In vivo efficacy of Dialium guineense fruit pulp on hemeoxygenase-1 and angiotensin converting enzyme in experimental diabetes

The study aims to investigate the effect of aqueous fruit pulp of Dialium guineense on hemeoxygenase, and insulin release, inhibition of angiotensin converting enzyme (ACE) and possible hypoglycemia in streptozotocin-induced diabetic rats. Twenty four male Wistar rats were grouped into control, diabetic (Dm), diabetic + 300 mg/kg body weight Dialium guineese (Dm+ DG) and diabetic + 100 mg/kg metformin (Dm +MET). Apart from the control, other rats were made diabetic by a single dose of 50 mg/kg streptozotocin injected intraperitoneally. Dialium and metformin were administered orally three days after induction of diabetes. Result showed significant (p<0.01) increase in serum ACE, blood glucose, and a decrease (p<0.01) in HO-1 and insulin in the Dm group. There was also an increase (p<0.01) in TC, TG and LDL-c. Tissue peroxidation (Heart and Kidney) was high in the diabetic untreated rats, superoxide dismutase and catalase activity was attenuated. Administration of aqueous fruit pulp and metformin significantly (p<0.001) increased HO-1 and insulin secretion, decreased ACE and blood sugar level (p<0.001) as well as the TC, TG and LDL-c. Antioxidant activities in the kidney and liver were potentiated. In conclusion, this study showed that Dialium guineese fruit pulp enhances HO-1 and insulin release and inhibits ACE activity. It is hypoglycemic, hypolipidemic and evokes antioxidant activity.


INTRODUCTION
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, hypercholesterolemia, liver and kidney dysfunction as well as derangement of pancreatic β-cell.It is also associated with profound changes in serum lipid, diabetic *Corresponding author.E-mail: nkanuee@yahoo.com;nkanuetah@gmail.com.Tel: +2347030299991; 08086127404.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 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.

Experimental design
Twenty four male Wistar rats weighing between 170-230 g were used for this study.The animals were randomly selected into four groups of six rats each.Group 1 was the control and received tap water.Group 2, 3 and 4 were injected with 50 mg/Kg body weight of streptozotocin intraperitoneally to render them diabetic.Groups 3 and 4 were then administered 300 mg/kg of D. guineense fruit pulp and 100 mg/kg body weight of metformin respectively.Administration of drugs lasted for three weeks.Blood samples were collected by cardiac puncture for biochemical analysis and the tissues removed and used for histological studies and tissue peroxidation.

Preparation of Dialium fruit pulp extract
The fruit pulp of D. guineense was purchased from Okuku Market, Yala Local Government Area of Cross River State.D. guineense fruits were collected and the dark coloured hard coats broken to expose the soft pink pulp of the fruit .The pulp was peeled from the water proof-like coat and then dried at room temperature by hot air oven (Amstel Hearson Oven, England) to evaporate its water content to a thick orange paste.Dried pulp was blended to powder and used when necessary.Animals received 300 mg/kg body weight of the suspension.

Induction of diabetes mellitus
The rats except the control group were rendered diabetic on a 12 h fast by a single intraperitoneal injection of 50 mg/kg body weight streptozotocin (SantaCruz Biotechnology, USA) dissolved in 0.01M citrate buffer, at a pH 4.5.All experiments on animals were carried out in absolute compliance with ethical guideline for research, care, and use of laboratory animals.After 3 days of streptozotocin injection, blood glucose concentrations were determined via AccuChek glucometer to confirm diabetes.Blood glucose levels

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 autooxidation 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

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

DISCUSSION
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  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 βcells 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 betacell 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 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 AT 2 level since ACE is needed to convert inactive AT 1 to the potent and active AT 2 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, 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.

Conclusion
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.

Figure 5 .
Figure 5. Showing effect of daily oral administration of aqueous D. guineense fruit pulp on blood glucose concentration in streptozotocin-induced diabetic rats.Values are expressed in Mean ± SEM n=5; **=p<0.01vs Control.

Figure 7 .
Figure 7. Showing effect of daily oral administration of aqueous D. guineense fruit pulp on insulin concentration in streptozotocin-induced diabetic rats.Values are expressed in Mean ± SEM n=5; **=p<0.01vs Control; b=p< 0.01 vs Dm, d=p<0.01vs Dm+ Met e=p0.01vs Met.

Figure 8 .
Figure 8. Showing effect of daily oral administration of aqueous D. guineense fruit pulp on insulin concentration in streptozotocin-induced diabetic rats.Values are expressed in Mean ± SEM n=5; **=p<0.01vs Control;b=p< 0.01 vs Dm, e=p<0.01vs Met.

Figure 9 .
Figure 9. Showing effect of daily oral administration of aqueous D. guineense fruit pulp on lipid peroxidation, superoxide dismutase and catalase activity inthe Kidney in streptozotocin-induced diabetic rats.Values are expressed in Mean ± SEM n=5; **=p<0.01vs Control;c=p< 0.01 vs Dm+DG, d=p<0.01vsDM+ Met.
Photomicrographs of cardiac tissue showing no inflammation of myocardium and infiltration of adipocytes in all groups.H &E X400 A C D B