One major disease that affects man in an alarming rate amongst others is diabetic mellitus. It comes with marked metabolic disorder resulting from different environmental and varied hereditary factors. Commonly associated complications  of   diabetes  mellitus  include  high  toll  of morbidity and mortality abnormal insulin secretion or insulin receptor inactivity, hyperglycemia, hyper-cholesterolemia, liver and kidney dysfunction as well as derangement of pancreatic β-cell.  It is also associated with    profound     changes    in    serum     lipid,   diabetic ketoacidosis and culminates in chronic renal failure, neuropathy and coronary heart disease (Arora, 2010). In recognition of all these life threatening factors, concerted efforts to ameliorate the upsurge of the disease have been on course and drugs that can manage diabetes more effectively sorted with vigour.

Research has shown that angiotensin converting enzyme involved in the control of blood pressure plays an essential role in the conversion of inactive angiotensin I to an active angiotensin II which causes vasoconstriction. Studies have also shown that randomized trials using angiotensin converting enzyme inhibitors (ACEI) example, natural ACE inhibitors like polyphenols, flavonoids, xanthines, terpenoids derived from herbs (Kang et el., 2003; Loizzo, et al., 2007) and AT2 receptor blockers significantly decreased the risk of DM (Andraws and Brown, 2007; Abuissa et al.2005), improved insulin sensitivity and glucose metabolism, and reduced plasma glucose in both experimental conditions and in humans with DM (Scheen, 2004).

Similarly, HO-I has been reported to be a key antioxidant enzyme that provents the development of diabetes by abating oxidative stress via suppression of macrophages and acting as an important component in anti-atherogenic activity (Orozco et al., 2007); it also plays a vital role in evoking insulin release (Ndisang et al., 2010; Mosen et al., 2005).

Substantive evidence indicates that factors such as hyperglycemia, free-fatty acids and adipokines that increase oxidative stress contribute to insulin resistance (Evans et al., 2003) even though the exact mechanism by which this occurs is not fully understood.  However, some available information has implicated oxidative stress in the development of different forms of insulin resistance (Evans et al., 2003; Vinayagamoorthi et al., 2008).

It is therefore generally believed that elevated oxidative stress may lead to the cascade of events that impairs insulin-signalling (Vinayagamoorthi et al., 2008), and as such strategies that cause reduction in oxidative stress as well as glucose/insulin intolerance may improve glucose metabolism. Today, attention is redirected to the use of medicinal plants to treat most chronic diseases such as diabetes and hypertension (Liu et al., 2003; Ullah et al., 2015) because of its recognized nutritional and medicinal properties.

Dialium guineense, otherwise referred to as black velvet tamarind is an indigenous tropical forest fruit tree that belongs to the family leguminosae. The plant is found in many countries in West Africa and is identified by different names. In countries like Sierra Leone, Senegal, and Guinea Bissau, it is called ‘Veludo.” In Nigeria, Dialium is called by different names depending on the ethnic group. The Igbos call it Icheku, while the Yorubas call it Awin.  In Hausa, it is called Tsamiyarkurm (Orwa et al., 2009). The Yakurr ethnic group in Cross River State, Nigeria calls it Okana gben gbenwen. D. guineense     has     been    convincingly    used    in    the management of various disease conditions such as severe cough, bronchitis, stomach aches, malaria fever, jaundice, antiulcer, hypertension and hemorrhoids (Lawal et al., 2010). 

Phytochemical components identified in the sticky pulp of D. guineense include gums, hemicelluloses, mucilage, pectin and tannins. It also contains some level of ascorbic acid, minerals (copper, potassium, calcium, iron, selenium, zinc and magnesium), vitamins like vitamin-A, folic acid, riboflavin, niacin and vitamin C, tartaric acid (an anti-oxidant), carbohydrates in the form of soluble sugars, cellulose, iron and lipids (Nahar et al.,1990; Herzog et al.,1994; Gideon et al., 2012), tannins, alkaloids, saponins, flavonoid, steroids and cardiac glycosides and some phenolic compounds (David et al., 2011; Ezeja et al., 2011). This study was therefore aimed at finding out the possible effect of Dialium giuneense fruit pulp consumption on heme oxygenase, insulin and ACE  activity in streptozotocin-induced diabetic Wistar rats since the fruit is being consumed  locally as  a socially and an alternative to Vitamin C.

Blood collection and analysis

Blood samples were collected from the animals through cardiac puncture. The blood samples were centrifuged at 3000 (rpm) revolutions for 10 min to obtain serum for lipid profile, insulin, angiotensin converting enzyme and hemeoxygenase-1 analysis. Tissues were homogenized, centrifuged and supernatant used to measure malondealdehyde concentration and antioxidant activity.

Catalase

The method of Sinha (1972) was used to estimate catalase activity. The major principle involves reduction of dichromate in acetic acid to chromic acetate when heated in the presence of hydrogen peroxide (H₂O₂).The perchromic acid formed is  an unstable intermediate. The chromic acetate finally produced is measured using the colorimeter.

Superoxide dismutase

The activity of SOD was assayed using the method of Mishra et al. (1972). The ability of superoxide dismutase to inhibit the auto-oxidation of epinephrine at pH 10.2 has been used as the basis of a convenient and sensitive assay for this enzyme.

Malondialdehyde

Plasma MDA was estimated by method of Jean et al. (1983). After the reaction of thiobarbituric acid with malondialdehyde, the reaction product was extracted in butanol and was measured.

Determination of ACE

ACE activity was determined using the method of Hooper et al. (1987) with modifications. In brief, a pre-incubation mixture

contained 100 mM Tris-HCl buffer with 300 mM NaCl and 10 μM ZnCl₂, pH 8.3/positive control/test sample of various concentrations and 2 mU of ACE enzyme. The reaction mixture was mixed and pre-incubated at 37°C for 10 min. Following pre-incubation, substrate (N-HippurylL-histidine-L–leucine tetrahydrate) was added to a final concentration of 5 mM. The reaction mixture was mixed and incubated at 37°C for 30 min. The reaction was heated up in boiling in water bath for 4 min. A control reaction was also carried out without the test samples. The reaction mixture was centrifuged at 15,000 rpm for 10 min at 25°C. The supernatant was transferred to HPLC vials and subjected to HPLC analysis.

Histological studies

Heart and kidney were removed, dissected and washed immediately on ice cold saline. A portion of these tissues was fixed in 10% neutral formal-saline fixative solution for histological studies. After fixation, tissues were embedded in paraffin. Solid sections were cut at 5 cm and stained with hematoxylin and eosins as described by Strate et al. (2005). The slides were viewed at magnification of X 400 and photomicrographs were taken.

Statistical analysis

All data obtained in this study were expressed as mean ± standard error of mean. Collected data were analyzed using ANOVA (analysis of variance) followed by Bonferronis multiple comparison post hoc tests to compare the level of significance between control and experimental groups. A value of p<0.05 was considered significant. All analysis was performed using the graph pad version 5 statistical software program.

The results of this study are presented in Figures 1-9 and Plates 1 and 2.

World over, research on ways to ameliorate developing cases of diabetes mellitus has been intensified. This diseases which is life threatening is said to be associated with increased oxidative stress, hypercholesterolemia and inflammatory activity with a resultant increase in high incidence of  liver  and  kidney  damage. The  aim  of  this study therefore, was to investigate the effect of aqueous fruit pulp of Dialium guineense on hemoxygenase-1, insulin      and angiotensin     converting    enzyme     in streptozotocin-induced diabetic rats, all of which are pointers to the etiology of cardiovascular and coronary heart disease. Streptozotocin used in this study has been reported to be specific in cytotoxicity, lipotoxicity, generation of hydroxyl free radicals, hyperglycemia and inflammation   that    may    cause    lipid   peroxidation  in pancreatic β-cells resulting in decreased endogenous insulin release (Donath et al., 2009).

In our study, results obtained showed an appreciable decrease in the level of insulin and hemoxygenase-I (HO-1) but with a concomitant increase in blood glucose, TC,

LDL-c and angiotensin converting enzyme (ACE) levels in the diabetic untreated group. Similar results have been reported by Erman et al. (1998) and Ustundag et al. (1998). Usually, with hyperglycemia, the pancreatic β-cells become easily destroyed by redox imbalance between free radical production and scavenging processes causing lipid peroxidation, β-cells  dysfunction (Lenzen 2008b; Donath, 1999; Djordjevic et al., 2004) and decreased insulin secretion (Robertson et al., 2003).

Treatment of the diabetic group with aqueous fruit pulp of D. guineese and a standard antidiabetic drug, metformin, respectively, resulted in a significantly (p<0.01) decreased blood glucose level and ACE but interestingly, increased serum hemoxygenase-1 (HO-1) and insulin concentration. HO-I and its derivative like carbon  monoxide,  ferritin   and    biliverdin    have   been reported to be  key antioxidant enzymes that prevent the development of diabetes by abating oxidative stress via suppression of macrophages and acting as an important component in antiatherogenic activity (Orozco et al., 2007); they play a vital role in evoking pancreatic beta-cell insulin release and improve glucose metabolism thus reducing hyperglycemia (Ndisang et al., 2010; Mosen et al., 2005; Ndisang et al., 2014).

The mechanism by which D. guineneese exerts hypoglycemia and induces HO-I release may not have been unconnected with the reported presence of such components as flavonoid, vitamin C and tanins (Lever et al., 1979; Arogba et al., 2006). There are numerous natural HMOX1 inducers originating from plants, including polyphenols that exert positive effect on diabetic subjects (Bonifaz et al., 2009) Flavonoids is reported to contain quercetin and rutin both of which prevent oxidative stress by scavenging free radicals (Larocca et al., 1995., Cox et al., 2000). Previous studies have shown that quercetin and rutin particularly lower blood glucose level  in  rats  (Vessal  et  al., 2003),  preserve  pancreatic beta cell integrity (Coskun et al., 2005), increase insulin secretion and prevent liver injury (Kobori et al., 2009).

Tanins and phenolic compounds on the other hand, available in several plant derivatives including D. guineense are reported to be anti-ACE and act as specific inhibitors of the enzyme (Liu et al., 2006). ACE activity in both experimental animals and humans is usually characteristically high and is required for the conversion of inactive angiotensin-I(AT-I) to the potent and pro-oxidative  angiotensin II(AT2). ACE is associated with increased superoxide production and impaired endothelium function that may lead to cardiovascular problems. Our study has shown that hyperglycemia predisposes to hypercholesterolemia and elevated triglyceride and LDL-cholesterol as earlier demonstrated by Arora et al. (2010). This factors are associated with enhanced AT receptor expression (Andraws and Brown,

2007; Abuissa et al., 2005) and are often remote cause of cardiovascular disease and atherosclerosis. The study of these risk factors is nevertheless imperative because they are intervening with the management of diabetes mellitus.

The registered decrease in ACE level due to oral administration of Dialium translates to a decrease in serum AT₂ level since ACE is needed to convert inactive AT₁ to the potent and active AT₂ whose effect is to cause systemic vasoconstriction and raise blood pressure. This decrease in ACE concentration is suggestive of a possible ACE inhibition by the fruit pulp. Studies have shown that randomized trials using angiotensin converting enzyme inhibitors (ACEI) and AT2 receptor blockers significantly decrease the risk of DM (Andraws and Brown, 2007; Abuissa et al., 2005), improve insulin sensitivity and glucose metabolism, and reduce plasma glucose in both experimental conditions and in humans with DM (Scheen, 2004).

Our results therefore strongly indicate that Dialium guineense fruit pulp contains agents that promote ACE inhibition. Indeed, some reports have shown that flavonoid, one of the components present in the fruit pulp of Dialium guineense presents an anti-atherogenic effect due to its inhibition of ACE in vitro (Loizzo et al., 2007). The reparative effect of the fruit pulp and maintenance of tissue integrity was further observed in the improved heart and kidney morphology. The antioxidant effect of Dialium guineense was further demonstrated by its ability to significantly (p<0.01) reduce lipid peroxidation in the liver and kidney by promoting superoxide dismutase and catalase enzyme activity. Many studies have shown that oxidative stress becomes apparent in diabetic subjects (Ceriello ,1997;, 2000;  ,2000; Waggiallah and Alzohairy, 2011).

Consistent with this view, our data provide further evidence that there is presence of oxidative stress with an alteration in antioxidant enzyme activities and increased lipid peroxidation (MDA levels) in diabetic condition. The reduction in serum SOD activity is thought to   be   as   a   result    of   excessive    autoxidation   and progressive glycation of enzymatic proteins. The reversal effect of Dialium fruit pulp on this unpleasant activity makes it convincing that it holds to an extent promising therapeutic properties.

The results of this study clearly demonstrated that Dialium guineense fruit pulp has a good antioxidant potential and decreases tissue lipid peroxidation, blood glucose level, induces hemeoxygenase and insulin release and acts as ACE inhibitor.

The authors have not declared any conflict of interests.

The authors of this research study wish to appreciate the technical assistance of Dr. Mrs Iya Eze of the Department of Medical Laboratory Science, University of Calabar and Dr. Sikirullai Olatunde Jeje Department of Physiology, Federal University of Technology, P.M.B 704, Akure, Nigeria.