Methoxyflavones from orthosiphon stamineus and their ptp1b inhibitory activities - Hoang Duc Thuan

Vietnam Journal of Science and Technology 56 (4A) (2018) 146-152 METHOXYFLAVONES FROM ORTHOSIPHON STAMINEUS AND THEIR PTP1B INHIBITORY ACTIVITIES Hoang Duc Thuan 3 , Nguyen Phi Hung 1, 2, * , Vu Quoc Trung 3 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 3 Hanoi National University of Education, 136 Xuan Thuy, Cau Giay, Viet, Ha Noi * Email: nguyenphihung1002@gmail.com Received: 23 July 2018; Accepted for publication: 10 October 2018 ABSTRACT Phytochemical analysis of the methanol extract of the aerial parts of Orthosiphon stamineus Benth. led to the isolation of four flavone compounds including 5-hydroxy-3,7,3′,4′- tetramethoxyflavone (1), 3,5,7,3′,4′-pentamethoxyflavone (2), 3,3′-dihydroxy-5,7,4′- trimethoxyflavone (3), and 3,5,3′-trihydroxy-7,4′-dimethoxyflavone (4). Their chemical structures were determined from the spectroscopic evidences, including 1D-NMR and MS, respectively. The inhibitory effects of the isolates (1-4) against protein tyrosine phosphatase 1B (PTP1B) were investigated in vitro using ursolic acid as positive control. Among the isolates, compound 4 exhibited potential activity with IC50 value of 10.12 ± 0.19 μM, the others showed weak activity. In this assay, ursolic acid displayed an IC50 value of 3.42 ± 0.25 μM. This is indicated that 3,3′-dihydroxy-5,7,4′-trimethoxyflavone (4) may be useful for discovery of PTP1B inhibitors as antidiabetic agent. Keywords: Orthosiphon stamineus Benth., flavone, ursolic acid, PTP1B inhibitor, type 2 diabetes. 1. INTRODUCTION Nowadays diabetes is a huge and growing problem. The most recent estimate in 2017 shows that 425 million people are living with diabetes and this number is set to rise beyond 625 million in less than 25 years [1]. Type 2 diabetes (T2D), or noninsulin-dependent diabetes mellitus, is the most common type accounting for approximately 90 % of the total cases among the three types of diabetes [2]. This type is characterized by a resistance to insulin, a peptide hormone produced by β-cells in the pancreas, which is responsible for glucose homeostasis [3, 4]. The insulin signaling pathway is negatively regulated by protein tyrosine phosphatases, most notably, protein tyrosine phosphatase 1B (PTP1B) [4]. The overexpression of PTP1B has been shown to inhibit the increased expression of insulin in insulin-resistant states [5]. Furthermore, recent genetic evidence has shown that PTP1B gene variants are associated with changes in Methoxyflavones from Orthosiphon stamineus Benth. and their PTP1B 147 insulin sensitivity [6]. At the genetic, molecular, biochemical, and physiological levels, PTP1B seems to be a promising drug target for the treatment of T2D and at-risk obese patients [7]. Natural products are rich sources of novel active agents for clinical uses [8]. Previous reports indicate that there are more than 1000 plant species being used to treat T2D all over the world [3] and various natural compounds display PTP1B inhibitory activity [9]. Orthosiphon stamineus Benth., belonging to Lamiaceae family, has common name as Cat’s Whiskers, Java Tea in America, Kumis Kuching in Indonesia, Misai Kuching in Malaysia, and “Râu Mèo” in Viet Nam. This is a fast-growing herbaceous shrub that can reach 1-2 meters tall and spread to a meter wide. The plant produces racemes that are 10-20 cm long with pretty tubular flowers that are uniquely shaped and bear 5-6 cm long stamens that look like cat’s whiskers, hence the common name. The plant is grown throughout Southeast Asia, Australia, and also Africa [10]. Traditional uses have trusted for many centuries for treating ailments of the kidney, bladder stone, urinary tract infection, liver and bladder problems, rheumatism, diabetes, and gout. In Viet Nam, it has been used for many decades in the treatment of renal inflammation, kidney stones and dysuria. The aerial parts are used as tea to reduce cholesterol and blood pressure. However, to the best of our knowledge, the chemical constituents of this plant have not been reported in detail. Therefore, in the interest of promoting drug discovery from natural sources, this research was conducted to identify bioactive compounds from the aerial parts of O. stamineus, focusing on PTP1B inhibitory activity. 2. MATERIALS AND METHODS 2.1. General experimental procedures The 1 H-NMR (500 MHz) and 13 C-NMR (125 MHz) spectra were recorded on a Bruker AM500 FT-NMR spectrometer, TMS was used as an internal standard. The electrospray ionization mass spectra (ESI-MS) were obtained on an Agilent 1260 series single quadrupole LC/MS system. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70- 230 mesh and 230-400 mesh, Merck) and YMC RP-18 resins (30-50 μm, Fuji Silysia Chemical Ltd.). Thin layer chromatography (TLC) used pre-coated silica gel 60 F254 (1.05554.0001, Merck) and RP-18 F254S plates (1.15685.0001, Merck). Compounds were visualized by spraying with aqueous 10 % H2SO4 and heating for 3–5 minutes. 2.2. Plant materials The aerial parts of Orthosiphon stamineus Benth. were collected in January, 2017 at Ngu Hiep, Thanh Tri, Ha Noi. The sample was identified by Dr. Nguyen Quoc Binh (Viet Nam National Museum of Nature, VAST). A voucher specimen (SH-164) was deposited at the Institute of Natural Products Chemistry (INPC), VAST. 2.3. Extraction and isolation The dried aerial parts of O. stamineus (2.1 kg) were cut into small pieces (1 to 2 cm long) before extracted with MeOH under sonication for 10 h, at 45 o C, each 5 L for 4 times. The MeOH-soluble extract was dried under reduced pressure to give a crude MeOH-extract (196.4 g). This crude extract was excessively fractionated with hexane and EtOAc to give the hexane (26 g) and EtOAc (11 g) fractions after vacuum evaporating under reduced pressure. The EtOAc fraction was further subjected on a silica gel column chromatography (10 × 60 cm I.D; 63–200 Hoang Duc Thuan, Nguyen Phi Hung, Vu Quoc Trung 148 μm particle size), using a gradient solvent system of hexane:acetone (15:1 → 0:1, v/v), to yield ten combined fractions (OS.EA1 to OS.EA10) according to their TLC profiles. Fraction OS.EA2 was further chromatographed on a silica gel column (3.5 x 60 cm), eluting with hexane:EtOAc (10:1 to 5:1, v/v) to give five subfractions (OS.EA2.1 to OS.EA2.5). Compounds 1 (50 mg) and 2 (13.2 mg) were purified from subfraction OS.EA2.3 by a C18 reversed-phase (RP-18) chromatography column (2.0 × 60 cm; 40–63 μm particle size) and eluted with a gradient solvent system of MeOH–H2O (from 6:4 to 8:2, v/v). Fraction OS.EA4 was also rechromatographed on a silica gel column (3.5 × 60 cm), eluting with hexane:EtOAc (6:1 to 1:1, v/v) to give ten subfractions (OS.EA4.1 to OS.EA4.10). Subfraction OS.EA4.7 was further chromatographed by a chromatographic column (2.0 × 80 cm) using reversed-phase (RP-C18) silica gel and eluting with MeOH–H2O gradient mixture (from 1:1.5 to 3:1, v/v), afforded compounds 3 and 4, respectively. 5-hydroxy-3,7,3',4'-tetramethoxyflavone (1): Yellow amorphous powder; 1 H-NMR (500 MHz, CDCl3) H ppm: 6.87 (1H, br s, H-6), 6.78 (1H, br s, H-8), 7.63 (1H, d, J = 2.0 Hz, H-2′), 7.15 (1H, d, J = 8.4 Hz, H-5′), 7.71 (1H, dd, J = 2.0, 8.4 Hz, H-6′), 3.80 (3H, s, 3-OCH3), 3.98 (3H, s, 7-OCH3), 3.93 (3H, s, 3′-OCH3), 3.96 (3H, s, 4′-OCH3), 12.95 (1H, s, 5-OH); 13 C-NMR (125 MHz, CDCl3) C ppm: 152.3 (C-2), 132.8 (C-3), 182.8 (C-4), 158.9 (C-5), 90.8 (C-6), 164.8 (C-7), 90.8 (C-8), 149.5 (C-9), 106.3 (C-10), 123.9 (C-1′), 111.3 (C-2′), 153.3 (C-3′), 153.4 (C- 4'), 108.9 (C-5'), 120.3 (C-6'), 61.1 (3-OCH3) 56.5 (7-OCH3), 56.3 (3′-OCH3), 56.3 (4′-OCH3). 3,5,7,3′,4′-pentamethoxyflavone (2): Yellow amorphous powder; 1H-NMR (500 MHz, acetone-d6) H ppm: 7.11 (1H, br s, H-6), 6.58 (1H, br s, H-8), 7.57 (1H, d, J = 2.0 Hz, H-2′), 7.13 (1H, d, J = 8.4 Hz, H-5′), 7.64 (1H, dd, J = 2.0, 8.4 Hz, H-6′), 3.82 (3H, s, 3-OCH3), 3.88 (3H, s, 5-OCH3), 4.00 (3H, s, 7-OCH3), 3.91 (3H, s, 3′-OCH3), 3.95 (3H, s, 4′-OCH3); 13 C-NMR (125 MHz, CDCl3) C ppm: 153.8 (C-2), 134.8 (C-3), 181.2 (C-4), 159.6 (C-5), 95.8 (C-6), 164.1 (C-7), 92.5 (C-8), 158.0 (C-9), 107.3 (C-10), 123.4 (C-1′), 111.3 (C-2′), 153.4 (C-3′), 153.1 (C- 4'), 110.9 (C-5'), 121.1 (C-6'), 61.1 (3-OCH3), 56.1 (5-OCH3), 56.3 (7-OCH3), 56.5 (3′-OCH3), 56.7 (4′-OCH3). 3,5,3′-trihydroxy-7,4′-dimethoxyflavone (3): Yellow amorphous powder; 1H-NMR (500 MHz, acetone-d6) H ppm: 6.90 (1H, br s, H-6), 6.66 (1H, br s, H-8), 7.52 (1H, d, J = 2.0 Hz, H- 2′), 7.14 (1H, d, J = 8.5 Hz, H-5′), 7.58 (1H, dd, J = 2.0, 8.5 Hz, H-6′), 8.09 (1H, s, 3-OH), 12.67 (1H, s, 5-OH), 4.00 (3H, s, 7-OCH3), 3.94 (3H, s, 4′-OCH3); 13 C-NMR (125 MHz, acetone-d6) C ppm: 164.9 (C-2), 131.1 (C-3), 183.5 (C-4), 165.0 (C-5), 91.7 (C-6), 154.9 (C-7), 104.4 (C-8), 157.2 (C-9), 106.5 (C-10), 125.1 (C-1′), 113.6 (C-2′), 147.9 (C-3′), 151.7 (C-4'), 112.5 (C-5'), 119.7 (C-6'), 56.9 (7-OCH3), 56.5 (4′-OCH3). 3,3'-dihydroxy-5,7,4′-trimethoxyflavone (4): Yellow amorphous powder; 1H-NMR (500 MHz, CDCl3) H ppm: 7.12 (1H, br s, H-6), 6.51 (1H, br s, H-8), 7.48 (1H, d, J = 2.0 Hz, H-2′), 7.13 (1H, d, J = 8.4 Hz, H-5′), 7.52 (1H, dd, J = 2.0, 8.4 Hz, H-6′), 3.87 (3H, s, 5-OCH3), 3.94 (3H, s, 7-OCH3), 4.01 (3H, s, 4′-OCH3), 12.95 (1H, s, 5-OH); 13 C-NMR (125 MHz, CDCl3) C ppm: 152.3 (C-2), 132.8 (C-3), 182.8 (C-4), 158.9 (C-5), 90.8 (C-6), 164.8 (C-7), 90.8 (C-8), 149.5 (C-9), 106.3 (C-10), 123.9 (C-1′), 111.3 (C-2′), 153.3 (C-3′), 153.4 (C-4'), 108.9 (C-5'), 120.3 (C-6'), 61.1 (3-OCH3) 56.5 (7-OCH3), 56.3 (3′-OCH3), 56.3 (4′-OCH3). 2.4. Protein tyrosine phosphatase 1B (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 tested samples were evaluated using the method described in the reported paper [11]. Methoxyflavones from Orthosiphon stamineus Benth. and their PTP1B 149 3. RESULTS AND DISCUSSION 3.1. Isolation and structural elucidation of isolated compounds The methanol extract of the aerial parts of Cat’s whiskers were partitioned with hexane and ethyl acetate. Phytochemical research of the ethyl acetate fraction led to the isolation of four natural products (14) (Fig. 1). Figure 1. Chemical structure of compounds 1-4 isolated from O. stamineus Benth. Compound 1 was obtained as yellow powder, the ESI mass spectrum of 1 exhibited an ion peak at m/z 359 [M+H] + , suggesting a molecular formula of C19H18O7 (M = 358). Its UV spectrum showed absorption bands of a typical flavone at 270 and 340 nm [12]. The 1 H NMR spectrum of 1 showed two broad singlet proton peaks at δH 6.87 (1H, br s, H-6) and 6.78 (1H, br s, H-8) that helped define ring A. An ABX-aromatic spin system at δH 7.63 (1H, d, J = 2.0 Hz, H-2′), 7.15 (1H, d, J = 8.5 Hz, H-5′), and 7.71 (1H, dd, J = 2.0, 8.5 Hz, H-6′), which were consistent with the substitution pattern assigned for ring B. The chemical shifts of C-3′ (δC 153.3) and C-4′ (δC 149.5) in the 13 C-NMR spectrum revealed oxygenation at these carbons. In addition, the 1 H and 13 C-NMR spectra of 1 gave four methoxy groups [δH 3.80 (3H, s, 3-OCH3), 3.98 (3H, s, 7-OCH3), 3.93 (3H, s, 3′-OCH3), and 3.96 (3H, s, 4′-OCH3), with corresponding carbon signals at δC 61.1 (3-OCH3) 56.5 (7-OCH3), 56.3 (3′-OCH3), and 56.3 (4′-OCH3)], all of these were found to be attached to C-3, C-7, C-3′, and C-4′ due to an conjugated hydroxyl group (δH 12.95, 1H, s) attached at C-5 found in the 1 H NMR spectrum [13]. A detailed comparison between the 1 H and 13 C-NMR data of 1 with published values led to the structurally identification of 1 as 5-hydroxy-3,7,3',4'-tetramethoxyflavone [14]. Compound 2 was also obtained as yellow powder. A molecular ion peak at m/z 373.12 [M+H] + obtained in the ESI-MS revealing a molecular formula of C20H20O7 for 2. The 1 H- and 13 C-NMR spectra of compound 2 were quite similar to compound 1 with four methoxy groups at δH 3.82 (3-OCH3), 4.00 (7-OCH3), 3.91 (3′-OCH3), and 3.95 (4′-OCH3), two singlet proton peaks at δH 7.11 (H-6) and 6.58 (H-8) of ring A, and an ABX-aromatic spin system of ring B at δH 7.57 (1H, d, J = 2.0 Hz, H-2′), 7.13 (1H, d, J = 8.5 Hz, H-5′), and 7.64 (1H, dd, J = 2.0, 8.5 Hz, H-6′). The difference between 1 and 2 was only the replacement of the 5-OH group in 1 by 5-OCH3 group in 2 (δH 3.88 and δC 56.1). Thus, chemical structure of compound 2 was determined as 3,5,7,3′,4′-pentamethoxyflavone [15]. Hoang Duc Thuan, Nguyen Phi Hung, Vu Quoc Trung 150 Compound 3 was obtained as yellow amorphous powder. The molecule formula of 3 was revealed as C17H14O7 based on a molecular ion peak at m/z 331.07 [M+H] + obtained from its ESI-MS. The 1 H-NMR spectrum of 3 also showed an aromatic ABX-spin system at δH 7.58 (1H, dd, J = 2.0, 8.5 Hz, H-6′), 7.14 (1H, d, J = 8.5 Hz, H-5′), and 7.52 (1H, d, J = 2.0 Hz, H-2′) assigning for the B ring, two broad singlet proton peaks at δH 6.90 (1H, br s, H-6) and 6.66 (1H, br s, H-8) of the A ring, and two singlet proton resonated at δH 12.94 (1H, s), which was assignable to 5-OH, and δH 8.09 (1H, br s) assignable to 3-OH [13]. In addition, two methoxy protons at δH 4.00 and 3.94 (each 3H, s) with corresponding carbons at δC 56.9 and 56.5 were displayed in the 1 H- and 13 C-NMR spectra of 3. The chemical shifts of C-3′ (δC 147.9) and C-4′ (δC 151.7) in the 13 C NMR spectrum revealed oxygenation at these carbons. In addition, the chemical shifts of C-3 appeared at δC 131.1 in the 13 C-NMR, revealing a hydroxyl group attached at C-3 position. Analysis of the HMBC data of 3 allowed us to assign the attachment of two methoxy group at C-7 and C-4′, respectively (Figure 2). Thus, the structure of compound 3 was established as 3,5,3′-trihydroxy-7,4′-dimethoxyflavone [16]. Compound 4 was also obtained as yellow powder. A molecular ion peak at m/z 345.09 [M+H] + was observed in the ESI-MS suggesting its molecular formula of C18H16O7. The 1 H- and 13 C-NMR spectra of compound 4 were quite similar to that of compound 3 except only for an additional methoxy signals at δH 3.87 and δC 56.3 in 4. In addition, the conjugated hydroxyl peak in the 1 H NMR spectrum of 3 was disappeared in 4. Two methoxy groups at δH 4.01 (7-OCH3) and 3.94 (4′-OCH3), two singlet proton peaks at δH 7.12 (H-6) and 6.51 (H-8) of ring A, an ABX-aromatic spin system of ring B at δH 7.48 (1H, d, J = 2.0 Hz, H-2′), 7.12 (1H, d, J = 8.5 Hz, H-5′), and 7.52 (1H, dd, J = 2.0, 8.5 Hz, H-6′) were also presented. Thus, chemical structure of compound 4 was determined as 3,3′-dihydroxy-7′,4′-dimethoxyflavone [17]. Figure 2. 1 H- 13 C (→) key HMBC correlations of compounds 3 and 4. 3.2. PTP1B inhibitory activity of isolated compounds The inhibitory effects of isolated compounds (1-4) on PTP1B enzyme activity were measured using ursolic acid as positive control (Table 1) [11]. All of the isolates (1-4) exhibited dose-dependent inhibition, among the isolates, 3,3′-dihydroxy-5,7,4′-trimethoxyflavone (4) possessed potential inhibitory activity with an IC50 value of 10.12 ± 0.19 µM. Compound 3 displayed weak activity with IC50 value of 52.64 ± 4.12 µM while compounds 1 and 2 showed no effect. The positive control, ursolic acid, showed an IC50 value of 3.42 ± 0.07 µM in this enzyme assay. Among these isolates, compound 1 with four methoxy groups at C-3, C-7, C-3, and C-4, and a hydroxyl group at C-5 showed no activity (IC50 > 100 µM), compound 2 with five methoxy group exhibited the same manner. In contrast, compound 3 with three hydroxyl groups at C-3, C-5, and C-3′, compound 4 with two hydroxyl groups at C-3 and C-3′ displayed stronger activity (IC50 value of 52.64 ± 4.12 and 10.12 ± 0.19 µM). This observation may suggest that the number of methoxy group and/or the position of the substitution of methoxy by hydroxy group in these flavonol-type compounds may be responsible to the diminishment of Methoxyflavones from Orthosiphon stamineus Benth. and their PTP1B 151 inhibitory activity of these compounds on PTP1B. In our knowledge, compounds 1-4 were first time isolated from O. stamineus, and that the PTP1B inhibitory activities of these compounds have also been investigated for the first time. Table 1. PTP1B inhibitory activity of isolated compounds (1-4) and ursolic acid. Compounds Inhibitory activity (IC50, µM) a 5-hydroxy-3,7,3',4'-tetramethoxyflavone (1) > 100 3,5,7,3′,4′-pentamethoxyflavone (2) > 100 3,5,3′-trihydroxy-7,4′-dimethoxyflavone (3) 10.12 ± 0.19 3,3′-dihydroxy-5,7,4′-trimethoxyflavone (4) 52.64 ± 4.12 Ursolic acid b 3.42 ± 0.07 a Results are expressed as IC50 values (µM), determined by regression analysis and expressed as the means ± SD of three replicates. b Positive control. 4. CONCLUSIONS Using combined chromatographic and spectroscopic methods, four flavones including 5- hydroxy-3,7,3',4'-tetramethoxyflavone (1), 3,5,7,3′,4′-pentamethoxyflavone (2), 3,5,3′- trihydroxy-7,4′-dimethoxyflavone (3), and 3,3′-dihydroxy-5,7,4′-trimethoxyflavone (4) were isolated and structurally identified from the methanol extract of the aerial parts of Orthosiphon stamineus Benth. 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