Alpha-Glucosidase Inhibitory and Antioxidant Activity of Solvent Extracts and Fractions of Typha domingensis ( Typhaceae ) Fruit

Purpose: To identify a solvent fraction with potent antiglucosidase and antioxidant activities from the fruit of Typha domingensis. Methods: Extracts were prepared using hexane, chloroform, ethyl acetate, acetone (AE), methanol, and water. Antiglucosidase and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities of extracts were assessed. The most active extract was partitioned into chloroform, ethyl acetate, butanol (BF) and water, and the antiglucosidase and radical scavenging activities of the fractions were determined. Mode of inhibition of the strongest antiglucosidase fraction was investigated. Polyphenol, coumarin, proanthocyanidin (TPro), and hydroxycinnamic acid contents of the extracts and fractions were evaluated. Results: AE had the highest antiglucosidase (EC50 = 12.36 μg/mL) and radical scavenging (EC50 = 8.57 μg/mL) activities. Solvent-partitioning of AE resulted in BF, which showed markedly stronger antiglucosidase activity (EC50 = 4.27 μg/mL) than quercetin (EC50 = 22.18 μg/mL). BF also had potent radical scavenging activity (EC50 = 7.20 μg/mL). BF was rich in TPro (735.65 mg/g) and was a competitive glucosidase inhibitor. TPro content correlated with antiglucosidase (R = 0.709) and DPPH scavenging activities (R = 0.838). Conclusion: TPro-rich BF of T. domingensis fruit is a highly potent glucosidase inhibitor and radical scavenger. The findings demonstrate a potential for the development of natural antihyperglycemic agents with antioxidant effect from T. domingensis fruit.


INTRODUCTION
Type 2 diabetes is a chronic metabolic disorder characterized by hyperglycemia.Postprandial hyperglycemia is also a key risk factor for microvascular and macrovascular complications in patients with type 2 diabetes [1].Thus, antihyperglycemic agents that target postprandial hyperglycemia are recommended for optimal diabetes management and reduction of diabetesassociated complications [1].α-Glucosidase is well-recognized as a therapeutic target for the modulation of postprandial hyperglycemia.α-Glucosidase inhibitors are used in therapy to delay or inhibit gastrointestinal digestion of oligosaccharides and the subsequent release of glucose.This consequently delays or reduces glucose absorption into the bloodstream following food uptake [2].Acarbose, miglitol, and voglibose are antiglucosidase drugs that are orally administered for the management of postprandial hyperglycemia.Owing to their undesirable side effects, there is great interest among researchers worldwide to search for alternative glucosidase inhibitors.Minimal side effects and low costs of herbal medicines in the treatment of diabetes have encouraged the search for glucosidase inhibitors of plant origin [2,3].
In this study, we evaluated the potential of medicinal plant Typha domingensis Pers.(Typhaceae) as a source of potent, natural αglucosidase inhibitors.
The fruit and inflorescence of several Typha species are traditionally used in the treatment of wounds and bleeding in different regions of the world [4,5].T. domingensis fruit is not traditionally used as an antidiabetic remedy.However, our preliminary screening has revealed moderate antiglucosidase and antioxidant activities in the crude water extracts of T. domingensis fruit, which were greater in potency compared with the male and female flowers [6].At present, the antiglucosidase and antioxidant capacities of the T. domingensis plant are underexplored in the literature.
Searching for concurrent antiglucosidase and antioxidant properties in the T. domingensis fruit has practical significance.Diabetic complications that are associated with postprandial hyperglycemia are known to be caused by the induction of oxidative stress [1].Hence, dual-function antidiabetic agents with concurrent antiglucosidase and antioxidant properties may provide additional benefits when compared with single-function glucosidase inhibitors.
As an extension to our previous screening work [6], a more detailed investigation was undertaken where bioassay-guided fractionation was performed on T. domingensis fruit.The antiglucosidase and radical scavenging activities of T. domingensis fruit extracts prepared by using extraction solvents differing in polarity were compared in this study.The most potent extract was further solvent-partitioned into different fractions and their antiglucosidase and radical scavenging activities were established.The contents of bioactive phytochemicals (polyphenols, coumarins, proanthocyanidins, and hydroxycinnamic acids) in the solvent extracts and fractions were determined and their correlations with antiglucosidase and radical scavenging activities were analyzed.Enzyme kinetic study was also performed to understand the possible mode of inhibition exerted by the solvent fraction exhibiting the most potent antiglucosidase activity.

EXPERIMENTAL Plant sample
Healthy, mature fruits of Typha domingensis were collected in May 2013 from a population of T. domingensis inhabiting a lakeshore at the Universiti Tunku Abdul Rahman (UTAR) campus.The species of the plant was authenticated by Professor Hean-Chooi Ong, a plant taxonomist at University of Malaya, Malaysia.A herbarium specimen has been stored at UTAR's Faculty of Science for future reference.

Preparation of solvent extracts
Fruits of T. domingensis were cleaned by rinsing in distilled water and then blotted dry with tissue paper.The samples were then oven-dried at 45 °C to constant weight.The dried sample was pulverized using a Waring blender.Solvent extracts were prepared by first mixing the pulverized samples with hexane, chloroform, ethyl acetate, acetone, methanol, or autoclaved deionized water at a 1:10 (dry weight: volume) ratio.Each of the mixtures was incubated at 25 o C for 48 h on an orbital shaker (100 rpm).The mixtures were subsequently vacuum-filtered and the filtrates were centrifuged at 8600 g and 4 °C for 10 min.The supernatants obtained from the hexane extract (HE), chloroform extract (CE), ethyl acetate extract (EAE), acetone extract (AE), methanol extract (ME), and water extract (WE) were collected, whereas the pellets were discarded.The organic solvent extracts were concentrated by rotary evaporation in vacuo and then taken to dryness in an oven at 35 o C; WE was freeze-dried.The solid residues recovered from HE, CE, EAE, AE, and ME were dissolved in dimethyl sulfoxide (DMSO), whereas the solid residue of WE was redissolved in water.Aliquots of 50 mg/mL were prepared and stored at -20 o C until further use.

Determination of antiglucosidase activity
Glucosidase inhibitory activity was determined as previously described [6].Quercetin was used as a reference compound as its effectiveness as a glucosidase inhibitor in vivo and in vitro has been established [7,8].EC 50 , defined as the extract/fraction concentration required to achieving 50 % antiglucosidase activity, was determined by using linear regression analysis.

Determination of 1, 1-diphenyl-2picrylhydrazyl (DPPH) radial scavenging activity
DPPH radical scavenging assay was carried out as previously described [9].Ascorbic acid was used as the positive control.EC 50 , defined as extract/fraction concentration required to achieve 50 % DPPH scavenging activity, was determined by using linear regression analysis.

Antiglucosidase and radical scavenging activities of different fractions
Fractionation of AE was carried out by suspending the solid residue of AE (2 g) in 100 mL of deionized water, followed by sequential partitioning into chloroform, ethyl acetate, and butanol by using a separatory funnel.Chloroform fraction (CF), ethyl acetate fraction (EAF), butanol fraction (BF), and water fraction (WF) were obtained.CF, EAF, and BF were concentrated by rotary evaporation in vacuo and then taken to dryness in an oven at 35 o C; WF was freeze-dried.The yield of CF, EAF, BF, and WF was 20.2, 20.1, 22.8, and 3.4 % w/w, respectively.The solid residues recovered from CF, EAF, and BF were dissolved in DMSO, whereas the solid residue of WF was redissolved in water.Aliquots of 50 mg/mL were prepared and stored at -20 o C until used.Antiglucosidase and DPPH scavenging activities of fractions were determined as described above.Their EC 50 values were also determined by using linear regression analysis.

Phytochemical contents and correlation with bioactivities
Total phenolic (TP), total proanthocyanidin (TPro), and total hydroxycinnamic acid (THA) contents of extracts/fractions were determined as described in [6].The three paramerets were expressed as mg gallic acid equivalents (GAE)/g sample, mg leucocyanidin equivalents (LE)/g sample and mg caffeic acid equivalents (CAE)/g sample, respectively.Total coumarin (TCou) content was determined as described in [10] and expressed as mg coumarin equivalents (CE)/g sample.Correlation analyses were carried out between phytochemical contents of all extracts and fractions and the 1/EC 50 values of antiglucosidase and DPPH scavenging activities.
Coefficient of determination (R 2 ) was determined from the analyses.

Evaluation of mode of α-glucosidase inhibition by butanol fraction
The antiglucosidase assay described above was carried out in the presence or absence of BF (4.27 and 5.34 µg/mL) using 0 -2 mM of the pnitrophenyl α-D-glucopyranoside (p-NPGP) substrate (S).The amount of p-nitrophenol formed during the reactions was determined by using a p-nitrophenol standard curve.Reaction velocity (v) was calculated as the amount of pnitrophenol formed (mM) per reaction duration (min).A Lineweaver-Burk plot was prepared.The mode of inhibition of glucosidase by BF was determined by analyzing the double reciprocal (1/v versus 1/[S]) plot using the Michaelis-Menten kinetics.V max (maximum velocity) and k m (substrate concentration that yields a halfmaximal velocity) were determined from the plot.

Data analysis
All experiments were carried out in triplicates and data are expressed as mean ± standard error of the mean (SEM).Statistical analyses were performed using SAS (Version 9.2).Data were analyzed by one-way ANOVA test and means of significant differences were separated using Fisher's Least Significant Difference (LSD) test or Student's t test at α = 0.05.Linear regression and correlation analyses were carried out using Microsoft Office Excel 2003.

RESULTS
All solvent extracts of T. domingensis fruit, except HE, exhibited concentration-dependent increases in antiglucosidase activity when tested at 20, 40, 60, 80, and 100 µg/mL (data not shown).No antiglucosidase activity was detected in HE.The EC 50 value of antiglucosidase activity of AE (12.36 µg/mL) was the lowest among all the solvent extracts (Table 1).The EC 50 values of AE and ME are 44 % and 37 % lower than that of quercetin, respectively.The EC 50 values of CE and EAE are 162 % and 488 % higher than that of quercetin.The EC 50 value of WE was not statistically different from that of quercetin.
All six extracts of T. domingensis fruit showed an increase in DPPH scavenging activity in a concentration-dependent manner when tested at 20, 40, 60, 80, and 100 µg/mL (data not shown).1).The EC 50 value of AE was 2.4-fold greater than that of ascorbic acid.HE had the highest EC 50 value among the extracts, which is also 260-fold higher than that of ascorbic acid.
Phytochemical analysis revealed that AE, ME, and WE generally contained higher levels of TP, TCou, TPro, and THA contents, relative to HE, CE, and EAE (Table 2).The four classes of phytochemicals were detected in all six solvent extracts, with the exception that THA was not detectable in HE.Among the six extracts, ME had the highest TCou, TPro, and THA contents, whereas AE had the highest TP content.
Among the organic and water fractions partitioned from AE, concentration-dependent increase in antiglucosidase activity was detected when BF, EAF, and WF were tested at 2, 4, 6, 8, 10, 15 and 20 µg/mL (data not shown).EC 50 values of the antiglucosidase activity of organic and water fractions of AE ranged between 4.27 and 28.68 µg/mL (Table 3).BF had the lowest EC 50 value, which is about 5-fold lower than the EC 50 value of quercetin.3).Among the fractions, BF had the lowest EC 50 value, which is about 2-fold greater than the EC 50 value of ascorbic acid.CF had the highest EC 50 value among the fractions, which is also 87-fold higher than that of ascorbic acid.
Different levels of TP, TCou, TPro, and THA contents were detected in the organic and water fractions of AE ( In order to determine the mode of inhibition of glucosidase by BF, a Lineweaver-Burk plot was prepared (figure not shown).The plot indicates that the inhibition of glucosidase by BF followed that of competitive kinetics.An increase in k m and unaltered V max values were detected when glucosidase-catalyzed cleavage of p-NPGP occurred in the presence of BF (Table 5).

DISCUSSION
Our results indicate that the active constituents of T. domingensis fruit which exhibited antiglucosidase and radical scavenging activities are likely to be relatively polar in nature.AE, ME, and WE had higher antiglucosidase and DPPH scavenging activities compared with extracts prepared with less polar solvents, such as HE and CE.The three extracts also had higher phytochemical contents relative to extracts prepared with the other solvents.When fractions of AE were compared, BF and EAF had higher antiglucosidase and DPPH scavenging activities than CF.With the exception of TCou, the phytochemical contents of BF and EAF were also higher compared with that partitioned into CF.Collectively, these results suggest that polar solvents were effective for extracting glucosidase inhibitors and radical scavengers from T. domingensis fruit.Similar to our findings, polar solvent ethyl acetate was more effective in extracting antiglucosidase agents from Callistephus chinensis in comparison with hexane and chloroform [11].By contrast, chloroform extract of Psoralea corylifolia was a more potent α-glucosidase inhibitor than extracts prepared with 50 % ethanol, ethanol, methanol, and water [12].Our observations, together with the aforementioned findings, suggest that optimal solvent polarity for the extraction of antiglucosidase agents from plant samples may not be easily predicted and is likely to be species or sample-dependent.
We found DPPH scavenging activity to be correlated with TP, THA and TPro contents to similar degrees.This concurs with previous reports of correlations between DPPH scavenging activity and TP and THA contents in other plants [13][14][15].Positive correlations between DPPH scavenging activity and TPro were also reported for rhizome extracts of highland ferns [14].When previously screening hot water extracts of T. domingensis fruit and flowers for antiglucosidase and antioxidant activity, we found the highest activities in the fruit extract, which also had the highest TPro content [6].In this study, the antiglucosidase activity of T. domingensis fruit extracts and fractions correlated more strongly with TPro content and weakly with THA content.Likewise, in highland ferns, THA content was either less strongly or not correlated to antiglucosidase activity when compared with TPro content [14].Taking into account our current results and reported evidence in the literature, TPro of T. domingensis fruit is a promising source of both glucosidase inhibitors and radical scavengers.On the other hand, THA of T. domingensis fruit is likely an important contributor of radical scavenging activity only.
Among all extracts and fractions analyzed, BF stood out as a dual-function antiglucosidase and radical scavenging agent of high potency.BF was a stronger inhibitor of α-glucosidase when compared with quercetin.In addition, based on the comparison of EC 50 values, the potency of BF as a radical scavenger was in the same order of magnitude as that of ascorbic acid.BF had the highest TPro content among all extracts and fractions analyzed.Thus, TPro is likely responsible for the potent dual bioactivities in the TPro-rich BF.Our findings corroborate with the reports of concurrent antiglucosidase and antioxidant activities in proanthocyanidins isolated from black chokeberry [16] and persimmon peel [17].
Our enzyme kinetic study suggests that TPro-rich BF contained active constituents that were possibly able to bind to the catalytic site of αglucosidase, thus acting as competitive inhibitors.Acetone extract of the leaves of Picralima nitida was also reported to be a competitive inhibitor of α-glucosidase [18], although there are more reports of plant extracts and natural products as non-competitive [19,20] and mixed-type [21] α-glucosidase inhibitors.Our study revealed that BF differs in its mode of inhibition from quercetin, which exerts either noncompetitive [22] or mixed-type [23,24] inhibition on α-glucosidase.Notably, the mode of glucosidase inhibition by BF is the same as that of acarbose, miglitol, and voglibose, three oral antihyperglycemic drugs [25].An advantage of competitive inhibitors is that their inhibitory action is reversible, thus allowing undesirable effects to be readily mitigated by decreasing the dosage of inhibitors used [26].
In this study, we used yeast α-glucosidase for the evaluation of antiglucosidase activity.Yeast α-glucosidase is available in pure form from commercial sources and has been widely used as a model for investigating antiglucosidase properties of natural products.In grape skin and pomace extracts, inhibition against yeast αglucosidase correlated with inhibition against mammalian α-glucosidase and suppression of postprandial hyperglycemia in Streptozocininduced diabetic mice [27].To assess the radical scavenging activity of T. domingensis fruit extracts and fractions, we have used the DPPH radical scavenging assay.The assay is widely used for antioxidant screening since it is rapid and simple, using DPPH radicals which are stable and commercially available.However, DPPH radicals have very little similarity to the highly reactive and transient oxygen radicals, such as hydroxyl, peroxyl and superoxide anion radicals, which occur in biological systems [28].Thus the ability of the T. domingensis extracts and fractions to scavenge biologically relevant radicals should be investigated.

CONCLUSION
In this study, we found that T. domingensis fruit is rich in polar bioactive constituents, as evidenced by the high phytochemical contents and strong bioactivities of extracts prepared with polar solvents.Correlation analysis suggests that proanthocyanidins of T. domingensis fruit are a promising source of both antiglucosidase inhibitors and radical scavengers.Proanthocyanidins-rich butanol fraction of acetone extract showed more potent antiglucosidase activity than quercetin and strong radical scavenging activity comparable to that of ascorbic acid.The results provide a strong rationale for future exploration of T. domingensis fruit as a novel source of antihyperglycemic agent with concurrent antioxidant activity.

Table 1 :
EC50 values of antiglucosidase and DPPH scavenging activities of solvent extracts Data are mean ± SEM (n = 3).The asterisks (*) denote values that are significantly different (p < 0.05) compared with the positive control, as determined by using Student's t test.nd = not determined.HE had no detectable antiglucosidase activity; hence, EC50 value was not calculated AE had the lowest EC 50 value for DPPH scavenging activity (8.57µg/mL) among all the solvent extracts (Table

Table 2 :
Phytochemical contents of organic solvent and aqueous extracts

Table 3 :
EC50 values of the antiglucosidase and DPPH scavenging activities of the solvent fractions of AE

Table 4 :
Phytochemical contents of the solvent fractions of AE

Table 5 :
km and vmax values for glucosidase activity in the presence or absence of BF a Data are mean ± SEM (n = 3).Values in the same column that are followed by different superscript letters are significantly different (p < 0.05), as determined by using Fisher's LSD test