Inhibitory potential and antidiabetic activity of leaf extracts of Vitex doniana

Pancreatic alpha (α)-amylase and intestinal α-glucosidase involved in the enzymatic breakdown of dietary carbohydrates are implicated in postprandial hyperglycemia. The inhibition of these two enzymes has been exploited in the design of antidiabetic drugs. This study was therefore designed to investigate the ability of the leaf extracts of Vitex doniana to inhibit α-amylase and α-glucosidase as well as the in-vivo antidiabetic potential of the ethanol leaf extract. The inhibitory potentials of the extracts on α-amylase were assayed through the method of starch-iodine colour change while αglucosidase inhibition was assessed by measuring the release of 4-nitrophenol by p-nitrophenyl-α-Dglucopyranoside (pNPG). Acarbose was used as the standard. The extracts displayed a dose-dependent significant inhibition of the enzymes. The aqueous leaf extract of V. doniana was the strongest inhibitor of α-amylase with half-maximal inhibitory concentration (IC50) of 3.09 ± 0.22 mg/ml while the ethanol leaf extract was the strongest inhibitor of α-glucosidase with IC50 of 17.12 ±1.42 mg/ml. The result also showed that both extracts contain mixed inhibitors of the two enzymes. The result of the in-vivo antidiabetic study conducted for 14 days showed a significant (p<0.05) reduction in the fasting blood glucose level of the extract treated groups of animals compared to the untreated diabetic group. These results strongly suggest that V. doniana can be exploited in the treatment of diabetes mellitus.


INTRODUCTION
Natural products of plant origin are a good source of intermediary metabolites some of which have been found to inhibit some enzymes (Rauf and Jehan, 2017). These plants contain compounds that are rich in polyphenols, which are known to interact with proteins and can inhibit enzyme activity (Oboh et al., 2012). The polyphenols *Corresponding author. E-mail: nnenna.ani@esut.edu.ng. have been reported to inhibit α-amylase and αglucosidase associated with lipid peroxidation and type 2 diabetes (Reddy et al., 2010). Natural products from plants have been used for the prevention and management of non-communicable diseases with minimal side effects and toxicity (Bhatia et al., 2019). Sangeetha and Vedasree (2012) reported that some herbal medicines used in treatment of diabetes act on the beta cells to secret insulin which mediates glucose metabolism.

Abbreviations
Pancreatic α -amylase hydrolyzes starch to long chain carbohydrate which in turn are degraded by intestinal αglucosidase to glucose and subsequently into the blood stream (Tundis et al., 2010). The breakdown of starch proceeds rapidly leading to the elevation of level of postprandial hyperglyceamia (PPHG) (Sudha et al., 2011). These two enzymes have therefore been found to be good targets in drug design for the treatment of diabetes (Subramanian et al., 2008). Some anti-diabetic drugs have been found to competitively inhibit alpha glucosidase by slowing down carbohydrate digestion (Nair et al., 2013).
In type 2 diabetes mellitus there is a dysfunction of endocrine system which is caused by low secretion of insulin by the pancreatic β-cells and insulin resistance or insensitivity of cells to insulin action to regulate blood glucose levels which leads to hyperglycemia (Ardisson Korat et al., 2014). Hyperglycemia is the characteristic feature of type 2 diabetes mellitus, thus, reduction of postprandial hyperglycemia is a good therapeutic approach in treatment of type 2 diabetes (Chipiti et al., 2017).
Diabetic drugs like acarbose, metformin, miglitol and voglibose (Kaissi and Sherbeeni, 2011) though effective for glycemic control can lead to liver disorders, flatulence and abdominal pain (Kwon et al., 2007). Plant products with the potentials of inhibiting α-amylase and αglucosidase can be used in the treatment of hyperglyceamia with little side effects.
Vitex doniana of the genus Verbenaceae, also known as black plum, is found in Southern Nigeria (Agbafor and Nwachukwu, 2011). It is the most widespread of the genus commonly found in savannah regions and useful in management of diseases (Dauda et al., 2011). In Nigeria, V. doniana is called dinyar in Hausa, galbihi in Fulani, ori nla in Yoruba and uchakoro in Igbo (Adejumo et al., 2013). The young leaves are cooked and prepared as vegetable soup and also added in some sauces. Phytochemical analyses revealed the presence of tannins, anthraquinones, flavonoids, resins, cardiac glycosides, saponins and alkaloids (Ezekwesili et al., 2012). Agbafor and Nwachukwu (2011) reported the antioxidant potential of the leaf extracts. Documented research work shows that V. doniana is used in the treatment of diabetes in traditional medicine and the extracts have exhibited anti-diabetic property in animal models (Obasi et al., 2013;Njoku et al., 2019). There is limited information on the V. doniana phytoconstituent's α-amylase and α-glucosidase inhibitory activity. This study will help establish the antidiabetic potential of V. doniana plant through their ability to inhibit activities of the two enzymes. Patients using α-glucosidase and αamylase inhibitors such as acarbose suffer from diarrhea, flatulence and meteorism. These side effects may be due to excessive inhibition of α-amylase. Therefore there is a search for a lead substance with a strong inhibitory activity on α-glucosidase, but mild inhibition on α-amylase activity. Therefore, this study was aimed to explore the antidiabetic potential of V. doniana sweet through elucidation of its possible α-amylase and α-glucosidase inhibitory activity and mechanism of inhibition. This study will help to validate the traditional uses of the plant in diabetes and also screen its specificity towards the pancreatic digestive enzymes.

Collection of plant material
V. doniana leaves were collected from farmlands in Obuoffia Awkunanaw in Nkanu West Local Government Area of Enugu State, Nigeria. It was authenticated in the Department of Botany, Nnamdi Azikiwe University Awka, Nigeria and deposited at the herbarium with a voucher specimen number 2/39.

Sample preparation
The leaves of V. doniana were shade-dried, ground into powder using a manual grinder and stored in air tight containers. The powder was divided into three parts (100 g each) and was extracted in various (500 ml) solvents, viz; methanol, ethanol and aqueous.

Ethical approval
This study was duly approved and was carried out strictly in compliance with the recommendations in the guide for the Institutional Animal Care and Use Committee of Nnamdi Azikiwe University, Awka, Nigeria in line with the protocols of Animal Care and Use in Research, Education and Testing (ACURET).

Aqueous extract
One part of the powdered sample was soaked in water. The extract was centrifuged at 10,000 rpm (Centurion centrifuge, Centurion Scientific LTD; West Sussex, United Kingdom, Model K220) for 10 min to get the supernatant from which the aqueous extract of V. doniana (VDAQ) was prepared at a concentration of 10 mg/ml.

Methanol extract
One part of the ground sample was soaked and extracted in methanol by stirring at 150 rpm at room temperature for 72 h. It was filtered using Whatman No.4 filter paper. The filtrate was evaporated to dryness at 40°C. The extracts were weighed, dissolved in dimethylsulphoxide (DMSO) to obtain methanol extract of V. doniana (VDM) at 10 mg/ml concentration, and afterwards kept in the refrigerator until use.

Ethanol extract
One part of the ground sample was extracted by stirring with ethanol at 150 rpm at room temperature for 72 h. It was filtered using Whatman No.4 filter paper. The filtrate was evaporated to dryness at 40°C. The extracts were weighed, dissolved in dimethylsulphoxide (DMSO) to obtain ethanol extract of V. doniana (VDE) at 10 mg/ml concentration, and afterwards kept in the refrigerator until use.

α-amylase inhibition assay
The α-amylase inhibitory activity was carried out using starch-iodine method as described by Mahomoodally et al. (2012). One hundred microliter (1.25-10 mg/ml) of the plant extracts was pre-incubated for fifteen minutes with 100 µl of the solution of enzyme at room temperature. Three milliliter (3 ml) soluble starch solution (1% starch) and 2 ml phosphate buffer were added to the reaction mixture. The reaction mixture was incubated at room temperature for 1 h. At timed interval, 0.1 ml from the reaction mixture was discharged into 10 ml iodine solution. After mixing, the absorbance of the starch-iodine solution was measured at 565 nm. α-amylase inhibitory activity was calculated as percentage inhibition thus: A0 and At being absorbance of starch-iodine solution at t = 0 min and t = 60 min, respectively.
Controls not containing the plant extracts are said to possess full enzyme activity. Acarbose was used as standard. IC50 values (Concentrations of extracts at 50% inhibition) were estimated from the graph and used to compare the plant extracts.

α-amylase inhibition kinetics
The mechanism of inhibition of α-amylase was carried out with the plant extract with the least IC50 (Ali et al., 2006); with some modifications. Thus, 250 μl of the plant extract (2 mg/ml) was preincubated for 10 min with 250 μl of α-amylase at room temperature in one group of test tubes. α-amylase was pre-incubated with phosphate buffer (250 µl; pH 6.9) in another group of test tubes. 250 μl of starch solution (0.30 -5.0 mg/mL) was added to the two groups of mixtures and the mixture was then kept at room temperature for 10 min. 500 μl of DNS was added to the reaction and then boiled for 5 min. The reducing sugars liberated were determined using spectrophotometer and converted to reaction velocities. Linweaver-Burk plot of 1/V against 1/[S] was plotted: V = reaction velocity and [S] is the substrate concentration. The mechanism of inhibition on α-amylase activity by the extract was assessed from Lineweaver-Burk plot. Km and Vmax were determined.

αglucosidase inhibition assay
The inhibitory activity of the extracts on α-glucosidase was assessed through measurement of the release of 4-nitrophenol by paranitrophenyl α-D glucopyranoside (PNPG) as described by Kim and Nho (2004). PNPG (5 mM, 0.3 ml) was added to 0.1 M sodium phosphate buffer (1.0 ml, pH: 6.9), 0.5 mg/ml of enzyme solution (0.2 ml) and the extract (0.2ml; 1.25-8 mg/ml), giving a total of 1.7 ml. After 30 minincubation (room temperature), 2.0 ml of 0.2 M sodium carbonate was added to stop the reaction. p-nitrophenol released was determined at 400 nm using spectrophotometer. The rates of inhibition were calculated as: The controls (inhibitor and reagent blank) were also run similarly and then subtracted. Reagent blank contained only substrate, buffer and enzyme and acarbose was used in place of the plant extract in positive controls. IC50 values were determined from the graph.

α-glucosidase inhibition kinetics
The mechanism of inhibition of α-glucosidase by the extracts was assessed by using PNPG in increasing concentration (Ali et al., 2006). The plant extract with the least IC50 was used. 50 μl of the extract (8 mg/ml) was added to 100 μl of α-glucosidase solution at 25ºC for 10 min in group of test tubes. α-glucosidase was incubated with phosphate buffer (50 µl; pH 6.9) in another group of test tubes. 50 μl of PNPG (0.63 -2.0 mg/ml) was added to the two groups of the test tubes. The reaction mixture was kept at 25°C for 10 min. 1000 μl of Na2CO3 was added to terminate the reaction. The liberated reducing sugars was measured spectrophotometrically, the amount determined using a paranitrophenol standard curve and converted to reaction velocities. Lineweaver-Burk plot was plotted. The mechanism of inhibition of the plant extract on α-glucosidase by the extract was determined by analyzing the Lineweaver-Burk plot.

Animal experiment
Thirty six (36) albino Wistar rats were used for the study. The animals were kept in a well-ventilated part of the animal house of the department of Applied Biochemistry of Enugu State University of Science and Technology under normal laboratory conditions of temperature and humidity in a 12 h light and 12 h dark cycle. They were allowed to acclimatize for a week and were fed with standard feed and distilled water ad libitum. At the end of the one week, the animals were weighed and their initial blood glucose were recorded using fine test glucometer.

Plant material extraction
The ground sample (1 kg) was soaked and extracted in 10 L of 70% ethanol for 48 h. It was first filtered using three layer muslin cloth and then with Whatman No. 4 filter paper with the aid of a vacuum pump. The filtrate was concentrated in a water bath at 60°C, redissolved at a concentration of 600 mg/ml and stored in refrigerator (4°C) until further use.

Study design
Six animals were randomly selected and placed separately as the normal control group (A). The remaining 30 animals were administered intraperitoneally with 120 mg/kg body weight of (Absorbance Control -Absorbance Test) % Inhibition = × 100 (Absorbance Control) alloxan dissolved in ice-cold normal saline. Forty-eight (48) hours after alloxan administration, fasting blood glucose levels were measured using Finetest glucometer to confirm diabetes and recorded as post-induction blood glucose level. Rats with a fasting blood glucose concentration greater than 200 mg/dl were considered diabetic and selected for the experiment. The diabetic animals were randomly placed into five groups (B-F) of six (6) animals each: Group A: The normal control group and was given the standard feed and distilled water only; Group B: This group received 200 mg/kg of V. doniana ethanol leaf extract orally, the standard feed and distilled water; Group C: This group received 400 mg/kg of V. doniana ethanol leaf extract orally, the standard feed and distilled water; Group D: This group received 600 mg/kg of V. doniana leaf extract orally, the standard feed and distilled water; Group E: This group received 5 mg/kg of glibenclamide orally, the standard feed and distilled water; Group F: This group was the positive control, they were diabetic (untreated) and were given standard feed and distilled water.
The treatment was given daily for 14 days and the blood glucose level of the animals in each group was taken daily by drawing blood from the tip of their tail and measured using Finetest glucometer for 14 days. Their weight was also measured on weekly basis using electronic balance.

Determination of body weight
The weights of the rats were measured before and after induction of diabetes and subsequently during treatment on weekly basis using an electronic weighing balance.

Statistical analysis
Graphpad prism version 7 was used for the kinetic data analysis while SPSS version 7 was used for other statistical analyses. Values were expressed as mean ± SD. Statistical significance of the results between groups was determined using multiple range test and one way analysis of variance (ANOVA) followed by LSD post hoc test. Differences between means were considered significant at P<0.05.

α-Amylase inhibition assay
The effect of the plant extracts of V. doniana on the activities of α-amylase are shown in Table 1. All the extracts significantly (p<0.05) inhibited α-amylase. The percentage inhibition increased with increase in concentration. Thus, while the standard acarbose showed an inhibition of 72.45%, the aqueous extract (VDAQ) showed 79.52% inhibition of the enzyme at the highest concentration tested. The inhibition effectiveness of the extracts on α-amylase was compared using IC 50 values. Aqueous extract of V. doniana (VDAQ) with IC 50 value of 3.09 mg/ml gave the highest inhibition followed by the ethanol extract (VDE), with an IC 50 value of 3.67 mg/ml while the methanol extract (VDM) was the least Ani et al. 75 (IC 50 = 4.02 mg/ml). Acarbose inhibited the activity with an IC 50 value of 4.07 mg/ml (Table 1).

α-Amylase inhibition kinetic studies
The mechanism of inhibition of α-amylase by the aqueous extract of V. doniana was assessed using Lineweaver-Burk plot (double reciprocal). It displayed patterns of mixed inhibition of the enzyme activity as shown in Figure 1. The Km was increased from 0.85 mg/ml without the extract to 1.2 mg/ml with the extract. The value of Vmax was reduced from 4.4 x 10 -1 1 to 4.0 x 10 -1 mg/ml.min -1 with and without the extract respectively.

α-Glucosidase inhibition assay
All the extracts exerted less than 50% inhibition of αglucosidase activity. The inhibition was found to increase with an increase in the concentration and it reached saturation after 8 mg. IC 50 values were used to compare inhibition effectiveness of the plant extracts (Table 2). However, at 8 mg/ml, the ethanol extract (VDE) was the most potent and exhibited a maximum of 24.63% inhibition (IC 50 of 17.12 mg/ml). This was followed by the aqueous extract (VDAQ), with an IC 50 value of 23.30 mg/ml achieving only 19.18% inhibition. The methanol extract (VDM) displayed an IC 50 of 23.33 mg/ml and was the least inhibitor (17.96% inhibition). Acarbose inhibited α-glucosidase with an IC 50 value of 9.0 mg/ml exhibiting 40% inhibition.

Kinetics of α-glucosidase inhibition
The mechanism of inhibition by the most active plant extract against α-glucosidase (VDE) displayed patterns of mixed inhibition as shown in Figure 2. The Km was increased from 1.9 x 10 -2 mg/ml in the absence of extract to 6.2 x 10 -2 mg/ml in the presence of extract while the value of Vmax was reduced from 2.72 to 2.39 mg/ml.min -1 with and without the extract respectively.

Weights of the animals
The weights of the animals recorded before the treatment, 48 h post-induction of diabetes and on weekly basis following treatment are presented in Table 3. Alloxan caused a reduction in the weight of the diabetic animals as seen 48 h post-induction. There was a significant improvement (p<0.05) in the weights of the animals following treatment with the ethanol leaf extract  Values are presented as means ± SD of the tests in triplicate. a Significant reduction in comparison with other groups. b Significant increase in comparison with Acarbose; c Significnat increase in comparison with all extract groups.
of V. doniana and maximum improvement was observed in the second week of the experiment compared to the weight in the first week and at the commencement of the experiment. The weight of the untreated diabetic group reduced when compared with the extract treated groups and their initial weight.

Antidiabetic activity
The in-vivo antidiabetic study was done using the ethanol leaf extract of V. doniana. The effects of treatment with all ethanol extract doses and glibenclamide on the blood glucose level of the diabetic rats in comparison with the normal control and diabetic control are presented in Table 4. The blood glucose levels were reduced considerably within 14 days of daily treatment. There was dose-dependent reduction in the blood glucose level of the treated groups. The diabetic groups treated with ethanol leaf extract of V. doniana showed significant (p<0.05) reduction in blood glucose levels when compared with the untreated group at all the doses tested. There was significant difference between the groups treated with the ethanol leaf extract of V. doniana and that treated with glibenclamide, a standard antidiabetic drug. The standard drug showed a better glucose lowering ability compared to the extract.

DISCUSSION
High blood sugar is the main characteristic feature of type 2 diabetes mellitus, thus, reduction of postprandial hyperglycemia is a good therapeutic approach in  Values are presented as means ± SD of the tests in triplicate. a Significant increase in comparison with the extract groups; b Significant decrease in comparison with other groups; c Significant increase in comparison with other extract groups.
treatment of type 2 diabetes (Chipiti et al., 2017). The leaf extracts of V. doniana showed potent inhibition of αamylase activity with a significant (p<0.05) increase in inhibition activity as the concentration increases. The aqueous extract was the most potent inhibitor of αamylase. The inhibitory activity increased significantly when compared with the standard. This might be one of the possible reasons for the reported traditional use of the plant in diabetes and their hypoglycemic activity. It might also be as a result of several phytochemicals such as flavonoids, tannins and saponins as reported (Sama et al., 2012;McEwan et al., 2010). Furthermore, the mechanism of inhibition of α-amylase by the aqueous extract of V. doniana was determined using Lineweaver-Burk plot. A pattern of mixed inhibition of the enzyme activity was displayed as shown in Figure 1. The Km was increased from 0.85 mg/ml with the extract to 1.2 mg/ml without the extract while the value of Vmax was reduced from 4.4 x 10 -1 to 4.0 x 10 -1 mg/ml.min -1 with and without the extract respectively. A mixed inhibitor prevents binding of the enzyme to its substrate thereby reducing efficiency. It is so because binding of the inhibitor at sites other than on the active site interferes with binding of the enzymes and subsequently, the rate at which products are formed. Mixed inhibition can lead to either a reduction in the affinity of the enzyme for the substrate (Km value  Values are presented as means ± SD of the tests in triplicate. a Significant increase in comparison with the extract groups; b Significant decrease in comparison with other groups; c Significant increase in comparison with other extract groups. Values are presented as mean ± SD. a Significant reduction in comparison with untreated diabetic group; b Significant increase in comparison with the normal control group; C significant increase in comparison with the treated groups; d Significant reduction in comparison with the extract treated groups. increases); that is where the free enzyme binds favorably to the inhibitor or to an increase in the affinity of the enzyme for the substrate (Km value decreases) if the inhibitor binds favorably to the enzyme-substrate complex. In any case, the apparent maximum rate of the enzyme reaction is reduced by the inhibition (Storey, 2004). In this study, the Vmax decreased with an increase in Km. This result shows that the inhibitory component of the plant extract do not bind on the active site of the enzyme competitively with the substrate rather the inhibitors bind to other site on the enzyme delaying the breakdown of carbohydrate (Mogale et al., 2001). A good strategy used in the treatment of type 2 diabetes is delaying glucose production and uptake (Picot et al., 2014).
Alpha-glucosidase inhibitors slow down hydrolysis of carbohydrate and reduce the postprandial glucose uptake in the diabetic (Kwon et al., 2007). With the low αglucosidase inhibition by V. doniana, it suggests that the plant is a mild α-glucosidase inhibitor but can still be used in treatment of postprandial hyperglycemia albeit not effectively. The ethanol extract of V. doniana was the strongest inhibitor of α-glucosidase. The inhibitory activity decreased significantly (p<0.05) in comparison with the standard (Acarbose). Acarbose, inhibited α-glucosidase with an IC 50 of 9.0 mg/ml and exhibited 40% inhibition. This finding is in line with other reports where acarbose exerted little inhibition on α-glucosidase (Shai et al., 2010). Furthermore, to ascertain the mechanism of inhibition of V. doniana on α-glucosidase, the ethanol leaf extract displayed mixed competitive inhibition. The Km was increased from 1.9 x 10 -2 mg/ml in the absence of extract to 6.2 x 10 -2 mg/ml in the presence of extract while the value of Vmax was reduced from 2.72 mg/ml.min -1 to 2.39 mg/ml.min -1 with and without the extract respectively. This also suggests that the inhibitor do not competitively bind on the active site of the enzyme with the substrate but binds to other site on the enzyme delaying turnover.
From the animal experiment, induction of alloxan produced loss of body weight in all the diabetic groups as seen 48 h post induction when compared to the normal control in Table 3. Following treatment with the ethanol leaf extract of V. doniana, there was significant improvement in body weight of the animals compared to the untreated diabetic control.
The result of the antidiabetic study showed that there was a significant (p<0.05) increase in the blood glucose levels of all the animals following administration of alloxan compared to the normal control as seen in Table  4. However, following treatment with ethanol leaf extract of V. doniana, there was significant reduction (p<0.05) in the fasting blood glucose level of the diabetic animals (groups B-D) compared to the untreated diabetic group (F). The blood glucose level was reduced significantly within 14 days of daily extract administration. The potent anti-diabetic effect of the ethanol leaf extract of V. doniana suggests that the plant extract contains potent antidiabetic active components, which produced antihyperglycemic effect in the diabetic rats. The reduction in the blood glucose level also led to increase in the body weight of the animals.
Therefore, leaf extracts of V. doniana have antidiabetic activity and the mechanism through which it exerts its antidiabetic effect is through the inhibition of the two carbohydrate metabolizing enzymes; α-amylase and αglucosidase.

Conclusion
Enzyme inhibitors disrupt biochemical processes and activities of enzymes and are thus produced as drugs.
Poisons are natural inhibitors that protect plants and animals from predators. V. doniana leaves extracts have displayed effective inhibition of α-amylase and a moderate inhibition of α-glucosidase. From the in-vivo study, the ethanol leaf extract has also confirmed that the plant has antidiabetic ability. This study has proven that one of the mechanisms by which V. doniana plant exerts its antidiabetic activity is through the inhibition of these two carbohydrate metabolizing enzymes. Diabetes is known as one of the world's greatest health problems and has become a serious medical concern worldwide. This prompts efforts in exploring for new antidiabetic agents. In adopting an alternative treatment approach to curb the Ani et al. 79 problems of drug resistance and its associated side effects, the leaf of V. doniana may be exploited as a good treatment option.