Effects of N-Butanol Portion of Indigofera Pulchra - Ahmadu Bello ...

European Journal of Scientific Research 

ISSN 1450-216X Vol.22 No.4 (2008), pp.501-507 

© EuroJournals Publishing, Inc. 2008 

http://www.eurojournals.com/ejsr.htm 

Effects of N-Butanol Portion of Indigofera Pulchra Leaves 

Extract on Blood Glucose Levels of Alloxan-Induced 

Diabetic and Normoglycemic Wistar Rats 

Tanko. Y 

Department of Human physiology Faculty of Medicine 

Ahmadu Bello University Zaria, Nigeria 

E-mail: yusuftanko@yahoo.com 

Abdelaziz. M.M 



Adelaiye. A.B 



Fatihu. M.Y 

Department of Vet. Pathology and Microbiology, Faculty of Vet. Medicine 

Ahmadu Bello University,Zaria. Nigeria 

Musa. K.Y 

Department of Pharmacognosy and Drug Development, Faculty of pharmaceutical 

Sciences Ahmadu Bello University Zaria, Nigeria 

Abstract 

The n-butanol portion of Indigofera pulchra leaves extract (papilionaceae) was 

assessed for blood glucose levels of normoglycemic and alloxan induced diabetes in Wistar 

rats. Different doses of the extract was tested after 18 hours fasted. And there blood glucose 

levels was measured at 0,2,4,8 and 24 hours after treatment. The highest dose (1000 mg/kg) 

administered did not significantly decreased the blood glucose levels of alloxan induced 

diabetic and normoglycemic rat after 4 and 8 hours of treatment. The doses of 250 and 500 

mg/kg significantly decrease the blood glucose levels of alloxan induced diabetic and 

normoglycemic Wistar rats. The activity resides more at the lowest dose of the extract 250 

mg/kg. The hypoglycemic and antihyperglycemic potentials of the n-butanol portion of the 

extract was comparable to that of biphasic isophane insulin (6.i.u/kg). The phytochemical 

screening revealed the presences of tannins, saponins and flavonoids. The median lethal 

dose (LD 50 ) in rats was calculated to be 2,154 mg/kg bodyweight. In conclusion, the doses 

of the extract has shown both significant (p

Effects of N-Butanol Portion of Indigofera Pulchra Leaves Extract on 

Blood Glucose Levels of Alloxan-Induced Diabetic and Normoglycemic Wistar Rats 502 

Keywords: Indigofera pulchra, n-Butanol, Hypoglycemic activity, Normoglycemic, 

Alloxan, Diabetes mellitus. 

1. Introduction 

Diabetes mellitus is a chronic metabolic disorder of impaired carbohydrates, fat and protein 

metabolism. It is characterized by hyperglycaemia expressed as abnormal glucose value, which is due 

to insulin deficiency and/or insulin resistance which results in decrease utilization of carbohydrate and 

excessive glycogenolysis and gluconeogenesis from amino acid by fatty acids (Pontiroli et al., 1994). It 

has been defined by the World Health Organization (WHO) on the basis of laboratory findings as a 

fasting venous plasma glucose concentration greater than 7.3 mmol/L (140mg/dl) or greater than 11.1 

mmol/L (200mg/dl) two hours after carbohydrate meal or two hours after oral ingestion of the 

equivalent of 7.5g of glucose. 

This disease is on the increase all over the world, especially in Africa where it was formerly 

regarded as the disease of the affluent (Amos et al., 1997). 

Diabetes is one of the most common non-communicable diseases worldwide when compared 

with the general population, mortality and morbidity is increased in diabetes, due to the associated 

chronic complications – both specific microvascular retinopathy, nephropathy) and non-specific 

macrovascular artherosclerosis). Acute metabolic complications (e.g. diabetic ketoacidosis) continue to 

be a cause of mortality in developing countries (Ayesha et al., 2002). 

Using current and projected estimates, it has been suspected that the global prevalence of type I 

diabetes mellitus will increase from 3.5 million in 1995 to 5.5 million in 2010 (Amos et al., 2000). For 

Africa, the projected increase is from 85,000 (1995) to 219,000 (2010). 

Recent estimates indicate there were 171 million people in the world with diabetes in the year 

2000 and this is projected to increase to 366 million by 2030 (WHO, 2007). It is associated with 

reduced life expectancy, significant mortality and diminished quality of life. In 2005 an estimated 1.1 

million people died from diabetes. The American Diabetes Association (ADA) estimated the national 

costs of diabetes in the USA for 2002 to be US$132 billion, increasing to US$192 billion in 2020 

(WHO, 2007). If this is the case in a developed country, talk more of a developing country like Nigeria 

where 30% of people with diabetes live in low and middle income countries causing 5% of the deaths 

globally each year and likely to increase by more than 50% in the next 10 years without urgent action 

(WHO, 2006). 

Because of the tendency of many professionals to cling to the belief that only pharmaceutical 

products are of value, therefore, possible source of new drugs to complement the action of oral 

hypoglycaemia agents. Renewed attention to alternative medicines and natural therapies has stimulated 

a new wave of research interest into traditional practice. The World Health Organization (WHO) 

Expert Committee on Diabetes listed as one of its recommendations; further investigations into 

traditional methods of treating diabetes (WHO, 1980). 

Indigofera pulchra(Willd) family: papilionaceae is an annual non climbing herbs or shrub that 

can grow up to 1m tall. It is widely distributed throughout west-Africa (Hepper,1976). In 

ethnomedicine, the leaves are used to treat infected wound (Hepper,1976;Burkhill, 1995) while the 

decoction of the aerial part is used as prophylactic against snake-bite (Sule et al.,2003) and as antiinflammatory 

(Abubakar et al.,2007). Previous pharmacological studies on the methanol extract of the 

aerial part of this plant showed that it exhibited venom detoxifying activities (Abubakar et al.,2006). 

The present study was designed to test the hypoglycemic effect of n-butanol portion of 

Indigofera pulchra leaves extract of on alloxan – induced and normoglycemic Wistar rats diabetes.

503 Tanko. Y, Abdelaziz. M.M, Adelaiye. A.B, Fatihu. M.Y and Musa. K.Y 

2. Materials and Methods 

2.1. Plant material 

Indigofera pulchra sample was collected from Samaru-Zaria in the month of October 2007 and was 

authenticated by A.U.Gallah of the Biological Sciences Department, Ahmadu Bello University Zaria- 

Nigeria where a voucher specimen (No.6558) was deposited. The leaves were air-dried and made into 

powder using pestle and mortar. 

2.2. Extraction 

The air- dried powdered plant material was extracted with 70% methanol using soxhlets apparatus, the 

solvent was removed in-vacuo to yeild a residue (100gms) referred to as methanol extract. 100 grams 

of the methanol extract was suspended in water and filtered, the water soluble part was extracted with 

n-butanol (4x500ml) to give 6gms n-butanol portion. 

2.3. Chemicals used 

All chemicals and drugs were obtained commercially and were of analytical grade. Alloxan 

monohydrate (Sigma).The Biphasic Isophane insulin AS Mixtard 30 HM Pen fill (Novo Nordisk AIS 

2880 Bagsvaerd, Denmark. NAFDAC Reg.no 04-1601). 

2.4. Acute toxicity study 

The lethal doses (LD 50 ) of the plant extract was determined by method of Lorke (1983) using 12 rats. 

In the first phase rats were divided into 3 groups of 3 rats each and were treated with the extract at 

doses of 10, 100 and 1000mg/kg body weight intraperitoneally. They were observed for 24 hours for 

signs of toxicity. In the second phase 4 rats were divided into 4 groups of 1 rat each and were also 

treated with the extract at doses of 1600, 2900 and 5000 mg/kg bodyweight (i.p).The median lethal 

dose (LD 50 ) was calculated using the second phase. 

2.5. Phytochemical screening 

The preliminary phytochemical screening of n-butanol portion of Indigofera pulchra leaves extract was 

carried out in order to ascertain the presence or absent of its constituents by utilizing standard 

conventional protocols (Trease and Evans, 1983). 

2.6. Animals and Induction of Diabetes Mellitus 

Wistar strain albino rats of both sexes weighing (180-200 g) bred in the Department of Pharmacology 

and Clinical Pharmacy, faculty of Pharmaceutical Sciences A.B.U Zaria, were used for the study of the 

effect of the n-butanol portion of Indigofera pulchra leaves extract on the blood glucose levels of the 

animals. The animals were fed ad libitum with pellet diet (Vital feeds, Jos, Nigeria) and water. They 

were kept and maintained under laboratory conditions of temperature, humidity and light (25 ± 1ºC and 

12 h light/dark cycle) respectively. We followed the Guide for the care and Use of Laboratory 

Animals, 1985, issued by the US Department of Health and Human Services, Public Health Service, 

National Institute of Health, NIH Publication No.86-23. The rats assigned to the diabetic groups were 

injected with alloxan monohydrate dissolved in sterile cold normal saline at a dose of 150 mg/kg body 

weight intraperitoneally as reported by earlier by (Kameswara Rao et al.,1999). Since alloxan is 

capable of producing fatal hypoglycemia as a result of massive pancreatic release of insulin, the rats 

were treated with 20% glucose solution intraperitoneally after 6 hours (Stanley et al., 2001).The were 

kept for the next 24 hours on 5% glucose solution bottles in their cages to prevent hypoglycemia .After 

a period of three days the rats with a blood glucose levels greater than 170mg/dl were considered 

diabetic and used for this research work (Stanley et al.,2001).



2.7. Experimental Design 

The alloxan -induced diabetic Wistar rats were randomly assigned into five groups (1-5) of five rats 

(n=5) each as follows, namely 

A. Group 1- Received normal saline i.p 

B. Group2- Received Biphasic Isophane Insulin 6 i.u/kg i.p(Stanley et al.,2001) 

C. Group3-Received 250mg/kg body weight of the n butanol portion of the extract i.p 

D. Group4- Received 500mg/kg body weight of the n-butanol portion of the extract i.p 

E. E.Group5- Received 1000mg/kg body weight of the n-butanol portion of the extract i.p 

The normoglycemic Wistar rats were also randomly assigned into five groups (6-10) with 

five rats (n=5) in each group as follows: 

F. Group 6- Received normal saline i.p 

G. Group7- Received Biphasic Isophane Insulin 6 i.u/kg i.p(Stanley et al.,2001) 

H. Group8-Received 250mg/kg body weight of the n-butanol portion of the extract i.p 

I. Group9- Received 500mg/kg body weight of the n-butanol portion of the extract i.p 

J. Group10- Received 1000mg/kg body weight of the n-butanol portion of the extract i.p 

2.8. Determination of blood glucose levels 

All blood samples were collected by cutting the tail-tip of the rats. Blood samples for blood glucose 

determination were collected from the tail at intervals of 0, 2, 4, 8 and 24 hours. Determination of the 

blood glucose level was done by the glucose-oxidase principle (Beach and Turner 1958) using the 

ONE TOUCH Basic (Lifescan, Milpitas, CA) instrument and results were reported as mg/dl (Rheney 

and Kirk, 2000). 

2.9. Statistical analysis 

Blood glucose levels were expressed in mg/dl as mean ± SEM. The data were statistically analyzed 

using ANOVA with multiple comparisons versus control group by Dunnett’s method. Values of 

p


3.1. Acute toxicity study (LD 50 ) 

The sign of toxicity were first noticed after 8-10 hours of extract administration. There was decreased 

locomotor activity, decreased feed intake, and prostration after 14 hours of extract administration. The 

median lethal dose (LD 50 ) in rats was calculated to be 2,154 mg/kg body weight. 

3.2. Blood Glucose levels of Alloxan-induced Diabetic Wistar rats 

Table 1 showed the results of the effects of three doses (250mg/Kg, 500mg/Kg and 1000mg/Kg) of n- 

butanol portion of the leaves extract of Indigofera pulchra, Insulin and control groups in alloxaninduced 

diabetic Wistar rats. The dose of Insulin and the three doses of the extract did not show any 

significant change in the blood glucose levels when compared to untreated control after 2 and 4 hours 

of treatments. However, after 8 and 24 hours of treatments the dose of insulin and 250mg/Kg of the 

extract showed a significant (p



results in decreased Insulin levels and hyperglycemia leading to type 1 diabetes mellitus. However, 

animal models of diabetes differ significantly from each other and none can be taken, without 

reservation, to reproduce the essentials of human diabetes (Bell and Hyde, 1983). 

In the alloxan-induced diabetic groups, the effect of three doses (250mg/Kg, 500mg/Kg and 

1000mg/Kg) of n-butanol portion of Indigofera pulchra, Insulin and control groups were evaluated. 

The dose of Insulin and the three doses of the extract did not show any significant change in the blood 

glucose levels when compared to untreated control after 2 and 4 hours of treatments. However, after 8 

and 24 hours of treatments the dose of insulin and 250mg/Kg of the extract showed a significant 

(p


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