Glycosylated haemoglobin values of alloxan-induced diabetic rats treated with graded doses of Cussonia arborea extract

ABSTRACT This study investigated glycosylated haemoglobin values and fasting blood glucose (FBG) levels of diabetic rats treated with methanol extract of Cussonia arborea. A total of 72 male wistar rats were assigned into 6 groups of 12 rats per group. Groups 1–5 rats were made diabetic by intraperitoneal injection of alloxan monohydrate at the dose of 160 mg/kg and treated with 62.5, 125, 250 mg/kg of the extracts, 2 mg/kg glibenclamide and 10 ml/kg distilled water (DW), respectively, while the non-diabetic group 6 rats received 10 ml/kg DW and served as normal control rats. The treatment was daily through the oral route for 84 days. The glycosylated haemoglobin values were measured on days 42, 56 and 84 post treatment while the FBG levels were determined on 2 weeks interval post treatment till the end of the experiment. The mean FBG level of rats treated with 125 mg/kg of the extract showed the most significant reduction (p < .05) in FBG from 315.33 ± 10.08 mg/dl to 93.00 ± 8.50 mg/dl and equally ameliorated haemoglobin glycosylation when compared to the negative control group. It was concluded that the methanol extract of C.arborea, at the dose of 125 mg/kg mitigated glycosylation of haemoglobin.


Introduction
Diabetes mellitus is a complex disease characterized by inability to regulate blood glucose as a result of relative or absolute deficiency in insulin (produced from pancreas) resulting to hyperglycaemia often accompanied by glucosuria, polydipsia and polyuria (Celik et al. 2002). Chronic hyperglycaemia and enhanced oxidative stress play major roles in diabetes pathogenesis. The disease is progressive and is associated with a high risk of complications (Dewanjee et al. 2008). It is one of the most common endocrine diseases and has a prevalence rate varying from 1 to 50% (King and Rewers 1993).
Glycosylated or glycated haemoglobin is the result of simple chemical reaction between haemoglobin and sugars after synthesis of haemoglobin is complete (Bunn et al. 1976). The reaction proceeds in two stages. Firstly, glucose combines with the alpha amino group of the valine residue N-terminus of beta (b) globin chains to form aldimine compound (Schiff base). This reaction is reversible and dissociation to native haemoglobin and glucose occurs readily. In the second phase, internal rearrangement of the aldimine intermediate by the Amadori reaction yields a stable ketoamine derivative. Assay of glycosylated haemoglobin helps to differentiate between diabetes mellitus and stress-induced hyperglycaemia (Goldstein et al. 1984).
Early workers confirmed that glycosylation begins during erythropoeiesis and continues slowly throughout the life of haemoglobin in the circulation. Concentrations reached in the red cell of diabetic subjects are consistent with their known life span of about 120 days (Bunn et al. 1976). An international expert committee (IEC), after an extensive review of both established and emerging epidemiological evidence, recommended the use of the glycosylated haemoglobin (HbA1c) test to diagnose diabetes with a threshold of ≥6.5% and American Diabetes Association (ADA) affirms this. The diagnostic HbA1c cut point of 6.5% is associated with an inflection point for retinopathy prevalence (IEC 2009).
Plants have been used since time immemorial in the management of diabetes mellitus; however their effects are usually exaggerated, unscientific or unstandardized. Cussonia arborea is a tropical plant belonging to the family of Araliacea with about 20 species (Tennant 2010). Folklorically, the plant is used in the treatment of malaria (De villers et al. 2010) fungi diseases, chronic diarrhoea (Kisangau et al. 2007) and diabetes (Amadou et al. 2008).
This study was therefore tailored to investigate the effects of treating alloxan-induced diabetic rats with graded doses of methanol root bark extract of C. arborea on glycosylated haemoglobin values.

Experimental animal
Male albino rats weighing between 100 and 105 g were obtained from the Department of Veterinary Physiology and Pharmacology, University of Nigeria, Nsukka laboratory animal house. The rats were randomly assigned into 6 groups of 12 rats per group. Diabetes was induced in groups 1-5 rats while the rats in group 6 served as normal control rats. The rats were acclimatized for two weeks. The environmental temperature where the animals were housed varied between 28 and 32°C. The animals were kept in stainless wire mesh cages and provided with good clean water ad libitum. They were fed with standard commercial feed (Guinea R growers). The experimental protocol used in this study was approved by the ethics committee of the University of Nigeria, Nsukka and conforms with the guide to the care and use of animals in research and teaching of the University of Nigeria, Enugu state, Nigeria (ECUN: 2015/58/702).

Plant material
The root bark of the plant material (C. arborea) used in this study was collected from Orukpa Local Government Area of Benue state and identified by a plant taxonomist, at the International Centre for Ethnomedicine and Drug Development, Echara, Aku road, Nsukka, Enugu state, Nigeria.

Preparation of the plant extract
Cold maceration method of extraction was employed. The root bark of C. arborea was air dried at a very low intensity of sunlight to avoid denaturation of the active ingredient. It was pulverized and stored in an air tight container pending its usage. About 1 kg of the powdered root bark was soaked in 5 l of 80% methanol (Sigma Aldrich, UK) with intermittent shaking every 2 h for 48 h. The mixture was filtered using Whatmann No 1 filter paper. The filtrate was concentrated using rotary evaporator and the extract stored as C. arborea extract (CAE) at 4°C.

Induction of experimental diabetes mellitus
Diabetes was induced in rats using the method described by Venugopal et al. (1998). The rats were injected with alloxan monohydrate (Sigma Aldrich, UK) dissolved in distilled water at a dose of 160 mg/kg body weight intraperitoneally, after overnight fasting (18 h). Before the injection with alloxan monohydrate, the FBG levels of the rats were taken using Accu-Check glucometer (Oh et al., 2013;Togashi et al., 2016 andWeitgasser et al., 1999). This was done by tail snip of the rats and allowing blood to drop on the glucometer strip. The value was read off on the screen of the glucometer. After induction, the rats were kept in clean stainless cages and fed with commercial feed and were also given clean water for about 2 days before they came down with diabetes. On the 2nd day, diabetes was confirmed. The rats were fasted overnight before the assessment of their blood glucose status on the 2nd day. The FBG values above 7 mMol/L (126 mg/dl) were considered diabetic (WHO 1980).

Treatments
The rats were treated as follows: Group 1. Diabetic rats treated with 62.5 mg/kg C. arborea extract (CAE). Group 2. Diabetic rats treated with 125 mg/kg CAE.
The rats were treated orally daily for 84 days and the FBG levels determined on days 14, 28, 42, 56, 70 and 84 while the glycosylated haemoglobin values were measured on days 42, 56 and 84 post treatments.

Assay principle
A haemolysed preparation of whole blood was mixed continuously for 5 min with a weakly binding cation-exchange resin. The labile fraction was eliminated during the hemolysate preparation and during the binding. During this mixing, non-glycosylated haemoglobin binds to the ion-exchange resin leaving glycosylated haemoglobin free in the supernatant. After the mixing period, a filter separator was used to remove the resin from the supernatant. The percent glycosylated haemoglobin was determined by measuring absorbances of the ratio of the absorbances of the glycosylated haemoglobin (GHb) and the total haemoglobin fraction (THb). The ratios of the absorbances of GHb and THb of the control and test were used to calculate the percent GHb of the sample.

Haemolysate preparation
Into a test tube was dispensed 0.5 ml of lysing reagent and labelled as test and control. Thereafter, 0.1 ml of the reconstituted control or well-mixed sample was added into the appropriately labelled tubes and mixed until complete lysis was evident.

Glycosylated haemoglobin separation
The cap from the ion exchange resin was removed and labelled as control and test and 0.1 ml of hemolysate from hemolysate preparation above was added into appropriately labelled ion exchange resin tubes. Thereafter, a resin separator was inserted into each tube so that the rubber sleeve was approximately 1 cm above the liquid level of the resin suspension and the tube was vortexed continuously for 5 min. The resin was allowed to settle and the resin separator was pushed until the resin was firmly packed and the supernatant decanted directly into a curvette and absorbance measured at 415 nm against distilled water.

Total haemoglobin fraction
Into test tubes labelled as 'test' and 'control' were dispensed 5 ml of distilled water and 0.02 ml of hemolysate from hemolysate preparation above and added to appropriately labelled tubes. The mixture was mixed well and the absorbance read against distilled water.

Calculations
Ratio of control (R c ) = Absorbance control GHb Absorbance control THb 10 is the value of the control.

Statistical analysis
Statistical package for social sciences (SPSS) version 20 was used for the analysis. One way Analysis of Variance (ANOVA) was used to compare the Means of the FBG levels and their difference separated using Duncans Multiple Range test while Data on glycosylated haemoglobin and FBG were correlated using Pearson parametric correlation. P-values <.05 were considered significant.

Results and discussion
The FBG of all the rats in groups 1-5 were significantly (p < .05) higher than that of the group 6 rats (normal control) post induction. On day 14 post treatment, the FBG levels of groups 2 and 4 rats were significantly (p < .05) lower than those of the group 5 rats but statistically comparable (p > .05) to that of the group 6 rats (normal control). These two groups (Groups 2 and 4) consistently compared very well (p > .05) with the normal control rats till the 84 d duration of the study (Table 1). The single intraperitoneal injection of alloxan monohydrate at the dose of 160 mg/kg body weight resulted in a significant (p < .05) elevation of the fasting blood glucose (FBG) levels when compared to the nondiabetic rat (Table 1). Alloxan monohydrate and its reduced product, dialuric acid establish a redox cycle with the formation of superoxide radicals (Szukudelski 2001). These radicals undergo dismutation to hydrogen peroxide with a simultaneous massive increase in cytosolic calcium ion concentration which causes rapid destruction of pancreatic beta cells of the islets of Langerhans resulting in a decrease in endogenous insulin secretion and attendant elevation of the blood glucose levels (Akuodor et al. 2014). Rats with elevated glucose levels of ≥126 mg/dl (7 mMol/L) were considered diabetic (WHO 1980). The results indicated that treatment of the diabetic rats with C. arborea extract (especially at the dose of 125 mg/kg body weight) significantly (p < .05) reduced the elevated glucose levels ( Table 1). The reductions achieved by the extract at this dose were comparable to that achieved by 2 mg/kg of glibenclamide, a known anti-diabetic drug. The result is in agreement with our earlier study (Aba et al. 2014) with methanol stem bark extract of C. arborea which demonstrated antihyperglycaemia. Other researchers (Khazraji et al. 1993;Lim et al. 2009;Liu et al. 2005) have also demonstrated hypoglycaemic potentials of root barks of different plants. Our earlier study indicated that the active principle responsible for the hypoglycaemic effect was a triterpenoid (Aba and Asuzu, 2016). Other researchers such as Min-jia et al., 2008;Santos et al., 2012 andCastellano et al., 2013 had earlier reported various degrees of hypoglycaemic activities of triterpenes isolated from different plant materials.
The glycosylated haemoglobin values of group 2 rats were significantly (p < .05) lower than that of the group 5 rats (negative control) but were statistically comparable (p > .05) to those of the normal control rats and to those treated with standard drug (Glibenclemide) 42 days post treatment. On days 56 and 84, all the treatment groups showed significantly (p < .05) lower values of glycosylated haemoglobin when compared with the negative control groups ( Table 2).
The glycosylated haemoglobin (HbA1c) values of the diabetic untreated rats were significantly (p < .05) higher than that of the normal control rats. This could be attributed to the effect of alloxan which induced hyperglycaemia by ultimately leading to decreases in insulin secretion consequent upon pancreatic beta cell destruction (Szkudelski, 2001). Persistent or prolonged increase in blood glucose leads to nonenzymatic adduction of glucose to the free amino groups at the N-terminal of beta chain of haemoglobin thereby forming glycosylated haemoglobin (Kilpatrick et al., 2009). However, all treatment groups recorded significantly (p < .05) lower HbA1c values at the end of the experiments (Table 2). Group 2 rats, treated with 125 mg/kg of the extract demonstrated the highest ameliorative effects with regards to glycosylation of haemoglobin. Glycosylated haemoglobin is a measurement that reflects both fasting and postprandial glucose concentrations over a 3-month period (Diana et al. 2010). Glycosylation of haemoglobin (non-enzymatic adduction of glucose moiety to haemoglobin) starts six weeks following persistent hyperglycaemia (Nagisa et al., 2003;IEC 2009). It then lasts till the life span of red blood cell (which is about 3 months). Reductions in the values of HbA1c by the treated groups indicate the hypoglycaemic effect of the extracts. The activities of the extract were dose-dependent with maximum activity at the dose of 125 mg/kg. The highest ameliorative effects shown by the rat group treated with 125 mg/kg of the extract compared to the other doses could be explained by drug-receptor occupancy theory (Rang, 2006). It is most likely that at the dose of 125 mg/kg body weight of the extract, the receptors for the extract have been fully occupied thus giving maximal effect. Increasing the dose beyond this did not lead to further effect instead it reduced efficacy probably due to deleterious side effects that may be associated with drug overdose. The mechanism by which C. arborea exhibited its effects on fasting blood glucose and on glycosylated haemoglobin values was not specifically investigated in this study, but different mechanisms such as increased insulin production, reduction in hepatic glucose output, increased antioxidant activities amongst others have been proposed as various mechanisms by which antidiabetic medicinal plants exhibit their effects (Aba and Asuzu, 2018). The result is in agreement with the findings of Anand et al.(2007) and Yu et al. (2006) who submitted the ameliorating effect of Brassica nigra and Astragalus membranaceus (hypoglycaemic plants) respectively on glycosylation of haemoglobin in diabetic rats. Oral hypoglycemic agents such as glibenclamide have also been reported (Diana et al. 2010) to lower HbA1c levels of diabetic rats.
The correlation between the glycosylated haemoglobin and fasting glucose levels indicated that FBG and HbA 1c significantly (p < .01) correlate with each other with R 2 value of 0.914 on day 84 post treatment. The implication of this strong positive correlation between fasting blood glucose and glycosylated haemoglobin is that, in the absence of glycosylated haemoglobin (which is a gold standard for diabetes monitoring), fasting blood glucose can be a reliable alternative. Measurement of HbA1c has advantage over fasting blood glucose since it does not depend on the erratic prandial flunctuations in the blood glucose (Musenge et al., 2016). However, since the average life span of RBC is about 3 months, HbA1c assay may encounter limitations when there are decreases in RBC counts following senescence and consequent lysis of RBCs (Roszyk et al., 2007). The finding of strong positive correlation between FBG and HbA1c suggests that both parameters can be used simultaneously to compensate for each other and increase accuracy.

Conclusion
It was concluded that treatment of diabetic rats with methanol extract of C. arborea (especially at the dose of 125 mg/kg) for 84 days lowered the FBG levels of the rats and ameliorated haemoglobin glycosylation. The effects on both FBG and glycosylated haemoglobin were positively and strongly correlated.

Aknowledgements
We wish to heartily appreciate the effort of Dr Ignatius Maduka, Department of Human Biochemistry, Nnamdi Azikiwe University, Nnewi campus, Anambra state, Nigeria for helping in procurement of glycosylated haemoglobin kit used in this study.

Disclosure statement
No potential conflict of interest was reported by the authors.