Phytochemical study of Euphorbia cyathophora collected in Dan Phuong, Hanoi and its antidengue activity against DENV1-4 virus serotypes

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In this study, the antidengue virus activity of the ethanol extract from the leaves and twigs of E. cyathophora on four virus serotypes DENV1-DENV4 was examined. The result showed that the ethanol extract displayed antidengue activity on DENV1-4 with PRNT50 values of 15.625-62.5 µg/mL. This extract was fractionated to the n-hexane and ethyl acetate fractions.

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Nội dung Text: Phytochemical study of Euphorbia cyathophora collected in Dan Phuong, Hanoi and its antidengue activity against DENV1-4 virus serotypes

Cite this paper: Vietnam J. Chem., 2023, 61(3), 371-377 Research article DOI: 10.1002/vjch.202300123 Phytochemical study of Euphorbia cyathophora collected in Dan Phuong, Hanoi and its antidengue activity against DENV1-4 virus serotypes Nguyen Thi Dung1, Pham Thi Ninh1, Nguyen Thi Luu1, Nguyen The Anh1, Vu Thi Bich Hau2, Nguyen Thi Thu Thuy2, Ho Ngoc Anh3, Phan Dieu Hang4, Tran Thi Phuong Thao1,5* 1 Institute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi 10000, Viet Nam 2 National Institute of Hygiene and Epidemiology, 1 Yersin Street, Hai Ba Trung, Hanoi 10000, Viet Nam 3 Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi 10000, Viet Nam 4 Hanoi University of Medicine, Nr. 1, Ton That Tung Street, Kim Lien, Dong Da, Hanoi 10000, Viet Nam 5 Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi 10000, Viet Nam Submitted March 30, 2023; Revised May 17, 2023; Accepted May 21, 2023 Abstract The ethanol extract from the leaves and twigs of Euphorbia cyathophora showed antiviral activity against four dengue virus serotypes (DENV1-4), with PRNT50 values of 15.625-62.5 µg/mL. Eight compounds including germanicol acetate (1), cymbopogone (2), 1-heptacosanol (3), ethyl α-L-arabinofuranoside (4), quercetin (5), 3-acetoxy-5,7,3’,4’- tetrahydroxyflavone (6), 3-acetoxy-5,7,4’-trihydroxyflavone (7), 4’-acetoxy-3,5,7-trihydroxyflavone (8) were isolated from this extract. Compounds 2-8 were isolated for the first time from Euphorbia cyathophora. Antiviral activity evaluation of compounds 1-3, 5-7 on DENV1-4 serotypes revealed that 7 was the most active compound with PRNT50 values of 23.8-95.2 µM. Keywords. Euphorbia cyathophora, antidengue activity, secondary metabolites. 1. INTRODUCTION terpenoids.[2,4,6,7] In this study, the antidengue virus activity of the ethanol extract from the leaves and Euphorbia cyathophora (Vietnamese name: Tieu twigs of E. cyathophora on four virus serotypes Trang Nguyen) is a plant belonging to Euphorbiaceae DENV1-DENV4 was examined. The result showed family.[1] This plant is widely distributed in tropical that the ethanol extract displayed antidengue activity and subtropical areas, especially in America and on DENV1-4 with PRNT50 values of 15.625-62.5 South East Asia. In Vietnam, it is cultivated along the µg/mL. This extract was fractionated to the n-hexane country as an ornamental or fence tree. Traditionally, and ethyl acetate fractions. Phytochemical the Vietnamese people used E. cyathophora leaves investigation of these fractions led to the isolation of for treatment of boil and scabies diseases, wound eight compounds including germanicol acetate (1), healing, menstrual disorder, detoxification and skin cymbopogone (2), 1-heptacosanol (3), ethyl α-L- bleeding. Its roots and barks are used to cure arabinofuranoside (4), quercetin (5), 3-acetoxy- respiratory disorder and diahera.[1,2] This plant 5,7,3’,4’-tetrahydroxyflavone (6), 3-acetoxy-5,7,4’- possesses diverse biological activities such as trihydroxyflavone (7), 4’-acetoxy-3,5,7- neuroprotection,[2] liver protection,[3] antimicrobial,[4] trihydroxyflavone (8). Except 1, the other seven anti-inflammation,[4] anti age, anticancer,[2] and anti- compounds have been isolated for the first time from hyperglycemic activity.[5] Up to now, there are few this plant. Evaluation of antidengue activity of studies on the chemical constitution of E. compounds 1-3, 5-7 showed that the flavonoid 7 was cyathophora. Phytochemical investigation of this the most active compound against DENV1-4, with plant showed the presence of flavonoids, phenols and PRNT50 values of 23.8 to 95.2 µM. This work 371 Wiley Online Library © 2023 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH
25728288, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300123 by Readcube (Labtiva Inc.), Wiley Online Library on [02/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Vietnam Journal of Chemistry Tran Thi Phuong Thao et al. reported for the first time antidengue activity of the (n-hexane/acetone 100/0→98/2) to yield seven above compounds against all dengue virus serotypes. fractions (ECH4.1→ECH4.7). The subfraction ECH4.3 (3.4 g) was repeatedly chromatographed 2. MATERIALS AND METHODS over silica gel column (n-hexane/EtOAc 99/1; 98/2; 95/5; 9/1), following purification with a Sephadex 2.1. Plant materials column to yield compound 1 (3.7 mg). Fraction ECH 4.7 (2.8 g) was separated over a silica gel column, Euphorbia cyathophora Murray leaves and twigs eluting with n-hexane/CH2Cl2 (100/0→9/1) to give were collected in Dan Phuong district, Hanoi in fourteen subfractions (ECH 4.7.1→ECH 4.7.14). January 2022. The plant was authenticated by Mr. Fraction ECH 4.7.7 (1.2 g) was chromatographed Nguyen The Anh, Institute of Chemistry, Vietnam over a Sephadex column (n-hexane/CH2Cl2/MeOH Academy of Science and Technology (VAST). A 1/1/2) to yield compound 3 (6 mg). Purification of voucher specimen was deposited in the Department fraction ECH 4.5 (2.5 g) over a silica gel column (n- of Organic Synthesis, Institute of Chemistry, VAST. hexane/CH2Cl2 100/0→9/1), following by Sephadex LH 20 (n-hexane/CH2Cl2/MeOH 1/1/2) to obtain 2.2. General experiment procedures compound 2 (4.4 mg). The ethyl acetate fraction (78.1 g) was The chemical and equipment’s using in this study chromatographed over a dianion column, eluting were described in our recent publication.[8] from 100% H2O to H2O/EtOH 7/3, 1/1 and 100% EtOH to obtain 8 fractions (ECE1→ECE8). Fraction 2.3. Antidengue virus bioassay ECE1 (2.9 g) was repeatedly purified by silica gel column chromatography (CH2Cl2/MeOH The Plaque Reduction Neutralization Test (PRNT) 100/0→0/100) and finally with a Sephadex LH20 method was applied for evaluation of the extract and column (MeOH 100%) to obtain compound 4 (7.3 the isolated compounds. The procedure of this mg). Due to the difficulties of separation, fraction method was described in Refs. [9-11]. Denge virus ECE5 (16 g) was acetylated with acetic anhydride (10 serotypes DENV-1, DENV-2, DENV-3 and DENV-4 mL) and pyridine (10 mL) at room temperature for 24 were bred in BHK-21 cell line. The test for the extract hours. Removal of acetic anhydride and pyridine was started at 500 µg/mL, diluted in log 2 to the under vacuo gave the residue, which was separated by corresponding concentrations of 250, 125, 62.5, dianion column (100% H2O, MeOH/H2O:1/1, 8/2, 31.25 and 15.625 µg/mL. The isolated compounds and 100% MeOH). As a result, seven fractions were evaluated at the concentrations of 62.5, 31.25, (AECE5.1→AECE5.7) were obtained. Fraction 15.625, 7.81 and 3.90 µg/mL. The concentration that AECE5.5 (4.0 g) was purified by silica gel column reduced the challenge virus plaque-forming by 50% chromatography with a gradient of CH2Cl2/MeOH to is defined as PRNT50 value. MTT method was used afford seven sub-fractions (AECE 5.5.1→AECE to determine cytotoxic activity of the tested sample 5.5.7). Fraction AECE5.5.4 (562.8 mg) was on BHK-21 cells.[10] repeatedly chromatographed over Sephadex columns (100% MeOH) to yield compound 5 (6.4 mg) and 2.4. Extraction and isolation compound 7 (4 mg) as a yellow solid. Correspondingly, compound 6 (3 mg) was obtained The dried E. cyathophora leaves and twigs (5 kg) from fraction AECE5.5.5 by the same process for the were powdered and extracted with ethanol/water purification of compound 5. Fraction AECE5.7 (9/1) (3x30 Lx24h). The solvents were combined and was subjected to a silica gel column, eluted with a evaporated under reduced pressure to give the residue gradient of n-hexane/acetone (100/0→9/1) to give (300 g). The ethanol extract was added with water eight fractions (AECE 5.7.1→AECE 5.7.8). Fraction (1.5 L) and partitioned successively with n-hexane AECE 5.7.3 (654.5 mg) was repeatedly purified over and ethyl acetate (3x2L for each solvent) to obtain the Sephadex columns (n-hexane/CH2Cl2/MeOH:1/1/2) corresponding extracts (131 g and 78.1 g, to give compound 8 (2.3 mg). respectively). The n-hexane fraction (131 g) was subjected to a silica gel column, eluting with a Compound 1 (Germanicol acetate): White gradient of n-hexane/CH2Cl2/MeOH (95/5/0.2; 8/2/1; amorphous powder, ESI-MS (m/z): 468.5 [M+H]+. 1H 7/3/1; 6/4/1; 100% CH2Cl2) to afford eight fractions NMR (CDCl3, 600 MHz), δH (ppm), J (Hz): 4.86 (1H, (ECH1→ECH8). Fraction ECH4 (14.8 g) was further s), 4.49 (1H, dd, J = 10.8, 6.0), 2.04 (3H, s), 1.07 (3H, purified by silica gel column chromatography s), 1.01 (3H, s), 0.94 (3H, s), 0.93 (3H, s), 0.90 (3H, © 2023 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 372
25728288, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300123 by Readcube (Labtiva Inc.), Wiley Online Library on [02/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Vietnam Journal of Chemistry Phytochemical study of E. cyathophora collected… s), 0.85 (3H, s), 0.84 (3H, s), 0.73 (3H, s). 13C NMR 8.4, H-5'), 6.38 (1H, s, H-8), 6.18 (1H, s, H-6). 13C (CDCl3,150 MHz), δC (ppm): 171.0 (CH3COO), NMR (150 MHz, CDCl3), δC (ppm): 177.2 (C-4), 142.7 (C-18), 129.8 (C-19), 80.9 (C-3), 55.6 (C-5), 166.2 (C-7), 162.4 (C-5), 158.3 (C-9), 148.7 (C-4'), 51.1 (C-9), 43.3 (C-14), 40.8 (C-8), 38.6 (C-1), 38.4 147.9 (C-2), 146.2 (C-3'), 137.1 (C-3), 124.1 (C-1'), (C-13), 37.8 (C-4), 37.7 (C-16), 37.4 (C-22), 37.1 (C- 121.6 (C-6’), 116.2 (C-5'), 115.9 (C-2'), 104.3 (C-10), 10), 34.5 (C-7), 34.3 (C-17), 33.3 (C-21), 32.3 (C-20), 99.4 (C-6), 94.5 (C-8). 31.3 (C-29), 29.2 (C-30), 27.9 (C-23), 27.5 (C-15), 26.2 (C-12), 25.2 (C-28), 23.7 (C-2), 21.3 Compound 6 (3-Acetoxy-5,7,3’,4’- (CH3COO), 21.1 (C-11), 18.1 (C-6), 16.7 (C-26); tetrahydroxyflavone): Yellow amorphous powder, 16.5 (C-24), 16.1 (C-25), 14.5 (C-27). ESI-MS (m/z) 366.9 [M+Na]+, 342.8 [M-H]-. 1H NMR (CD3OD, 600 MHz) δH (ppm), J (Hz): 7.37 (1H, Compound 2 (Cymbopogone): White crystal, ESI- d, J = 2.4, H-2’), 7.33 (1H, dd, J = 8.4, 2.4, H-6’), MS (m/z): 427.2 [M+H]+. 1H NMR (CDCl3, 600 6.92(1H, d, J = 8.4, H-5’), 6.45 (1H, d, J = 1.8, H-8), MHz), δH (ppm), J (Hz): 2.39 (1H, m, H-2a), 2.30 6.25 (1H, d, J = 1.8, H-6), 2.34 (s, 3H, CH3COO). 13 (1H, m, H-2b), 2.25 (1H, q, J = 6.6, H-4), 1.97 (1H, C NMR (150 MHz, CDCl3), δC (ppm): 177.2 (C-4), m, H-1a), 1.84 (H-12a), 1.75 (1H, m, H-6a), 1.70 (2H, 169.8 (CH3COO), 166.5 (C-7), 163.0 (C-5), 158.7 m, H-1b, H-11a), 1.60 (3H, m, H-10, H-18, H-11b), (C-9), 158.3 (C-2), 150.5 (C-4’), 146.6 (C-3’), 131.4 1.48 (4H, m, H-21a, H-15a, H-20, H-22a), 1.43 (5H, (C-3), 122.1 (C-6’), 121.9 (C-1’), 116.5 (C-2’), 116.0 m, H-8, H-16a, H-7a, H-21b, H-22b), 1.36 (4H, m, H- (C-5’),105.5 (C-10), 100.2 (C-6), 95.1 (C-8), 20.4 6b, H-16b, H-15b, H-7b), 1.23 (3H, m, H-12b, H-19), (CH3COO). 0.98 (3H, s, H-27), 0.91 (3H, s, H-25), 0.90 (3H, s, H- 26), 0.89 (3H, d, J = 6.6, H-29), 0.88 (3H, d, J = 6.6, Compound 7 (3-acetoxy-5,7,4’- H-23), 0.83 (3H, d, J = 6.6, H-30), 0.80 (3H, s, H-28), trihydroxyflavone): Yellow amorphous powder, 0.71 (3H, s, H-24). 13C NMR (CDCl3, 150 MHz), δC HR-ESIMS (m/z): 329.0622 (calcd. for C17H13O7 (ppm): 213.1 (C-3), 60.1 (C-19), 59.7 (C-10), 58.2 329.0661 [M+H]+), 351.0445 (calcd. for C17H12O7Na (C-4), 51.7 (C-18), 49.5 (C-8), 42.7 (C-17), 42.1 (C- 351.0481 [M+Na]+). 1H NMR (CD3OD, 600 MHz) δH 5), 41.5 (C-2), 41.0 (C-6), 40.1 (C-9), 39.0 (C-13), (ppm), J (Hz): 7.76 (1H, d, J = 8.4, H-2’, H-6’), 6.94 37.9 (C-14), 35.6 (C-11), 35.5 (C-22), 30.7 (C-20), (1H, d, J = 8.4, H-3’, H-5’), 6.41 (1H, d, J = 1.2, H- 29.1 (C-16), 28.4 (C-12), 28.3 (C-15), 22.8 (C-30), 8), 6.22 (1H, d, J = 1.2, H-6), 2.32 (s, 3H, CH3COO). 13 22.3 (C-1), 21.9 (C-29), 20.1 (C-26), 19.9 (C-7), 18.2 C NMR (150 MHz, CDCl3), δC (ppm): 177.0 (C-4), (C-21), 16.3 (C-25), 16.2 (C-28), 15.6 (C-27), 14.5 169.8 (CH3COO), 164.0 (C-7), 162.9 (C-5), 162.2 (C- (C-24), 6.7 (C-23). 4'), 158.8 (C-9), 158.2 (C-2), 131.3 (C-3), 131.1 (C- 2', 6'), 121.6 (C-1'), 116.8 (C-3',5'), 104.7 (C-10), Compound 3 (1-Heptacosanol): White amorphous 100.8 (C-6), 95.5 (C-8), 20.3 (CH3COO). powder, ESI-MS (m/z): 793.3 [2M+H]+, 1H NMR (CDCl3, 600 MHz) δ (ppm), J (Hz), δH (ppm): 3.63 Compound 8 (4’-acetoxy-3,5,7- (2H, t, J = 6.6 Hz), 1.56-1.25 (m, 50 H, 25xCH2), 0.88 trihydroxyflavone): Yellow amorphous powder, (3H, t, J = 7.2). 13C NMR, (CDCl3 150 MHz), δC HR-ESIMS (m/z): 327.0514 (calcd. for C17H11O7 (ppm): 63.1, 32.8, 31.9, 29.6, 25.6, 14.1. 327.0505 [M-H]-). 1H NMR (Acetone-d6, 600 MHz) δH (ppm), J (Hz): 8.29 (1H, d, J = 8.4, H-2', H-6'), Compound 4 (Ethyl α-L-arabinofuranoside): 7.32 (d, J = 8.4, H-3’, H-5’), 6.56 (1H, s, H-8), 6.28 White amorphous powder, αD25, (c 0.1, MeOH) = + (1H, s, H-6), 2.30 (s, 3H, CH3COO). 13C NMR (150 33.8. ESI-MS (m/z): 415.0 [2M+2H2O+Na]+. 1H MHz, Acetone-d6), δC (ppm): 177.0 (C-4), 169.4 NMR (CDCl3, 600 MHz) δH (ppm), J (Hz): 4.88 (1H, (CH3COO), 165.4 (C-7), 162.3 (C-9), 157.9 (C-5), d, J = 1.8), 3.95 (1H, dd, J = 4.2, 1.8), 3.93 (1H, m), 153.0 (C-4'), 145.5 (C-2), 137.8 (C-3), 129.7 (C-2', C- 3.84 (1H, dd, J = 6.6, 4.2), 3.79 (2H, m), 3.65 (1H, 6'), 129.5 (C-1'), 122.8 (C-3', C-5'), 104.2 (C-10), dd, J = 12.0, 5.4), 3.50 (1H, m), 1.22 (3H, t, J = 6.6). 99.3 (C-6), 94.6 (C-8), 20.9 (CH3COO). 13 C NMR (CDCl3, 150 MHz), δC (ppm): 109.2 (C-1’), 85.2 (C-4’), 83.6 (C-2’), 78.8 (C-3’), 64.2 (C-1), 63.0 3.RESULTS AND DISCUSSION (C-5’), 15.4 (C-2). 3.1. Antidengue virus activity of the ethanol Compound 5 (Quercetin): Yellow amorphous extract powder, (+)-ESI-MS (m/z) 302.9 [M+H]+. 1H NMR (CD3OD, 600 MHz) δH (ppm), J (Hz): 7.74 (1H, s, H- Evaluation of antidengue activity of the ethanol 2’), 7.65 ( 1H, dd, J = 8.4, 1.2, H-6’), 6.90 (1H, d, J = extract of E. cyathophora indicated PRNT50 values of © 2023 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 373
25728288, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300123 by Readcube (Labtiva Inc.), Wiley Online Library on [02/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Vietnam Journal of Chemistry Tran Thi Phuong Thao et al. 15.625, 15.625, 62.5 and 31.25 µg/mL against methylene carbons. The detail assignment for each DENV-1, DENV-2, DENV-3 and DENV-4 virus carbon and proton was verified by means of 2D NMR serotypes, respectively. The ethanol extract displayed spectra. The appearance of a methyl group (C-23) was the CC50 value > 500 µg/mL on BHK21 cell line, attributed to C-4, due to the correlation in HMBC determining the non-cytotoxicity of this extract in this spectra between δH 2.25 (H-4)/δC 213.1 (C-3), 6.7 (C- experiment. 23), δC 14.5 (C-24) and δC 42.1 (C-5). The proton- proton correlation in COSY spectrum between δH 3.2. Structural elucidation of the isolated 2.25 (H-4)/0.88 (H-23) further confirmed this compounds observation. The isopropyl group was attached at C- 19, based on the correlation of δH 0.89 (H-29), δH 0.83 Compound 1 was obtained as a white amorphous (H-30), and δH 1.48 (H-20)/δC 60.1 (C-19). The powder. Its ESI-MS spectrum showed an ion location of the methyl groups at C-24 and C-25 was molecular peak at m/z 468.5 [M+H]+. The NMR established by the correlation between δH 0.71 (H- spectrum 1 displayed the signals of an olean-type 24)/δC 42.1 (C-5), 58.2 (C-4), 41.0 (C-6) and 59.7 (C- triterpene, including eight methyl singlets at δH 1.07 10) as well as 0.91 (H-25)/δC 40.1 (C-9), 49.5 (C-8), (3H, s), 1.01 (3H, s), 0.94 (3H, s), 0.93 (3H, s), 0.90 59.7 (C-10) and 35.6 (C-11). The assignment of the (3H, s), 0.85 (3H, s), 0.84 (3H, s) and 0.73 (3H, s). methyl protons at C-26, C-27 and C-28 was based on The signal of an olefin proton at δH 4.86 (1H, s, H-19) the long range correlation between δH 0.90 (H-26)/ deduced the presence of an olefin bond at C-18 and 37.9 (C-14), δC 49.5 (C-8), 28.3 (C-15), 0.98 (H-27)/ C-19 position. In addition, the chemical shift of δC 39.0 (C-13), 28.3 (C-12), 51.7 (C-18) and 0.80 (H- proton H-3 shifted to the downfield at δH 4.49 (1H, 28)/δC 42.7 (C-17), 51.7 (C-18), 35.5 (C-22), dd, J = 10.8, 6.0, H-3), indicating the location of an respectively. The relative configuration of 2 was ester group at C-3. This is further demonstrated by the confirmed by NOESY spectra. The correlation appearance of an acetyl group at δH 2.04 (3H, s, between δH 2.25 (H-4)/δH 1.60 (H-10) indicated that CH3COO)/δC 171.0 CH3COO). The 13C NMR and they are on the same face of the molecule. DEPT spectra show the presence of 32 carbons, Conversely, the methyl proton H-23 (δH 0.88) including one carbonyl ester at δC 171.0, one methyl correlated to H-24 (δH 0.71), H-24 (δH 0.71) to H-25 group at δC 21.3 (CH3COO), two olefin carbons at δC (δH 0.91) determined that they are closed to each 142.7 (C-18) and 129.8 (C-19), eight methyl carbons, other. The spectral data of compound 2 were identical ten methylene carbons, three methine carbons at δC with those reported for cymbopogone, which was 55.6 (C-5), 51.1 (C-9), 38.4 (C-13), six quaternary previously isolated from lemongrass Cymbopogon carbons, and one oxygenated methine carbon at δC citratus Stapf.[13] This is the first time this compound 80.9 (C-3). Comparison of the NMR and MS spectral has been isolated from Euphorbia cyathophora. data of 1 with those reported for 3β-acetoxy-olean- Compound 3 was yielded as a white amorphous 18-ene gave complete agreement.[12] Therefore, the powder. The NMR spectra indicated that this structure of 1 was confirmed to be 3β-acetoxy-olean- compound is a saturated aliphatic alcohol. The 1H 18-ene or germanicol acetate. This compound was NMR spectrum gave the signals of an oxygenated previously isolated from Euphorbia cyathophora.[7] methylene group at δH 3.63 (2H, t, J = 6.6 Hz), Compound 2 was isolated as a white crystal. Its together with 25 methylene groups at δH 1.56-1.25 ESI-MS spectrum indicated a molecular ion peak at (m, 50H, 25xCH2), and a methyl group at δH 0.88 m/z 427.2 [M+H]+. The NMR spectra of 2 suggested (3H, t, J = 7.2). The 13C NMR spectra was in that this compound possessed a triterpene type accordance with 1H NMR data, including methylene lupane. The 1H NMR spectra showed the signals of carbons at δC 32.8-22.6, a methyl carbon at δC 14.1, five methyl singlets at δH 0.98 (3H, s, H-27), 0.91 and an oxygenated methylene carbon at δC 63.1. (3H, s, H-25), 0.90 (3H, s, H-26), 0.80 (3H, s, H-28), Comparison of the spectral data of 3 with Ref. [14], 0.71 (3H, s, H-24) and three methyl doublets at δH 3 was elucidated to be 1-heptacosanol. 0.89 (3H, d, J = 6.6, H-29), 0.88 (3H, d, J = 6.6, H- Compound 4 was afforded as a white amorphous 23) and 0.83 (3H, 3H, d, J = 6.6, H-30). The rest powder. This compound contained the feature signals were attributed to the methylene and methine structure of a monosaccharide, based on the spectral protons. The 13C NMR data of 2 gave 30 signals, data. The ESI-MS spectrum of 1 gave a pseudo- including one ketone carbon at δC 213.1 (C-3), six molecular ion peak at m/z 415.0 [2M+2H2O+Na]+. methines at δC 60.1 (C-19), 59.7 (C-10), 58.2 (C-4), The 1H NMR spectrum indicated the signals of eight 51.7 (C-18), 49.5 (C-8) and 30.7 (C-20), five protons attached to the oxygenated carbons in the quaternary carbons at δC 42.7 (C-17), 42.1 (C-5), 40.1 range of δH 3.49-4.88, of which one proton anomer (C-9), 39.0 (C-13), 37.9 (C-14), together with ten appeared at δH 4.88 (1H, d, J = 1.8, H-1'). In addition, © 2023 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 374
25728288, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300123 by Readcube (Labtiva Inc.), Wiley Online Library on [02/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Vietnam Journal of Chemistry Phytochemical study of E. cyathophora collected… a methyl group in the highfield region was resonated sugar moiety and an ethanolic group. The optical at δH 1.22 (3H, t, J = 6.6). The 13C NMR spectrum rotation [αD 25oC, c 0.1 MeOH = +33.8] and the corresponded to the 1H NMR one, with the spectral data of 4 were compatible with those reported appearance of four oximethine carbons at δC 109.2 for α-L-arabinofuranoside.[15] Thus, compound 4 was (C-1', C-anomer), 83.0 (C-2'), 78.8 (C-3') and 85.2 characterized as ethyl α-L-arabinofuranoside. This (C-4'). Besides, two oxymethylene carbons at δC 64.2 compound was previously isolated from Dichomitus (C-1) and 63.0 (C-5'), and a methyl group at δC 15.4 squalens. This is the first-time ethyl α-L- were also observed. Analysis of the above spectral arabinofuranoside have been isolated from E. data revealed that compound 4 possessed a furanose cyathophora. 5: R1 = R2 = R3 = R4 = R5 = OH 6: R1 = R2 = R4 = R5 = OH; R3 = OCOCH3 7: R3 = OCOCH3; R1 = R2 = R5 = OH; R4 = H 8: R1 = R2 = R3 = OH; R4 = H; R5 = OCOCH3 Figure 1: The compounds isolated from n-hexane (1-3) and EtOAc extract (4-8) of Euphorbia cyathophora Compound 5 was isolated as a yellow amorphous CH3COO)/δC 169.8 (CH3COO) and 20.4 (CH3COO). powder. The ESI-MS spectrum of 5 indicated a This was further confirmed by the observation of a pseudo-molecular ion peak at m/z 302.9 [M+H]+. molecular ion peak at m/z 366,9 [M+Na]+. The NMR spectral data of this compound displayed the location of the acetyl group was determined to be at signals characteristic of a flavonoid. The protons C-3, evidenced by the shielding chemical shift of this resonated at δH 7.74 (1H, s, H-2’), 7.65 (1H, dd, J = carbon (δC 131.4), compared with the one in 8.4, 1.2, H-6’), 6.90 (1H, d, J = 8.4, H-5') belonging compound 5 (δC 137.1). The spectral data of 6 were to an aromatic ring with ABX substitution system. in agreement with those reported for 3-acetoxy- Two protons at δH 6.38 (1H, s, H-8) and 6.18 (1H, s, 5,7,3’,4’-tetrahydroxyflavone.[17] This compound H-6) were assigned at the meta position with each was elucidated to be as 3-acetoxy-5,7,3’,4’- other. The 13C NMR spectra gave 15 carbons, tetrahydroxyflavone. including five aromatic methine carbons at δC 99.4 Compound 7 was formed as a yellow amorphous (C-6), 94.5 (C-8), 115.9 (C-2'), 116.2 (C-5'), 121.6 powder, affording from an acetylated fraction. The (C-6'). The remaining signals were quaternary NMR spectra of 7 were almost identical with carbons in the range of δC 104.3-177.2. The above compound 6. The only difference was the lack of one spectral data of compound 5 matched with those hydroxyl group at C-3’ of the B ring in compound 7, reported for quercetin.[16] Therefore, compound 5 was comparing with 6. This was confirmed by the identified as quercetin. presence of four aromatic protons at δH 7.76 (1H, d, J Compound 6 was obtained as a yellow = 8.4, H-2’, H-6’)/δC 131.1 (C-2',6'), δH 6.94 (1H, d, amorphous powder. This compound was isolated J = 8.4, H-3’, H-5’)/δC 116.8 (C-3',5'). The from an acetylated fraction of the ethyl acetate attachment of the acetyl group at C-3 led to the extract. The NMR data of 6 were similar to 5, except shifting of the chemical shift to the downfield [δC the appearance of an acetyl group at δH 2.34 (s, 3H, 131.3 (C-3)]. In combination with the molecule ion © 2023 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 375
25728288, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300123 by Readcube (Labtiva Inc.), Wiley Online Library on [02/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Vietnam Journal of Chemistry Tran Thi Phuong Thao et al. peak at m/z 329.0622 (calculated for C17H13O7 antidengue activity of quercetin against DENV-1, 329.0661 [M+H]+) in HR-ESIMS spectrum, the DENV-3 and DENV-4. In silico studies indicated that structure of 7 was determined to be 3-acetoxy-5,7,4’- quercetin was able to interact with E, NS1, NS3 and trihydroxyflavone.[18] NS5 proteins of dengue virus and block the viral Compound 8 was isolated as yellow amorphous replication cycle.[21,22] Compound 6, an acetylated powder. The NMR data of 8 demonstrates the signals derivative of compound 5 at C-3 position was in similar to 7, except the attachment of an acetyl group active, indicating the important role of the hydroxyl at C-4’ of B ring. This was determined from the group at C-3 of quercetin. Compound 7 (3-acetoxy- upfield shift of C-4’ (δC 153.0) in compound 8, when 5,7,4’-trihydroxyflavone) possessed a similar compared with those for compound 7 (δC 162.2). The structure to compound 6, only with the absence of a spectral data of 8 were in agreement with those for hydroxyl group at C-3’. This compound showed 4’-acetoxy-3,5,7-trihydroxyflavone.[19] Based on the significant activity against all serotypes DENV1-4, HR-ESIMS spectra (m/z 327.0514, calculated for even much better than 5 (quercetin). 3-Acetoxy- C17H11O7 327.0505 [M-H]-) and detailed analysis of 5,7,4’-trihydroxyflavone (compound 7) is a potential NMR data, compound 8 was identified as 4’-acetoxy- antidengue compound that needs to be further studied 3,5,7-trihydroxyflavone. for identification of antiviral drug against dengue infection. 3.3. Antidengue activity of the isolated compounds Acknowledgement. The authors are grateful to the Institute of Chemistry, Vietnam Academy of Science Compounds 1-3, 5-7 were evaluated for antiviral and Technology for financial support [Nr: activity against four virus serotypes DENV1-4. The VHH.2022.02]. result showed that two of them were active on all virus serotypes DENV1-4. 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