Comparative Phytochemical Analysis and Antiradical Activity of Five Plants Used for the Treatment of Type 2 Diabetes in Benin

Background: Diabetes remains a real public health problem in the world today. Although considered a disease of rich countries, today diabetes is increasingly a major concern in developing countries and particularly in sub-Saharan Africa. In Benin, its prevalence in 2015 was 12.4%. Objective: This work aimed to compare the secondary metabolites, the content of phenolic compounds (total phenol, flavonoid) and the antiradical power of five plants ( Bambusa vulgaris Schrad. Ex Wendel; Parkia biglobosa (Jacq.) R. Br. Ex G. Don; Mangifera indica L.; Saccharum officinarum L.; and Annona muricata L. ) used in Benin by traditional healers to treat type 2 diabetes. Materials and Methods: Secondary metabolites were identified by coloration and precipitation reactions specific to each family of metabolites. Total phenols were determined by Folin Ciocalteu method. The aluminum trichloride method has been used to quantify total flavonoids. The antiradical capacity was evaluated by 2,2-diphenyl-l-picrylhydrazyl (DPPH). Results: Plants which leaves were higher in total phenols were Mangifera indica, Parkia biglobosa and Bambusa vulgaris while Annona muricata and Parkia biglobosa were the richest plants in flavonoids. The hydroethanolic extract of leaves of Bambusa vulgaris (IC 50 =0.28mg/mL), Parkia biglobosa (IC 50 =0.3mg/mL) and Mangifera indica (IC 50 =2.5mg/mL) showed more pronounced antiradical activity than vitamin C (IC 50 =3.2 mg/mL) which were synthetic antioxidant. Conclusion: Our results showed that among the five plants studied Mangifera indica, Parkia biglobosa and Bambusa vulgaris were the richest in total phenols and also those whose leaf extracts had the highest antiradical activities. These three plants could therefore be considered as potential remedies for type 2 diabetes and its complications.


INTRODUCTION
Diabetes is a global public health problem [1]. Over 422 million adults were living with diabetes in 2014 [2]. In 2012, 1.5 million deaths worldwide were directly attributable to diabetes, the eighth leading cause of death in humans [3]. More than 80% of diabetes deaths occur in low-income countries. [1] The number of diabetics in sub-Saharan Africa were estimated at 19.8 million in 2013 [4]. Diabetes is on the rise in Benin and its prevalence increased from 2.9% in 2008 to 12.4% in 2015 and generated a mortality rate of 2% [5]. Type 2 diabetes and its associated complications are accompanied by oxidative stress, production of free radicals and reactive oxygen species. The chronicity of hyperglycaemia characteristic of type 2 diabetes generates oxidative stress responsible for increased glycolysis which, by increasing mitochondrial membrane potential, increases the production of radicals and inhibits glyceraldehyde-3-phosphate dehydrogenase [6][7]. Oral antidiabetics lead to the normalization of blood sugar only in less than 50% of cases. They have no regressive effect on established lesions and they are contraindicated in renal and hepatocellular insufficiencies [8]. In addition, there are the problems of intolerance, side effects, hypersensitivity and resistance related to antidiabetic drugs [9]. Moreover, in developing African countries and particularly in Benin, the medical care of diabetes is limited by the inaccessibility of certain populations to health centers and the high cost of conventional medicine drugs. In these conditions, populations often resort to medicinal plants for treatment. The Beninese flora is rich and diversified in plants used to treat type 2 diabetes [10]. Bambusa vulgaris, Parkia biglobosa, Mangifera indica, Saccharum officinarum and Annona muricata are some of the plants that are used to treat type 2 diabetes in Benin. It therefore seems essential to direct scientific studies on these plants, which are in high demand in traditional medicine to treat diabetes. It was in this sense that this work compared the quantity of phenolic compounds and the antiradical activity of these five plants.

Material
The

Pretreatment of plants
After harvesting, five batches of the leaves of the five plants were dried at laboratory temperature until their plant mass stabilized and then reduced to powder.

Plant extraction
The extraction was made with hydroethanolic. 5g of powdered biomass were mixed with 100 mL solvent for 72 hours. Further, all the extracts were filtered through Whatman No.1 filter paper and concentrated. The residues were dried to constant weight and stored in the darkness at 4℃ to avoid the degradations until use.

Preliminary phytochemical screening
Secondary metabolites were carried out by coloration and precipitation reactions specific to each family (Table1: Methods for the identification of secondary metabolites of plants) [12][13].

Determination of phenolic compounds
Total phenol content: Total phenolic content was determined using the Folin-Ciocalteu colorimetric method. This method consisted of using a mixture of phosphotungstic and phosphomolybdic acids, which were reduced during the oxidation of phenols into a mixture of tungsten blue oxide and molybdenum. Finally, the absorbance was measured at 765 nm using a spectrophotometer and the total phenol content are expressed in micrograms of gallic acid equivalence per milligram of dry matter (mgGAE/gEx) [14-15].
Total flavonoids content: The method of aluminum trichloride (AlCl 3 ) was used to quantify the total flavonoids. This technique was based on the formation of the aluminum complex flavonoids. The absorbance was read at 415 nm using a spectrophotometer and the Total flavonoid content are expressed in micrograms quercetin equivalence per milligram of dry matter (μgQE/gEx) [16].

Evaluation of antiradical activity
The antiradical activity was evaluated by the DPPH method. The principle of this method was based on measuring the trapping free radicals in a solution of DPPH. This trapping was indicated by the disappearance of the purple color of DPPH. The mixture of DPPH solution and the sample was left in the darkness for an hour and the absorbance measured at 517 nm.

Phenolic compound content
The calibration curves for determining the contents of total phenols and total flavonoids were shown in Figure 1. The calibration curves for measuring the total phenols content was obtained with the equation y = 0.0252x+ 0.0189 with the coefficient of determination of R 2 = 0.98. The calibration curves for determining the content of total flavonoids was y = 0.1099x+0.0136 with the coefficient of determination R 2 =0.99.

Anti-radical activity of plant extracts
The curves in Figure 4 showed the change in trapping percentage as a function of the concentrations of the hydroethanolic plant extract. At the level of the five curves, there was a gradual increase in trapping percentage with the increase in the concentration of the hydroethanolic extract of leaves of Bambusa vulgaris, Parkia biglobosa, Mangifera indica, Saccharum officinarum and Annona muricata. The concentrations (IC 50 ) of the hydroethanolic extract trapping 50 of the DPPH radical were determined. The IC 50 were listed in Table 3.
From the analysis of this table, it appeared that the IC 50 of Bambusa vulgaris, Parkia biglobosa and Mangifera indica were respectively 0.28 mg/mL, 0.3 mg/mL and 2.50 mg/mL. The hydroethanolic extract of Bambusa vulgaris, Parkia biglobosa and Mangifera indica showed a more interesting antiradical activity than vitamin C (IC50=3.20 mg/mL) which was a synthetic compound. The IC 50 of the hydroethanolic extract of Annona muricata and Saccharum officinarum were 11.00 mg/mL and 39.00 mg/mL.

Discussion
The leaves of Bambusa vulgaris, Parkia biglobosa, Mangifera indica, Saccharum officinarum and Annona muricata were rich and diversified in secondary metabolites. However, it should be noted that it had been identified in the leaves of Bambusa vulgaris collected in Benin, coumarins and leuco anthocyanins which were absent in our sample [19]. On the other hand, our results were in agreement with those who studied the sample from Ivory Coast [20]. Concerning the leaves of Parkia biglobosa harvested in Nigeria, the absence of the sterols and terpenes were noted whereas that were present in the sample from Benin Our results showed that the leaves of Mangifera indica, Parkia Biglobosa and Bambusa vulgaris were the richest in total phenols while Annona muricata and Saccharum officinarum were the least rich. It was reported that phenolic compounds were useful in the prevention of type 2 diabetes [27][28][29][30][31][32].
The hydroethanolic extract of the leaves of the five plants (Bambusa vulgaris, Parkia biglobosa, Mangifera indica, Saccharum officinarum and Annona muricata) showed interesting antiradical activities. It should be noted that the hydroethanolic leaf extract of Bambusa vulgaris, Parkia biglobosa and Mangifera indica showed more pronounced antiradical activity than vitamin C, which is a synthetic antioxidant. Our results were consistent with previous work on the antiradical activity of these plants [29][30][31][32][33]. The plants which had the weakest IC 50 and therefore had the highest antioxidant activities are those which contained the significant quantities of total phenols. With the exception of Annona muricata which contains few total phenols but was rich in vitamin C a good antioxidant [34]. The role of polyphenols as an antioxidant was no longer to be demonstrated, [35,36;37,38] so it was consistent that these plants have high antioxidant activities. Oxidative stress had also been shown to induce insulin resistance which leads to type 2 diabetes [39-40]. Hence Bambusa vulgaris, Parkia biglobosa, Mangifera indica and Annona muricata leaves with their high antioxidant activities could prevent insulin resistance and therefore prevent type 2 diabetes and its complications which were also linked to oxidative stress.    and Bambusa vulgaris were the richest in total phenols and also those whose leaf extracts had the highest antiradical activities. These three plants could therefore be considered as potential remedies for type 2 diabetes and its complications.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

ACKNOWLEDGEMENT
All participants in this work are authors.