Bangladesh Journal of Pharmacology Volume: 14; Number 4; Year 2019 Cite this article as: Kim DD, Nguyet VT, Anh HX, Trang NTT, Chuyen NH, Huong LM, Ha TTH, Ho DH, Dat NT. Cytotoxic phenolic constituents from the leaves of Ehretia asperula. Bangladesh J Pharmacol. 2019; 14: 196- 97. Cytotoxic phenolic constituents from the leaves of Ehretia asperula Sir, The genus Ehretia is mainly distributes in tropical areas of Asia, Africa, Northern America and exhibited valuable pharmacologial properties (Li et al., 2010; Shukla and Kaur, 2018). In Vietnam, Ehretia asperula Zoll. & Mort. has been used in traditional medicine for the treatment of ulcer, tumors, liver disease and inflammation (Nguyen et al., 2017). To date, there are few reports about the biological activities and chemical composition of this plant. In the present study, the leaves of E. asperula were collected from the Bai Rong Hamlet, Thuong Tien Commune, Kim Boi District, Hoa Binh Province. The air- dried and powdered material (5.0 kg) was extracted in methanol at room temperature for 3 days and repeated 3 times. The combined extracts were evaporated under reduced pressure to obtain a crude residue (320 g), which was then re- dissolved in water (2 L) and successively extracted with n- hexane and ethyl acetate, respectively. The organic layers were dried in vaccuo to give n-hexane and 34.4 g ethyl ace- tate residues, respectively. Ethyl acetate was chromato- graphed on a silica gel column eluted with solvent mixtures of dichloromethane and methanol (100/1, 50/1, 10/1, 5/1, 1/1 and 1/100, v/v) to give 6 fractions F1-F6, respectively. The fraction F2 was subjected to a silica gel column eluted with dichloromethane-methanol (5/1) to afford two subfractions F2.1 and F2.2). Subfraction F2.1 was separated on a silica gel column, eluting with n-hexane-dichloromethane-methanol (5/15/1, v/v/v) to obtain compound 1 (7.1 mg) and caffeic acid (2) (37.5 mg). Methyl caffeate (3) (5.0 mg) was purified from subfraction F2.2 by a C18 column using methanol-water (1/2, v/v). Fraction F3 was subjected to fractionation over silica gel column, eluted with dichloromethane-acetone (10/1, v/v) to give three subfractions F3.1-F3.3. Subfraction F3.1 was separated using silica gel column and further purified with C18 column to yield methyl rosmarinate (6) (3.2 mg), oresbiusin B (7) (8.1 mg) and dimethyl lithospermate (8) (100 mg). Subfraction F3.2 was isolated over silica gel column eluted with dichloromethane-methanol(2:1, v/v) to provide rosmarinic acid (5) (3.2 mg). Astragalin (4) (11.0 mg) was isolated from subfraction F3.3 using C-18 column chromatography eluted with methanol-water (1/1, v/v). Compound 1 was isolated as a white amorphous powder and had the molecular formula C15H14O4, as determined by the HR-ESI-MS molecular ion peak at m/ z 259.0975 [M+H]+. The 1H NMR spectrum of 1 showed an aromatic ABX spin system at δH 7.04 (1H, d, J = 8.0 Hz, H-5), 7.32 (1H, d, J = 2.0 Hz, H-2) and 7.34 (1H, dd, J = 8.0, 2.0 Hz, H-6), an aromatic A2B2 protons at δH 7.15 (2H, d, J = 8.0 Hz, H-3′, 5′) and 6.84 (2H, d, J = 8.0 Hz, H -2′, 6′). In addition, an aldehyde proton at δH 9.71 (1H, s, H-7) and two methylene signals at δH 2.73 (2H, t, J = 7.0 Hz, H-7′), 3.71 (2H, t, J = 7.0 Hz, H-8′) were also recognized. The 13C-NMR combined with the HSQC experiments indicated the presence of two aromatic rings [δC 131.1 (C, C-1), 118.3 (CH, C-2), 147.2 (C, C-3), 151.8 (C, C-4), 116.1 (CH, C-5), 128.8 (CH, C-6), 158.5 (C, C-1′), 119.5 (CH, C-2′, 6′), 131.0 (CH, C-3′, 5′), 133.7 (C, C -4′)], an aldehyde group [δC 193.1 (CH, C-7)] and a 2- subsituted ethanol moiety [δC 39.4 (CH2, C-7′), 64.0 (CH2, C-8′)]. The HMBC correlations showed the coupling from H-7 to C-1, C-2 and C-6, from H-2 and H -6 to C-7 suggesting the aldehyde group attached to C-1 of the ABX aromatic ring. These data were similar to those of 3,4-dihydroxybenzadehyde (protocatechuic aldehyde) moiety (Ooike et al., 1997). The methylene protons H-7′ coupled to C-4′, C-3′ and C-5′ indicated the ethanol moiety attached to C-3′ of the A2B2 aromatic ring, which suggested the structure of 4-(2-hydroxyethyl) -phenol (tyrosol) moiety (Hussain et al., 2014). Based on these data, the structure of 1 consisted of protocatechuic aldehyde and tyrosol fragments. The NOESY spectrum showed the cross peak between H-2 and H-2′, 6′ confirming that two fragments linked via C-3 and C-1' positions (Figure 1). Thus, compound 1 was newly elucidated to be 4-hydroxy- 3-[4-(2-hydroxyethyl)-phenoxy]-benzaldehyde. Cytotoxicity of the isolated compounds was evaluated against human lung (Hep-G2, LU-1), cervical (HeLa), breast (MCF-7), and rhabdomyosarcoma (RD) cancer cell lines (Table I). Compound 1 exhibited the strongest effect to Hep-G2, LU-1, HeLa and RD cells with IC50 values in the range of 7.1-10.2 μM. Compounds 2, 6 and 7 showed moderate activity while 3, 4, 5 and 8 were inactive. Interestingly, all compounds did not affect the viability of normal cells (monkey kidney VERO cell line) up to 30 μM. Methyl caffeate (2) has been reported as a promising anti-cancer agent by inducing apoptosis (Balachandran et al., 2015; Kim et al., 2019). In this study, compound 1 showed stronger cytotoxic effect than methyl caffeate. Further studies are needed to clarify the anti-cancer ability of Ehretia asperula as well as compound 1. A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2019; 14: 196-197 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v14i4.42414 Letter to the Editor This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor This work was supported by Department of Science and Technology, Hoabinh Province, and Vietnam Academy of Science and Technology (NCVCC30.01/19-19). Dang Dinh Kim1, Vu Thi Nguyet1, Ha Xuan Anh1, Nguyen Thi Thu Trang1, Nguyen Hong Chuyen1, Le Mai Huong2, Tran Thi Hong Ha2, Do Hai Ho3, Nguyen Tien Dat4* 1Institute of Environmental Technology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam; 2Institute of Natural products Chemistry, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam; 3Department of Science and Technology, Hoabinh, Vietnam; 4Center for research and technology transfer, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam. *Corresponding author: email: ngtiend@imbc.vast.vn References Balachandran C, Emi N, Arun Y, Yamamoto Y, Ahilan B, Sangeetha B, Duraipandiyan V, Inaguma Y, Okamoto A, Ignacimuthu S, Al-Dhabi NA, Perumal PT. In vitro anticancer activity of methyl caffeate isolated from Solanum torvum Swartz. fruit. Chem Biol Interact. 2015; 242: 81-90. 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Photochemistry 1997; 46: 123-26. Shukla A, Kaur A. A systematic review of traditional uses bio- active phytoconstituents of genus Ehretia. Asian J Pharm Clin Res. 2018; 11: 88-100. Bangladesh J Pharmacol 2019; 14: 196-197 197 Table I Cytotoxicity (μM) of the isolated compounds Compounds Hep-G2 LU-1 HeLa MCF-7 RD Vero 1 7.1 ± 0.7 8.5 ± 1.1 10.2 ± 0.9 >30 7.9 ± 0.8 >30 2 14.5 ± 2.0 >30 17.4 ± 2.16 22.6 ± 2.69 >30 >30 6 22.1 ± 3.6 >30 24.9 ± 2.63 15.1 ± 1.23 >30 >30 7 25.4 ± 3.5 >30 >30 >30 >30 >30 Ellipticine 3.2 ± 0.2 3.0 ± 0.4 1.3 ± 0.1 4.00 ± 0.4 3.3 ± 0.3 >30