Clinical Evaluation to Assess the Efficacy of Ethanolic Extract of Avocado Fruit on Diabetic Dyslipidemia Studied in STZ- Induced Experimental Albino Rats

 

U. S. Mahadeva Rao1*, R. Babujanarthanam2 and B. Arirudran3 

1Asso.Professor and Head, P.G. Department of Biochemistry, SRM College of Arts and Science, Chennai, India.

2Head, Department of Biochemistry, KMG College of Arts and Science, Gudiyattam. India.

3Assistant  Professor,  P.G. Department of Biochemistry, SRM College of Arts and Science, Chennai, India. *Corresponding Author E-mail: raousm@gmail.com

 

ABSTRACT:

In Diabetes mellitus, the insulin defect reflects in elevated gluconeogenic metabolite accumulation, which leads to excess acetyl Co-A storage, and, in turn, the acetyl Co-A  acts as a precursor of lipids directly, and  lipoprotein indirectly, synthesis. Thus, hyperlipidemia is an associated complication of diabetes mellitus. Recently, we have reported that ethanolic extract of Persea americana lowered the blood glucose and oxidative stress in diabetic rats. In this present study, the hypolipidemic effect of Persea americana fruit was investigated in streptozotocin-induced diabetic rats. Oral administration of fruit extract (300 mg/kg b.w.) for 30 days resulted in significant reduction in serum and tissue cholesterol, triglycerides, phospholipids and free fatty acids in STZ-diabetic rats. In addition to that, significant decrease in high density lipoprotein while significant increase in low density lipoprotein  and very low density lipoprotein, were observed in diabetic rats, which were brought to near normal after 30 days of herbal extract treatment. The results obtained are comparable with glyclazide, a standard drug. Results of the present study indicate that the fruits of Avocado showed antihyperlipidemic effect in addition to its antidiabetic and free radical scavenging activity properties in type 2 diabetic rats.

 

KEYWORDS: Avocado; Acetyl Co-A; hyperlipidemia; lipoproteins; gluconeogenic; Persea americana

 


 

INTRODUCTION:

Diabetes mellitus is a common disease in man. A predisposition to the disease is probably inhabited as an autosomal recessive trait. About 25% of family relatives of diabetics show diminished glucose tolerance as compared to 1% in the general population. It is a chronic disorder generally due to disordered principal essential energy nutrients metabolism, cause of which is deficiency, or depleted effectiveness of insulin, resulting in hyperglycemia and glycosuria, in particular1.

 

Thus, Diabetes mellitus (DM) is the world’s largest endocrine disorder as old as mankind affecting carbohydrate, fat and protein metabolism. It is associated with a two to four fold increased risk of coronary artery disease. Studies indicate that patients with type 2 diabetes mellitus who have no history of coronary artery disease have the same risk for cardiac events as do nondiabetic patients with pre-existing coronary artery disease.

 

This emphasizes the extensive but silent nature of coronary artery disease in patients with diabetes mellitus2.

 

Therefore, search for a drug having twofold properties, that is lowering of blood lipids and glucose together is in great demand. Despite of remarkable advancement in the management of diabetes by synthetic drugs, there has been a renewed interest in medicinal plants because they do not elicit any side effects3.

 

Persea americana Miller (syn. P.gratissima Gaertn), belong to Lauraceae family commonly called avocado or alligator pear, is a small tree with gray trunk. Originating from Central America, it was introduced in East Africa (Ethiopia).  The fruit of this plant are used as an herbal medicine.  The amount of simple sugars in the avocado fruit is low, but in contrast, it contains appreciable levels of dietary fiber (DF)4. These modifications may resolve constipation, reduce fat absorption, lower glycemic index and plasma insulin levels, alter colon fermentation and microbial proliferation, and reduce plasma cholesterol5. Therefore, adding recommended levels of DF to the diet is considered vital for normal intestine performance, good health, and for controlling major risk factors for diabetes, obesity, gallstones, hypercholesterolemia and heart disease6.

However, scientific evidences for the pharmacological properties of the avocado fruit are limited. Recently, we have evaluated the antidiabetic and antioxidant potential of avocado fruits in streptozotocin-induced experimental diabetes in rats. The present study was aimed to investigate the hypolipidemic properties of avocado fruits in diabetic rats.

 

MATERIALS AND METHODS:

Plant Material and Processing:

Samples of ripe alligator pear fruits were purchased and authenticated from Tepi Agricultural Research Center, Tepi. The samples were washed with clean tap water to remove dirt on the fruits. Known amount of dry powder was repeatedly extracted by the process of maceration in an aspirator using 95% ethanol as menstruum. The extract was concentrated under reduced pressure by rotary evaporator to obtain thick syrup mass and stored at 40C. The yield was approximately 65% of fresh fruit.  The aqueous extract was prepared freshly at the time of administration. (vide certificate No. ETARC 102/08/02).

 

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 160 – 180 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.

 

Induction of experimental diabetes:

Rats were fasted overnight and experimental diabetes was induced by single intra peritoneal injection of STZ with a dose of 50mg/kg b.w. STZ was dissolved in a freshly prepared 0.1M cold citrate buffer pH 4.57. Control rats were similarly injected with 0.1M cold citrate buffer; pH 4.5. Because STZ is capable of inducing fatal hypoglycemia as a result of massive pancreatic insulin release, STZ-administrated rats were provided with 10% glucose solution after 6h for the next 24h to prevent hypoglycemia. Neither death nor any other adverse effect was observed. After 3 days for the development and aggravation of diabetes, rats with moderate diabetes (i.e., blood glucose concentration >250mg/dl) that exhibited glycosuria were selected for the experiment8.

 

Experimental design:

The animals were divided into four groups, comprising of six animals in each group as follows: Control rats (Group I), STZ-induced diabetic control rats (Group II), Diabetic rats treated with avocado fruit extract (300mg / kg b.w. / day) in aqueous solution for 30 days (Group III), and Diabetic rats treated with reference drug, glyclazide (5mg/kg b.w. /day) in aqueous solution for 30 days (Group IV)9. After 30days of treatment, rats were fasted overnight and sacrificed by cervical dislocation. Blood was collected with and without anticoagulant.

 

Biochemical parameters:

Fasting blood glucose was estimated by the method of O-toludine10, glycosylated hemoglobin by the method Nayak and Pattabiraman11 and plasma insulin was assayed by RIA assay kit (for rats) supplied by Linco Research, Inc., USA. The liver and kidney tissues of control and experimental groups of rats were excised, rinsed in ice cold saline and homogenized in 0.1M Tris-HCl buffer, pH 7.4 with a Teflon homogenizer. The total lipids were extracted from the liver and kidney tissues according to the methods of Folch et al12. Total cholesterol of plasma, liver and kidney was estimated by the method of Parekh and Jung13, triglycerides was estimated by the method of Rice;14 free fatty acids was estimated by the method of Hron and Menahan15, and phospholipids was estimated by the method of Bartlette16. After digestion with perchloric acid and the phosphorous liberated was estimated by the method of Fiske and Subbarow17. Serum HDL-cholesterol (HDL-C) was estimated by the method of Burstein et al.18 Serum VLDL-cholesterol (VLDL-C) and LDL-cholesterol (LDL-C) were calculated according to method of Friedewald et al19. VLDL-C = TG/5 and LDL-C = Total cholesterol – (HDL-C + VLDL-C) both values are expressed as mg/dl. Analysis of fatty acid composition in the lipid extract of liver and kidney tissues were performed in gas chromatography according to the method of Morrison and Smith20.

 

Statistical analysis:

All the grouped data were statistically analyzed with SPSS v16.0 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 AND DISCUSSION:

Table 1 depicts the levels of fasting blood glucose, glycosylated hemoglobin and plasma insulin in the control and experimental groups of rats. The elevated levels of blood glucose and glycosylated hemoglobin in the diabetic group of rats were significantly normalized by the oral administration of avocado fruit extract for 30 days, while the decreased plasma insulin level also elevated notably.

 

Table 2 represents the serum lipoproteins levels in the control and experimental groups of rats. The levels of VLDL-C and LDL-C were elevated significantly whereas the HDL-C was declined significantly in the diabetic group of rats. Oral treatment with avocado fruit extract as well as glyclazide to diabetic group of rats reverted back the altered levels to near normalcy.

Table 1. Levels of fasting Blood Glucose, Glycosylated hemoglobin and Plasma Insulin in the control and experimental groups of rats

Group

Fasting blood

glucose (mg/dl)

Glycosylated hemoglobin (%Hb)

Plasma

insulin (µU/ml)

Group-I

94.46 ± 5.18

5.58± 0.48

13.12±1.14

Group-II

269 ±14.18a

12.29 ±0.39a

3.68 ±0.85a

Group-III

129.89 ±8.69b

7.57 ±0.75b

9.35 ±0.68b

Group-IV

114.27 ±5.19b

9.69 ±0.63b

12.55 ±0.19b

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 within the groups as follows: a rats compared with Group-I; b rats compared with Group-II.

 

Table 2. Levels of Lipoprotein profile in the control and experimental groups of rats

Group

VLDL-Cholesterol (mg/dl)

LDL-Cholesterol (mg/dl)

HDL-Cholesterol (mg/dl)

Group-I

14.64 ± 0.18

45.68 ±3.48

23.92±2.14

Group-II

59.87 ±2.18a

119.39 ±8.39a

13.48 ±1.85a

Group-III

29.89 ±1.69b

77.07 ±1.75b

19.75 ±0.98b

Group-IV

25.37 ±3.19b

59.89 ±3.63b

22.59 ±3.17b

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 within the groups as follows: a rats compared with Group-I; b rats compared with Group-II.

 

Figure1. Levels of Fatty acid composition in the liver tissues of control and experimental groups of rats.

( 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 within the groups as follow: a rats compared with Group-I; b rats compared with Group-II).

 

The levels of lipids profile in the serum, liver and kidney tissues of control and experimental groups of rats are shown in Tables 3, 4, 5. The levels of total cholesterol, triglycerides, phospholipids and free fatty acids were significantly increased in diabetic group of rats. However, the oral treatment with avocado fruit extract as well as glyclazide significantly ameliorated these elevated levels into near normal levels. Figure 1 and 2 represent the levels of palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linolenic acid (18:3) and Arachidonic acid (20:4) in liver and kidney tissues respectively, of control and experimental groups of rats. A significant increase in the levels of palmitic acid, stearic acid and oleic acid were noticed both in liver as well as kidney tissues of diabetic rats. Conversely, the levels of essential fatty acids such as, linolenic acid and arachidonic acid were decreased in the diabetic group of rats. However, these altered levels were brought back to near normal level after treatment with herbal extract as well as reference drug.

 

Figure2. Levels of Fatty acid composition in the kidney tissues of control and experimental groups of rats

(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 within the groups as follow: a rats compared with Group-I; b rats compared with Group-II).

 

Cardiovascular complications are the leading cause of morbidity and mortality in diabetes and are often associated with hyperlipidemia. Hyperlipidemia is an important modifiable risk factor for the development and progression of cardiovascular disease. Compelling evidence also supports an independent link between low HDL-C levels21 and high TG levels22, 23 and atherosclerosis. Epidemiological studies also provide the largest body of evidence for the relationship between serum total cholesterol and cardiovascular complications. Statins, the preferential lipid lowering drug for majority of diabetic patients with dyslipidemia are associated with incidence of adverse effects24. Hence search for a noval therapeutic agent, which is capable of acting as both antidiabetic as well as antihyperlipidemic continues. Plants are important source of potentially useful structures for the development of drugs. STZ-induced experimental diabetes in rats is frequently used to study the disturbances in lipid metabolism under diabetic conditions25.  Based on our earlier study26 the treatment dosage schedule was fixed as 300 mg/kg b.w./rat/day for 30 days. Glyclazide, a universally practiced sulfonylurea used in the treatment of diabetes, was used as a reference drug27.

The observed decrease in the levels of blood glucose and glycosylated hemoglobin with a concomitant improvement in the level of plasma insulin in the avocado fruit extract treated group of rats indicates the antihyperglycemic activity of avocado fruit extract. Similarly the observed increase in the level of plasma insulin in the present study indicates that avocado fruit extract stimulates insulin secretion from the remnant ß-cells and or from regenerated ß-cells.


 

Table 3. Levels of Serum Lipid profile in the control and experimental groups of rats

Group

Total cholesterol

Triglycerides

Phospholipids

Free fatty acids

Group-I

83.89 ± 16.12

78.47±17.78

112.23±8.82

1 4.87±1.34

Group-II

234.67 ± 23.19a

144.87±20.72a

212.28±7.23a

49.34±2.45a

Group-III

134.75±23.82b

107.66±13.25b

163.04±4.57b

23.56±0.99b

Group-IV

133.15±18.42b

115.63±17.85b

159.12±6.23b

23.29±1.55b

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 within the groups as follows: a rats compared with Group-I; b rats compared with Group-II.

 

Table 4. Levels of Lipid profile in the hepatic tissue of control and experimental groups of rats

Group

Total cholesterol

Triglycerides

Phospholipids

Free fatty acids

Group-I

9.29 ± 0.12

8.07±0.98

22.23±0.62

2.89.09±0.24

Group-II

14.37 ±0 .23a

14.83±0.92a

52.24±3.13a

9.04±0.65a

Group-III

10.55±0.92b

8.96±0.35b

39.07±1.97b

4.96±0.79b

Group-IV

11.15±0.82b

10.43±0.87b

28.16±3.03b

3.99±0.25b

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 within the groups as follows: a rats compared with Group-I; b rats compared with Group-II.

 

Table 5. Levels of Lipid profile in the kidney tissue of control and experimental groups of rats

Group

Total cholesterol

Triglycerides

Phospholipids

Free fatty acids

Group-I

5.19 ± 0.42

7.01±0.34

16.88±1.02

2.99±0.28

Group-II

15.17 ±1 .02a

13.38±0.82a

32.29±2.03a

9.79±0.61a

Group-III

11.05±0.41b

9.06±0.48b

29.67±0.67b

5.91±0.29b

Group-IV

8.95±0.66b

8.13±0.29b

22.56±2.04b

4.09±0.51b

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 within the groups as follows: a rats compared with Group-I; b rats compared with Group-II.

 

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In this context, a number of medicinal plants have been reported to have antihyperglycemic and insulin releasing stimulatory effect28-30.

 

During diabetes significant alterations in the concentrations and compositions of lipids has been widely reported31 and is characterized by marked hyperlipidemia. It has also been well documented that the experimental rats induced with STZ had increased plasma cholesterol and triglyceride levels through insulin deficient and hyperglycemic states32,33.

 

Hypercholesterolemia and hypertriglyceridemia are the associated features of diabetes34,35 Elevated level of cholesterol is a powerful risk factor for many coronary diseases. The degree of hypercholesterolemia is directly proportional to severity in diabetes [36]. In the present study, we have observed elevated levels of cholesterol in the serum of diabetic group of rats. The abnormal high concentrations of plasma lipid components in diabetes is mainly due to the increase in the mobilization of free fatty acids from the peripheral depots in the absence or deficiency of insulin, since insulin inhibits hormone sensitive lipase. On the other hand, glucagon, catecholamine and other hormones enhance lipolysis. Our finding of increased plasma lipids correlated with the above finding of hyperlipidemia in diabetes. Treatment with avocado fruit extract to diabetic rats resulted in the correction of hyperlipidemia and this may be attributed to the enhanced glucose utilization. Avocado fruit extract, which contain many biologically active phytochemicals, such as alkaloids, glycosides, polyphenols, amino acids, tannins, saponins, sterols, triterpenes etc., enhances glucose metabolism by increasing glycolysis and glycogen synthesis and decreasing gluconeogenesis and glycogenolysis. Thus, the oral administration of avocado fruit extract to diabetic group of rats optimizes glucose utilization and decreases mobilization of fat depots, which decreases hyperlipidemia.

Virtually every lipid and lipoprotein component is affected by insulin resistance and diabetes mellitus37,38. The abnormalities mainly include increased levels of VLDL-C, LDL-C and low level of HDL-C39,40. These abnormalities appear to result from increased hepatic secretion of VLDL particles due to increased concentration of free fatty acids and glucose, reduced VLDL clearance due to reduced activity of lipoprotein lipase and reduced LDL clearance due to glycation of ligand proteins41. An increase in the levels of LDL-C and VLDL-C and concomitant decrease in HDL-C level in serum of diabetic rats as reported by us is in par with the above studies. Triglyceride enrichment changes the composition of all lipoproteins and makes them better substrates for hepatic lipase. This leads not only to decreased levels of HDL-C but also to small, dense LDL particles42. The atherogenicity of small LDL particles is attributed to their increased susceptibility to oxidation.43. Hypertriglyceridemia is a marker for insulin resistance and the constellation of atherogenic metabolic abnormalities that exists in this condition. The triglyceride enriched lipoproteins in patients with insulin resistance and type 2 diabetes can, because of their relatively small size, easily enter the blood vessel wall, where they are oxidized; they then bind to specific receptors on macrophages and are phagocytized, leading to the sequences of events that cause the atherosclerotic lesions44.

 

In addition, hyperglycemia brings other factors into the scenario that aggravates pre-existing lipoprotein abnormalities. These include, glycation of apoproteins and lipoprotein receptors, decreased levels of lipoprotein lipase and increased activity of hepatic lipase, alterations in the matrix composition of the blood vessel, increased lipoprotein oxidation and other changes in vascular biology that contribute to the development of atherogenesis2. Avocado fruit extract through its normoglycemic effect, decreases glycation of proteins and restores lipoprotein levels.

 

Phospholipids are integral part of biomembranes rich with polyunsaturated fatty acid, which are susceptible substrate for free radicals. Increased phospholipid contents in tissues and serum were reported in STZ-induced diabetic rats45. Further, the observed elevation in the phospholipid levels is consequence of elevated lipoproteins. The restoration of phospholipids by avocado fruit extract may be due to improvement in the levels of insulin secretion and free radical scavenging activity (FRSA).

 

Diabetes results in a decrease in glucose utilization and increase in glucose production in insulin-dependent tissues46. The liver tissue regulates the uptake, oxidation and metabolic conversion of free fatty acids, synthesis of cholesterol and phospholipids and secretion of specific classes of serum lipoproteins. Persistent hyperlipidemia alters lipid metabolism in kidney,47, 48 which ultimately leads to initiation and progression of renal injury. A marked increase in the hepatic and renal lipid concentration has been observed in the present study. During diabetes the hormone sensitive lipase is activated and increases the removal of free fatty acids from adipose tissues, thereby providing substrates for triglycerides synthesis in the liver49. Earlier reports also suggested that the lipid profile in diabetic liver tissues were severely altered due to the diminished activity of the lipogenic enzyme glucose-6-phosphate dehydrogenase50 which substantially reduces the oxidation of the hydrogen shuttle system and the redox state of the cell46 and hence increased lipolysis. The effect of avocado fruit extract on lipids in diabetes could be mainly through its control over hyperglycemia.

 

Fatty acids form an important component of cell membranes, eicosanoid precursors and are therefore required for both the structure and function of every cell in the living system. Diabetes produces specific alterations in the fatty acid profile of several tissues and membranes in both humans and the STZ induced diabetic animals that have been related to the reduced activities of insulin sensitive hepatic 9 and 6-desaturases51. These alterations in fatty acid composition could be an important factor in the development of diabetic complications such as cataracts, neuropathy and coronary heart diseases. Oral administration of avocado fruit extract was found to decrease the concentrations of total saturated fatty acids in the tissues of diabetic rats.

 

A significant decrease in the levels of polyunsaturated fatty acids in diabetic rat tissues may be attributed to the diminished activity of 5 desaturase activity. This results in the impaired formation of linolenic acid and stearidonic acid from linoleic acid. Thus, the reduced availability of essential fatty acid intermediates in diabetes is further exacerbated by increased destruction due to increased generation of reactive oxygen species (ROS)52. The increased generation of potential free radicals induced by hyperglycemia leads to damage of plasma membrane, which results in degradation of phospholipids and polyunsaturated fatty acids. Mitigation of free radical mediated oxidative stress by avocado fruit extract may help in the restoration of essential fatty acids in diabetic tissues. The n-6 and n-3 PUFA are known to decrease thrombosis and are known to lower the incidence of cardiovascular complications. Thus, normalization of the fatty acid composition in diabetic tissues treated with avocado fruit extract may prevent vascular complications in diabetes.

 

CONCLUSION:

In conclusion, the anti-hyperlipidemic properties of avocado fruit extract may be attributed to the normalization of glucose metabolism, inhibition of lipolysis in addition to its potential FRSA. It could also be speculated that the observed hypoglycemic and hypolipidemic effects of avocado fruit extract might be related to the rich phytochemicals such as flavonoids, polyphenols, alkaloids, steroids, amino acids, and vitamins with strong antioxidant properties. The process of extraction and identification of active principles responsible for the observed pharmacological actions of avocado fruit through bioactivity guided fraction is under progress to understand the possible mechanism of action of avocado fruits.

 

ACKNOWLEDGEMENT:

The authors thank SRM Management especially Dr. R. Shivakumar Provice-Chancellor for his motivation and constant encouragement

 

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Received on 05.05.2011        Modified on 28.05.2011

Accepted on 12.06.2011        © AJRC All right reserved

Asian J. Research Chem. 4(7): July, 2011; Page 1131-1136