莖花蔥臭木化學成分的研究

唐 霆，納 智，許又凱，3*

1中國科學院西雙版納熱帶植物園熱帶森林生態(tài)學重點實驗室，勐臘666303;2中國科學院研究生院，北京100049; 3中國科學院西雙版納熱帶植物園熱帶植物資源開放實驗室，勐臘666303

Introduction

Dysoxylum cauliflorum Hiern(Meliaceae)is a plant from the genus Dysoxylum originated in Fiji，Papua New Guinea，and New Zealand.Many species of the genus have been used as medicines to relieve fever，rigid limbs，convulsions，haemorrhage，and facial distortion in children［1-3］.Various bioactive compounds have been obtained by previous studies on the chemical constituents of the Dysoxylum genus，such as cytotoxic tirucallane-type alkaloids［4］，antifeeding limonoids［5］，cytotoxic diterpenes［6］，antileukemic triterpene glucosides［7］，and antibacterial triterpenoids［8，9］.The phytochemical investigation of D.cauliflorum has only afforded several dammarane triterpenoids and one sesquiterpene［10，11］.Thus，this plant was selected cultured in China for further investigation looking forward to get more bioactive compounds.The present study led to the discovery of five known compounds isolated from this plant for the first time.

Fig.1 The chemical structures of compounds 4 and 5

Experimental

General

1H NMR and13C NMR spectra were recorded on a Bruker AVANCE III-600，Bruker DRX-500 and Bruker AM-400 spectrometer using TMS as internal standard.EIMS spectra were measured on a AutoSpec Premier P776 spectrometer.Column chromatography was performed on silica gel(200-300 mesh，Qingdao Meigao Chemical Co.Ltd.，Qingdao，China).All solvents were distilled prior to use.

Plant material

The seeds of D.cauliflorum were collected at Xishuangbanna Tropical Botanical Garden，Chinese Academy of Sciences，Yunnan Province，in November 2008，and identified by Xiao Chunfen of the Xishuangbanna Tropical Botanical Garden，Chinese Academy of Sciences.A voucher specimen(No.02059)was deposited in the Herbarium of the Xishuangbanna Tropical Botanical Garden，Chinese Academy of Sciences.

Extraction and isolation

The air-dried powder of the seeds(8.1 kg)were extracted with 95%EtOH three times(15 L×3))at room temperature.The crude extracts(1.6 kg)were suspended in water，and then partitioned with petroleum ether and EtOAc，respectively.The petroleum ether extract(130 g)was subjected to silica gel column chromatography eluted with petroleum ether/Me2CO (from 98∶2 to 0∶100)to give eight fractions(1-8) and afford compounds 1(42.4 mg)，3(1.23 g)，and 4(5 mg)purified by crystallization and recrystallization with Me2CO and CH3OH，respectively.Fraction 6 (3.3 g)was subjected to silica gel column eluted with CHCl3/Me2CO(from 100∶0 to 85∶15)to afford 2 (1.21 g).Fraction 7(8.8 g)was further purified on silica gel column(CHCl3/Me2CO from 100∶0 to 85∶15)to yield 5(1.20 g).

Identification

β-sitosterol(1) C29HO，white needle crystal (Me2CO).It was confirmed by comparing it with the standard sample.

Ethylgallate (2) C9H10O5，white powder (Me2CO)，1H NMR(pyridine-d5，500 MHz)δ:7.57 (2H，s，H-2，H-6)，4.26(2H，q，J=7.0 Hz，H-8)，1.16(3H，t，J=7.2 Hz，H-9);13C NMR(pyridine-d5，100 MHz)δ:167.1(s，C-7)，147.7(s，C-3，C-5)，141.0(s，C-4)，121.4(s，C-1)，110.2(d，C-2，C-6)， 60.5(t，C-8)，14.4(q，C-9).The NMR data were identical to those of literature［12］.

Daucosterol(3) C35H60O6，white powder (Me2CO)，1H NMR(pyridine-d5，500 MHz)δ:5.34 (1H，br.d，J=4.5 Hz，H-6)，3.99(1H，m，H-3)，2.47(1H，m，H-17)，0.65(3H，s，H-18)，0.92(3H，s，H-19)，0.88(3H，d，J=6.5 Hz，H-21)，0.98，0.86 (each 3H，d，J=6.5 Hz，H-26，H-27)，0.90(3H，t，J =7.5 Hz，H-29)，5.06(1H，d，J=8.0 Hz，Glu-H-1)，4.07(1H，t，J=8.0 Hz，Glu-H-2)，4.30(2H，m，Glu-H-3，Glu-H-4)，3.98(1H，m，Glu-H-5)，4.57 (1H，d，J=11.5 Hz，Glu-H-6a)，4.43(1H，br.d，J= 13.0 Hz，Glu-H-6b).The 1H NMR date were accorded with the literature［13］.

1-O-β-D-glucopyranosyl-(2S，3S，4R，8Z)-2-N-(2'-hydroxytetracosanoyl)octadecasphinga-8-ene(4) C48H93NO10，white powder(Me2CO)，1H NMR(pyridine-d5，500 MHz)δ:8.56(1H，d，J=9.0 Hz，NH)，5.58-5.39(2H，m，H-8，H-9)，5.28(1H，m，H-2)，4.95(1H，br.s，H-1″)，4.70(1H，m，H-1b)，4.57 (1H，br.s，H-2')，4.50(1H，m，H-1a)，4.47(1H，m，H-6″b)，4.34(1H，m，H-6″a)，4.28(1H，m，H-3)，4.19(3H，m，H-4，H-3″，H-4″)，4.01(1H，t，J=7.5 Hz，H-2″)，3.86(1H，br.s，H-5″)，2.23(2H，m，H-7)，2.06(2H，m，H-10)，1.95(2H，m，H-5)，1.23-1.29(54H，br.d，J=32.0 Hz，27×CH2)，0.85(6H，t，J=6.5 Hz，H-18，H-24');13C NMR(pyridine-d5，150 MHz)δ:176.1(s，C-1')，130.9(d，C-9)，130.7 (d，C-8)，106.1(d，C-1″)，79.1(d，C-3″)，78.9(d，C-5″)，76.3(d，C-3)，75.7(d，C-2″)，72.9(d，C-2')，72.8(d，C-4)，71.8(d，C-4″)，71.0(t，C-1)，62.9(t，C-6″)，52.1(d，C-2)，36.0(t，C-3')，34.4(t，C-5)，32.6(t，C-10)，32.1(t，C-7)，30.6-30.1(t，n× CH2)，27.3(t，C-4')，23.4(t，C-17，C-23')，14.7(q，C-18，C-24').The NMR date were consistent with the literature［14］.

2，3，2″，3″-tetrahydroamentoflavone(5) C30H22O10，yellow powder(CHCl3)，EI-MSm/z:542［M］+.1H NMR(DMSO-d6，400 MHz)δ:7.20-7.18 (4H，m，H-2'，H-6'，H-2″'，H-6″')，6.82(1H，d，J= 8.0 Hz，H-5')，6.68(2H，d，J=8.4 Hz，H-3″'，H-5″')，6.04(1H，s，H-6″)，5.87(2H，s，H-6，H-8)，5.39(2H，d，J=12.8 Hz，H-2，H-2″)，2.57-3.23 (4H，m，H-3，H-3″);13C NMR(DMSO-d6，100 MHz) δ:197.2(s，C-4″)，196.7(s，C-4)，166.9(s，C-7)，164.8(s，C-7″)，163.7(s，C-5)，163.4(s，C-5″)，162.5(s，C-9)，160.3(s，C-9″)，157.6(s，C-4″')，156.3(s，C-4')，131.6(s，C-1')，129.2(s，C-1″')，128.4(d，C-6')，128.3(d，C-2″'，C-6″')，127.4(d，C-2')，120.3(s，C-3')，115.3(d，C-5'，C-3″'，C-5″')，106.2(s，C-8″)，102.0(s，C-10，C-10″)，96.1(d，C-6，C-6″)，95.2(d，C-8)，78.9(d，C-2)，78.1(d，C-2″)，42.5(t，C-3)，41.6(t，C-3″).The NMR data were equal to those of literature［15］.

Compound 5 was evaluated for its cytotoxicity against five human cancer cell lines using the MTT method［16］，which included human myeloid leukemia HL-60，hepatocellular carcinoma SMMC-7721，lung cancer A-549 cells，breast cancer MCF-7，and colon cancer SW480.Cisplatin(Sigma，USA)was used as the positive control and the IC50value was calculated by Reed and Muench’s method［17］.The result showed compound 5 was inactive with its IC50values＞40 μM.

Acknowledgment We appreciate Prof.Li Yan，Kunming Institute of Botany，Chinese Academy of Sciences，for cytotoxicity assays.

1 Aalbersberg W，Singh Y.Dammarne triterpenoids from Dysoxylum richii.Phytochemistry，1991，30:921-926.

2 Chen JL，Kernan MR，Jolad SD，et al.Dysoxylins A-D，tetranortriterpenoids with potent anti-RSV activity from Dysoxylum gaudichaudianum.J Nat Prod，2007，70:312-315.

3 Russell GB，Hunt MB，Bowers WS，et al.A sesquiterpenoid ant repellent from Dysoxylum spectabile.Phytochemistry，1994，35:1455-1456.

4 Zhang XY，Li Y，Wang YY，et al.Tirucallane-type alkaloids from the bark of Dysoxylum laxiracemosum.J Nat Prod，2010，73:1385-1388.

5 Luo XD，Wu SH，Wu DG，et al.Novel antifeeding limonoids from Dysoxylum hainanense.Tetrahedron，2002，58:7797-7804.

6 Duh CY，Wang SK，Cheng IS.Cytotoxic prenyleudesmane diterpenes from the fruits of Dysoxylum kuskusense.J Nat Prod，2000，63:1546-1547.

7 Kashiwada Y，F(xiàn)ujioka T，Chang JJ，et al.Antitumor agents 136.Cumingianosides A-F，potent antileukemic new triterpene glucosides，and Cumindysoside-A and Cumindysoside-B，trisnortriterpene and tetranortripene glucosides with a 14，18-cycloapoeuphane-type skeleton from Dysoxylum cumingianum.J Org Chem，1992，57:6946-6953.

8 He XF，Wang，XN，Gan LS，et al.Two novel triterpenoids from Dysoxylum hainanense.Org Lett，2008，10:4327-4330.

9 He XF，Wang XN，Yin S，et al.Ring A modified novel triterpenoids from Dysoxylum hainanense.Eur J Org Chem，2009: 4818-4824.

10 Huang R，Harrison LJ，Sim KY.A triterpenoid with a novel abeo-dammarane skeleton from Dysoxylum cauliflorum.Tetrahedron Lett，1998，40:1607-1610.

11 Benosman A，Richomme P，Roussakis C，et al.Effects of triterpenes from the stem bark of Dysoxylum cauliflorum on a non-small-cell bronchopulmonary carcinoma cell line (NSCLC-N-6).Anticancer Res，2000，20:1855-1859.

12 Du ZZ，He HP，Wu B，et al.Chemical constituents from the pericarp of Trewia nudiflora.Helv Chim Acta，2004，87:758-763.

13 Yang H，Xie JL，Sun HD.Study on chemical constituents of Saussurea lappaⅡ.Acta Bot Yunnan，1997，19:92-96.

14 Qi SH，Wu DG，Zhang S，et al.A new tetranortriterpenoid from Dysoxylum lenticellatum.Z Naturforsch(B)，2003，58: 1128-1132.

15 Ahmad I，Ishratullah K，Ilyas M，et al.Tetrahydroamentoflavone from nuts of Semecarpus prainii.Phytochemistry，1981，20:1169-1170.

16 Mosmann T.Rapid colorimetric assay for cellular growth and survival:Application to proliferation and cytotoxicity assays.J Immunol Methods，1983，65:55-63.

17 Reed L，Muench H.A simple method of estimating fifty percent endpoints.Am J Hygiene，1938，27:493-497.