INTRODUCTION:
Plant medicine or phytomedicine (the sum total of practices, using herbal preparations produced by physical or biological processes, to prevent, cure, or control diseases relying on past experience/ observations handed down from generation to generation) has been used in healthcare delivery in many parts of East Africa and the rest of the world. Effective health cannot be achieved in East Africa, unless orthodox medicine is complemented with traditional medicine1. At least 80% of African depends on plant medicine for their healthcare2-11. Today, medicinal plants are increasingly being used in most parts of the world as: hypolipidemic12-14; contraceptive, abortifacients, emmenagogues or oxytocic15; antihypertensive16-19; treatment for skin diseases10, wound healers20; antimicrobial6,11,16,21; antiulcer22,23 and hypoglycemic13,14,24-32.
Hyperlipidemia has been implicated in atherosclerosis, which is the primary cause of heart disease and stroke. Many hypolipidemic drugs have already been proved to be useful in lowering serum lipid levels in patients. However, its side effects in long-term treatment have been frequently reported and its prices are still pricey. Thus, efforts to develop effective and better hypolipidemic drugs had led to the discovery of natural agents.
The alligator pear unflatteringly known as avocado, midshipman's butter, vegetable butter, or sometimes as butter pear, is the only important edible fruit of the laurel family, Lauraceae belongs to Dicotyledoneae class. It has traditionally been used for antibacterial, antifungal, hypotensive, and anti-inflammatory and anti-immune enhancer effect. Furthermore, avocado juice, which is made from the ripe fruit, was very popular of its numerous health benefits. The avocado tree may be erect, usually to 30 ft (9 m) but sometimes to 60 ft (18 m) or more, with a trunk 12 to 24 in (30-60 cm) in diameter, (greater in very old trees) or it may be short and spreading with branches beginning close to the ground. Almost evergreen, being shed briefly in dry seasons at blooming time, the leaves are alternate, dark-green and glossy on the upper surface, whitish on the underside; variable in shape (lanceolate, elliptic, oval, ovate or obovate), 3 to 16 in (7.5-40 cm) long. Because limited reports on avocado were available in the literature, it was thought to be desirable to carry out systematic investigations on the leaf and fruit of this plant33. Information provided by practitioners of Ethiopian traditional medicine suggests that Avocado fruit and leaf possesses hypolipidemic activity. The aim of this present study is to investigate and evaluate the hypolipidemic effect of avocado leaf and fruit extract on Tyloxapol- induced hyperlipidemia in rats, to provide scientific evidence for development of P.americana as a potential natural oral hypolipidemic agent or functional food.
Chemical composition: According to Kadam, and Salunkhe, the avocado has a high lipid content of around 18%. Among the saturated fatty acids, myristic level may be, 1%, palmitic, 7.2%; stearic, 1.7%. Of the unsaturated fatty acids, palmitoleic may range from 5.5 to 7.5%; oleic may be 60.9%, linoleic, 14.3%. Non saponifiable represents 1.6 to 2.4%. Iodine number is 94.4. Amino acids of the pulp (N = 16 p. 100) are recorded as: arginine, 3.4; cystine, 0.1: histidine, 1.8; isoleucine, 3.4; leucine, 5.5; lysine, 4.3; methionine, 2.1; phenylalanine, 3.5; threonine, 2.9; tryptophan, 0.1 ; tyrosine, 2.3; valine, 4.6; aspartic acid, 22.6; glutamic acid, 12.3; alanine, 6.0; glycine, 4.0; proline, 3.9; serine, 4.1 .
MATERIALS AND METHODS:
Plant Materials:
1. P.americana leaves extract preparation (PAL)
Alligator pear leaves were selectively collected and validated in the Tepi Agricultural Research Center, Tepi. The leaves were washed with water, shade dried and then oven dried at 40 0C. The dried leaves were later pulverized. The powder leaves were extracted in soxhlet apparatus using respective two solvents, dichloromethane and methanol. The solid extracts thus obtained were preserved in a refrigerator until ready for use. The dose of the extract administered was given on a kg b.w. basis.
2. P.americana fruits extract preparation (PAF)
Fresh fruits of P.americana were selectively collected and authenticated in the Tepi Agricultural Research Center, Tepi. The skin was peeled off and the edible part was chopped into thin pieces, dried at 50-600C, and ground into powder. Known amount of dried amount was exhaustedly extracted by the process of maceration in an aspirator using respective two solvents, dichloromethane and methanol as menstruum. The extracts were concentrated under reduced pressure by rotary evaporator to obtain thick syrup mass, and stored at 40C. The yield was approximately 65% of fresh fruit. Working concentration of the two different extracts were made in non-pyrogenic distilled water before use in the experiments.
Chemicals /Reagents:
Tyloxapol (Triton WR-1339) was purchased from Sigma-Aldrich Chemie GMBH, simvastatin 20 mg from Merck Sharp and Dohme, cholesterol reagent and triglyceride reagent from Biolabo, France
Experimental animals:
The animal experiments were designed and conducted according to the ethical norms approved by Ethiopian Government and Institutional Animal Ethical Committee (IAEC) for the investigation of experimental pain in conscious animals. Before beginning the experiments, the albino rats were allowed to acclimatize to animal house condition for a period of one week. Male Wistar Albino rats weighing 180 ± 20 g were used. Throughout the experimental period, the rats were fed with balanced commercial pellet diet with composition of 5% fat, 21% protein, 55% nitrogen-free extract and 4% fiber (w/w) with adequate mineral and vitamin levels for the animals. Diet and water were provided ad libitum.
Toxicity and dosage fixation studies:
Acute toxicity studies on P.americana fruit and leaf extracts were performed in experimental animals. Graded doses of Methanolic and dichloromethane solvent extracts of avocado fruits and leaves (100, 250, 500 and 1000mg dry extract/kg.b.w.) were administered orally and the animals were monitored for two weeks following administration, and observed nil mortality of experimental animals.
Dosage fixation studies were carried out by administering graded doses of avocado fruit and leaf extracts (100, 200, 300, 400 and 500mg extracts/ kg b.w. / rat /day) to Tyloxapol-induced hyperlipidemic experimental rats for various time periods. It was found that both the extracts showed optimum hypolipidemic efficacy at a concentration of 300mg/kg b.w. administered orally for 30days. Hence the dosage schedule was fixed as 300mg/kg b. w./rat/day for 30 days.
Experimental design:
Animals were randomly divided into seven groups of six rats each. The rats in Group-I received saline as control group (0.1 ml/ Kg body weight) while those of other groups were injected intra-peritoneally 3% Tyloxapol solution with a single dose of 300 mg/kg b.w. to induce hyperlipidemia. Treatment was conducted immediately after Tyloxapol injection. The rats in Group -II were untreated while in Group -III were administered orally 80 mg of simvastatin /kg b.w., once per day. Animals in Group IV and V were administered orally dichloromethane (PAL-I) and methanol (PAL-II) extracts from leaf, respectively at doses of 300mg of dried extract/kg, once per day. Animals in Group VI and VII were administered orally dichloromethane (PAF-I) and methanol (PAF-II) extracts from fruit, respectively at doses of 300 mg of dried extract/kg once per day.
Biochemical parameters:
Blood samples were taken from tail vein. Serum was analyzed for total cholesterol estimation by Parekh and Jung34, and triglyceride levels by Rice35 after 24-hour Tyloxapol injection by using the enzymatic-colorimetric method.
Statistical Analysis:
All the grouped data were statistically analyzed with SPSS/ 16.0 software. Hypothesis testing methods included one way analysis of variance followed by post hoc test least significant difference (LSD) test. The p < 0.05 was considered to indicate statistical significance. All the results were expressed as mean ± SEM for six rats in each group.
RESULTS:
Lipid profiles of serum total cholesterol and triglycerides of experimental rats from various groups are summarized in Table 1.The results show that total cholesterol and triglyceride levels were significantly increased in tyloxapol injected rats as compared to those of saline control rat after 24-hour injection of tyloxapol or saline solution. Cholesterol and triglycerides levels of tyloxapol injected rats were more than two folds higher compared to those of control rats. Treatment with any herbal extracts caused a considerable reduction in the total cholesterol and triglyceride levels, comparable to simvastatin.
Table 1 . Total cholesterol and triglyceride levels in control and experimental groups of rats
|
Groups |
n |
Cholesterol(mg/dl) |
Triglyceride (mg/dl) |
|
Saline Control |
6 |
110.68 |
189.32 |
|
Tyloxapol (3%) |
6 |
245.45 |
513.43 |
|
Simvastatin (reference drug) |
6 |
117.12 |
210.04 |
|
PAL-I extract |
6 |
111.84 |
202.09 |
|
PAL-II extract |
6 |
113.18 |
212.63 |
|
PAF-I extract |
6 |
148.52 |
216.85 |
|
PAF-II extract |
6 |
149.66 |
300.01 |
Values are given as mean ± SEM for groups of six rats each; Values are statistically significant at p<0.05. Statistical significance (*) determined by ANOVA was compared as follows: a rats compared with saline control group; b rats compared with reference drug group.
Figure1. Hypolipidemic effect of alligator pear leaf extracts in control and experimental groups of rats. *p<0.05 as compared to tyloxapol injected group.
Hypolipidemic Effect of Extracts from Leaves
Our data indicated that the two extracts from leaves reduced remarkably cholesterol and triglyceride levels in experimental rats (Figure 1). No difference was found between cholesterol and triglyceride levels of PAL-I and PAL-II treated groups and those of simvastatin treated group, suggesting the equivalence of hypolipidemic effect of PAL-I, PAL-II and simvastatin. Thus, these three experimental treatments had action in diminution of high cholesterol and high-triglyceride levels induced by tyloxapol.
Hypolipidemic Effect of Extracts from Fruit:
Results show that cholesterol and triglyceride levels of treated groups either with simvastatin or with the extracts from fruit PAF-I and PAF-II, reduced significantly as compared to tyloxapol-induced hyperlipidemic untreated group (Figure 2). There was no notable difference among cholesterol levels of PAF-I and PAF-II treated rats and that of simvastatin treated rats. However, hypotriglyceridemic activity of PAF-II was less than that of simvastatin.
Figure2. Hypolipidemic effect of alligator pear fruit extracts in control and experimental groups of rats.*p<0.05 as compared to tyloxapol injected group. ≠p<0.05 as compared to Simvastatin treated group.
Table 2. Reduction in cholesterol and triglyceride levels in treated rats as compared to tyloxapol injected rats
|
Groups |
Reduction in Cholesterol level (%) |
Reduction in Triglyceride level (%) |
|
Simvastatin (reference drug) |
61.11 |
62.94 |
|
PAL-I extract |
59.743a |
63.52a |
|
PAL-II extract |
58.01a |
61.12a |
|
PAF-I extract |
44.82a |
59.98a |
|
PAF-II extract |
43.09a |
43.67a |
Values are given as mean ± SEM for groups of six rats each; Values are statistically significant at p<0.05. Statistical significance (*) determined by ANOVA was compared as follows: a rats compared with reference drug group.
Comparison of Hypolipidemic Effect of Extracts from Leaf and Fruit:
A notable reduction in cholesterol levels of rats treated with the extracts was observed 24 hours after tyloxapol injection. Cholesterol levels decreased 59.74%, 58.01%, 44.82% and 43.09%, respectively, in groups orally administered PAL-I, PAL-II, PAF-I and PAL-II as compared to the Tyloxapol injected group 61.11% (Table 2). Furthermore, there was no significant difference among groups. With regards to triglyceride levels, the statistic results show that PAF-II treated group was significantly different in comparison with the other three extract treated groups (p < 0.05). Hence, hypotriglyceridemic activity of PAF-II was the least among all extracts.
DISCUSSION:
This study was performed to investigate the hypolipidemic effect of extracts from P.americana on tyloxapol-induced hyperlipidemia in experimental albino rats. Preliminary experiments demonstrated that 24 hours after administration, all four extracts from leaves by dichloromethane (PAL-I) and by methanol (PAL-II), as well as, from fruits by dichloromethane (PAF-I) and by methanol (PAF-II) remarkably reduced the cholesterol and triglyceride levels in the plasma of hyperlipidemic rats. The hypocholesterolemic effect of all extracts was comparable to the effect of simvastatin. The hypotriglyceridemic effect of PAL-I, PAL-II, and PAF-I was also comparable to the effect of simvastatin, while that of PAF-II was lower than simvastatin.
On the other hand, according to experimental data, cholesterol and triglyceride levels in PAL-I and PAL-II treated groups were lower than those of other treated groups including simvastatin treated group. This hypothesizes an orientation about the extraction and the purification of principal ingredients possessing good hypocholesterolemic and hypotriglyceridemic activity from leaf extracts of avocado.
Considering cholestyramine (bile acid-binding, cholesterol-lowering drug) as 100% bound, the relative in vitro bile acid binding on dry matter and total dietary fiber basis was 20% and 60%, respectively. Bile acid binding for P.americana was significantly higher than for all the other vegetal. The results put forward its hypolipidemic effect by decreasing absorption of cholesterol from diet.
In the present study, tyloxapol−a detergent agent was used to produce hyperlipidemia in rats. Its mechanism of action is known to accelerate the hepatic cholesterol synthesis in phase I (after 24-hour tyloxapol injection).36, 37, Hence antihyperlipidemic effect of extracts from leaf and fruit of P.americana could be due to interfering with cholesterol biosynthesis. More studies are needed to elucidate hypolipidemic activity of these extracts for the purpose of application of P.americana in hyperlipidemia treatment and prevention.
CONCLUSION:
In conclusion, extracts from leaf of P.americana by dichloromethane (PAL-I) or methanol (PAL-II) and extracts from fruit by dichloromethane (PAF-I) or methanol (PAF-II) possessed hypolipidemic activity in tyloxapol-induced hyperlipidemia in rats. The extracts may be useful in lowering cholesterol and triglyceride levels in hyperlipidemia. It could also be speculated that the observed hypolipidemic effects of P.americana fruit as well as leaf extracts might be related to the rich phytochemicals such as flavonoids, phenolic acids, alkaloids, steroids, proteins and vitamins with strong free radical scavenging properties. The process of extraction and identification of active principles responsible for the observed pharmacological actions of P.americana fruit as well as leaf extracts through bioactivity guided fraction is under progress to understand the possible mechanism of action of P.americana fruits and leaves.
ACKNOWLEDGMENT:
The authors thank the President, Vice Presidents and Campus Dean of Mizan -Tepi University, Tepi, for their motivation and constant encouragement. The authors extend their thanks to Mr. G. Manoharan, Lecturer, who assisted in calculating the statistical data.
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Received on 11.10.2010 Modified on 09.11.2010
Accepted on 28.11.2010 © AJRC All right reserved
Asian J. Research Chem. 4(4): April, 2011; Page 574-578-706