Hepatic, Pancreatic, and Renal Histo-Morphologic Alterations in Administration of Aqueous and Ethanol Seed Extract of Buchholzia Coriacea in Alloxan-Induced Diabetic Rats

Primarily, effect of insulin on glucose breakdown is to aid its efficient uptake and utilization by most cells of the body, except those of the brain [1,2]. Defects in insulin secretion or its action leads to an increase in blood glucose level with consequence in derangement of carbohydrate, protein and fat metabolism [3]. Diabetes is characterized by increased thirst, hunger and loss of weight [4,5]; resulting in chronic complications like macrovascular, microvascular, and neuropathic disorders [6]. Long term consequences of diabetes are diseases of the coronary arteries and cerebrovascular system; renal failure, neuropathy, retinopathy and premature death [4].

There is a rapid increase in the prevalence of diabetes globally and the World Health Organization (WHO) has predicted that by 2030 the number of adults with diabetes would have almost doubled globally, from 177 million in 2000 to 370 million [7]. According to Rowley et al., experts have estimated that the cases of adults with diabetes is set to increase rapidly by 64% by 2025‚ which means that a shocking 53.1 million people will be affected by the disease. The prevalence of diabetes among adults worldwide in 2010 was 285 million (6.4%) and this value is predicted to rise to around 439 million (7.7%) by 2030 [8].
In Nigeria, according to Olatunbosun et al, the prevalence of diabetes ranged from low level of 0.8% among adults in rural highland dwellers to over 7% in urban Lagos with an average of 2.2% nationally [9]. Despite the availability of several oral anti-hyperglycemic agents and insulin for the management of diabetes, there is still an increasing demand by sufferers to use herbal drugs even when their biologically active compounds are unknown due to their effectiveness, fewer side effects, and relative low cost [10]. This has prompted active research efforts to provide a more effective, safer and cheaper alternative agent of plant origin that possess anti-hyperglycemic properties.
Buchholzia Coriacea (Wonderful cola) is an evergreen shrub belonging to the family Capparidaceae. It is found in many tropical countries like Ghana, Gabon, Cameroon, Central African Republic, Congo, Angola, Nigeria, among others [11]. Previous studies have shown that the ethanol extract of Buchholzia Coriacea elicit hypoglycemic effects that exhibits synergistic actions with the hypoglycemic agent metformin. Traditional medicine and scientific research on different parts of the plant has claimed that it is a valuable alternative therapy for treatment of Diarrhea [12], malaria [13], worm infection [14], rheumatism [15], diabetes [10], hypertension, psychiatric disorders, asthma and cough, impotence, among others.

Aim of Study
Study sought to investigate the effect of Buchholzia Coriacea (wonderful kola) seed extract on the histo-architecture of selected organs (liver, kidney and pancreas), in alloxan-induced diabetic wistar rats. Specifically, study:

i.
Examined the effect(s) of wonderful kola on blood glucose level

ii.
Determined the effect(s) of wonderful kola on body weight iii. Examined the effect(s) of wonderful kola on liver, pancreas, and renal histo-architectures

Study design
Fourty rats, weighing an average of 128.6 g were divided into eight groups of five rats each as follows:

Normoglycemic groups
Normal control: neither given Alloxan monohydrate, nor administered with Buchholzia Coriacea extract. They received normal feed and water ad libitum for the duration of study.

Preparation of Plant's extract
Fresh seeds of Buchholzia Coriacea were collected and identified by renown botanists from the Department of Botany, Faculty of Science, Delta State University, Abraka. The seeds were immediately cleaned off debris, peeled, chopped and shade-dried for one week in laboratory trays. The dried seeds were pulverized into powder with sterilized machine and weighed. 500 g of the powder was then divided into two equal parts and macerated in 4500 ml distilled water and 4500 ml of ethanol respectively, following intermittent shaking for 48 hours. Extract was then obtained with rotary evaporator (an electrical evaporator extraction apparatus). The solvent was extracted at temperature of 45ºC and pressure of 60 mmhg of water. Paste-like extract was obtained and oven-dried to complete solid, then, grinded to smooth powdered form and stored in a refrigerator till use.

Procedure
Ethical clearance: Ethical clearance was obtained from the Research and Ethics Committee of the Faculty of Basic Medical Sciences, College of Health Sciences, Delta State University, Abraka, Delta State.
Acute toxicity test: Graded doses (250, 500, 1000, 2000, and 5000) mg/kg of the aqueous and ethanol extracts of wonderful kola was administered to the different groups orally. They were then observed for acute toxicity signs like behavioural changes or death over 24 h. though no deaths were recorded, dizziness was however observed at the dose of 2,000 and 5000 mg/kg body weight.
Sample collection: Blood glucose levels were checked every seven days (weekly), using the ACCUCHEK glucometer. To achieve this, blood sample was taken from tail vein of the rats on each occasion. After an overnight fast on the last day of the experiment, a final blood glucose check was done. Then rats were sacrificed by cervical decapitation and each rat was placed on its dorsal surface, and a laparotomy was carried out to expose the internal organs. Blood was collected by cardiac puncture, using 5 ml syringes and 21 G needle. Obtained blood samples were centrifuged at a rate of 4000 rpm for 10 minutes, and serum was collected and stored in a refrigerator at 4 o C for analysis of the liver enzymes.
Inducing diabetes: To induce diabetes, Alloxan monohydrate was dissolved in 0.9% sodium chloride buffer (pH 7) intraperitoneally at a dose of 50 mg/kg body weight. Rats in control group were administered with equal volume of 0.9% sodium chloride, pH 7 (that was used to prepare the Alloxan monohydrate solution). Thereafter, rats were fed with normal feed and water. Three days (72 hours) after induction, diabetes was confirmed at a random blood glucose level of ≥ 200 mg/ dl, using the ACCUCHEK glucometer.

Preparation of tissue for microscopic examination:
The process of preparation of tissue for histological examinations was done for liver, kidney and pancreas; First, the tissues were processed by impregnating the specimen into embedding medium to provide support and suitable consistency for microtomy sectioning using different graded solutions of alcohol from 70% to 100% to dehydrate, them (tissues). Thereafter tissues were processed with paraffin wax, using an automatic tissue processor. Samples were then embedded in paraffin wax at 70 degrees centigrade and cut with a rotary microtone to 4 µ. The staining technique employed in this study was the haematoxylin and eosin staining techniques (Plates 1-24).
Photomicrography: Stained tissue images were captured with digital microscopic eyepiece 'Scoptek' Dcm 500, 5.0 mega pixels connected to USB 2.0 computer.

Statistical approach
Results were expressed as Mean ± SEM (standard error of mean) and statistical significance of the treatment effect was analysed with one way analyses of variance (ANOVA), followed by post Hoc LSD's test for multiple comparison, using software social science (SPSS) version 22. P-values <0.05 were considered to be statistically non-significant.

Results
See Figures 1 and 2 for results.

Discussion
Numerous advantageous effects of Buchholzia coriacea seed extracts have been identified in literatures, earning it the name "wonderful kola [16,17].
The body weight of normal and diabetic rats before induction and after of treatment is shown in Figure 1. There was a significant elevation in the body weight of normal control rats when compared baseline (0 day before induction) body weight to final (day 14 after treatment). The administration of Metformin (50 mg/kg) and aqueous 250 mg/kg and ethanol 250 mg/kg extracts also showed moderate increase (p>0.05) in the body weight of normal rats as compared to day 0 and day 14 after treatment. There was a significant (p<0.05) reduction in the body weight of rats in diabetic control group when compared baseline (0 day after induction) body weight to final (day 14 after treatment). The treatment with Metformin (50 mg/kg) and aqueous 250 mg/kg and ethanol 250 mg/kg extracts showed a significant decrease in the body weight of diabetic rats as compared baseline (0 day after induction) body weight to final (day 14 after treatment). There was no significant difference (p>0.05) in the body weight of normoglycemic treated groups when compared with the normoglycemic control group (normal control) before induction and after treatment. A mild decrease (p<0.05) in metformin diabetes treated group, aqueous extract diabetes treated group and ethanol extract diabetes treated group when compared to the diabetes control group Figure 2 shows changes in glucose levels of normal and diabetic rats administered with Metformin (50 mg/kg) and aqueous 250 mg/ kg and ethanol 250mg/kg extracts in. Here, there was a mild change in fasting blood glucose level in normal control rats when compared with baseline (0 day before induction) body weight to final (day 14 after treatment). There was also a mild but not significant (p>0.05) decrease in the metformin normoglycemic treated group, AEBC normoglycemic treated group and EEBC normoglycemic treated group when compared to the normal control group after day 14 treatment. The administration of Metformin (50 mg/kg) and aqueous 250 mg/kg and ethanol 250mg/ kg extracts also shows mild alteration (p>0.05) in the fasting blood glucose level of normal rats after day 14 treatment as compared to day 0. Findings from this study showed a significant (p<0.05) increase in the glucose level of diabetic rats when compared to glucose level of the rats at the commencement (0 day before induction) fasting blood glucose level to day 4 (after induction) of the experiment ranging from 72.04 to 76.43%.
It was also observed that the values of glucose level of the diabetic rats treated with Metformin (50mg/kg) and aqueous 250 mg/kg and ethanol 250mg/kg extracts were not up to the glucose level of diabetic control rats. Despite this, there was statistical significance (p<.05) in percentage changes between Metformin (50mg/kg) and aqueous ethanol (250mg/kg) extracts. The metformin treated group showed more effect (-189.60 ± 23.36 * c), followed by ethanol extract treated group (-161.96 ± 9.81 * a) and aqueous extract treated group (-151.10 ± 14.92 * a) when compared to diabetic control rats (-109.78 ± 26.33).

Relevance of Study
Since diabetes became a global monster to earthlings, any study that is directed towards its amelioration is not only Jermaine, but hugely recommended. This study will provide basic information on histologic changes in pancreas, liver, and kidneys; being the major organs that directly or indirectly part-take in glucose homeostasis as a result of graded-dose use of Buchholzia coriacea seed extracts. This implies that administration of this extract with dose and administration duration considered will be of be scientific merit in the folkloric use of the extract for management of this ailment.

Conclusion
Administration of aqueous and ethanol seed extract of Buchholzia coriacea for treatment of Alloxan-Induced diabetic rats modulated key carbohydrate metabolic enzymes, resulting in normal blood glucose homeostasis. This implies that administration of this extract with dose considered and the duration of administration has anti-hyperglycemic and glycolytic effects with adverse effects on liver and kidney functions. Thus, there could be scientific merit in the folkloric use of the extract in the management of diabetes.

Recommendations
Being just a preliminary work, we recommend further study on this, however with more sophisticated and high-tech stains/equipment that can reveal deeper tissue structures and cellular components.