Annona muricata leaves were collected from Mowe, Ogun State, Nigeria in February 2006. The plant was identified by Dr. Folorunso of the Department of Botany, Obafemi Awolowo University, Ile Ife and a voucher specimen was deposited in the Herbarium of the Department

A. muricata leaves were air dried at room temperature for four weeks. The air-dried leaves were powdered in a warring blender (Christy and Norris – 47362, England) at the Department of Pharmacognosy Obafemi Awolowo University, Ile Ife. A 600g of the powdered leaf leaves was soaked in 5 litres of 70% methanol for 72 hours at room temperature. The mixture was filtered and the filtrate was evaporated at 60°C using a vacuum rotary evaporator (RE 100B, Bibby Sterilin, United Kingdom). The wet residue was freeze-dried using a vacuum freeze drier (FT33- Armfield, England) and was stored until ready to use.

Thirty healthy adult Wistar rats (Rattus norvegicus) of both sexes, weighing between 150g and 250g were used for the experiment. The rats were bred in the animal holding of department of Anatomy and Cell Biology Obafemi Awolowo University Ile Ife, were maintained on standard rat pellets (Ladokun feeds, Ibadan, Nigeria), and were given water ad libitum. The animals were randomly assigned into three groups A, B, and C of ten rats each. Group A was the control, non-diabetic group of rats, group B was the experimentally induced diabetic group without A. muricata treatment while group C was the experimentally induced diabetic group treated with methanolic extracts of A. muricata. There was a pre-experimental period of four weeks during which the serum lipid profiles was monitored in the animals before the commencement of the experiment. The rats receive humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health.

Forty adult male Wistar rats (150 – 200g) were randomly assigned into six groups (T1, T2, T3, T4, T5 and T6) of eight animals in each group. T1 was treated with distilled water and is considered as control. The other five groups were treated with methanolic extract of A. muricata given intraperitoneally in increasing dosage of 25, 50, 100, 200, and 400mg/kg body weight. The extract was dissolved in distilled water and the average volume injected was 0.3ml. The control group was given equivalent volume of distilled water used in dissolving the extract. All the rats were returned to their cages and given free access to food and water. The mortality in each cage was assessed 24 hours, 48 hours and 72 hours after administration of extract. The percentage mortality in each group was calculated and plotted against the log₁₀ of the extract dose. Regression line was fitted by method of least squares and confidence limits for the lethal dose (LD₅₀) values were calculated by method of Abdel-Barry et al ₁₆.

Diabetes mellitus was experimentally induced in groups B and C by a single intraperitoneal injection of 80mg/kg body weight. streptozotocin (Sigma, St. Louis, USA) dissolved in 0.1M sodium citrate buffer pH 6.3. The control (group A animals) were injected intraperitoneally with equivalent volume of the citrate buffer. The rats were fasted overnight before STZ administration. The serum lipid profiles were monitored weekly in the animals for the next four weeks. After four weeks of experimental-induction of diabetes, group C rats were given daily intraperitoneal injection of 100mg/kg of extracts of A.muricata dissolved in distilled water for two weeks and the animals were monitored for another four weeks.

The serum levels of triglyceride (TGL), total cholesterol (TC) and high-density lipoprotein-cholesterol (HDLC) were determined spectrophotometrically, using enzymatic colorimetric assay kits (Randox, Northern Ireland) while low-density lipoprotein cholesterol (LDLC) and very low-density lipoprotein cholesterol (VLDLC) were calculated. Animals were fasted for 12-16 hours before blood samples were obtained. About two milliliters of blood was collected from the tail vein of each rat into an ice-cold centrifuge tubes. The blood samples were centrifuged in a Denley BS400 centrifuge (England) at 5000 R.P.M for 5-minutes. The supernatant (serum) collected was assayed for the serum levels of TGL, TC and HDL-C using the Randox Biochemical kits while LDL-C and VLDL-C were calculated.

The serum level of TGL was determined by the method of Treitz ₁₇. 1000 μl of the reagent was added to 10μl each of the sample and standard. This was incubated for 10 minutes at 20-25°C and the absorbance of the sample (A _sample) and standard (A _standard) was measured against the reagent blank within 30 minutes.

Figure 1

The serum level of TC was determined after enzymatic hydrolysis and oxidation of the sample as described by Richmond ₁₈ and Roeschlau et al., ₁₉. 1000 μl of the reagent was added to 10μl each of the sample and standard. This was incubated for 10 minutes at 20-25°C and the absorbance of the sample (A _sample) and standard (A _standard) was measured against the reagent blank within 30 minutes.

Figure 2

Low-density lipoproteins (LDL and VLDL) and chylomicron fractions in the sample were precipitated quantitatively by the addition of phosphotungstic acid in the presence of magnesium ions. The mixture was allowed to stand for 10 minutes at room temperature centrifuged for 10 minutes at 4000rpm. The supernatant represented the HDLC fraction. The cholesterol concentration in the HDL fraction, which remains in the supernatant, was determined.

The concentration of LDL cholesterol was calculated mmol/L using Friedewald’s equation ₂₀ as stated below.

Figure 3

The concentration of VLDL cholesterol was calculated mmol/L using Friedewald’s equation ₂₀ as stated below.

Figure 4

The antiatherogenic index was calculated according to the method of Guido and Joseph₂₁. AAI was calculated from total cholesterol and HDL cholesterol using the formula below;

Figure 5

The values were expressed as a percentage

The data were analysed using descriptive and inferential statistics. All values are presented as mean ± standard error of mean (SEM) for ten rats in each of the three group of rats. The significance of difference in the means of all parameters reported for the three groups of animals was determined using paired sample student t – test and a p – value of < 0.05 (two tailed) was considered as significant.

Tables 1 and 2 illustrate the effects of A. muricata on the levels of total cholesterol, triglycerides, HDLC, LDLC, VLDLC and AAI in the serum of experimentally induced diabetic rats. The levels of total cholesterol, triglycerides LDL-C and VLDL-C were significantly (p < 0.05) increased in diabetic rats whereas the level of HDL-C and the percentage of AAI (ratio of HDL to total cholesterol) were significantly (p < 0.05) reduced in diabetic rats when compared to the control normal rats. Administration of A. muricata to STZ diabetic rats restored all these changes to near normal levels by significant (p < 0.05) reduction of the level of total cholesterol, triglycerides, LDLC and VLDLC of diabetic rats and significant increase in the level of HDL-C and percentage of AAI.

Figure 6

Table 1: Effects of on the Serum Total Cholesterol, Triglycerides and High Density Lipoprotein Cholesterol (HDLC) of Experimentally-Induced Diabetic Rats

Values are given as mean ± SEM for ten rats in each group.

a, b within column signifies that means with different letters differs significantly at P < 0.05 (two tailed T-test) while means with the same letters does not differ significantly at P <0.05 (two tailed T-test)

Figure 7

Table 2: Effects of on the Serum Low Density Lipoprotein Cholesterol (LDLC) Very Low Density Lipoprotein Cholesterol (VLDLC) and Antiartherogenic index (AAI) of Experimentally-Induced Diabetic Rats

Values are given as mean ± SEM for ten rats in each group.

a, b within column signifies that means with different letters differs significantly at P < 0.05 (two tailed T-test) while means with the same letters does not differ significantly at P <0.05 (two tailed T-test

The authors thank Dr. O.O. Komolafe for his assistance.