Chemical constituents of the ethyl acetate extract from the leaf of mugwort (Artemisia vulgaris L)

Science & Technology Development Journal, 22(4):352- 355 Open Access Full Text Article Research Article 1Faculty of Chemistry, University of Science, VNU-HCM 2Central Laboratory for Analysis, University of Science, VNU-HCM Correspondence Truong Van Nguyen Thien, Faculty of Chemistry, University of Science, VNU-HCM Email: ngttvan@hcmus.edu.vn History  Received: 27-12-2018  Accepted: 22-3-2019  Published: 30-11-2019 DOI : 10.32508/stdj.v22i4.1744 Copyright © VNU-HCM Press. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Chemical constituents of the ethyl acetate extract from the leaf of mugwort (Artemisia vulgaris L.) Truong Van Nguyen Thien1,*, Thien Tai Phan1, Tung Thanh Phan1, Lien Kim Thi Tran1, Nhu Tiet Thi Tran1, Phu Hoang Dang1, Linh Phi Nguyen2, Quang That Ton1 Use your smartphone to scan this QR code and download this article ABSTRACT Introduction: Mugwort (Artemisia vulgaris L.) is a familiar herbal medicine and also a daily veg- etable. It is one of the ingredients in the famous remedy "Cao ích mẫu" specializing in men- strual disorders or the omelet with mugwort that helps save blood flow to the brain to treat headaches. In both traditional medicine and the new drugs, diseases are usually treated by mugwort as diabetes, epilepsy combination for psychoneurosis, depression, irritability, insom- nia, anxiety, and stress. To demonstrate the medicinal uses, the chemical constituents of this herbal were continually studied. Methods: The leaves of mugwort were collected in Ba Ria - Vung Tau province, Vietnam. The plant was identified by the late pharmacist and botanist Binh Duc Phan. A voucher specimen (AV001) was deposited in the herbarium of the Department of Organic Chemistry, VNUHCM–University of Science. Dried leaf powder of A. vulgaris (11 kg) was extracted with methanol and evaporated under reduced pressure to give a methanol ex- tract (910 g), which was dissolved in methanol-water (1:9) and then successively partitioned with petroleum ether, chloroform, and ethyl acetate. From the previously researched ethyl acetate frac- tion, nine compounds were isolated: six known phenolic compounds (luteolin, 6-methoxyluteolin, eupatilin, o-coumaric acid, vanillic acid, and protocatechuic acid), sinapyl alcohol diisovalerate, vulgarin, and one new compound (artanoic acid). Results: In this research, ethyl acetate frac- tion was also studied. From subfraction EA4, six compounds were isolated by three skeletons: phenolic compounds (5,40 -dihydroxyflavone and 4-hydroxyphenyl acetate), phenyl propanoid (methyl 2-O-b -D-glucopyranosylcoumarate and 2-O-b -D-glucopyranosylcoumaric acid) and uracil (5-methyluracil and uridine). The structure of the isolated compounds was determined to base on 1D, 2D NMR spectra, HR-ESI-MS, and comparison with published data. Conclusion: Particularly, four compounds (methyl 2-O-b -D-glucopyranosylcoumarate, 2-O-b -D-glucopyranosylcoumaric acid, 5-methyluracil, and uridine) were known for the first time from this species. Key words: mugwort, Artemisia vulgaris L., flavone, coumarin, and uracil INTRODUCTION Artemisia vulgaris L., a familiar herbal species in Viet Nam, is used in both traditional medicine and new drugs. Diseases are usually treated by mugwort as di- abetes, epilepsy combination for psychoneurosis, de- pression, irritability, insomnia, anxiety, and stress1. The primary responsibility for these activities are con- stituents, such as flavonoids, coumarins, sesquiter- pene lactones, volatile oils, inulin, and traces of al- kaloids2. In the previous research of my group, three flavonoids (luteolin, 6-methoxyluteolin, and eu- patilin), four phenolic compounds (o-coumaric acid, vanillic acid, protocatechuic acid, and sinapyl alcohol diisovalerate), and two sesquiterpene lactones (vul- garin and artanoic acid) are isolated from the ethyl acetate fraction3. In this study, the ethyl acetate fraction is continu- ously researched and six compounds are isolated, in- cluding methyl 2-O-b -D-glucopyranosylcoumarate (1), 2-O-b -D-glucopyranosylcoumaric acid (2), 5- methyluracil (3), uridine (4), 5,40-dihydroxyflavone (5), and 4-hydroxyphenyl acetate (6). METHOD General experimental procedures The NMR spectra were acquired on a Bruker Avance III 500 MHz spectrometer with tetramethylsilane (TMS) as an internal standard, with chemical shifts expressed in d (ppm) values. The HR-ESI-MS were determined with aMicrOTOFQIImass spectrometer (Bruker Daltonics). Analytical and preparative thin- layer chromatography (TLC) were performed on pre- coated Merck Kieselgel 60 F254 or RP-18 F254 plates (0.25 mm or 0.5 mm thickness). Cite this article : Nguyen Thien T V, Tai Phan T, Thanh Phan T, Kim Thi Tran L, Tiet Thi TranN, HoangDang P, Phi Nguyen L, That TonQ.Chemical constituentsof theethyl acetate extract fromthe leaf ofmugwort (Artemisia vulgaris L.). Sci. Tech. Dev. J.; 22(4):352-355. 352 Science & Technology Development Journal, 22(4):352-355 Plant material The leaves of Artemisia vulgaris L. were collected at Lang Dai, Dat Do, Ba Ria - Vung Tau province, Viet- nam onMay 2011. The plant was identified by the late pharmacist and botanist Binh Duc Phan. A voucher specimen (AV001) was deposited in the herbarium of the Department of Organic Chemistry, VNUHCM– University of Science. Extraction and isolation From the ethyl acetate fraction in the previous re- search3, fraction EA4 (6.14 g) was subjected to silica gel column chromatography eluting with petroleum ether –ethyl acetate, followed by ethyl acetate – methanol with increasing polarity to yield six frac- tions (EA4.1 – 6). Fraction EA4.3 (300.6 mg) was separated over a silica gel column eluted with chloroform-methanol (from 9.5:0.5 to 0:10), as well as preparative TLC, eluted with petroleum ether– acetone (4:6) to afford 1 (6.0 mg), 2 (5.5 mg), and 6 (8.2 mg). Fraction EA4.4 (207.5 mg) was subjected to Sephadex LH-20 column eluted with methanol, and further fractionated by silica gel column chromatog- raphy eluted with chloroform– ethyl acetate (from 9:1 to 3:7) to obtain 3 (6.3mg), 4 (4.0mg), and 5 (4.5mg). RESULTS ANDDISCUSSION Six compounds (1 – 6) were isolated from the ethyl acetate fraction of the leaves of Artemisia vulgaris L. Compound 1 was obtained as a white amorphous powder, and its molecular formula was determined as C16H20O8 by HR-ESI-MS analysis atm/z 341.1161 [M+H]+. The 1H-NMR spectrum of 1 showed four signals of four aromatic protons at dH 7.02 (1H, dd, 7.5, 7.5 Hz, H-5), dH 7.19 (1H, d, 8.5 Hz, H-3), dH 7.37 (1H, ddd, 8.5, 7.5, 1.5 Hz, H-4), dH 7.71 (1H, dd, 7.5, 1.5 Hz, H-6). Furthermore, the 13C-NMR and HSQC spectra showed aromatic carbon signals at dC 115.5 (C-3), 122.3 (C-5), 128.7 (C-6), 132.1 (C-4), and two signals of quartet carbon at dC 123.4 (C-1), dC 156.1 (C-2). It demonstrated that 1 had a 1,2-di sub- stituted benzene. There were two signals of two olefin protons, (E) configuration, at dH 7.95 (1H, d, 16.0 Hz, H-7), and dH 6.64 (1H, d, 16.5 Hz, H-8) with carbon signals at dC 139.8 (C-7), dC 118.7 (C-8); and a car- boxyl group at dC 167.3 (C-9). HMBC correlations between H-7/C-2, C-8, C-9; H-8/C-9 showed that 1 had the 2-h ydroxycinnamoyl skeleton. The signals of amethoxy group at dH 3.70 (3H, s, H-10) and dC 51.7 (C-10), correlated with C-9 in the HMBC spectrum (Figure 1). Therefore, the methoxy group linked to the carboxyl group of the cinnamoyl skeleton. Figure 1: Significant HMBC (!) correlations of 2- hydroxycinnamoyl skeleton. 1H-NMR and 13C-NMR spectra showed an anomer proton at dH 5.00 (1H, d, 8.5 Hz, H-10) and dC 100.4 (C-10). The HMBC correlation between H-10/C-2, C- 30 showed that the sugar moiety linked to cinnamoyl skeleton at C-2. The above data compared with the published one indicated that 1 was methyl 2-O-b -D- glucopyranosylcoumarate4. Compound 2 was obtained as a white amorphous powder, and its molecular formula was determined as C15H18O8 by HR-ESI-MS analysis atm/z 326.1033 [M]+, calcd 326.1002. The 1H-NMR, 13C-NMR spectra of 2 were similar to those of compound 1. However, the lack of themethoxy group in compound 2 showed that it was a carboxylic acid. The compar- ison of the above data with the one in the literature5 assigned 2 as O-coumarico glucosidase acid. Compound 3 was obtained as a white amorphous powder and its molecular formula was determined as C5H6N2O2 by HR-ESI-MS analysis at m/z 127.0451 [M+H]+. The 1H-NMR spectrum displayed four sig- nals including two amide protons at dH 10.56 (br), and dH 10.97 (br), a methyl group at dH 1.71 (3H, d, 1.0 Hz, H-7), and an olefin proton signal at 7.23 (1H, s). The 13C-NMR andHSQC spectra showed two sig- nals of two carbonyl carbons at dC 165.2 (C-2) and 151.7 (C-4), two olefin signals at dC 108.2 (C-5) and 138.2 (C-6) and a methyl group at dC 12.2 (C-7). The above information showed an uracil skeleton in com- pound 3. The HMBC correlations between H-7/C-5, C-6 confirmed the position of themethyl group on the C-5 of the uracil skeleton (Figure 2). Based on the above discussions and the comparison with the published ones5, the structure of 3 was 5- methyluracil. Compound 4 was obtained as a white amorphous powder and its molecular formula was determined as C9H12N2O6 by HR-ESI-MS analysis atm/z 267.0628 [M+Na]+ andm/z 245.0825 [M+H]+.1H-NMR, 13C -NMR, HSQC spectra showed that compound 4 had the uracil skeleton as in compound 3. The 1H-NMR spectrum showed an amide proton at dH 11.29 (br), two olefin protons with the (Z) configuration at dH 7.88 (1H, d, 8.0 Hz, H-6), 5.64 (1H, d, 8.0 Hz, H-5). 353 Science & Technology Development Journal, 22(4):352-355 Figure 2: Significant HMBC (!) correlations of uracil skeleton. The 13C-NMR spectrum showed two olefin carbons at dC 102.2 (C-5) and dC 141.2 (C-6); two carbonyl groups at dC 163.6 (C-2) and dC 151.2 (C-4). In addition, there was an anomer proton at dH 5.77 (1H, d, 5.5 Hz, H-10) and five oxymethine proton of a sugar moiety at dH 3.53–5.36 in the 1H-NMR spec- trum. The 13C-NMR spectrum showed an anomer carbon at dC 88.1 (C-10), and four oxynated carbons at dC 85.4 (C-4 0), 73.8 (C-2 0), 70.3 (C-3 0) and 61.2 (C-5 0). It demonstrated a sugar moiety in compound 4. The HMBC correlations between proton H-6/C-2, C- 4, C-5 and C-10, proton H-5/C-2, C-6, C-10, and the anomer proton H-10/C-2, C-5, C-6, C-20, C-30, C-40 showed that the sugar moiety linked to the first nitro- gen of the uracil skeleton (Figure 3). The above data compared with the published one in- dicated that 4 was uridine6. Compound 5 was obtained as a yellow amorphous powder and its molecular formula was determined as C15H10O4 by HR-ESI-MS analysis at m/z 254.0593 [M]+, calcd 254.0579. The 1H-NMR spectrum dis- played two signals of four aromatic protons of a 1,4- disubstituted benzene at dH 7.91 (2 H, d, J= 8.5 Hz, H-20 and H-60) and 6.91 Hz (2H, d, J = 8.5 Hz, H-30 andH-50). Moreover, there were three signals of three aromatic protons of a 1,2,3-trisubstituted benzene at dH 7.32 (1H, s, J = 2.5 Hz, H-8); 7.53 (2H, d, J = 7.5 Hz, H-6 andH-7) and an olefin proton at dH 7.08 (1H, s, J = 2.5 Hz, H-3). The 13C-NMR spectrum showed fifteen carbons: seven aromatic quaternary carbons (dC 182.9, 167.4, 162.4, 158.3, 149.8, 120.7, 109.9), eight aromatic methine carbons (dC 115.9 (C-30, C- 50), 132.7 (C-20, C-60), 110.8, 130.2, 107.1 and 102.5). Figure 3: Significant HMBC (!) correlations of compound 4. Based on the above discussions and the comparison with the published one7, the structure of 5 was 5,4 0 -dihydroxyflavone. Compound 6 was obtained as a white amorphous powder, and its molecular formula was determined as C8H8O3 by HR-ESI-MS analysis at m/z 175.0377 [M+Na]+, calcd 175.0371. The 1H-NMR spectrum showed two signals of four aromatic protons of a 1,4- disubstituted benzene at dH 7.44 (2H, d, J = 9.0 Hz) and 6.74 (2H, d, J = 9.0 Hz), one methyl group at dH 2.01 (3H, s), and one hydroxyl at dH 8.93 (1H, s). The 13C-NMR spectrum showed four signals of six aromatic carbons (dC 154.2, 121.7, 121.6, 115.8), one methyl group (dC 24.0), and one carboxyl group (dC 171.4). On the basis of the above discussions and the comparison with the published one8, the structure of 6 was 4-hydroxyphenyl acetate. CONCLUSION From the leaves of Artemisia vulgaris L. col- lected at Ba Ria - Vung Tau province, six compounds were isolated, including methyl 2-O-b -D-glucopyranosylcoumarate (1), 2-O-b -D- glucopyranosylcoumaric acid (2), 5-methyluracil (3), uridine (4), 5,40-dihydroxyflavone (5), and 4-hydroxyphenyl acetate (6). Compounds 1, 2, 3, 4 were known for the first time from this species. LIST OF ABBREVIATIONS 1D NMR: One-dimensional nuclear magnetic reso- nance; 2D NMR: Two-dimensional nuclear magnetic reso- nance; 354 Science & Technology Development Journal, 22(4):352-355 13 C-NMR: Carbon-13 nuclear magnetic resonance; 1 H-NMR: Proton nuclear magnetic resonance; HR-ESI-MS: High-resolution electrospray ionization mass; TMS: Tetramethylsilane; TLC: Thin-layer chromatography; EA: Ethyl acetate; HMBC: Heteronuclear Multiple Quantum Coher- ence. COMPETING INTERESTS The authors declare that they have no conflicts of in- terest. AUTHORS’ CONTRIBUTIONS Truong Van Nguyen Thien, Thien Tai Phan, Tung Thanh Phan, Kim Lien Tran Thi, Nhu Tiet Thi Tran, and Phu Hoang Dang have contributed in conduct- ing experiments, getting hold of data and writing the manuscript. Linh Phi Nguyen, Quang Ton That have contributed significantly explanation of data and revising the manuscript. ACKNOWLEDGMENT This work was supported by Grant T2018-11 from VNUHCM - University of Science. REFERENCES 1. Walter HL,Memory PF, Elvin L. Medical Botany, 2nd Ed. vol. 345. New Jersey: John Wiley and Sons; 2003. 2. Bamoniri A, Mirjalili BBF, Mazoochi A, Batooli HJ. Chemical composition of Artemisia Vulgaris L. from Kashan area isolated by nano scale injection. Iranian Journal of Organic Chemistry. 2010;2:533–536. 3. Thien TVN, Tran LTK, Tran NTT, Duc TP, Do LTM, Tu DD, et al. A new eudesmane-type sesquiterpene from the leaves of Artemisia vulgaris. 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