Nutritional and Industrial Research Laboratories, Department of Biochemistry, Faculty of Basic Medical Science, College of Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria

Corresponding Author: Sarah Onyenibe Nwozo, Nutritional and Industrial Research Laboratories, Department of Biochemistry, Faculty of Basic Medical Science, College of Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria. E-mail: sonwozo@yahoomail.com, onyenibe.nwozo@mail.ui.eu.ng

	ABSTRACT
	INTRODUCTION
	MATERIALS AND METHODS
	RESULTS
	DISCUSSION
	CONCLUSION
	ACKNOWLEDGMENT
	CONFLICT OF INTEREST
	REFERENCES

	ABSTRACT

The aim of this study is to evaluate the co-administration of aqueous leaf extracts of Momordica charantia and Boerhaavia diffusa on Streptozotocin induced Diabetes in Male Wistar Rats. A total of 36 male wistar rats between the weights of 100-120g were divided into six (6) groups (n=6), five (5) groups received I.P. Streptozotocin (60mg/kg) and the remaining group served as the normal control. Rats body weights, blood sugar levels, glucose 6 phosphate dehydrogenase, key liver enzymes, enzymatic antioxidant and liver tissue histology were examined using standard procedures and the results demonstrated that, administration of leaf extract of M.charantia, B.diffusa and particularly co-administration of M.charantia+B.diffusa significantly p<0.05 aided weight reduction, lowered blood sugar levels, enhanced the levels of liver enzymes and antioxidant parameters. Thus, M.charantia and B.diffusa are excellent therapeutic candidates for the management of diabetes mellitus and its related complication.

	INTRODUCTION

   Diabetes mellitus (DM) is a major global health concern with a projected rise in prevalence from 171 million in 2010 to 366 million 2030 (1). In the same light, of course, it is a serious, chronic disease that occurs either when the pancreas does not produce enough insulin (a hormone that regulates blood sugar, or glucose), or when the body cannot effectively use the insulin it produces. (DM) is one of four (2) priority non-communicable diseases (NCDs) targeted for action by world leaders. Both the number of cases and the prevalence of diabetes have been on a steady increase over the past few decades (3). Importantly, complications associated with DM account for increased morbidity, disability, and mortality and represent a threat for the economies of all countries, especially the developing ones (4).

   Among the many challenges faced by developing countries in the face of rapid urbanization is the need for medications to debilitate the silent killer DM. To address this need, indigenous knowledge is often referred to, including the use of extracts from medicinal plants (5). Although, insulin and oral hypoglycemic agents like sulphonylureas and biguanides are still the key players in the management of DM. However, various harmful side effects, exorbitant cost and reported weak effectiveness of some conventional treatment regimen has resulted to a switch to a safer, more effective and viable alternative. It has also being recognizes that traditional medicine is an accessible, affordable and culturally acceptable form of healthcare trusted by numerous people, which stands out as a way of coping with the relentless rise of chronic non-communicable diseases in the midst of soaring health-care costs and nearly universal austerity’ (6, 7). Medicinal plants and isolated compounds are increasingly being used in most parts of the world as hypoglycemic agents (8, 9) and it has continued to play an important role in the management of DM, especially in developing countries, where many people do not have access to conventional anti-diabetic therapies (10, 11). Ethno-pharmacological surveys indicate that more than 1,200 plants are used in traditional medicine systems following claims of their hypoglycemic properties (10, 12, 13).

   Momordica charantia is one of most important plants that have been traditionally used for the treatment of diabetes (14). A number of studies have evaluated the hypoglycemic ability of the different extracts of M. charantia in both human and experimental animals (15, 16, 17) and possible medicinal properties, acting alone or in combination, have been isolated from bitter melon fruit, seeds, leaves, stems, pericaps, endosperm, callus tissues, and cotyledons (18). Among these, the most actively studied constituents shown to improve glycemic control include charatin, polypeptide-p, vicine and momordin (19). Boerhaavia diffusa is a small perennial creeping herb, commonly known as “Red hogweed” and can be found in Nigeria and many other countries as well and reasonable number of studies have also demonstrated it hypoglycemic ability (20, 21, 22).

   Currently, combination therapies are employed for the treatment of critical diseases, such as cancer, acquired immunodeficiency syndrome (AIDS) and pulmonary tuberculosis, in order to achieve enhanced therapeutic effects. The modern approach of combination therapy is a renewal of what was advocated in Chinese medicine that started thousands of years ago on the use of herb-herb combination for improvement of therapeutic outcome (23). It is believed that each active component of a plant will be strengthened by the presence of another plant that has such active ingredient (synergism) or can aid its effectiveness in the body (24). On this note, the objective of this study is to evaluate and compare the therapeutic potency of M. charantia, B. diffusa and co-admimstration of M. charantia+B. diffusa on STZ induced diabetes in male Wistar rats.

	MATERIALS AND METHODS

   Plant material

   Fresh leaves sample of M. charantia and B. diffusa were collected from surrounding areas of University of Ibadan, Ibadan North LGA and were identified and authenticated by Mr Donatus Eseimukhai, the Herbarium at Botany Department, University of Ibadan. The fresh leaves were air dried for two weeks in Nutritional Biochemistry postgraduate students’ laboratory and ground into coarse powder using Hammer mill in Pharmacognosy Research Laboratory. M. charantia and B. diffusa powder 1450 g and 630 g respectively was extracted in the cold by maceration in water for 72 h, it was filtered using Whatman filter paper No 1 and filtrate was then concentrated using Bucchi rotary evaporator model R-200 at 30°C and was further concentrated using a vacuum oven model VF-220 set at 30°C and a pressure of 700 mg/Hg, yielding a brownish residue weighing 119.3061 g (8.23%) and 127.7631 g (20.28%) respectively. They extracts were kept in air tight bottles and stored in a refrigerator.

   Animals

   A total of 36 male Wistar rats between the weights of 100-120 g were procured from the central animal house, department of physiology, University of Ibadan, Nigeria for this study and were allowed to acclimatize for two weeks before commencement of experimentation. They male Wistar rats were kept in well kempt and ventilated cages and their beddings changed every three days and they were fed rat normal pellet diet and allowed free access to clean drinking water. All the processes involved in the handling and experiment were carried out according to standard protocols approved by the animal ethics committee of the department.

   Induction of diabetes with streptozotocin

   Diabetes was induced by single dose intraperitoneal injection of 60 mg/kg streptozotocin (Sigma Chemical, St Louis, MO, USA) dissolved in a citrate buffer (0.1 M, pH 4.5) and after 48hours blood samples were collected from caudal vein for determination of fasting blood sugar level using ACCU-CHEK Glucometer. Rats with values between (301-344 mg/dl) were considered diabetic and (80-92mg/dl) were considered normal in this study.

   Experimental design

   Following two weeks of acclimatization, administration of the extracts was done by oral intubation using corn oil as vehicle and treatment duration was for a period of 30 days.

   Group 1: Normal control received corn oil

   Group 2: Negative control received 60 mg/kg STZ and remained untreated (Diabetic untreated)

   Group 3: Positive control received 60 mg/kg STZ and treated with 100mg/kg metformin (Diabetic untreated)

   Group 4: Received 60 mg/kg STZ and treated with 200 mg/kg aqueous leaf extract M. charantia (Diabetic+M.charantia)

   Group 5: Received 60 mg/kg STZ and treated with 200 mg/kg aqueous leaf extract of B. diffusa (Diabetic+B.diffusa)

   Group 6: Received 60 mg/kg STZ and co-administered with 200 mg/kg aqueous leaf extracts M.charantia+B.diffusa (Diabetic+MC+BD)

   Sample collection

   Blood was collected in plain tubes and allowed to stand for a period of 10-15 minutes for coagulation and then centrifuged at 3000 rpm using centrifuge (model R-8C) for 10 minutes. The supernatant (serum) was transferred into sample bottles for liver function enzyme test and glucose-6-phosphate dehydrogenase (G6PD) determinations. The liver were quickly excised, washed in ice cold 1.15% KC1 solution, blotted with filter paper and weighed. They were then chopped into bits and homogenized in four volumes of the homogenizing phosphate buffer (pH 7.4) using a Teflon homogenizer. The resulting homogenate was centrifuged with (Labnet cold centrifuge model 2366) at 10,000 rpm for 15 minutes in a cold centrifuge (4°C). The supernatant was then collected and used for antioxidant enzyme assays.

   Biochemical analysis

   Blood glucose level was measured using ACCU-CHEK glucometer, Superoxide dismutase (SOD) activity was determined by the method of (25), catalase (CAT) activity was determined by the method of (26), reduced glutathione (GSH) concentration estimated using the method described by (27) and Glutathione-S-transferase activity was determined according to (28). Serum level of alanine and aspartate aminotransferases (ALT and AST), alkaline phosphatase (ALP), and glucose-6-phosphate dehydrogenase (G6PD) were quantified by spectroscopy using Randox commercial assay kits.

   Histopathological Studies

   Small pieces of heart and kidney tissues were fixed in 10% formalin solution, followed by embedding in melted paraffin wax. Histopathological assessment and photomicrography of the prepared slides was done by using an Olympus light Microscope with attached Kodak digital camera.

   Statistical analysis

   Data were treated by ANOVA (analysis of variance) and mean separation was done using Turkey HSD and Duncan. Paired T-test was used to establish difference in timely events among same individual group animals and ρ<0.05 were considered significant. Data was expressed as means ± standard deviation. All statistical analysis was done using IBM SPSS Version 22 and Microsoft Excel.

	RESULTS

   Effect on body weight

   Animals with body weight ranging 90-100 g was obtained and fed with normal pellet diet for a period of two weeks and the body weight was measured and recorded as initial body weight ranged 123.7-130.8 g. Experimental groups eventually received 60mg/kg i.p STZ injection and after 30days of treatment the body weight was also measured and recorded as final body weight ranged 180.8-224. They result in Figure 1 showed a significant increase p<0.05 in the mean body weight of the rats across all groups (final body weight) when compared to the initial body weight. However the groups that were treated with 100 mg/kg metformin and co-administration of 200mg/kg aqueous leaf extracts of (M.charantia+B.diffussa) outstandingly had an increased weight gain relative to the remaining groups.

   Effect on blood sugar levels

   Figure 2 shows effect of co-administration of 200 mg/kg leaf extracts of M.charantia+B.diffusa and only 200 mg/kg leaf extract of M. charantia and B. diffusa on blood sugar levels. Blood sugar levels was determined after 60 mg/kg i.p. injection of STZ and represented as initial FBSL and at the end of treatment blood sugar levels was also measured and represented as final FBSL. Final FBGL had no significant difference p>0.05 relative to the initial FBGL in the normal control and diabetic untreated groups although initial fasting blood sugar levels was obviously higher in the diabetic untreated group relative to the normal control. However, treatment with 200 mg/kg aqueous leaf extracts of M. charantia, B. diffusa, co-administration and 100mg/kg metformin significantly lowered p<0.05 blood sugar levels (Final FBGL versus Initial FBGL) but co-administration of (M.charantia+B.diffusa) showed a better hypoglycemic potency similar to the reference drug (metformin).

   Effect on Glucose 6 Phosphate Dehydrogenase (G6PD)

   At the end of this study, serum G6PD was measured and as shown in Figure 3 G6PD levels was significantly p<0.05 lower in the negative control (diabetic untreated) when compared with the normal control and treatment with 200 mg/kg aqueous leaf extracts of M. charantia and B. diffusa significantly elevated p<0.05 the levels of G6DH relative to the diabetic untreated. Co-administration of 200 mg/kg leaf extracts of (M.charantia+B.diffusa) also had significant increase p<0.05 in G6PD level relative to the diabetic untreated however it showed a greater ability in elevation of G6PD levels when compared to the administration of only M.charantia and B.diffusa respectively.

   Effect on liver enzymes

   Table 1 show the effect of co-administration of (M.charantia+B.diffusa), M. charantia and B. diffusa in STZ induced diabetic rats. ALP levels was significantly elevated p<0.05 in the negative control (Diabetic untreated) relative to the normal control and all the treatment groups significantly lowered ALP levels when compared to the negative control. However, co-administration of 200 mg/kg aqueous leaf extract of (M.charantia+B.diffusa) exhibited a better potency as they was a significant decrease p<0.05 in ALP levels relative to only 200 mg/kg aqueous leaf extract of M. charantia and B. diffusa treated groups and similar effect was observed in ALT and AST levels.

   M.charantia and B.diffusa on antioxidant parameters

   Rat liver antioxidant markers were examined as shown in Table 2 (CAT, SOD, GSH and GST) and the levels was significantly p<0.05 lower in the negative control (Diabetic untreated) when compared with the other groups. Treated groups showed significant increase p<0.05 in antioxidant markers levels relative to the negative control (untreated-diabetic). However, there was no significant difference p>0.05 between the group co-administered with M.charantia+B.diffusa and the groups individually treated with only M. charantia and B. diffusa.

   Effect on Liver tissues histology (Fig. 4A-F)

   (A) Negative control (untreated diabetic): The hepatic tissue shows severe disseminated necrosis of all the three zones. There is moderate macrovesicular steatosis and there are periportal and portal inflammation by acute or chronic inflammatory cells. Small white arrow: Steatosis, Small black arrow: Inflammatory cell, Big blue arrow: Necrosis.

   (B) STZ + M. charantia: The hepatic tissue shows moderate Periportal inflammation and moderate congestion of the portal vein and central vein. Big white arrow: Congestion, Small black arrow: Inflammatory cell, Big black arrow: Eosinophilic fluid.

   (C) STZ + B. diffusa: The hepatic tissue shows slight inflammation of portal triad and central vein, mild haemorrhage and moderate congestion of the blood vessels. There are scanty binucleated hepatocytes. Big white arrow: Congestion, Small white arrow: Binucleated hepatocytes, Small black arrow: Inflammatory cell.

   (D) STZ + Metformin: There is moderate periportal inflammation and focal areas of infiltration by acute or chronic inflammatory cells. Big white arrow: Congestion, Small black arrow: Inflammatory cell.

   (E) STZ+M.charantia+B.diffusa:The hepatic tissue show moderate multiple focal areas of necrosis with slight inflammation by acute and chronic inflammatory cells. There is mild congestion of the central vein. Big white arrow: Congestion, Small black arrow: Inflammatory cell, Big blue arrow: Necrosis.

   (F) Normal control: The renal tissue shows adequate amount of renal corpuscles in the cortex. No inflammation or necrosis seen.

View larger version : [in a new window] Figure 1. Effects of co-administration and only M. charantia and B. diffusa on body weight in STZ-induced diabetic rats. *Significant (P<0.05) vs Initial body weight.

 

View larger version : [in a new window] Figure 2. Effects of co-administration and only M. charantia and B. diffusa on blood sugar levels in STZ-induced diabetic rats. *Significant (P<0.05) vs Initial FBGL.

 

View larger version : [in a new window] Figure 3. Effects of co-administration and only M. charantia and B. diffusa on Glucose 6 phosphate dehydrogenase in STZ-induced diabetic rats. Bars with same alphabet as superscript are non-significantly (p>0.05).

 

View larger version : [in a new window] Figure 4. A to F Showing liver tissue histology.

	DISCUSSION

   DM is a serious, progressive endocrine disorder affecting approximately 5% of the world’s population and it is characterized by hyperglycemia (29). DM causes reduction in body weight (30). In this light, our study revealed that the diabetic status was associated with a reduction in body weight, as there was an observed decrease in the body weight of untreated-diabetic rats (Fig. 1). This observation is consistent with experimental evidence as STZ induced diabetes is associated with a severe reduction in body weight, due to the degradation or loss of structural proteins that are obviously known to contribute to the body weight (31, 32). The extracts of M. charantia or B. diffusa mitigated weight loss but the group co-administered with M.charantia+B.diffusa extract had a greater impact in controlling the loss of body weight caused by DM similar to the reference drug (100 mg/kg metformin). Additionally, the result of this study (Fig. 2) revealed that, M. charantia, B. diffusa and co-administration of M.charantia+B.diffusa showed fasting blood glucose lowering ability. Observed hypoglycemic activity of the leaves of M. charantia and B. diffusa could be due to the presence of terpenoid, which have been shown to be involved in the stimulation of the ß-cells and the subsequent secretion of preformed insulin (33). Our finding corroborate the reports of (15, 16, 34) on M. charantia, (20-22) on B. diffusa but there is no existing data on the synergistic hypoglycemic ability of co-administration of both extracts together as practiced by herbalist thus highlighting our result to be the first.

   To further confirm the hypoglycemic ability of leaf extract of M. charantia and B. diffusa, glucose-6-phosphate dehydrogenase (G6PD) levels was evaluated (Fig. 3). G6PD catalyzes the first step in the hexose monophosphate (HMP) shunt an alternative pathway for the catabolism of glucose to yield pentose sugar (35) and (G6PD) is a good predictor for diabetes. In this light, the result of the present study (Fig. 3) revealed that, G6PD activity was significantly lower p<0.05 in negative control group (diabetic untreated) relative to the other groups respectively. However, treatment with only M. charantia and B. diffusa showed a similar ability, reference drug (metformin) and co-administration of M.charantia+B.diffusa had a similar ability in elevation of G6PD activity. The observed increase in G6PD activity in the treatment groups might indicate an improvement in glucose utilization by an alternative pathway for glucose utilization and associated NADPH for the amelioration of oxidative stress induced by STZ intoxication.

   Enzyme activities in the tissues are often used as ‘marker’ to ascertain early toxic effects of administered foreign compounds to experimental animals. ALP is a membrane bound enzyme while ALT and AST are cytosolic enzymes and high levels of ALP, ALT and AST respectively in the serum are indicators of cell membrane permeability and consequent degree of damage to the liver (10). The observed significant p<0.05 increase in the serum levels of liver enzymes (ALP, ALT, AST) Table 1 in the negative control relative to the treatment groups indicates that, STZ exerts some levels of liver dysfunction (36-38). However, administration of leaf extract of M. charantia, B. diffusa and co-administration of M.charantia+B.diffusa particularly restored the liver integrity, as there was a significant p<0.05 decrease in liver biomarkers (ALP, ALT, AST). Histopathology result gives credence to hepatoprotective role of the plant extracts. STZ-induced diabetes (Fig. 4A) caused severe disseminated necrosis of all the three zones, moderate macrovesicular steatosis and chronic inflammatory cells. In diabetic-treated groups (Fig. 4B-E) with either Metformin or M. charantia or B. diffusa or M.charantia+B.diffusa only moderate inflammation was seen thus showing the various therapy conferred protection against STZ-induced liver tissue damage.

   Antioxidants are proven essential tools in the investigation of oxidant stress-related diabetic pathologies and the activities of the antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase have been described as reduced in diabetics (39, 40). Also, hyperglycemia in DM leads to increased lipid peroxidation in the body, followed by the development of chronic complications due to oxidative stress (41). In the light of this, our result on Table 2 corroborates this finding. Untreated-diabetic group had a significant reduced p<0.05 antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase and reduced glutathione) levels relative to the diabetic-treated groups. However, treatment with extracts of M. charantia, B. diffusa and co-administration of both extracts, counteracted the oxidative stressed caused by STZ and this is not a surprising as the plant drug have shown antioxidant activity 42, 43).

	CONCLUSION

   The results of this study showed that leaf extract of M. charantia, B. diffusa and particularly simultaneous of extracts aided weight gain, lowered elevated blood sugar levels, decreased the levels of liver enzymes and enhanced tissue antioxidant parameters caused by DM. Thus, M. charantia and B. diffusa are excellent therapeutic candidates for the management of DM and its related complication.

	ACKNOWLEDGMENT

   The authors are very grateful to Nutritional and Industrial Unit, Department of Biochemistry, University of Ibadan for providing the material resources and an enabling environment for the successful completion of this study.

	CONFLICT OF INTEREST

   The authors declare that no conflicting interests exist.

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