Isolation and Characterization of an α-Glucosidase Inhibitor from Musa spp. (Baxijiao) Flowers

The use of α-glucosidase inhibitors is considered to be an effective strategy in the treatment of diabetes. Using a bioassay-guided fractionation technique, five Bacillus stearothermophilus α-glucosidase inhibitors were isolated from the flowers of Musa spp. (Baxijiao). Using NMR spectroscopy analysis they were identified as vanillic acid (1), ferulic acid (2), β-sitosterol (3), daucosterol (4) and 9-(4′-hydroxyphenyl)-2-methoxyphenalen-1-one (5). The half maximal inhibitory concentration (IC50) values of compounds 1–5 were 2004.58, 1258.35, 283.67, 247.35 and 3.86 mg/L, respectively. Compared to a known α-glucosidase inhibitor (acarbose, IC50 = 999.31 mg/L), compounds 3, 4 and 5 showed a strong α-glucosidase inhibitory effect. A Lineweaver-Burk plot indicated that compound 5 is a mixed-competitive inhibitor, while compounds 3 and 4 are competitive inhibitors. The inhibition constants (Ki) of compounds 3, 4 and 5 were 20.09, 2.34 and 4.40 mg/L, respectively. Taken together, these data show that the compounds 3, 4 and 5 are potent α-glucosidase inhibitors.


Introduction
Diabetes mellitus has become an alarming global problem in recent years. Postprandial hyperglycemia plays an important role in the development of diabetes mellitus type II and the resulting complications. One therapeutic approach to treat postprandial hyperglycemia is to retard the cleavage of glucose from disaccharide via inhibition of α-glucosidase in the digestive organs [1]. α-Glucosidase (EC 3.2.1.20) is a glucosidase that acts on 1,4-α bonds, breaking down starch and disaccharides into glucose. This enzyme is ubiquitous in plants, microorganisms, and animal tissues, although the substrate specificity of α-glucosidase differs greatly depending on the source [2]. α-Glucosidase inhibitors can decrease the postprandial increase in blood glucose and in turn help avoid the onset of late diabetic complications [3]. From this perspective, researchers have focused on finding more effective α-glucosidase inhibitors from natural materials for use as antidiabetic compounds, such as triterpene glycoside from Acanthopanax senticosus Harm leaves [4], flavonoid glycosides in Microctis folium [5] and polyphenols from green tea [6].
The banana planting area in China covers nearly 412,800 hectares, with an annual production of more than 1,085 million tons in 2012,which represents a huge economic value. Many banana flowers are produced; to date, in China they have only been used as organic material and fertilizer in the plantations. Some prior works have shown that crude extracts of banana flowers exhibited biological activity, including antihyperglycemic activity, advanced glycation end product (AGE) inhibitory activity [7], antimalarial activity [8], regulation of altered antioxidant and lysosomal enzyme activities [9] and wound-healing potential [10]. Our previous study showed that the banana flowers has tremendous nutritional value [11], antioxidant activity, and can be consumed as a food additive [12]. Despite a large body of studies on banana flowers, there is limited information on their chemical constituents. Thus, this study was conducted to determine the potential value of banana flowers as a routine and inexpensive source of useful biologically active compounds. The objectives of this project were to isolate, elucidate, and biologically evaluate phytochemicals found in banana flowers for α-glucosidase inhibitory activity. This is the first phytochemical and biological study of flowers of Musa spp. (Baxijiao).

Isolation of α-Glucosidase Inhibitors
The banana flowers were milled and extracted with 95% ethanol at room temperature. The crude extract was evaporated under vacuum, and the concentrated extracted was dispersed in water and partitioned successively with petroleum ether, ethyl acetate and n-butanol to obtain the PEF, EAF, BUF and AF fractions, respectively, which were all evaluated for their α-glucosidase inhibitory activities. Among the four fractions, the results suggested that the α-glucosidase inhibitory activities were mainly contained in the PEF and EAF fractions, which were therefore were subjected to successive chromatographic fractionation and purification to yield compounds 1-5, as shown in Figure 1.

Structural Elucidation of Isolated Compounds
The isolated compounds were identified by one-dimensional NMR and comparison with previously reported data. The chemical structures of these isolates are shown in Figure 2. Vanillic acid (1) and ferulic acid (2) are organic acids, β-sitosterol (3) and daucosterol (4) are sterols, and 9-(4ꞌ-hydroxyphenyl)-2-methoxyphenalen-1-one (5) is a phenylphenalenone. This is the first report of each of these compounds being isolated from banana flowers [13].

α-glucosidase Inhibitory Activity of Extracts Fractionated from Flowers of Musa spp. (Baxijiao)
The inhibitory activities of the crude ethanol extract (CEE) of banana flowers were determined at the concentration of 1.5 mg/mL against α-glucosidase ( Figure 3). The percent inhibition of the ethanol extract against α-glucosidase was 88.56% ± 2.3% (IC 50 = 343.09 ± 4.35 mg/L). This result suggested that the extract of banana flower of Musa spp. (Baxijiao) might be a promising antidiabetes drug candidate. After the ethanol extract was extracted with H 2 O, petroleum ether, ethyl acetate and n-butanol, four fractions (AF, PEF, EAF and BUF) were obtained, and the inhibitory activities of these four extracts against α-glucosidase were also determined. Figure 3 shows that at 1.5 mg/mL, the inhibitory effect against α-glucosidase had the following order: PEF > acarbose (reference) > EAF > BUF > AF. The percent inhibition of the PEE, acarbose, EAF, BUF and AF fractions against α-glucosidase were 74.0% ± 2.5%, 56.4% ± 1.1%, 52.8% ± 1.5% and 44.1% ± 2.4%, respectively. The bioactive PEF (IC 50 = 788.36 ± 19.32 mg/L) and EAF (IC 50 = 1877.77 ± 12.53 mg/L) fractions were chromatographed over a silica column and further purified using Sephadex LH-20 to afford the isolated α-glucosidase inhibitors. The IC 50 value of extract was lower than that of the fractionated compounds, because besides the isolated compounds, other phytochemical polyphenolics in the crude ethanol extracts might also contribute the α-glucosidase inhibitory activity.

Evaluation of α-Glucosidase Inhibitory Activity
Compounds 1-5 purified from Musa spp. (Baxijiao) showed α-glucosidase inhibitory properties, which were compared with those of acarbose, used in this study as the standard inhibitor. Acarbose decreases the hydrolysis of 4-nitrophenyl-α-D-glucopyranoside (4-NPGP) by inhibiting the action of α-glucosidase. As shown in Table 1, all of the constituents investigated exhibited a certain degree of α-glucosidase inhibitory activity, and compounds 3, 4 and 5 showed more remarkable inhibitory effects on α-glucosidase activity than the positive control acarbose, which is commonly used for therapeutic purposes. Compound 5 in particular demonstrated excellent in vivo activity (IC 50 = 3.86 mg/L).
According to our results, the IC 50 values of these compounds exhibited the following order: compound 5 > 4> 3 > acarbose > 2 > 1. Tabussum, et al. [14] reported that β-sitosterol isolated from Chrozophora plicata exhibited a strong inhibitory effect on α-glucosidase with an IC 50 value of 287.12 ± 0.75 μM. Mbaze, et al. [15] isolated vanillic acid from the stem bark of Fagara tessmannii (Rutaceae), which exhibited a strong inhibitory effect on α-glucosidase with an IC 50 value of 69.4 ± 0.8 μM. Because the inhibition is dependent on the concentration of the substrate, the enzyme and the duration of incubation with the enzyme, the α-glucosidase inhibitory effects of the same compounds in different reports are different [14]. Therefore, compounds 3, 4 and 5 have the potential to be clinically effective α-glucosidase inhibitors.

Mode of Inhibition of α-Glucosidase for Compounds 3, 4 and 5
The mechanism of the binding of compounds 3, 4 and 5 to α-glucosidase was further studied. Figure  4 shows a Lineweaver-Burk plot of the α-glucosidase inhibitory activities of compounds 3, 4 and 5 and in the absence of the inhibitor with different substrate concentrations of pNPG. It is evident from the results ( Figure 4A) that the presence of compound 5 caused a decrease in V max values compared to the control with a comparative (without much change in the K m values) increase in K m value, indicative of typical reversible, mixed-type inhibition.

Plant Materials
Musa spp. (Baxijiao), the most popular and accessible species in Hainan was chosen for this study. The banana flowers used were grown in the experimental field at the Haikou Experimental Station of the Chinese Academy of Tropical Agricultural Sciences (Chengmai City, Hainan Province, China) from January to December 2012. The plants were managed using commercial practices with standard fertilization and culture management. Mature banana plants from four plots of approximately 30 square meters per plot were collected, and the flowers were manually separated from the plant. After separation, the samples were thoroughly washed in running water, cut into small pieces, dried overnight in an air dryer at 40 °C, ground to a particle size of 40 meshes using a grinder, packed in black polyethylene bags and stored at −20 °C until further analysis.

α-Glucosidase Inhibitory Activity Assay
The α-glucosidase inhibitory activities were calculated by the reported method with slight modifications [4][5][6]. One hundred microliters of 10 mg/L α-glucosidase (50 U/mg) was premixed with 0.5 mL of the test sample in 0.5 mL phosphate buffer (pH 6.8) and incubated at 37 °C for 15 min. Then 0.5 mL of 2.5 mmol/L p-nitrophenyl-α-D-glucopyranoside as the substrate was added to the mixture to start the reaction. The reaction was incubated at 37 °C for 15 min and stopped by adding 1 mL 0.2 mol/L sodium carbonate solution. The α-glucosidase activity was determined by measuring the release of PNP at 405 nm. For the inhibitory activity assay, crude ethanol extracts were dissolved in 50% aqueous dimethyl sulfoxide, while the chloroform, ethyl acetate, and n-butanol fractions and purified compounds were dissolved in dimethyl sulfoxide. Acarbose was used as positive control. The inhibition of the test sample on α-glucosidase was calculated as follows: where A control : Absorbance for 100% enzyme activity (+enzyme), A sample : Test sample (+enzyme + inhibitor) and A background : Inhibitor background absorbance (−enzyme + inhibitor).

Determination of the Inhibitory Mode of Action of the Active Compounds
To determine the kinetic mode of inhibition of isolated α-glucosidase inhibitors, Lineweaver-Burk plot analysis was performed. This kinetics study was carried out in the presence and absence of inhibitors with varying concentrations of 4-NPGP as the substrate. The mode of inhibition of the test compounds was assessed on the basis of the inhibitory effects on K m (dissociation constant) and V max (maximum reaction velocity) of the enzyme determined using a Lineweaver-Burk plot, which is the double reciprocal plot of the enzyme reaction velocity (V) versus the substrate (p-nitrophenyl β-D-glucopyranoside, pNPG) concentration (1/V versus 1/[pNPG]) [20].