In Vitro Α- Glucosidase Inhibitory and In Vivo Glucose Digestion Activities of Ethanol Leaf Extract of Acalypha Wilkesiana in Normoglycaemic Rats

In Vitro Α-Glucosidase Inhibitory and In Vivo Glucose Digestion Activities of Ethanol Leaf Extract of Acalypha Wilkesiana in Normoglycaemic We observed the ethanol leaf extract of Acalypha wilkesiana to be a rich source of phytochemicals and vitamins of therapeutic benefits. Considering the search for medicinal plants and products with hypoglycemic potentials, we conducted in vitro and in vivo studies to respectively investigate α- glucosidase inhibitory and glucose digesting activities of A. wilkesiana extract. The α-glucosidase inhibitory activity was determined following previously described methods at concentrations of 0.1-0.9μg/ mL using acabose as standard drug. Oral glucose tolerance test was determined in 24 normoglycemic adult male Wistar rats of (140‐160g) weight, assigned to four groups (n=6). At 0 minute, Group1 (control) and 2 (glucose treated) received 10ml/ kg mL distilled water while 3 (standard hypoglycemic treated) and 4 ( A.wilkesiana extract treated) received glimepiride (0.1mg/kg) and A. wilkesiana extract (250mg/ kg) respectively. 30 minutes after, group 1 received distilled water (10ml/kg mL) while groups 2, 3 and 4 received 5% glucose water (10ml/kg) and blood glucose level measured at 30 minutes interval for 180 minutes. The results showed a dose dependent % inhibition of α- glucosidase activity from 51.12±0.14 to 58.01±0.11 with acarbose and 50.48±0.29% to 56.14±0.56% with ethanol leaf extract of A. wilkesiana . A. (69.50±4.71mg/dl; p<0.05) and glimepiride (74.33±5.10mg/ dl; p>0.05) administrations concealed the rise in blood glucose following glucose loading (97.67±3.25mg/dl) and significantly lowered blood glucose levels compared with the glucose treated group at 30 through 180 minutes. These findings suggest that ethanol leaf extract of possesses hypoglycemic potentials by inhibiting α- glucosidase activity and increasing glucose digestion or inhibiting absorption in normoglycemic rats.

We observed the ethanol leaf extract of Acalypha wilkesiana to be a rich source of phytochemicals and vitamins of therapeutic benefits. Considering the search for medicinal plants and products with hypoglycemic potentials, we conducted in vitro and in vivo studies to respectively investigate α-glucosidase inhibitory and glucose digesting activities of A. wilkesiana extract. The α-glucosidase inhibitory activity was determined following previously described methods at concentrations of 0.1-0.9μg/ mL using acabose as standard drug. Oral glucose tolerance test was determined in 24 normoglycemic adult male Wistar rats of (140-160g) weight, assigned to four groups (n=6). At 0 minute, Group1 (control) and 2 (glucose treated) received 10ml/ kg mL distilled water while 3 (standard hypoglycemic treated) and 4 (A.wilkesiana extract treated) received glimepiride (0.1mg/kg) and A. wilkesiana extract (250mg/ kg) respectively. 30 minutes after, group 1 received distilled water (10ml/kg mL) while groups 2, 3 and 4 received 5% glucose water (10ml/kg) and blood glucose level measured at 30 minutes interval for 180 minutes. The results showed a dose dependent % inhibition of α-glucosidase activity from 51.12±0.14 to 58.01±0.11 with acarbose and 50.48±0.29% to 56.14±0.56% with ethanol leaf extract of A. wilkesiana. A. wilkesiana (69.50±4.71mg/dl; p<0.05) and glimepiride (74.33±5.10mg/ dl; p>0.05) administrations concealed the rise in blood glucose following glucose loading (97.67±3.25mg/dl) and significantly lowered blood glucose levels compared with the glucose treated group at 30 through 180 minutes. These findings suggest that ethanol leaf extract of A. wilkesiana possesses hypoglycemic potentials by inhibiting α-glucosidase activity and increasing glucose digestion or inhibiting absorption in normoglycemic rats. condition of individuals with diabetes, not only by hypoglycemic effects, but also by improving lipid metabolism, antioxidant status and capillary function [9]. In our recent investigation, we observed the ethanol leaf extract of Acalypha wilkesiana to be a rich source of phytochemicals, macro-elements and vitamins known to have several therapeutic benefits [10]. Though this plant is not edible, it is found to contain alkaloids, tannins, resins [11]as well as plenty of terpenoids and vitamin C and moderate level of flavonoids [10].
Therefore, this study investigates the in vitro and in vivo αglucosidase inhibitory and glucose digestion activities of ethanol leaf extract of A. wilkesiana using normoglycemic adult male Wistar rats.

Collection, Processing and Extraction of Plant Materials
Samples of Acalypha wilkesiana leaves were collected from Benin City, Nigeria and authenticated at the Herbarium Unit of Forestry Research Institute, Ibadan, Nigeria. The leaves were sorted, air-dried for 7 days and then pulverized and packaged in polyethene air tight bags. 200g of the powder leave was added into a container containing 1.5L of 70% ethanol and used to prepare the ethanolic extract as described in Majekodunmi and Nubani [22] with few modifications.

In Vitro Anti-Diabetic Activity of Ethanolic Leaves Extract of A. Wilkesiana
The α-glucosidase inhibitory activity of the ethanol leaf extract of A. wilkesiana was determined as described by Shai et al. [23] with slight modifications. Briefly, ethanol leaf extract of A. wilkesiana or acarbose at different concentrations (0.1-0.9 μg/mL) was incubated with 500 μL of porcine pancreatic amylase (2 U/mL) in phosphate buffer (100 mM, pH 6.8) at 37 °C for 20 minutes. 250 μL of 1% starch dissolved in 100 mM phosphate buffer (pH 6.8) was added to the reaction mixture and incubated at 37° C for 1 hour. Dinitrosalicylate colour reagent (1 mL) was added and boiled for 10 minutes. The absorbance of the resulting mixture was read at 540 nm and the inhibitory activity was expressed as percentage of a control without the inhibitors. All assays were carried out in triplicate.
The inhibitory activities of the fractions on the α-glucosidase were calculated using the following formula: Whereas is the absorbance in the presence of the sample and Ac is the absorbance of the control.

Experimental Animals
Twenty-four (24) adult male Wistar rats with average weight of 140-160g were obtained from the Animal House of the College of Medicine, Ambrose Alli University, Ekpoma. They were housed at room temperature on a 12 hour dark-light cycle and acclimatized for a 14-day period with ad libitum access to clean water and food (rat chow; Vital Feed Nig. Ltd, Jos, Nigeria).

Oral Glucose Tolerance Test in Normoglycemic Rats
The 24 rat which were normoglycemic were used for the study after acclimatizing for two weeks. They were fasted overnight for 12 hours before the experiment. Their fasting blood glucose levels The blood glucose levels were measured at 0, 30 min, 60 min, 90 min, 120 min, 150 min and 180 min after glucose loading. The mean increment of glyceamia in each group was calculated as the sum of increases in blood glucose divided by the number of animals after each period according to the method of Madar [24]. Data were expressed in means ± SEM. Paired sample "t"-test and one-way analysis of variance (ANOVA) was used to compare the group means between treatments in the in vitro and in vivo studies respectively.

Discussion
The major goal in diabetes management is the near normal maintenance of fasting and postprandial blood glucose levels [25]. In fact, Matsui et al. [26] and Wassmann and Nickenig [27], to have digestibility action on glucose similar to acarbose. This assertion is based on the fact that α-glucosidase is involve in the digestion of starch into various oligosaccharides, and disaccharides to produce glucose and other monosacharides [28]. In support of this finding, Fonkoua et al. [29] has reported similar findings with hydroethanolic extract of A. wilkesiana but reported no effect with aqueos extract. Both murine model [30] and clinical [31] studies have shown inhibitors of α-amylase and α-glucosidase suppress the production and absorption of glucose in the small intestine.
Acarbose is a complex oligosaccharide that binds competitively to the α-glucosidases at the brush border of the small intestine to delay sucrose and starch breakdown and the absorption of  [29] has reported hypoglycaemic effect of 400mg/kg hydroethanolic extract of A. wilkesiana in streptozotocin diabetic rats. However, Al-Attar [20] reported orally aqueous A. wilkesiana leaves extract to cause a duration dependent decrease in blood glucose in streptozotocin induced diabetic rats.

Conclusion
Overall, this study showed that ethanolic leave extract of A.
wilkesiana possesses hypoglycemic activity in normaglycemic state. Since a similar dose dependent difference was observed between A. wilkesiana leaves extract and acarbose in suppressing α-glucosidase, A. wilkesiana leaves extract therefore showed hypoglycemic potential via the inhibition of α-glucosidase and stimulation of glucose digestion mechanisms.