Insulin-Mimetic biflavones from a vietnamese medicinal plant selaginella tamariscina - Nguyen Dinh Tuan

Vietnam Journal of Science and Technology 56 (4A) (2018) 22-29 INSULIN-MIMETIC BIFLAVONES FROM A VIETNAMESE MEDICINAL PLANT SELAGINELLA TAMARISCINA Nguyen Dinh Tuan 1, 2 , Nguyen Phi Hung 1, 2, * , Do Huu Nghi 1 1 Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Ha Noi Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Ha Noi * Email: nguyenphihung1002@gmail.com Received: 23 July 2018; Accepted for publication: 12 October 2018 ABSTRACT During the search for antidiabetic agents from natural sources, four biflavones including amentoflavone (1), robustaflavone (2), cupressuflavone (3), and 3,8′′-biapigenin (4) have been isolated from the methanol extract of Vietnamese medicinal plant Selaginella tamariscina by using combined chromatographic experiments. The chemical structures of isolated compounds were determined and elucidated by the interpretation of NMR spectral data, mass spectra as well as comparison with data from the literature. All isolated compounds showed significant stimulatory effects on 2-NBDG glucose uptake in 3T3-L1 adipocyte cells. At a concentration of 10 μM, compounds 3 and 4 exhibited potential stimulatory effects by 1.47 and 1.44 fold of inductions as compared with the control (DMSO), respectively. The result suggests that these biflavonoids maybe potential as insulin mimetics for developing antidiabetic agents. Keywords: Selaginella tamariscina, Selaginellaceae, 2-NBDG, insulin, biflavonoids, antidiabetes. 1. INTRODUCTION Diabetes is one of the largest global health issues of the 21 st century. The number of people with diabetes anticipate rising from current estimate of 425 million in 2017, and 625 million in 2040 (IDF) [1]. Recently, a number of synthetic small molecules, such as zinc (II) complexes and vanadium compounds, have been shown to mimic the action of insulin in cell culture and animal models of diabetes. In addition, many natural products, such as antibiotics (e.g., anisomycin), fungal metabolites (e.g., L-783,281), as well as plant, extracts promote glucose uptake in cells [2, 3]. However, clinical tests have shown that none of these compounds or extracts can replace insulin in the treatment of diabetes. Therefore, there is still the need to search for new antidiabetic agents that can mimic the effect of insulin. The genus Selaginella comprises more than 740 species distributed around the world. This is the only surviving genus of the plant family Selaginellaceae [4]. It has common name as Insulin-mimetic bioflavones from a Vietnamese medicinal plant Selaginella tamariscina 23 spikemoss and “Quyen ba” in Viet Nam. In traditional medicine, the plant has been used to treat dysmenorrhea, metrorrhagia, hematuria, prolapse of the anus, abdominal lumps in women, and acute and chronic hepatitis [5a]. Biological studies have demonstrated the effects on blood glucose lowering and facilitating the repair of injured pancreatic islet β-cells [5b]. Phytochemical researches on the species have identified selaginellins and biflavonoids as main chemical constituents along with lignans and phenylpropanoids [5c]. In Viet Nam, Quyen ba is found growing on rocks and/or sandy arid land along the country at an elevation of less than 1000 m above sea level. Among thirty nine recognized species, thirteen of them are common appearance including S. tamariscina. The plant has been used in Vietnamese folk medicine to treat acute and chronic hepatitis; it has been used in folk medicine to treat acute hepatitis, cholecystitis, inflammation of the intestines, dysentery, pulmonary tuberculosis, and hyperglycemia [6, 7]. In this study, the plant was collected on a mountain area at an altitude of about 250 m above sea level. We reported here the purification, structural determination, and potential antidiabetic properties of the chemical constituents of this medicinal plant. 2. MATERIALS AND METHODS 2.1. Plant materials The whole parts of Selaginella tamariscina (Beauv.) Spring were collected in Oct, 2017 at Phan Rang, Ninh Thuan. The sample was identified by Dr. Nguyen Quoc Binh (Viet Nam National Museum of Nature, Vietnam Academy of Science and Technology (VAST). A voucher specimen (QB-BT01) was deposited at the Institute of Natural Products Chemistry (INPC), VAST. 2.2. Extraction and isolation The dried whole parts of S. tamariscina (1.85 kg) were cut into small pieces before extracted with MeOH under sonication for 10 h, at 45 o C, each 5 L for 4 times. The filtered MeOH-soluble extract was combined and dried under reduced pressure to give a crude MeOH extract (150.1 g). This crude extract was further partitioned with EtOAc to give EtOAc (56.3 g) fraction after vacuum evaporating under reduced pressure. The EtOAc fraction was further subjected to a silica gel column chromatography (CC), using a gradient solvent system of hexane:acetone (15:1 → 0:1, v/v), to yield twenty fractions (ST.EA-1 to ST.EA-10) according to their thin layer chromatography (TLC) profiles. Fraction ST.EA12 was further chromatographed on a silica gel column, using a gradient solvent system of CH2Cl2–MeOH with increasing polarity, to afford ten subfractions (ST.EA12-1 to ST.EA12-10). The subfraction ST.EA12-3 were combined and subjected to a C18 reversed-phase (RP-18) CC and eluted with MeOH–H2O (from 4:6 to 4:1, v/v), resulting in the isolation of compounds 1 (27.6 mg), 2 (7.8 mg), and 3 (5.6 mg), respectively. Fraction ST.EA14 was also chromatographed on an open silica gel column, eluting with hexane–EtOAc (gradient, v/v) with increasing polarity, to afford ten subfractions (ST.EA14-1 to ST.EA14-10). Purification of subfraction ST.EA14-5 by an open C18 reverse- phase column, eluting with a gradient solvent system of MeOH–H2O (v/v), resulted in the isolation of compound 4 (4.5 mg). Amentoflavone (1): Yellow powder; Low-FAB-MS m/z 538.88 [M] + (C30H18O10); 1 H- NMR (500 MHz, Acetone-d6) and 13 C-NMR (125 MHz, Acetone-d6) are given in Table 1. Nguyen Dinh Tuan, Nguyen Phi Hung, Do Huu Nghi 24 Robustaflavone (2): Yellow powder; Low-FAB-MS m/z 538.90 [M] + (C30H18O10); 1 H- NMR (500 MHz, Pyridine-d5) and 13 C-NMR (125 MHz, Pyridine-d5) are given in Table 1. Cupressuflavone (3): Yellow amorphous powder; Low-FAB-MS m/z 538.08 [M] + (C30H18O10); 1 H-NMR (500 MHz, Acetone-d6) H (ppm): 6.81 (1H, s, H-3), 6.49 (1H, br s, H-6), 7.52 (2H, d, J = 8.8 Hz, H-2′/H-6′), 6.76 (2H, d, J = 8.8 Hz, H-3′/H-5′), 6.81 (1H, s, H-3′′), 6.49 (1H, br s, H-6′′), 7.52 (2H, d, J = 8.8 Hz, H-2′′′/H-6′′′), 6.76 (2H, d, J = 8.8 Hz, H-3′′′/H-5′′′), 13.17 (1H, s, 5-OH), 13.01 (1H, s, 5′-OH); 13C-NMR (125 MHz, Acetone-d6) C (ppm): 163.1 (C-2), 102.1 (C-3), 181.8 (C-4), 160.5 (C-5), 100.0 (C-6), 161.0 (C-7), 99.9 (C-8), 154.5 (C-9), 102.9 (C-10), 121.0 (C-1′), 127.5 (C-2′/C-6′), 115.6 (C-3′/C-5′), 161.0 (C-4′), 163.1 (C-2′′), 102.1 (C-3′′), 181.8 (C-4′′), 160.5 (C-5′′), 100.0 (C-6′′), 161.0 (C-7′′), 99.9 (C-8′′), 154.5 (C-9′′), 102.9 (C-10′′), 121.0 (C-1′′′), 127.5 (C-2′′′/C-6′′′), 115.6 (C-3′′′/C-5′′′), 161.0 (C-4′′′). 3,8′′-biapigenin (4): Yellow amorphous powder; Low-FAB-MS m/z 538.09 [M]+ (C30H18O10); 1 H-NMR (500 MHz, Acetone-d6) H (ppm): 6.73 (1H, s, H-3), 6.52 (1H, br s, H-6), 7.66 (2H, d, J = 8.8 Hz, H-2′/H-6′), 6.83 (2H, d, J = 8.8 Hz, H-3′/H-5′), 6.66 (1H, s, H-3′′), 6.24 (1H, br s, H-6′′), 6.45 (1H, br, s, H-8′′), 7.52 (2H, d, J = 8.8 Hz, H-2′′′/H-6′′′), 6.76 (2H, d, J = 8.8 Hz, H-3′′′/H-5′′′), 13.2 (1H, s, 5-OH), 13.0 (1H, s, 5′-OH); 13C-NMR (125 MHz, Acetone-d6) C (ppm): 165.5 (C-2), 103.4 (C-3), 182.3 (C-4), 162.4 (C-5), 100.1 (C-6), 164.2 (C-7), 102.8 (C-8), 156.6 (C-9), 104.6 (C-10), 121.9 (C-1′), 129.3 (C-2′/C-6′), 115.1 (C-3′/C-5′), 162.0 (C-4′), 165.6 (C-2′′), 103.6 (C-3′′), 182.6 (C-4′′), 163.5 (C-5′′), 99.9 (C-6′′), 162.4 (C-7′′), 94.8 (C-8′′), 157.3 (C-9′′), 104.8 (C-10′′), 122.2 (C-1′′′), 127.5 (C-2′′′/C-6′′′), 115.6 (C-3′′′/C-5′′′), 156.6 (C- 4′′′). 2.3. Cell culture and induction of 3T3-L1 adipocytes 3T3-L1 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in DMEM with 10 % FCS. To induce differentiation, 3T3-L1 preadipocytes were cultured until confluence was reached (0 day), and the culture medium was replaced with a fresh induction medium containing 5 μg/mL insulin, 0.5 mM 3-isobutyl-1- methylxanthine (IBMX), and 1 μM dexamethasone (DEX) in DMEM with 10 % FBS for 2 days. The medium was then replaced with a differentiation medium containing 5 μg/mL insulin and DMEM with 10 % FBS every 2 days for up to 8 days until the cells were harvested [8]. 2.4. Adipocyte-based measurement of 2-NBDG uptake 3T3-L1 adipocytes grown in black 96-well plates were incubated with each sample for 24 h at 37 o C in a 5 % CO2 atmosphere. Subsequently, 250 µM of 2-NBDG (dissolved in PBS with 1 % BSA) was added to the cells and incubated for a further 30 min. After incubation, cells were washed two times with PBS to remove excess fluorescence in the wells. Then, fluorescence retained by the cells was measured using a PerkinElmer Victor3 V 1420 Multilabel Plate Counter at an excitation and emission wavelength of 485 nm and 535 nm, respectively [9]. 3. RESULTS AND DISCUSSION 3.1. Isolation and structural elucidation of isolated compounds The methanol extract of the spikemoss was partitioned with ethylacetate. Phytochemical research of this fraction led to the isolation of four natural products (14) (Figure 1). Insulin-mimetic bioflavones from a Vietnamese medicinal plant Selaginella tamariscina 25 Compound 1 was obtained as yellow powder. Its 1 H- and 13 C-NMR spectra showed a pair of signal with a ratio of 1:1. Of which, two proton resonances at H 12.8 and 12.9 (each 1H, s) and two quaternary carbon resonances at C 183.0 and 183.3 were characteristic of conjugated 5- OH, 5ʹʹ-OH, C-4 and C-4ʹʹ of a flavonoid skeleton (Table 1 and Figure 1). This indicates the structure of a biflavone-type skeleton [10]. An ABX aromatic spin system [H 7.17 (d, 8.8, H- 5ʹ), 7.88 (dd, 2.0, 8.8, H-6ʹ), and 8.15 (d, 2.0, H-2ʹ)], an AAʹBBʹ system [H 7.51 (d, 8.8, H- 2ʹʹʹ/H-6ʹʹʹ) and 6.77 (d, 8.8, H-3ʹʹʹ/H-5ʹʹʹ)], and five singlet/broad singlet signals which assignable to H-3 (H 6.64), H-6 (H 6.20), H-8 (H 6.55), H-3ʹʹ (H 6.54), and H-6ʹʹ (H 6.41) were observed in 1 H-NMR spectrum (Table 1). In contrast, ten quaternary oxygenated aromatic carbon signals were also observed in the 13 C-NMR spectrum, in addition to two ketone, six quaternary carbons, and twelve aromatic carbons. Further analyses of 2D-NMR including COSY, HSQC and HMBC data allowed to elucidate each proton-carbon group, as well as the arrangement of each proton-carbon position in the structure of compound 1. The linkage of two flavone-skeletons at C-3ʹ―C-8ʹʹ bond was characterized by the cross-cross correlation between H-2ʹ (H 8.15) and C-8ʹʹ (104.2) in the HMBC spectrum (Figure 2). The LR-FAB-MS showed a molecular ion peak at m/z 538.88 [M] + revealing the molecular formula of C30H18O10 for compound 1. From the above data and comparing the NMR data of 1 with the reported values led to the structural characterization of compound 1 as amentoflavone [11]. Figure 1. Chemical structure of compounds 1‒4 isolated from S. tamariscina. Compound 2 was also obtained as yellow powder, the molecular formula, C30H18O10, was obtained from a molecular ion peak at m/z 538.90 [M] + on its LR-FAB-MS. Its 1 H and 13 C NMR spectra of 2 were similar to that of 1 with two proton resonances at H 13.89 and 13.77 (each 1H, s) and two quaternary carbon resonances at C 181.9 and 181.5 were characteristic of conjugated 5-OH, 5ʹʹ-OH, C-4 and C-4ʹʹ of a flavonoid skeleton (Table 1 and Figure 1). The 1H-NMR spectrum also exhibited an ABX aromatic spin system for ring B of the first flavone unit and an AAʹBBʹ system for the second, five singlet/broad singlet signals which assignable to H-3, H-6, H-8, H-3ʹʹ, and H-6ʹʹ (Table 1). In the 13C NMR spectrum, 10 quaternary oxygenated aromatic carbon signals were observed, in addition to two ketones, six quaternary carbons, and twelve aromatic carbons (Table 1). Datailed comparision of its 1 H- and 13 C-NMR data with reported values led to the establishment of the structure of compound 2 as robustaflavone [12]. Nguyen Dinh Tuan, Nguyen Phi Hung, Do Huu Nghi 26 Table 1. 1 H- and 13 C-NMR spectroscopic data for compounds 1 and 2. Position 1 2 H (ppm) (J in Hz) C (ppm) H (ppm) (J in Hz) C (ppm) 1 2 165.0 165.3 3 6.64 (s) 104.0 7.06 (s) 104.1 4 183.0 181.9 5 163.1 166.4 6 6.20 (br, s) 99.9 6.77 (d, 2.2) 100.3 7 165.1 166.4 8 6.55 (br, s) 94.9 6.85 (d, 2.2) 95.3 9 158.7 159.6 10 105.2 105.5 1ʹ 123.3 122.1 2ʹ 8.15 (d, 2.0) 132.6 8.59 (br, s) 128.9 3ʹ 120.7 123.4 4ʹ 160.3 161.5 5ʹ 7.17 (d, 8.8) 117.4 7.17 (d, 8.6) 117.9 6ʹ 7.88 (dd, 2.0, 8.8) 128.9 7.29 (br d, 8.6) 132.9 1ʹʹ 2ʹʹ 165.1 165.0 3ʹʹ 6.54 (s) 103.4 6.93 (s) 103.5 4ʹʹ 183.3 181.5 5ʹʹ 162.6 164.7 6ʹʹ 6.41 (s) 100.0 105.9 7ʹʹ 156.0 162.6 8ʹʹ 104.2 6.81 (s) 95.3 9ʹʹ 162.7 156.7 10ʹʹ 105.4 105.4 1ʹʹʹ 123.0 123.2 2ʹʹʹ 7.51 (d, 8.8) 129.1 7.29 (d, 8.6) 129.5 3ʹʹʹ 6.77 (d, 8.8) 116.7 7.17 (d, 8.6) 117.0 4ʹʹʹ 161.8 162.7 5ʹʹʹ 6.77 (d, 8.8) 116.7 7.17 (d, 8.6) 117.0 6ʹʹʹ 7.51 (d, 8.8) 129.1 7.29 (d, 8.6) 129.5 5-OH 12.83 (s) 13.89 (s) 5ʹʹ-OH 12.90 (s) 13.77 (s) Compound 3 was obtained as yellow amorphous powder. The molecule formula of 3 was established as C30H18O10 based on the molecular ion peak at m/z 538.08 [M] + obtained from its LR-FAB-MS. Its 1 H- and 13 C-NMR spectra displayed characteristic signals for a flavone skeleton with a proton signal at H 6.81 (1H, s, H-3) and carbon signal assigned for C-4 at C 181.8. These observations suggested a structure of a flavone-type skeleton [10]. However, two proton resonances at H 13.17 (1H, s) and 13.01 (1H, s), which assignable for conjugated 5-OH Insulin-mimetic bioflavones from a Vietnamese medicinal plant Selaginella tamariscina 27 and 5′ʹ-OH were found in its 1H-NMR (Figure 1). Thus, the above obtained data also revealed the structure of 3 as biflavone-type skeleton, named cupressuflavone [10]. Compound 4 was also obtained as yellow amorphous powder. Its molecular formula was deduced as C30H18O10 from a molecular ion peak at m/z 538.09 [M] + in the LR-FAB-MS. The 1 H- and 13 C-NMR spectra of 4 were similar to that of compounds 1 and 2 except for two AAʹBBʹ aromatic spin systems presented in 4 [δH 7.66 (2H, d, J = 8.8 Hz, H-2′/H-6′), 6.83 (2H, d, J = 8.8 Hz, H-3′/H-5′), 7.52 (2H, d, J = 8.8 Hz, H-2′′′/H-6′′′) and 6.76 (2H, d, J = 8.8 Hz, H-3′′′/H-5′′′). In addition, the conjugated hydroxy peaks at δH 13.2 (5-OH) and 13.0 (5ʹʹ-OH) in the 1 H NMR spectrum of 4 was also presented (Figure 1). Two singlet/broad singlet proton peaks at δH 6.24 (H-6ʹʹ) and 6.45 (H-8ʹʹ) of ring A, δH 6.73 (H-3) and 6.52 (H-6) of the second unit were presented. Detailed comparison of the 1 H- and 13 C-NMR data with reported values led to the identification of compound 4 as 3,8ʹʹ-biapigenin [13]. Figure 2. 1 H‒13C (→) key HMBC correlations of compounds 1 and 2. 3.2. 2-NBDG glucose uptake stimulatory activity of isolated compounds 2-NBDG has been reported as a useful fluorescent-tagged glucose probe for discovering insulin mimetic compounds [14]. Thus, the stimulatory effects of compounds 1–4 were further evaluated on glucose uptake using 2-NBDG in 3T3-L1 adipocyte cells [15]. As presented in Figure 3, all the isolates showed stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. Figure 3. Stimulatory effects of the isolated compounds 1–4 on glucose uptake in 3T3-L1 adipocyte cells (Insulin: positive control; Control: DMSO). At a concentration of 10 M, cupressuflavone (3) and 3,8′′-biapigenin (4) significantly induced 2-NBDG uptake by 1.47 and 1.44 fold induction as compared with the control (DMSO). Amentoflavone (1) and robustaflavone (2) showed weak activity with 1.08 and 1.13 fold of Nguyen Dinh Tuan, Nguyen Phi Hung, Do Huu Nghi 28 induction. The positive control (insulin) showed an induction of 1.54 fold at a concentration of 100 nM. 4. CONCLUSIONS Using combined chromatographic and spectroscopic methods, four biflavones including amentoflavone (1), robustaflavone (2), cupressuflavone (3), and 3,8′′-biapogenin (4) were isolated and structurally identified from the methanol extract of Selaginella tamariscina. All of the isolates (1‒4) were investigated for their stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. At a concentration of 10 μM, cupressuflavone (3) and 3,8′′-biapigenin (4) exhibited potential stimulatory effects by 1.47 and 1.44 fold of inductions as compared with the control (DMSO), respectively. In this assay, the positive control (insulin) showed an induction of 1.54 fold at a concentration of 100 nM. The result suggests that these biflavonoids maybe potential as insulin mimetics for developing antidiabetic agents. Acknowledgements. 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