ANTIHYPERGLYCEMIC AND ANTI-OXIDANT POTENTIAL OF ETHANOL EXTRACT OF VITEX THYRSIFLORA LEAVES ON DIABETIC RATS

The antihyperglycemic effect of an ethanol extract of Vitex thyrsiflora leaves was investigated in normal male rats and streptozotocin‐induced diabetic male rats and its antioxidant potential was evaluated. After preparation of the extract, it was subjected to a phytochemistry screening, and tested on male rats made hyperglycemic in the Oral Glucose Tolerance Test (OGTT) and in streptozotocin diabetic rats. Glibenclamide (10 mg/kg) was served as a positive control in both experiments. During both experiments, the extract was tested at 200 and 300 mg/kg. The evaluation of the antioxidant potential was done through the determination of the total polyphenols and total flavonoid contents, and by using the ferric reducing antioxidant power assay and the free radical scavenging activity on DPPH method. The results show that the extract contains alkaloids, flavonoids, steroids, saponins and phenolic compounds. The test on hyperglycemic rats in OGTT showed that, the extract was effective (at a dose of 200 mg/kg) to significantly decreased glucose-induced hyperglycemia (**p ˂ 0.01). The extract was ineffective on streptozotocin‐induced diabetic rats. The study of the antioxidant potential showed that, polyphenols and flavonoids increase with the concentration of the extracts. IC50 value was found to be 65.97, based on the log (inhibitor) vs. normalized response-Variable slope. FRAP appears to be significantly highly correlated with total polyphenols content and total flavonoids content. This shows that the ethanol extract of V. thyrsiflora leaves could be served to prevent acute hyperglycema, but not a chronic hyperglycemic state.


INTRODUCTION
The genus Vitex (Verbenaceae) is constituted by 250 species of small trees and shrubs which occur in tropical and subtropical regions 1 . Vitex thyrsiflora Baker (Verbenaceae) is a glabrous under shrub that is widely distributed in Cameroon 2 . This plant is reported to be useful in the treatment of orchitis 2 . The root barks are used in the treatment of stomach pains, sexual sterility and wounds 3 . Previous chemical investigations on the leaves and fruits resulted in the isolation of phytoedysteroids 3 . However, the investigation of some other Vitex species have resulted in the isolation of iridoids 4,5,6 , diterpenes 7,8 , steroids 9 , flavonoids 6 and triterpenoids 9 . Several of previous compounds isolated from Vitex genus have shown antioxidants activities 10,11 . Among all secondary metabolites, phenolic antioxidants appear to be the most important since they have shown promising antioxidant activity in both in vivo and in vitro investigations. Plant phenolics are mainly classified into five major groups, phenolic acids, flavonoids, lignans, stilbenes and tannins 12,13,14 .The antioxidant activities of these compounds have been attributed to various mechanisms which have been ISSN: 2456- 8058 20 CODEN (USA): UJPRA3 established by various assay procedures 15 . Antioxidant activity has a fundamental role in cellular protection during an inflammation process. The identification of phytochemical compounds in plant species has been exploited in recent years, due to the growing popularity of herbal medicines and consumers growing enthusiasm for foods with bioactive characteristics that aid in preventing and fighting disease 16,17 . Diabetes is characterized by chronic hyperglycemia, a source of increased oxidative stress and tissue oxidative damage. In particular, hyperglycemia promotes the glycation of proteins resulting in the formation of advanced glycation products (AGE). Thus antioxidant molecules appear as an opportunity for a strategy to fight not only against hyperglycemia, but also against complications related to diabetes mellitus 18,19 . Then, the objectives of this study based on these findings, was first to evaluate the antihyperglycemic effect of an ethanol extract of V. thyrsiflora leaves in glucose loaded rats and in STZ-diabetic rats and second to determine the Total Polyphenols Content (TPC), Total Flavonoids Content (TFC), the Ferric Reducing Antioxidant Power Assay (FRAP) and the DPPH free radical scavenging assay of this extract.

Induction of diabetes
Rats were fasted overnight (16h) before inducing diabetes with streptozotocin. Streptozotocin was prepared in freshly prepared sodium chloride solution 0.9% and was injected intraperitonially at a concentration of 55 mg/kg body weight in a volume of saline of about 500µl/200g b.w by applying the protocol of Szkudelski 22 . Control rats were injected with saline solution only. The diabetic state was confirmed 72 h after streptozotocin injection. Threshold value of fasting blood glucose was taken as ≥ 200mg/dL. Diabetic rats were weighed, matched for body weight and divided into 5 groups consisting of 5 animals each.

Antihyperglycaemic effect of ethanol extract of V. thyrsiflora leaves on diabetic rats
The approval of the Institutional Animal Ethics Committee was obtained before starting the study. An International protocol for conducting experiments on animals were followed.

Experimental design
The Diabetic rats were divided into 5 groups with five rats each: (A, B, C and D). Group A rats received DMSO 3%, those of group B and C were treated with ethanol extract of V. thyrsiflora at the doses of 200 and 300 mg/kg b.w respectively, and Group D received glibenclamide (10 mg/kg b. w). Blood samples were collected before the commencement of treatment with the extract and then after, at 1h, 3h and 5h intervals.

Oral Glucose Tolerance Test on normoglycemic rats
This study was carried out on normal male rats with normal blood glucose level, according to the method of Schoenfelder et al. 23 . The animals were fasted for 16 h prior to the study. Five groups with 5 animals each were constituted and the animals received a dose of 3 g/kg of glucose by oral route 60 min after haven been treated with the extract as follows: Group I: vehicle (DMSO 3%; 10 ml/kg b. w, negative control), Group II: water-ethanol extract (200 mg/ kg b.w), Group III: ethanol extract (300 mg/ kg b. w), Group IV: ethanol extract (400 mg / kg b. w) Group V: glibenclamide (10 mg/ kg b. w), was served as positive control. Blood was collected from the animals before administration of the extract (-60 min), and then after at 0, 30, 60, 90 and 150 min post administration.

Blood Glucose Estimation
Blood samples were obtained by tail prick and fasting blood glucose levels were estimated with a One Touch Ultra glucometer (Life Scan, Inc., Milpitas, CA, USA) in all animals. Blood glucose levels were expressed in mg/dL.

Dosage of phenolic compounds and antioxidants evaluation Determination of Total phenolic content (TPC)
The ability of the extracts to reduce the phosphomolybdic-tungstate chromogene in Folin Ciocalteu with maximum absorbance at 760 nm (Total phenolic content, TPC) was assessed as earlier described by Vinson et al. 24 . Data were reported as mean±SD for triplicate measurements. Catechin was used as control and the results were expressed as mg Catechin equivalent/g (mg CE/g).

Determination of Total flavonoid content (TFC)
The Total Flavonoid Content (TFC) was measured as earlier described 25 . Total Flavonoid Content of the extract were expressed as mg Quercetin Equivalent/g (mg QE/g) through the calibration curve with quercetin. Data were reported as mean± SD for triplicate measurements.

Determination of ferric reducing antioxidant power (FRAP) assay
The Ferric Reducing Antioxidant Power (FRAP) of an extract measures the ability of the extract to reduce ferric tripyridyltriazine to ferrous tripyridyltriazine, yielding a blue coloration with maximum absorbance at 593 nm. FRAP assay was performed like previously described by Benzie and Strain 26 . The FRAP value was calculated and expressed as mg Catechin Equivalent/g (mg CE/g) based on a plotted calibration curve, using Catechin as standard, at a concentration ranging from 50 to 600 μmol.

DPPH free radical scavenging assay
The ability of the extract to scavenge free radicals by converting DPPH (purple in color) into di-phenyl hydrazine (yellow in color) was measured at 517 nm as earlier described 27 .
The percentage (%) radical scavenging effect (RSE) of extract was calculated as follows: Where Abs1 is the absorbance of the control (containing all reagents except for the extract), and Abs2 is the absorbance of plant extract (is the optical density in the presence of the extract). All tests were performed in triplicates. The parameter IC 50 (concentration at which the DPPH radicals were scavenged by 50%) (mg/mL) was also calculated. IC 50 values were estimated from the % inhibition versus concentration plot, using a non-linear regression algorithm. The data was presented as mean values ± standard deviation (n = 3).

Statistical Analysis
Results were expressed as mean± SD. Statistical analysis were carried out using one way Analysis of variance (ANOVA) followed by Dunnet test for comparison to vehicle control or followed by Newman-Keuls Multiple Comparison Test for antioxidant parameters, using GraphPad Prism 5.03 software. A value of p≤0.005. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 was considered to be significant.

RESULTS
Phytochemical screening revealed that the ethanol extract of V. thyrsiflora leaves contained alkaloids, flavonoids, steroids, phenolics compound, saponosids, catechic tanins and anthraquinones (  Figure 1b). The administration of ethanol extract of V. thyrsiflora leaves at 200mg/kg or 300mg/kg to diabetic rats didn't show any difference in the blood glucose level, according to the AUC associated with each group tested. Even glibenclamide didn't show a significant effect on lowering the high blood glucose level state induces by streptozotocin (Figure 2). The Total Polyphenol Content (TPC) appears to be significantly dose dependent on the concentrations of the extract, until 40 mg/kg (79.36±0.3717 mg Catechin/ g), where the TPC became stable and not different to that of 50 mg/kg (Figure 3). The Total Flavonoid Content (TFC) evaluated in V. thyrsiflora ethanol extract showed a significant dose dependent increase when compared to 2.5mg/kg. The content was the same at doses of 5mg/kg and 10 mg/kg ( Figure 4). The correlation between Ferric Reducing Antioxidant Power (FRAP) and Total Flavonoid Content (TFC) showed a very strong positive correlation (Pearson r = 0.9223 with R 2 = 0.8507, p** = 0.0088) ( Figure 5). The correlation between FRAP and TPC was significantly high (p** = 0.0010) ( Figure 6).

DISCUSSION
The aim of this work was to evaluate the antioxidant and the antihyperglycemic potential of the ethanol extract of V. thyrsiflora leaves. The phytochemical screening reveals that the ethanol extract of V. thyrsiflora leaves possesses alkaloids, flavonoids, steroids, phenolic compounds, catechic stanins, anthraquinones and saponosids in traces. The evaluation of the effect of V. thyrsiflora ethanol leaves extract on hyperglycemic normal rats in OGTT showed that, the 200mg/kg dose was the most active to lower the AUC induced by glucose loaded in normal rats male (**p ≤ 0.01). Glibenclamide (10 mg/kg), a widely used antidiabetic drug was effective to decrease the AUC observed in the vehicle group (**p ≤ 0.01).
The results of OGTT in normal rats could be correlated with the ability of the extract to probably enhance the secretion of insulin in the likely manner of sulfonylureas and inhibit αglucosidases present in the border brush of the small intestine 28,29 . Enhanced tissue uptake of blood glucose induced by V. thyrsiflora might also be taken into consideration as an alternative possibility. In our case, the decrease in blood glucose at 200 mg/kg and not at 300 mg/kg could be explained by the presence of hyperglycemic compounds which have become the majority at this dose. Some studies showed that, antagonistic relationship among phytochemicals would affect the efficacy of crude extracts as used in traditional medicine, like observed by Milugo et al 30 on antagonistic effect of alkaloids and saponin. Furthermore, the results show that administration of the extract is associated with no effect on blood glucose level in STZ-diabetic rats. Since streptozotocin selectively destroys ß-cells of the pancreas, we would expect the extract to exert no effect on plasma glucose concentrations in STZ diabetic rats if the mode of action is mediated through insulin production. Furthermore, the observations in the present study indicate that the hypoglycaemic effect of the extract as well as that of glibenclamide were far more pronounced in OGTT rats than in STZ-diabetic rats. This result is probably in line with the early suggestion that glibenclamide was effective in moderately streptozotocin-induced diabetic animal and ineffective in severe diabetic rats 31,32 ; the extract could act in the same way as glibenclamide. These observations taken together suggest that ethanol extract of V. thyrsiflora leaves has a potential in the management of acute hyperglycaemia. Antioxidant parameters showed a dose-dependent rise in the concentration of the extract. The TPC and the TFC showed a positive correlation with the FRAP test, which could suggest that this method would act through polyphenols and flavonoids. The Pearson correlation coefficient (R 2 ) is often used for measuring and describing the degree of linear regression between two continuous quantitative variables that are normally distributed. In our study, the R 2 value of the correlation between the iron reduction technique "FRAP" (Ferric Reducing Antioxidant Power) and the Total Phenolic Content was equal to 0.9477 with p** = 0.0010 and R 2 = 0.8507 with p** = 0.0088 for Total Flavonoid Content respectively. These results suggested that a great part of the antioxidant capacity of the ethanol extract of V. thyrsiflora leaves is attributed to the Total Polyphenol Content, mainly to flavonoid content in the extract, which have the hydrogen-donor ability to reduce iron. Similar studies suggested a linear relationship between antioxidant capacity and flavonoid contents of the plant extract 33,34 . The DPPH assay is often used to evaluate the ability of antioxidant to donate hydrogen or to scavenge free radicals. However, DPPH scavenging activity is best represented by IC 50 value, defined as the concentration of the antioxidant needed to scavenge 50% of DPPH present in the test solution 35 . In our study IC 50 was 65.97 mg/mL, R 2 = 0.9945 for the V. thyrsiflora leaves ethanol extract based on the log (inhibitor) vs normalized response -variable slope, which can be considered to be low antioxidant activity (IC 50 from 50.62 to 110.46 mg/mL) according to the scale of Surinut et al. 36 ; this might suggest that the antioxidant activity of this extract would not be mediated by donating hydrogen to scavenge free radicals but by the reduction of iron.

CONCLUSION
Based on the results of the present study, we conclude that the plant extract possesses antioxidant potential.
The findings of the present study also suggest that V. thyrsiflora ethanol leaf extract could be a potential natural source of antioxidants and could have greater importance as therapeutic agent in preventing or slowing oxidative stress related degenerative diseases. However, this work is the first report which evaluates antihyperglycemic effect of ethanol extract of V. thyrsiflora leaves and its antioxidant potential. Further studies should be carried out to evaluate α-amylase inhibitory and beta-glucosidase inhibitory activities isolate and identify active compounds, to understand the mechanism of action against hyperglycemia.

CONFLICT OF INTEREST
"No conflict of interest associated with this work".   Data are expressed as means ± S.D (n = 3); a significantly different from 2.5 mg/mL, b significantly different from 2.5 mg/mL and 5 mg/mL, c significantly different from 2.5 mg/mL, 5 mg/mL and 10 mg/mL, d significantly different from 2.5 mg/mL, 5 mg/ml, 10 mg/mL and 20 mg/mL. Data are expressed as means ± S.D (n = 3); a significantly different from 2.5 mg/mL, b significantly different from 2.5 mg/mL, 5 mg/mL and 10 mg/mL, c significantly different from 2.5 mg/mL, 5 mg/ml, 10 mg/mL and 20 mg/mL, d significantly different from 2.5 mg/mL, 5 mg/mL, 10 mg/mL, 20 mg/mL and 40 mg/mL.