Components with Anti-Diabetic Activity Isolated from the Leaves and Twigs of Glycosmis pentaphylla Collected in Vietnam

Abstract

A phytochemical investigation of the leaves and twigs of Glycosmis pentaphylla (Rutaceae), collected in Vietnam, yielded three new compounds named glyfuran (1), glyphyllamide (2), and glyphyllazole (3), along with twenty-five known compounds (4–28). The structures of isolates were determined by IR, MS, NMR, and UV data analyses. In the anti-diabetic activity screening, (+)-isoaltholacton (4), glycoborinine (17), 2′,4′-dihydroxy-4,6′-dimethoxychalcone (24), and flavokawain A (25) simultaneously exhibited inhibition of dipeptidyl peptidase-4 (DPP4) and stimulation of the glucagon-like peptide-1 (GLP-1) secretion on the murine intestinal secretin tumor cell line (STC-1).

1. Introduction

Diabetes mellitus is a metabolic disease characterized by excessive amounts of blood sugar, leading to a family of diseases that includes hypertension, osteoporosis, retinopathy, and urethritis. Along with the increasing popularity of a sedentary lifestyle, diabetes has gradually become a global epidemic. According to the World Health Organization, approximately 422 million people worldwide suffer from this disease, with up to 1.5 million dying yearly [1]. Owing to the eating habits of East Asians (rice is the staple food), the prevalence of diabetes is much higher than in other areas of the world. It is estimated that 8.5% of adults in Taiwan are afflicted with this disease, and the number of patients is predicted to increase in the future [2]. Anyway, the treatment of many diabetes complications is very costly and has become an economic burden for governments everywhere. Moreover, people with type 2 diabetes are twice as likely to develop liver or pancreatic cancer [3,4].

The leading causes of diabetes are the pancreas being unable to produce enough insulin (juvenile diabetes) or when the body becomes resistant to insulin (adult-onset diabetes). There is no cure for diabetes, but it can be treated and controlled, with treatment being divided into two kinds: subcutaneous insulin injection and oral hypoglycemic drugs. Oral hypoglycemic drugs induce effects that include the promotion of insulin secretion (Diamicron) with an increase in insulin receptor sensitivity (Metformin), α-glucosidase inhibitors (Acarbose), and dipeptidase-4 (DDP-4) inhibitors (Galvus). These medications often cause unpleasant side effects such as flatulence, diarrhea, hypoglycemia, weight gain, and even liver toxicity [1]. As a result, many people are turning to herbal medicines to treat diabetes [5]. So far, many Traditional Chinese Medicines or folk herbs have shown significant effects on lowering blood sugar or reducing the side effects of western medicines. For example, Gastrodia elata water extract can increase the quality of islet β cells and reduce cell apoptosis [6]; polyacetylene from Bidens pilosa var. radiata can decrease blood sugar and increase insulin release [7], while the polysaccharides in the flower buds of Lonicera japonica can lower blood sugar [8]. In terms of pure compounds, iminosugars and sugar derivatives were regarded as important antidiabetic agents [9,10,11,12,13].

Glucagon-like peptide-1 (GLP-1) is an incretin that can decrease blood sugar levels in a glucose-dependent manner by enhancing insulin secretion; moreover, the action of GLP-1 is preserved in patients with type 2 diabetes, and substantial pharmaceutical research has therefore been directed towards developing GLP-1-based treatment. However, endogenous GLP-1 is rapidly degraded primarily by dipeptidyl peptidase-4 (DPP-4) [14]. In our pre-screenings for anti-diabetes, the extract of Glycosmis pentaphylla (Retz.) DC. was able to simultaneously inhibit DPP-4 and stimulate the secretion of GLP-1 (Figure S1) in a dose-dependent manner (Figure S2), so G. pentaphylla has been proven to have the potential for development of an anti-diabetes drug and was accordingly selected for the following studies.

G. pentaphylla, a species of plant belonging to the family Rutaceae, is a shrub or small tree, 1.5–5.0 m high, and is widely distributed over India, Malaysia, Southern China, and the Philippine Islands to Vietnam. This plant is a Traditional Chinese Medicine (TCM) that can strengthen the stomach and relieve pain. In Vietnam, the roots, leaves, and branches of G. pentaphylla are collected year-round to relieve pain, to treat rheumatism, body aches, boils, impetigo, and snakebite. It is also used for postpartum women to cure uterine bleeding, eating indigestion, and abdominal distention [15,16]. Herein, the details of extraction, isolation, structural elucidation, anti-diabetes, and cytotoxicity of isolated compounds are described.

2. Results

The air-dried twigs and leaves of G. pentaphylla were extracted with 95% ethanol. After partition and column chromatography, three new compounds named glyfuran (1), glyphyllamide (2), and glyphyllazole (3), along with 25 known compounds, (+)-isoaltholactone (4) [17], (+)-altholactone (5) [18], 6R-goniothalamin (6) [19], (6R,7R,8S)-8-chlorogoniodiol (7) [20], (6R,7S,8R)-8-chlorogoniodiol (8) [21], 7-epi-(+)-gonidiol (9) [22], 8-epi-(+)-goniodiol (10) [23], (6S,7S,8S)-goniodiol (11) [24], (+)-9-deoxygoniopypyrone (12) [25], (−)-8-epi-9-deoxygoniopyrone (13) [25], leiocarpin C (14) [26], dictamine (15) [27], 2-hydroxy-6,8-dimethoxy-3-methyl-9H-carbazole (16) [28], glycoborinine (17) [29], N-(4-hydroxyphenethyl)cinnamamide (18) [30], uvariadiamide (19) [31], alpinetin (20) [32], tsugafolin (21) [33], naringenin trimethyl ether (22) [33], 4′,6′-dihyroxy-2′,4-dimethoxydihydrochalcone (23) [34], 2′,4′-dihyroxy-4,6′-dimethoxychalcone (24) [35], flavokawain A (25) [36], glycothiomin-A (26) [37], penangin (27) [38], and ellipeiopsol B (28) [39], were obtained (Figure 1).

Compound 1 was observed as a white amorphous powder with ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ + 54 (c 0.05, MeOH). The molecular formula C₁₇H₂₂O₅ (seven indices of hydrogen deficiency) of 1 was deduced from a sodium adduct peak at m/z 329.13605 in the HRESIMS. The IR spectrum indicated the presence of hydroxy (3380 cm⁻¹) and ester carbonyl (1684 cm⁻¹) groups. The ¹H NMR data (Table 1) of 1 clearly demonstrated the presence of one mono-substituted benzene ring [δ_H 7.29 (m, H-4), 7.36 (2H, m, H-3/H-5), 7.39 (2H, m, H-2/H-6)], two olefinic protons [δ_H 6.00 (dd, J = 11.8, 1.8 Hz, H-12), δ_H 6.46 (dd, J = 11.8, 6.5 Hz, H-11)], four oxymethines [δ_H 4.16 (t, J = 5.0 Hz, H-8), δ_H 4.60 (t, J = 5.0 Hz, H-9), 5.00 (d, J = 5.6 Hz, H-7), and 5.65 (ddd, J = 6.5, 5.6, 1.8 Hz, H-10)], and one methyl group [δ_H 0.95 (t, J = 7.5 Hz, H-4′)]. The ¹³C NMR and DEPT spectra (Table 1) exhibited seventeen carbons, containing one ester group (δ_C 167.2), one olefinic quaternary carbon (δ_C 140.2), seven olefinic methines (δ_C 120.9, 125.6, 125.6, 127.8, 128.5, 128.5, and 148.2), four oxymethines (δ_C 73.7, 78.7, 79.0, and 84.0), one oxymethylene (δ_C 64.8), two methylenes (δ_C 19.1 and 30.6), and one methyl group (δ_C 13.6).

In the COSY spectrum (Figure 2) of 1, three proton sequences of H-2/H-3/H-4/H-5/H-6, H-7/H-8/H-9/H-10/H-11/H-12, and H₂-1′ (δ_H 4.15)/H₂-2′ (δ_H 1.66)/H₂-3′ (δ_H 1.40)/H₃-4′ (δ_H 0.95) were observed. The proton spin-spin coupling systems of H-2/H-3/H-4/H-5/H-6 and the HMBC correlations (Figure 2) of H-7 to C-1 (δ_C 140.2) and H-2/H-6 to C-7 (δ_C 84.0) revealed the presence of a monosubstituted benzene ring (ring A), while the sequences of H-7/H-8/H-9/H-10 and the HMBC correlation from H-10 to C-7 (δ_C 84.0) suggested 1 contained a tetrahydrofuran ring (ring B). The n-butyl ester attached at C-12 was constructed by the COSY consequences H₂-1′/H₂-2′/H₂-3′/H₃-4′ and the HMBC correlation from H-11, H-12, and H-1′ to C-13 (δ_C 167.2). On the basis of these definitive 2D NMR analyses, the planar structure of 1 was established.

Moreover, in the NOESY spectrum of 1, the presence of NOESY correlations (Figure 2) between H-11 and H-12 validated the cis conformation of Δ₁₁. The NOESY cross-peaks of H-8/H-9/H-10 suggested they were β-orientated; on the other hand, the NOESY correlations of H-1/H-7 revealed these protons were α-orientated.

Additionally, compound 4 is the major component of G. Pentaphylla (0.006%), suggesting compound 1 might be derived from 4 (Figure 3). After hydrolysis, 4 might become intermediate A, and A was esterificated to form 1. Through comparison between compounds 1 and 4, the absolute configuration of 1 was established and assigned the trivial name glyfuran.

Compound 2 was isolated as a white amorphous powder with ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ − 10 (c 0.05, MeOH). The high-resolution ESIMS data showed a protonated molecule peak at [M + H]⁺ at m/z 336.18067, which indicated the molecular formula of C₁₈H₂₅NO₅ and seven degrees of unsaturation. The IR spectrum of 2 showed absorption bands at 1736 and 1671 cm⁻¹, revealing the presence of ester and amide functionality. The ¹H NMR data (Table 2) of 2 possessed the signals of one monosubstituted benzene ring [δ_H 7.29 (2H, m, H-3/H-5), 7.31 (m, H-4), and 7.37 (2H, m, H-2/H-6)], two methylenes [δ_H 4.02 (q, J = 7.1 Hz, H-1‴) and δ_H 4.19 (q, J = 7.1 Hz, H-1″)], and two methyl groups [δ_H 0.93 (t, J = 7.4 Hz, H-4‴) and δ_H 1.24 (t, J = 7.1 Hz, H-2″)]. The ¹³C NMR and DEPT spectra exhibited eighteen carbons, including two ester carbonyls (δ_C 170.5 and 170.8), one amide carbonyl (δ_C 170.7), one olefinic quaternary carbon (δ_C 134.4), five olefinic methines (δ_C 127.4, 128.9, 128.9, 129.3, and 129.3), two oxymethylenes (δ_C 61.8 and 64.9), one N-bearing methine (δ_C 48.6), four methylenes (δ_C 19.0, 36.2, 30.4, and 43.6), and two methyl groups (δ_C 13.6 and 14.0). The planar structure of 2 was elucidated by dividing it into three partial structures, X, Y, and Z (Figure 2). A proton spin system of H-2 (δ_H 7.37)/H-3 (δ_H 7.29)/H-4 (δ_H 7.31)/H-5 (δ_H 7.29)/H-6 (δ_H 7.37) was observed from the COSY spectrum. In the HMBC spectrum, H₂-7 (δ_H 3.62) showed correlations to C-1 (δ_C 134.4) and C-8 (δ_C 170.7), and the correlation from H-2/H-6 to C-1 revealed the presence of partial structure X. For the partial structure Y, a butyl group attached to an ester group was identified by the COSY correlations of H-1‴ (δ_H 4.02)/H-2‴ (δ_H 1.54)/H-3‴ (δ_H 1.33)/H-4‴ (δ_H 0.93) and the HMBC correlations from H₂-3′ (δ_H 2.82 and 3.00) and H-1‴ to C-4′ (δ_C 170.8). Additionally, the HMBC correlations from H-1″ (δ_H 4.19) to C-1′ (δ_C 170.5), along with the proton spin system between H-1″ (δ_H 4.19) and H-2″ (δ_H 1.24) established the partial structure Z. These three parts were connected by the HMBC correlations from H-2′ (δ_H 4.83) to C-8, C-1′, and C-4′. According to the above 2D NMR analyses, the planar structure of 2 was established (Figure 2). Structurally, 2 showed similar ¹H and ¹³C NMR data to a synthetic compound N-PhAc-_D-Asp(OEt)OEt [40]. In addition, the negative optical rotation values of 2 (−10) and N-PhAc-_D-Asp(OEt)OEt (−39) suggested the R configuration of C-2′.

Compound 3 was isolated as a brown-yellow solid and had a molecular formula of C₁₅H₁₅NO₃ based on HRESIMS data (m/z 280.09466 [M + Na]⁺), accounting for nine degrees of unsaturation. The IR spectrum showed the absorption bands due to hydroxy (3412 cm⁻¹), amide (1625 cm⁻¹), and phenyl (1512 and 1443 cm⁻¹) functional groups. The UV spectrum displayed absorption maxima at 307 and 259 nm; therefore, 3 was defined to have a carbazole skeleton [41]. The ¹H NMR spectrum (Table 3) of 3 showed the presence of three singlet protons, one for a phenolic hydroxy group at δ_H 7.94 (brs, 2-OH) and the other two for aromatic methines at δ_H 6.83 (s, H-1) and 7.67 (s, H-4), as well as a couple of aromatic protons [δ_H 6.84 (d, J = 8.5 Hz, H-6) and 7.56 (d, J = 8.5 Hz, H-5)]. The ¹H NMR spectrum also displayed a methyl at δ_H 2.39 (s, 3-Me), along with two methoxy groups at δ_H 3.96 (s, 7-OMe) and δ_H 4.00 (s, 8-OMe). The ¹³C NMR exhibited the signals of fifteen carbons, being twelve aromatic carbons (δ_C 96.8, 106.2, 114.3, 116.2, 117.8, 119.5, 121.4, 133.7, 134.0, 139.6, 149.4, and 152.5), one methyl carbon (δ_C 16.1), and two methoxy carbon groups (δ_C 56.9 and 60.9). The COSY correlation showed one fragment of H-5/H-6. In the HMBC experiment, ³J correlations from δ_H 3.96 to δ_C 149.4 (C-7) and from δ_H 4.00 to δ_C 133.7 (C-8) indicated the positions of two methoxy groups. In addition, the correlations from 3-Me to C-2 (δ_C 152.5), C-3 (δ_C 116.2), and C-4 (δ_C 121.4) revealed the position of a methyl group. The HMBC correlations from H-4 to C-5a (δ_C 119.5)/C-1a (δ_C 139.6) and from H-5 to C-4a (δ_C 117.8) were used to construct the pyrrole moiety. Therefore, compound 3 was determined to be 7,8-dimethoxy-3-methyl-9H-carbazol-2-ol, and the trivial name glyphyllazole was given.

The anti-diabetic activity of isolated secondary metabolites was evaluated by stimulating the secretion of GLP-1 and inhibiting DPP-4. As shown in Figure 4A, compounds 1, 4, 6, 7, 17, 24, and 25 exhibited stimulatory effects on GLP-1 secretion from murine intestinal secretin tumor cell line (STC-1) at a concentration of 100 μM. In the acute toxicity evaluation (Figure 4B), except for compound 7, all tested compounds retained the cell viability of STC-1 cells at the concentration of 100 μM. On the other hand, all tested compounds provided inhibitory effects on DPP-4 enzyme activities (Figure 4C); significantly, compounds 2–4, 9, 10, 16–18, 20, 24, and 25 demonstrated inhibition rates over 50%.

3. Materials and Methods

3.1. General Experimental Procedures

The optical rotations, UV, and IR spectra were recorded on a Jasco P-2000 digital polarimeter, a Jasco V-530 UV/VIS spectrophotometer, and a Jasco FT-IR 4600 spectrometer, respectively. NMR spectra data were corrected in CDCl₃ (δ_H 7.26 and δ_C 77.0) using solvent peaks as the internal standard on Bruker AVIII HD 700X NMR and Varian VNMRS 600 MHz spectrometers. HRESIMS data were obtained by using a Bruker 7T solariX spectrometer. Column chromatography was performed using Merck silica gel 60 (0.040–0.063 mm), C₁₈ silica gel (0.040–0.075 mm), and Sephadex LH-20 material. NP-HPLC was composed of a Hitachi L-6000 pump and a Hitachi L-4000 UV detector with Phenomenex Luna silica (5 μm, 250 × 10 mm) and CN (5 μm, 250 × 10 mm) columns. RP-HPLC was carried out on a Shimadzu chromatography system consisting of a LC-20AT pump, a SPD-M20A PDA detector, and a CBM-20A system controller with Phenomenex Luna C₁₈ (5 μm, 250 × 10 mm), phenyl-hexyl (5 μm, 250 × 10 mm), and Kinetex biphenyl (5 μm, 250 × 10 mm) columns.

3.2. Plant Material

The leaves and twigs of G. pentaphylla were purchased in July 2018 from Cu Lao Cham Island (Hoi An City, Quang Nam province, Vietnam) by Associate Professor Quang Vinh Nguyen (Institute of Biotechnology and Environment, Tay Nguyen University, Vietnam) and Associate Professor Chia-Hung Yen (Graduate Institute of Natural Products, Kaohsiung Medical University, Taiwan). A voucher specimen (code No. KMU-VN016) was deposited at Kaohsiung Medical University.

3.3. Extraction and Isolation

The dried leaves and twigs of G. pentaphylla (0.7 kg) were extracted by 95% EtOH (5 L × 3, each for 3 days) at room temperature to give an ethanolic extract, which was then partitioned between EtOAc and H₂O (1:1) to provide an EtOAc layer. The EtOAc layer was further partitioned by hexanes/MeOH/H₂O (4:3:1) to obtain a 75% MeOH_(aq) layer (13.3 g). This methanol extract was subjected to a silica gel flash column (hexanes/CH₂Cl₂/MeOH, 60/10/1 to 0/0/1) to afford subfractions GP-1 to GP-6. GP-3 (5.2 g) was separated by a silica gel open column stepwise-eluted with hexanes/EtOAc (10/1 to 0/1) and EtOAc/MeOH (5/1 to 0/1) to yield 18 subfractions (GP-3-1 to GP-3-18). GP-3-7 (80.5 mg) was subjected to a Sephadex LH-20 open column eluting with CH₂Cl₂/MeOH (1/1) to obtain four fractions (GP-3-7-1 to GP-3-7-4). Compound 25 (2.1 mg) was purified from GP-3-7-4 (17.8 mg) by semi-preparative RP-HPLC (phenyl-hexyl column, flow = 2.0 mL/min, 65% MeCN_(aq), isocratic, 360 nm). GP-3-10 (342.6 mg) was applied to chromatography on a silica gel open column (hexanes/CH₂Cl₂/MeOH, 1/1/0 to 0/0/1) to give 6 (32.5 mg) and fractions GP-3-10-2 to GP-3-10-4. GP-3-10-3 (220.9 mg) was further separated on a Sephadex LH-20 column (CH₂Cl₂/MeOH, 1/1) to afford seven fractions (GP-3-10-3-1 to GP-3-10-3-7). GP-3-10-3-5 (12.4 mg) was isolated by semi-preparative NP-HPLC (silica column, flow = 2.0 mL/min, hexanes/CH₂Cl₂/MeOH = 60/10/1, isocratic, 280 nm) to obtain 2 (3.1 mg). Compounds 1 (2.5 mg), 15 (1.1 mg), and 23 (38.1 mg) were purified from GP-3-10-3-6 (108.4 mg) by repeated semi-preparative NP-HPLC (silica and CN columns, flow = 2.0 mL/min, hexanes/CH₂Cl₂/MeOH = 70/10/1, isocratic, 280 nm). GP-3-11 (1.8 g) was fractionated by a Sephadex LH-20 column eluted with CH₂Cl₂/MeOH (1/1) to give five fractions (GP-3-11-1 to GP-3-11-5). GP-3-11-3 (167.3 mg) was subjected to semi-preparative RP-HPLC (C₁₈ column, flow = 2.0 mL/min, 30% MeCN_(aq), isocratic, 280 nm) to afford 4 (38.6 mg), 5 (4.0 mg), 7 (12.9 mg), 8 (1.5 mg), and 18 (1.4 mg). GP-3-11-4 (237.2 mg) was isolated by semi-preparative RP-HPLC (phenyl-hexyl column, flow = 2.0 mL/min, 50% MeCN_(aq), isocratic, 280 nm) to yield 21 (1.4 mg) and 24 (43.2 mg). GP-3-11-5 (23.1 mg) was purified by semi-preparative RP-HPLC (C₁₈ column, flow = 2.0 mL/min, 50% MeCN_(aq), isocratic, 280 nm) to obtain 3 (3.5 mg), 16 (1.9 mg), and 17 (2.9 mg). GP-3-12 (315.4 mg) was subjected to Sephadex LH-20 column eluted with CH₂Cl₂/MeOH (1/1) to obtain five fractions (GP-3-12-1 to GP-3-12-5). Compound 22 (6.2 mg) was purified from GP-3-12-3 (55.8 mg) by semi-preparative RP-HPLC (C₁₈ column, flow = 2.0 mL/min, 60% MeCN_(aq), isocratic, 280 nm). Compound 28 (1.7 mg) was isolated from GP-3-12-4 (84.1 mg) by semi-preparative RP-HPLC (phenyl-hexyl column, flow = 2.0 mL/min, 30% MeCN_(aq), isocratic, 210 and 280 nm). GP-3-13 (218.5 mg) was processed using a Sephadex LH-20 open column eluted with CH₂Cl₂/MeOH (1/1). GP-3-13-2 (153.3 mg) was fractionated over C₁₈ open column (H₂O/MeOH, 100/0 to 0/100) to give GP-3-13-2-1 to GP-3-13-2-5. Compounds 11 (15.3 mg) and 13 (4.0 mg) were obtained from GP-3-13-2-1 (24.1 mg) by semi-preparative RP-HPLC (phenyl-hexyl column, flow = 2.0 mL/min, 20% MeCN_(aq), isocratic, 254 nm). Compounds 10 (12.7 mg) and 14 (13.5 mg) were isolated from GP-3-13-2-2 (65.2 mg) by repeated semi-preparative RP-HPLC (phenyl-hexyl and biphenyl columns, flow = 2.0 mL/min, 25% MeCN_(aq), isocratic, 254 nm). GP-3-13-2-3 (17.5 mg) was purified by semi-preparative RP-HPLC (phenyl-hexyl column, flow = 2.0 mL/min, 40% MeOH_(aq), isocratic, 254 nm) to yield 9 (4.7 mg). GP-3-13-2-4 (13.7 mg) was isolated by semi-preparative RP-HPLC (phenyl-hexyl column, flow = 2.5 mL/min, 60% MeOH_(aq), isocratic, 280 nm) to afford 20 (3.2 mg). GP-3-14 (282.1 mg) was subjected to Sephadex LH-20 (CH₂Cl₂/MeOH, 1/1) and ODS (H₂O/MeOH, 100/0 to 0/100) open columns, respectively. Compound 12 (2.9 mg) was purified from GP-3-14-2-2 (18.8 mg) by semi-preparative RP-HPLC (C₁₈ column, flow = 2.0 mL/min, 30% MeCN_(aq), isocratic, 254 nm). GP-3-15 (364.1 mg) was chromatographed by a Sephadex LH-20 open column eluted with CH₂Cl₂/MeOH (1/1) to obtain three fractions (GP-3-15-1 to GP-3-15-3). GP-3-15-3 (163.5 mg) was applied to a C₁₈ gel open column (H₂O/MeOH, 100/0 to 0/100) to obtain fractions GP-3-15-3-1 to GP-3-15-3-5. Compounds 26 (18.8 mg) and 27 (44.8 mg) were purified by semi-preparative RP-HPLC (biphenyl column, flow = 2.0 mL/min, 10% MeCN_(aq), isocratic, 280 nm). GP-3-15-3-3 (18.6 mg) was isolated by semi-preparative RP-HPLC (C₁₈ column, flow = 2.0 mL/min, 30% MeCN_(aq), isocratic, 280 nm) to give 19 (8.8 mg).

Glyfuran (1): White amorphous powder; ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ + 54 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 276 (3.23), 226 (3.20) nm; IR (KBr) v_max 3380, 2923, 2853, 1684, 1456, 1259, 1192, 1026 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 1; HRESIMS m/z 329.13605 [M + Na]⁺ (calcd for C₁₇H₂₂NaO₅, 329.13594).

Glyphyllamide (2): White amorphous powder; ${\left[\alpha \right]}_{\mathrm{D}}^{24}$ − 10 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 285 (2.83), 213 (3.53) nm; IR (KBr) v_max 2925, 2854, 1736, 1671, 1457, 1179 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 2; HRESIMS m/z 336.18067 [M + H]⁺ (calcd for C₁₈H₂₆NO₅, 336.18055).

Glyphyllazole (3): Brown-yellow solid; UV (MeOH) λ_max (log ε) 307 (3.80), 259 (3.96), 237 (2.23), 212 (4.0) nm; IR (KBr) v_max 3142 (OH), 2925, 1626, 1513, 1443, 1296, 1229, 1140, 1068 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 3; HRESIMS m/z 280.09468 [M + Na]⁺ (calcd for C₁₅H₁₅NNaO₃, 280.09441).

3.4. Anti-Diabetic Assays

3.4.1. Cell Culture

Murine enteroendocrine cell line (STC-1) purchased from American Type Culture Collection (ATCC) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 15% (v/v) horse serum (HS) and 5% (v/v) fetal bovine serum (FBS).

3.4.2. GLP-1 Secretion

Cells were seeded into a 24-well plate at 1.5 × 10⁵ cells/well. After 72 h, cells were washed with glucose-free DMEM at 0.1% BSA three times and then replaced with DMEM (5.5 mM glucose) with/without VN016 or testing compound preparations for 1 h. At the end of treatment, the supernatant was collected and measured for GLP-1 using an active GLP-1 assay kit (Cisbio). The viability of cells after extract or compound treatment was measured by neutral red assay according to the previous description [42].

3.4.3. DPP-IV Activity Assay

According to the manufacturer’s instructions, the measurement of the activity and potential inhibition of DPP-IV, a type II membrane glycoprotein, was performed using the DPP-IV GloTM Protease Assay (Promega).

4. Conclusions

As part of an ongoing program to search for bioactive compounds from Vietnam’s medicinal plants, a chemical study on the leaves and twigs of G. pentaphylla has been carried out, resulting in the purification of three new (1−3) and twenty-five known (4–28) compounds. This research led to the isolation of eleven styryl-lactones (4–14) and five alkaloids (15–19), which proved the genus Glycosmis is a rich source of both alkaloid and styryl-lactone. In anti-diabetic evaluation, compounds 4, 17, 24, and 25 displayed dual activities by stimulating GLP-1 secretion while inhibiting DPP-4 (which can rapidly degrade GLP-1) without cytotoxicity in STC-1 cells. Consequently, our pharmacological data supports G. pentaphylla as being a folk medicine with a hypoglycemic effect.

Author Contributions

Conception and design of the work: I.-C.H. and Y.-B.C.; purification, analysis and interpretation of the NMR data, structure elucidation, and manuscript preparation: M.T.T.N., Y.-C.L., S.-R.C. and F.-R.C.; bioactive assay: H.-K.L.; critical revision of the manuscript: Y.-B.C. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and in the Supplementary Materials.

Acknowledgments

The authors wish to thank Quang Vinh Nguyen (Institute of Biotechnology and Environment, Tay Nguyen University, Buon Ma Thuot, Vietnam) for purchasing the plant material from Vietnam.

Conflicts of Interest

The authors declare that they have no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

Figure 1. Structures of compounds 1−28.

Figure 2. Key COSY (bold), HMBC (arrow), and NOESY (double arrow) correlations of 1–3.

Figure 3. The plausible biosynthetic pathway of 1 from 4.

Figure 4. The anti-diabetes effect of isolated compounds in STC-1 cells. (A) GLP-1 secretory effects of the methanol layer of G. pentaphylla (VN16) and isolated compounds. (B) Acute toxicity evaluation of the methanol layer of G. pentaphylla (VN16) and isolated compounds in STC-1 cells. (C) The effects of the methanol layer of G. pentaphylla (VN16) and isolated compounds on DPP4. Data represent mean ± SEM (n = 3–4). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test. * p < 0.05, *** p < 0.001 when compared to None.

Table 1

¹H NMR (600 MHz) and ¹³C NMR (150 MHz) spectroscopic data of 1 in CDCl₃.

No.	1
	δH (Mult, J in Hz)	δC, Type
1	–	140.2, C
2	7.39, m	125.6, CH
3	7.36, m	128.5, CH
4	7.29, m	127.8, CH
5	7.36, m	128.5, CH
6	7.39, m	125.6, CH
7	5.00, d (5.6)	84.0, CH
8	4.16, t (5.0)	78.7, CH
9	4.60, t (5.0)	73.7, CH
10	5.65, ddd (6.5, 5.6, 1.8)	79.0, CH
11	6.46, dd (11.8, 6.5)	148.2, CH
12	6.00, dd (11.8, 1.8)	120.9, CH
13	–	167.2, C
1′	4.15, t (5.0)	64.8, CH2
2′	1.66, m	30.6, CH2
3′	1.40, m	19.1, CH2
4′	0.95, t (7.5)	13.6, CH3

Table 2

¹H NMR (700 MHz) and ¹³C NMR (175 MHz) spectroscopic data of 2 in CDCl₃.

No.	2
	δH (Mult, J in Hz)	δC, Type
1	–	134.4, C
2	7.37, m	128.9, CH
3	7.29, m	129.3, CH
4	7.31, m	127.4, CH
5	7.29, m	129.3, CH
6	7.37, m	128.9, CH
7	3.62, s	43.6, CH2
8	–	170.7, C
1′	–	170.5, C
2′	4.83, m	48.6, CH
3′	2.82, dd (17.0, 4.6)	36.2, CH2
	3.00, dd (17.0, 4.3)
4′	–	170.8, C
1″	4.19, q (7.1)	61.8, CH2
2″	1.24, t (7.1)	14.0, CH3
1‴	4.02, t (7.1)	64.9, CH2
2‴	1.54, m	30.4, CH2
3‴	1.33, m	19.0, CH2
4‴	0.93, t (7.4)	13.6, CH3
NH	6.46, d (7.6)	–

Table 3

¹H NMR (600 MHz) and ¹³C NMR (150 MHz) spectroscopic data of 3 in CDCl₃.

No.	3
	δH (Mult, J in Hz)	δC, Type
1	6.83, s	96.8, CH
1a	–	139.6, C
2	–	152.5, C
3	–	116.2, C
4	7.67, s	121.4, CH
4a	–	117.8, C
5	7.56, d (8.5)	114.3, CH
5a	–	119.5, C
6	6.84, d (8.5)	106.2, CH
7	–	149.4, C
8	–	133.7, C
8a	–	134.0, C
2-OH	7.94, br s	–
3-Me	2.39, s	16.1, CH3
7-OMe	3.96, s	56.9, CH3
8-OMe	4.00, s	60.9, CH3

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph15121543/s1, Figure S1: Preliminary screening of dual biological activities related to modulation of GLP-1 levels; Figure S2: dose-dependent effects of VN016-MeOH; Figures S3–S27: HR-ESI-MS, IR, NMR (¹H, ¹³C, COSY, HSQC, HMBC, and NOESY), and UV spectra of compounds 1–3.

References

1. World Health Organization. Diabetes. Available online: https://www.who.int/health-topics/diabetes#tab=tab_1 (accessed on 25 August 2020).

2. Health Promotion Administration, Ministry of Health and Welfare. The Information about the Diabetes Patients in Taiwan. Available online: https://www.hpa.gov.tw/Pages/List.aspx?nodeid=359 (accessed on 25 August 2020).

3. Coughlin, S.S.; Calle, E.E.; Teras, L.R.; Petrelli, J.; Thun, M.J. Diabetes mellitus as a predictor of cancer mortality in a large cohort of US adults. Am. J. Epidemiol.; 2004; 159, pp. 1160-1167. [DOI: https://dx.doi.org/10.1093/aje/kwh161] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15191933]

4. Li, D.; Yeung, S.C.; Hassan, M.M.; Konopleva, M.; Abbruzzese, J.L. Antidiabetic therapies affect risk of pancreatic cancer. Gastroenterology; 2009; 137, pp. 482-488. [DOI: https://dx.doi.org/10.1053/j.gastro.2009.04.013] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19375425]

5. Covington, M.B. Traditional Chinese Medicine in the treatment of diabetes. Diabetes Spectr.; 2001; 14, pp. 154-159. [DOI: https://dx.doi.org/10.2337/diaspect.14.3.154]

6. Yang, H.J.; Kim, M.J.; Kwon, D.Y.; Kim, D.S.; Lee, Y.H.; Kim, J.E.; Park, S. Anti-diabetic activities of Gastrodia elata Blume water extracts are mediated mainly by potentiating glucose-stimulated insulin secretion and increasing β-cell mass in non-obese type 2 diabetic animals. Nutrients; 2016; 8, 161. [DOI: https://dx.doi.org/10.3390/nu8030161]

7. Chien, S.C.; Young, P.H.; Hsu, Y.J.; Chen, C.H.; Tien, Y.J.; Shiu, S.Y.; Li, T.H.; Yang, C.W.; Marimuthu, P.; Tsai, L.F. et al. Anti-diabetic properties of three common Bidens pilosa variants in Taiwan. Phytochemistry; 2009; 70, pp. 1246-1254. [DOI: https://dx.doi.org/10.1016/j.phytochem.2009.07.011]

8. Wang, D.; Zhao, X.; Liu, Y. Hypoglycemic and hypolipidemic effects of a polysaccharide from flower buds of Lonicera japonica in streptozotocin-induced diabetic rats. Int. J. Biol. Macromol.; 2017; 102, pp. 396-404. [DOI: https://dx.doi.org/10.1016/j.ijbiomac.2017.04.056]

9. Nash, R.J.; Kato, A.; Yu, C.-Y.; Fleet, G.W.J. Iminosugars as therapeutic agents: Recent advances and promising trends. Future Med. Chem.; 2011; 3, pp. 1513-1521. [DOI: https://dx.doi.org/10.4155/fmc.11.117]

10. Yang, L.-F.; Shimadate, Y.; Kato, A.; Li, Y.-X.; Jia, Y.-M.; Fleet, G.W.J.; Yu, C.-Y. Synthesis and glycosidase inhibition of N-substituted derivatives of DIM. Org. Biomol. Chem.; 2020; 18, pp. 999-1011. [DOI: https://dx.doi.org/10.1039/C9OB02029B]

11. Chennaiah, A.; Dahiya, A.; Dubbu, S.; Vankar, Y.D. A stereoselective synthesis of an imino glycal: Application in the synthesis of (−)-1-epi-adenophorine and a homoiminosugar. Eur. J. Org. Chem.; 2018; 2018, pp. 6574-6581. [DOI: https://dx.doi.org/10.1002/ejoc.201801241]

12. Chennaiah, A.; Bhowmick, S.; Vankar, Y.D. Conversion of glycals into vicinal-1,2-diazides and 1,2-(or 2,1)-azidoacetates using hypervalent iodine reagents and Me₃SiN₃. Application in the synthesis of N-glycopeptides, pseudo-trisaccharides and an iminosugar. RSC Adv.; 2017; 7, pp. 41755-41762. [DOI: https://dx.doi.org/10.1039/C7RA08637G]

13. Rajasekaran, P.; Ande, C.; Vankar, Y.D. Synthesis of (5,6 & 6,6)-oxa-oxa annulated sugars as glycosidase inhibitors from 2-formyl galactal using iodocyclization as a key step. ARKIVOC; 2022; 2022, pp. 5-23.

14. Green, B.D.; Liu, H.K.; McCluskey, J.T.; Duffy, N.A.; O’Harte, F.P.; McClenaghan, N.H.; Flatt, P.R. Function of a long-term, GLP-1-treated, insulin-secreting cell line is improved by preventing DPP IV-mediated degradation of GLP-1. Diabetes Obes. Metab.; 2005; 7, pp. 563-569. [DOI: https://dx.doi.org/10.1111/j.1463-1326.2004.00430.x]

15. Ahmed, I.; Islam, R.; Sikder, M.A.A.; Haque, M.R.; Al Mansur, M.A.; Rashid, M.A. Alkaloid, sterol and triterpenoids from Glycosmis pentaphylla (Retz.) DC. Dhaka Univ. J. Pharm. Sci.; 2014; 13, pp. 115-118. [DOI: https://dx.doi.org/10.3329/dujps.v13i2.21887]

16. Do, H.B.; Dang, Q.C.; Bui, X.C.; Nguyen, T.D.; Do, T.D.; Pham, V.H.; Vu, N.L.; Pham, D.M.; Pham, K.M.; Doan, T.N. et al. Medicinal Plants and Medicinal Animals in Vietnam Episode 1 (Cây thuốc và động vật làm thuốc ở Việt Nam tập 1); Science and Technology Press: Hanoi, Vietnam, 2006; 541.

17. Colegate, S.M.; Din, L.B.; Latiff, A.; Salleh, K.M.; Samsudin, M.W.; Skelton, B.W.; Tadano, K.; White, A.H.; Zakaria, Z. (+)-Isoaltholactone: A furanopyrone isolated from Goniothalamus species. Phytochemistry; 1990; 29, pp. 1701-1704. [DOI: https://dx.doi.org/10.1016/0031-9422(90)80153-8]

18. Prasad, K.R.; Gholap, S.L. Stereoselective total synthesis of bioactive styryllactones (+)-goniofufurone, (+)7-epi-goniofufurone, (+)-goniopypyrone, (+)-goniotriol, (+)-altholactone, and (−)-etharvensin. J. Org. Chem.; 2008; 73, pp. 2-11. [DOI: https://dx.doi.org/10.1021/jo0702342]

19. Cavalheiro, A.J.; Yoshida, M. 6-[ω-Arylalkenyl]-5,6-dihydro-α-pyrones from Cryptocarya moschata (Lauraceae). Phytochemistry; 2000; 53, pp. 811-819. [DOI: https://dx.doi.org/10.1016/S0031-9422(99)00532-4]

20. Sharada, A.; Rao, K.L.S.; Yadav, J.S.; Rao, T.P.; Nagaiah, K. First stereoselective synthesis of (6R,7R,8S)-8-chlorogoniodiol. Synthesis; 2017; 49, pp. 2483-2487. [DOI: https://dx.doi.org/10.1055/s-0036-1588972]

21. Lan, Y.H.; Chang, F.R.; Yu, J.H.; Yang, Y.L.; Chang, Y.L.; Lee, S.J.; Wu, Y.C. Cytotoxic styrylpyrones from Goniothalamus amuyon. J. Nat. Prod.; 2003; 66, pp. 487-490. [DOI: https://dx.doi.org/10.1021/np020441r]

22. Yoshida, T.; Yamauchi, S.; Tago, R.; Maruyama, M.; Akiyama, K.; Sugahara, T.; Kishida, T.; Koba, Y. Syntheses of all stereoisomers of goniodiol from yeast-reduction products and their antimicrobiological activity. Biocis. Biotechnol. Biochem.; 2008; 29, pp. 2342-2352. [DOI: https://dx.doi.org/10.1271/bbb.80262]

23. Ramachandran, P.V.; Chandra, J.S.; Ram Reddy, M.V. Stereoselective syntheses of (+)-goniodiol, (−)-8-epigoniodiol, and (+)-9-deoxygoniopypyrone via alkoxyallylboration and ring-closing metathesis. J. Org. Chem.; 2002; 67, pp. 7547-7550. [DOI: https://dx.doi.org/10.1021/jo0259358]

24. Ramesh, P.; Rao, T.P. Biosynthesis-inspired total synthesis of bioactive styryllactones (+)-goniodiol, (6S,7S,8S)-goniodiol, (−)-parvistone D, and (+)-parvistone E. J. Nat. Prod.; 2016; 79, pp. 2060-2065. [DOI: https://dx.doi.org/10.1021/acs.jnatprod.6b00386] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27518479]

25. Tai, B.H.; Huyen, V.T.; Huong, T.T.; Nhiem, N.X.; Choi, E.M.; Kim, J.A.; Long, P.Q.; Cuong, N.M.; Kim, Y.H. New pyrano-pyrone from Goniothalamus tamirensis enhances the proliferation and differentiation of osteoblastic MC3T3-E1 cells. Chem. Pharm. Bull.; 2010; 58, pp. 521-525. [DOI: https://dx.doi.org/10.1248/cpb.58.521] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20410636]

26. Chandra Rao, D.; Shekhar, V.; Kumar Reddy, D.; Chinnababu, B.; Venkateswarlu, Y. Concise stereoselective total synthesis of leiocarpin C. Helv. Chim. Acta; 2013; 96, pp. 2179-2184. [DOI: https://dx.doi.org/10.1002/hlca.201200656]

27. Tanaka, H.; Woong Ahn, J.; Katayama, M.; Wada, K.; Marumo, S.; Osaka, Y. Isolation of two ovicidal substances against two-spotted spider mite, Tetranychus urticae Koch, from Skimmia repens Nakai. Agric. Biol. Chem.; 1985; 49, pp. 2189-2190. [DOI: https://dx.doi.org/10.1271/bbb1961.49.2189]

28. Schuster, C.; Roennefahrt, M.; Julich-Gruner, K.K.; Jaeger, A.; Schmidt, A.W.; Knoelker, H.J. Synthesis of the pyrano[3,2-a]carbazole alkaloids koenine, koenimbine, koenigine, koenigicine, and structural reassignment of mukonicine. Synthesis; 2016; 48, pp. 150-160.

29. Chakravarty, A.K.; Sarkar, T.; Masuda, K.; Shiojima, K. Carbazole alkaloids from roots of Glycosmis arborea. Phytochemistry; 1999; 50, pp. 1263-1266. [DOI: https://dx.doi.org/10.1016/S0031-9422(98)00666-9]

30. Mahindroo, N.; Connelly, M.C.; Punchihewa, C.; Kimura, H.; Smeltzer, M.P.; Wu, S.; Fujii, N. Structure-activity relationships and cancer-cell selective toxicity of novel inhibitors of glioma-associated oncogene homologue 1 (Gli1) mediated transcription. J. Med. Chem.; 2009; 52, pp. 4277-4287. [DOI: https://dx.doi.org/10.1021/jm900106f]

31. Zarecki, A.P.; Kolanowski, J.L.; Markiewicz, W.T. Microwave-assisted catalytic method for a green synthesis of amides directly from amines and carboxylic acids. Molecules; 2020; 25, 1761. [DOI: https://dx.doi.org/10.3390/molecules25081761]

32. Itokawa, H.; Morita, M.; Mihashi, S. Phenolic compounds from the rhizomes of Alpinia speciosa. Phytochemistry; 1981; 20, pp. 2503-2506. [DOI: https://dx.doi.org/10.1016/0031-9422(81)83082-8]

33. Seidel, V.; Bailleul, F.; Waterman, P.G. (Rel)-1β,2α-di-(2,4-dihydroxy-6-methoxybenzoyl)-3β,4α-di-(4-methoxyphenyl)-cyclobutane and other flavonoids from the aerial parts of Goniothalamus gardneri and Goniothalamus thwaitesii. Phytochemistry; 2000; 55, pp. 439-446. [DOI: https://dx.doi.org/10.1016/S0031-9422(00)00346-0]

34. Somsrisa, J.; Meepowpan, P.; Krachodnok, S.; Thaisuchat, H.; Punyanitya, S.; Nantasaen, N.; Pompimon, W. Dihydrochalcones with antiinflammatory activity from leaves and twigs of Cyathostemma argenteum. Molecules; 2013; 18, pp. 6898-6907. [DOI: https://dx.doi.org/10.3390/molecules18066898] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23752472]

35. Thuy, T.T.; Porzel, A.; Ripperger, H.; Van Sung, T.; Adam, G. Chalcones and ecdysteroids from Vitex leptobotrys. Phytochemistry; 1998; 49, pp. 2603-2605. [DOI: https://dx.doi.org/10.1016/S0031-9422(98)00411-7]

36. Zenger, K.; Agnolet, S.; Schneider, B.; Kraus, B. Biotransformation of flavokawains A, B, and C, chalcones from Kava (Piper methysticum), by human liver microsomes. J. Agric. Food Chem.; 2015; 63, pp. 6376-6385. [DOI: https://dx.doi.org/10.1021/acs.jafc.5b01858]

37. Wu, T.S.; Chang, F.C.; Wu, P.L. Flavonoids, amidosulfoxides and an alkaloid from the leaves of Glycosmis citrifolia. Phytochemistry; 1995; 39, pp. 1453-1457. [DOI: https://dx.doi.org/10.1016/0031-9422(95)00171-3]

38. Hinterberger, S.; Hofer, O.; Greger, H. Synthesis and corrected structures of sulphur-containing amides from Glycosmis species: Sinharines, penimides, and illukumbins. Tetrahedron; 1994; 50, pp. 6279-6286. [DOI: https://dx.doi.org/10.1016/S0040-4020(01)80647-5]

39. Kijjoa, A.; Bessa, J.; Pinto, M.M.; Anatachoke, C.; Silva, A.M.; Eaton, G.; Herz, W. Polyoxygenated cyclohexene derivatives from Ellipeiopsis cherrevensis. Phytochemistry; 2002; 59, pp. 543-549. [DOI: https://dx.doi.org/10.1016/S0031-9422(01)00465-4]

40. Conde, S.; López-Serrano, P. Regioselective lipase-catalysed amidation of N-blocked L-and D-aspartic acid diesters. Eur. J. Org. Chem.; 2002; 2002, pp. 922-929. [DOI: https://dx.doi.org/10.1002/1099-0690(200203)2002:5<922::AID-EJOC922>3.0.CO;2-E]

41. Pudlo, M.; Csányi, D.; Moreau, F.; Hajós, G.; Riedl, Z.; Sapi, J. First Suzuki–Miyaura type cross-coupling of ortho-azidobromobenzene with arylboronic acids and its application to the synthesis of fused aromatic indole-heterocycles. Tetrahedron; 2007; 63, pp. 10320-10329. [DOI: https://dx.doi.org/10.1016/j.tet.2007.07.068]

42. Sathyanarayana, A.R.; Lu, C.-K.; Liaw, C.-C.; Chang, C.-C.; Han, H.-Y.; Green, B.D.; Huang, W.-J.; Huang, C.; He, W.-D.; Lee, L.-C. et al. 1,2,3,4,6-Penta-O-galloyl-d-glucose interrupts the early adipocyte lifecycle and attenuates adiposity and hepatic steatosis in mice with diet-induced obesity. Int. J. Mol. Sci.; 2022; 23, 4052. [DOI: https://dx.doi.org/10.3390/ijms23074052]