Phenolic and flavonoid compounds isolated from ethyl acetate fraction of helicteres hirsuta lour. Collected in Thua Thien Hue

T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 122 PHENOLIC AND FLAVONOID COMPOUNDS ISOLATED FROM ETHYL ACETATE FRACTION OF Helicteres hirsuta Lour. COLLECTED IN THUA THIEN HUE Hoang Dac Thang1, Truong Thi Thu Hien1, Nguyen Thi Thu Hien1 Tran Minh Duc1, Do Thanh Tuan1, Nguyen Phuc Khanh Nhi2 Le Canh Viet Cuong5, Hoang Le Tuan Anh5 SUMMARY Objectives: To isolate and determine chemical structure of compounds from Helicteres hirsuta Lour. Subjects and methods: The aerial part of the Helicteres hirsuta Lour. collected in A Luoi district, Thua Thien Hue province in March 2019 were powdered and extracted with different solvents including ethyl acetate. Compounds structures were elucidated by 1H-, 13C-NMR, HSQC, and HMBC spectral as well as in comparison with those reported in the literature. Results and conclusions: From the ethyl acetat extract of Helicteres hirsuta Lour, four compounds including methyl-2-O-β-D-glucopyranosyl benzoate (1), syringin (2), 3,4ʹ,7,8- tetrahydroxyflavone (3), and tiliroside (4) were isolated. Compounds 1, 2 and 3 were reported from genus Helicteres for the first time. * Keywords: Helicteres hirsuta; Flavonoid; Phenolic. INTRODUCTION Helicteres hirsuta Lour. is a shrub, distributed mainly in Asian countries such as Vietnam, India, Myanmar, China, Philippines, Malaysia, Cambodia, Indonesia and Thailand [1]. According to traditional experience, its leaves and roots are used as pain relievers, and for treatment of dysentery, flu, measles, boils, dribbling and liver-related diseases [1, 2]. The main chemical components of this plant are flavonoids, phenolics, lignans and triterpenoids [2, 3]. Additionally, there are other compounds such as steroids, benzofuran [4]. Helicteres hirsuta Lour. is a valuable medicinal material that receives much attention from domestic and international scientists. Biological activity studies have shown that extract and compounds isolated from Helicteres hirsuta have anticancer, antibacterial and 1Vietnam Military Medical University 2Hanoi University of Mining and Geology 3Vietnam University of Traditional Medicine 4Thai Binh University of Medicine and Pharmacy 5Mientrung Institute for Scientific Research, Vietnam Academy of Science and Technology Corresponding author: Hoang Le Tuan Anh (hltanh@misr.vast.vn) Date received: 14/8/2020 Date accepted: 10/10/2020 T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 123 antioxidants activity [5, 6]. We conducted this study: To describe the results of the isolation and chemical structure of four compounds were isolated from ethyl acetate fraction of Helicteres hirsuta Lour. collected in Thua Thien Hue province. In particular, the compounds methyl-2-O-β- D-glucopyranosyl benzoate (1), siringin (2) and 3,4ʹ,7,8-tetrahydroxyflavone (3) were first isolated from the genus Helicteres. MATERIALS AND METHODS 1. Materials The aerial part of the Helicteres hirsuta Lour. was collected in A Luoi district, Thua Thien Hue province in March 2019. The scientific name is identified by MSc Le Tuan Anh, Mientrung Institute for Scientific Research, Vietnam Academy of Science and Technology (VAST). The voucher specimens are kept at Mientrung Institute for Scientific Research, VAST and Vietnam Military Medical University. 2. Solvents and equipments Thin-layer chromatography (TLC) performed on Aluminum TLC plate coated normal phase (DC-Alufolien 60 F254, Merck); reversed-phase C-18 (150 µm, Fujisilica Chemical Ltd). Substances are detected by two-wavelength 254 nm and 368 nm ultraviolet light or using reagents that are 10% H2SO4 solution sprayed evenly on the thin plate, dry and heat slowly until the color appears. Column chromatography is conducted with adsorbent normal phase and reversed-phase. The particle size of the normal phase silica gel is 0.040 - 0.063 mm (240 - 430 mesh, Merck) and reversed-phase of YMC (30-50 µm, Fujisilica Chemical Ltd). Nuclear magnetic resonance (NMR): Measured on the Bruker AM500 of the Institute of Chemistry, VAST. 3. Extraction and isolation The dried aerial parts of Helicteres hirsuta (5.0 kg) were powdered and extracted three times with methanol (3 × 10 liters); distill the solvent to obtain 300g of methanol extract. This residue is distributed into 2 liters of water and extracted in turn with n-hexane, dichloromethane and ethyl acetate. After removal of the solvent under reduced pressure, n-hexane extract (HHH, 230g), dichloromethane extract (HHD, 17.0g), ethyl acetate extract (HHE, 26.0g) and water layer (HHW) were obtained. The ethyl acetate residue (HHE, 26.0g) crudely separated on a silicagel gel of normal phase column chromatography eluting with dichloromethane/methanol (20/1, v/v) obtained 3 fractions, HHE1-HHE3. The fraction HHE2 continued to be separated on the reversed-phase chromatography column (RP-18) with methanol/water elution solvent to give 6 fractions, HHE2.1-HHE2.6. Compound 1 (7.0 mg) was obtained after purifying the fraction HHE2.2 on normal phase CC with dichloromethane/methanol elution solvent (15/1, v/v). Compound 3 (5.0 mg) was obtained when splitting HHE2.4 fractions on Sephadex LH20 column chromatography with methanol/water (1/1, v/v). Compound 2 (4.5 mg) and 4 (6.0 mg) were obtained by splitting the HHE2.6 fraction on the Sephadex LH20 column chromatography with methanol/water eluting solvent system (1/1, v/v). T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 124 RESULTS AND DISCUSSIONS * Isolated compounds: Compound 1: White powder with molecular formula: C14H18O8 (M = 314). 1H-NMR (500 Hz, MeOD-d4) δH (ppm): 7.78 (1H, dd, J = 2.0, 7.5 Hz, H-3), 7.15 (1H, td, J = 1.0, 7.5 Hz, H-4), 7.56 (1H, ddd, J = 2.0, 7.5, 8.5 Hz, H-5), 7.42 (1H, dd, J = 1,0, 8.5 Hz, H-6), 4.92 (1H, d, J = 7.5 Hz, H-1ʹ), 3.55 (1H, m, H-2ʹ), 3.48 (1H, m, H-3ʹ), 3.43 (1H, m, H-4ʹ), 3.50 (1H, m, H-5ʹ), 3.74 (1H, dd, J = 5.5, 12.0 Hz, Ha-6), 3.93 (1H, dd, J = 2.0, 12.0 Hz, Hb-6), 3.91 (3H, s, OCH3). 13C-NMR (125 Hz, MeOD-d4) δC (ppm): 158.7 (C-1), 122.4 (C-2), 132.0 (C-3), 123.7 (C-4), 135.1 (C-5), 119.0 (C-6), 168.6 (C-7), 104.1 (C-1ʹ), 75.0 (C-2ʹ), 78.5 (C-3ʹ), 71.3 (C-4ʹ), 77.6 (C-5ʹ), 62.6 (C-6ʹ), 52.8 (OCH3). Compound 2: White powder with molecular formula: C17H24O9 (M = 372). 1H-NMR (500 Hz, MeOD-d4) δH (ppm): 6.77 (2H, s, H-2, H-6), 6.57 (1H, d, J = 16.0 Hz, H-7), 6.35 (1H, d, J = 16.0 Hz, H-8), 4.24 (1H, dd, J = 2.0, 11.5 Hz, H-9), 4.89 (1H, d, J = 7.5 Hz, H-1ʹ), 3.50 (1H, m, H-2ʹ), 3.44 (1H, m, H-3ʹ), 3.43 (1H, m, H-4ʹ), 3.23 (1H, m, H-5ʹ), 3.69 (1H, dd, J = 5.5, 12.0 Hz, Ha-6ʹ), 3.80 (1H, dd, J = 2.5, 12.0 Hz, Hb-6ʹ), 3.88 (6H, s, 3ʹ, 5ʹ-OCH3). 13C-NMR (125 Hz, MeOD-d4) δC (ppm): 135.3 (C-1), 105.5 (C-2, C-6), 154.4 (C-3, C-5), 135.9 (C-4), 131.3 (C-7), 130.1 (C-8), 63.6 (C-9), 105.4 (C-1ʹ), 75.8 (C-2ʹ), 77.9 (C-3ʹ), 71.4 (C-4ʹ), 78.4 (C-5ʹ), 62.6 (C-6ʹ), 57.1 (3ʹ, 5ʹ-OCH3). Compound 3: Yellow powder with molecular formula: C15H10O6 (M= 286). 1H-NMR (500 Hz, MeOD-d4) δH (ppm): 7.57 (1H, d, J = 8.5 Hz, H-5), 6.98 (1H, d, J = 8.5 Hz, H-6), 8.26 (2H, d, J = 8.5 Hz, H-2ʹ, H-6ʹ), 8.95 (2H, d, J = 8.5 Hz, H-3ʹ, H-5ʹ). 13C-NMR (125 Hz, MeOD-d4) δC (ppm): 147.7 (C-2), 138.2 (C-3), 174.9 (C-4), 116.7 (C-5), 115.1 (C-6), 156.9 (C-7), 134.1 (C-8), 151.4 (C-9), 116.4 (C-10), 124.2 (C-1ʹ), 131.0 (C-2ʹ, C-6ʹ), 116.3 (C-3ʹ, C-5ʹ), 160.5 (C-4ʹ). Compound 4: Yellow powder with molecular formula: C30H26O13 (M = 549). 1H-NMR (500 Hz, MeOD-d4) δH (ppm): 6.15 (1H, d, J = 2.0 Hz, H-6), 6.32 (1H, d, J = 2,0 Hz, H-8), 8.01 (2H, d, J = 9.0 Hz, H-2ʹ, H-6ʹ), 6.84 (2H, d, J = 9.0 Hz, H-3ʹ, H-5ʹ), 5.24 (1H, d, J = 7,5 Hz, H-1ʺ), 3.49 (1H, m, H-2ʺ), 3.47 (1H, m, H-3ʺ), 3.35 (1H, m, H-4ʺ), 3.49 (1H, m, H-5ʺ), 4.21 (1H, dd, J = 6.5, 12.0 Hz, Ha-6ʺ), 4.32 (1H, dd, J = 2.0, 12.0 Hz, Hb-6ʺ), 6.95 (1H, d, J = 15.5 Hz, H-2‴), 7.42 (1H, d, J = 15.5 Hz, H-3‴),7.33 (2H, d, J = 8.5 Hz, H-5‴, H-9‴), 6.82 (2H, d, J = 8.5 Hz, H-6‴, H-8‴).13C-NMR (125 Hz, MeOD-d4) δC (ppm): 159.3 (C-2), 135,.2 (C-3), 179.4 (C-4), 162.9 (C-5), 100.3 (C-6), 166.8 (C-7), 95.0 (C-8), 158.5 (C-9), 105.4 (C-10), 122.8 (C-1ʹ), 132.2 (C-2ʹ, C-6ʹ ), 116.1 (C-3ʹ, C-5ʹ), 161.5 (C-4ʹ), 104.1 (C-1ʺ), 75.7 (C-2ʺ), 78.0 (C-3ʺ), 71.7 (C-4ʺ), 75.8 (C-5ʺ), 64.3 (C-6ʺ), 168.8 (C-1‴), 114.8 (C-2‴), 146.6 (C-3‴), 127.1 (C-4‴), 131.2 (C-5‴, C-9‴), 116.8 (C-6‴, C-8‴), 161.2 (C-7‴). * Structural identification of isolated compounds: The 1H-NMR spectrum of compound 1 appear four protons of the aromatic ring 1, 2 potential at δH 7.78 (1H, dd, J = 2.0, 7.5 Hz), 7.15 (1H, td, J = 1.0, 7.5 Hz), 7.56 (1H, ddd, J = 2.0, 7.5, 8.5 Hz), 7.42 (1H, dd, J = 1.0, 8.5 Hz); an anomer proton at T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 125 δH 4.92 (1H, d, J = 7.5 Hz); and three protons of a methoxy group at δH 3.91 (3H, s). Besides, on 13C-NMR and HSQC observed signals of 14 carbon including six carbons of aromatic ring 1, 2 potential at δC 158.7, 135.1, 132.0, 123.7, 122.4 and 119.0; six carbons of glucopyranosyl at δC 104.1, 78.5, 77.6, 75.0, 71.3 and 62.6; one carbon carboxyl at δC 168.6 and one methoxy group δC 52.8. The interaction constant of the proton anome JH1ʹ/H-2ʹ = 7.5 Hz, besides the value of the chemical displacement of the six carbons in the saccharine section allowed the determination of the saccharine part of compound 1 was β-glucopyranosyl. The location of carbon carboxyl connection with the aromatic ring at C-1 was determined based on the HMBC interaction between H-6 (δH 7.42) and carbon C-7 (δC 168.6). Similarly, the position of the saccharine section attached to the aromatic ring at C- 2 and the methoxy group at C-7 were determined based on HMBC interactions between H-1ʹ (δH 4.92) and C-2 (δC 122.4) and methoxy protons (δH 3.91) with C-7 (δC 168.6) (figure 1). From the above analysis, combined with the previous published data, compound 1 was identified as methyl-2-O-β-D-glucopyranosyl benzoate [7]. According to our search, this is the first time compound 1 which has been isolated from the genus Helicteres. This compound has been reported to have anti-inflammatory activity [8]. The 1H-NMR spectrum of 2 appear signals of four olefin protons at δH 6.77 (2H, s), 6.57 (1H, d, J = 16.0 Hz), 6.35 (1H, d, J = 16.0 Hz); one proton anome at δH .89 (1H, d, J = 7.5 Hz) and six protons of two methoxy groups at δH 3.88 (6H, s). Analysis of signals on 13C-NMR and HSQC spectra allowed to determine the presence of 17 carbon including four non- hydrogen carbons at δC 154.4 (2C), 135.9, 135.3; nine methine groups at δC 131.3, 130.1, 105.5 (2CH), 105.4, 78.4, 77.9, 75.8, 71.4; two methylene groups at δC 63.6, 62.6; and two methoxy groups at δC 57.1 (2OCH3). A sharp increased in the intensity of carbon signals δC 154.4 (2C), 105.5 (2CH) and 57.1 (2OCH3), allowed to suggest an aromatic ring with a symmetrical structure. The interaction constant of the proton anome JH1ʹ/H-2ʹ = 7.5 Hz, along with the chemical shift of the six carbons in the saccharine section (105.4, 75.8, 77.9, 71.4, 78.4 and 62.6) allowed the determination of the saccharine portion of compound 2 was β-glucopyranosyl. The chemical shift values of carbon δC 131.3 (C-7), 130.1 (C-8) and the interaction constant of the corresponding protons 6.57 (1H, d, J = 16.0 Hz, H-7) and 6.35 (1H, d, J = 16.0 Hz, H-8) allowed us to determine the presence of CH = CH double bonds trans configuration. Interactions on the HMBC spectrum between H-7 (δH 6.57) and C-9 (δC 63.6) and H-9 (δH 4.24) and C-7 (δC 131.3) determined the presence of hydroxypropenyl group. The position of this group associated with the aromatic ring at C-1 was determined based on the HMBC interaction between H-7 (δH 6.57) and C-1 (δC 135.3)/C-2/C-6 (δC 105.5) and H-8 (δH 6.35) with C-1 (δC 135.3). In addition, HMBC interactions between proton anomers (δH 4.89) with C-4 (δC 135.9) and protons of two methoxy groups (δH 3.88) with C-3/C-5 (δC 154.4) in turn determine the location of the saccharine section associated with the T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 126 aromatic ring at C-4 and the two methoxy groups at C-3, C-5 (figure 1). Moreover, the results of comparing NMR spectra of compound 2 with published data [9] of syringin compound illustrated that the data were consistent at the corresponding locations. From the above analysis, compound 2 was determined to be syringin. According to our search, this is also the first time compound 2 has been isolated from the genus Helicteres. Biological activity studies showed that syringin has anti-inflammatory, cytotoxic, and osteoporotic effects [10]. Figure 1: Chemical structure and HMBC interactions of compounds 1, 2. Compound 3: The 1H-NMR spectrum of 2, signals of six aromatic protons appeared at δH 8.95 (2H, d, J = 8.5 Hz), 8.26 (2H, d, J = 8.5 Hz), 7.57 (1H, d, J = 8.5 Hz), 6.98 (1H, d, J = 8,5 Hz). Analysis of signals on the 13C-NMR and HSQC spectra determined the presence of 15 carbon including 1 carbon carbonyl at δC 174.9, eight carbons not associated with hydrogen at δC 147.7, 138.2, 156.9, 134.1, 151.4, 116.4, 124.2, 160.5; six methin carbons including four symmetric methin carbons at δC 131.0 (2CH), 116.3 (2CH) and two methin carbons at δC 116.7, 115.1. NMR spectral data of compound 3 suggested this was a flavon. HMBC interactions from H-5 (δH 7.57) to C-4 (δC 174.9)/C-7 (δC 156.9)/C-9 (δC 151.4) and H-6 (δH 6.98) to C-8 (δC 134.1)/C-10 (δC 116.4) determine the positions of the two hydroxyl groups at C-7 and C-8. Similarly, HMBC interactions from H-2ʹ/H-6ʹ (δH 8.26) to C-2 (δC 138.2)/C-4ʹ (δC 160.5) and H-3ʹ/H-5ʹ (δH 8.95) to C-1ʹ (δC 124.2)/C-4ʹ (δC 160.5) determined the presence of hydroxyl group at C-4ʹ. In addition, T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 127 the presence of a non-hydrogen-associated carbon signal (C-3) with chemical shift of δC 138.2 allowed predicting the presence of the hydroxyl group at this location (figure 2). The above analysis combined with previous published data [11], compound 3 was identified as 3,4ʹ,7,8- tetrahydroxyflavone. According to our investigation, the compound 3,4ʹ,7,8- tetrahydroxyflavone was also isolated for the first time from Helicteres genus. This compound has been reported to have antioxidant and potential compound in the development of anticancer products [12]. Figure 2: Chemical structure and HMBC interactions of compounds 3, 4. In a similar way, compound 4 was identified as tiliroside [13]. This compound has been reported to be isolated from Helicteres hirsuta [2]. Tiliroside is a flavon that is often present in the chemical composition of plants with many remarkable bioactive such as cytotoxin, antioxidant, inhibition of β-glucoside enzymes related to diabetes, anti- inflammatory, anti-osteoporosis... [14]. CONCLUSION From the ethyl acetate fraction of Helicteres hirsuta Lour. collected in Thua Thien Hue province, we isolated and determined the chemical structure of four compounds including methyl-2-O-β-D- glucopyranosyl benzoate (1), syringin (2), 3,4ʹ,7,8-tetrahydroxyflavone (3) and tiliroside (4). In which compounds 1, 2 and 3 were the first time isolated from the genus Helicteres. REFERENCES 1. VV Chi. 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