Abstract: The study examined the microbial purities, acute and chronic toxicities of a poly-herbal formulation A prepared with Ocimum gratissimum, Momordica charantia and Vernonia amygdalina leaves and formulation B prepared with Ocimum gratissimum, Citrus medica and Momordica charantia leaves used in the treatment of diabetes. The microbial purity was evaluated on some bacterial and fungal organisms using appropriate diagnostic media. Toxicity of the polyherbal preparation was evaluated on Swiss albino mice by administering to the animals oral graded doses of the lyophilized drug in the ranges of 1.0 to 20.0 g kg-1 body weight and observed continuously for the first 4 h and hourly for the next 12 h, then 6 for 56 h (72 h, acute toxicity). Also the effects on the biochemical and haematological parameters were evaluated on the two formulations. The formulations were found to contain heavy loads of both pathogenic and non pathogenic microorganisms higher than officially accepted limits with formulation A having more microbial loads than B. The median acute toxicity values (LD50) of the formulations were, respectively determined to be 15.0 and 15.65 g kg-1 body weight (b.wt.). There was dose dependent significant (p≤0.05) decrease in plasma glucose levels in the animals treated with the two doses (250 and 500 mg kg-1 b.wt.) of the formulations/glibenclamide compared to the diabetic control. The photomicrograph of pancreatic tissues treated with formulations A and B showed significant beta cells survivor compared to diabetic control. There was however, significant increase (p≤0.05) in aspartate aminotransferases (AST) and creatinine levels in the group treated with highest dose of formulation A and B. Significant increase (p≤0.05) in alanine aminotransferases (ALT) levels was observed in the groups treated with formulation B while marginal increase occurred in the group treated with formulation A. The formulations also ameliorated dislipidemia.
INTRODUCTION
Diabetes Mellitus (DM) is a clinical syndrome characterized by hyperglycemia due to absolute or relative deficiency of insulin. Lack of insulin affects the metabolism of carbohydrate, protein and fat. Therefore it may present with hyperglycaemia, hypertriglyceridaemia and hypercholesterolaemia (Walter, 1977). DM is a major cause of morbidity and mortality in the world today afflicting many lives both in the developed and developing countries as a result of macrovascular complications i.e., coronary artery disease and cerebrovascular disease and the microvascular complications such as diabetic nephropathy, a major cause of kidney failure and diabetic retinopathy, a major cause of blindness (Sharon and Marvin, 1975; Walter, 1977, Barnett and O’Gara, 2003).
There are two main types of DM namely juvenile onset or insulin dependent diabetes and type II diabetes or Non Insulin Dependent Diabetes Mellitus (NIDDM). Type II is usually seen in adults and is associated with relative insulin deficiency or insulin resistance with hyperinsulinaemia (Grundy et al., 1999).
Diabetes has been conventionally treated with orthodox medicines which function as hypoglycaemic agents or insulin production modulators and/or lipoprotein lowering agents (Ogbonnia et al., 2008). Insulin, sulfonylureas, biguanides, alpha glucosidase inhibitors and glitazones have been empirically used in the control of NIDDM but have their short comings in terms of unfavorable pharmacokinetic profile, adverse drug reactions such as hypoglycaemia as seen in insulin and sulphonyureas, lactic acidosis in biguanides and unwanted weight gain and expensive cost in case of glitazones usage. Furthermore they are often unable to lower glucose concentrations to within the normal range or to reinstate a normal pattern of glucose homeostasis when used as a monotherapy in trying to limit the adverse drug reactions (Senthilvel et al., 2006; El Naggar et al., 2005). Apart from insulin which is usually being administered by injections all the oral hypoglycaemic agents are not suitable for use during pregnancy (Senthilvel et al., 2006; Sushruta et al., 2006).There is the need, therefore, to encourage the use of alternative therapy with plants and plant derived products being considered to have less side effect and low toxicity. Herbal medicine is now recognized as the most common form of alternative medicine and is used by about 60% of the world population both in the developing countries as well as in the developed countries where modern medicines are predominantly used (Rickert, 1999). Following increasing use of herbal formulations in the treatment of diseases particularly metabolic disorders by many Nigerians, it became imperative to evaluate the antimicrobial purity, toxicity profile and the efficacy of some of the polyherbal plants already being used by the natives in the management of metabolic disorders like DM. Ocimum gratissimum, Citrus medica and Momordica charantia and Vernonia amygdalina are widely and commonly employed in the traditional medicine in the treatment of various diseases including diabetes. The antidiabetic activity of the individual plant products has been reported in various literatures (Akah and Okafor, 1992; Srivastava et al., 1993; Mohammed et al., 2007; Conforti et al., 2007). However, to the best of our knowledge, the two herbal formulations for this investigation have not been verified scientifically for antimicrobial purity, toxicity profile and antidiabetic activity.
In this light, the present study was aimed at evaluating antimicrobial purity, toxicity profile as well as comparing the antidiabetic effects of two locally and commonly consumed antidiabetic formulations; one prepared with leaves of Ocimum gratissimum, Momordica charantia and Vernonia amygdalina (formulation A) and the other prepared with leaves Ocimum gratissimum, Citrus medica and Momordica charantia (formulation B) on an alloxan-induced diabetes in animals.
MATERIALS AND METHODS
| • | A polyherbal formulation, with a dark brownish colour and a slightly thick non viscous liquid formulation 1.5 L sealed in a plastic container (Formulation A) was ceded by Dastops Alternative Therapy of No. 6, Owokoniran Street Iyana-Ipaja, Lagos. Formulation A ingredient of production was unspecified quantities of Ocimum gratissimum, Momordica charantia and Vernonia amygdalina leaves. The dosage, batch number and expiry dates were not stated and was prepared on the 1st June, 2010 |
| • | A polyherbal formulation, a slightly thick, non viscous dark brownish liquid formulation (Formulation B, 1.5 L) sealed in a plastic container was ceded by a traditional practitioner Ade and Ade Anti-diabetes, of No. 4 Adeyemi-Moye Street Ijegun-Ikotun, Lagos, Nigeria. Formulation B ingredient of production was unspecified quantities of Ocimum gratissimum, Citrus medica and Momordica charantia leaves |
There was no specification of the quantity of each plant material used. The batch number and expiry dates were not stated except the date of production which was on the 1st June, 2010.
The plants samples used for the preparations were obtained from the practitioners. Each plant specimen was authenticated by a taxonomist Mr. Odewo of the Department of Botany and Microbiology, University of Lagos, Lagos and voucher specimens, respectively prepared. Citrus medica with voucher No. LUH 2771, Momordica charantia with voucher No. LUH 2773, Vernonia amygdalina with voucher No. LUH 2774 and Ocimum gratissimum with voucher No. LUH 2775 were deposited at the Department of Botany Herbarium, University of Lagos, Akoka, Lagos.
The formulations were selected on the basis of matching the contents maximally and are consumed locally by many diabetic patients. The un-tampered procured polyherbal formulation bottles were stored in a refrigerator at 4-6°C until the quantity needed for the purity test was aseptically taken. For the toxicity and diabetic studies 1000 mL of each formulation as was procured was filtered using Whatman’s no 4 filter and the respective resulting 785 and 704 mL filtrates were subsequently freeze dried to yield 37.7 and 34.7 g gels which were stored in an air tight container in a fridge until they were needed.
Animals: Swiss albino mice (22.5±2.5 g) and Wistar rats (135±15 g) of either sex were obtained and kept under standard experimental conditions. They were housed in the roomy plastic cages with enough ventilation (5 animals per cage) and maintained on standard animal cubes (Livestock Feeds Nigeria Ltd) and provided with water ad libitum. They were allowed to acclimatize for seven days to the laboratory conditions before the experiment. The use of the animals and the experimental protocol were in strict compliance with the Institute of Laboratory Animal Research (ILAR) (ILAR, 1996).
Purity test evaluation of formulations A and B: The microbial load of the preparation was determined using the standard plate method (Fontana et al., 2004). The antimicrobial activity of the preparation was investigated using the cup diffusion method on Nutrient Agar (NA) for bacterial organisms and Sabouraud Dextrose Agar (SDA) for fungal organisms (Raghavendra et al., 2006). The cultures were incubated for 24 h and observations were made for zones of incubation (NCCLS, 1997).
Acute toxicity study: The toxicity study was carried out using thirty-five Swiss albino mice (22.5±2.5 g) of both sexes. The animals were randomly distributed into one control group and six treated groups, containing five animals per group. After depriving them of food overnight, the control group received 0.3 mL of 2% acacia solution orally while each treated group received orally solution of the lyophilized extract in 2% acacia solution in the doses of 1.0, 2.5, 5.0, 10.0, 15.0 and 20.0 g kg-1 body weight (b.wt.), respectively (Shah et al., 1997; Burger et al., 2005). Log dose and probit analysis were used to determine the LD50 of the formulations.
Biochemical assay: After overnight fasting, diabetes was experimentally induced in the animals by administering intraperitoneally (i.p.) alloxan monohydrate at 150 mg kg-1 body weight (b.wt.) dissolve in normal saline (Mbaka et al., 2008). The blood sugar level was monitored with a glucometer (Accu-chek, Roche Diagnostics) after 72 h and the rats with plasma glucose levels >200 mg dL-1 were classified as diabetic and were used for the study. Animals were divided into five groups, all comprised of five rats, respectively. Groups II and III had two sub groups for formulations A and B. Group IV was glibenclamide treated while groups I and V were the control. Daily treatment which lasted for 30 days were administered as follows:
| Group I: | Alloxan induced rats not treated |
| Group II: | Alloxan induced rats treated with 500 mg kg-1 b.wt. |
| Group III: | Alloxan induced rats treated with 250 mg kg-1 b.wt. |
| Group IV: | Alloxan induced and treated with glibenclamide, 600 μg kg-1 b.wt. (Mahdi et al., 2003) |
| Group V: | Normal not treated (positive control), (0.5 mL 2% acacia suspension) |
Groups I and V were common to the formulations and the procedure was the same for the two formulations.
During the course of treatment, the animals were weighed every seven days from the beginning of the treatment. The animals were sacrificed under mild diethyl ether anaesthesia after 24 h of fast and blood was obtained via cardiac puncture into the heparinized bottle (biochemical analysis), fluoride oxalate (glucose analysis) and EDTA tube for haematological analysis. The blood collected with fluoride oxalate and heparinized bottle were centrifuged within 5 min of collection at 4000 rpm for 10 min to obtain blood plasma used to determine the plasma glucose levels and biochemical parameters. The total cholesterol, total triglyceride, HDL-cholesterol levels were estimated using precipitation and modified enzymatic procedures from Sigma Diagnostic (Wasan et al., 2001; Egwim, 2005). LDL-cholesterol level was calculated using Friedwald equation (Crook, 2006). Plasma was analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinines by standard enzymatic assay analysis and the plasma protein and glucose contents were determined using enzymatic spectroscopic methods (Hussain, 2002).
Effects on haematological parameters: The blood sample in EDTA tube was analyze for Red Blood Cells (RBC) by haemocytometic method (Dacie and Lewis, 1984); the haemoglobin (Hb) content was by Cyanmethaemoglobin (Drabkin) method (Dacie and Lewis, 1984); packed cell volume (PCV) was according to Ekaidem et al. (2006) while White Blood Cells (WBC) and its differentials (neutrophil, eosinophil, basophil, lymphocyte and monocyte) were as described by Dacie and Lewis (1984).
Electrolytes: The electrolytes were analyzed using automated microprocessor analytic instrument (ISE 6000 analyzer-SFRI sail ©-Berganton-France) that uses ISE (Ion Selective Electrode) technology in measuring various combinations of sodium, potassium, chloride, calcium and bicarbonate.
Histopathology: The pancreatic, renal and hepatic tissues harvested from each group were fixed in Bouin’s fluid for ten days before embedding in paraffin wax. The embedded tissues were sectioned at 5 μm, mounted on a slide and stained with Haematoxylin and Eosin (H and E) (Mbaka and Owolabi, 2011).
Statistical analysis: Student’s t-test was used and differences were considered significant at p<0.05 or p<0.01. All data are expressed as Mean±Standard error of the mean.
RESULTS
Purity test evaluation of formulations A and B: The results were summarized in Table 1.
The formulations A and B were observed to have high loads of the following undesirable microbial and fungal contaminants, Escherichia coli, Klebsiella species, Pseudomonas aeruginosa, Candida albican, a non lactose fermenter, Salmonella typhi and Staphyllococus species which were higher than officially accepted limits. Formulation A had more contaminant loads than formulation B.
Acute toxicity study: The acute toxicity result as summarized in Table 2, showed that all the animals that received 5000 mg kg-1 b.wt of formulation A extract survived beyond 24 h while 20, 80 and 100% death were recorded in the animals that received 10,000, 15000, 20000 mg kg-1 b.wt. of the extract, respectively.
Also all the animals that received 10,000 mg kg-1 b.wt. of formulation B extract survived beyond 24 h while 20% and 40% death were recorded in the animals that received 15000 and 20000 mg kg-1 b.wt. orally, respectively. LD50 values were calculated to be 15,000 and 15.625 mg kg-1, respectively for formulations A and B.
Weight variation result: The percentage increases in the weight of the animals treated with the two formulations compared to the control were shown in Fig. 1 and 2, respectively. Generally, there was insignificant (p≥0.05) increase in b.wt. of the animals treated with the respective formulations while a significant increase in weight was observed in the animals treated with the reference drug, glibenclamide, compared to the normal control.
Activity of the formulations on organs: The macroscopic examinations of the different organs of the animals treated with the various doses of the respective formulations and glibenclamide did not indicate any visible colour changes of the organs.
The results of the effects of the formulations on various organ weights were summarized in Table 3. There were no significant changes observed in the various organs weight examined compared to the normal control except increase in the weight of the liver (p≤0.01) of the animals treated with highest doses of formulation A and B.
Biochemical assay: Table 4 showed the summary of the results of the effects of the respective formulations on the biochemical parameters. There was dose dependent significant (p≤0.05) reduction in plasma glucose levels in the animals treated with the two formulations compared to the diabetic control. The total protein level showed marked increment in 250 and 500 mg kg-1 b.wt. dose treatments with formulation B and 250 mg kg-1 b.wt. treatment with formulation A compared to the normal. AST level increased markedly in the highest dose (500 mg kg-1 b.wt.) treatment with the two formulations compared to normal.
Significant increase in ALT level was observed in the animals treated with the formulation B while marginal increase occurred in the two dose treatment with formulation A compared to the normal. Significant rise (p≤0.05) in creatinine level was observed only in animals treated with highest doses of the respective formulations compared to the normal. The urea level decreased markedly in 250 and 500 mg kg-1 b.wt. treatment with formulation A and 500 mg kg-1 b.wt. of formulation B while treatment at 250 mg kg-1 b.wt. of the formulation recorded no significant change. The albumin level increased significantly (p≤0.05) in all the treated groups except for the group that received 250 mg kg-1 b.wt. of formulation B while total bilirubin recorded marked increase in the two dose treatment with formulation B compared to normal.
Significant decrease (p≤0.05) in Total Cholesterol (TC) and triglyceride (TG) levels occurred in all the dose treatments with both formulations compared to the diabetic control. On the other hand, LDL-cholesterol was observed to have risen significantly only in 250 mg kg-1 b.wt. treatment with formulation B while showing marked decrease in the two dose treatments of formulation A and 500 mg kg-1 dose treatment of formulation B. On the contrary, significant increase (p≤0.05) in HDL cholesterol level occurred in all the animals treated with the various doses of the respective formulations compared to the diabetic control.
Haematological study: The effects of the respective formulations on the haemoglobin, Red Blood Cells (RBC) components and the RBC differentials were presented in Table 5. Significant decrease (p≤0.01) was observed in the haemoglobin content and PCV volume. RBC count showed marked decrease in the animals that received highest doses of formulations A and B while at 250 mg kg-1 dose treatments with formulation A and B insignificant decrease occurred compared to the normal. White Blood Cells (WBC) count increased markedly in the animals that received the various doses of formulations A and B.
There was fluctuation in values of RBC differentials. The Mean Corpuscular Volume (MCV) and Mean Corpuscular Haemoglobin Concentration (MCHC) showed no appreciable changes while Mean Corpuscular Haemoglobin (MCH) level recorded marked increase in all the animals treated with the formulations compared to the normal. The lymphocyte level showed significant variation except for the group that received 500 mg kg-1 b.wt. of formulation A.
Electrolytes profiles: The effect of the formulations on the electrolytes was summarized in Table 6. There was insignificant change in HCO3- and Cl- levels in all the animals treated with various doses of the respective formulations compared to the normal. However, significant increase (p≤0.01) in Na+ level was observed in all the treated animals compared to normal while K+ level increased markedly only in the animals treated with 500 mg kg-1 b.wt. of formulation B.
Histopathology: The tissue histology of normal pancreatic tissue (Fig. 3a) showed the pancreatic acini and islet cells. The beta cells appeared darkly spotted and the most numerous of the islet cells. The diabetic control (Fig. 3b) showed severe beta cell necrosis and many coalesced vacuoles. The animals post treated with formulation A, (Fig. 3c) showed considerable increase in beta cells with few coalesced vacuoles compared to diabetic control. Pancreatic tissue of animals post treated with formulation B (Fig. 3d) showed more survivor beta cells compared to formulation A. In glibenclamide treated (Fig. 3e), there was mild lesion in the islet cells and many vacuolations.
The histology of the normal renal tissue (Fig. 4a) showed the cortical area in which were the renal corpuscles. The corpuscles contained dense round mass of glomerular apparatus separated by Bowman’s space from the surrounding structures. Renal tissue of diabetic control (Fig. 4b) showed mild edematous changes within the interstitial cells. The photomicrograph of tissue post treated with formulation A (Fig. 4c) indicated no pathological changes. The photomicrograph of tissue post treated with formulation B (Fig. 4d) showed normal appearance. The photomicrograph of renal tissue post treated with glibenclamide (Fig. 4e) indicated mild inflammatory changes.
The photomicrograph of normal hepatic tissue (Fig. 5a) showed the portal tracts at the periphery of indistinct hepatic lobule. The hepatocytes radially arranged continued from the lobular margins towards the centre vein with each column interspaced by sinusoids. The photomicrograph of hepatic tissue of diabetic control (Fig. 5b) showed no abnormality. The tissue morphology of post treatment with formulations A (Fig. 5c) and B (Fig. 5d) and glibenclamide (Fig. 5e) indicated no abnormalities.
DISCUSSION
The use of herbal formulations in treating metabolic disorder is widely practiced amongst the rural populace and it is gradually gaining wider acceptance because of the successes recorded. Of great concern is the safety expressed by the purity, the contents of pathogenic and nonpathogenic microorganisms in these formulations, especially the commercial liquid herbal formulations widely consumed by the increasing number of diabetic patients.
In this study, the formulations used were observed to have high loads of various microbial contaminants at levels higher than officially accepted limits. More worrisome was the high load of E. coli 45.5x103 cfu mL-1 and 10.4x103 cfu mL-1 found, respectively in the two formulations indicating the use of faecal or soil contaminated and untreated raw materials or water for the preparations. Also the presence of other microorganisms especially the pathogenic organisms revealed the unhygienic nature of the manufacturing process and non compliance to the good manufacturing practice. The presence of the bacterial and fungal microorganisms above officially accepted limits for the two liquid herbal formulations made the preparations unsafe and unfit for human consumption. However, according to a previous study (Ogbonnia et al., 2010), some of the herbal formulations might be microbiologically safe for consumption.
The median acute toxicity values (LD50) of the preparations were determined to be 15,000 mg kg-1 b.wt. for extract A and 15, 625 mg kg-1 b.wt. for extract B. The poly-herbal medicine can be classified as been non-toxic, since the LD50 by oral route for both formulations were found to be much higher than World Health Organization (WHO) toxicity index of 2 g kg-1 (Ghosh, 1984; Klaasen et al., 1995).
Although increase in appetite and water consumption was observed in the diabetic animals treated with the respective formulations, there was however no significant weight gain by the animals. The non significant weight increase observed clearly suggested that the formulations might not have the obesity forming tendencies which is one of the undesirable side effects normally associated with sulphonylureas and glitazones. Although there were very high levels of microbial contaminants present, the macroscopic examinations of the various organs of the diabetic animals treated with the formulations or glibenclamide (a suiphonyurea) did not reveal any changes.
The extracts of the formulations were observed to be more effective in lowering the plasma glucose levels of the diabetic animals than the reference drug, glibenclamide. The tissue histology showed the formulations to have more survivor beta cells which activity may have accounted for quantitative increase in insulin production that promotes glucose uptake and utilization by other tissues. The effective decrease of blood glucose by the formulations means that they could minimize the risk of other diseases complications often associated with diabetes. The effective lowering of blood sugar levels demonstrated by the respective formulations supported their local use as hypoglycaemic agents.
The formulations were also observed to have a significant decreasing effect on the plasma Total Cholesterol (TC), triglycerides (TG) and low density lipoprotein-cholesterol (LDL-cholesterol) levels and significant increase in high density lipoprotein cholesterol (HDL-cholesterol) level in all the treated animals which clearly demonstrated the presence of hypolipidaemia agents in the formulations. The ability of the formulations to manage dyslipidaemia demonstrated their potential beneficial effects on reducing cardiovascular risk factors which are the major causes of death in the patients suffering diabetes mellitus (Valli and Giardina, 2002).
The hepatic and cardiac tissues release aspartate transaminase (AST) and alanine transaminase (ALT) and the elevation of plasma concentrations of these enzymes is indicative of deleterious effects to hepatic and cardiac tissues (Crook, 2006). There was significant increase in AST level of the animals treated with highest doses of the respective formulations while significant increase in ALT level was observed only in the animals treated with formulations B. This implies that the two formulations might be injurious to the heart and toxic to liver particularly at a very high dose. However, hepatic tissue morphology indicated no abnormality which suggested that the toxic level of the two formulations were below the threshold that could have precipitated pathological changes on the organ.
Increase in plasma creatinine levels coupled with decrease in the protein levels is often a sign of impaired renal function (Tietz, 1982). The extracts of the respective formulations at various doses significantly increased the creatinine levels suggesting that the formulations might contain compound that are deleterious to the renal system. The appreciable recovery in RBC count compared to the diabetic control suggested that the formulations may have potentiated erythropoietin release in the kidney known to enhance RBC production (erythropoiesis) (Polenakovic and Sikole, 1996; Sanchez-Elsner et al., 2004). However, at 500 mg kg-1 b.wt. treatment, RBC showed less effective recovery. Decreases in PCV and Hb levels were recorded in the diabetic animals treated with the formulations which could be probably due to decrease in iron absorption. Usually WBC show increase in activity in response to toxic environment (Robins, 1974). The increase in WBC count observed could be due to toxic challenge from the formulations. There were no significant changes in MCHC and MCV signifying that the polyherbal medicine did not regenerative anemia. The importance of calculated blood indices in anaemia diagnosis has been reported (Agbor et al., 1999).
CONCLUSION
The high LD50 value was also an indication that each of the formulations has a high safety margin. However, the high load of microbial contaminant present in both formulations has negated there safety. The results showed that the respective formulations had both good hypoglycaemic activity and beneficial effects on the plasma lipid profile by ameliorating dislipidaemia. It was also obvious that the active principles of the different plant extracts exhibited synergistic activity. The considerably high number of survivor beta cells observed in the treated groups was indicative that both formulations ameliorated beta cells damage.