|Year : 2011 | Volume
| Issue : 2 | Page : 230-235
Antihyperlipidemic effect of Trichilia connaroides in hypercholesterolemic rats and its possible mechanism
Prasanna Gurunath Subbarao, Purnima Ashok
Department of Pharmacology, KLE University's College of Pharmacy, 2nd Block, Rajaji Nagar, Bangalore - 560 010, Karnataka, India
|Date of Submission||13-Aug-2010|
|Date of Decision||19-Oct-2010|
|Date of Acceptance||24-Nov-2010|
|Date of Web Publication||12-May-2011|
Prasanna Gurunath Subbarao
Department of Pharmacology, KLE University's College of Pharmacy, 2nd Block, Rajaji Nagar, Bangalore - 560 010, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: The primary objective of this study was to investigate the antihyperlipidemic effect of the chloroform (CETC) and methanol (METC) extracts of leaves of Trichilia connaroides in hypercholesterolemic rats and, subsequently, to evaluate the possible mechanism of its antihyperlipidemic effect. Materials and Methods: The antihyperlipidemic activity of CETC and METC (100 mg/kg) in hypercholesterolemic rats was investigated by recording the serum lipid profile after a month-long oral treatment of these extracts. Further, hypercholesterolemic regression test and hypercholesterolemic progression test were carried out to understand the possible mechanism of its antihypercholesterolemic effect. The data were analyzed for statistical significance by one-way ANOVA, followed by Dunnet's test. Results and Conclusion: Hypercholesterolemic rats treated with CETC and METC produced a significant fall (P<0.05) in plasma triglyceride, total cholesterol, very low density lipoprotein (VLDL )-cholesterol and low density lipoprotein (LDL)-cholesterol and rise (P < 0.05) in high density lipoprotein (HDL) -cholesterol. A significant reduction (P < 0.01) in atherogenic index, increase (P < 0.05) in body weight and an insignificant influence on food intake were also observed at the end of the study. A hypercholesterolemic regression test revealed a significant reduction (P < 0.05) in the serum cholesterol level in both CETC and METC extract-treated animals. During the hypercholesterolemic progression test, a similar reduction in the serum cholesterol level was observed only in the METC extract-treated animals. The antihyperlipidemic effect was similar to fenofibrate and ezitimibe. Significant changes in the lipid profile in hypercholesterolemic animals confirm a potential antihyperlipidemic activity of the extracts. The CETC and METC extracts influenced the absorption and metabolism of dietary cholesterol to elicit the antihyperlipidemic effect.
Keywords: Antihyperlipidemia, high-fat diet, hypercholesterolemia, hypercholesterolemic progression test, hypercholesterolemic regression test, Trichilia connaroides
|How to cite this article:|
Subbarao PG, Ashok P. Antihyperlipidemic effect of Trichilia connaroides in hypercholesterolemic rats and its possible mechanism. J Pharm Bioall Sci 2011;3:230-5
|How to cite this URL:|
Subbarao PG, Ashok P. Antihyperlipidemic effect of Trichilia connaroides in hypercholesterolemic rats and its possible mechanism. J Pharm Bioall Sci [serial online] 2011 [cited 2020 Oct 27];3:230-5. Available from: https://www.jpbsonline.org/text.asp?2011/3/2/230/80777
Atherosclerosis is a leading cause of disability, and two-thirds of these cases are associated with dyslipidemia.  The lipid abnormalities are among the key risk factors for cardiovascular diseases. The direct correlation between an increased concentration of LDL in serum and the risk of developing clinically obvious atherosclerosis, particularly coronary artery disease, is well known.  Further, the concentration of HDL-cholesterol has been correlated strikingly and inversely with coronary heart diseases. , Management of atherogenic dyslipidemia is focusing on reducing the LDL-cholesterol level, and elevating the HDL-cholesterol level is now a new paradigm in the treatment of atherosclerotic cardiovascular diseases. ,
Non-pharmacological treatments in the management of hyperlipidemia include dietary modification and use of certain foods and dietary additives that interfere with cholesterol absorption and reducing plasma LDL-cholesterol levels by approximately 10-15%. 
In the recent past, there is a growing interest in screening herbs for their potential lipid-lowering effects. Trichilia connaroides (Wight and Arn.) Bentv. [(Syn: Zanthoxylum connaroides (Wight and Arn.) Bentv. Heynea trijuga (Roxb.ex sims) (Family: Meliaceae)] is found in moist forests throughout the greater part of India and is referred to as Karai, karaivilangu (Tamil), korakudi, kuravatti (Malayalam), Kora (Kannada) etc.  Two tetracyclic triterpenes (Heynic acid I and II),  tetranorterpenoides (Trijugin-A and Trijugin-B)  and Trijugin B acetate from the chloroform extract of the defatted leaves and fruits have been isolated and characterized,  and pentanortriterpenoids with a novel carbon skeleton (Trijugin C)  from the ethanol extract of leaves have been isolated and characterized.
A preliminary investigation of the hexane, chloroform and methanol extract of leaves of Trichilia connaroides has reported significant hypocholesterolemic and antihypercholesterolemic activity and methanol extract producing an increase in the serum HDL-c level (up to 50%) in normocholesterolemic rats.  The current study was envisaged to investigate the antihyperlipidemic effect of chloroform (CETC) and methanol (METC) extracts of leaves in hypercholesterolemic animals and the possible mechanism of antihypercholesterolemic effect.
| Materials and Methods|| |
Collection and processing of plant material
Fresh leaves of Trichilia connaroides were collected from the Chaurla ghat section of Jhamboti village of Belgaum district of Karnataka and were authenticated by a competent botanist of ICMR, and a voucher specimen of the same has been deposited at the Regional Research Centre, Indian Council of Medical Research, Belgaum and also in the Department of Pharmacology of our institution (H-1/Tc/2007-8).
Shade-dried leaves were coarse powdered, and around 400 g of the leaves was defatted with n-hexane (2.5L) by leaving the powder with the solvent overnight, with occasional shaking, and successively extracted with chloroform and methanol (2.5 L of each) in a soxhlet extractor. The CETC (8% yield) METC (10% yields) were stored in airtight containers in a cool, dry place, away from sunlight till further use.
Drugs and chemicals
Solvents, namely n-hexane, chloroform and methanol, were of analytical grade (M/s Qualigens ,Mumbai, India), commercial diagnostic kits for the estimation of lipid profile were from M/s Agapee Diagnostics, Kerala, India and ingredients for the high-fat diet (HFD) were locally procured and cholesterol and bile acid were of analytical grade (M/s Spectrochem, Mumbai and M/s LOBA Chemie , Mumbai, India). Rosvuastatin, fenofibrate and ezitimibe were gift samples from industry.
Preparation of the HFD
The HFD was prepared according to the formula standardized in our laboratory, containing cholesterol up to 6%, bile acid 2% and adequate amounts of carbohydrate and vitamin mixture.
Healthy, male, Wistar albino rats were purchased from a registered breeder and maintained in the animal house facility of this institution, in accordance with the guidelines of Committee for Purpose of Control and Supervision of Experimental Animals, Ministry of Environment and Forestry, Government of India. During the study, the test animals either received a commercial pelleted diet (M/s Gold Mohr , Pune, India) or HFD and water ad libitum. Animals were allowed a week's time for acclimatization. Ethical Clearance was taken from the Institutional Animal Ethics Committee of this institution prior to experimentation (626/02/a/CPCSEA).
Induction of hypercholesterolemia
Healthy, male, Wistar albino rats weighing 130-150 g (whose initial serum cholesterol level was recorded) had free access to HFD (average consumption was around 60 g for six animals per day) for 6 weeks. At the end of the 6 th week, animals with a serum cholesterol level above 150 mg/dl  were selected for the study.
Evaluation of the antihyperlipidemic activity of the extracts in hypercholesterolemic rats
Hypercholesterolemic animals were randomly assigned to four groups (n = 8). Group I was a vehicle control group, treated with vehicle (0.2 ml /animal), Group II and III animals were treated with CETC (100 mg/kg) and METC (100 mg/kg), respectively. Group IV and V animals were treated with Rosvuastatin (1 mg/kg) and Fenofibrate (65 mg/kg),  respectively.
Test animals received the respective treatment, orally, once each day, for 30 days and, during this period, had free access to HFD and water ad libitum. The serum lipid profile was measured on day 1 and day 32, after overnight fasting.
The body weight and food consumption of all test animals were monitored regularly.
Hypercholesterolemia progression test
The hypercholesterolemia progression test was carried out to evaluate the possible inhibitory influence of CETC and METC on the dietary lipid or cholesterol absorption, responsible for the observed antihypercholesterolemic effect during this test.
Thirty Wistar albino rats weighing between 200 and 220g (not more than 10% difference, within the groups) were randomly assigned to three groups (n = 6). Group I was a vehicle control group and was treated with vehicle (each animal 0.2 ml), Group II and III animals were treated with CETC (100 mg/kg b.w.) and METC (100 mg/kg b.w., p.o.). Group IV animals were treated with Ezitimibe (10 mg/kg b.w.). 
The test animals received the respective treatment orally, once a day, for a period of 10 days. During this period, they had free access to HFD (in place of a normal pelleted diet) and water ad libitum. The serum cholesterol level was measured on days 1 and 11, respectively, in overnight-fasted animals.
Hypercholesterolemia regression test
The hypercholesterolemia regression test was carried out to evaluate the possible acceleratory influence of CETC (100 mg/kg) and METC (100 mg/kg) on the metabolism of cholesterol and/or the inhibitory influence on cholesterol synthesis responsible for the observed antihypercholesterolemic effect.
Around 30-35 male, Wistar albino rats had free access to HFD for a month to induce hypercholesterolemia and, after confirming hypercholesterolemia, the selected animals were assigned to four groups (n = 6). Group I animals were treated with vehicle (0.2 ml /animal), Group II, III and IV animals were treated with CETC (100 mg/kg b.w.), METC (100 mg/kg b.w.) and Fenofibrate (65 mg/kg b.w.).respectively.
The test animals received their respective treatment, orally, once a day, for a period of 15 days and had free access to a normal pelleted diet and water ad libitum. The serum cholesterol level was measured on days 1 and 16, respectively, in overnight-fasted animals.
For the estimation of serum triglyceride (TG), total cholesterol (TC) and HDL-cholesterol, blood was collected from overnight-fasted animals by retro-orbital puncture under mild ether anesthesia and the serum was separated. Serum TG was measured by GPO-PAP, TC, according to CHOD-PAP and HDL-cholesterol by precipitation methods. All estimations were performed as per the instructions supplied with the diagnostic kit.
Estimation of LDL-cholesterol and VLDL-cholesterol 
LDL-cholesterol and VLDL-cholesterol was calculated using the following formula:
LDL-cholesterol = Total cholesterol - HDL-c + TG / 5 and
VLDL-cholesterol = TG / 5
Calculation of Atherogenic index 
Atherogenic index was calculated for each animal in various groups receiving either extract or standard lipid-lowering drug treatment at the end of the chronic study using following formula:
All data were expressed as mean ± SEM of n = 6. The statistical significance was evaluated by one-way ANOVA, followed by Dunnet's multiple tests using Graph Pad Prism (3.0). A value of P <0.05 and less was considered to be statistically significant.
| Results|| |
Changes in the lipid profile of hypercholesterolemic animals at the end of the study are tabulated [Table 1]. The vehicle-treated hypercholesterolemic animals recorded a significant increase (P < 0.05) in TG, TC, LDL-c and VLDL-c levels and a significant reduction in the HDL-c level.
|Table 1: Changes in the lipid profile of hypercholesterolemic animals due to various treatments|
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The extract-treated animals recorded a significant decrease (P < 0.05) in TG, TC, LDL-c and VLDL-c levels and a significant increase in the HDL-c level. Similar changes were also recorded by the statin- and fibrate-treated groups of animals. The lipid-lowering activity of the extracts at 100 mg/kg was comparable with that of statin and fibrate.
The results of the hypercholesterolemia progression test are tabulated in [Table 2]. The level of serum cholesterol in the vehicle-treated hypercholesterolemic animals continued to rise, and a significant reduction (P < 0.05) of the same was observed in the METC- and ezitimibe-treated animals. In the CETC-treated animals, the reduction in the serum cholesterol level was insignificant.
|Table 2: Effect of extracts and standard lipid-lowering drug treatment during the hypercholesterolemic progression test and the hypercholesterolemic regression test|
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The results of the hypercholesterolemia regression test are tabulated in [Table 2]. The vehicle-treated hypercholesterolemic animals recorded a slight fall in the serum cholesterol level at the end of the study, despite changing the diet to normal. On the contrary, in the CETC- and METC-treated animals, a significant (P < 0.05) reduction in serum cholesterol was observed and in the fenofibrate-treated animals, a similar effect was observed.
Changes in body weight, including the percentage change in body weight of hypercholesterolemic animals due to various treatments, are tabulated [Table 3]. At the end of the study, the vehicle-treated hypercholesterolemic animals recorded a 24% gain in body weight, and in the extract-treated animals, this was 15% and 11%, respectively. The rosvuastatin- and fenofibrate-treated animals' recorded a 13% and 15% gain in body weight, respectively. There was no change in the amount of feed consumed in any group of animals, except for a marginal reduction in the METC-treated hypercholesterolemic animals (not shown in the Table).
|Table 3: Change in body weight and food intake of hypercholesterolemic animals due to various treatments|
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The Atherogenic index of the test animals undergoing treatment with the extracts and the lipid-lowering drugs are tabulated [Table 4]. Untreated hypercholesterolemic control animals recorded a significantly (P < 0.01) higher atherogenic index compared with the normal vehicle-treated animals. On the contrary, the extract- and lipid-lowering drug-treated groups of animals recorded a significantly lower (P < 0.01) atherogenic index (compared with the hypercholesterolemic control animals).
|Table 4: Atherogenic index of hypercholesterolemic animals treated with extracts and lipid-lowering drugs|
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| Discussion|| |
The current study investigated the antihypercholesterolemic activity in hypercholesterolemic animals in the light of the reported hypocholesterolemic activity, and the study was extended to understand the possible mechanism of its antihypercholesterolemic effect by carrying out the hypercholesterolemia progression and regression tests. These tests evaluate the inhibitory influence of the extracts on the dietary lipid or cholesterol absorption, accelerating the influence of the extracts on lipid metabolism and or inhibitory influence on synthesis, respectively.
Hypercholesterolemic animals had a significantly elevated serum TC and LDL-c levels, which is likely to promote atherosclerosis, mimicking one of the risk factor for cardiovascular diseases. A significant reduction in elevated lipid levels and a rise in HDL-c level were observed, which may be beneficial in preventing atherosclerosis.
LDL carries cholesterol from the liver to the peripheral and smooth muscle cells of the arteries. A rise in LDL-c may cause deposition of cholesterol in the arteries and aorta, hence, a direct risk factor for coronary artery disease. , Further, low levels of HDL-c are also associated with a high risk of coronary artery disease  and, logically, raising the levels of lipoprotein HDL-c is an important target for therapy. A significantly reduced atherogenic index of the extract-treated hypercholesterolemic animals, in addition, was a beneficial effect. Further, an insignificant change in feed intake and survival of animals during and post-study indicates the safety of extracts in long-term use.
It is now a well-known fact that plasma lipid levels are determined by exogenous lipid absorption, endogenous lipid synthesis and its metabolism; therefore, are the common targets for synthetic lipid-lowering agents (statins and fibrates). The hypercholesterolemia progression test and the hypercholesterolemia regression test essentially considered these aspects, with the intention of understanding the influence of extracts on influencing the absorption of cholesterol or metabolism or both.
In the hypocholesterolemic progression test, the TC level was largely determined by an externally supplemented diet rich in cholesterol and in the hypocholesterolemic regression test, it was largely determined by endogenous cholesterol metabolism, when diet rich in cholesterol was replaced with a normal, pelleted diet.
The CETC- and METC- (100 mg/kg) treated hypocholesterolemic rats had a significant (P < 0.05) change in the entire lipid profile. Further tests, namely hypercholesterolemic progression and regression test, carried out to understand the possible mechanism of the observed antihyperlipidemic effect, reveled distinct as well as a possible mechanism shared by these extracts. Results of the hypercholesteromic progression test revealed that CETC had an insignificant effect on the serum cholesterol level, indicating an insignificant inhibitory influence on cholesterol absorption. On the contrary, METC treatment had resulted in a significant (P < 0.05) reduction in the TC level suggesting, a potential cholesterol absorption inhibitory property, like ezitimibe (cholesterol absorption inhibitor). This confirms the conclusions made in the preliminary report that CETC and METC treatment are likely to influence the endogenous biosynthesis of cholesterol, leading to hypocholesterolemic and antihypercholesterolemic effects. 
Results of the hypercholesterolemic regression test reveled that CETC as well METC treatment significantly (P < 0.05) reduced the serum cholesterol concentration, confirming that the observed antihyperlipidemic effect produced by both the extracts is likely to be due to a significant acceleratory influence on the metabolism of cholesterol and/or inhibitory influence on cholesterol biosynthesis.
In the light of the above findings, it is proposed that the observed antihypercholesterolemic nature of the extracts of Trichilia connaroides is probably due to a combination of the inhibitory effects on cholesterol absorption and accelerating its metabolism. Both CETC and METC extracts produced the observed antihyperlipidemic effect by mechanism that were similar to fenofibrate and, in addition, METC, like ezitimibe.
An increase in HDL-c and decrease in LDL-c due to chronic treatment, especially in hypercholesterolemic animals, is a specific benefit, considering the role of HDL-c in overall cardiovascular health. The chloroform extract of leaves and fruits of Trichilia connaroides is reported to contain tetracyclic triterpenes and tetranortriterpenoids. The significant health benefits of such phenolic compounds possessing a significant influence on human metabolism and on well-defined and appropriate biomarkers of oxidative stress, cardiovascular diseases, cancer etc., have been reviewed.  The methanol extract of the leaves is reported to contain a flavone glucoside;  therefore, the observed effect is likely to be due to this secondary metabolite. Trichilia connaroides is a promising herb with a potential in the management of dyslipidemia and prevention of cardiovascular consequences.
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[Table 1], [Table 2], [Table 3], [Table 4]
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