Evaluation of antihyperglycemic , antiradical and acute oral toxicity activities of aqueous leaves extract of Moringa oleifera Lam ( Moringaceae ) from Benin in normal rats

1 Laboratory of Enzymology and Proteins Biochemistry, Faculty of Sciences and Techniques, University of AbomeyCalavi, 05 BP 972 Cotonou, Republic of Benin. 2 Laboratory of Biotechnology and Animal Breeding, Faculty of Agricultural Sciences, University of Abomey-Calavi, 01 BP 526 Cotonou, Republic of Benin. 3 Laboratory of Cytogenetic, Faculty of Health Sciences, University of Abomey-Calavi, 01 BP 188 Cotonou, Republic of Benin. 4 Laboratory of Ethnopharmacology and Animal Health, Animal Production Department, Faculty of Agricultural Sciences, University of Abomey-Calavi, 01 BP 526, Cotonou, Republic of Benin. 5 Multidisciplinary Laboratory, University of Agriculture of Ketou, BP 95 Ketou, Republic of Benin.


INTRODUCTION
Diabetes mellitus is a major degenerative disease in the world today (Ogbonnia et al., 2008).The fundamental defect in diabetes mellitus is an absolute or relative lack of biologically active insulin, which results in the impairment of uptake and storage of glucose, and reduced glucose utilization for energy purpose (Tiwari and Rao, 2002).According to World Health Organization projection, the prevalence of diabetes is likely to increase by 35% (Sangeeta et al., 2010).Currently, there are over 150 million diabetics world-wide and this is likely to increase to 300 million or more by the year 2025.The prevalence of diabetes mellitus in Benin is 2.9% (Djrolo et al., 2011).
Although, anti-diabetic drugs are accessible, in many cases, the treatment is not suitable because these drugs are not able to heal this infection and present side effects.In the same way, the high cost of the anti-diabetic drugs directs the populations of low and middle-income countries to resort to the medicinal plants.Medicinal plants constitute an important source of potential therapeutic agents for diabetes and hyperlipidemia (Adisakwattana et al., 2011).Most of the plants prescribed for diabetes mellitus are not edible and therefore, the studies on edible plants which have an antihypoglycemic effect would be of great value in the dietary management of the disease.M. oleifera is used in traditional medicine all over the world to cure many diseases (india, Pakistans, ancient Rome, Greece and Egypt).
Different parts of this plant such as the leaves, roots, seed, bark and flowers were used in indigenous medicine for the treatment of some pathologies.Leaves were known to act as cardiac and circulatory stimulants, possess antitumor, antipyretic, antiepileptic, antiinflammatory, antiulcer, antispasmodic, diuretic, antihypertensive, cholesterol lowering, antioxidant, antidiabetic, hepatoprotective, antibacterial and antifungal activities, and are being used for the treatment of different ailments in the indigenous system of medicine, particularly in South Asia (Farooq et al., 2007).Fresh root is acrid and vesicant (has the taste of horse-radish).Internally, it is used as stimulant, diuretic and antilithic.Seeds are acrid and stimulant.Bark is emmenogogue and even abortifacient, antifungal and antibacterial.Flowers are cholagogue, stimulant, tonic and diuretic, and useful to increase the flow of bile.The plant is also a cardiac circulatory tonic and antiseptic.
Ethnobotanical studies along different phytodistricts in Benin showed the use of M. oleifera as one of the plants involved in the traditional medicine in the treatment of diabetes pathology (Laleye et al., 2015).As in most cases, a lot of plants are used in indigenous medicine to treat diseases, it is very difficult to say either if the results are from action of a single plant or a synergy of all plants.
To elucidate this aspect, there is a need to evaluate the action of this plant extract in order to provide scientific proof and its role in the treatment of pathologies.In the same way, the phytochemical screening of aqueous extract of M. oleifera was done to evaluate its action on diabetes.

Plant material
The leaves of M. oleifera were collected from Abomey-Calavi (Benin) city during the month of January to February, and were certified at the National Herbarium of Benin.A voucher specimen (AA6533/HNB) was reserved in this herbarium.The plant material was air dried at room temperature in the laboratory and further dried in the oven for 1 week at a temperature of 24°C.The leaves were then powdered using mortar and pestle.

Preparation of the water extract
The powder was hot macerated with distilled water and then the extract obtained was filtered, concentrated by rotary vacuum pump to get the solid mass because the main methods of M. oleifera remedy preparations in Benin is the maceration.

Animal treatments
In this study, albino Wistar rats (male and female) weighing 150 to 200 g and 4 months old were used.Animals were maintained under standard conditions and guarded in iron cages with easy access to water and food.The temperature of the room was maintained at 25°C and the relative humidity is between 35 and 60%.Animals were submitted to an alternated cycle of light (12 h) and dark (12h).

Phytochemical screening
The freshly prepared crude leaves extract of M. oleifera was subjected to qualitative phytochemical analysis for the presence of *Corresponding author.E-mail: verickaz@yahoo.fr.Tel: +229 966408110.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License various classes of active chemical constituents such as tannins, saponins, glycosides, flavonoids, alkaloids using the methods described by Houghton and Raman (1998).

Quantification of the bioactive molecules
Determination of total phenolic content Singleton et al. (1999) was used to determine total phenolic content.125 μl of aqueous extract at the concentration of 1 to 5 mg/ml was diluted into 250 µl of distilled water.Then, 625 μl of Folin -Ciocalteu reagent was added and gently mixed.After 2 min of homogenization, 500 μl of 20% sodium carbonate was added.The contents were mixed and allowed to stand for 2 h.The optical density of the blue colored samples was measured at 760 nm.Gallic acid was used as standard.The total phenolic content was expressed in mg/g of gallic acid equivalent of mixture.

Determination of total flavonoid content
Total flavonoid content of the extract was measured based on method described by Kim et al. (2003) with slight modifications.According to this method, 400 μl of extract was mixed with 500 μl of distilled water and then 120 μl of 5% sodium nitrite was added to the mixture and allowed to stand for 5 min, followed by the addition of 120 μl of 10% of AlCl3 to the mixture which was mixed using a vortex.800 μl of 1 M NaOH was added after 6 min and the mixture was incubated for 15 min in darkness.Rutin solution was used as reference and the content in flavanoids was expressed as mg of rutin equivalent/1 mg of sample.The calibration curve established with the reference solution was used to read directly the concentration values.Broadhurst and Jones (1978) method modified by Heimler et al. (2006) was used to appreciate condensed tannins content.1.5 ml of vanillin solution initially dissolved in methanol for a final concentration at 4% was mixed with 500 µl of extract, 2 ml of methanol and 1.5 ml of concentrated hydrochloric acid.The mixture was incubated for 15 min and the absorbance was taken at 500 nm.Catechin solution was used as reference and the content of condensed tannins was expressed as mg of catechin equivalent/mg of extract.

Total antioxidant capacity assay (TAO)
The ability of the plant extract to trap free radicals explains their antioxidant capacity.The method described by Lamien-Meda et al. (2008) was used.0.75 ml of different concentrations (from 0.5 to 3.5 mg/ml) of leaves extract were mixed with 1.5 ml of DPPH solution (0.4 mg/ml in methanol).Then, the mixture was incubated at room temperature for 15 min in a dry bath room.Ascorbic acid solution is used as standard and the total antioxidant activity was expressed as millimol of ascorbic acid equivalent per gram of aqueous extract.

Acute toxicity studies
This assay was done in accordance with Organization for Economic Cooperation and Development (OECD, 2001) guideline.Three female and non-gravid Wistar rats weighing 150 to 200 g received by gavage, 2000 mg/kg aqueous extract of M. oleifera after being kept on fasting the previous night.The animals are observed closely during the first four hours and daily for 14 days (for weight change, tremors, convulsion, salivation, diarrhea, lethargy, sleep, coma, and death, changes in the skin, fur, eyes and behavioral pattern).At the end of 14 days, a histological study was performed on some organs (liver and kidney) and biochemical parameters were measured.

Collection of blood and serum samples
Anesthetized rats underwent cervical decapitation.The blood was collected in heparinized bottles for hematological studies.Blood samples taken from non-heparinized bottles were allowed to coagulate.The serum was separated from the clot and centrifuged in clean bottles for biochemical analysis.

Hematological measurements
The hematological examinations were done using blood samples collected from retro-orbital of the experimental rats and conserved in capillary tubes (EDTA).These examinations using Toro and Ackermann (1975) and Duncan et al. (1994) included red blood cell counts, white blood cells and platelets hemoglobin, hematocrit, mean globular volume (VGM), average corpuscular content in hemoglobin (TCMH), the mean corpuscular concentration in hemoglobin (CCMH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT).Creatinine levels and serum urea were measured using the method described by Coles (1986).
Anti-hyperglycemic activity of M. oleifera Pareek et al. (2009) method was used to evaluate antihyperglycemic activity by oral glucose tolerance test (OGTT).30 rats divided into 6 lots of five rats were constituted before the experiment (OGTT), all the rats were beforehand submitted to a non-hydric fast for 16 h.500 and 1000 mg/kg of aqueous extract were administered to the lots 3 and 4. Lots 1 and 2 received each, a dose of 5 ml/kg of Glibenclamide.The control received distilled water with a dose of 10 ml/kg.D-glucose was administrated to the rats with a dose of 3 g/kg after half an hour.SD CHECK glucometer was used to take the glycemia in a sequential way every 30 min from start point corresponding to T0 up to 120 min corresponding to T120.The final measure was taken at T 180 min.

Statistical analysis
Statistical analysis of the data was done and expressed as a mean using Minitab Version 1.0 software.The differences obtained from the univariate variance analysis (ANOVA) were reported using the Kruskal-Wallis test.Correlations between different values were expressed graphically using Graph Pad Prism version 5.For p ≤ 0.05, the results are statistically significant.

Phytochemical screening and bioactive molecules quantification
Only three families of compounds were absent from the ten important families of compounds sought in the extract (Table 1).They are leucoanthocyan, coumarin and cyanogenic glycoside.The content of phenolic compounds,  flavonoids and condensed tannins respectively in the aqueous leaves extract of M. oleifera are 46.242±2.44,10.80±0.64 and 2.85±1.018(Figure 1).

Total antioxidant capacity assay (TAO)
The antioxidant capacity of the plant leaves extract was 2.47 ± 0.26 mmol equivalents of ascorbic acid per mg of leaves extract.Total antioxidant capacity represents both oil soluble and water soluble antioxidants that are capable of scavenging reactive oxygen species and protects from chronic diseases such as cancer, diabetics and arthritics (Mrudula et al., 2014).

Acute toxicity studies
It was observed that no rat that received the aqueous extract of the plant at a dose of 2000 mg/kg died.This result shows that the concentration administered to the rats is below the toxicity threshold, 2000 mg/kg which is therefore lower than the LD50.It was observed that the rats used in this test gained weight as those of the control group.This is contrary to the effects of toxicity.Figure 2 presents the evolution of animal's weight during the 14 days.

Determination of hematological and biochemical parameters
Table 2 presents the results obtained from different measurements of hematological parameters.No differences were observed between control animals and experimental animals.These results showed that the aqueous extract of M. oleifera leaves has no impact on the metabolic parameters of rats.Table 3 presents evolution of some biochemical parameters in comparison with control rats.Maintaining of metabolic equilibrium requires that these parameters  are constant.As compared to the 2000 mg/kg dose, no significant differences were observed in these parameters, as the p>0.005.For both hematological and biochemical parameters, no significant difference (p>0.005) was observed.

Anti-hyperglycemic activity of M. oleifera
From the analysis of the graphs presented in Figure 3, distilled water showed a high level of hyperglycemic activity from T0 to T90, after which a low decrease up to T180 was noticed.For plant leaves extract, the two concentrations (500 and 1000 mg/kg) presented the same effect on the hyperglycemic activity.An increase of the hyperglycemic level was observed only for the first half of time.Then, from T30, hyperglycemic activity decreased progressively until T180 for both concentrations.

DISCUSSION
Traditional medicine is the oldest medicine used for the treatment of many diseases and by rural as well as urban people.This medicine was based mostly on the use of diverse plants which revealed different pharmacological properties.These properties were attributed to the presence of some secondary plants metabolites.Indeed, the phytochemical screening carried out on the aqueous extract of M. oleifera leaves revealed the presence of phenolic compounds, sugar and other substances.Their presence confirms in certain proportions, their efficiency in the treatment of many diseases.Most medicinal plants are the important source of bioactive molecules that contribute to the reduction of risk and progression of certain acute and chronic diseases such as heart diseases, cancer, diabetes, etc. (Ali et al., 2008).Farooq et al. (2007) also revealed the presence of alkaloids, tannins and flavonoids in M. oleifera leaves extract but in different proportions (Teteh et al., 2013).The age of the plant, the harvest period of the plant, the climate, the method of extraction, the type of soil could explain the differences noticed.In this case, phenolic compounds content was higher than the remainder and indicated that it was the most important compound in M. oleifera leaves extract.This result could probably justify the antioxidant activity of the leaves.Phenolic compounds were reported to have multiple biological effects, including antioxidant activity (Jaehak et al., 2014).The antioxidant activity of phenolic compounds is mainly due to their redox properties, which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Singleton et al., 1965).
Regarding antihyperglycemic activity, it was shown that leaves extract presented this activity.A maximum action on blood glucose level decrease was obtained with a dose of 500 mg/kg.The findings are in agreement with the result of Edoga et al. (2013) who showed that the leaves extract of M. oleifera reduced blood sugar levels in the normoglycemic rats, this effect was dose dependent.The antihyperglycemic effect of the plant may be due to the presence of alkaloids, flavonoids, steroids and other constituents present in the leaves extract which could act synergistically or independently in lowering the blood sugar level.Some alkaloids are known hypoglycemic agents.For instance berberine, a quaternary ammonium salt from the protoberberine group of isoquinoline alkaloids has been used successfully in experimental models of diabetes mellitus, berberine was shown to possess insulin sensitizing effect (Wang et al., 2010).Also, flavonoids, being polyphenolics, are known to be hypoglycemic.The antidiabetic activity of four flavonoidsboswellic acid, ellagic acid, quercetin and rutin were demonstrated in rats and the proposed mechanism of action was by increasing the peripheral utilization of glucose and inhibiting the glucose transporter activity from intestine (Jadhav and Puchchakayala, 2012).Thus, M. oleifera might decrease the velocity of gastric emptying, a major determinant of postprandial glycemia (Horowitz et al., 2002).Some herbal medicines were reported to inhibit gastric emptying and also glucose uptake in the small intestine (Matsuda et al., 1998).High fiber diets decrease postprandial blood glucose by slowing the rate of food passage from the stomach to the small intestine (Tsai and Peng, 1981).
Administration of M. oleifera leaves extract did not affect blood biochemical parameters.In the same way, the toxicity test on Wistar rats revealed a gain of body weight with all groups of rats even when the dose was 2000 mg/kg.The increase in the body weight of rats might be due to the fact that M. oleifera is rich in amino acids, vitamins and minerals, particularly iron (Booth and Wickens, 1988;Mori et al., 2009).The results obtained show that the LD50 is greater than 2000 mg/kg.This result is in concordance with the work of Ojo et al. (2013) who used the dose of 5000 mg/kg.At doses above this level; however, the animals may exhibit some toxic changes.The plant is safe for consumption and for medicinal uses.

Conclusion
The result obtained from this research study revealed that aqueous leaves extract of M. oleifera exhibited diverse bioactive compounds with different properties.The plant leaves extract presented no acute toxicity and could be safe for consumption as traditional vegetable and for medicinal uses.With its contribution in the decrease of blood glucose level, this result confirmed its use by traditional therapist for treatment of diabetes.However, it is important that future study should contribute to identifying and characterizing the crude extract specific molecules involved in the treatment of each type of the disease.

Figure 1 .
Figure 1.Total phenolic, flavonoids and condensed tannins content of M. oleifera leaf extract.

Figure 2 .
Figure 2. Effects of M. oleifera on the body weights of rats.

Figure 3 .
Figure 3.Effect of M. oleifera leaves extract on blood glucose level in rats.T0, T30, T60, T90, T120 and T180 stands for time interval of hyperglycemic activity in minutes.

Table 1 .
Phytochemical analysis of M. oleifera leaves extract.

Table 2 .
Hematological parameters in rats after administering M. oleifera extract.

Table 3 .
Determination of biochemical parameters of rats after treatment with M. oleifera extract.