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Chemical Constituents from the Leaves of Datura metel L.
Li Ting, Guo Rui,Yang Bingyou,Kuang Haixue* Heilongjiang University of Chinese Medicine,Harbin,China
Abstract: Objective To study the chemical constituents in the leaves of Datura metel L. Methods The chemical constituents of ethanol extract from the leaves of Datura metel L. were isolated and purified by chromatography over silica gel, AB-8 macroperous resin, ODS, Sephadex LH-20 columns and RP-preparative HPLC, The structures were elucidated on the basis of physicochemical properties and spectral data analyses. Results Eight compounds were isolated and identified asNaphthisoxazol A (1), 1-O-ß-D-glucopyranosyl(1^-2)-ß-D-glucopy-ranosyl-(E)-3-hexene (2), L-Tryptophan (3), quercetin3-O-2-(E-caffeoyl)-a-Larabinopyranosyl-(1^-2)-ß-D-glucopyranoside-7-O-ß-D-glucopyranoside (4), N-butyl-O-alpha-D-Fructofuranosidase (5), anoectochine (6), dihydrovomifoliol-O-ß-D-glucopyranoside (7), (6S,7E,9S)-9-[(ß-D-glucopyranosyl)-oxy]megastigma-4,7-dien-3-one (8). Conclusion Compounds 1-8 are firstly found in the Solanaceae.
Key words: leaves of Datura metel L.; chemical constituents; Solanaceae; Naphthisoxazol A; congmuyaglyeoside I; L-Tryptophan
Introduction
Flos Daturae(Chinese name:Yangjinhua),the dry flower of Datura metel L. It has an obvious effect on psoriasis and anesthesia for clinical use in China.The leaves of Datura metel L. are extremely rich. In order to further expand the medicinal resources, be conducive to the protection of the medicinal herbs and the sustainable utilization of Chinese medicine.This report conducts on the chemical constitude of the leaves .We discovered 8 compounds for the first time in our research.Their structures were elucidated to be Naphthisoxazol A (1), 1-O-ß-D-glucopyranosyl(1^2)-ß-D-glucopy-ranosyl-(E)-3-hexene (2), L-Tryptophan (3), quercetin3-O-2-(E-caffeoyl)-a-Larabinopyranosyl-(1^2)-ß-D-glucopyranoside-7-O-ß-D-glucopyranoside (4), N-
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butyl-O-alpha-D-Fructofuranosidase (5), Anoectochine (6), dihydrovomifoliol-O-ß-D-glucopyranoside (7), (6^,7£,9^)-9-[(ß-D-glucopyranosyl)-oxy]megastigma-4,7-dien-3-one (8).All of them are firstly found in the Solanaceae.
Material and methods
The UV and NMR spectra were recorded on Bruker DPX 400 (400 MHz for 1H NMR and 100
13
MHz for C NMR), respectively. Chemical shifts are given as 5 values with reference to tetramethylsilane (TMS) as an internal standard, and coupling constants are given in Hz. The ESI-MS analyses were conducted on Finnigan MAT LCQ mass spectrometer. Preparative HPLC (Waters 2414-2998) was performed on Waters C18 (10pm, 10x250mm, Atlantis) .A Hypersil ODS II (5pm, 4.6x250mm, SunFire) for analytical HPLC (Waters 2695-2996). Macroporous absorption resin (AB-8 Crosslinked Polystyrene, Nankai, Tianjin, China) and silica gel ( 200-300 mesh, Qingdao, China) were employed for column chromatography. SephadexTM LH-20 (GE Healthcare Bio-Sciences AB).
The dry leaves of D.metel were collected in the medical plants garden of heilongjiang University of Chinese medicine in September 2010, and identified by prof. Zhenyue Wang. A voucher specimen(2010056) is deposited at the Herbarium of Heilongjiang University of Chinese Medicine,China.
Extraction and Isolation
The dried leaves of D.metel (12kg) were quenched with 95% EtOH , the residue were extracted with 95% EtOH under reflux(2x100L) for 2h, then combined quenched and refluxed solution ,and evaporated under vacuum to syrup, followed by suspension in H2O. The suspension was extracted with petroleum, then removed the petroleum fraction and the water layer was passed through AB-8 Crosslinked Polystyrene,andsequentially eluted with H2O ,30% EtOH,70% EtOH,respectively.30% EtOH elution was subjected to silica gel and eluted successively with CH2Cl2/MeOH(12:1-0:100) gradient eluted to give 5 fractions(Fr.1-5). Fr.1 was purified by preparative HPLC, obtained Compound 1(10.07mg),2(50.67mg); Fr.2 continues silica gel chromatography obtained Compound 4(30.67mg); Compound
3(20.57mg),5(35.88mg),6(10.56mg),7(33.25mg) were obtained by ODS column chromatography. Fr.4 continues silica gel chromatography elution with CH2Cl2/MeOH(8:1-0:100) gradient eluted to afford a number of sub-fraction, Fr.4-2 was purified by Sephadex LH-20 obtained Compound 7(5.87mg).
Result and discussion
Compound 1 Pale yellow oilD Its molecular formula,CnH9O2N. ESI-MS m/z 187[M]+/H-NMR (400 MHz, CD3OD) S: 7.69 (1H, br.d, J = 7.8 Hz, H-5), 7.36 (1H, br.d, J = 7.8 Hz, H-8), 7.19 (1H, s, H-4), 7.11 (1H, ddd, J = 7.8, 6.9 Hz, 0.9 Hz, H-7) , 7.04 (1H, ddd, J = 7.8, 6.9, 0.9 Hz, H-6), 3.87 (1H, dd, J = 9.2 Hz, 3.7 Hz, H-9a), 3.51 (1H, dd, J = 15.1, 3.7 Hz, H-9b), 3.15 (1H, dd, J = 15.1, 9.2 Hz, H-9a) ; 13C-NMR (100 MHz, CD3OD) S: 174.6 (C-3), 138.4 (C-8a), 128.5 (C-4a), 125.2 (C-8), 122.8 (C-7), 120.1 (C-6), 119.4 (C-5), 112.5(C-4), 109.6 (C-3a), 56.7 (C-9a), 28.5(C-9)0 Compound 1 was characterized as Naphthisoxazol A[1]0
Compound 2 White amorphous powder. Its molecular formula,Ci8H32On. ESI-MS m/z 424 [M]+0 1H-NMR (400 MHz, C5D5N) S: 3.72 (2H, m, H-1), 2.23 (2H, br.q, J = 6.7, H-2), 5.50 (1H, m, H-3), 5.43 (1H, m, H-4), 1.92(2H, br.tq, J = 6.6, 6.6), 0.89 (3H, t, J = 6.6), 4.24 (1H, d, J = 7.7, H-1'), 3.21 (m, H-2'), 3.18 (m, H-3'), 3.10 (m, H-4'), 3.16 (m, H-5'), 3.63 (m, H-6'), 4.35 (1H, d, J = 7.3, H-1''), 2.99 (m, H-2''), 3.11 (m, H-3''), 3.41 (m, H-4''), 3.76 (m, H-5''), 3.54 (m, H-6'') ; 13C-NMR (100 MHz, C5D5N) S: 69.4 (C-1), 28.3 (C-2), 125.6 (C-4), 133.6 (C-4), 20.8 (C-5), 14.4 (C-6), 103.1 (C-1'), 84.4 (C-2'), 78.8 (C-3'), 71.4 (C-4'), 77.9 (C-5'), 62.6 (C-6'), 106.6 (C-1''), 76.9 (C-2''), 78.1 (C-3''), 71.3 (C-4''), 78.4 (C-5''), 62.5 (C-6'')0 Compound 2 was characterized as 1-O-ß-D-glucopyranosyl(1^2)-ß-D-glucopy-ranosyl-(E)-3-hexene[2]Q
Compound 3 yellowish powder, Its molecular formula,C11H12N2O2, ESI-MS m/z 216 [M]+. 1H-NMR (400 MHz, DMSO-4) S: 10.98 (1H, s, NH), 7.56 (1H, d, J = 7.8 Hz, H-5), 7.34 (1H, d, J
= 8.1 Hz, H-7), 7.2 (1H, s, H-2), 7.05 (1H, t, J = 7.5 Hz, H-6), 6.96 (1H, t, J = 7.4Hz, H-4), 3.42 (1H, dd, J = 3.9, 8.9Hz, H-11), 3.34 (1H, dd, J = 3.6, 15.0 Hz, H-10a), 2.95 (1H, dd, J = 9.0, 15.0Hz, H-10b); 13C-NMR (100 MHz, DMSO-d6) S: 123.9 (C-2), 109.7 (C-3), 118.2 (C-4), 118.3 (C-5), 120.8 (C-6), 111.3 (C-7), 136.3 (C-8), 127.2 (C-9), 27.2 (C-10), 54.8 (C-11), 170.5 (COOH)o Compound 3 was characterized as L-Tryptophan[3]0
Compound 4 Yellow amorphous powderD Its molecular formula,C41H44O24, ESI-MS m/z 919 [M-H]-, 1H-NMR (400 MHz, CD3OD) S: 6.44 (2H, d, J = 2.2 Hz, H-6), 6.54 (2H, d, J = 2.2 Hz, H-8), 7.54 (1H, d, J = 2.2 Hz, H-2'), 6.0 (1H, t, J = 8.0 Hz, H-5'), 7.38 (1H, dd, J = 2.0, 8.0 Hz, H-6'), 5.92 (1H, d, J = 7.6 Hz, H-1''), 3.67 (1H, dd, J = 7.5, 9.0 Hz, H-2''), 3.65 (1H, t, J = 9.0 Hz, H-3''), 3.35 (1H, t, J = 9.0 Hz, H-4''), 3.26 (m, H-5''), 3.93 (1H, dd, J = 3.0, 12.0 Hz, H-6a''), 3.63 (1H, dd, J = 5.0, 12.0 Hz, H-6b''), 5.12 (1H, d, J = 7.0 Hz, H-1'''), 4.87 (1H, dd, J = 7.0, 9.0 Hz, H-2'''), 3.64 (1H, dd, J = 9.0, 2.5 Hz, H-3'''), 3.63 (1H, m, H-4'''), 4.02 (1H, dd, J = 12.0, 2.0 Hz, H-5a'''), 3.36 (1H, dd, J = 12.0, 3.0 Hz, H-5b'''), 6.14 (1H, d, J = 16.0 Hz, H-a), 7.39 (1H, d, J = 16.0 Hz, H-ß), 6.74 (1H, d, J = 2.0, H-2''''), 6.56 (1H, d, J = 8.0Hz, H-5''''), 6.60 (1H, dd, J = 2.0, 8.0 Hz, H-6''''),
5.08 (1H, d, J = 7.5 Hz, H-1.....), 3.52 (1H, dd, J = 7.5, 9.0 Hz, H-2.....), 3.52 (1H, t, J = 9.0 Hz, H-
3.....), 3.40 (1H, t, J = 9.0 Hz, H-4.....), 3.56 (1H, m, H-5.....), 3.80 (1H, dd, J = 12.0, 3.0 Hz, H-6a.....),
3.65 (1H, dd, J = 12.0, 5.0 Hz, H-6b.....); 13C-NMR (100 MHz, DMSO-d6) S: 158.3 (C-2), 135.5 (C-
3), 179.4 (C-4), 162.1 (C-5), 100.7 (C-6), 164.0 (C-7), 95.4 (C-8), 157.6 (C-9), 107.7 (C-10), 123.1 (C-1'), 117.4 (C-2'), 145.8 (C-3'), 149.0 (C-4'), 116.0 (C-5'), 123.3 (C-6'), 98.4 (C-1''), 79.6 (C-2''), 76.1 (C-3''), 71.3 (C-4''), 78.3 (C-5''), 62.4 (C-6''), 99.1 (C-1'''), 74.8 (C-2'''), 75.1 (C-3'''), 71.3 (C-4'''), 64.4 (C-5'''), 168.6 (COOH), 115.1 (C-a), 146.5 (C-ß), 127.3 (C-1''''), 114.7 (C-2''''), 146.4 (C-
3''''), 149.7 (C-4''''), 16.2 (C-5''''), 122.2 (C-6''''), 101.6 (C-1.....), 77.9 (C-2.....), 77.8 (C-3.....), 72.5 (C-
4'''''), 78.1 (C-5'''''), 61.9 (C-6'"'% Compound 4 was characterized asquercetin3-O-2-(E-caffeoyl)-a-Larabinopyranosyl-(1^2)-ß-D-glucopyranoside-7-O-ß-D-glucopyranoside[4].
Compound 5 Colorless syrups 0 Its molecular formula,C11H22O6, ESI-MS m/z: 251 [M+H]+. 1H-NMR (400 MHz, CD3OD) S: 3.49 (1H, dt, J = 9.2, 6.4 Hz, H-1a), 3.66 (1H, dt, J = 9.2, 6.8 Hz, H-1b), 1.48~1.55 (2H, m, H-2), 1.33~1.42 (2H, m, H-3), 0.92 (3H, t, J = 7.3 Hz, H-4), 3.53 (1H, d, J = 11.9 Hz, H-1'a), 3.67 (1H, d, J = 11.9 Hz, H-1'b), 4.09 (1H, d, J = 4.9 Hz, H-3'), 3.89 (1H, dd, J = 7.0, 4.9 Hz, H-4'), 3.76 (1H, ddd, J = 7.0, 2.8, 4.9 Hz, H-5'), 3.62 (1H, dd, J = 12.0, 4.9 Hz, H-6'a), 3.77 (1H, dd, J = 12.0, 2.8 Hz, H-6'b); 13C-NMR (100 MHz, CD3OD) S: 62.2 (C-1), 33.5 (C-2), 20.4 (C-3), 14.3 (C-4), 62.2 (C-1'), 105.2 (C-2'), 78.4 (C-3'), 77.3 (C-4'), 83.4 (C-5'), 65.0 (C-6')0 Compound 5 was characterized asN-butyl-O-alpha-D-Fructofuranosidase[5].
Compound 6 Yellow crystalline solidD Its molecular formula,C12H12N2O2, ESI-MS m/z: 217 [M+H]+. 1H-NMR (400 MHz, DMSO) S: 10.9 (1H, S, NH), 7.43 (1H, d, J = 7.92, H-4), 6.98 (1H, dd, J = 7.3, 7.5 Hz, H-5), 7.06 (1H, dd, J = 7.4, 7.5 Hz, H-6), 7.32 (1H, d, J = 8.0 Hz, H-7), 4.14 (1H, d, J = 15.5 Hz, H-8a), 4.22 (1H, d, J = 15.3 Hz, H-8b), 3.64 (1H, dd, J = 5.0, 10.5 Hz, H-10), 2.81 (1H, dd, 10.5, 16.0 Hz, H-12a), 3.13 (1H, dd, J = 5.0, 16.0 Hz, H-12b); 13C-NMR (100 MHz, C5D5N) S: 127.7 (C-2), 106.6 (C-3), 117.8 (C-4), 118.7 (C-5), 121.3 (C-6), 111.1 (C-7), 126.2 (C-7a), 136.1 (C-3a), 40.4 (C-8). 169.3 (C-10), 56.6 (C-11), 22.9 (C-12)0 Compound 6 was characterized as Anoectochine[6]0
Compound 7 Colorless oilyD Its molecular formula,C2oO8H33, ESI-MS m/z 424 [M+H]+. 1H-NMR (400 MHz, MeOD) S: 1.01 (3H, s, 5-Meb), 1.09 (3H, s, 5-Mea), 1.17 (3H, d, J = 6.16 Hz, 3'-Me), 1.48 (m, H-2'b), 1.78 (m, H-2'a), 1.82 (m, H-l'b), 1.98-2.08 (m,H-l'a), 2.03 (3H, d, J = 1.28 Hz, 3-Me), 2.14 (1H, dd, H-6b), 2.60 (1H, d, J = 18.2 Hz, H-6a), 3.10-3.90 (m, H-2"-H-6"), 3.81 (m, H-3'), 4.30 (1H, d, J = 7.8 Hz, H-1"), 5.83 (1H, dq, H-2); 13C-NMR (100 MHz, MeOD) S: 201.0 (C-1), 171.7 (C-3), 126.7 (C-2), 102.3 (C-1"), 79.3 (C-4), 78.1 (C-5"), 77.9 (C-3"), 76.2 (C-3'), 75.1 (C-2"), 71.8 (C-4"), 62.9 (C-6"), 51.1 (C-6), 42.9 (C-5), 34.9 (C-2'), 33.6 (C-1'), 24.7 (C-5Mea), 24.1 (C-5Meb), 21.8 (C-3'Mea), 20.1 (C-3'Meb)D Compound 5 was characterized as dihydrovomifoliol-O-ß-D-glucopyranoside[7].
Compound 8 White amorphous powder. Its molecular formula,C19H30O8, ESI-MS m/z 387 [M+H]+ . 1H-NMR (400 MHz, CD3OD) S: 4.34 (1H, d, J = 7.76 Hz, H-1'), 1.01 (3H, s, Me-11),
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1.03 (3H, s, Me-12), 1.28 (3H, d, J = 6.36 Hz, Me-10), 4.15 (2H, 2dd, J = 17.8, 1.4 Hz, H-13), 6.15 (1H, br.s, H-4), 5.78 (1H, dd, J = 15.4, 5.92 Hz, H-7), 5.71 (1H, dd, J = 15.5, 8.4 Hz, H-8), 2.50 (2H, d, J = 16.8 Hz, H-2), 2.69 (1H, d, J = 8.36 Hz, H-6), 4.35 (1H, dq, J = 7.76, 6.2 Hz, H-9); 13C-NMR (100 MHz, MeOD) S: 37.2 (C-1), 52.1 (C-2), 202.1 (C-3), 122.4 (C-4), 168.2 (C-5), 49.9 (C-6), 138.1 (C-7), 128.9 (C-8), 77.1 (C-10), 21.1 (C-10), 27.9 (C-11), 27.6 (C-12)0 Compound 8 was characterized as(6S,7E,9S)-9-[(ß-D-glucopyranosyl)-oxy]megastigma-4,7-dien-3-one[8].
References
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[2] Mang Zhi Qiang, Zhang Yang, Song Shaojiang, Xu Suixu. Two new glycoside in liaodong wood bud[J]. Chinese Journal of Medicinal Chemistry, 2008, 18(4): 300-316.
[3] LI Guo-qiang,DENG Zhi-wei,LI Jun,FU Hong-zheng,and LIN Wen-han. Che mical Constituents from Starfish Asterias rollestoni [J]. Journal of Chinese Pharmaceutical Sciences 2004, 13(2): 81-86.
[4] Sara Vitalini, Alessandra Braca, Gelsomina Fico. Study on secondary metabolite content of Helleborus niger L. leaves [J]. Fitoterapia , 2011, 82(2): 152-154.
[5] Yang Minghui, Yang Xueqiong, Zhang Fengmei, Yang Yabin, Cai Le, Ding Zhongtao. The chemical composition of Woodwardia unigemma ta (Makino)Nakai [J]. Chinese herbal medicine journal, 2009, 40(10): 1546-1548.
[6] MEI-HUA HAN, XIU-WEI YANG, and YAN-PING JIN. Novel triterpenoid acyl esters and alkaloids from Anoectochilus roxburghii [J]. Phytochemical Analysis Phytochem. Anal. 2008, 19(5): 438-443.
[7] Rolf and Dersson and Lennart n. Lundgren. Monoaryl and cyclohexe none glycosides from needles of Pinus Sylves Tris*[J]. Phytochemistry, 1988, 27(2): 559-562.
[8] Xiao-Hong Xua,Chang-Heng Tana,Shan-Hao Jianga,and Da-Yuan Zhu. Debilosides A-C: Three New Megastigmane Glucosides from Equisetum debile [J]. Verlag Helvetica Chimica Acta AG, Zürich, 2006, 89(7): 1422-1426.
Study on Antifungal Activities of Endophytic Fungi from Gentian against Alternaría sp.
Duo Liu, Xiaolin Shang, Xiaowei Du Pharmacognosy, Heilongjiang University of Chinese Medicine, Harbin, China.
Abstract
Two endophytic fungi ,LD421 and LD455, which is isolated and purified in the laboratoryshow obvious inhibition effect on Alternaria sp.. Mixed fermentation culture of the two endophytic fungi is carried out in different ways for the selection of the optimal mixed culture method. Meanwhile, the inoculation proportion and fermentation time are also optimized. The results show that on the condition that the inoculation proportion between LD421 and LD455 equals to 3:1 and the fermentation lasts for ten days, the inhibitory rate can reach 88.20±0.05% together with the fact that the viscosity of the mixed-cultured fermentation broth is lower than that of purely cultured endophytic fungus LD421, leading to easier acquisition of metabolism broth.
Key words: endophytic fungus, mixed culture,
Introduction
During the development of microbiology, isolation and pure culture is a big step forward. Modern industries concerning microbial fermentation are mostly based on pure culture. With further research, multi-fungus mixed culture has become one of the most important research
