Antihyperhomocysteinemic and antihyperlipidemic effect of Trichilia connaroides in methionine-induced hyperhomocysteinemic animals.

The current study investigates the antihyperhomocysteinemic and antihyperlipidemic effect of chloroform and methanol extracts of the leaves of Trichilia connaroides in methionine-induced hyperhomocysteinemic rats. Hyperhomocysteinemia was induced in albino Wistar rats by oral administration of L-Methionine (1 gm / kg) and they were treated simultaneously with chloroform and methanol extracts (100 mg / kg) from the leaves of Trichilia connaroides. Serum homocysteine, lipid profile, and products of lipid peroxidation (MDA) in the heart homogenate were recorded and treated for statistical significance. Hyperhomocysteinemic animals recorded significantly elevated serum homocysteine changes in lipid profile (P < 0.01) and Thibarbituric acid reactive substances (P < 0.01), compared to the vehicle control animals. Animals treated with chloroform and methanol extracts recorded significantly (P < 0.01) lower serum homocysteine, entire lipid profile, LPO (P < 0.01), except a significant increase in HDL-cholesterol (P < 0.01) compared to hyperhomocysteinemic animals. Thus, we conclude that chloroform and methanol extracts of Trichilia connaroides have significant antihyperhomocysteinemic and antihyperlipidemic effects on methionine-induced hyperhomocysteinemic animals. Trichilia connaroides, therefore, holds promise as a cardioprotective herb.

Introduction

Methionine is one of the essential amino acids and methionine metabolism is the only known source for homocysteine in mammals. Excessive methionine uptake might lead to hyperhomocysteinemia (Hhcy).[1] Hhcy is regarded as an independent risk for cardiovascular events, a risk factor for atherosclerotic cardiovascular diseases, and is also associated with renal dysfunction and neuronal diseases among others.[2-5] Hhcy produces a direct toxic effect on the heart and is likely to cause oxidative stress in the heart and aorta, leading to atherosclerotic changes in experimental laboratory animals, fed with a methionine-rich diet.[67] There is a growing interest in the screening of herbs for potential antihyperhomocysteinemic activity.

Trichilia connaroides (Wight and Arn.) Bentv. [(Syn: Zanthoxylum connaroides (Wight and Arn.) Bentv., Heynea trijuga (Roxb.ex sims) (Family: Meliaceae)] is a rich source of bioactive tetracyclic triterpene (Heynic acid I and II) tetranorterpenoides (Trijugin-A and Trijugin-B), Trijugin B acetate,and pentanortriterpenoids, with a novel carbon skeleton (Trijugin C).[8-11] We have reported earlier that chloroform and methanol extracts of leaves have hypocholesterolemic, antihypercholesterolemic effects and cause elevated HDL-cholesterol in experimental animals.[12] Considering the potential role of this herb as being cardioprotective, the current study has been envisaged to investigate the antihyperlipidemic and antihyperhomocysteinemic effect of chloroform and methanol extract from the leaves of Trichilia connaroides in methionine-induced hyperhomocysteinemic animals.

Materials and Methods

Collection and processing of plant material

Fresh leaves of Trichilia connaroides were collected from the Jhamboti village near Belgaum (Karnataka), duly authenticated, and preserved (H1 / TC / 2007-8). Shade-dried leaves were coarsely powdered, and around 400 g of the powder was defatted with n-hexane (2.5 L), by leaving the powder with the solvent overnight, with occasional shaking, and filtered. The filtrate was shade-dried to evaporate the trace amount of hexane that remained. Next, it was successively extracted with chloroform and methanol (2.5 L of each), respectively, in a soxhlet extractor. The chloroform (CETC) and methanol extracts (METC) yielded 8 and 10%, respectively, and these were stored in airtight containers in a cool dry place, away from sunlight, till further use.

Drugs and Chemicals

Analytical grade solvents for extraction, namely n-hexane, chloroform, and methanol were purchased from M/s Qualigens, Mumbai. Other chemicals like Bovine serum albumin were from M/s Hi-Media, Mumbai, L-Methionine was from M/s Spectrochem, Mumbai. Folic acid, Thiobarbituric acid, Folin- Caecoltieu reagent, and Trichloroacetic acid were obtained from M/s CDH, New Delhi. Tween-80 was obtained from SD Fine Chemicals, Mumbai. Diagnostic kits for the estimation of lipid profile and homocysteine were from M/s Agapee Diagnostics, Kerala and M/s IBL-America, respectively.

Animals

Healthy, male, Wistar albino rats[13] (180–200 g) (not more than 10% difference within the groups) were purchased from a registered breeder (M/s Venkateshwara Enterprises, Bangalore) and maintained in the animal house facility of this institution, with temperature maintained between 18 and 29ºC and relative humidity maintained between 30 and 70%, with the light and dark cycle (12: 12) in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forestry, Government of India, The test animals received a commercial pelleted diet (M/s Gold Mohr, India) and water ad libitum. The animals were allowed a week for acclimatization. Ethics clearance was taken from the Institutional Animal Ethics Committee of this institution prior to experimentation (626 / 02 / a / CPCSEA).

Antihyperhomocysteinemic and antihyperlipidemic activity in hyperhomocysteinemic animals

The test animals were randomly assigned to five groups (Group I – V) (n = 8). Group I was a vehicle control group, which received 0.2 ml of Tween 80 in distilled water, group II was a hyperhomocysteinemic control animal group, which received L-Methionine (1 g / kg)[14] only. Groups III, IV, and V animals received L-Methionine (1 g / kg) + CETC (100 mg / kg), L-Methionine (1 g / kg) + METC (100 mg / kg), and L-Methionine (1g / kg) + Folic acid (100 mg / kg),[14] respectively, orally, each day for a period of ten weeks.

Estimation of serum homocysteine and lipid profile

Twenty-four hours after the last dose of the respective treatment, blood was collected from overnight fasted animals by retro-orbital puncture under mild ether anesthesia. The serum was separated and the lipid profile and homocysteine were estimated using a diagnostic kit. All estimations were performed as per instructions supplied with the commercial diagnostic kit.

Measurement of total protein content and lipid peroxidation (LPO) of the heart homogenate[1516]

The test animals were sacrificed by decapitation and the heart was carefully dissected, washed, cleared of blood clots, if any, and homogenized, and the protein content in the homogenized sample (0.5 ml) was estimated using the modified Lowry method suggested by Pomory, using standard albumin (BSA), and expressed as g / dL. The LPO was measured by estimating the thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation and expressed as n mol MDA / mg protein.

Statistical analysis

All data were expressed as mean ± SEM of n = 8. The statistical significance was evaluated by One- way ANOVA, followed by Dunnet's multiple tests using the Graph Pad Prism. Values of P < 0.05 and less were considered as statistically significant.

Results

Effect of extracts on serum homocysteine

The effects of extract treatment on serum homocysteine are shown in Table 1. Serum homocysteine levels were significantly elevated (P < 0.01) in methionine-induced hyperhomocysteinemic animals compared to normal vehicle-treated animals. CETC, METC, and folic acid-treated animals recorded significantly lower levels (P < 0.01) of serum homocysteine compared to hyperhomocysteinemic control animals. METC-treated animals recorded a greater fall in serum homocysteine level than CETC-treated animals.

Table 1

Effect of extracts treatment and folic acid on serum homocysteine and LPO / TBARS levels in hyperhomocysteinemic animals

Effect of extract treatment on lipid profile and LPO

Changes in the lipid profile and LPO of extract-treated animals are shown in Tables 1 and 2. Hyperhomocysteinemic animals had shown significantly elevated (P < 0.01) serum triglyceride, LDL-cholesterol, total cholesterol, and lowered HDL-cholesterol levels and products of LOP compared to the vehicle-treated animals. Extract-treated animals recorded significantly lower (P < 0.01) levels of triglyceride, LDL-cholesterol, and total cholesterol, as also products of LPO, and an elevated (P < 0.01) HDL-cholesterol level. A similar, significant (P < 0.01) reduction in lipid profile and products of LPO was also observed in folic acid-treated hyperhomocysteinemic animals.

Table 2

Effect of extract treatment on serum lipid profile of hyperhomocysteinemic animals

Discussion

Methionine-induced Hhcy in laboratory animals is one of the routinely used animal models to study various aspects of Hhcy. Our study reflects that methionine (1 mg / kg) has maximal dietary intake in human individuals, to elicit Hhcy, which is confirmed by the significantly elevated serum homocysteine levels. Homocysteine from the demethylation of dietary methionine is likely to generate partially reduced reactive oxygen species (ROS), capable of stimulating lipid peroxidation, involved in atherosclerotic processes. A significantly elevated level of serum homocysteine, lipid profiles, reduced levels of HDL-cholesterol, and LPO levels in the heart homogenate have been observed in hyperhomocysteinemic animals. Free radicals generated by Hhcy initiate the LPO of membrane-bound polyunsaturated fatty acids and impair the membrane structures and their functional integrity.[17] Elevated serum homocysteine and LPO in hyperhomocysteinemic animals confirms the same.

The current study has established the beneficial role of CETC and METC as antihyperhomocysteinemic and antihyperlipidemic in hyperhomocysteinemic animals. Considering the observed effect together, the results are suggestive of the cardioprotective role of these extracts in Hhcy animals. The observed activity is likely to be due to the presence of several bioactive constituents and / or flavonol glucoside.[18]

Conclusion

Chloroform and methonolic extracts of Trichilia connaroides possess a significant amount of antihyperhomocysteinemic and antihyperlipidemic activity in hyperhomocysteinemic animals.