Chemical constituents of Datura metel L - Nguyen Thi Mai

Compound 7 had a molecular formula C51H82O22, as deduced by the HRESIMS at m/z 1081.4988 [M+Cl]- (calcd. for C51H82ClO22, 1081.4986) and the 13C NMR data. The 1H and 13C NMR spectra, in combination with DEPT, HSQC, COSY, and HMBC spectra showed the presence of two tertiary methyls at δH 0.83 (s, H3-18) and 1.07 (s, H3-19), one secondary methyl group at H 1.00 (d, J = 7.0 Hz, H3-21), a trisubstituted olefinic proton at δH 5.40 (d, J = 3.0 Hz, H-6), and an acetalic carbon signal at C 110.8 (C-22), implying that 7 possesses the 5,6-spirostane skeleton [21]. The 1H NMR spectrum of 7 contained signals for four anomeric protons at H 4.52 (d, J = 8.0 Hz, H- 1 ), 5.22 (br s, H-1 ), 4.86 (H-1 ), and 4.22 (d, J = 7.5 Hz, H-1 ), that showed HSQC correlations with anormeric carbons at C 100.4 (C-1 ), 102.3 (C-1 ), 102.9 (C-1 ), and 104.7 (C-1 ), respectively, indicating that 7 possesses four sugar units. Comparison of the 13C NMR data of the sugar units with those reported previously suggested the presence of two glucopyranoses and two rhamnopyranoses. The relatively large couplingVJC, 55(2) constant (J > 7.5 Hz) of the anomeric proton of the glucose revealed the -configuration, whereas the - oriented anomeric form of the rhamnose was defined based on the chemical shift values of its C-3 and C-5 positions [22]. In the HMBC spectrum, the correlation between the anomeric proton of a glucose unit at H 4.22 (H-1 ) and C 72.8 (C-27) indicated that this glucose was located at C-27 position of the aglycone (figure 2). The sugar sequence at C-3 of the aglycone was identified as - L-rhamnopyranosyl-(1 4)[ -L-rhamnopyranosyl- (1 2)]- -D-glucopyranoside by the HMBC correlations between H 4.86 (H-1 ) and C 79.9 (Glc C-4), between H 5.22 (H-1 ) and C 79.3 (Glc C-2), and between H 4.52 (H-1 ) and C 79.3 (C-3). This assignment was also supported by the ROESY correlations between H 4.86 (H-1 ) and H 3.54 (H- 4 ), between H 5.22 (H-1 ) and H 3.42 (H-2 ), and between H 4.52 (H-1 ) and H 3.61 (H-3) (figure 3). On the basis of the above analysis, along with comparison of the NMR data of 7 with those of the very recently reported spirostane-type saponin, the structure of compound 7 was established as shown in Figure 1, namely dioscoroside D [20]. In summary, our phytochemical study on the acidic methanol extract of D. metel resulted in the isolation and identification of seven compounds, including pterodontriol B (1), disciferitriol (2), scopolamine (3), adenosine (4), thymidine (5), ilekudinoside C (6), and dioscoroside D (7). Among the isolated compounds, pterodontriol B, disciferitriol, ilekudinoside C, and dioscoroside D were reported for the first time from the Datura genus

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Vietnam Journal of Chemistry, International Edition, 55(2): 188-195, 2017 DOI: 10.15625/2525-2321.2017-00442 188 Chemical constituents of Datura metel L. Nguyen Thi Mai 1 , Nguyen Thi Cuc 2 , Tran Hong Quang 2* 1 Hanoi University of Transport and Communications 2 Institue of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST) Received 17 October 2016; Accepted for publication 11 April 2017 Abstract Chemical investigation of an acidic methanol extract of the whole plant of Datura metel resulted in the isolation of seven compounds, including pterodontriol B (1), disciferitriol (2), scopolamine (3), adenosine (4), thymidine (5), ilekudinoside C (6), and dioscoroside D (7). Their structures were elucidated by extensive spectroscopic methods, including 1D and 2D NMR and mass spectra and in comparison with reported data in the literature. Among the isolated compounds, pterodontriol B, disciferitriol, ilekudinoside C, and dioscoroside D were reported for the first time from the Datura genus. Keywords. Datura metel, Solanaceae, sesquiterpene, triterpenoid saponin, steroidal saponin 1. INTRODUCTION Datura metel L. is an annual herb that belongs to the Solanaceae family. It has tropical American origin and is widely cultivated in many tropical and temperate regions. In the Vietnamese traditional medicine, D. metel has been used for the treatment of coughs, bronchial asthma, and rheumatism [1]. Its leaves have been used as anesthetics in surgery, a fumigant in bronchial asthma, and anti-contractive agents in the stomach ulcers [1]. The flowers of D. metel have been used widely in the Chinese traditional medicine for the treatment of asthma, convulsions, pain, and rheumatism for centuries [2]. Previous studies of the pharmacological effects have shown that D. metel seeds exhibits a hypoglycemic activity in normal and alloxan-induced diabetic rats [3], the chloroform extract of D. metel displays an antifungal effect toward several pathogenic species of Aspergillus [4], and the seeds and fruit pulps of D. metel have a high antioxidant activity [5]. Chemical studies have demonstrated that the major chemical components of D. metel are withanolide- type steroids [6-12], which have been shown to suppress NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells [11, 12], and exhibit cytotoxicity against HCT-116, A549, DLD- 1, BGC-823, and K562 cancer cell lines [6, 7, 10]. In addition, the isolation of some megastigmane sesquiterpenes and amide alkaloids from D. metel was also reported [13, 14]. In the present study, we report the isolation and structural elucidation of seven compounds from the acidic methanol extract of the whole plants of D. metel. 2. MATERIAL AND METHODS 2.1. Plant material The whole plants of D. metel were collected in Thai Binh province, Vietnam during May 2015, and identified by Dr. Bui Van Thanh, Institute of Ecology and Biological Resources. A voucher specimen (NCCT-CDM-5.2015) was deposited at the Herbarium of the Institute of Marine Biochemistry, VAST. 2.2. General experimental procedures All NMR spectra were recorded on a Bruker AM500 FT-NMR spectrometer (500 MHz for 1 H and 125 MHz for 13 C-NMR), chemical shifts are reported in ppm using TMS as an internal standard. ESIMS spectra were recorded on Agilent 1100. Column chromatography (CC) was performed on silica gel 230-400 mesh or RP-18 resins (150 μm, Fuji Silysia Chemical Ltd.). Compounds were visualized by spraying with aqueous 10% H2SO4 and heating for 5 minutes. 2.3. Extraction and isolation VJC, 55(2) 2017 Tran Hong Quang et al. 189 The whole plant of D. metel was dried (5 kg), ground, and extracted with MeOH/acetic acid (pH = 5.0) under sonication at room temperature. After concentration under reduced pressure, the MeOH extract (300 g) was suspended in water and partitioned with CHCl3 to give CHCl3 and aqueous fractions. The aqueous fraction was alkalinized by adding NH4OH until pH = 9.0, and then partitioned successively with CH2Cl2 and EtOAc to provide CH2Cl2, EtOAc, and aqueous fractions, respectively. The CH2Cl2 and EtOAc fractions were combined and subjected to a reversed phase (RP) C18 column chromatography (CC), eluted with MeOH-H2O (10:17, v/v) to provide three subfractions (DME1- DME3). Subfraction DME1 was fractionated using a silica gel CC, being eluted with EtOAc-MeOH-H2O (20:1:0.01, v/v/v) to give 3 (15 mg). The aqueous fraction was neutralized and subjected to fractionation through a Diaion HP-20 column, being eluted with a stepwise gradient of MeOH in water (25–100 %) to give four fractions (DMW1–DMW4). Fraction DMW2 was fractionated using reversed phase (RP) C18 column chromatography (CC), being eluted with MeOH-H2O (1:3, v/v) to yield subfractions DMW21–DMW25. Subfraction DMW22 was then fractionated on a silica gel column, being eluting with CH2Cl2-MeOH-H2O (5:1:0.05, v/v/v) to give 4 (6 mg) and 5 (8 mg). Fraction DMW4 was subjected to a RP C18 CC, being eluted with a stepwise gradient of MeOH-H2O (1:2–4:1, v/v) to yield five subfractions (DMW41– DMW5). Fraction DMW42 was separated using a silica gel CC, being eluted with CH2Cl2-MeOH-H2O (6:1:0.05, v/v/v) to provide four subfractions (DMW421–DMW424). Subfraction DMW421 was fractionated using a silica gel CC, being eluted with EtOAc-MeOH-H2O (13:1:0.05, v/v/v) and further purified by a silica gel CC, being eluted with CH2Cl2-MeOH-H2O (6:1:0.1, v/v/v) to give 2 (7 mg). Subfraction DMW424 was fractionated by a RP C18 CC, eluted with acetone-H2O (1:3, v/v) and further purified by a silica gel CC, being eluted with CH2Cl2-MeOH-H2O (6.5:1:0.05, v/v/v) to give 1 (7 mg). Subfraction DMW44 was fractionated using a silica gel CC, being eluted with EtOAc-MeOH-H2O (2.5:1:0.1, v/v/v) to provide five subfractions (DMW441–DMW445). Subfraction DMW443 was fractionated by a RP C18 CC, being eluted with MeOH-H2O (2:1, v/v) and further purified by a silica gel CC, being eluted with CH2Cl2-MeOH-H2O (3:1:0.1, v/v/v) to yield 7 (12 mg), and 6 (10 mg). Pterodontriol B (1): white, amorphous powder; C15H28O3, M = 256; ESIMS: m/z 255 [M H] ; 1 H (CD3OD, 500 MHz) and 13 C NMR data (CD3OD, 125 MHz), see table 1. Disciferitriol (2): white, amorphous powder; C15H28O3, M = 256; ESIMS: m/z 255 [M H] ; 1 H (CD3OD, 500 MHz) and 13 C NMR data (CD3OD, 125 MHz), see table 1. Scopolamine (3): white, amorphous powder; C17H21NO4, M = 303; ESIMS: m/z 304 [M+H] + ; 1 H (CD3OD, 500 MHz) and 13 C NMR data (CD3OD, 125 MHz), see table 2. Adenosine (4): white, amorphous powder; C10H13N5O4, M = 267; ESIMS: m/z 268 [M+H] + ; 1 H (CD3OD, 500 MHz) and 13 C NMR data (CD3OD, 125 MHz), see table 2. Thymidine (5): white, amorphous powder; C10H14N2O5, M = 242; ESIMS: m/z 241 [M H] ; 1 H (CD3OD, 500 MHz) and 13 C NMR data (CD3OD, 125 MHz), see table 2. Ilekudinoside C (6): white, amorphous powder; C41H66O14, M = 782; ESIMS: m/z 805 [M+Na] + ; 1 H (CD3OD, 500 MHz) and 13 C NMR data (CD3OD, 125 MHz), see table 3. Dioscoroside D (7): white, amorphous powder; C51H82O22, M = 1046; HRESIMS: m/z 1081.4988 [M+Cl] (calcd. for C51H82ClO22, 1081.4986); 1 H (CD3OD, 500 MHz) and 13 C NMR data (CD3OD, 125 MHz), see table 3. 3. RESULTS AND DISCUSSION Compound 1 was obtained as a white, amorphous powder. Its molecular formula was identified as C15H28O3 by an ESIMS ion peak at m/z 255 [M H] , along with the 13 C NMR data. The 1 H NMR of 1 showed the signals of four tertiary methyl groups at δH 1.26 (6H, s, H3-12 and H3-13), 1.11 (3H, s, H3-14), and 0.93 (3H, s, H-15), and one oxymethine proton at δH 3.26 (1H, m, H-1) (Table 1). The 13 C NMR and DEPT spectra contained signals for 15 carbons, including three non- protonated carbons (two of which were oxygenated), three methines (of which one was oxygenated), five methylenes, and four methyl carbons. Comparison of the 1 H and 13 C NMR data of 1 with those of the reported ent-eudesmane sesquiterpene, pterodontriol B revealed that the structures of these compounds are similar [15]. The minor differences between the 13 C chemical shifts of these compounds observed at C-3, C-9, and C-11 might be due to the different solvents that these compounds were recorded in (1: in CD3OD; pterodontriol B: in C5D5N). In the HMBC spectrum, the HMBC correlations from δH 1.26 (H3-12 and H3-13) to δC 43.1 (C-7) and 75.2 (C- 11), from δH 1.11 (H3-14) to δC 42.0 (C-3), 72.8 (C- 4), and 48.4 (C-5), and from δH 0.93 (H3-15) to δC 80.8 (C-1), 48.4 (C-5), 39.2 (C-9), and 39.8 (C-10) indicated that four methyl groups and three hydroxyl VJC, 55(2) 2017 Chemical constituents of Datura metel L. 190 groups are located at C-11, C-4, and C-10 positions (figure 2). Thus, compound 1 was determined to be pterodontriol B. Compound 2 was isolated as a white, amorphous powder and its molecular formula was established as C15H28O3 by the presence of an ion [M H] at m/z 255 in the ESIMS. The 1 H and 13 C NMR spectra of 2 were found nearly identical with those of 1, except for significant difference of the chemical shift of C-7 (2: δH 1.33/ δC 50.7 vs 1: δH 1.70/ δC 43.1) (table 1), suggesting that these compounds have different configuration at C-7. This was supported by a good agreement when comparing the 1 H and 13 C NMR data of 2 with those reported for the 7-epimer of 1, disciferitriol (table 1) [15]. The different 13 C chemical shift between these compounds at C-11 could be explained by influence of the different solvents used (2: in CD3OD; disciferitriol: in C5D5N). Therefore, compound 2 was identified as disciferitriol. The molecular formula of compound 3 was determined to be C17H21NO4 by the observation of an ion [M+H] + at m/z 304 in the ESIMS and 13 C NMR spectroscopic analysis. The 1 H NMR spectrum contained signals for five aromatic protons at H 7.32 (3H) and 7.37 (2H) which were characteristic of a phenyl ring. The 1 H NMR spectrum further exhibited signals for oxygenated proton signals at δH Table 1: 1 H and 13 C NMR data for compounds 1 and 2 C C #1 1 C #2 2 C a,b H a,c C a,b H a,c 1 80.1 80.8 3.26 (m) 79.5 80.3 3.24 (m) 2 30.1 29.4 1.63 * 30.4 29.3 1.66 * 3 39.0 42.0 1.75 (m) 1.53 * 42.8 41.9 1.75 (m) 1.53 (m) 4 71.7 72.8 71.7 72.5 5 48.2 48.4 1.64 * 54.0 54.0 1.26 * 6 21.8 21.7 2.03 (br d, 13.5)/1.53 * 22.3 23.1 1.67 (m) 1.29 (m) 7 42.8 43.1 1.70 (m) 50.6 50.7 1.33 (m) 8 21.9 21.6 1.80 (m) 1.63 (m) 23.1 22.6 1.95 (m) 1.15 (m) 9 42.4 39.2 1.60 * 1.53 * 42.4 42.0 1.95 * 1.09 * 10 39.6 39.8 40.1 40.1 11 74.0 75.2 71.0 73.4 12 29.8 28.7 d 1.26 (s) d 27.9 26.8 d 1.19 (s) d 13 30.4 29.5 d 1.26 (s) d 28.0 27.4 d 1.20 (s) d 14 23.1 22.0 1.11 (s) 23.4 22.5 1.11 (s) 15 14.4 14.1 0.93 (s) 14.0 13.7 0.88 (s) a Recorded in CD3OD, b 125 MHz, c 500MHz, d Signals are interchangeable; * Overlapped signal; #1 C of pterodontriol B [15] in C5D5N; #2 C of disciferitriol [15] in C5D5N Figure 1: Chemical structures of compounds 1-7 from D. metel VJC, 55(2) 2017 Tran Hong Quang et al. 191 Table 2: 1 H and 13 C NMR data for compounds 3-5 C C #1 3 C #2 4 C #3 5 C a,b H a,c C d,b H d,c C a,b H a,c 1 57.3 58.6 3.17 (m) 2 55.8 56.4 3.57 (d, 3.5) 152.4 152.3 8.13 (s) 152.37 152.4 4 55.4 56.1 3.03 (d, 3.5) 149.0 149.0 138.16 138.1 7.83 (d, 1.0) 5 59.2 58.7 3.28 (m) 119.3 119.3 111.51 111.5 6 30.4 29.4 1.50 (d, 16.0) 2.13 (m) 156.2 156.1 166.42 166.5 7 66.3 67.1 4.99 (t, 5.5) 8 30.2 29.6 1.71 (d, 16.0) 2.22 (m) 139.9 139.8 8.34 (s) 10 41.6 39.1 2.52 (s) 1′ 171.2 172.8 87.9 87.9 5.87 (d, 6.5) 86.25 86.2 6.30 (t, 7.0) 2′ 53.8 55.9 3.79 (m) 73.4 73.4 4.59 (dd, 5.5, 6.5) 41.22 41.1 2.26 (m) 3′ 63.4 64.4 3.77 (m) 4.15 (t, 11.0) 70.6 70.6 4.14 (dd, 3.0, 5.5) 72.21 72.2 4.42 (m) 4 85.9 85.8 3.96 (m) 88.82 88.8 3.93 (m) 5 61.6 61.6 3.53 (br d, 12.0) 3.67 (br d, 12.0) 62.84 62.8 3.82 (dd, 12.0, 3.0) 3.76 (dd, 12.0, 3.5) 1′′ 135.4 137.5 2′′, 6′′ 128.3 129.9 7.32* 4′′ 127.6 128.8 7.32* 3′′, 5′′ 127.3 129.2 7.37* 5-CH3 12.44 12.4 1.90 (d, 1.0) a Recorded in CD3OD, b 125 MHz, c 500MHz, d in DMSO-d6; * Overlapped signal; #1 C of scopolamine [16] in CDCl3; #2 C of adenosine [17] in DMSO-d6; #3 C of thymidine [18] in CD3OD. 3.57 (d, J = 3.5 Hz, H-2), 3.03 (d, J = 3.5 Hz, H-4), 4.99 (t, J = 5.5 Hz, H-7), 3.77 (m, H-3 a), and 4.15 (t, J = 11.0 Hz, H-3 b), and two protons bearing nitrogens at δH 3.17 (m, H-1) and 3.28 (m, H-5), and the down-filed signal of a methyl group bearing nitrogen at δH 2.52 (H3-10). Analysis of the 13 C NMR and HSQC indicated the presence of one carbonyl carbon at δC 172.8 (C-1 ), five aromatic methines at δC 137.5 (C-1 ), 129.9 (C-2 and 6 ), 128.8 (C-4 ), and 129.2 (C-3 and 5 ), one epoxy group at δC 56.4 (C-2) and 56.1 (C-4), one oxymethine at δC 67.1 (C-7), one oxymethylene at δC 64.4 (C-3 ), two methines bearing nitrogen at δC 58.6 (C-1) and 58.7 (C-5), two methylenes, and one methyl group. The 1 H and 13 C NMR data of 3 (in CD3OD) showed a similarity with those of scopolamine (in CDCl3), suggesting that the structures of both compounds are identical (table 2) [16]. By the HMBC correlations observed between δH 2.52 (H3- 10) and δC 58.6 (C-1) and between δH 3.57 (H-2) and δH 3.03 (H-4) and δC 58.7 (C-5), the positions of the methyl and epoxy groups were assigned to C-10 and C-4/C-5, respectively (figure 2). The overall structure of 3 was subsequently assigned by the HMBC correlations between δH 3.79 (H-2 ) and δC 137.5 (C-1 ), 129.9 (C-2 and C-6 ), between δH 3.77 and 4.15 (H2-3 ) and δC 172.8 (C-1 ), 55.9 (C- 2 ), and 137.5 (C-1 ), and between δH 4.99 (H-7) and δC 172.8 (C-1 ). Based on the above analysis, compound 3 was determined to be scopolamine. The ESIMS of compound 4 exhibited an ion [M+H] + at m/z 268, corresponding with the molecular formula C10H13N5O4. The 1 H NMR spectrum showed signals of two down-field aromatic protons at δH 8.13 (s, H-2) and 8.34 (s, H-8), suggesting that these protons are connecting with nitrogen atoms. The 1 H NMR further displayed a signal for one anomeric proton at δH 5.87 (d, J = 6.5 Hz) revealing that 4 has one sugar moiety. The 13 C NMR contained 10 carbon signals, including two down-field aromatic methines at δC 152.3 (C-2) and 139.8 (C-8), three non-protonated carbons at δC 149.0, 119.3 (C-5), and 156.1, suggesting that 4 VJC, 55(2) 2017 Chemical constituents of Datura metel L. 192 possesses the purine nucleus. The five remaining carbon signals, including four oxymethines and one oxymethylene group were assigned to a - ribofuranose by comparing with the data reported in the literature (table 2) [17]. Thus, compound 4 was identified as adenosine. Table 3: 1 H and 13 C NMR data for compounds 6 and 7 C C #1 6 C #2 7 C C #1 6 C #2 7 C a,b H a,c C a,b H a,c C a,b H a,c C a,b Ha,c 1 47.3 47.3 0.88 * 2.05 * 37.5 38.5 1.10 * /1.90 * 1′ 106.6 106.2 4.31 (d, 7.5) 100.3 100.4 4.52 (d, 8.0) 2 66.9 68.0 3.81 * 30.1 30.7 1.62 * /1.94 * 2′ 73.1 72.9 3.60* 77.9 79.3 3.42 (dd, 7.5, 9.5) 3 88.4 88.5 3.48 (d, 9.5) 78.1 79.3 3.61 (m) 3′ 74.9 74.6 3.53* 77.8 77.9 3.61* 4 44.7 45.2 39.0 39.5 2.31 (t, 12.0)/2.48 (dd, 3.0, 13.0) 4′ 69.7 70.0 3.84* 78.5 79.9 3.54 (t, 9.5) 5 47.7 47.7 1.22 * 140.8 141.8 5′ 67.8 67.8 3.65* 3.92 (d, 10.5) 76.9 76.5 3.34 * 6 18.2 18.7 1.39 * /1.49 * 121.8 122.6 5.40 (d, 3.0) 6 61.3 61.9 3.67 * 3.82 (br d, 11.0) 7 33.2 33.6 1.33 * /1.68 * 32.3 33.1 1.58 * /2.01 * 1″ 95.7 95.7 5.37 (d, 8.0) 102.0 102.3 5.22 (br s) 8 40.8 41.0 31.7 32.7 1.68 * 2″ 74.1 73.9 3.33* 72.5 72.1 3.95* 9 48.2 48.8 1.64 * 50.3 51.6 0.99 * 3″ 78.9 78.2 3.42* 72.8 72.4 3.68* 10 37.8 38.5 37.1 38.0 4″ 71.4 71.2 3.38* 74.1 73.9 3.43 (dd, 9.0, 9.5) 11 23.9 24.5 1.98 * 21.1 21.9 1.56 * 5″ 79.2 78.5 3.36* 69.5 69.7 4.14* 12 126.2 127.0 5.27 (br s) 39.8 40.9 1.23 * /1.79 * 6″ 62.5 62.5 3.82*/3.71* 18.6 17.8 1.27 (d, 6.5) 13 138.4 139.3 40.4 41.4 1″ 102.9 102.9 4.86* 14 42.6 43.4 56.6 57.7 1.18 * 2″ 72.5 72.1 3.86* 15 28.7 29.2 1.11 * /1.96 * 32.2 32.7 1.31 * /2.01 * 3″ 72.7 72.3 3.65* 16 24.7 25.2 1.78 * /2.09 * 81.1 82.2 4.42 (q, 7.5) 4″ 73.9 73.7 3.43 (dd, 9.0, 9.5) 17 48.4 48.4 62.8 63.7 1.78 5″ 70.4 70.6 3.95* 18 53.4 54.1 2.27 (d, 12.0) 16.3 16.7 0.83 (s) 6″ 18.5 17.9 1.28 (d, 6.0) 19 39.2 40.2 1.00 * 19.4 19.8 1.07 (s) 1′″ 105.0 104.7 4.22 (d, 7.5) 20 39.4 40.3 1.41 * 42.0 42.9 1.93 * 2′″ 75.1 75.1 3.19* 21 30.8 30.6 1.31 * 15.0 14.8 1.00 (d, 7.0) 3′″ 78.6 78.0 3.36* 22 36.8 37.5 1.67 * / 1.79 (br d, 13.5) 109.5 110.8 4′″ 71.6 71.6 3.29 * 23 63.8 64.0 3.28 (d, 11.5)/3.71 * 31.3 31.9 1.63 * 1.73 * 5′″ 78.5 78.0 3.28* 24 14.7 14.4 0.76 (s) 23.9 24.2 1.57 * /1.68 * 6′″ 62.8 62.7 3.69*/ 3.88 * 25 17.4 17.8 1.07 (s) 36.7 37.1 1.91 * 26 17.6 17.9 0.86 (s) 63.6 64.4 3.52 (br d, 11.0) 3.76 * 27 23.8 24.0 1.15 (s) 72.0 72.8 3.33 * /3.80 * 28 176.2 177.9 29 17.8 17.6 0.92 (d, 6.5) 30 21.3 21.5 1.30 (d, 6.5) a Recorded in CD3OD, b125 MHz, c500MHz; *Overlapped signal; #1 C of ilekudinoside C [19] in C5D5N; #2 C of dioscoroside D in [20] C5D5N VJC, 54(5) 2016 Chemical constituents of Datura metel 193 The molecular formula of thymidine (5), C10H14N2O5 was deduced by its ESIMS ion at m/z 241 [M H] and 1 H and 13 C NMR spectra. The 1 H NMR spectrum showed signals for one aromatic proton at δH 7.83 (d, J = 1.0 Hz, H-4), one methyl group at δH 1.90 (d, J = 1.0 Hz, 5-CH3), and one anomeric proton at δH 6.30 (t, J = 7.0 Hz, H-1 ). The 13 C NMR spectrum displayed 10 carbon signals, including two carbonyl carbons at δC 152.4 (C-2) and 166.5 (C-6), one aromatic methine at δC 138.1 (C-4), one non-protonated aromatic carbon at δC111.5 (C-5), and one methyl at δC 12.4 (5-CH3), suggesting the presence of a methyl-pyrimidinedione structural moiety. The remaining carbon signals, including three oxymethines, one oxymethylene, and one methylene carbons were assigned to a deoxy-β- D-ribofuranoside by comparing with the reported values (table 2) [18]. So the structure of thymidine (5) was established as shown in figure 1. Ilekudinoside C (6) was isolated as a white, amorphous powder. Its molecular formula was C53H86O21, as deduced by ESIMS at m/z 805 [M+Na] + and its 13 C NMR spectrum. The 13 C NMR spectrum exhibited 41 carcbon signals, of which 30 were assigned to a triterpenoid alglycone and 11 to a saccharide moiety. The 1 H NMR spectrum of 6 contained signals for six methyl groups at δH 0.76 (s, H3-24), 1.07 (s, H3-25), 0.86 (s, H3-26), 1.15 (s, H3- 27), 0.92 (d, 6.5, H3-29), and 1.30 (d, 6.5, H3-30), a trisubstituted olefinic proton at δH 5.27 (br s), and two anomeric protons at δH 4.31 (d, 7.5, H-1 ) and 5.37 (d, 8.0, H-1 ). The signals at δC 127.0 and 139.3 in the 13 C NMR spectrum, assignable to C-12 and C-13, suggested the presence of a 12 -ursane- type triterpene. Signals at δC 88.5 (C-4) and 177.9 (C-28) in the 13 C NMR spectrum suggested that 6 is a bisdesmosidic ursane-type saponin. The sugar units were identified as one glucopyranose and one arabinopyranose based on comparing the 13 C NMR data of 6 with those reported previously in the literature [19]. The relatively large spin couplings of the anomeric protons (J > 7.5 Hz) were indicative of the -arabinopyranose and -glucopyranose. In the HMBC spectrum, the HMBC correlation from H 3.48 (H-3) to C 68.0 (C-2) suggested that a hydroxyl group is attached to C-2 position (Figure 2). The HMBC correlations between δH 4.31 (H-1 ) and δC 88.5 (C-3) and between δH 5.37 (H-1 ) and δC 177.9 (C-28) indicated that the arabinose and glucose were located at C-3 and C-28, respectively. Based on the data obtained and comparing with those of the reported compound (table 3) [19], the structure of ilekudinoside C (6) was elucidated as shown in figure 1. Figure 2: Selected HMBC correlations of compounds 1-3, 6, and 7 and COSY correlations of compound 7 Figure 3: Selected ROESY correlations of compound 7 Compound 7 had a molecular formula C51H82O22, as deduced by the HRESIMS at m/z 1081.4988 [M+Cl] - (calcd. for C51H82ClO22, 1081.4986) and the 13 C NMR data. The 1 H and 13 C NMR spectra, in combination with DEPT, HSQC, COSY, and HMBC spectra showed the presence of two tertiary methyls at δH 0.83 (s, H3-18) and 1.07 (s, H3-19), one secondary methyl group at H 1.00 (d, J = 7.0 Hz, H3-21), a trisubstituted olefinic proton at δH 5.40 (d, J = 3.0 Hz, H-6), and an acetalic carbon signal at C 110.8 (C-22), implying that 7 possesses the 5,6 -spirostane skeleton [21]. The 1 H NMR spectrum of 7 contained signals for four anomeric protons at H 4.52 (d, J = 8.0 Hz, H- 1 ), 5.22 (br s, H-1 ), 4.86 (H-1 ), and 4.22 (d, J = 7.5 Hz, H-1 ), that showed HSQC correlations with anormeric carbons at C 100.4 (C-1 ), 102.3 (C-1 ), 102.9 (C-1 ), and 104.7 (C-1 ), respectively, indicating that 7 possesses four sugar units. Comparison of the 13 C NMR data of the sugar units with those reported previously suggested the presence of two glucopyranoses and two rhamnopyranoses. The relatively large coupling VJC, 55(2) 2017 Chemical constituents of Datura metel L. 194 constant (J > 7.5 Hz) of the anomeric proton of the glucose revealed the -configuration, whereas the - oriented anomeric form of the rhamnose was defined based on the chemical shift values of its C-3 and C-5 positions [22]. In the HMBC spectrum, the correlation between the anomeric proton of a glucose unit at H 4.22 (H-1 ) and C 72.8 (C-27) indicated that this glucose was located at C-27 position of the aglycone (figure 2). The sugar sequence at C-3 of the aglycone was identified as - L-rhamnopyranosyl-(1 4)[ -L-rhamnopyranosyl- (1 2)]- -D-glucopyranoside by the HMBC correlations between H 4.86 (H-1 ) and C 79.9 (Glc C-4), between H 5.22 (H-1 ) and C 79.3 (Glc C-2), and between H 4.52 (H-1 ) and C 79.3 (C-3). This assignment was also supported by the ROESY correlations between H 4.86 (H-1 ) and H 3.54 (H- 4 ), between H 5.22 (H-1 ) and H 3.42 (H-2 ), and between H 4.52 (H-1 ) and H 3.61 (H-3) (figure 3). On the basis of the above analysis, along with comparison of the NMR data of 7 with those of the very recently reported spirostane-type saponin, the structure of compound 7 was established as shown in Figure 1, namely dioscoroside D [20]. In summary, our phytochemical study on the acidic methanol extract of D. metel resulted in the isolation and identification of seven compounds, including pterodontriol B (1), disciferitriol (2), scopolamine (3), adenosine (4), thymidine (5), ilekudinoside C (6), and dioscoroside D (7). Among the isolated compounds, pterodontriol B, disciferitriol, ilekudinoside C, and dioscoroside D were reported for the first time from the Datura genus. Acknowledgment. This research was supported by NAFOSTED under grant number 104.01-2014.69. REFERENCES 1. V. V. Chi. 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Solanum incanum and S. heteracanthum as sources of biologically active steroid glycosides: confirmation of their synonymy, Fitoterapia, 83, 1115-1119 (2012). 22. R. Kasai, M. Okihara, J. Asakawa, K. Mizutani, O. Tanaka. 13 C NMR study of alpha- and beta-anomeric pairs of d-mannopyranosides and l- rhamnopyranosides, Tetrahedron, 35, 1427-1432 (1979). Corresponding author: Tran Hong Quang Institute of Marine Biochemistry Vietnam Academy of Science and Technology No. 18, Hoang Quoc Viet, Cau Giay District, Hanoi E-mail: quangth2004@yahoo.com.

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