Hypoglycaemic and hypolipidaemic effects of crude extracts and Chromatographie fractions of Morinda morindoides root bark in diabetic rats

Hypoglycaemic and hypolipidaemic effects of different extracts and fractions of root bark from the plant Morinda morindoides (Baker) Milne-Redh of the family Rubiaceae were evaluated in alloxan-induced diabetic rats. The aqueous and methanolic extracts were administered to 48 rats orally at a dose of 400 mg kg ' for 21 days. Fractions (hydromethanol, hexane, chloroform and ethyl acetate) from bio-activity guided fractionation and Chromatographie sub fractions (CsF) A-F from accelerated gradient chromatography were also evaluated in 45 rats for the hypoglycaemic activity at doses of 400 mg-kg"', 200 mg-kg ' and 100 mg-kg ' of solvent fractions and (CsF) A-F, respectively. Glibenclamide was used as positive control. Polyoxyethylene sorbitan monooleate and distilled water administered to rats were used as negative controls. The dose of 400 mg kg ' of aqueous and methanolic extracts and 100 mg kg ' of chloroform CsF B of Morinda morindoides caused (62.8%, 56% and 74%, respectively) reductions in blood glucose level (BGL). The aqueous extract caused significant {P < 0.05) decreases in the values of serum cholesterol (133.48 ± 1.1) mg-dl'', low density lipoprotein (66.38 mg-dl'' and significant (P < 0.05) increase in the value of high density lipoprotein (51.03 ± 3.0) mg ' when compared to the control. These results confirm the folkloric claim of the hypoglycaemic and hypolipidaemic activities of Morinda morindoides root bark.

Diabetes mellitus which causes hyperglycaemia is a disease caused by insufficient insulin production by the pancreatic ß cells or inability of the body to utilize insulin due to the peripheral tissue resistance. This leads to a serious damage of many parts of the body's systems, especially nerves and blood vessels (WHO 2010).
The chemical agents used in the treatment of diabetes mellitus type 2 are expensive and could lead to adverse side effects. Development of alternative strategies for the prevention and treatment of diabetes is therefore necessary especially in countries with poor economy (Fertigetal. 1995).
Morinda morindoides (Baker) Milne-Redh of the family Rubiaceae has medicinal application in some African countries, especially in Nigeria, where it is used for the treatment of different diseases. It is called brimstone tree in English and "oju ologbo" in Yoruba (Southwest, Nigeria). M morindoides is used in the form of infiasion against malaria (Tona et al. 2001), diarrhoea, haemorrhoids, gonorrhoea, amoebiasis and rheumatism (Kambu 1990;Tona et al. 1999;Cimanga et al. 2006). The extract from root bark is used for the treatment of diabetes mellitus in South Western part of Nigeria.
Though many studies have been carried out on the leaf extract of this plant, less work has been done on the root bark extract in the treatment of diabetes mellitus as claimed by the traditional healers in Southwest Nigeria. This study is therefore aimed at investigating the hypoglycaemic and hypolipidaemic properties of aqueous and methanol extracts, and the bioactive solvent Chromatographie sub filetions of M morindoides root bark in alloxan-induced diabetic rats.

Plant material and preparation of extract
Fresh root bark of Morinda morindoides was identified at the Forestry Research Institute of Nigeria (F.R.I.N), Ibadan, and Botany Department, College of Natural Sciences, University of Agriculture, Abeokuta, Nigeria where a specimen was deposited.
Five hundred grams of the root bark were air-dried, pulverized and soaked in 2 1 of distilled water for 48 h, filtered and freeze-dried. Then, two kilograms of the air-dried and pulverized root bark were soaked in 5 I of methanol for 72 h and concentrated at 55 °C in water bath. The samples were stored at -4 °C until the experiment.
Two grams of the freeze-dried solid M. morindoides were dissolved in 20 ml of distilled water (100 mg ml'). Two g of the solid residue from concentrated methanol filtrate were dissolved in 20 ml of Tween-80 (100 mg ml'). These solutions were used for administration to the rats.

Bio-activity guided fractionation
One hundred grams of the crude methanol extract of the root bark M. morindoides were suspended in 1 : 3 ratio with methanol-water mixture (hydromethanol), -n-Hexane, -chloroform, and -ethyl acetate. This dilution was done in a separatory funnel at each stage. The organic fractions (phases) were collected and the solvent was removed by concentrating the fraction in water bath at 55 °C. This procedure was carried out for all n-Hexane, chloroform, ethyl acetate and MeOH. The bioactive solvent-fractions were subjected to purification by further fractionation using a standard procedure of accelerated gradient chromatography (Svoronos and Sarlo 1993) and the Chromatographie sub-fractions were pooled into six (A-F) according to their TLC. All fractions and reference glibenclamide were suspended in Tween-80 and used for oral administration to the rats.

Animals
Ninety three, 8-10 week-old Wistar rats of both sexes, weighing 150-200 g were provided with rodent feed from Vital feeds Limited, Ibadan, Nigeria and supplied with water ad libitum. The animals were acclimatized to laboratory conditions for two weeks before the experiment with controlled lighting of 12 : 12 h of light : dark cycles, temperature of 26 ± 2 °C and relative humidity of 55%.

Induction of diabetes mellitus
The fasting blood glucose levels were measured at intervals of 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h after a single dose of the extract and glibenclamide in rats. Diabetes mellitus was induced by a single injection of 5% alloxan monohydrate (Sigma-Aldrich, Belgium) at a dose rate of 125 mg kg ' intraperitoneally (Trivedi et al! 2004). Diabetes mellitus was confirmed after 48 h. Only rats with blood glucose level of 150 mg dl' and more were included in the study. The control group of rats was injected with normal saline intraperitoneally. The third day to the alloxan injection was taken as Day 0. The rats were fasted for 12 h before the experiment but were allowed unrestricted access to water. Polyoxyethylene sorbitan monooleate (Tween 80, Sigma-Aldrich, Belgium) was administered orally as delivery vehicle for the plant extract, glibenclamide (Nigerian-German Chemical PLC) and the negative control group.
Effect of multiple administration of aqueous extract of M. morindoides on blood glucose level and lipids -Experiment 1 The animals were divided into 4 groups of 6 rats in one group. Group 1 included diabetic rats administered 400 mgkg' of aqueous extract of M morindoides. Group 2 included diabetic rats administered 10 mg kg ' of glibenclamide; Group 3 included diabetic rats given distilled water at 10 ml kg ' and Group 4 included control rats (non-diabetic) administered 10 ml-kg ' of distilled water orally. All administration was done once daily for 21 days and the blood glucose level was measured at Day 0 and at Day 21.
The serum cholesterol, triglycérides, the high density lipoprotein (HDL) and low density lipoprotein (LDL) were also measured at Day 21 from blood collected from the retro-orbital plexus of ether-anaesthetized rats using standard procedures (Bucolo and David 1973;Allain et al. 1974).
The effect of single dose of the methanol extract of M morindoides in rats -Experiment 2 Rats used for this experiment were as described above except for Group 1 that included diabetic rats administered 400 mg kg-' methanol extract of M morindoides and Group 4 of rats administered 5 ml kg ' Tween-80 serving as control; all 4 groups were treated only once.  (Fuentes et al. 2004).
Hypoglycaemic effect of the Chromatographie fractions of Af. morindoides -Experiment 3 In this experiment, 9 groups of 5 rats per group were used with Groups 1-8 consisting of diabetic rats and Group 9 consisting of non-diabetic rats. Groups 1-6 were given 100 mg-kg ' of the Chromatographie sub-factions (CsF) A-F. Rats in Group 7 were treated with 10 mg-kg ' glibenclamide and Groups 8-9 rats were given Tween-80 at a dose of 5 ml-kg' body weight. The fasting blood glucose levels were measured at intervals of 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h after the administration.

Statistical analysis
Results were expressed as mean ± SEM. Analysis of the data was done using the one-way Analysis of Variance (ANOVA) followed by the Duncan multiple range test. P value < 0.05 was considered significant in all cases.

Effect of multiple administration of aqueous extract of M. morindoides on BGL and lipids
After 21 days of treatment, there was a significant {P < 0.05) reduction in blood glucose level of the rats treated with 400 mg kg' aqueous extract of M morindoides (62.9% reduction in BGL). The group of rats treated with 10 mg-kg' of glibenclamide showed a significant {P < 0.05) decrease in the blood glucose level (63.2% reduction) after 21 days of treatment compared to the control rats with 269.50 ± 10.6 mg-dl' at day 0 to 324.53 ± 22.3 mg-dl ' at day 21 ( 20.1% increase in BGL) ( Table 1).

Effect of single dose methanolic extract of M. morindoides
Methanolic extracts oí Morinda morindiodes at a single dose of 400 mg-kg ' body weight produced a significant {P < 0.05) reductions in the blood glucose level of the rats at 1 h, 2 h, 4 h, 6 h, 8 h and 24 h post treatments ( Table 2). The percentage hourly reductions in blood glucose level were from 2.5% in 30 min to 17.7%, 33.1%, 45.9%, 40.4% and 41.8% in 1 h, 2 h, 4 h, 6 h, 8 h and 24 h, respectively. The extract produced a significant reduction {P < 0.05) of BGL (56%) at the 24* h after treatment when compared to the untreated diabetic control group ( Table 2).

i o Discussion
In this study, the hypoglyeaemic and hypolipidaemic activities of the crude aqueous extract, solvent fi-actions and Chromatographie sub-fi-actions of M. morindoides were evaluated in alloxan-induced diabetic rats. The 21-day treatment with aqueous extract of M. morindoides caused a significant decrease in the blood glucose of hyperglycaemie rats, and in serum triglycéride, cholesterol and LDL while increasing the serum HDL level. These effects were comparable to those obtained for glibenelamide a standard anti diabetic agent.
It was also observed that the methanol extract of M. morindoides caused significant reduction in blood glucose levels compared to glibenclamide. It was further observed that the hypoglycaemic effect of the chloroform fraction M morindoides was higher than that of glibenclamide. This may suggest that the extract of M. morindoides is anti-diabetic as WHO (2010) associated the treatment of diabetes with the lowering of blood glucose and the concentrations of other known risk factors that damage blood vessels and nervous tissues. Plants such as Laportea ovaliofolia have been reported to be anti-diabetic as they cause the reduction of blood glucose level and serum lipids of diabetic animals (Mom o h et al. 2006). The hypoglycaemic and hypolipidaemic actions of M. morindoides may be similar to those of insulin because insulin is hypoglycaemic and lowers lipid levels (Ahmed et al. 2001). The hypoglycaemic activity of M morindoides may be attributable to its flavonoids content and flavonoid o-glycosides (Cimanga et al. 1995;Harisolo et al. 2009). Flavonoids are reported to potentiate the increase of pancreatic secretion of insulin from ß-cells and to increase the peripheral utilization of glucose. Ves sal et al. (2003), reported that quercetin, a flavonoid with antioxidant properties brings about the regeneration of the pancreatic islets and probably increases insulin release in streptozocin-induced diabetic rats. This study suggests the hypoglycaemic and hypolipidaemic potential of Morinda morindoides.