In vitro Studies on the Inhibition of α -Amylase and α Glucosidase by Leaf Extracts of Picralima nitida (Stapf)

Purpose: To investigate the anti-diabetic potential of Picralima nitida leaf extracts in vitro. Methods: The current study evaluated the anti-diabetic potential of Picralima nitida leaf via in vitro inhibition of α -amylase and α -glucosidase using the acetone, water and ethanol extracts. Preliminary phytochemical analysis was performed on the acetone, aqueous and ethanol extracts of Picralima nitida leaf. The α -amylase inhibitory potentials of the extracts were investigated by reacting different concentrations of the extracts with α -amylase and starch solution while α -glucosidase inhibition was determined by pre-incubating α -glucosidase with different concentrations of the extracts followed by the addition of p-nitrophenylglucopyranoside (pNPG). The mode(s) of inhibition of both enzymes was determined using Lineweaver-Burke plot. Results: The acetone extract of Picralima nitida displayed the most effective inhibition of both α amylase and α -glucosidase activities with half-maximal inhibitory concentration (IC 50 ) of 6.50 and 3.00 mg/ml, respectively. The results also showed that the extracts contain both non-competitive and competitive inhibitors of α -amylase and α -glucosidase respectively. The results of phytochemical analysis of the extract revealed the presence of saponins, flavonoids, tannins, terpenoids and reducing sugars. Conclusion: The observed inhibitions of α -amylase and α -glucosidase suggest that the leaf extracts of P. nitida may be useful in the management of diabetes mellitus. This buttresses the traditional use of P. nitida for the management of sugar-related disorders in some parts of Nigeria.


INTRODUCTION
Diabetes mellitus is a complex disease characterized by gross derangement in carbohydrate, fat and protein metabolism due to deficiency in insulin secretion and/or action [1].Mammalian α-amylase is a prominent enzyme in the pancreatic juice which breaks down large and insoluble starch molecules into absorbable molecules ultimately maltose [2].α-glucosidase, on the other hand, anchored in the mucosal brush border of the small intestine catalyzes the end step of digestion of starch and disaccharides that are abundant in human diet [3].Inhibitors of α-amylase and α-glucosidase delay the breakdown of carbohydrate in the small intestine and decrease the postprandial blood glucose excursion levels in diabetic patients [4].The inhibition of these two prominent enzymes has been found as a useful and effective strategy to lower the levels of postprandial hyperglycemia [6].The incidence of diabetes mellitus in the world is increasing at an alarming rate, affecting close to 5 % of its population [5].
Although several studies have been conducted on the biological and pharmacological potentials of Picralima nitida, there is no report on its effect on α-amylase and α-glucosidase activities in vitro Yet the species is used in the traditional management of sugar-related disorders in Nigeria.The objective of this study, therefore, was to evaluate the α-amylase and αglucosidase inhibitory potentials of Picralima nitida leaf extracts

Plant material
The leaves of Picralima nitida were obtained from Ikoga-Zebbe village, Badagry Local Government Area of Lagos State, Nigeria in May 2012, and authenticated by Dr AB Kadiri of the Department of Botany, University of Lagos, Nigeria.A voucher specimen (LUH 4725) was deposited in the University herbarium of the Department of Botany, University of Lagos.

Chemicals and reagents
α-Amylase from Aspergillus oryzae, αglucosidase from Saccharomyces cerevisiae and para-nitrophenyl-glucopyranoside were products of Sigma-Adrich Co, St Louis, USA while soluble starch (extra pure) was obtained from JT Baker Inc, Phillipsburg, USA.Other chemicals and reagents used were of analytical grade while the water used was glass-distilled.

Preparation of plant extracts
Fresh leaves of Picralima nitida were cut and washed under running tap to remove all foliar contaminants, dried at room temperature and pulverized to fine powder.The powdered leaves were divided into three portions of 50 g each and extracted with 500 ml each of acetone, ethanol or water and kept on orbital Labcon Platform shaker (Laboratory Consumables, PTY, Durban, South Africa) in covered 1000 ml flasks for 24 h at room temperature.The resulting infusions in each case was decanted, filtered and evaporated to dryness in a rotary evaporator (Cole Parmer SB 1100, Shangai, China) in respect of the organic solvent extracts.For the water extract, it was freezedried using Virtis Bench Top freeze dryer (SP Scientific Series, USA).The yield was 3.99, 6.62 and 2.41 g for acetone, ethanol and water extracts, respectively.The dried crude extract was dissolved in 10 % dimethylsulphoxide (DMSO) to yield a stock solution from which lower concentrations were prepared.

Phytochemical screening
The phytochemical composition of each extract was determined using methods variously described by Trease and Evans [13] and Sofowara [14].

Test for tannins
In the test for tannins, 0.5 g of dried powdered sample was boiled in 20 ml of water in a test tube and filtered.A few drops of 0.1 % ferric chloride were added and observed for brownish green or a blue black colouration.

Test for saponin
Approximately 2 g of powdered material was boiled in 20 ml of distilled water in a water bath and filtered.Next, 10 ml of the filtrate was mixed with 5 ml of distilled water and shaken vigorously and observed for a stable persistent froth.The frothing was mixed with 3 drops of olive oil and shaken vigorously again and then observed for the formation of emulsion.

Test for flavonoids
A portion of the powdered material was heated with 10 ml of ethyl acetate over a steam bath for 3 min.The mixture was filtered and 4 ml of the filtrate was shaken with 1 ml of dilute ammonia solution.Development of yellow colouration is an indication of the presence of flavonoids.

Test for steroids
In this test, 2 ml of acetic anhydride was added to 0.5 g of extract with 2 ml concentrated H 2 SO 4 .The colour change from violet to blue or green is indication of steroids.

Test for terpenoids
In brief, 5 ml of extract (0.5 mg/ml) was mixed with 2 ml chloroform and 3 ml concentrated H 2 SO 4 was carefully added to form a layer.A reddish brown colouration of the interface was indication of terpenoids.

Test for anthraquinones
5 ml of chloroform was added to 0.5 g of the extracts of each specimen.The resulting mixture was shaken for 5 min after which it was filtered.The filtrate was then shaken with equal volume of 10 % ammonia solution.The presence of a bright pink colour in the aqueous layer indicated the presence of anthraquinones.

Test for reducing sugar
To about 1 g of each sample in the test tube was added 10 ml distilled water and the mixture boiled for 5 min.The mixture was filtered while hot and the cooled filtrate made alkaline to litmus paper with 20 % sodium hydroxide solution.The resulting solution was boiled with an equal volume of Benedict qualitative solution on a water bath.The formation of a brick red precipitate depicted the presence of reducing compound.

α-Amylase inhibitory assay
This assay was carried out using a modified procedure of McCue and Shetty [15].A total of 250 µL of extract (1.25 -10 mg/ml) was placed in a tube and 250 µL of 0.02 M sodium phosphate buffer (pH 6.9) containing α-amylase solution (0.5 mg/ml) was added.The content of the tubes were pre-incubated at 25 o C for 10 mins, after which 250 µL of 1 % starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added at timed intervals.The reaction mixtures were incubated at 25 °C for 10 min.The reaction was terminated by adding 500 µL of dinitrosalicylic acid (DNS) reagent and further incubated in boiling water for 5 min and cooled to room temperature.The content of each test tube was diluted with 5 ml distilled water and the absorbance measured at 540 nm in a spectrophotometer (Spectrumlab S23A, Globe Medical, England).A control was prepared using the same procedure except that the extract was replaced with distilled water.The α-amylase inhibitory activity was calculated as in Eq 1.
The concentration of extract resulting in 50 % inhibition of enzyme activity (IC 50 ) was determined graphically using Microsoft Excel 2007.

Mode of α-amylase inhibition
The mode of inhibition of the leaf extract was conducted using the extract with the lowest IC 50 according to the modified method described by Ali et al., [16].Briefly, 250 µL of the (5 mg/mL) extract was pre-incubated with 250 µL of αamylase solution for 10 mins at 25 ºC in one set of tubes.In another set of tubes, 250 µL of phosphate buffer (pH 6.9) was also preincubated with 250 µL of α-amylase solution.Starch solution (250 µL) of increasing concentrations (0.30 -5.0 mg/mL) was added to both sets of reaction mixtures to initiate the reaction.The mixture was then incubated for 10 min at 25 °C, and then boiled for 5 min after addition of 500 µL of DNS to stop the reaction.The amount of reducing sugars released was determined spectrophotometrically using a maltose standard curve and converted to reaction velocities.A double reciprocal plot (1/v versus 1/[S]) where v is reaction velocity and [S] is substrate concentration was plotted.The type (mode) of inhibition of the crude extract on αamylase activity was determined by analysis of the double reciprocal (Lineweaver-Burk) plot using Michaelis-Menten kinetics.

α-Glucosidase inhibitory assay
The effect of the plant extracts on α-glucosidase activity was determined according to the method described by Kim et al., [17], using α-glucosidase from Saccharomyces cerevisiae.The substrate solution p-nitropheynyl glucopyranoside (pNPG) (3.0 mM) was prepared in 20 mM phosphate buffer, pH 6.9. 100 µL of α-glucosidase (1.0 U/ml) was pre-incubated with 50 µL of the different concentrations of the extracts (acetone, ethanol and water) for 10 mins.Then 50 µL of 3.0 mM (pNPG) as a substrate dissolved in 20 mM phosphate buffer (pH 6.9) was added to start the reaction.The reaction mixture was incubated at 37 o C for 20 mins and stopped by adding 2 ml of 0.1 M Na 2 CO 3 .The α-glucosidase activity was determined by measuring the yellow colored para-nitrophenol released from pNPG at 405 nm.The results (% Inhibition) are expressed as percentage of the blank (control) as in Eq 2. % Inhibition = {(Ac -Ae)/Ac}100 …………… (2) where Ac and Ae are the absorbance of the control and extract, respectively.
The concentration of extract resulting in 50 % inhibition of enzyme activity (IC 50 ) was determined graphically using Microsoft Excel.

Mode of α-glucosidase inhibition
The mode of inhibition of the extracts was determined using the extract with the lowest IC 50 according to the modified method described by Ali et al [16].Briefly, 50 µL of the (5 mg/mL) extract was pre-incubated with 100 µL of αglucosidase solution for 10 mins at 25 ºC in one set of tubes.In another set of tubes αglucosidase was pre-incubated with 50 µL of phosphate buffer (pH 6.9).50 µL of PNPG at increasing concentrations (0.63 -2.0 mg/mL) was added to both sets of reaction mixtures to start the reaction.The mixtures were then incubated for 10 mins at 25 °C, and 500 µL of Na 2 CO 3 was added to stop the reaction.The amount of reducing sugars released was determined spectrophotometrically using a paranitrophenol standard curve and converted to reaction velocities.A double reciprocal plot (1/v versus 1/[S]) where v is reaction velocity and [S] is substrate concentration was plotted.The type (mode) of inhibition of the crude extract on αglucosidase activity was determined by analysis of the double reciprocal (Lineweaver-Burk) plot using Michaelis-Menten kinetics.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 5 statistical package (GraphPad Software, USA).The data were analysed by oneway analysis of variance (ANOVA) followed by Bonferroni test.All the results were expressed as mean ± SE of triplicate determinations.The level of significance was taken at 5 % confidence interval (p < 0.05)

RESULTS
The results obtained from the phytochemical screening conducted on the P. nitida leaf extracts are presented in Table 1.Anthraquinone was absent in all the extracts while reducing sugar was common to all.The acetone extract contains only saponin and terpenoids, whereas the ethanol extract contains flavonoids, steroids and terpenoids.
The inhibitory potentials of different extracts of P. nitida on α-amylase activity are presented on Figure 1.At lower concentrations (1.25 -2.5 mg/ml), there were no significant differences (p > 0.05) among the values generated for all the extracts.However, there was significant difference (p < 0.05) between the percentage inhibition of acetone and ethanolic extract at concentration of 5.00 mg/ml.
When the concentration of the extract was increased to 10 mg/ml, acetone extract exhibited very high inhibitory activity (88.39%) against α-amylase which is significantly different (p < 0.05) when compared with ethanol and aqueous extract.The α-amylase inhibition is dose-dependent but the acetone extract displayed most effective inhibition against α-amylase with an IC 50 value of 6.50 mg/ml (Table 2).The mode of inhibition of α-amylase by acetone extract of P. nitida leaf was determined by Lineweaver-Burke plot which showed that the mode of inhibition of this enzyme by the extract is non-competitive (Fig. 2).Similar to α-amylase inhibition, there was no significant difference in α-glucosidase inhibition at lower concentrations of the extracts and the results.The inhibitions of α-glucosidase by all the plant extracts at lower concentrations (0.63 -1.25 mg/mL) were not significantly different (p > 0.05) from one another.At 2.50 mg/mL, the percentage inhibition of acetone and aqueous extract were significantly different (p < 0.05) when compared to ethanolic extract.The acetone extract displayed the highest inhibition (67.3 %) at the highest concentration (5.0 mg/mL) while it was between 40.0 and 45.0 % for aqueous and ethanol extracts, respectively.Further investigation into the mode of αglucosidase inhibition by the plant extracts provided a characteristic inhibition profile in which acetone extract was the most potent αglucosidase inhibitor with an IC 50 value of 3.00 mg/ml (Table 2).The mode of inhibition of αglucosidase by this acetone extract of P. nitida leaf was determined using Lineweaver-Burke plot and the plot displayed near competitive inhibition of the enzyme activity.

DISCUSSION
Diabetes mellitus is a common metabolic disorder which may eventually lead to multiple organ damage and syndromes [18].Elevated postprandial glucose level increases the risk of cardiovascular disease which is the most common cause of death among the people with diabetes.Postprandial spikes can also result into micro-vascular damage through oxidation of low density lipoprotein (LDL) and pro-atherogenic mechanism [19].Management of the blood glucose level is a critical strategy in the control of diabetes and its complications.
Diet rich in carbohydrate causes sharp rise in the blood glucose level as the complex carbohydrate in food is rapidly absorbed in the intestine aided by the α-amylase and α-glucosidase enzyme which break carbohydrate to simple absorbable sugars (monosaccharides) [19].Inhibitors of saccharide hydrolyzing enzymes (α-amylase and α-glucosidase) have been useful as oral hypoglycemic drugs for the control of hyperglycemia especially in patients with Type II diabetes mellitus [20].These inhibitors delay carbohydrate digestion and prolong overall carbohydrate digestion time causing reduction in the rate of glucose absorption and consequently reducing the post prandial plasma glucose rise [21].amylase and α-glucosidase inhibitors from plants with minimal or no side effects [22].
In the present study, the acetone extract showed the most potent inhibition of α-amylase activity at all concentrations tested.However, at high concentration (10.00 mg/ml), the inhibition was strong which is undesirable of α-amylase inhibitor.This was also attested to by the high IC 50 for the α-amylase inhibition.In order for hypoglycemic agents to elicit their pharmacological effects and prevent the side effects experienced with the use of synthetic drugs, medicinal plants should be mild inhibitors of α-amylase [19].To determine the mode of inhibition of this enzyme, the double reciprocal plot indicated a non-competitive inhibition of αamylase by P. nitida extracts.This suggests that the active component of the extract binds to a site other than the active site of the enzyme and combine with either free enzyme or enzymesubstrate complex possibly interfering with the action of both [23].
The in vitro α-glucosidase inhibitory results demonstrated that all the extracts of P. nitida leaf possess α-glucosidase inhibitory activities.However, acetone extract exhibited the strongest inhibition of the enzyme as attested to by the lowest IC 50 generated.This is in agreement with previous reports that phytochemicals from plants are strong inhibitors of α-glucosidase [19].The curve generated from the Lineweaver-Burke plot of this inhibition showed a near competitive mode of inhibition of the α-glucosidase.This implies the active components of the extract compete with the substrate for binding to the active site of the enzyme, thereby preventing or slowing down the breakdown of oligosaccharides to disaccharides [22].The phytochemical screening revealed the presence of saponins and terpenoids.The inhibitory effect of the extracts of P. nitida may not be unconnected with the presence of saponins and terpenoids in it.

CONCLUSION
Although the acetone extract exhibited the most potent α-amylase and α-glucosidase inhibitory activity which is an indication that the solvent is capable of extracting the active constituents in P. nitida, the use of infusion and decoction is to be encouraged for a mild to moderate activity since a higher dosage of the alcohol extract can lead to undesirable α-amylase and α-glucosidase inhibitory effects in humans.The inhibitory effect of these extracts on both enzymes may be linked to the presence of saponins and terpenoids which may be acting individually or in synergy.This study further validates the folkloric use of P. nitida in the management of sugar related disorders in Nigeria.

Fig 2 :
Fig 2: α-Glucosidase inhibitory activity of Picralima nitida leaf extracts (a), and mode of inhibition of αglucosidase by acetone extract of Picralima nitida leaf (b)