Antidyslipidemic Property of Annona Squamosa Leaves Extract Studied in Streptozotocin-Induced Experimental Diabetes in Rats

 

S. K. Hayath Basha and S. Subramanian*

Department of Biochemistry, University of Madras, Guindy Campus, Chennai – 600 025, Tamilnadu, India.

*Corresponding Author E-mail: subbus2020@yahoo.co.in

ABSTRACT:

 

 

INTRODUCTION:

Diabetes mellitus is a chronic metabolic disorder, characterized by hyperglycemia associated with impaired metabolism of glucose as well as lipid and protein. The prevalence and progression of diabetes is increased as the result of pancreatic beta cell dysfunction leading to defective insulin secretion and or resistance to insulin action in peripheral tissues, such as skeletal muscles, adipose tissues and liver (ADA, 2010). More than 200 million people worldwide have diabetes and this number is likely to more than double by the year of 2030 (van Dieren et al., 2010).  Several epidemiological studies have demonstrated the strong causal relations between dyslipidemia and cardiac diseases, a major cause of morbidity and mortality in diabetes. The characteristic features of diabetic dyslipidemia are a high plasma triglyceride concentration, low HDL cholesterol concentration and increased concentration of small dense LDL-cholesterol particles (Muačević-Katanec and Reiner, 2011).

 

Though there is a large class of drugs used in the treatment of dyslipidemia, none of them is found to be effective and free from side effects (Pahan, 2006; Gaist et al., 2001). Thus, it is essential to develop and utilize effective and natural agents that may be beneficial in correcting the lipid metabolism and preventing vascular complications. Hence, attention to alternative medicines and natural therapies has raised researchers’ interest in traditional herbal medicine. Because of their perceived effectiveness, with minimal side effects in clinical experience and relatively low costs, herbal drugs are prescribed widely, even when their contents of the biologically active constituents are unknown.

 

Annona squamosa L., commonly known as custard apple, is cultivated throughout India, mainly for its edible fruit. The plant extract is traditionally used for the treatment of cardiac problems, constipation, haemorrhage, bacterial infection, ulcer, tumour and hyperthyroidism (Yadav et al., 2011; Panda and Kar, 2007; Dholvitayakhun et al., 2011).  In India, many of the tribes and villagers use the leaves of Annona squamosa for the management of diabetes. Recently, we have reported the antihyperglycemic and antioxidant property of Annona squamosa leaves extract in the experimental diabetic rats (Hayath Basha and S. Subramanian, 2011). Hence, in the present study, we aimed to evaluate the role of Annona squamosa leaves extract on diabetic dyslipidemia in streptozotocin-induced experimental diabetes in rats.

 

MATERIALS AND METHODS:

Plant material

Fresh, mature Annona squamosa leaves were collected from a tree in Kolli Hills, Tamil Nadu, India. The plant was identified at the Herbarium of Botany, CAS in Botany, University of Madras. An exemplar specimen was deposited in the department herbarium.

 

Preparation of plant extract

The Annona squamosa leaves were first washed well with distilled water and shadow dried at room temperature. The dried leaves were powdered in an electrical grinder and stored at 5°C until further use. One hundred grams of powder was extracted with petroleum ether (60-80°C) to remove lipids. It was then filtered, and the filtrate was discarded. The residue was extracted with 95% ethanol by soxhlation. The ethanol was evaporated in a rotary evaporator at 40-50°C under reduced pressure. The yield was 1.5 g/100 g.

 

Animals

Adult male albino rats of Wistar strain weighing approximately 160–180 g were procured from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India. Animals were acclimatized to animal house conditions, fed with commercial pelleted rat chow (Hindustan Lever Ltd., Bangalore, India) and had free access to water. All the animal experiments were designed and conducted according to the ethical norms approved by the Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee guidelines.

 

Experimental protocol

Diabetes was induced in rats with a single intraperitoneal dose of streptozotocin (50 mg/kg body weight) in 0.1 M of cold citrate buffer of pH 4.5 (Rakieten et al., 1963). Rats were provided with 5% glucose solution for 48 h after streptozotocin injection in order to prevent severe hypoglycemia. After a week’s time for the development and aggravation of diabetes, the rats with moderate diabetes having persistent glycosuria and hyperglycemia (blood glucose range of above 250 mg/dl) were considered as diabetic rats and further experiments were conducted.

The rats were grouped into four of a minimum of six animals in each group as follows:

Group 1: Normal control rats

Group 2: Diabetic rats

Group 3: Diabetic rats treated with Annona squamosa leaves extract (100 mg/kg b.w. /day) for 30 days

Group 4: Diabetic rats treated with gliclazide (5 mg /kg body weight/rat/day) orally for 30 days

 

At the end of the experimental period, the animals were fasted overnight and then sacrificed by cervical decapitation. Blood was collected without anticoagulant to separate serum. Liver and kidney were dissected out and immediately washed in ice-cold saline and homogenized in tris-HCl buffer, pH 7.4 (0.1 M) with a Teflon homogenizer. The total lipids were extracted from the tissue homogenates by the method of Folch et al. (1957).

 

Assay of lipid profile

Cholesterol content in serum, liver and kidney were estimated by the method of Parekh and Jung (1970); triglycerides were estimated by the method of Foster and Dunn (1973) and free fatty acids by the method of Itaya (1977). Total phospholipids content was estimated by the method of Bartlett (1959) after digestion with perchloric acid and the phosphate liberated was estimated by the method of Fiske and Subbarow (1925). High-density lipoproteins (HDL) and low-density lipoproteins (LDL) were separated from the serum according to dual precipitation technique (Burstein and Scholnick, 1972) and the cholesterol content of the lipoproteins was estimated.

 

Statistical analysis

All the grouped data were statistically evaluated with SPSS software. Hypothesis testing methods included one way analysis of variance (ANOVA) followed by least significant difference (LSD) test. The p values of less than 0.05 were considered to indicate statistical significance. All the results were expressed as mean ± SD for six animals in each group.

 

RESULTS:

The serum lipid and lipoprotein profiles of control and experimental groups of rats are shown in Tables 1 and 2 respectively. There was an increase in the levels of serum cholesterol, phospholipids, triglycerides and free fatty acids in STZ-induced diabetic rats when compared with control rats. Simultaneously, LDL, VLDL-cholesterol levels were significantly increased and HDL-cholesterol levels were decreased in the diabetic groups. In diabetic rats treated with A. squamosa leaves extract as well as gliclazide, the serum lipid profiles were restored to near normal level when compared with diabetic rats.

 

 

Table  1. Serum lipid profile in the experimental groups of rats

Serum lipids (mg/dl)	Control	STZ-Diabetes	STZ + A. squamosa	STZ + gliclazide
Cholesterol	88.74 ± 1.33	202.11 ± 4.19 a*	92.18 ± 2.68 b*	88.97 ± 1.41 b*
Triglycerides	77.58 ± 2.73	138.82 ± 4.37 a*	79.42 ± 1.88 b*	79.81 ± 2.43 b*
Phospholipids	108.35 ± 4.47	171.15 ± 3.28 a*	126.27 ± 3.73 b*	117.55 ± 4.04 b*
Free fatty acids	23.20 ± 1.69	50.70 ± 2.79 a*	32.24 ± 2.24 b*	25.98 ± 2.06 b*

Results are expressed as mean ± S.D. (n=6).  One way ANOVA followed by post hoc test LSD, *p <0.05. The results were compared with ^a Control; ^b STZ-Diabetes.

 

Table 2. Serum lipoproteins cholesterol levels in the experimental groups

Lipoprotein cholesterol (mg/dl)	Control	STZ-Diabetes	STZ + A. squamosa	STZ +gliclazide
HDL-Cholesterol	24.60 ± 1.46	13.24 ± 0.54a*	21.84 ± 1.30 b*	21.81 ± 1.14 b*
LDL-Cholesterol	48.62 ± 1.66	161.27 ± 4.40 a*	54.35 ± 3.25 b*	51.19 ± 2.52 b*
VLDL-Cholesterol	15.51 ± 0.54	27.76 ± 0.87 a*	15.98 ± 0.49 b*	15.94 ± 0.48 b*

Results are expressed as mean ± S.D. (n=6).  One way ANOVA followed by post hoc test LSD, *p <0.05. The results were compared with a Control; b STZ-Diabetes.

Table 3. Levels of cholesterol, triglycerides, phospholipids and free fatty acids in liver and kidney tissues of experimental groups of rats

Lipids (mg/dl)	Control	STZ-Diabetes	STZ + A. squamosa	STZ + gliclazide
Cholesterol levels In liver In kidney	7.98 ± 0.24 5.83 ± 0.51	12.67 ± 0.99 a* 9.36 ± 038 a*	8.78 ± 0.61 b* 6.24 ± 0.32 b*	8.37 ± 0.32 b* 5.87 ± 0.64 b*
Triglyceride levels In liver In kidney	6.20 ± 0.54 5.22 ± 0.41	8.62 ± 0.81 a* 8.95 ± 0.13 a*	6.42 ± 0.72 b* 5.51 ± 0.34 b*	5.81 ± 0.25 b* 4.77 ± 0.32 b*
Phospholipids levels In liver In kidney	22.01 ± 1.25 16.34 ± 0.34	40.92 ± 1.06 a* 27.92 ± 0.49 a*	22.16 ± 1.75 b* 16.35 ± 0.46 b*	22.06 ± 0.88 b* 15.82 ± 0.37 b*
Free fatty acids levels In liver In kidney	3.52 ± 0.34 4.08 ± 0.20	6.07 ± 0.24 a* 7.24 ± 0.46 a*	4.12 ± 0.16 b* 3.96 ± 0.15 b*	3.62 ± 0.36 b* 3.37 ± 0.33 b*

Results are expressed as mean ± S.D. (n=6).  One way ANOVA followed by post hoc test LSD, *p <0.05.  The results were compared with ^a Control; ^b STZ-Diabetes.

 

The levels of cholesterol, phospholipids, triglycerides and free fatty acids in liver and kidney of control and experimental groups of rats are represented in Table 3. The diabetic rats showed a significant increase in cholesterol, phospholipids, triglycerides and free fatty acids of liver and kidney when compared with control rats. These alterations were restored to near normalcy upon treatment with A. squamosa leaves extract as well as gliclazide.

 

DISCUSSION:

Hypertriglyceridemia and hypercholesterolemia are the common characteristic features of diabetes (Chahil and Ginsberg, 2006). Cardiovascular disease is the leading cause of morbidity and mortality in diabetes and is often associated with hyperlipidemia. Changes in the serum lipid concentration are a frequent complication in patients with diabetes mellitus, which certainly contributes to the developments of vascular disease. The abnormal high concentrations of plasma lipids 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. Hence, the marked hyperlipidemia that characterizes the diabetic state may therefore be regarded as a consequence of the uninhibited actions of lipolytic hormones on fat depots (Goldberg, 2001).

 

Diabetes is also known to be associated with an increase in the synthesis of cholesterol, which may be due to the increased activity of HMG CoA reductase (Goodman et al., 1982). A number of observations indicate that plasma HDL cholesterol is low in untreated insulin-deficient diabetics (Glasgow et al., 1981). HDL cholesterol levels are inversely related to cardiovascular diseases whereas LDL-cholesterol is positively associated. Increased level of LDL-cholesterol may arise from glycosylation of the lysyl residues of apoprotein B as well as from decreasing affinity for the LDL receptor and hence, decreased metabolism (Golay et al., 1987).

 

Serum HDL-cholesterol levels falls following chemically induced diabetes, which was associated with a decline in HDL-turnover rate (Golay et al., 1987). Also serum VLDL-cholesterol level of diabetic patients was reported to be very high. The above reports are on par with our findings. The HDL-cholesterol levels correlate with lipoprotein lipase (LPL) levels in IDDM patients (Nikkila et al., 1977). This was interpreted to reflect the role of triglyceride rich lipoproteins as a source of HDL. It is shown that VLDL and chylomicrons contribute surface apoproteins and lipids to HDL during hydrolysis by LPL. Since LPL activity is regulated by insulin, this provides a mechanism by which insulin might affect HDL cholesterol levels. Oral administration of A. squamosa leaves extract normalized these effects, possibly by controlling the hydrolysis of certain lipoproteins and their selective uptake and metabolism by different tissues.

 

In the present study, increased levels of triglycerides were observed in diabetic rats and were subsequently decreased by A. squamosa leaves extract treatment. Hypertriglyceridemia is a common finding in patients with diabetes mellitus, particularly in those who have vascular complications (Kudchodkar et al., 1988). Bruan and Severson (1992) reported that a deficiency of lipoprotein lipase (LPL) activity might contribute significantly to the elevation of triglycerides in diabetes. Also an excessive lipolysis in diabetic adipose tissue leads to increased free fatty acids in circulation, which upon  entering the liver are esterified to form triglycerides (Lewis et al., 2002). Studies on insulin-dependent diabetes mellitus and streptozotocin-induced diabetes in experimental animals have suggested that the increase in circulating VLDL and their associated triglycerides are largely due to defective clearance rate of these substances from the circulation (Reaven and Reaven, 1974; Van Tol, 1977). Lopes-Virella et al. (1983) reported that the treatment of diabetes with insulin helped to lower plasma triglyceride levels by returning lipoprotein lipase levels to normal. The observed restoration of triglyceride levels following A. squamosa leaves extract treatment is supported by above reports on insulin treatment.

 

The increased level of phospholipids observed in diabetic rats was brought down by treatment with A. squamosa leaves extract. The elevated serum phospholipids levels are a consequence of elevated lipoproteins. Jain et al. (2004) suggested that, the levels of glycemic control and elevated levels of HDL cholesterol and triglycerides in the blood are significantly correlated with the phospholipid levels. The serum cholesterol/phospholipid ratio is one of the important markers of development of atherosclerosis. Keelan et al. (1985) have observed a significant increase of phospholipids in diabetics. The restoration of phospholipids by A. squamosa leaves extract may be due to controlled mobilization of serum triglycerides, cholesterol and phospholipids is presumably mediated by controlling the tissue metabolism and improving the level of insulin secretion and action.

 

An increase in the levels of total lipids in liver and kidney of STZ-induced diabetic rats may indicate an increased mobilization of lipids from these tissues and storage capacity, which may have caused an increase in serum triglycerides and phospholipids. Rajalingham et al. (1993) reported that variety of derangements in metabolic and regulatory mechanisms, due to insulin deficiency, is responsible for the observed accumulation of lipids. The increase in total lipids observed in diabetic rats was due to the impairment of insulin secretion, which resulted in enhanced mobilization of lipid from the adipose tissue to the serum.

 

The excessive lipolysis in diabetic adipose tissue leads to increased free fatty acids in circulation. They enter the liver and are esterified to form triglycerides (Lewis et al., 2002). In alloxan-induced diabetic rats the tissue cholesterol, phospholipids and triglycerides were significantly increased and insulin treatment had an appreciable effect on the lipids of these tissues. Changes in the fatty acids during diabetes are closely associated with the activity of Na+/K+-ATPase in the kidney. Accumulation of fatty acids results in higher levels of their metabolites such as acyl carnitine and long chain acyl CoA and this may be a cause for diabetic nephropathy (Parving and Hommel, 1989). Thus the diabetic complications associated with renal tissue may be partly due to abnormalities in lipid metabolism.

 

Fatty acids are important components of cell membranes, which are eicosanoid precursors and are therefore required for both the structure and function of every cell in the body. Fatty acids in the form of esters are generally present as triglycerides, phospholipids and cholesterol esters. As esters of phospholipids, they form an important part of the cell membrane, while as triglycerides they constitute an important source of stored energy.

 

Fatty acids undergo changes during the process of injury, repair and cell growth (Cameron and Cotter 1997). Faas et al. (1988) found that there is an alteration in the erythrocyte membrane and plasma fatty acid composition in diabetic patients. Seigneur et al. (1994) reported that there is a significant alteration in the fatty acid composition of serum and variety of tissues in experimental diabetes. The alteration of the membrane phospholipid composition appears to be responsible for the biochemical alterations produced during long-term diabetes.

 

Thus, the alterations in lipids, lipoproteins cholesterol and fatty acid levels in streptozotocin-induced diabetic rats were restored to near normal levels by A. squamosa leaves extract administration. Hence, it may be concluded that the hypolipidemic effect produced by the A. squamosa leaves extract might be due to the synergistic action of phytoconstituents such as flavonoids, saponins, and triterpenoids in the extract.

 

 

REFERENCES:

1.      American Diabetes Association.  Diagnosis and classification of diabetes mellitus. Diabetes Care. 33 (Suppl 1); 2010: S62-69.

2.      van Dieren S, Beulens JW, van der Schouw YT, Grobbee DE and Neal B. The global burden of diabetes and its complications: an emerging pandemic. European Journal of Cardiovascular Prevention and Rehabilitation. 17 (Suppl 1); 2010: S3-8.

3.      Muačević-Katanec D and Reiner Z. Diabetic dyslipidemia or 'diabetes lipidus'? Expert Review of Cardiovascular Therapy. 9(3); 2011: 341-348.

4.      Pahan K. Lipid-lowering drugs. Cellular and Molecular Life Sciences. 63(10); 2006: 1165-1178.

5.      Gaist D, García Rodríguez LA, Huerta C, Hallas J and Sindrup SH. Are users of lipid-lowering drugs at increased risk of peripheral neuropathy? European Journal of Clinical Pharmacology. 56(12); 2001: 931-933.

6.      Yadav DK, Singh N, Dev K, Sharma R, Sahai M, Palit G and Maurya R. Anti-ulcer constituents of Annona squamosa twigs. Fitoterapia. 82(4); 2011: 666-675.

7.      Panda S and Kar A. Annona squamosa seed extract in the regulation of hyperthyroidism and lipid-peroxidation in mice: possible involvement of quercetin. Phytomedicine. 14(12); 2007: 799-805.

8.      Dholvitayakhun A, Tim Cushnie TP and Trachoo N. Antibacterial activity of three medicinal Thai plants against Campylobacter jejuni and other foodborne pathogens. Natural Product Research. 2011. [Epub ahead of print]

9.      Hayath Basha SK and Subramanian S. Biochemical evaluation of antidiabetic and antioxidant potentials of annona squamosa leaves extracts studied in stz induced diabetic rats. International Journal of Pharmaceutical Sciences and Research. 2(3); 2011: 643-655.

10.    Rakieten N, Rakieten ML and Nadkarni MV. Studies on the diabetogenic action of streptozotocin (NSC-37917). Cancer Chemotherapy Reports. 29; 1963: 91-98.

11.    Folch J, Lees M and Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. The Journal of Biological Chemistry. 226(1); 1957: 497-509.

12.    Parekh AC and Jung DH: Cholesterol determination with ferric acetate –uranyl acetate and sulphuric acid - ferrous sulphate reagents. Analytical Chemistry. 42; 1970: 1423–1427.

13.    Foster LB and Dunn RT: Stable reagents for determination of serum triglycerides by a colorimetric Hantzsch condensation method. Clinical Chemistry. 19(3); 1973: 338–340.

14.    Itaya K: A more sensitive and stable calorimetric determination of free fatty acids in plasma. Journal of Lipid Research. 18(5); 1977: 663–665.

15.    Bartlett GR: Phosphorus assay by column chromatography. The Journal of Biological Chemistry. 234(3); 1959: 466–468.

16.    Fiske CH and Subbarow Y: The colorimetric determination of phosphorus. The Journal of Biological Chemistry. 66; 1925: 375–400.

17.    Burstein M and Scholnick HR: Precipitation of chylomicron and very low density protein from human serum with sodium lauryl sulphate. Life Sciences. 11(4); 1972: 177–184.

18.    Chahil TJ and Ginsberg HN. Diabetic dyslipidemia. Endocrinology and metabolism clinics of North America. 35(3); 2006: 491-510.

19.    Goldberg IJ. Clinical review 124: Diabetic dyslipidemia: causes and consequences. The Journal of clinical endocrinology and metabolism. 86(3); 2001: 965-971.

20.    Goodman MW, Michels LD and Keane WF. Intestinal and hepatic cholesterol synthesis in the alloxan diabetic rats. Proceedings of the Society for Experimental Biology and Medicine. 170; 1982: 286-290.

21.    Glasgow AM, August GP and Hung W. Relationship between control and serum lipids in juvenile onset diabetes. Diabetes Care. 4(1); 1981: 76-80.

22.    Golay A, Zech L, Shi MZ, Jeng CY, Chiou YA, Reaven GM and Chen YD. Role of insulin in regulation of high density lipoprotein metabolism. Journal of lipid research. 28(1); 1987: 10-18.

23.    Nikkila EA, Huttunen JK and Ehnholm C. Postheparin plasma lipoprotein lipase and hepatic lipase in diabetes mellitus. Diabetes. 26(1); 1977: 11-21.

24.    Kudchodkar BJ, Lee JC, Lee SM, DiMarco NM and Lacko AG. Effect on cholesterol homeostasis in diabetic rats. Journal of lipid research. 29(10); 1988: 1272-1287.

25.    Bruan JEA and Severson DL. Lipoprotein lipase release from cardiac myocytes is increased by decavandate but not insulin. The American journal of physiology. 262(5 Pt 1); 1992: E663-E670.

26.    Lewis GF, Carpentier A, Adeli K and Giacca A. Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes. Endocrine reviews. 23(2); 2002:201-229.

27.    Reaven EP and Reaven GM. Mechanisms for development of diabetic hypertriglyceridemia in streptozotocin-treated rats. Effect of diet and duration of insulin deficiency. The Journal of clinical investigation. 54(5); 1974: 1167-1178.

28.    Van Tol A. Hypertriglyceridemia in the diabetic rat. Defective removal of serum very low density. Atherosclerosis. 26(1); 1977: 117-128.

29.    Lopes-Virella MF, Whitmann HJ, Mayfield PK, Loadholt CB and Colwell JA. Effect of metabolic control on lipid, lipoprotein and apolipoprotein levels in 55 insulin-dependent diabetic patients: a longitudinal study. Diabetes. 32(1); 1983: 20-25.

30.    Jain M, Cui L, Brenner DA, Wang B, Handy DE, Leopold JA, Loscalzo J, Apstein CS and Liao R. Increased myocardial dysfunction after ischemia-reperfusion in mice lacking glucose-6-phosphate dehydrogenase. Circulation. 109(7); 2004: 898-903.

31.    Keelan M, Walker K and Thomson AB. Intestinal brush border membrane marker enzymes, lipid composition and villus morphology: Effect of fasting and diabetes mellitus in rats. Comparative biochemistry and physiology. 82(1); 1985: 83-89.

32.    Rajalingham R, Srinivasan N and Govindarajulu P. Effect of alloxan - induced diabetes on lipid profiles in renal cortex and medulla of mature albino rats. Indian journal of experimental biology. 31(6); 1993: 577-579.

33.    Parving HH and Hommel E. Prognosis in diabetic nephropathy. British medical journal. 299(6693); 1989: 230-233.

34.    Cameron NE and Cotter MA. Effects of antioxidants on nerve and vascular dysfunction in experimental diabetes. Diabetes research and clinical practice. 45(2-3); 1997: 137-146.

35.    Faas FH, Dang AQ, Norman J and Carter WJ. Red blood cell and plasma fatty acid composition in diabetes mellitus. Metabolism. 37(8); 1988: 711-713.

36.    Seigneur M, Freyburger G, Gin H, Claverie M, Lardeau D, La-Cape G, Moigne F, Crockett R and Boisseass MR. Serum fatty acid profiles in type 1 and type 2 diabetes : Metabolic alterations of fatty acids of the main serum lipids. Diabetes research and clinical practice. 23(3); 1994: 169-177.

 

 

 

Received on 26.12.2011         Modified on 28.01.2012

Accepted on 12.02.2012         © AJRC All right reserved