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Adesegun S. A., et al., /Journal of Natural Products, Vol. 6(2013): 90-95 α-amylase inhibition and antioxidant activity of Pterocarpus osun Craib
1*Adesegun S. A., 1Fayemiwo, O., 1Odufuye, B. 2Coker, H.A.B.
1Department of Pharmacognosy, 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Lagos, PMB 12003, Lagos, Nigeria (Received 08 December 2012; Revised 13 Dec. 2012-05 February 2013; Accepted 08 February 2013) ABSTRACT
In this study, methanol leaf extract of Pterocarpus osun and its fractions were
assessed for anti-diabetic ability using in vitro α-amylase inhibitory potential and
antioxidant activities determined by DPPH radical scavenging effect, reducing power
and total phenolic content. Our results showed that the extract had a dose dependent
prevention of digestion of carbohydrates by inhibiting α-amylase. The maximum
effect was observed at 2.0mg/ml was (74.98±4.35%, P<0.05). At 0.2mg/ml, the
aqueous fraction demonstrated 71.87±3.41% inhibition which is comparable to
88.10±4.59% of acarbose used as reference drug. The hexane and chloroform
fractions demonstrated lower (P<0.05) inhibitory activity; 16.16 and 14.34%
respectively. The total phenolic content of the P. osun extract measured using Folin
Ciocalteu reagent in terms of gallic acid equivalent (GAE) was found to be
97.01±0.02mg/g. The DPPH radical scavenging activity of the extract increased with
concentration but was significantly less than (P<0.05) ascorbic acid used as positive
control. The radical scavenging activity of the aqueous fraction (94.60±3.45%) at
0.2mg/ml was however higher than that of reference drug. The hexane and chloroform
fractions however showed lower radical scavenging activity; 12.35±0.07 and
46.20±1.23% respectively. The extract showed dose-dependent increase in reducing
power. The aqueous fraction however showed highest reducing ability while the n-
hexane fraction had lowest. This study indicates that α-amylase inhibitory and
antioxidant activities of the methanol extract of P. osun resides in the aqueous
fraction.
Keywords: Pterocarpus osun; α-amylase inhibiton; Antioxidant activity; Antidiabetes.
INTRODUCTION
Diabetes mellitus (DM) is fast becoming leading cause of mortality in the developed and developing world. It is often due to excessive calorie intake, hyperglycemia and oxidative stress. One of steps for managing DM is the management of postprandial hyperglycemia by reducing the digestion of carbohydrates by α-glucosidases (Fowler, 2007). Medicinal plants have been reported to contribute to the management of type 2 diabetes by inhibiting carbohydrate hydrolyzing enzymes like pancreatic amylase. This delays carbohydrate digestion and prolong carbohydrate digestion time thus reducing glucose absorption rate and postprandial plasma glucose level (Jung, et al., Copyright 2013, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved 90 Adesegun S. A., et al., /Journal of Natural Products, Vol. 6(2013): 90-95 2006; Kwon, et al., 2006). Oxidative stress and free radicals have also been implicated in type 2 diabetes (Droge, 2002). In Nigeria, the sap of the bark of Pterocarpus osun (Leguminosae) is used as dye and the wood is used in carpentry (Burkill, 1995). Antidiabetic properties of other species like P. indicus and P. marsupium had been established (Joshi, et al., 2004; Ragasa, et al., 2005) however there were very little reports on the activities of the leaf of P. osun. In the present study, we report for the first time antioxidant and anti-amylase activities of the plant. MATERIALS AND METHODS
Plant Material: The fresh leaves of P. osun were collected at Ikire, Oyo State, Nigeria
in May 2010 and identified by Mr. I. K. Odewo of the Herbarium Unit, Department of
Botany, University of Lagos after comparing with voucher specimen LUH 3153. The
leaves were dried at 40°C in an oven and milled to produce fine powder.
Extraction and Fractionation: Previous studies on another species (Joshi, et al.,
2004) and preliminary studies in our laboratory showed methanol extract to be
effective. About 400g of the powdered plant material was extracted in May 2010 with
methanol (2.5L) using Soxhlet apparatus for 48h. The extract was concentrated under
reduced pressure using rotatory evaporator at 40°C until a semi-solid sticky mass was
obtained. The yield was 7.64%w/w. Extract (20g) was dissolved in a mixture of
methanol and water then partitioned between n-hexane and chloroform successively
to obtain hexane, chloroform and aqueous fractions that were concentrated and
subjected to in- vitro α-amylase inhibitory and antioxidant investigations.
Amylase assay: The amylase inhibition assay was carried out by a method previously
described (Kwon, et al., 2006). A total of 500µl of extract (0.05-2.0mg/ml) and 500µl
of 0.02M sodium phosphate buffer (pH 6.9 with 0.006M sodium chloride) containing
porcine pancreatic α-amylase (0.5mg/ml; Sigma Chemical Company, St. Louis, MO)
were mixed at 32°C for 10min, then 500µl of 1% starch solution in 0.02M sodium
phosphate buffer (PH 6.9 with 0.006M sodium chloride) was added to each tube at
timed intervals. The reaction mixtures were incubated at 32°C for 10min. The
reaction was stopped with 1.0ml of dinitrosalicylic acid (DNS) color reagent. The test
tubes were then incubated in a boiling water bath for 5min and then cooled to room
temperature. The reaction mixture was diluted with 10ml distilled water and
absorbance was measured at 540nm. The α-amylase inhibitory activities of the
hexane, chloroform and aqueous fractions were tested at 0.2mg/ml concentration
using similar procedure. The inhibition of α-amylase was calculated as follows:
% inhibition= Acontrol – Aextract X 100 Determination of Total Phenolic Content: The total phenolic content of the extract
was determined in terms of gallic acid equivalent using Folin-Ciocalteu reagent
(Adesegun, et al., 2009). The method depends on the transfer of electrons in alkaline
medium from phenolic compounds to phosphomolybdic/phosphotungstic acid
complexes. Gallic acid equivalent was determined from a calibration concentration
curve. The extract (100mg/ml, 1.0ml) was mixed thoroughly with 5ml Folin-
Ciocalteu reagent (1:9 in distilled water) and after 5min, 4.0ml of sodium carbonate
(0.7M) was added and the mixture was allowed to stand for 1h with intermittent
shaking. The absorbance was measured at 765nm in a spectrophotometer. All
determinations were carried out in triplicate.
Copyright 2013, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved 91 Adesegun S. A., et al., /Journal of Natural Products, Vol. 6(2013): 90-95 Determination of Radical Scavenging Activity: The method of Koleva, et al., (2002)
with some modifications was used for the determination of free radical scavenging
activity of the extract. The extract (1.0ml, 0.1-5.0mg/ml) was diluted to 20% of the
original concentration with methanol and 1.0ml of methanolic solution of 1,1-
diphenyl-2-picrylhydrazyl (DPPH) (1mM) was added. The mixture was shaken
vigorously and allowed to stand at 32°C in dark for 30min. The decrease in
absorbance at 517nm was then measured using spectrophotometer. The radical
scavenging ability of the hexane, chloroform and aqueous fractions were tested at
0.2mg/ml using similar procedure. Ascorbic acid was used as reference standard and
deionized water in place of extract or the control was used as blank. All analysis was
performed in triplicate and the ability to scavenge the DPPH radical was calculated
using the following equation:
Scavenging Effect (%) = [(Ao − A1)/Ao] × 100
• Ao was absorbance of the control; A1 was absorbance in the presence of the sample of extract or standard. Evaluation of Reducing Ability: The reducing power of the extract was determined
by the method of Yen and Chen (1995) with some modifications. The ability of the
extract and its fractions to reduce the ferric-ferricyanide complex to the ferrous-
ferricyanate complex of Prussian blue was estimated by recording the absorbance at
700nm after incubation. The extract (1.0ml, 0.1-5.0mg/ml) was mixed with 2.5ml of
phosphate buffer (0.2M, pH 6.6) and 2.5ml of potassium ferricyanide (1%) was added
then the mixture was incubated at 50°C for 20min. A portion (2.5ml) of
trichloroacetic acid (10%) was added to the mixture and centrifuged at 1000g for 10
min. The upper layer of the solution (2.5ml) was mixed with distilled water (2.5ml)
and FeCl3 (0.5ml, 0.1%) and the absorbance was measured at 700nm against a blank
in the spectrophotometer. The reducing power of hexane, chloroform and aqueous
fractions were tested at 0.2mg/ml concentration using similar procedure Ascorbic acid
was used as positive controls. Higher absorbance of the reaction mixture indicated
increased reducing power.
Statistical Analysis: All data were expressed as Mean±Standard error of mean.
Analysis of variance was performed by ANOVA procedures and P<0.05 was
considered to be statistically significant.
Alpha-amylase inhibitory activity: The extract of P. osun possessed concentration
dependent inhibitory effect on the starch breakdown in vitro as shown in Table 1. The
highest inhibitory effect was at 2.0mg/ml and the IC50 was 0.6mg/ml compared to
acarbose 0.04mg/ml used as positive control. The hexane, chloroform and aqueous
fractions of P. osun showed α-amylase inhibitory effects 16.16±0.94, 14.34±0.48 and
71.87±3.41% respectively at 0.2mg/ml.
Total Phenolic content: of the P. osun leaf extract determined using Folin Ciocalteu
reagent in terms of gallic acid equivalent (GAE) was found to be 97.01±0.02mg/g.
DPPH radical scavenging activity: The scavenging activity of the DPPH radical was
tested by reduction of the stable radical DPPH to the yellow colored diphenyl picryl
hydrazine. The scavenging effect of extract and ascorbic acid are depicted in Table 2.
As the concentration increases, the scavenging potential of the crude extract increased
by combating formation of the DPPH free radical. The extract and ascorbic acid
scavenged the radical with IC50; 0.45 and 0.07mg/ml respectively. At 0.2mg/ml, the
free radical scavenging ability of hexane, chloroform and aqueous fractions were
12.35±0.07, 46.20±1.23 and 94.6±3.45% respectively while ascorbic acid was
88.26±3.88%.
Copyright 2013, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved 92 Adesegun S. A., et al., /Journal of Natural Products, Vol. 6(2013): 90-95 Reducing power: The crude extract showed a dose-dependent increase in reducing
power (Table 3). The hexane, chloroform and aqueous fractions of P. osun gave
0.034, 0.234, 0.948 absorbance respectively at 0.2mg/ml. Ascorbic acid had the
highest 0.987. The reducing power thus follows the order ascorbic acid> aqueous>
chloroform > hexane.
DISCUSSION
Medicinal plants are natural sources of antioxidants and bioactive compounds useful to health. Postprandial hyperglycemia is one of the risk factors associated with type 2 diabetes mellitus. Digestion of dietary starch by α-glucosidases like pancreatic α-amylase plays significant role in elevated blood glucose thus inhibition of these enzymes is very useful tool in management of hyperglycemia (Kwon, et al., 2006). The anti-diabetic activity of medicinal plants could be evaluated using several methods; in vitro α-amylase inhibitory assay is one of such techniques. The extract of P. osun inhibited α-amylase but its activity was significantly less than that of positive control acarbose (P<0.05). The inhibitory effect of aqueous fraction was comparable to than that of the positive control, indicating that the α-amylase inhibitory ability resides in this fraction. Alpha-amylase is an enzyme found in the salivary, intestinal mucosal and pancreatic secretions responsible for breaking down of α-1,4-glycosidic bonds in starch. Therefore, the enzyme increases the availability of glucose in the blood. P. osun extract could be useful in post-prandial hyperglyceamia by reducing the hydrolysis of carbohydrates. The observed activity may be due to the presence of chemical constituents such as phenolic compounds (tannins and flavonoids), terpenes and triterpenes in the extract and fractions (Ebi and Ofoefule, 2000; Kim, et al., 2000). Phenolics have been reported to inhibit α-amylase activities. They also have anti-hyperglycemic activity and inhibit the development of diabetes (Hanamura, et al., 2006; Zunino, et al., 2007). Free radicals play a significant role in biological damages and DPPH is commonly used in evaluation free radical scavenging properties of natural antioxidants (Adesegun, et al., 2007). The crude extract of P. osun showed DPPH radical activity but this is significantly less (P<0.05) than that ascorbic acid used as positive control. The scavenging power of the aqueous fraction was significantly higher (P<0.05) than that of ascorbic acid and the crude. But chloroform and hexane fractions showed weaker activity compared to control. This suggested that the plant is natural source of antioxidants. The DPPH radical changes color from purple to yellow after reacting with antioxidants which scavenge the radical by hydrogen donation. The extent of the reaction depends on the hydrogen donating ability of the antioxidant. The extract of P. osun has a high phenolic content which may be responsible for the observed α-amylase inhibitory and antioxidant properties. Plant phenolics constitute one of the major groups of compounds acting as primary antioxidants or free radical terminators due to the ability of their hydroxyl groups to scavenge radicals. This activity is reported to be due to their redox properties which play a significant role in ion adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposition of peroxides (Tsao and Akhtar, 2005). The results also showed P. osun leaf extract as a reducing agent although the activity was lower than ascorbic acid (Table 3). The reducing ability of aqueous fraction was comparable to ascorbic acid. The result suggest that the extract could react with free radicals and convert them to more stable compounds. In the reducing power assay, the antioxidants in the extract cause the reduction by conversion of the Fe3+/ferricyanide complex to the ferrous form. The phenolic compounds present in Copyright 2013, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved 93 Adesegun S. A., et al., /Journal of Natural Products, Vol. 6(2013): 90-95 extract as well as flavonoids and tannins reported in this genus may responsible for reported activity. Antioxidants are known to have reducing power (Duh, et al., 1999). CONCLUSION
This study shows that the leaf extract of Pterocarpus osun possess α-amylase inhibitory activity and antioxidant properties. The fractionation of the extract revealed that the observed activities reside mainly in the aqueous fraction. The extract could be a veritable source of natural antioxidant and antidiabetic agent. Acknowledgements: The authors are grateful to Mr. T. I. Adeleke and Mr. Solomon Julius of
Pharmacognosy Department, University of Lagos, for their technical assistance.
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Adesegun, S.A., Elechi, N.A., Coker, H.A.B., (2007): Antioxidant power of Macaranga
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Concentration
Inhibitory Effect (%)*
Acarbose
• *Each value represents mean ± SEM (n = 3). Table -2: Radical scavenging effect of extract and ascorbic acid.
Concentration
Inhibitory Effect (%)*
Ascorbic acid
• *Each value represents mean ± SEM (n = 3). Table -3: Reducing power of extract and ascorbic acid .
Concentration
Absorbance (nm)
Ascorbic acid
• *Each value represents mean ± SEM (n = 3). Copyright 2013, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved 95

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