A Determination of Potential α-Glucosidase Inhibitors from Azuki Beans (Vigna angularis)

1. Introduction

2. Results and Discussion

2.1. Isolation of Active Compounds and Structural Determination

Itoh et al. [7] investigated the antidiabetic effects of azuki beans on streptozotocin (STZ)-induced diabetic rats. We also observed that azuki beans showed the highest α-glucosidase inhibition ability among sixteen legumes (data not shown). However, the active components of the extract were not ascertained. In this study, two pure compounds were separated from the EtOAc-soluble fraction by the method mentioned above; they were identified as vitexin and isovitexin (structures are shown in Figures 1 and 2), by comparison of their spectral data with those in the literature [8,9].

Figure 1. Chemical structures of vitexin.

Figure 2. Chemical structures of isovitexin.

Vitexin (1): yellow powder. ¹H NMR (500 MHz, DMSO-d6) d: 13.15 (1H, s, OH-5), 8.01 (2H, d, J = 8.7 Hz, H-2′, 6′), 6.86 (2H, d, J = 8.7 Hz, H-3′, 5′), 6.76 (1H, s, H-3), 6.23 (1H, s, H-6), 4.69 (1H, d, J = 9.8 Hz, H-1′ of glu), 3.85–3.22 (6H, m, glucosyl H). ¹³C NMR (DMSO-d 6) d: 182.0 (C-4), 164.8 (C-2), 162.7 (C-7) 161.3 (C-4′), 160.4 (C-5),156.09 (C-9), 128.5 (C-2′, 6′), 121.2 (C-1′), 115.8 (C-3′, 5′), 104.7 (C-10), 104.1 (C-8),102.4 (C-3), 98.2 (C-6). Positive ESI-MS: m/z 433 [M + H]⁺.

Isovitexin (2): yellow powder. ¹H NMR (500 MHz, DMSO-d6) d: 13.55 (1H, s, OH-5), 7.93 (2H, d, J = 8.8 Hz, H-2′, 6′), 6.93 (2H, d, J = 8.8 Hz, H-3′, 5′), 6.78 (1H, s, H-3), 6.51 (1H, s, H-8), 4.58 (1H, d, J = 9.8 Hz, H-1 of glu), 4.03–3.11 (6H, m, glucosyl H). ¹³C NMR (DMSO d6) d: 181.9 (C-4), 163.5 (C-2), 163.3 (C-7), 161.6 (C-4′), 160.6 (C-5), 158.2 (C-9), 128.4 (C-2′, 6′), 121.1 (C-1′), 116.06 (C-3′, 5′), 108.9 (C-6), 103.4 (C-10), 102.8 (C-3), 93.64 (C-8). Positive ESI-MS: m/z 433 [M + H]⁺.

2.2. Alpha-Glucosidase Inhibition Activities

To determine the α-glycosidase inhibition ability in vitro, we calculated the IC₅₀ values (Table 1). The EtOAc-soluble fraction had the highest α-glucosidase inhibitory activity of the four partition parts. Vitexin was the most active (IC₅₀ of 0.4 mg·mL⁻¹), followed by isovitexin (IC₅₀ of 4.8 mg·mL⁻¹).

Table 1. Alpha-glucosidase inhibitory activity of CH₂Cl₂-soluble, EtOAc-soluble, n-BuOH-soluble, residual extract, vitexin and isovitexin.

2.3. Fluorescence Spectra

Fluorescence quenching can be divided into two types: dynamic quenching and static quenching. Dynamic quenching stems from the collision between two fluorescent luminophors, while static quenching arises from the formation of a new nonfluorescent complex that forms between the fluorescent luminophors and quencher [10]. Dynamic quenching follows the Stern–Volmer equation [11]:

$$F_0/F=1+K_{\text{sv}}[Q]=1+K_{\text{q}}{\tau }_0[Q]$$

(1)

where F₀ and F are the fluorescence intensities of the fore-and-aft interaction between α-glucosidase and flavonoid, [Q] is the concentration of quencher and flavonoid, and τ₀ is the average life of the fluorescent substance without the quencher, valued at approximately 10⁻⁸ s. K_sv and K_q are the dynamic quenching constant and rate constant in the process of double molecule quenching [12].

The quenching fluorescence spectra of α-glucosidase by flavonoids were recorded at 25 and 37 °C (Figures 3 and 4). The values of K_sv and K_q were obtained with the Stern-Volmer equation from plots of linear equations obtained by F₀/F vs. [Q]. The values of K_sv decreased with the increase of temperature, and K_q was greater than 2.0 × 10¹⁰ (Table 2). Therefore, the process of quenching is a static quenching by the formation of a complex.

Figure 3. The effect of vitexin on fluorescence spectrum of α-glucosidase after they were added to the enzyme solution.

Figure 4. The effect of isovitexin on fluorescence spectrum of α-glucosidase after they were added to the enzyme solution.

Table 2. Constants of K_sv and K_q of the interaction between α-glucosidase and vitexin, isovitexin.

Static quenching follows the equations [13]:

$$\text{lg}\frac{F_0-F}{F}=\text{lg}\hspace{0.17em}{K}_{\text{A}}+\text{n}\hspace{0.17em}\text{lg}\hspace{0.17em}{[Q]}_{\text{f}}$$

(2)

$$[\text{flavonoid}]\text{f}=[\text{flavonoid}]-n\hspace{0.17em}[\alpha -\text{glucosidase}-\text{flavonoid\hspace{0.17em}n}]$$

(3)

$$[\alpha -\text{glucosidase}-\text{flavonoidn}]=\frac{F_0-F}{F_0-F_{\infty }}[\alpha -\text{glucosidase}]$$

(4)

Here, [Q]_f is the concentration of free flavonoid, [flavonoid]_f; and [α-glucosidase-flavonoid _n] is the concentration of α-glucosidase bound with the flavonoid.

The vitexin binding constant (K_A) is higher than the isovitexin constant (Table 3). The number of binding sites (n) was close to one at 37 °C, which is the most suitable temperature for the flavonoid molecules to bind with α-glucosidase.

Table 3. Values of K_A and n of the interaction between α-glucosidase and vitexin, isovitexin.

3. Materials and Methods

4. Conclusion

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