Chemical constituents of ethyl acetate extract from taxus wallichiana zucc (taxaceae) leaves collected in Ha Giang province

110 HNUE JOURNAL OF SCIENCE DOI: 10.18173/2354-1059.2020-0035 Natural Sciences 2020, Volume 65, Issue 6, pp. 110-115 This paper is available online at CHEMICAL CONSTITUENTS OF ETHYL ACETATE EXTRACT FROM Taxus wallichiana ZUCC. (TAXACEAE) LEAVES COLLECTED IN HA GIANG PROVINCE Pham Huu Dien1, Pham Thi My1 and Bui Tuan Nam2 1Faculty of Chemistry, Hanoi National University of Education 2Yen Minh High School, Ha Giang province Abstract. Investigation on the chemical constituents of ethyl acetate extract from leaves of Taxus wallichiana Zucc. (Taxaceae), collected in Ha Giang province, has resulted in the isolation of three metabolites for the first time: sciadopitysin (1), β-sitosterol (2), and isoquercitrin (3). Their structures were determined by spectroscopic methods. Keywords: Taxus wallichiana Zucc., sciadopitysin, isoquercitrin. 1. Introduction Taxus wallichiana Zucc. (Taxaceae), named as “cay thong do”, is widely distributed in higher than 1,500 m in the mountains of Northern (such as Ha Giang, Cao Bang provinces) and Western Vietnam (such as Da Lat, Lam Dong provinces) [1, 2]. According to Vietnam traditional medicine, this plant is used for curing asthma, bronchitis, hiccough, indigestion, epilepsy, ascarid [1]. The fascinating molecule taxol [3] an important antitumor and anti-leukemic drug and its close analogs have been isolated from the plant. The genus Taxus was intensively investigated for the content not only of taxoids but also of other constituents, for instance, biflavones [4], flavones [5], lignans [6], diterpenoids [7]. This paper describes the isolation and structural elucidation of three metabolites from the leaves of Taxus wallichiana Zucc., collected in Ha Giang province. 2. Content 2.1. Materials and methods * Plant material Leaves of Taxus wallichiana Zucc. were collected in Bat Dai Son commune, Quan Ba distr., Ha Giang (July 2017), and identified by Prof. Dr. Do Huu Thu (Institute of Ecology, Natural Resource and Biology, VAST, Vietnam). Voucher specimens are deposited at the faculty of Chemistry, Hanoi University of Education (TW201707). Received June 12, 2020. Revised June 23, 2020. Accepted June 29, 2020. Contact Pham Huu Dien, e-mail address: dienph@hnue.edu.vn Chemical constituents of ethyl acetate extract from Taxus wallichiana Zucc 111 * General procedure Thin layer chromatography (TLC) was carried out on precoated Si gel GF254 (Merck Co., Germany) and TLC spots were viewed at 254, 302, and 366 nm and visualized by spraying with vanillin-10% H2SO4 solution. Column chromatography (CC) was carried out on silica gel 60 (60 - 100 M, Merck). NMR (1H, 13C NMR, HSQC and HMBC) spectra were recorded on a Bruker Avance 500MHz instrument. The chemical shift (δ) values are given in ppm with TMS as internal standard, coupling constant J - by Hz. Mass spectra, including high resolution MS were recorded on an HP 5989B mass spectrometer and FT-ICR-MS (Varian 910-MS TQFTMS-7 Tesla). * Extraction and Isolation Dried powder of T.wallichiana leaves (2,500 g) was extracted with methanol. The methanolic extract was concentrated to give a residue (175 g) which was further partitioned into n-hexane, EtOAc, BuOH, and water. The ethyl acetate crude extract (13.7 g) was subjected to CC over silica gel and eluted gradient with n-hexane - ethyl acetate from 4:1 to 1:1, ethyl acetate - methanol from 10:1 to 0: 10. Eight fractions were successively obtained. Fraction 3 (37 mg) was chromatographed on CC using n-hexane - ethyl acetate as the solvent system to yield compounds 1 (4 mg), 2 (8 mg), and 3 (15 mg). Compound 1: yellow crystals; ESI-MS positive: m/z [M+H]+ calcd for C33H25O10: 581.0 found 580.9, negative m/z [M-H]- calcd for C33H23O10 579.0 found 578.7; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3): Table 1. Compound 2: white needle crystals; ESI-MS positive: m/z [M-H2O+H]+ calcd for C29H49: 397.0 found 397.1; 1H NMR (500 MHz, CDCl3) δ (ppm), J (Hz): 5.35 (t 2.5, H-6), 3.52 (m, H-3), 1.01 (s, H18), 0.92 (d 6.5, H-21), 0.87 (t 7.5, H-29), 0.83 (d 6.5, H-26), 0.81 (d 6.5, H-27), 0.68 (s, H-19); 13C NMR (125 MHz, CDCl3) δ (ppm): 140.8 (C-5), 121.7 (C-6), 71.8 (C-3), 56.8 (C-14), 56.1 (C-17), 50.2 (C-9), 45.9 (C-24), 42.34 (C-4), 42.32 (C-13), 39.8 (C-12), 37.3 (C-1), 36.5 (C-20), 36.2 (C-10), 34.0 (C-22), 31.9 (C-7), 31.9 (C-8), 31.7 (C-2), 29.7 (C-25), 28.2 (C-16), 26.1 (C-23), 24.3 (C-15), 23.1 (C-28), 21.1 (C-11), 19.8 (C-18), 19.4 (C-26), 19.1 (C-27), 18.8 (C-21), 12.0 (C-19), 11.9 (C-29). Compound 3: yellow powder; ESI-MS negative: m/z [M-H]- calcd for C21H19O12: 463.0, found 462.8; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6): Table 2. 2.2. Results and discussion Compound 1 was obtained from ethyl acetate extract of Taxus wallichiana leaves. Its molecular formula was identified as C33H24O10 based on its pseudo molecular ion peaks from ESI-MS, together with 1H- and 13C NMR data. The 1H NMR spectrum (Table 1) shows two hydroxyl signals at δH 12.89 and 13.04 (each, s) shifted to the lower magnetic field due to intramolecular hydrogen bonding to the carbonyl groups and also three methoxyl groups (H 3.75 3.80 3.83, each s), twelve olefinic protons which are the three pairs of ortho coupling protons, one pair of meta coupling protons, and four isolated protons. The 13C NMR spectrum of 1 exhibited thirty-three carbons including two carbonyl signals for flavones at δC 181.9, 182.1, three methoxyl signals, and twenty-eight olefinic carbons which include twelve tertiary carbons and sixteen quaternary carbons. From 1H and 13C NMR spectral analysis 1 was assumed to be a Pham Huu Dien, Pham Thi My and Bui Tuan Nam 112 biflavonoid. The HMBC spectrum of 1 shows the crosslink peak H-2’/C-8” revealed that two flavones connected between C-3’ and C-8”. From the above analysis of 1D, 2D NMR and MS spectra, 1 was assumed to be sciadopitysin [8]. This compound was isolated for the first time from Vietnamese Taxus wallichiana Zucc. According to Gu Q. et al, sciadopitysin can inhibit the Aβ aggregation and reduce Aβ-induced toxicity in the primary cortical neurons, becomes a promising active component for anti-Alzheimer’s disease [9]. Figure 1. HSQC, HMBC and +MS spectra of compound 1 Figure 2. Structures of compounds 1, 2 and 3 OH3CO OH O OCH3 OHO OH O OCH32 3 45 6 7 8 9 10 1' 2' 3' 4' 5' 6' 2" 3" 4"5" 6" 7" 8" 9" 10" 1''' 2''' 3''' 4''' 5''' 6''' HO 1 2 3 4 5 6 7 8910 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 O OH OH HO HO O O OOH HO OH OH2 6 1' 2' 1" 6" 3 45 7 8 9 10 3' 4' 5' 6' 2" 3"4" 5" 1 2 3 Chemical constituents of ethyl acetate extract from Taxus wallichiana Zucc 113 Table 1. 1H NMR and 13C NMR data of compounds 1 (δ ppm, J Hz) No 1H NMR 13C NMR No 1H NMR 13C NMR No 1H NMR 13C NMR 2 - 163,1 5’ 7.36 d 9.0 111.8 3’’’ 6,92 d 9,0 114,5 3 6.98 s 103,7 6’ 8.22 dd 9.0 2.5 128.3 4’’’ - 165,2 4 - 181.9 2” - 163.6 5’’’ 6.92 d 9.0 114.5 5 - 160.6 3” 6.88 s 103.2 6’’’ 7.59 d 9.0 127.8 6 6.35 d 2.0 98.1 4” 182.1 7-OCH3 3.75 s 55.5 7 - 162.2 5” - 161.1 4’-OCH3 3.80 s 55.9 8 6.78 m 92.7 6” 6.42 s 98.6 4’’’-OCH3 3.83 s 56.0 9 - 157.3 7” - 161.8 HO-5 13.04 s - 10 - 103.6 8” - 103.8 HO-5” 12.89 s - 1’ - 122.8 9” - 154.3 HO-7” 10.85 s - 2’ 8.08 d 2.5 130.9 10” - 104.8 3’ - 122.4 1’’’ - 121.6 4’ - 160.6 2’’’ 7.59 d 9.0 127.8 Compound 2 was obtained from ethyl acetate extract of Taxus wallichiana leaves. Its molecular formula was identified as C29H50O based on its pseudo molecular ion peak [M-H2O+H]+ from ESI-MS, together with 1H- and 13C NMR data. The 1H NMR spectrum of 2 shows the presence of two methyl singlets at H 0.68, and 1.01 ppm; three methyl doublets that appeared at H 0.81, 0.83 and 0.92 ppm; and a methyl triplet at H 0.87 ppm. The 1H NMR spectra also showed the presence of one olefinic proton at H 5.35 ppm suggesting them belonged to a double bond, >C=CH-. The proton that appeared as a multiplet at  3.52 ppm was assigned for a methine proton bonded to carbinol carbon (CHOH). From the above analysis, we suggested that compound 2 should belong to the group of sterols. The 13C NMR spectrum of compound 2 shows the presence of six methyl (C 11.9, 12.0, 18.8, 19.1, 19.4, 19.8), eleven methylene, nine methine, and three quaternary carbons. Among them, two carbon signals at C 140.8 and 121.7 were assigned for a double bond, >C=CH-. The carbon signal at C 71.8 was assigned for cyclic carbinol carbon of a sterol (C-3). The above spectral data supported the presence of sterol skeleton having hydroxyl group at C-3 position with one double bond at Pham Huu Dien, Pham Thi My and Bui Tuan Nam 114 C-5/C-6 with six methyl groups. Thus, compound 2 was assigned as the known - sitosterol [10]. This compound is very popular in many plants. The molecular formula of compound 3 was found to be C21H20O12 by FT-ICR-MS. Analysis of its 1H NMR spectra revealed that it has five aromatic protons at H 7.58, 7.57, 6.84 6.40 and 6.20 ppm; seven O-glycoside protons from H 3.09 to 5.45 ppm, suggesting that 3 should be an O-glycoside flavonoid (Table 2). The 13C NMR spectrum has resonances of 21 carbons (Table 2) including one carbonyl signal for flavones at δC 177.4, fourteen olefinic carbons which include five tertiary carbons and nine quaternary carbons, six aliphatic carbons of monosaccharide. The HMBC spectrum of 3 shows the crosslink peak between C-3 and H-1”, revealed that monosaccharide moiety should connect to flavone by C-3/C-1” bond. Compound 3 has very similar spectral data with those of isoquercitrin [11]. Therefore, compound 3 was determined as isoquercitrin. This compound was isolated for the first time from Vietnamese Taxus wallichiana Zucc. According to Chen Q. et al, isoquercitrin can inhibit the progression of pancreatic cancer in vivo and in vitro by regulating opioid receptors and the mitogen- activated protein kinase signaling pathway [12]. Table 2. 1H NMR and 13C NMR data of compounds 3 (δ ppm, J Hz) No 1H NMR 13C NMR No 1H NMR 13C NMR No 1H NMR 13C NMR 2 - 156.3 10 - 103.9 1” 5.45 d 7.5 100.9 3 - 133.3 1’ - 121.1 2” 3.26 m 74.1 4 - 177.4 2’ 7.58 d 2.0 116.2 3” 3.23 m 76.5 5 - 161.2 3’ - 144.7 4” 3.09 m 69.9 6 6.20 d 2.0 98.6 4’ - 148.4 5” 3.09 m 77.5 7 - 164.1 5’ 6.84 d 9.0 116.2 6” 3.58 d 9.0 3.32 brd s 60.9 8 6.40 d 2.0 93.4 6’ 7.57 m 121.5 HO- 5 12.63 s - 9 - 156.13 3. Conclusions Chemical composition of ethyl acetate extract of the leaves of Taxus wallichiana Zucc. collected in Ha Giang province, has been investigated. Three metabolites, sciadopitysin (1), β-sitosterol (2), and isoquercitrin (3) were isolated for the first time and structurally elucidated by MS, 1D, and 2D NMR spectroscopies. Acknowledgement: Authors would like to thank Prof. Dr. Do Huu Thu (Inst. of Ecol. and Biol. Resources, VAST) for identifying the sample. 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