (M. vulgare) herb extracts using Triton WR-1339-induced hyperlipidemia in mice. Methods: Hyperlipidemia was developed by intraperitoneal injection of Triton (200 mg/kg body weight ). The animals were divided into main four groups of eight mice each: normal control group, hyperlipidemic control group, hyperlipidemic plus tween-40 control and treated group. The fourth one was divided into four subgroups, petroleum ether extract group, chloroform extract group, ethyl acetate extract group and methanol extract treated group each of them contains two sub-sub group for treating animals with two doses at 0.1 and 0.25 LD50. Results: After 7 h and 24 h of treatment, the intragastric administration of all extracts caused a signifi cant decrease of plasma total cholesterol. Triglyceride levels were also signifi cantly lowered by all extracts while petroleum ether produced the lowest decreasing level. Similar results were observed for LDL-cholesterol concentrations. Furthermore, more polar extracts (methanol and ethyl acetate)-soluble fractions showed a signifi cant ameliorative action on elevated atherogenic index (AI) and LDL/HDL-C ratios, while these atherogenic markers were not statistically suppressed by the chloroform and petroleum ether-soluble extract. Conclusion: The fi ndings indicated that Marrubium may contain polar products able to lower plasma lipid concentrations and might be benefi cial in treatment of hyperlipidemia and atherosclerosis.

ARTICLE INFO

Article history:

Received 15 January 2016

Received in revised form 16 February 2016

Accepted 15 March 2016

Available online 20 May 2016

Keywords:

Anti-hyperlipidemia

Marrubium vulgare

Triton

Antioxidant

Atherogenic index

1. Introduction

Experimental and epidemiological studies have shown that the plasma hypercholesterolemic state could contribute to the development of atherosclerosis and related cardiovascular system diseases (CVD) which are the most common cause of death in both western and eastern societies [1].

Triton WR-1339, a non-ionic detergent (oxyethylated tertiary octyl phenol formaldehyde polymer), has been widely used to produce acute hyperlipidaemia in animal models in order to screen natural or chemical drugs[2] and to study cholesterol and triacylglycerol metabolism [3]. The accumulation of plasma lipids by this detergent appears to be especially due to the inhibition of lipoprotein lipase activity [4]. Hyperlipidemia is a highly predictive risk factor for atherosclerosis, coronary artery diseases and cerebral vascular diseases [5]. Coronary heart disease, stroke, atherosclerosis and hyperlipidemia are the primary cause of death [6]. Hyperlipidemia is characterized by elevated serum total cholesterol. Low density and very low-density lipoprotein cholesterol and decreased high density lipoprotein levels. Hyperlipidemia associated lipid disorders are considered to cause atherosclerotic cardiovascular disease [7]. Atherosclerosis (Sclrero-hardening) of arteries is a generalized disease of arterial network known as a progressive and silent killer disease characterized by the formation of lesions called atherosclerosis plaques in the walls of large and or medium sized coronary arteries and reduces blood fl ow to the myocardium called coronary artery diseases (CAD) [8]. Hyperlipidemia is classifi ed into a primary and a secondary type, which indicates the complexities associated with disease. The primary disease may be treated using anti-lipidemic drugs but the secondary type originating from diabetes, renal lipid nephrosisorhypothyroidism demands the treatment of the original disease rather than hyperlipidemia[9]. Medicinal plants play amajor role in hypolipidemic activity, literature suggests that the lipid lowering action is mediated through, inhibition of hepatic cholesterol biosynthesis and reduction of lipid absorption in the intestine [10].

In many developing countries, most hyperlipidemic individuals use medicinal plants as folk medicine to treat hyperlipidaemia and prevent atherosclerosis. Therefore, there is a strong interest, locally, in natural hypolipidemic substances derived from medicinal plants. Vast numbers of plants have received attention in this regard and have been shown to lower plasma lipid levels [11]. Marrubium vulgare L. (M. vulgare), white horehound, is an aromatic medicinal plant belongs to the family Lamiaceae and native to Europe, northern Africa and Asia [12]. Marrubiin is the main active compound isolated from M. vulgare L., a medicinal plant used in folk medicine to cure several diseases particularly gastroenteric and respiratory[13]. Aqueous extract of M. vulgare L. provides a source of natural antioxidants, which inhibit LDL oxidation and enhance reverse cholesterol transport and thus can prevent cardiovascular diseases development [14]. The goal of this work is evaluation of antihyperlipidemic eff ect of M. vulgare successive extracts in Triton WR-1339 model as indicator for their eff ect on fatty acid biosynthesis and blood lipid removal.

2. Material and methods

2.1. Preparation of M. vulgare extracts

The areal parts of the cultivated M. vulgare were collected and air dried in the shade, ground and then successively and exhaustively extracted by gradual solvents as follows, petroleum ether (40-60 ℃), chloroform, ethyl acetate and methanol. Plant extracts were prepared by continuous extraction using soxhlet apparatus. The four extracts were then concentrated under reduced pressure to remove solvent completely and lyophilized to be free from any solvent and water.

2.2. Antioxidant properties of Marrubium extracts

2.2.1. Total polyphenols content

Total polyphenols of M. vulgare extracts, petroleum ether, chloroform, ethyl acetate and methanol, were determined by the Folin–Ciocalteu procedure [15] to aliquots of 0.5 mL were added 0.25 mL of Folin–Ciocalteu reagent and 1.25 mL 20% aqueous sodium carbonate solution. Samples were vortexed and absorbance of blue colored mixtures recorded after 40 min at 725 nm against a blank containing 0.5 mL of water or 4% DMSO in water, 0.25 mL of Folin–Ciocalteu reagent and 1.25 mL of 20% aqueous sodium carbonate solution. The amount of total polyphenols was calculated as gallic acid equivalents from the calibration curves of gallic acid standard solutions and expressed as mg gallic/g dry plant extract. All measurements were done in triplicate.

2.2.2. Free radical scavenging activity

The free radical scavenging activity of M. vulgare extracts were measured by 1, 1-diphenyl-2-picryl-hydrazil (DPPH•) using the method of Yamaguchi et al., [16]. Briefl y, 0.1 mM solution of DPPH•in ethanol was prepared. Then, 1 mL of this solution was added to 3 mL of M. vulgare extracts and standards solution at diff erent doses (50, 100, 250, 500 and 1 000 μg/ mL). The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then the absorbance was measured at 517 nm in a spectrophotometer (Jasco, serial No. C317961148, Japan). Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The The reduction in DPPH• radical concentration in the reaction medium was calculated from the following equation:

DPPH• scavenging eff ect (%) = 100 - [(A0- A1)/ A0)伊100]

where A0was the absorbance of the control reaction and A1was the absorbance in the presence of the sample [17].

2.2.3. Reducing power

The reducing power of marrubium extracts was determined according to the method of Oyaizu M[18]. M. vulgare extracts and standard solution at diff erent concentrations (50, 100, 250, 500 and 1 000 μg/mL) in 1 mL of methanol were mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe (CN)6] (2.5 mL, 1%). The mixture was incubated at 50 ℃ for 20 min. A portion (2.5 mL) of TCA (10%) was added to the mixture, which was then centrifuged for 10 min at 1 000 g. The upper layer of solution (2.5 mL) was mixed with methanol (2.5 mL) and FeCl3(0.5 mL, 0.1%), and the absorbance was measured at 700 nm. Vitamin C was used as a control. Higher absorbance of the reaction mixture indicated greater reducing power.

2.2.4. Metal chelating activity

The chelating of ferrous ions by the M. vulgare extracts and standards was estimated by the method of [19]. Briefl y, M. vulgare extracts and standards solution at diff erent concentrations (50, 100, 250, 500 and 1 000 μg/mL) were added to a solution of 2 mM FeCl2(0.05 mL). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL) and the mixture was shaken vigorously and left standing at room temperature for ten minutes. After the mixture had reached equilibrium, the absorbance of the solution was then measured spectrophotometrically at 562 nm. The percentage of inhibition of ferrozine-Fe2+complex formation was given by the formula:

Percent of inhibition = [(A0-A1)/ A0] 伊100

Where A0was the absorbance of the control, and A1was the absorbance in the presence of the sample of M. vulgare extracts and standard. The control contains FeCl2and ferrozine [20].

2.2.5. Lipid peroxidation- Ammonium thiocyanate method

The antioxidant activity of M. vulgare extracts and standards were determined according to the method of [21] with some modifi cations. A pre emulsion was prepared by mixing 175 μg Tween 20, 155 μL linoleic acid, and 0.04 M potassium phosphate buff er (pH 7.0). A 1 mL of sample (1 mg/mL) in 99.5% ethanol was mixed with 4.1 mL linoleic emulsion, 0.02 M phosphate buffer (pH 7, 8 mL) and distilled water (7.9 mL). The mixed solutions of all samples (21 mL) were incubated in screw cap-tubes under dark conditions at 40 ℃ at certain time intervals. To 0.1 mL of this mixture was pipetted and added with 9.7 mL of 75% and 0.1 mL of 30% ammoniumthiocyanate sequentially. After 3 min, 0.1 mL of 0.02M ferrous chloride in 3.5% hydrochloric acid was added to the reaction mixture. The peroxide level was determined by reading daily of the absorbance at 500 nm. Antioxidant assay of vitamin C was also assessed for comparison. All test data was the average of three replicate analyses. The inhibition of lipid peroxidation in percentage was calculated by the following equation:

% Inhibition = [(A0- A1) / A0] 伊 100

where A0was the absorbance of the control reaction and A1was the absorbance in the presence of extracts or standard compounds.

2.3. Acute toxicity study

The acute toxicity test for the M. vulgare gradual extracts was carried out to evaluate any possible toxicity. Male albino mice (n = 8) of either eight were tested by administering diff erent doses in intraperitoneal route of Marrubium extracts by increasing or decreasing the dose, according to the response of animal [22]. The dosing patron was 500, 1 000, 1 500, 2 000, 2 500, 3 000 and 3 500 mg/kg body weight. All groups were observed for any gross eff ect or mortality during 48h. Death of half of examined animals was observed at 2, 2.5, 2.5 and 3 g/kg b. wt. for petroleum ether, chloroform, ethyl acetate and methanol extracts, respectively.

2.4. Triton model of hyperlipidemia

Triton WR-1339 (Sigma–Aldrich) was dissolved in normal saline (pH 7.4) and administered intraperitoneally to mice (200 mg/kg b. wt.) in order to develop an acute hyperlipidemia in experimental animals.

2.4.1. Animals housing

One hundred and sixty adult male albino mice, weighing 27–30 g, bred in the animal house of National Research Centre, Egypt, were provided ad libitum access only to tap water throughout the experimental duration (24 h). Animals were fed on standard diet; temperature through the housing was controlled at 22-24 ℃, relative humidity (60±5)% and light/dark cycles (12/12hrs). Animals were divided into groups and housed in polypropylene cages; each cage was contained eight mice adapted for one week before starting the experiment.

2.4.2. Experimental design

Overnight fasted mice were randomly divided into twenty groups of eight animals each. The fi rst group, serving as a normal control (NCG), received an intraperitoneal administration of normal saline and water by gavage. The second include two subgroups, gavaged with tween at 0.72% and 25% in saline that used as solvents control -ve (SCG). The third, hyperlipidemic control group (HCG) was treated with Triton and gavaged by distilled water. The fourth include eight treated subgroup, animals administered Marrubium extracts at 10% and 25% of LD50for each extract in intragastric route with intraperitoneal injection of sterilized saline, served as positive control group. The fi fth group contained eight treated sub-groups in which animals were administered Marrubium extracts at 10% and 25% of LD50for each extract in intragastric route with intraperitoneal injection of Triton, served as treated group. After treatments (7 h and 24 h), animals were anaesthetized briefl y with diethyl ether and blood was taken from their tail vein using a heparinized capillary. The blood samples were immediately centrifuged (2500 rpm/10 min) and the plasma were used for biochemical analysis also sera were collected for determination of liver and kidney function.

2.4.3. Analytical procedures

Triglycerides in plasma were quantifi ed by an enzymatic method using Bio-diagnostic kits (Bio-diagnostic, Egypt). Briefly, after enzymatic hydrolysis with lipases, the formation of quinoneimine from hydrogen peroxide, 4-aminophenazone and 4-chlorophenol under the catalytic effect of peroxidase, was followed spectrophotometrically at 540 nm. Total cholesterol levels were determined by the cholesterol oxidase enzymatic method, using Biodiagnostic Kits (Bio-diagnostic, Egypt); cholesterol was hydrolyzed and, in the presence of phenol, the quinoneimine as indicator was formed from hydrogen peroxide and 4-aminoantipyrine via peroxidase catalysis and spectrophotometrically measured at 510 nm. HDL-cholesterol concentrations were quantifi ed by the same method as used to determine total cholesterol after removal of other lipoproteins by precipitation with phosphotungstic acid (PTA) and MgCl2(Bio-diagnostic kit, Egypt). The total lipid content was determined by the reaction between lipids and sulfuric, phosphoric acid, and vanillin to form pink colored complex quantified spectrophotometrically at 545 nm. The LDL-cholesterol was calculated by the Friedwald formula [23]:

LDL-Cholesterol = Total cholesterol - [HDL-Cholesterol +

(triglycerides/5)]

The atherogenic index (AI) was calculated by the following formula:

AI = (total cholesterol- HDL-C)/HDL-C

The LDL-C/ HDL-C ratio was calculated as the ratio of plasma LDL-C to HDL-C levels.

2.5. Statistical analysis

The results obtained in all analysis were expressed in median ± SD (standard deviation). The levels of statistical signifi cance (P<0.05) were calculated based one-way ANOVA test for comparisons among means.

3. Results

3.1. Polyphenol content of M. vulgare extracts and their antioxidant activity

The polyphenolic compounds content as mg gallic acid/ g dry plant extract of M. vulgare extracts, radical scavenging activity, reducing power, metal chelation eff ect as well as lipid peroxidation inhibition activity are shown in Table 1. M. vulgare extracts were scavenged DPPH radicals by diff erent ef fecting level. Methanol and ethyl acetate extracts produced the same eff ect in an insignifi cant manner (IC50= 22.37 and 23.57 μg/mL, respectively) also chloroform extract and petroleum ether showed nearly the same eff ect as radical scavenger (IC50= 28 and 32 μg/mL, respectively). Methanolic extract was the most powerful extract as reducing agent followed with ethyl acetate extract in an insignifi cant diff erence (IC50= 13.99 and 15.49 μg/ mL, respectively ) and then chloroform extract (IC50= 18.41 μg/ mL) while petroleum ether extract was the weak one (IC50=26.57 μg/mL) but all Marrubium extract remained more ef fective than vitamin C (IC50= 29 μg/mL). The same trend of radical scavenger results was observed with ferrous ion chelation. Methanolic and ethyl acetate extracts had nearly the same least IC50(24.45 μg/mL and 25.46 μg/ mL, respectively) followed with chloroform extract (IC50= 39.24 μg/ mL) while petroleum ether was the weakest extract in chelating ion metals. These activities were refl ected on plant extract ef fect on lipid peroxidation. The most effective extracts as scavenger, reducing agent and metal chelator were the most inhibitor for lipid peroxidation. Methanol and ethyl acetate extracts inhibited lipid peroxidtion with low IC50(50.05 and 67.04 μg/ mL, respectively). However, chloroform produced the middle eff ect (IC50=73.14 μg/ mL) followed with petroleum ether (IC50=78.15 μg/ mL) and all of them were eff ective than vitamin C (IC50=85 μg/ mL). Polyphenolic content of diff erent plant fractions were estimated by folin reagent. The methanolic extract contained the maximum amount of polyphenolic compounds (385 mg/g) followed with ethyl acetate (320 mg/g) then chloroform fraction (140 mg/g) while petroleum ether had the lowest concentration (30 mg/g).

Table 1Polyphenol content and antioxidant activity of M. vulgare extracts.

Table 2Eff ect of M. vulgare extracts on lipid parameters in Triton WR-1339-induced hyperlipemia in mice after 7 h.

3.2. Effect of M. vulgare extracts as hypolipidemic agent in Triton model induced hyperlipidemia in mice

Triton and tween magnify the cholesterol production and LDL concentration in experimental animals after 7h from administration while M. vulgare extracts signifi cantly reduced all determined lipid parameters. Methanol extract at 0.1 and 0.25 LD50significantly reduced cholesterol production in hyperlipidemic animals (150 and 145 mg/ dL) with signifi cant reduction in LDL (30 and 28 mg/dL) and induction of HDL concentration (131 and 122 mg/dL) with reduction in plasma tri-glyceride concentration (Table 2). The second eff ective extract in inhibiting cholesterol production is ethyl acetate at the tested two doses (165 and 164 mg/dL for 0.1 and 0.25 LD50, respectively) which showed significant decreasing in LDL level (30 and 22 mg/dL, respectively) and tri-glycerides level (79 and 67 mg/dL, respectively). Also, chloroform extract at 0.1 and 0.25 LD50showed favorable eff ect in reducing the cholesterol level (188 and 153 mg/dL, respectively) with signifi cant reduction in triglyceride (113 and 97 mg/dL, respectively) and decreasing LDL level (28 mg/dL) as well as signifi cant increment in plasma HDL (92 and 87 mg/dL). The lowest mean values in cholesterol level were recorded with petroleum ether extract (220 and 256 mg/dL). It is clearly that increasing pet. ether extract to 0.25 LD50produced lower eff ect than 0.1 LD50, this trend was true with all estimated lipid parameters. It decreased LDL level to reach 33mg/dl at 0.1 LD50and reached 49 mg/dL at 0.25 LD50while increased HDL to be 74 mg/dL at 0.1 LD50and 65 mg/dL at 0.25LD50.

Administration of Triton elevated animal cholesterol, triglycerides and low density lipoprotein with a significant reduction in high density lipoprotein. However, treating hyperlipidemic animals with methanol significantly reduced cholesterol after 24h of administration (160 mg/dL at 0.1LD50and 116 mg/dL at 0.25LD50) and triglycerides (104 and 95 mg/dL for the two doses, respectively) as well as significant reduction in LDL-cholesterol (41.48 and 35.17 mg/dL for the two doses, respectively) accompanied with significant induction in HDL-cholesterol (161 and 169.6 mg/dL for the two doses, respectively) (Table 3). The ethyl acetate extract showed the same effect on all assessed parameters. It increased good lipids, HDL-cholesterol, to be near the –ve control group (146 and 157.8 mg/dL for the two doses, respectively) with signifi cant reduction in cholesterol (175.6 and 168 mg/dL for the two doses, respectively), triglycerides (100 and 90 mg/dL for two used doses, respectively) and LDL-cholesterol (48.6 and 38 mg/dL for two doses, respectively). Cholesterol was also decreased to reach lowerlevel than –ve control when animal treated with chloroform extract at 0.1 and 0.25LD50(198.8 and 183 mg/dL for the two doses, respectively). This decreasing was accompanied with significant reduction in triglycerides (130 and 120 mg/dL for the low and high doses, respectively) and LDL-cholesterol (52.49 and 49 mg/dL, respectively) as well as favorable effect on HDL-cholesterol (112 and 117.89 mg/dL). The petroleum ether extract produced the lower eff ect against hyperlipidemia as compared to the other three extracts with the same trend of data.

It lowered the total cholesterol in hyperlipidemic animals at the 0.1 and 0.25 LD50(250 and 290 mg/dL, respectively), triglycerides level (200 and 255 mg/dL for the two doses, respectively) and LDL-cholesterol (53 and 79 mg/dL, respectively) with increasing the production of HDL-cholesterol level (104 and 93 mg/ dL).

The changes of atherogenic index (AI) and LDL-C/HDL-C ratio in control and treated mice were shown in Table 4. The Triton administration signifi cantly aff ects the cardiovascular risk markers. Indeed, the AI was statistically increased in both tween control (0.85) and hyperlipidemic with tween group (2.67) when compared with values found in their relative normal lipidemic control (0.32) at 7 h. Similar results were noticed at 24 h.

Besides, there were significant further increases of LDL-C/ HDL-C ratios in Triton-injected animals (2.39). In contrast to normal lipidemic mice (0.24), 7 h after Triton treatment produced an elevated ratio either in hyperlipidemic group animals (2.19) by many times. This changing pattern was maintained until 24 h whenthe ratios were increased in animals compared to normal group. Methanol extract highly reduced AI (0.15 and 0.19 for low and high doses, respectively) lower than normal control group (0.32). The ethyl acetate extract showed the same trend with signifi cant increments than methanol extract (0.42 and 0.53, respectively) with decreasing LDL-C/HDL-C ratios (0.19 and 0.28, respectively).

The atherogenic index was lowered with chloroform administration (1.04 and 0.76 for two doses, respectively) accompanied with reduction in HDL/LDL-cholesterol ratio at 7h and the same trend of data was recorded at 24h. The minimum recorded effect was of petroleum ether. It decreased AIat low level of administration (1.97) didn’t produce this eff ect at high dose (2.97) with signifi cant decrease in HDL/LDL- cholesterol ratio (0.45 and 0.75 for two doses, respectively). This trend was also observed at 24hwith minor increments in values.

Table 3Eff ect of M. vulgare extracts on plasma lipid parameters in Triton WR-1339-induced hyperlipemia in mice after 24 h.

Table 4Eff ects of M. vulgare on atherogenic index (AI) and LDL/HDL-C ratio in Triton WR-1339-induced hyperlipemia in mice after 7 h and 24 h.

4. Discussion

Hyperlipidemia is characterized as elevation in triglycerides and cholesterol in the blood therefore the most plausible strategy in prevention or treating hyperlipidemia is using drugs that contains array of compounds which can delay fat digestion and absorption through gastrointestinal tract by inhibiting pancreatic lipase and pancreatic cholesterol esterase activities. The non-ionic detergent, Triton WR-1339, has been widely used to block the uptake of triacylglycerol-rich lipoproteins from plasma by peripheral tissues in order to produce acute hyperlipidemia in animal models which are often used for a number of objectives, in particular for screening natural or chemical hypolipidemic drugs [2]. With this aim, many medicinal plants have been assessed for their hypolipidemic activity in a Triton WR-1339-induced hyperlipidemic model [11]. This work was aimed to assess the possible hypocholesterolemic and hypotriglyceridemic activities of diff erent M. vulgare extracts. It is clear from our results that the methanolic and ethyl acetate fractions from this plant decrease plasma total cholesterol in a marked manner, either 7 h or 24 h after Triton treatment. The reduction of plasma total cholesterol was associated with a decrease of its LDL fraction which is a major, potentially modifi able risk factor of cardiovascular diseases and the target of many hypocholesterolemic therapies. This fi nding suggests that the cholesterol-lowering activity of these extracts appears to be due to the enhancement of LDL-C catabolism, as demonstrated by[11].

It is evident from recorded results that fractions rich in polyphenolic compounds are those ameliorate the hyperlipidemic eff ect of triton, these fi ndings are in accordance of Mäkynen et al. [24]

Recently, a number of clinical studies suggest that the increased risk of coronary heart disease is associated with a high serum concentration of TC, LDL-C and triglyceride. The abnormally high concentration of serum lipids is mainly due to the increase in the mobilization of free fatty acids from the peripheral depots[25]. On the other hand, low serum concentration of HDL-C is also responsible for coronary heart disease [25]. Methanolic and ethyl acetate fractions appears highly ameliorative eff ect in lowering LDL and increasing HDL-cholesterol followed with chloroform and then the lowest eff ective one, petroleum ether fraction.

Preclinical observations demonstrate that hyperlipidemia promotes accumulation of oxidatively modifi ed low density lipoproteins in the arterial wall, promoting endothelial dysfunction and development of atherosclerosis and congestive heart diseases [26, 27]. Polyphenolic compounds considered as health promoting compounds that included in most functional foods for many diseases as antihyperlipidemic and antihyperglycemic foods [28]. The physiological effect of polyphenolic compounds include possible antioxidant activity, therefore suggestion their role in prevention of coronary heart disease including atherosclerosis [29]. They may also work by making liver cells more effi cient to remove LDL-C from blood by increasing the LDL-C receptor densities in liver and by binding to apolipoprotein B[30].

The methanol and ethyl acetate fractions contained high amount of polyphenol. On the other hand, the in-vitro antioxidant estimated properties of four fractions showed the potent effect as radical scavenger hydrogen donator and metal chelator agent that refl ected on their effect on inhibiting lipid peroxidation. Plant fractions arranged in their activities as antioxidant had the same pattern of activity in hyperlipidemic animals; methanol, ethylacetate, chloroform and then petroleum ether.

Free radicals and other reactive oxygen species are considered to be important causative factors in the development of diseases of aging such as neurodegenerative diseases, cancer and cardiovascular diseases. Phytochemicals have long been recognized to possess many properties including antioxidant, anti-allergic, anti-infl ammatory, antiviral, anti-proliferative and anti-carcinogenic. This relationship has led to considerable interest in assessing the antioxidant capacity of foods, botanicals and other nutritional antioxidant supplements. As plants produce signifi cant amount of antioxidants to prevent the oxidative stress caused by photons and oxygen, they represent a potential source of new compounds with antioxidant activity. Thus, continued research is being undertaken all over the world on diff erent plant species and their therapeutic principles [31]. The four plant fractions showed potent eff ect as radical scavenger when assessed against DPPH radicals with reduction capability. They also play as excellent metal ion chelator as they tested against ferrous ions. These activities were accompanied with inhibition of lipid peroxidation.

The possible mechanism of this activity may result from the enhancement of lecithin cholesteryl acyl transferase (LCAT) and inhibition of hepatic triglyceride lipase (HTL) on HDL which may lead to a rapid catabolism of blood lipids through extrahepatic tissues [32]. It is also reported that triglycerides play a key role in the regulation of lipoprotein interactions to maintain normal lipid metabolism [33]. Indeed, the elevated plasma TGs levels were associated with an increased incidence of coronary artery disease. Moreover, these higher plasma TG levels have been attributed mainly to an increased population of small, dense LDL deposits which are very atherogenic and enhanced cholesteryl ester mass transfer from apolipoprotein B-containing lipoproteins (VLDL and LDL). TGs have also been proposed to be a major determinant of cholesterol esterifi cation, its transfer and HDL remodelling in human plasma [34].

Methanol and ethyl acetate fractions from M. vulgare signifi cantly suppressed the elevated blood concentrations of TGs. This result suggests that the extracts are able to restore, at least partially, the catabolism of triglycerides. The underlying mechanism of this activity is not elucidated by the present study. However, as hypothesized by many works with other plants [35, 36] the restoration of catabolic metabolism of triglycerides could be due to an increased stimulation of the lipolytic activity of plasma lipoprotein lipase (LPL).

Administration of M. vulgare provides a benefi cial eff ect on mice lipid metabolism in regard to the reduction of AI. In fact, the AI was deceased in fraction treated groups. This ameliorative action was due to the plasma lipid-lowering activity of diff erent fractions. It is also desirable to have higher plasma HDL and lower LDL-cholesterol to prevent atherogenesis, since there is a positive correlation between an increased LDL-C/HDL-C ratio and the development of atherosclerosis [37]. The results found clearly demonstrate that the bioactive compound (s) contained in this plant have a polar character since the polar extracts were more active than the other extracts. These results are in accordance with those of [14] who reported that aqueous extract of M. vulgare L. inhibit LDL oxidation and enhance reverse cholesterol transport with antioxidant properties which increased the anti-atherogenic potential of HDL. Plant methanol and ethyl acetate extracts possess cholesterol-suppressive capacities and an ability to attenuate the accelerated development of atherosclerosis in hypercholesterolemic models.

Conflict of interest statement

Authors want to declare that they have no confl ict of interest

References

[1] Nilsson PM, Olsen MH, Laurent S. Early vascular aging (EVA): New directions in cardiovascular protection. J Hypertens 2014; 32(7): 1470A7.

[2] Schurr PE, Schultz JR, Parkinson TM. Triton induced hyperlipidaemia in rats as an animal model for screening hypolipideamic drugs. Lipids 1972; 7: 69- 74.

[3] Ghatak SB, Panchal SJ. Anti-hyperlipidemic activity of oryzanol isolated from crude rice bran oil, on triton WR-1339-induced acute hyperlipidemia in rat. Revista Brasileira de Farmacognosia 2012; 22(3): 642- 648.

[4] Scharwey M, Tatsuta T, Langer T. Mitochonderial lipid transport at a glance. J Cell Sci 2013; 126: 5317- 5323.

[5] Elhissi JH, Sukkar DN, El-Sakka MA. Nutritional attributes as predictors of coronary heart disease. Pharmacol Pharm 2014; 5: 1171- 1179.

[6] Smith GD, Song F, Sheldon TA. Cholesterol lowering and mortality: the importance of considering initial level of risk. Br Med J 1993; 306: 1367.

[7] Saravanan R, Rajendra Prasad N, Pugalandi KV. Eff ect of Piper bettle leaf extract on alcoholic toxicity in the rat brain. J Med Food 2003; 6: 261-265.

[8] Mohale DS, Dewani AP, Saoji AN, Khadse CD. Antihyperlipidemic activity of isolated constituents from the fruits of Lagenaria siceraria in albino rats. Int J Green Pharm 2008; 2: 104- 107.

[9] Suzuki T, Suzuki Y. Current topics of lipid dynamics and pathobiology in membrane lipid rafts. Biol Pharmaceut Bull 2006; 29(8):1538 -1541.

[10] Gramza A, Korczak J. Tea constituents (Camellia sinensis L.) as antioxidants in lipid systems. Trends Food Sci Tech 2005; 16: 351- 358.

[11] Khanna AK, Rizvi F, Chander R. Lipid lowering activity of Phyllanthus niruri in hyperlipemic rats. J Ethnopharmacol 2002; 82: 19- 22.

[12] Everist DL. Poisonous plants of Australia. 3rd ed. Sydney:Angus and Robertson; 1981,p. 449- 450.

[13] Rodrigues CA, Savi AO, Schlemper V, Reynaud F, Cechinel Filho V. An improved extraction of Marrubium vulgare. Chromatographia 1998; 47: 7.

[14] Berrougui H, Isabelle M, Cherki M, Khalil A. Marrubium vulgare L. extract inhibits human-LDL oxidation and enhance HDL-mediated cholesterol effl ux in THP-1 macrophage. Life Sci 2006; 80(2): 105- 112.

[15] Hagerman AE, Butler LG. Protein precipitation method for the quantitative determination of tannins. J Agr & Food Chem 1978; 26: 809-812.

[16] Yamaguchi T, Takamura H, Matoba T, Terao J. HPLC method for evaluation of the free radical-scavenging activity of foods by using 1, 1-diphenyl-2-picrylhydrazyl. Biosci Biotechnol Biochem 1998; 62: 1201-1204.

[17] Oktay M, Gülçin ˙I, Küfrevioˇglu Ö˙I. Determination of in vitro antioxidant activity of fennel (Foeniculum vulgare) seed extracts. Lebensmittel-Wissenchaft und Technologie 2003; 36: 263- 271.

[18] Oyaizu M. Studies on products of browning reaction: Antioxidative activities of products of browning reaction prepared from glucosamine. Jpn J Nutr 1986; 44: 307- 315.

[19] Dinis TCP, Madeira VMC, Almeida LM. Action of phenolic derivatives (acetaminophen, salicylate, and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch Biochem Biophys 1994; 315: 161- 169.

[20] Gülçin ˙I, Büyükokuroˇglu ME, Küfrevioˇglu ÖI. Metal chelating and hydrogen peroxide scavenging eff ects of melatonin. J Pineal Res 2003a; 34: 278- 281.

[21] Gülçin I, Oktay M, Kirecci E, Küfrevioˇglu ÖI. Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chem 2003b; 83: 371- 382.

[22] Bruce RD. An up-and-down procedure for acute toxicity testing. Fund & Appl Toxicol 1985; 5: 151- 157.

[23] Fiedwald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma without use of preparative ultracentrifuge. Clin Chem 1972; 18: 499- 502.

[24] Mäkynen K, Jitsaardkul S, Tachasamran P, Sakai N, Puranachoti S, Nirojsinlapachai N, et al. Cultivar variations in antioxidant and antihyperlipidemic properties of pomelo pulp [Citrus grandis (L.) Osbeck] in Thailand. Food Chem 2013; 139: 735–743.

[25] Ahmed I, Lakhani MS, Gillet M, John A, Raza H. antidiabetic Momordica charantia (karela) fruit extract in streptozotocin induced diabetic rats. Diabetes Res Clin Pract 2001; 51 (3): 155-161.

[26] Parab SR, Mengi SA. Hypolipidemic activity of Acorus calamus L. in rats. Fitoterapia 2002; 73 (6): 451- 455.

[27] Takahashi Y, Zhu H, Yoshimoto T. Essential roles of lipoxygenases in LDL oxidation and development of atherosclerosis. Antioxid & Redox Signa 2005; 7: 425- 431.

[28] Sharma I, Aaradhya M, Kodikonda M, Naik PR. Antihyperglycemic, antihyperlipidemic and antioxidant activity of phenolic rich extract of Brassica oleraceae var gongylodes on streptozotocin induced Wistar rats. Research 2015; 4:212.

[29] El-Beshbishy HA, Singab ANB, Sinkkonen J, Pihlaja K. Hypolipidemic and antioxidant eff ects of Morus alba L. (Egyptian mulberry) root bark fractions supplementation in cholesterol-fed rats. Life Sci 2006; 78: 2724- 2733.

[30] Mansoor M, Mohan T, Srinivasa Rao D. Antioxidant and antihyperlipidemic activities of etnanolic leaves extract of Hibiscus cannabinus. Int J Adv in Pharm Med Bioallied Sci 2015; 3(2): 89-92.

[31] Auddy B, Ferreira M, Blasina F, Lafon L, Arredondo F, Dajas F, et al. Screening of antioxidant activity of three Indian medicinal plants, traditionally used for the management of neurodegenerative diseases. J Ethnopharmacol 2003; 84: 131-138.

[32] Anila L, Vijayalakshmi NR. Flavonoids from Emblica officinalis and Mangifera indica- effectiveness for dyslipidemia. J Ethnopharmacol 2002; 79 (1): 81- 87.

[33] Hokanson JE, Austin MA. Plasma triglyceride level is a risk factor to cardiovascular disease independent of high-density lipoprotein cholesterol level: a meta-analysis of population based prospective studies. J Cardiovasc Risk 1996; 3: 213- 219.

[34] Supekar AR, Kale AJ. Anti-atherosclerosis activity of seed oil of Punica granatum Linn in Triton X-100 induced hyperlipidemic rat. Int J Adv Res 2015; 3(10): 1276- 1280.

[35] El-Tantawy WH, Temraz A, Hozaien HE, El-Gindi OD, Taha KF. Antihyperlipidemic activity of an extract from roots and rhizomes of Panicum repens L. on high cholesterol diet-induced hyperlipidemia in rat. Z Naturforsch C 2015; 70(5- 6):139-144.

[36] Xie W, Wang W, Su H, Xing D, Cai G, Du L. Hypolipidemic Mechanisms of Ananas comosus L. leaves in mice: Different from Fibrates but Similar to Statins. J Pharmacol Sci 2007; 103: 267- 274.

[37] Harnafi H, Hana SC, Nour el Houda B, Mohammed A, Souliman A. Hypolipemic activity of polyphenol-rich extracts from Ocimum basilicum in Triton WR-1339-induced hyperlipidemic mice. Food Chem 2009; 108: 205- 221.

E-mail: abeeryousry@yahoo.com

doi:Document heading 10.1016/j.apjtm.2016.03.038

*Corresponding author:Abeer Y Ibrahim, Medicinal and Aromatic Plants Researches Dept., Pharmaceutical and Drug Industries Division, National Research Centre, Egypt, 12622.