α-Amylase and α-Glucosidase Inhibitory Activities of Chemical Constituents from Wedelia chinensis (Osbeck.) Merr. Leaves

Thao, Nguyen Phuong; Binh, Pham Thanh; Luyen, Nguyen Thi; Hung, Ta Manh; Dang, Nguyen Hai; Dat, Nguyen Tien

doi:https://doi.org/10.1155/2018/2794904

Journal of Analytical Methods in Chemistry

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Research Article | Open Access

Volume 2018 | Article ID 2794904 | https://doi.org/10.1155/2018/2794904

α-Amylase and α-Glucosidase Inhibitory Activities of Chemical Constituents from Wedelia chinensis (Osbeck.) Merr. Leaves

Nguyen Phuong Thao,¹Pham Thanh Binh,¹Nguyen Thi Luyen,^1,2Ta Manh Hung,³Nguyen Hai Dang,^1,2and Nguyen Tien Dat^2,4

Academic Editor: Federica Pellati

Received12 Feb 2018

Accepted04 Apr 2018

Published09 May 2018

Abstract

As part of an ongoing search for new natural products from medicinal plants to treat type 2 diabetes, two new compounds, a megastigmane sesquiterpenoid sulfonic acid (1) and a new cyclohexylethanoid derivative (2), and seven related known compounds (3–9) were isolated from the leaves of Wedelia chinensis (Osbeck.) Merr. The structures of the compounds were conducted via interpretation of their spectroscopic data (1D and 2D NMR, IR, and MS), and the absolute configurations of compound 1 were determined by the modified Mosher’s method. The MeOH extract of W. chinensis was found to inhibit α-amylase and α-glucosidase inhibitory activities as well as by the compounds isolated from this extract. Furthermore, compound 7 showed the strongest effect with IC₅₀ values of 112.8 ± 15.1 g/mL (against α-amylase) and 785.9 ± 12.7 g/mL (against α-glucosidase). Compounds 1, 8, and 9 showed moderate α-amylase and α-glucosidase inhibitory effects. Other compounds showed weak or did not show any effect on both enzymes. The results suggested that the antidiabetic properties from the leaves of W. chinensis are not simply a result of each isolated compound but are due to other components such as the accessibility of polyphenolic groups to α-amylase and α-glucosidase activities.

1. Introduction

In recent years, the number of diabetic patients is rapidly rising in most parts of the world, especially in developing Southeast Asian countries [1, 2]. The control of blood glucose concentrations near the normal range is mainly based on the use of oral hypoglycaemic/antihyperglycaemic agents and insulin. However, all of these treatments have limited efficacy and are associated with undesirable side effects [3, 4], leading to increasing interest in the use of medicinal plants for the alternative management of type 2 diabetes mellitus. An effective suggestion for type 2 diabetes management is the inhibition of α-glucosidase and α-amylase [5].

Natural health-care products derived from medicinal plants or herbs have been developed as alternative or complementary treatments for many common disorders [6]. Several medicinal plants have been the useful sources of novel biologically active compounds. Many pharmaceutical agents have been discovered by screening natural products from plants, many of which have been developed as new leads for pharmaceuticals [7]. Predominantly herbal drugs have been widely used globally for diabetic treatment over thousands of years due to their traditional acceptability and lesser side effects. Therefore, screening of α-amylase (1,4-α-D-glucan glucanohydrolases; EC. 3.2.1.1) and α-glucosidase (α-D-glucoside glucohydrolase, EC 3.2.1.20) inhibitors in medicinal plants has received much attention [6, 7].

Among the traditionally used important medicinal plants, the genus Wedelia (Asteraceae) contains approximately 107 species in the world, and among them, about 6 species are in Vietnam. They are all herbal plants and distributed in tropical and subtropical regions of Asia and Pacific Islands [8]. Wedelia chinensis (Osbeck.) Merr. (Asteraceae) is a deciduous shrub, widely distributed in several Asian countries such as China, Japan, and mainland Vietnam. The leaves, stems, and fruits of this species have been traditionally used in fold medicine for the treatment of chin cough, diarrhoea, diphtheria, faucitis, hemorrhoids, and injuries due to falls, jaundice, and pertussis and are often consumed as tea in the form of infusion [9]. Phytochemically, up to date, a number of secondary metabolites have been identified, including various types of compounds belonging to chemical classes of flavonoids, diterpenoids, and triterpenoids. Besides, several other compounds including common saponins and phytosterol derivatives were also reported in the species, but they appear to have a more limited distribution. Recently, several studies have demonstrated the exploration of pharmacological potential, such as analgesic [10], androgen-suppressing [11], anticancer [12, 13], antibacterial, anticonvulsant, antifungal [14], antioxidant [13], anti-inflammatory [10, 15], antiosteoprotic [16], antiulcerogenic, antistress [17], hepatoprotective [18], and sedative activities [19]. However, the investigation about the chemical constituents of W. chinensis is not sufficient compared to the other plants in the genus Wedelia.

As part of an ongoing search for new biologically active natural products from Vietnamese medicinal plants, we found that a MeOH extract of W. chinensis leaves showed significant in vitro α-amylase and α-glucosidase inhibitory activities. Previously, there have been no reports on either the extracts or isolated components from this species against α-amylase and α-glucosidase activities. Reported herein are the isolation and structural elucidation of a new megastigmane sesquiterpenoid sulfonic acid (1), a new cyclohexylethanoid derivative (2), and 7 known compounds (3–9) as well as the evaluation of α-amylase and α-glucosidase activities.

2. Materials and Methods

2.1. General Experimental Procedures

Optical rotations were measured using a JASCO P-2000 polarimeter (JASCO, Oklahoma, OK, US). IR spectra were obtained on a Bruker TENSOR 37 FT-IR spectrometer (Bruker, Billerica, MA, USA). The ¹H and ¹³C, HMQC, HMBC, NOESY/ROESY, and COSY NMR spectra were recorded on a 500 MHz Bruker DRX spectrometer (Bruker, Tupper Hall, CA, USA), and the chemical shift (δ) was expressed in ppm with reference to the TMS signals. The HRESIMS were obtained using an Agilent 6550 iFunnel Q-TOF LC/MS system (Emeryville, CA, USA). Medium-pressure liquid chromatography (MPLC) was carried out on a Biotage-Isolera One system. Column chromatography (CC) was conducted using 65–250 or 230–400 mesh silica gel (Sorbent Technologies, Atlanta, GA, USA), porous polymer gel (Diaion® HP-20, 20–60 mesh, Mitsubishi Chemical, Tokyo, Japan), Sephadex™ LH-20 (Supelco, Bellefonte, PA, USA), octadecyl silica (ODS, 50 m, Cosmosil 140 C₁₈-OPN, Nacalai Tesque), and YMC RP-C₁₈ resins (30–50 m, Fuji Silysia Chemical). Analytical thin-layer chromatography (TLC) systems were performed on precoated silica gel 60 F₂₅₄ plates (1.05554.0001, Merck) and RP-18 F_254S plates (1.15685.0001, Merck), and the isolated compounds were visualized by spraying with 10% H₂SO₄ in water and then heating for 1.5–2 minutes. All procedures were carried out with solvents purchased from commercial sources, which were used without further purification.

2.2. Plant Material

The leaves of W. chinensis (Osbeck.) Merr. were collected from Ba Dinh, Ha Noi, Vietnam, in April 2017 and taxonomically identified by Professor Tran Huy Thai (Institute of Ecology and Biological Resources). A voucher specimen (NCCB-2016.55.01) was deposited at the Herbarium of Institute of Marine Biochemistry and Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology.

2.3. Compounds

From the methanolic extract of W. chinensis, 9 compounds (1–9) were isolated and structurally elucidated. Stock solutions of tested compounds in DMSO were prepared kept at −20°C and diluted to the final concentration in fresh media before each experiment. To not affect the cell growth, the final DMSO concentration did not exceed 0.5% in all experiments.

2.4. Extraction and Isolation

The dried leaves of W. chinensis (4.4 kg) were cut into pieces and extracted with 95% aqueous MeOH (3 × 6.5 L) under ultrasonic agitation at 90 Hz. The methanol solution was removed of solvent under a vacuum and was filtered through a Büchner funnel to produce a dried brown extract (160 g, A). Since the MeOH extract significantly reduced α-amylase and α-glucosidase inhibitory activities, it was suspended in distilled water and partitioned between EtOAc (1 L × 3) to obtain EtOAc (16.9 g, B) and aqueous soluble fractions (W).

The EtOAc fraction was separated on silica gel MPLC (column: Biotage® SNAP Cartridge, KP-SIL, 340 g) using the mobile phase of n-hexane-acetone (gradient 30 : 70, 50 : 50, 70 : 30, 0 : 100, 15 mL/min, 90 min) to give six fractions (B-1 to B-6). This MPLC procedure was repeated 5 times using the same conditions before further isolation. Fraction B-3 was chromatographed by Sephadex® LH-20 CC (Φ25 mm, L 1250 mm) eluted with acetone-H₂O (gradient 95 : 5, 70 : 30, 50 : 50, v/v) to give three subfractions (B-3.1 to B-3.3) and further purified by YMC RP-C₁₈ CC (Φ15 mm, L 700 mm) using acetone-H₂O (1 : 2) as the eluent to pomonic acid (8, crystalline powder, 11.2 mg) and pomolic acid (9, crystalline powder, 15.4 mg). Next, fraction B‒6 was chromatographed over a silica gel CC (Φ12 mm, L 600 mm) eluted with n-hexane-EtOAc (2 : 1) to obtain jaceosidin (7, pale yellow crystals, 56.8 mg).

The H₂O fraction was separated using a Diaion HP-20 column (Φ100 mm, L 500 mm) and was eluted with a gradient solvent mixture of MeOH-H₂O (gradient 25 : 75, 50 : 50, 65 : 35, 75 : 25, to pure MeOH, stepwise) to yield five fractions (W-1 to W-5), based on TLC analysis. The fractionation W-4 was separated via silica gel CC (Φ30 mm, L 750 mm) and eluted repeatedly with CH₂Cl₂-MeOH (0 → 100%) to yield five subfractions (W-4.1 to W-4.5). Subfraction W-4.1 was subjected to a silica gel CC (Φ20 mm, L 800 mm with a solvent mixture of CH₂Cl₂-MeOH, 9 : 1) and passed over a Sephadex LH-20 column (Φ15 mm, L 950 mm) and then through an open YMC RP-C₁₈ silica gel column (Φ15 mm, L 800 mm, 65 → 100%, H₂O-MeOH) to afford wednenol (2, colorless oil, 12.5 mg), cleroindicin E (3, colorless oil, 15.2 mg), and rengyol (4, white solid, 46.6 mg). Finally, when the same steps were repeated as above, wednenic (1, white powder, 10.6 mg), cornoside (5, brown oil, 28.7 mg), and benzyl β-D-glucopyranoside-2-sulfate (6, amorphous white powder, 86.1 mg) were also obtained by purifying subfraction W-4.5 on YMC RP-C₁₈ silica gel (Φ20 mm, L 700 mm) and followed by passing over a Sephadex LH-20 column (Φ15 mm, L 900 mm) using mixtures of MeOH-H₂O (1 : 5).

2.5. Physical and Spectroscopic Data of Compounds

Wednenic (1): white powder; −26.5 (c 0.25, MeOH); IR ν_max (KBr) 3395, 2968, 2930, 1648, 1580, 1511, 1372, 1226, 1163, 1075, and 1040 cm⁻¹; HRESIMS (positive-ion mode) m/z 345.0987 [M + H]⁺ (cald. for C₁₃H₂₃O₇S, 345.0984) and 367.0801 [M + Na]⁺ (cald. for C₁₃H₂₂NaO₇S, 367.0803) and (negative-ion mode) m/z 225.1482 [M − SO₄Na]⁻ (cald. for C₁₃H₂₁O₃, 225.1490); for ¹H NMR (CD₃OD, 500 MHz) and ¹³C NMR (CD₃OD, 125 MHz) spectroscopic data, see Table 1.

Wednenol (2): colorless oil; −47.2 (c 0.22, MeOH); IR ν_max (KBr) 3341, 2950, 1455, 1270, 1168, and 920 cm⁻¹; HRESIMS (positive-ion mode) m/z 185.1170 [M + H]⁺ (cald. for C₁₀H₁₇O₃, 185.1177); for ¹H NMR (CD₃OD, 500 MHz) and ¹³C NMR (CD₃OD, 125 MHz) spectroscopic data, see Table 1.

Benzyl β-D-glucopyranoside-2-sulfate (6): amorphous white powder; 60.8 (c 0,25, MeOH); IR ν_max (KBr): 3595, 3100, 2952, 2850, 1647, 1575, 1511, 1362, 1228, 1153, 1055, and 1010 cm⁻¹; ESIMS (negative-ion mode) m/z 349,1 [M − H]⁻; ¹H NMR (500 MHz, CD₃OD): δ_H 3.31 (1 H, m, H-5′), 3.44 (1 H, t, J = 9.0 Hz, H-3′), 3.66 (1 H, t, J = 9.0 Hz, H-4′), 3.71 (1 H, d, J = 1.5, 12.0 Hz, H-6′b), 3.91 (1 H, d, J = 1.5, 12.0 Hz, H-6′a), 4.17 (1 H, t, J = 8.0 Hz, H-2′), 4.53 (1 H, d, J = 7.5 Hz, H-1′), 4.74 (1 H, d, J = 12.0 Hz, H-7b), 4.93 (1 H, d, J = 12.0 Hz, H-7a), 7.25 (1 H, d, J = 7.5 Hz, H-4), 7.33 (2 H, d, J = 7.5 Hz, H-3 and H-5), and 7.47 (2 H, br t, J = 7.5 Hz, H-2 and H-6); ¹³C NMR (125 MHz, CD₃OD): δ_C 138.9 (C-1), 128.8 (C-2 and C-6), 129.1 (C-3 and C-5), 128.4 (C-4), and 71.5 (C-7); Glc: 101.0 (C-1′), 81.4 (C-2′), 77.4 (C-3′), 71.5 (C-4′), 77.6 (C-5′), and 62.6 (C-6′).

2.6. Preparation of (S)- and (R)-MTPA Ester Derivatives of 1

Compound 1 (3.0 mg) was dissolved in 2.5 mL of anhydrous CH₂Cl₂. Dimethylaminopyridine (35 mg), triethylamine, and (R)-MTPA chloride (30 L) were then added in sequence. The reaction mixture was stirred for 3 h at room temperature and then quenched by the addition of 1 mL of aqueous MeOH. The solvents were removed under vacuum, and the residue was passed through a small silica gel column using CH₂Cl₂-MeOH (100 : 1) as the eluent to provide the (S)-MTPA ester of 1 (1a, 1.2 mg). The (R)-MTPA derivative (1b, 1.5 mg) was prepared with (S)-MTPA chloride and purified in the same manner.

(S)-MTPA ester derivative of 1 (1a): 1 H·NMR (CD3OD, 500 MHz): δ_H 7.613–7.421 (10 H, overlap, aromatic protons), 3.630 (3 H, s, OCH3), 1.501 (1H, dd, J = 3.5, 12.5 Hz, H-2a), 1.859 (1 H, t, J = 12.5 Hz, H-2b), 4.693 (1 H, dt, J = 3.5, 12.5 Hz, H-3), 5.767 (1 H, d, J = 3.5 Hz, H-4), 6.418 (1 H, dd, J = 1.0, 16.5 Hz, H-7), 5.901 (1 H, dd, J = 6.0, 16.5 Hz, H-8), 5.983 (1 H, dd, J = 6.0, 12.5 Hz, H-9), 1.431 (3 H, d, J = 6.0 Hz, H-10), 1.202 (3 H, s, H-11), 1.050 (3 H, s, H-12), and 1.315 (3 H, s, H-13).

(R)-MTPA ester derivative of 1 (1b): 1 H·NMR (CD3OD, 500 MHz): δ_H 7.613–7.401 (10 H, overlap, aromatic protons), 3.58 (3 H, s, OCH3), 1.516 (1 H, dd, J = 3.5, 12.5 Hz, H-2a), 1.878 (1 H, t, J = 12.5 Hz, H-2b), 4.676 (1 H, dt, J = 3.5, 12.5 Hz, H-3), 5.767 (1 H, d, J = 3.5 Hz, H-4), 6.395 (1 H, dd, J = 1.0, 16.5 Hz, H-7), 5.846 (1 H, dd, J = 6.0, 16.5 Hz, H-8), 5.982 (1 H, dd, J = 6.0, 12.5 Hz, H-9), 1.463 (3 H, d, J = 6.0 Hz, H-10), 1.174 (3 H, s, H-11), 1.014 (3 H, s, H-12), and 1.328 (3 H, s, H-13).

2.7. Assay for α-Amylase Inhibition

The porcine pancreas α-amylase (A3176, Sigma-Aldrich) enzyme inhibitory activity was carried out according to the standard method with minor modifications [20–22]. The substrate was prepared by boiling 100 mg potato starch in 5 mL phosphate buffer (pH 7.0) for 5 min and then cooling to room temperature. The sample (2 mL dissolved in DMSO) and substrate (50 mL) were mixed in 30 mL of 0.1 M phosphate buffer (pH 7.0). After 5 min preincubation, 5 mg/mL α-amylase solution (20 mL) was added, and the solution was incubated at 37°C for 15 min. The reaction was stopped by adding 50 mL of 1 M·HCl, and then, 50 mL of iodine solution was added. The absorbances were measured at 650 nm by a microplate reader. Acarbose was used as a positive control. The inhibitory activity was calculated by the following equation: α-amylase inhibitory activity (%) = (1–A/A0) × 100, where A is the absorbance of the sample and A0 is the absorbance of the blank, respectively. The IC₅₀ value was calculated by GraphPad Prism.

2.8. Assay for α-Glucosidase Inhibition

The yeast α-glucosidase (G0660, Sigma-Aldrich) inhibition assay was performed using the substrate p-nitrophenyl-α-D-glucopyranoside according to the previously described method [21–23]. Briefly, samples and acarbose were prepared by dissolving at 2 mg/mL (with extracts) or 0.8 mM (with pure compound) in DMSO, and 0.5 U/mL α-glucosidase (40 mL) was mixed in 120 mL of 0.1 M phosphate buffer (pH 6.8). After 5 min preincubation, 5 mM p-nitrophenyl-α-D-glucopyranoside solution (40 mL) was added, and the solution was incubated at 37°C for 30 min. Pipette the following reagents into a 96-well plate. Each concentration of samples was carried out in triplicate. The absorbance of released 4-nitrophenol was measured at 405 nm by using a microplate reader (xMark, Bio-Rad, USA). Acarbose was used as the positive control. The inhibitory activity was calculated by the following equation: α-glucosidase inhibitory activity (%) = (1–A/A0) × 100, where A is the absorbance of the sample and A0 is the absorbance of the blank, respectively. The IC₅₀ value was calculated by GraphPad Prism.

2.9. Data Expression and Statistical Analysis

Data were expressed as mean value ± standard deviation (SD) of blood glucose. Data were evaluated using two-way ANOVA followed by Dunnett’s multiple comparison test, and groups were considered significantly different if . All data are presented as mean ± SD.

3. Results and Discussion

3.1. Identification of Compounds 1–9

A MeOH extract from the leaves of W. chinensis was suspended in H₂O and fractionated successively with EtOAc-soluble fraction, and then, each fraction was evaluated for α-amylase and α-glucosidase activities. The EtOAc-soluble fraction and water layer were chosen for subsequent studies, which resulted in the isolation of two new compounds (1–2), together with seven known compounds (3–9; Figure 1). Moreover, compounds 1–6 were reported for the first time from this species and from the genus Wedelia.

Wednenic (1) was obtained as a white powder with a negative optical rotation (−26.5, c 0.25, MeOH), and the molecular formula, C₁₃H₂₂O₇S, was determined by HRESIMS, with a protonated molecular ion peak at m/z 345.0987 [M + H]⁺ and a sodium adduct molecular ion peak at m/z 367.0801 [M + Na]⁺. The fragment ion peak at m/z 225.1482 [M − SO₄Na]⁻ in the (−)HRESIMS spectrum showed the presence of a sulfate group in 1. A detailed assessment of the NMR data indicated that 1 is a megastigmane, a compound class known as components of plant species.

The ¹H NMR spectrum (Table 1) of 1 exhibited signals for four methyl groups [δ_H 1.24 (d, J = 6.0 Hz, H-10), 1.03 (s, H-11), 1.13 (s, H-12), and 1.28 (s, H-13)], a pair of methylene protons [δ_H 1.47 (dd, J = 3.5, 12.5 Hz, H-2a) and 1.84 (t, J = 12.5 Hz, H-2b)], three oxygenated methines [δ_H 4.60 (ddd, J = 3.0, 3.5, 12.5 Hz, H-3), 4.27 (dd, J = 1.0, 3.0 Hz, H-4), and 4.31 (dd, J = 6.0, 12.5 Hz, H-9)], and two olefinic protons [δ_H 5.92 (dd, J = 1.0, 16.5 Hz, H-7) and 5.69 (dd, J = 6.0, 16.5 Hz, H-8)]. The larger coupling constant (J = 16.5 Hz) between H-7 and H-8 indicates that the geometry of the Δ⁷ double bond is E. The ¹³C NMR and DEPT spectra (Table 1) showed 13 carbon signals of four methyls [δ_C 23.7 (C-10), 24.8 (C-11), 29.5 (C-12), and 17.1 (C-13)], a methylene [(δ_C 37.9 (C-2)], an oxygenated methine bearing a sulfate group [δ_C 75.5 (C-3)], two oxygenated methines [δ_C 71.6 (C-4) and 68.5 (C-9)], and three nonprotonated carbons [δ_C 35.5 (C-1), 69.4 (C-5), and 71.3 (C-6)], together with a pair of trans-olefinic methine carbons [δ_C 125.6 (C-7) and 139.3 (C-8)]. Based on these data, a megastigmane sesquiterpenoid sulfonic acid has been determined for 1.

Interpretation of the COSY and HSQC spectra of 1 revealed the presence of two partial structures, “C-2/C-3/C4” and “C-7/C-8/C-9/C-10” (Figure 2). These two partial structures were connected through a nonprotonated carbon (C-6) on the basis of HMBC correlations of H-4, H-7, H-8, H-12, and H-13 to C-6. The HMBC correlations from H₃-11 and H₃-12 to C-1, C-2, and C-6 indicated that C-2, C-11, C-12, and C-6 were all connected with C-1. The chemical shifts and coupling constants of 1 were in good agreement with those of (3S,4S,5R,6S,9S,7E)-megastigman-7ene-5,6-epoxy-3,4,9-triol 9-O-β-D-glucopyranoside recorded in the same deuterated solvent [24] but quite different from the data of the C-4. The substitution at C-3 and C-4 was tentatively identified as a sulfonate group and a hydroxyl group based on the HMBC correlations, which supported the total structure of 1 (Figure 2).

The relative configuration of 1 was deduced from analysis of coupling constants and the NOESY spectrum (except for C-9), which were both consistent with a chair conformation for the cyclohexane ring. The α-orientation of H-3 was deduced from the cross-peak of H-3 to H-2a/H₃-12 and H-7 and H-2b to H-11 and H-8 in the NOESY spectrum, and the larger coupling constant J = 12.5 Hz of H-3 (δ_H 4.60) with one of the H-2a protons (δ_H 1.84) indicated that H-3 was axial. Moreover, the NOESY correlation between H-4 and H-3 indicated that H-4 and H-3 were axial bonds, which established its α-orientation. Additionally, the coupling constants at H-3 [δ_H 4.60 (1 H, ddd, J = 3.0, 3.5, 12.5 Hz)] and H-4 [δ_H 4.27 (1 H, dd, J = 1.0, 3.0 Hz)] of 1 were in good agreement with those of (3S,4S,5R,6S,9S,7E)-megastigman-7ene-5,6-epoxy-3,4,9-triol 9-O-β-D-glucopyranoside [δ_H 3.79 (1 H, ddd, J = 3.0, 3.0, 12.0 Hz, H-3) and 3.88 (1 H, dd, J = 1.0, 3.0 Hz, H-4)] recorded in the same deuterated solvent. These findings confirmed that two compounds have the same configurations at C-3 and C-4 [24]. The chemical shifts of C-5 (δ_C 69.4) and C-6 (δ_C 71.3) corresponded well to the similar signals observed in the ¹³C NMR spectra (in CD₃OD) with (3S,4S,5R,6S,9S,7E)-megastigman-7ene-5,6-epoxy-3,4,9-triol 9-O-β-D-glucopyranoside [δ_C 69.7 (C-5) and 71.6 (C-6)] [24] and the major difference from the ¹³C NMR spectrum (in CD₃OD) with 5S,6R configurations of (3S,4S,5S,6R,7E,9S)-5,6-epoxy-3,4,9-trihydroxy-7-megastigmen-3-O-β-D-glucopyranoside (komaroveside C) [δ_C 68.2 (C-5) and 70.4 (C-6)] [25]. These data clearly indicated the presence of 5R,6S configurations in 1. To determine the absolute configuration of C-9 in 1, (S)- and (R)-MTPA esters (1a and 1b) were prepared. Significant Δδ values (Δδ = δ_S-MTPA-ester − δ_R-MTPA-ester) were observed for the proton signal adjacent to C-9, as shown in Figure 3. According to the rule of the modified Mosher’s method [26, 27], the absolute configuration at C-9 in 1 was assigned S-form. On the basis of the abovementioned data, the structure of 1 was elucidated to be (3S,4R,5R,6S,9S,7E)-megastigman-5,6-epoxy-7-ene,4,9-diol,3-sulfonic acid.

A molecular formula of C₁₀H₁₆O₃ was established for wednenol (2) based on the presence of 10 signals in its ¹³C NMR spectrum and the HRESIMS protonated molecular ion peak at m/z 185.1170 [M + H]⁺ (cald. 185.1178). In the ¹H NMR spectrum, signals were observed for a secondary methyl group [δ_H 1.15 (d, J = 6.5 Hz, H-10)], two oxygen-bearing methine groups [δ_H 4.00 (dd, J = 6.0, 9.5 Hz, H-2) and 3.51 (q, J = 6.5 Hz, H-9)], oxygenated methylene groups [δ_H 3.95 (ddd, J = 3.0, 8.0, 9.5 Hz, H-8a) and 4.05 (dd, J = 8.0, 8.5 Hz, H-8b)], and four methylene groups (Table 1). Its ¹³C NMR spectrum exhibited ten carbon signals, including a methyl [δ_C 17.0 (C-10)], two methines [δ_C 83.3 (C-2) and 74.3 (C-9)], an oxygenated methylene [δ_C 66.4 (C-8)], and four methylenes [δ_C 37.5 (C-3), 29.2 (C-5), 30.5 (C-6), and 36.0 (C-7)], suggesting that 2 was a cyclohexylethanoid derivative [28].

Analysis of the ¹H and ¹³C NMR data of 2 (Table 1) suggested that this compound shares several structural similarities with cleroindicin E (3) [28], except for major differences in the resonances associated with the additional methyl and oxygenated methine groups. The ¹H-¹H COSY spectrum of 2 was observed to exhibit proton correlations between H-5 and H-6, between H-7 and H-8, between H-2 and H-3, and between H-9 and H-10 (Figure 2). In the HMBC spectrum, cross-peaks between H-10 (δ_H 1.15) and C-4 (δ_C 75.4), and H-10 (δ_H 1.15) and C-9 (δ_C 74.3), between H-9 (δ_H 3.51) and C-3 (δ_C 37.5), C-4 (δ_C 75.4), and C-5 (δ_C 29.2) clearly indicated the positions of the methyl and oxygenated methine groups. Moreover, the C-1 resonance was shifted downfield, from δ_C 75.8 in 3 to δ_C 79.4 in 2. The downfield shift of C-1 may be explained by the presence of a C-1‒C-9 ether linkage in 2. These findings suggested that one cyclohexylethanoid unit in 2 should have a 1,2,4-trioxygenated-cyclohexylethanoid structure. Detailed analysis of the other COSY and HSQC spectra unambiguously identified the planar structure of 2 (Figure 2). In the NOESY spectrum of 2, the doublet of doublets belonging to H-2 [δ_H 4.00 (1H dd, J = 6.0, 9.5 Hz)] was observed, which correlated with H-3a and H-7a, and the coupling constant J = 6.0 Hz of H-2 (δ_H 4.00) with one of the H-3a protons (δ_H 1.39) indicated that H-2 was equatorial orientation. Additionally, the NOESY correlation of H-3a and H-9 and their coupling constant (J = 6.5 Hz) suggested that H-9 was also an equatorial bond. Thus, the structure of wednenol was elucidated as 2.

On comparison of their physical and spectroscopic data with published values, the known compounds were identified as cleroindicin E (3) [29], rengyol (4) [30], cornoside (5) [28], benzyl β-D-glucopyranoside-2-sulfate (6) [31], jaceosidin (7) [32], pomonic acid (8) [33], and pomolic acid (9) [34].

3.2. α-Amylase and α-Glucosidase Inhibitory Activities of Compounds 1–9

The inhibitory effects of the isolated compounds against porcine pancreas α-amylase and yeast α-glucosidase were evaluated in comparison with the antidiabetic acarbose. α-Glucosidase is the key catalyzing enzyme involved in the process of carbohydrate digestion and glucose release. Inhibition of α-glucosidase is one very effective way of delaying glucose absorption and lowering the postprandial blood glucose level, which can potentially suppress the progression of DM.

The isolated flavonoid (7) showed the most active α-amylase and α-glucosidase inhibitory activities with IC₅₀ values of 112.8 ± 15.1 and 785.9 ± 12.7 g/mL, respectively (Table 2 and Figure 4). This is in agreement with a recent report of the α-amylase and α-glucosidase inhibitory activities in other flavonoids [35–37]. Compounds 1, 8, and 9 showed moderate inhibitory effects against α-amylase and α-glucosidase, when compared with those of a standard reference drug, acarbose, with IC₅₀ values of 124.0 ± 21.3 g/mL (against α-amylase) and 642.6 ± 46.4 g/mL (against α-glucosidase). In contrast, other compounds (2–6) showed weak or did not show any effect on both enzymes. These results indicated that the megastigmane, flavonoids, and triterpenoids exhibited high inhibitory activity but were not sufficient to clarify the structure-activity relationships between derivatives. Further research is required to clarify their potential selective α-amylase and α-glucosidase activities.

4. Conclusion

In conclusion, the present work reported for the first time the α-amylase and α-glucosidase inhibitory effects of W. chinensis, in support of their ethnomedicinal use for diabetes. This report partly defines the reason on why these medicinal plants possess antidiabetic properties and could provide a scientific warrant for their application as health supplementary herbal products for diabetes treatment and prevention.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments

This research was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under Grant no. 104.01-2016.55.

Supplementary Materials

HRESIMS, ¹H, ¹³C, and 2D NMR spectra of new compounds 1–2; ¹H and ¹³C NMR data of compounds 3–5 and 7. (Supplementary Materials)

References

http://www.who.int/diabetes/facts/world_figures/en/index5.html.
“Addressing Asia’s fast growing diabetes epidemic,” Bulletin of the World Health Organization, vol. 95, no. 8, pp. 550-551, 2017.
View at: Publisher Site | Google Scholar
Y. I. Kwon, E. Apostolidis, and K. Shetty, “Inhibitory potential of wine and tea against α-amylase and α-glucosidase for management of hyperglycemia linked to type 2 diabetes,” Journal of Food Biochemistry, vol. 32, no. 1, pp. 15–31, 2008.
View at: Publisher Site | Google Scholar
R. K. Campbell, J. R. White Jr., and B. A. Saulie, “Metformin: a new oral biguanide,” Clinical Therapeutics, vol. 18, no. 3, pp. 360–371, 1996.
View at: Publisher Site | Google Scholar
A. J. Krentz and C. Bailey, “Oral antidiabetic agents: current role in type 2 diabetes mellitus,” Drugs, vol. 65, no. 3, pp. 385–411, 2005.
View at: Publisher Site | Google Scholar
M. Ekor, “The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety,” Frontiers in Pharmacology, vol. 4, no. 177, pp. 1–10, 2013.
View at: Publisher Site | Google Scholar
A. G. Atanasov, B. Waltenberger, E.-M. Pferschy-Wenzig et al., “Discovery and resupply of pharmacologically active plant-derived natural products: a review,” Biotechnology Advances, vol. 33, no. 8, pp. 1582–1614, 2015.
View at: Publisher Site | Google Scholar
““V. The Plant List,” http://www.theplantlist.org/.
View at: Google Scholar
The Wealth of India, A Dictionary Indian Raw Materials and Industrial Products, The Wealth of India, Raw Materials, Council of Scientific and Industrial Research, New Delhi, India, 2005.
H. Wagner, B. Geyer, Y. Kiso, H. Hikino, and G. S. Rao, “Coumestans as the main active principles of the liver drugs Eclipta alba and Wedelia calendulacea,” Planta Mededica, vol. 34, no. 5, pp. 370–374, 1986.
View at: Google Scholar
F. M. Lin, L. R. Cheu, E. H. Lin, F. C. Ke, and M. J. Tsai, “Compounds from W. chinensis synergistically suppress androgen activity and growth in prostate cancer,” Carcinogenesis, vol. 28, no. 12, pp. 2521–2529, 2007.
View at: Publisher Site | Google Scholar
C. H. Tsai, S. F. Tzeng, S. C. Hsieh et al., “Development of a standardized and effect-optimized herbal extract of Wedelia chinensis for prostate cancer,” Phytomedicine, vol. 22, no. 3, pp. 406–414, 2015.
View at: Publisher Site | Google Scholar
A. Manjamalai and B. Grace, “Antioxidant activity of essential oils from Wedelia chinensis (Osbeck) in vitro and in vivo lung cancer bearing C57BL/6 mice,” Asian Pacific Journal of Cancer Prevention, vol. 13, no. 7, pp. 3065–3071, 2012.
View at: Publisher Site | Google Scholar
I. Nomani, A. Mazumder, and G. S. Chakraborthy, “Wedelia chinensis (Asteraceae)-an overview of a potent medicinal herb,” International Journal of PharmTech Research, vol. 5, no. 3, pp. 957–964, 2013.
View at: Google Scholar
W.-C. Lin, C.-C Wen, Y.-H. Chen et al., “Integrative approach to analyze biodiversity and anti-inflammatory bioactivity of Wedelia medicinal plants,” PLoS One, vol. 10, no. 6, Article ID e0129067, 2015.
View at: Publisher Site | Google Scholar
A. Shirwaikar, R. G. Prabhu, and S. Malini, “Activity of Wedelia calendulacea Less. in post-menopausal osteoporosis,” Phytomedicine, vol. 13, no. 1-2, pp. 43–48, 2006.
View at: Publisher Site | Google Scholar
N. Verma and R. L. Khosa, “Effect of Costus speciosus and Wedelia chinensis on brain neurotransmitters and enzyme monoamine oxidase following cold immobilization stress,” Journal of Pharmaceutical Sciences, vol. 1, no. 2, pp. 22–25, 2009.
View at: Google Scholar
P. Murugaian, V. Ramamurthy, and N. Karmegam, “Hepatoprotective activity of Wedelia calendulacea against acute hepatotoxicity in rats,” Research Journal of Agriculture and Biological Sciences, vol. 4, no. 6, pp. 685–687, 2008.
View at: Google Scholar
T. Prakash, N. R. Rao, and A. H. M. V. Swamy, “Neuropharmacological studies on Wedelia calendulacea Less stem extract,” Phytomedicine, vol. 15, no. 11, pp. 959–970, 2008.
View at: Publisher Site | Google Scholar
R. Kusano, S. Ogawa, Y. Matsuo, T. Tanaka, Y. Yazaki, and I. Kouno, “α-Amylase and lipase inhibitory activity and structural characterization of acacia bark proanthocyanidins,” Journal of Natural Products, vol. 74, no. 2, pp. 119–128, 2011.
View at: Publisher Site | Google Scholar
E. A. H. Mohamed, M. J. A. Siddiqui, L. F. Ang et al., “Potent α-glucosidase and α-amylase inhibitory activities of standardized 50% ethanolic extracts and sinensetin from Orthosiphon stamineus Benth as anti-diabetic mechanism,” BMC Complementary and Alternative Medicine, vol. 12, no. 176, 2012.
View at: Publisher Site | Google Scholar
T. T. H. Hanh, N. M. Chau, L. H. Tram et al., “Inhibitors of α-glucosidase and α-amylase from Cyperus rotundus,” Pharmaceutical Biology, vol. 52, no. 1, pp. 74–77, 2014.
View at: Publisher Site | Google Scholar
M.S. Ali, M. Jahangir, S. S. Hussan, and M. I. Choudhary, “Inhibition of alpha-glucosidase by oleanolic acid and its synthetic derivatives,” Phytochemistry, vol. 60, no. 3, pp. 295–299, 2002.
View at: Publisher Site | Google Scholar
Q. Yu, K. Katsunami, H. Otsuka, and Y. Takeda, “Staphylionosides A-K: megastigmane glucosides from the leaves of Staphylea bumalda DC,” Chemical and Pharmaceutical Bulletin, vol. 53, no. 7, pp. 800–807, 2005.
View at: Publisher Site | Google Scholar
I. K. Lee, K. H. Kim, S. Y. Lee, S. U. Choi, and K. R. Lee, “Three new megastigmane glucopyranosides from the Cardamine komarovii,” Chemical and Pharmaceutical Bulletin, vol. 59, no. 6, pp. 773–777, 2011.
View at: Publisher Site | Google Scholar
I. Ohtani, T. Kusumi, Y. Kashman, and H. Kakisawa, “High-field FT NMR application of Mosher’s method. The absolute configurations of marine terpenoids,” Journal of the American Chemical Society, vol. 113, no. 11, pp. 4092–4096, 1991.
View at: Publisher Site | Google Scholar
J. M. Seco, E. Quiñoá, and R. Riguera, “The assignment of absolute configuration by NMR,” Chemical Reviews, vol. 104, no. 1, pp. 17–118, 2004.
View at: Publisher Site | Google Scholar
T. Hase, Y. Kawamoto, K. Ohtani, R. Kasai, K. Yamasaki, and C. Picheansoonthon, “Cyclohexylethanoids and related glucosides from Millingtonia hortensis,” Phytochemistry, vol. 39, no. 1, pp. 235–241, 1995.
View at: Publisher Site | Google Scholar
M. Honzumi, T. Kamikubo, and K. Ogasawara, “A stereocontrolled route to cyclohexylethanoid natural products,” Synlett, vol. 1998, pp. 1001–1003, 1998.
View at: Publisher Site | Google Scholar
H. Abdullahi, E. Nyandat, C. Galeffi, I. Messana, M. Nicoletti, and G. B. M. Bettolo, “Cyclohexanols of Halleria lucida,” Phytochemistry, vol. 25, no. 12, pp. 2821–2823, 1986.
View at: Publisher Site | Google Scholar
K. Ohtani, R. Kasai, K. Yamasaki et al., “Lignan glycosides from stems of Salvadora persica,” Phytochemistry, vol. 31, no. 7, pp. 2469–2471, 1992.
View at: Publisher Site | Google Scholar
A. Tapia, J. Rodriguez, C. Theoduloz, S. Lopez, G. E. Feresin, and G. Schmeda-Hirschmann, “Free radical scavengers and antioxidants from Baccharis grisebachii,” Journal of Ethnopharmacology, vol. 95, no. 2-3, pp. 155–161, 2004.
View at: Publisher Site | Google Scholar
D. L. Cheng and X. P. Cao, “Pomolic acid derivatives from the root of Sanguisorba officinalis,” Phytochemistry, vol. 31, no. 4, pp. 1317–1320, 1992.
View at: Publisher Site | Google Scholar
C. Hata, M. Kakuno, K. Yoshikawa, and S. Arihara, “Triterpenoid saponin of aquifoliaceous plants.V. Ilexosides XV-XIX from the barks of Ilex crenata Thunb,” Chemical and Pharmaceutical Bulletin, vol. 40, no. 8, pp. 1990–1992, 1992.
View at: Publisher Site | Google Scholar
D. F. Pereira, L. H. Cazarolli, C. Lavado et al., “Effects of flavonoids on α-glucosidase activity: potential targets for glucose homeostasis,” Nutrition, vol. 27, no. 11-12, pp. 1161–1167, 2011.
View at: Publisher Site | Google Scholar
A. H. Zeid, M. AliFarag, M. A. AzizHamed, Z. A. AzizKandil, R. HassanEl-Akad, and H. MohamedEl-Rafie, “Flavonoid chemical composition and antidiabetic potential of Brachychiton acerifolius leaves extract,” Asian Pacific Journal of Tropical Biomedicine, vol. 7, no. 5, pp. 389–396, 2017.
View at: Publisher Site | Google Scholar
P. V. A. Babu, D. Liu, and E. R. Gilbert, “Recent advances in understanding the anti-diabetic actions of dietary flavonoids,” Journal of Nutritional Biochemistry, vol. 24, no. 11, pp. 1777–1789, 2013.
View at: Publisher Site | Google Scholar

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Copyright © 2018 Nguyen Phuong Thao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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