Effects of Ethanolic Leaf Extract of Solenostemon Monostachyus on Blood Glucose level and Histopathological Parameter in STZ induced Diabetic Wistar Albino Rats

One of the most dangerous and life threatening diseases in the world today, involving the pancreas, is diabetes mellitus [1]. It is a condition primarily defined by the level of hyperglycemia giving rise to risk of micro vascular damage (retinopathy, nephropathy and neuropathy). It is associated with reduced life expectancy, significant morbidity due to specific diabetes related micro vascular complications, increased risk of macro vascular complications (ischemic heart disease, stroke and peripheral vascular disease) and diminished quality of life [2]. The record of its prevalence in the world today and the increase in morbidity and mortality rates calls for an urgent attention from the health professionals and policy makers in tackling this disease. In CôtedIvoire, the rate of morbidity is 5.7%. Traditional medical herbs used for strengthening the body immune system and for therapeutic purposes are known to have many essential and nutritional elements. Many plants have been documented to have the efficacy to treat diabetes in CôtedIvoire [3].


Introduction
One of the most dangerous and life threatening diseases in the world today, involving the pancreas, is diabetes mellitus [1]. It is a condition primarily defined by the level of hyperglycemia giving rise to risk of micro vascular damage (retinopathy, nephropathy and neuropathy). It is associated with reduced life expectancy, significant morbidity due to specific diabetes related micro vascular complications, increased risk of macro vascular complications (ischemic heart disease, stroke and peripheral vascular disease) and diminished quality of life [2]. The record of its prevalence in the world today and the increase in morbidity and mortality rates calls for an urgent attention from the health professionals and policy makers in tackling this disease. In CôtedIvoire, the rate of morbidity is 5.7%. Traditional medical herbs used for strengthening the body immune system and for therapeutic purposes are known to have many essential and nutritional elements. Many plants have been documented to have the efficacy to treat diabetes in CôtedIvoire [3].
Diets rich in fruits, cereals and vegetables have been shown to play a crucial role in the prevention of a lot of disorders such as cardiovascular diseases, certain cancer types and even ageing [4,5]. Thus efforts have been made towards the elucidation of bioactive potentials of many plants of recent. This has however not translated to utilization of these plants coupled with the fact that the bioactive potentials of a lot of them are yet to be investigated. Solenostemon monostachyus sp. Beauv (family Lamiaceae), common name (African dead nettle), Effik name (Ntorikwot, Awakmmon) is an important herb that is widespread in West and central Africa. It occurs as an annual weed in anthropogenic habitat and rocky savannahs. It is slightly succulent, aromatic and grows up to 100cm tall [6].
It has been reported that plant has been traditionally used in the past for ritual purposes related to pregnancy. The decoction of the leaves is also taken as a diuretic [7]. Research has also shown that the leaves possess antimicrobial activity [8].
(The health promoting properties of plants are ascribed to the possession of various photochemical especially phenolics and this beneficial activity is related to their antioxidant activity [9]. Herbal medicine has improved as alternative effective solution to health problems and cost of pharmaceutical products. However, little work has been done to match ethanobotanical information with analytical research to identify active chemical compounds. Plants used in traditional medicine can offer potential sources of new biological active compounds, many of them as antidiabetic, anti-HIV, and antifungal agents. Examples of these compounds include flavonoids, sapononins, phenolics, glucosinolates, and cynagenic glycosides [10]. (Potential natural or synthetic substances with biocide activity are considered candidates for developing new drugs for the treatment of various chronic as well as infectious diseases. Indigenous medicinal plants continue to be rich source of therapeutic drugs. The active principles of many drugs are usually found in plants as secondary metabolites. The search for new pharmacologically active agents obtained by screening natural sources such as microbial fermentations and plant extracts has led to the discovery of many clinically useful drugs that play a major role in the treatment of human diseases ( Figure 1).

Materials and Method
Chemicals and Reagents: Several chemicals and analytical grade reagents were purchased, and used for this research which includes: Ethanol (98%), sodium hydroxide, sodium chloride and glucometer strips which were purchased from Globus Chemical, Mayne Avenue, Calabar, and Cross River State, Nigeria. The beta cell destructive agent, streptozotocin (STZ) was obtained from sigma, St

Identification and preparation of Plant Materials:
Fresh leaves of Solenostemon monostachyus were collected from local garden at the University of Calabar, Nigeria. The sample of the plant specimen was identified and authenticated by a Botanist from the botanical garden, and the Voucher specimen was deposited in the herbarium of the same University. The leaves were sorted to eliminate any dead matter and other unwanted particles. The leaves were air-dried for 2 weeks and the leaves were blended with a manual hand blender and 150g of the plant was weighed and soaked in 700ml of ethanol. The mixture was then placed in a water bath at about 60-80 0 C for 10 minutes for through extraction of the plant active component and allowed to cool. The extract was then filtered with a chess material and later a watchman no 1 filter paper to obtain a homogenous filtrate. The filtrate was then concentrated in vacuum at low temperature of 37-40 0 C.
The concentrate was then allowed open in a water bath for complete dryness. The extract was refrigerated at 2-5 0 C until when used. Appropriate concentration of the extract was subsequently made by dilution with distilled water into 250/ mg/kg body weight and administered to the animals (Table 1). Handling and treatment of Animals: A total of 24 adult male albino rats weighing between 150-250g obtained from the disease free stock of the animal house, Biochemistry Department, College of Medical Sciences University of Calabar, Nigeria, were used for the study. The rats were divided into four groups with six rats each, as follows: Group A (normal control group receiving distilled water as placebo), Group B (diabetic control group receiving distilled water as placebo), Group C (insulin group receiving 5ᶸ/kg bodyweight of insulin). Group D (diabetic test group will received oral dose of Solenostemon monostachyus leaves extract juice).

Induction of experimental diabetes:
Prior to diabetes induction, the rats were subjected to 12hr fast, and then diabetes was induced by intraperitoneal injection of 40mg/kg b.

Anatomy Physiology & biochemistry international journal (APBIJ)
STZ treatment, diabetes was confirmed in STZ treated rats with a fasting blood sugar (FBS) concentration ≥ 200mg/dl. This was estimated using One Touch Glucometer (Life scan, Inc. 1996 Milpas, California, U.S.A) with blood obtained from the tail vein of the rats.
The rats were acclimatized in the experimental animal house for one week before the commencement of the experiment. The animals, housed in stainless steel cages under standard conditions (ambient temperature, 28.0±2.0º C and humidity, 46%, with a 12 hr light/dark cycle), were fed with the normal rat pellets. All the rats in both test and control groups were allowed free access to food and water ad libitum, throughout the experimental period. Good hygiene was maintained by constant cleaning and removal of faeces and spilled feed from cages daily. The animals in test groups III will received insulin 5ᶸ/kg body weight and IV will received oral daily doses of aqueous extract of Solenostemon monostachyus leaves respectively, using orogastric tubes and syringes.
This lasted for a period of 21 days and the experiments were conducted between the hours of 7.00 am and 7.00pm daily. Rats in the control groups I were administered, by oral gavage, with 5ml of distilled water (placebo). All the animal experiments were carried out in accordance with the guidelines of the Institution's Animal Ethical Committee.
Collection and analysis of blood: All the animals were anaesthetized with chloroform vapor, twenty-four (24) hours after last day of extract administration, and dissected for blood collection. Blood samples were collected by cardiac puncture into a set of plain and fluoride oxalate sample bottles. Determination of LD50/Acute toxicity tests. This was done orally using albino mice. Biochemical tests were carried out, which include: liver enzymes-alanine aminotransferase and aspartate aminotransferase according to the method described by Reithman (1957). Serum alkaline Phosphatase using Randox test kits. Lipoprotein profile-Total cholesterol, HDL-C, TG, serum electrolytes, urea and creatinine Statistical Analyses: The results obtained from this study were analyzed by one-way analysis of variance (ANOVA), followed by Student's t-test to evaluate the significance of the difference between the mean value of the measured parameters in the respective test and control groups using SPSS windows. A significant change was considered acceptable at P <0.05.

Results
Changes in blood glucose of diabetic and non-diabetic test animals: Daily changes in blood glucose that was monitored following daily treatment with extract and insulin in diabetic and non-diabetic rats. The blood glucose levels ranged between 60-108 mg/dl in the normal control rats (NC) that were not given any extract. Fasting blood glucose of other treatment groups varied differently, the treatment groups tended toward lowering the blood glucose levels when compared with the diabetic control (DC), but the Solenostemon monostachyus (SM) group was closely related to the insulin treated group. The diabetic control group range was between 601 and 295 mg/dl. Differences in DC showed sustained elevations in blood glucose level of untreated diabetic rats ( Table 2).          showing numerous atrophic glomeruli with prominent Bowman's capsules, dense acellular mesangium and glomeruli. The cortical tubules have ghost-like appearance with prominent lumen and intensely stained atrophic cells with scanty interstitium. Mild pathology. The histological and/or architectural integrity of the liver and kidney tissues in the experimental group was examined and compared with the controls. Results revealed that for liver tissues there was no evidence of liver cell injury for the controls, because sections of the liver tissues showed a preserved architecture with dilated and congested central veins. The hepatocytes were prominent and radiated from the central vein. The hepatic cells had an eosinophilic cytoplasm and ovalto-ovoid basophilic nuclei. The sinusoidal spaces are prominent and contain portal tracts were prominent and contain the bile duct, portal vein and hepatic arteries.

Effect of treatment on the histological architecture of liver and kidney tissues using Haematoxylin and Eosin technique
For normal control kidney, there was no indication of kidney lesion since sections of the kidney showed a distinct cortex and medulla. The Glomeruli were prominent with a distinct Bowman Space. The mesangium was hypercellular and with abundant interstitium. The medulla consisted of numerous renal tubules of various sizes and shapes. The tubules were lined with cuboidal to columnar epithelium with a distinct lumen (Figure 3).   The kidney tissues for INS revealed a section showing a distinct cortex and medulla. Numerous glomeruli showed as hyalinized mesangium that is acellular and some show glomerulosclerosis. Majority of the glomeruli were viable. The cortical renal tubules had a patent lumen surrounded by deeply stained cuboidal epithelium. The intervening interstitium was sparse. The medulla consisted of numerous collecting tubules of various sizes and shapes lined by an intact epithelium ( Figures  6 & 7). were seen. For section of the kidney tissue in this Solenostemon monostachyus (SM) group numerous atrophic glomeruli with prominent Bowman capsules and dense oesinophilic a cellular mesangium and glomeruli were seen. The cortical tubules had a ghostlike appearance with prominent lumen and intensely stained atrophic cells. The intervening interstitium was scanty (Figures 8 & 9).