Triterpenoids from selaginella rolandi - Principis and their ptp1b inhibitory activity

Vietnam Journal of Science and Technology 56 (4A) (2018) 69-73 TRITERPENOIDS FROM SELAGINELLA ROLANDI-PRINCIPIS AND THEIR PTP1B INHIBITORY ACTIVITY Nguyen Phi Hung 1, 2, * , Nguyen Dinh Tuan 1, 2 , Pham Quoc Long 1 1 Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Ha Noi 2 Graduate University of Science and Technology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Ha Noi * Email: nguyenphihung1002@gmail.com Received: 30 July 2018; Accepted for publication: 10 October 2018 ABSTRACT As part of an ongoing search for new protein tyrosine phosphatese 1B (PTP1B) inhibitors from medicinal plants, the methanol extract of Selaginella rolandi-principis was found to possess PTP1B inhibition. Thus, bioassay-guided separation of this active extract led to the isolation of four compounds (1–4) as active principles. Their chemical structures were identified by extensive analysis of spectroscopic and physicochemical data. Among the isolates, compounds 1–3 exhibited potential inhibitory activities against PTP1B with IC50 values of 18.8 ± 0.7, 5.6 ± 0.9, and 28.4 ± 1.2 µM, respectively, against PTP1B. In this assay, ursolic acid as positive control displayed an IC50 value of 3.46 0.07 µM. Keywords: Selaginella, Selaginella rolandi-principis, PTP1B inhibitor, tritepenoid, type-2 diabetes. 1. INTRODUCTION Protein tyrosine phosphatases 1B (PTP1B), a member of the PTP family, is thought to function as a negative regulator of insulin signal transduction. PTP1B directly interacts with activated insulin receptor or insulin receptor substrate-1 (IRS-1) to dephosphorylate phosphotyrosine residues, resulting in down regulation of insulin action [1]. PTP1B overexpression in rat primary adipose tissues and 3T3/L1 adipocytes has been shown to decrease insulin-sensitive Glut4 translocation [2] and insulin receptor and IRS-1 phosphorylation [3], respectively. Therefore, it has been suggested that compounds that reduce PTP1B activity or expression levels cannot only be used for treating type 2 diabetes but also obesity [4, 5]. As part of an ongoing search for new protein tyrosine phosphatese 1B inhibitors and insulin mimetic agents from medicinal plants, the methanol extract of S. rolandi-principis was found to possess stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells and PTP1B inhibition. Thus, it was chosen for further phytochemical and biological investigation in detail. Nguyen Phi Hung, Nguyen Dinh Tuan, Pham Quoc Long 70 Selaginella, also known as spikemoss, is the only surviving genus within the plant family Selaginellaceae. Selaginella includes more than 740 species widely distributed around the globe [6]. It has been used in oriental medicine to treat inflammation, amenorrhea, dysmenorrhea, metrorrhagia, hematuria, prolapse of the anus, abdominal lumps in women, chronic hepatitis and hyperglycemia [7] A number of flavonoids, biflavonoids, lignans, selaginellins, and phenols have been reported as chemical constituents of the genus [8]. Among the genus, Selaginella rolandi-principis Alston was first recorded in 1934 9. In Vietnam, Selaginella 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. rolandi-principis. The plant has been used in Vietnamese folk medicine to treat acute hepatitis, cholecystitis, inflammation, dysentery, pulmonary tuberculosis and hyperglycemia [10, 11]. In this study, we report the isolation and evaluation of antidiabetic activity of isolated compounds from S. rolandi-principis. 2. MATERIALS AND METHODS 2.1. Plant materials The aerial parts of S. rolandi-principis Alston were collected in 2017 at Pu Hoat Nature Reserve, Hanh Dich Commune, Que Phong Districst, Nghe An Province. The sample was identified by Dr. Nguyen Quoc Binh, Vietnam National Museum of Nature, VAST. Voucher specimens were deposited at the Vietnam National Museum of Nature (SH-172) and Institute of Natural Products Chemistry (QB-NA02SR-P), VAST. 2.2. Extraction and isolation The aerial parts of S. rolandi-principis were naturally dried, cut into small spieces, and extracted with 96 % ethanol at 40 o C under sonication for 10 h (repeated for 3 times). After filtration, the extracted were combined and evaporated under reduced pressure to give a crude EtOH extract (150 g). This extract was then suspended in H2O (1.5 L) and partitioned successively with hexane, dichloromethane and ethyl acetate. The resulting fractions were concentrated under diminished pressure to give the corresponding solvent-soluble fractions of hexane (39.0 g, Hx), dichloromethane (45.0 g, DM), ethyl acetate (31.0 g, EA) and water (26.9 g, W) residue, respectively. The fraction DM (40.0 g) was chromatography over silica gel column (400-630 mesh), eluted with hexane-acetone (10:1 to 0:1 v/v) to afford 10 fractions (DM1-DM10). The fraction DM2 (2.2 g) was subjected to a silica gel chromatography column (CC) eluting with solvent mixture of chloroform-acetone (100:1 to 0:1, v/v) to produce five sub- fractions (DM2.1-DM2.5). The subfraction DM2.3 (819 mg) was further separated by normal silica gel CC, eluting with hexane-acetone (9:1, v/v) to afford eight sub-fractions (DM2.3A- MD2.3H). Compounds 1 (22.6 mg), 2 (15.4 mg), and 3 (10.5 mg) were obtained from subfraction DM2.3B-D, respectively by silica gel CC eluting with hexane-acetone (8:1, v/v). Compound 4 (32.0 mg) was isolated from the sub-fraction DM2.5 (0.3 g) by silica gel CC eluting with hexane-acetone (6:1, v/v). 2.3. PTP1B inhibitory assay Protein tyrosine phosphatase 1B (human recombinant) was purchased from Biomol International LP, Plymouth Meeting, PA, USA, and the inhibitory activities of the isolated Triterpenoids from a Vietnamese medicinal plant Selaginela rolandi-principis 71 compounds against PTP1B were evaluated using p-nitrophenyl phosphate (p-NPP) as substrate [12]. Briefly, to each of 96 wells (final volume: 200 μL) was added 2 mM pNPP and PTP1B (0.05-0.1 μg) in a buffer containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol (DTT) with or without test compounds. Following incubation at 37 °C for 30 min, the reaction was terminated with 10 N NaOH. The amount of produced p-nitrophenol was estimated by measuring the absorbance at 405 nm. The nonenzymatic hydrolysis of 2 mM pNPP was corrected by measuring the increase in absorbance at 405 nm obtained in the absence of PTP1B enzyme. The Sigma plot program (SPCC Inc., Chicago, IL) was used to analyze both the double reciprocal Lineweaver–Burk plot, which is the most straight forward means of diagnosing an inhibitor model and the Dixon plot by plotting 1/v as a function of inhibitor [I] for each substrate concentration. 3. RESULTS AND DISCUSSION Phytochemical study of the EtOH extract of S. rolandi-principis using chromatographic methods led to the isolation of three triterpenoids (1‒ 3) and a sterol (4) (Figure 1). Their chemical structures were determined by extensive analysis of the physicochemical and spectroscopic data, as well as by comparison with reported literatures. Figure 1. Chemical structure of compounds 1- 4 isolated from S. rolandi-principis. Betulinic acid (1), lupeol (2), and epi-lupeol (3) are lupane-type triterpenes of natural origin isolated from various plants [13-16]. They have been exhibited a variety of biological and medicinal properties such as anti-HIV, anti-bacterial, anti-malarial, anti-inflammatory, anthelmintic, antinociceptive, anti-HSV-1, and anti-cancer activities. Among these, lupeol (2) possessed wide range of anticancer effect against pancreatic, prostate, ovarian, colorectal and myeloma, breast, stomach, cervical, lymphoma, leukemia, melanoma, and lung cancers [16]. This is the first time that these lupane-type triterpenes have been isolated from Selaginella genus. β-sitosterol (4) is the dominant phytosterol, which may undergo oxidative process just like cholesterol, resulting in β-sitosterol oxides. To our knowledg, this is the first time that these compounds (1–4), except for β-sitosterol (4), were isolated and identified from Selaginella genus. And that S. rolandi-principis has been phytochemically studied for the first time. The PTP1B inhibitory activity of the isolated compounds (1–4) were evaluated using p- NPP as a substrate described previously [12], and the results were showed in Table 1. Ursolic acid was used as a positive control in this assay and displayed an IC50 value of 3.46 0.07 μM and mixed-competitive inhibition mode. Among the isolates, compounds 1 and 2 exhibited the most potent inhibitory activity with IC50 values of 18.86 ± 0.79 and 5.60 ± 0.89 µM, Nguyen Phi Hung, Nguyen Dinh Tuan, Pham Quoc Long 72 respectively. Compound 3 displayed significant activity with IC50 values of and 28.42 ± 1.24 μM, while compound 4 had weak activity (IC50 > 30 μM). In lupan-type tritepenes (1–3), lupeol (2) with β-OH at C-3 displayed the strongest activity (IC50 = 5.60 ± 0.89 µM) while epi-lupeol (3) bearing an α-OH at C-3 showed weak activity (IC50 = 28.42 ± 1.24 µM). Betulinic acid (1) also bearing an β-OH at C-3, however exhibited weaker activity (IC50 = 18.86 ± 0.79 µM) as compared with compound 2. From our data obtained, it might suggest that the orientation of the OH functional group at C-3 could affect the inhibitory activity of these triterpenes against PTP1B and that the COOH functional group at C-28 may not improve the activity but affected the inhibition type. To determine the inhibition mode of the active compounds, the inhibitory properties of several appropriate concentrations of the active compounds (1–3) were evaluated at various concentrations of p-NPP [12]. Table 1 shows the result of inhibition type of the active compounds (1–3) and ursolic acid. Lupeol (2) and epi-lupeol (3) showed non-competitive inhibition of PTP1B while betulinic acid (1) and ursolic acid possessed mixed-competitive inhibition. Table 1. Inhibitory effects of isolated compounds (1–4) on PTP1B enzyme. Compound Inhibitory effect (IC50, μM) a (Ki value, μM) Inhibition type 1 18.86 ± 0.79 15.00 ± 0.59 Mixed-competitive 2 5.60 ± 0.89 6.13 ± 0.26 Non-competitive 3 28.42 ± 1.24 - c Non-competitive 4 > 30 - - Ursolic acid b 3.46 0.07 6.46 0.15 Mixed-competitive a Results are expressed as IC50 values (μM), determined by regression analysis and expressed as the means ± SD of three replicates. b Positive controls. c Data not determined. 4. CONCLUSIONS Using combined chromatographic and spectroscopic methods, three lupan-type triterpenoids including betulinic acid (1), lupeol (2), epi-lupeol (3) and a sterol, β-sitosterol (4) were isolated from the methanol extract of Selaginella rolandi-principis. All of the isolates compounds (1–4) were investigated for their inhibitory effects on PTP1B enzyme activity. Lupan-type tritepenes (1–3) exhibited potential inhibitory activities with IC50 values of 18.86 ± 0.79, 5.60 ± 0.89, and 28.42 ± 1.24 µM against PTP1B, while β-sitosterol (4) was weak active. Among the active compounds, lupeol (2) and epi-lupeol (3) without carboxyl group (COOH) at C-28 showed non-competitive inhibition type while lupan-type tritepene containing COOH at C- 28 (betulinic acid, 1) displayed mixed-competitive inhibition. Acknowledgments. This study was supported by the National Foundation for Science and Technology Development of Viet Nam, Ministry of Science and Technology (NAFOSTED-104.01-2017.50). We wish to thank the Center for Applied Spectroscopy, Institute of Chemistry (VAST) for the spectroscopic measurements. Triterpenoids from a Vietnamese medicinal plant Selaginela rolandi-principis 73 REFERENCES 1. Goldstein B. J., Bitter-Kowalczyk A., White M. 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