Norclerodane diterpenoids from rhizomes of Dioscorea bulbifera
Hai Liu a,b,c ,Gui-Xin Chou a,b ,Yin-Long Guo c ,Li-Li Ji a,b ,Jun-Ming Wang a,b ,Zheng-Tao Wang a,b,*
a
The MOE Key Laboratory for Standardization of Chine Medicines,Institute of Traditional Chine Medicine,Shanghai University of Traditional Chine Medicine,1200Cailun Road,Shanghai 201210,China b
Shanghai R&D Centre for Standardization of Chine Medicines,1200Cailun Road,Shanghai 201210,China c
Shanghai Mass Spectrometry Center,Shanghai Institute of Organic Chemistry,Chine Academy of Sciences,345Lingling Road,Shanghai 200032,China
a r t i c l e i n f o Article history:
Received 31August 2009
Received in revid form 7January 2010Available online 7May 2010Keywords:
Dioscorea bulbifera Dioscoreaceae
Norclerodane diterpenoid Diosbulbins K–M Diosbulbinoside G Mosher’s method
a b s t r a c t
Three norclerodane diterpenoids,diosbulbins K–M,and one analogous enolglycoside,diosbulbinoside G,together with four norclerodane diterpenoids,diosbulbins B,E,F and G,were isolated from rhizomes of Dioscorea bulbifera .Their structures were established by spectroscopic and chemical methods,including 1
H and 13C NMR,NOESY,HSQC,HMBC,and HRMS analys.The relative configurations of diosbulbins K and L,and diosbulbin F were confirmed by X-ray crystallographic diffraction analysis,and the absolute stereochemistry of diosbulbin K was determined by a modified Mosher’s method.The 13C NMR spectro-scopic data for diosbulbins E,F and G were also measured for the first time.The compounds did not show significant cytotoxic and anti-bacterial activities.
Ó2010Elvier Ltd.All rights rerved.
1.Introduction
Dioscorea bulbifera L.(Dioscoreaceae)is a liana widely distrib-uted throughout the tropics and temperate regions.The rhizome of D.bulbifera has been ud in folk and traditional Chine medicine for the treatment of thyroid dias and cancer (Gao et al.,2002;Guo,1992;Lin et al.,2002;Xu and Ding,1998).In Bangladesh,it is ud by indigenous people for treatment of lep-rosy and tumors (Murray et al.,1984).Earlier chemical investiga-tion of its tubers afforded a ries of norclerodane diterpenoids and enolglycosides (Ida et al.,1978a,b;Kawasaki et al.,1968;Komori,1997;Komori et al.,1968,1973;Murray et al.,1984;Yonemitsu et al.,1993).Recently,nine clerodane diterpenoids (Teponno et al.,2006,2007,2008;Wang et al.,2009)and three apianen lactones from this plant were reported (Zheng et al.,2003).Clerodane-type diterpenes also show broad biological and pharmacological properties,such as anti-tumor,anti-bacterial,anti-feedant,anti-fungal,and hallucinogenic activities (Harding et al.,2006;Komori,1997;Roengsumran et al.,2002;Shriram et al.,2008;Teponno et al.,2006;Urones et al.,1995).
In our recent screening of natural products with anti-tumor activities,the ethyl acetate fraction of the ethanol extract of D.bulbifera showed potent anti-tumor activity using a tumor-embed-ded mice mod
el.Phytochemical investigation of this fraction led to isolation of eight norclerodane diterpenoids including four previ-ously unreported compounds.This paper reports the structure elu-cidation of the diterpenoids and 13C NMR spectroscopic data of three other constituents 6,7and 8which have not been previously documented.
2.Results and discussion
无人机航拍Air-dried and pulverized rhizomes of D.bulbifera were extracted with EtOH–H 2O (4:1).The concentrated extract was suspended in water and extracted successively with petroleum ether (60–90°C)and EtOAc.The EtOAc soluble portion was fractionated by silica gel and Sephadex LH-20column chromatography to give four compounds (1–4)and diosbulbins B (5)(Yonemitsu et al.,1993),E (6),F (7),and G (8)(Ida et al.,1978a )(Fig.1).Their structures were determined by means of spectroscopic and chemical methods.Compound 1was obtained as colourless crystals from MeOH.Its molecular formula C 20H 24O 7was established by HRESIMS bad on its [M+Na]+ion at m /z 399.1416(calc.399.1419).The IR spectrum of 1showed absorption peaks for carbonyl (1726,1721,and 1707cm À1),hydroxyl (3494cm À1),and furan (873cm À1)function-alities (Murray et al.,1984).The 1H NMR spectrum of 1showed characteristic signals of a b -substituted furan ring at d H 7.54(s ,H-16),7.44(d ,J =1.0Hz,H-15),6.49(d ,J =1.0Hz,H-14)(Teponno et al.,2006,2008)and two m
ethyl resonances at d H 3.62(s ,H-20)and 1.24(s ,H-19).The one proton signal at d H 5.63(dd ,J =12.5,3.8Hz,H-12)together with the resonances at d H 1.96(dd ,湖北名茶
0031-9422/$-e front matter Ó2010Elvier Ltd.All rights rerved.doi:10.1016/j.phytochem.2010.04.002
*Corresponding author at:The MOE Key Laboratory for Standardization of Chine Medicines,Institute of Traditional Chine Medicine,Shanghai University of Traditional Chine Medicine,1200Cailun Road,Shanghai 201210,China.Tel.:+862151322507;fax:+862151322519.
E-mail address: ,wangzht@shutcm.edu (Z.-T.Wang).Phytochemistry 71(2010)
1174–1180
Contents lists available at ScienceDirect
Phytochemistry
j o u r n a l h o m e p a g e :www.elvier.c o m /l o c a t e /p h y t o c h e
m
J=14.7,12.5Hz,H-11b),and2.33(dd,J=14.7,3.8Hz,H-11a)were consistent with an ABX system formed by a–CHO–CH2–moiety. Furthermore,the downfield signal obrved at d H4.17(1H,m,H-2)was ascribed to the methine proton H-2(Table1).The13C NMR spectrum of1exhibited20resonances,of which19were from the diterpene skeleton and one from the esterified methyl
Table1
1H NMR spectroscopic data[d(J Hz)]of compounds1–4.
1a2b3c4b 1a 1.90(brdd,13.2,2.2) 1.94(m) 2.10(m) 1.92(m)
1b 1.42(td,13.2,2.4) 1.45(td,13.2,2.4) 1.59(t,13.3) 1.49(m)
2 4.17(m) 4.14(m) 4.75(m) 4.15(m)
3a 2.45(brdd,14.5,2.4) 2.44(m) 2.30(m) 2.42(m)
3b 1.75(ddd,14.5,6.1,2.4) 1.81(m) 1.77(d,12.3) 1.68(ddd,14.2,6.8,2.2)
4 3.35(m) 3.29(m) 2.74(m) 4.11(dd,6.8,1.6)
5 2.18(dd,12.2,3.8) 2.24(dd,12.0,4.0)
6
7a 3.32(m) 2.62(m) 3.21(d,17.6) 3.24(m)
7b 2.65(dd,17.4,7.3) 2.62(m) 2.67(m) 2.41(m)
8 2.80(dd,7.3,2.5) 3.33(m) 2.67(m)
9
10 3.02(td,12.2,2.4) 2.90(m) 2.30(m) 3.10(brd,12.4)
好雨知时节的全诗11a 2.33(dd,14.7,3.8) 2.36(m) 2.10(m) 2.43(m)
11b 1.96(dd,14.7,12.5) 1.87(m) 1.88(dd,13.8,12.4) 1.92(m)
12 5.63(dd,12.5,3.8) 5.58(dd,11.6,4.8) 5.55(dd,12.8,2.2) 5.69(dd,11.4,6.0)
13
14 6.49(d,1.0) 6.56(s) 6.61(s) 6.05(d,1.1)
157.44(d,1.0)7.54(s)7.67(s)7.48(d,1.1)
167.54(s)7.63(s)7.75(s)7.56(s)
17
18
19 1.24(s) 1.08(s) 1.09(s)0.99(s)
20 3.62(s)
6-Glc
10 4.75(d,7.8)
20 3.28(m)
30 3.29(m)
40 3.30(m)
50 3.35(m)
60 3.80(m)
3.60(m)
a Measured in CD
3
OD and trace CDCl3.
b Measured in CD
3
OD.
c Measure
d in DMSO.
H.Liu et al./Phytochemistry71(2010)1174–11801175
group.Signals for a b-substituted furan ring[d C143.9(C-15),140.2 (C-16),125.2(C-13)and108.6(C-14)]were obrved.Other salient features of this spectrum included resonances assigned to a ketone group at d C207.7(C-6),two ester carboxyl groups at d C172.1(C-17)and175.0(C-18),and two oxygenated methines[d C65.0(C-2),71.2(C-12)](Table2).The remaining signals were due to one angular methyl,four methylene,four methine groups and one qua-ternary carbon(Table2).From the above data,1contained a norc-lerodane diterpenoid skeleton(Murray et al.,1984;Komori,1997). Comp
arison of the NMR spectroscopic data of1with tho of dios-bulbin F(7)established that structure1was identical to7except for the different configuration of d-lactone at C-8.The downfield shift of C-8by3.4ppm and the upfield shift of C-10by9.4ppm, C-9by1.6ppm and C-11by1.7ppm in ring B of1,compared to the data for7,must ari from the different configuration at C-8. The d-lactone(ring C)was therefore cis-fud to ring B and adopted a chair-like conformation with the furyl residue at the C-12equa-torial.This was further proved by the correlations of Me-19(d H 1.24)/H-8(d H2.80)and H-12(d H5.63)/H-10(d H3.02)in the NOESY spectrum.A NOESY correlation of H-5(d H2.18)with Me-19(d H 1.24)indicated that the ring A was trans-fud to ring B.Final proof of the structure and relative configuration of compound1was ob-tained from a single crystal X-ray analysis using direct methods (Fig.2).The single crystal X-ray analysis of diosbulbin F(7)is also shown in Fig.2.The absolute configuration at C-2of1was assigned using a modified Mosher’s method.Where the compound(2mg) was derivatized with Mosher’s reagents,(R)and(S)methoxytriflu-oromethylphenylacetic chloride in an NMR tube using pyridine-d5 as solvent(Ohtani et al.,1991;Su et al.,2002).The difference in chemical shift values(D d SR)of the diastereomeric esters,(R)-MTPA (1R)and(S)-MTPA(1S),indicated the absolute configuration of C-2 as R and allows assignment of the absolute configuration as C-4R, C-5S,C-8R,C-9S,C-10R,and C-12S.From the this evidence,com-pound1was deduced as methyl15,16-epoxy-6-keto-2a-hydro-xyl-8b,5b-norclerodane-13(16),14(15)-dien-17,12b-olide-18-car-boxylic acid and was designated diosbulbin K.
Compound2was obtained as colourless needles with a molec-ular formula of C19H22O7determined by positive-ion HRESIMS bad on its[M+Na]+ion at m/z385.1260(calc.385.1263).The IR spectrum showed absorption peaks at3429cmÀ1(hydroxyl group),1734,1707,1691cmÀ1(carbonyl group)and875cmÀ1 (furan).The1H,13C,and DEPT NMR spectroscopic data of2were similar to tho of diosbulbin F(7)with characteristic signals for ketone carbonyl,b-substituted furan ring and d-lactone,except for the lack of a methyl group.The positions of the carboxyl at C-
Table2
13C NMR spectroscopic data(d)of compounds1–4and6–8.
1a2b3c4b6a7a8a
133.332.929.434.229.534.331.8
265.065.375.966.879.866.066.5
335.034.333.235.739.835.730.2
437.537.044.638.144.738.043.1
549.649.773.6118.844.551.340.9
6207.7208.0205.6145.270.1208.678.0
737.236.634.733.230.637.925.0
849.044.245.875.042.345.643.5
934.734.932.939.537.136.336.0
1031.839.942.932.440.841.238.4
1140.241.641.634.843.442.942.5
1271.270.569.673.671.971.971.9
13125.2124.3125.2127.7126.0125.0126.0
14108.6108.3109.0110.1109.8109.4109.8
15143.9143.6143.7145.2145.1144.8145.1
day0
16140.2140.0140.1141.5141.4140.9141.3
17172.1174.6171.4174.4177.6175.2176.6
18175.0176.3175.7177.9181.4176.1180.7
1921.017.227.117.418.819.118.3
2051.852.4
6-Glc
10102.7
2075.3
3078.4
4071.7
5078.3
6063.1
a Measured in CD
3
欢跃OD and trace CDCl3.
b Measured in CD
3
OD.
撤单是什么意思c Measure
d in
DMSO.
Fig.2.X-ray crystal structures of compounds1,3and7. 1176H.Liu et al./Phytochemistry71(2010)1174–1180
4,the ketone carbonyl at C-6,and the hydroxyl group at C-2were assigned by HMBC correlations bet
ween the proton resonances at d H3.29(H-4)and2.24(H-5)with the carbon at d C176.3(C-18), the signal at d H2.24(H-5)with the ketone carbonyl signal at d C 208.0,and the proton resonance at d H3.29(H-4)with the carbon at d C65.3(C-2),respectively.The COSY and key HMBC correlations of2are shown in Fig.3.The NOESY correlations of H-1b(d H1.45) and H-5(d H2.24)with Me-19(d H1.08),H-3b(d H1.81)with H-5(d H 2.24),and H-12(d H5.58)with H-8(d H3.33)indicated that both the A/B and B/d-lactone rings were trans-fud(Fig.3).Thus,2was elu-cidated as15,16-epoxy-6-keto-2a-hydroxyl-8a,5b-norclerodane-13(16),14(15)-dien-17,12b-olide-18-carboxylic acid and named diosbulbin L.
我的芭比娃娃Compound3was obtained as colourless crystals from MeOH with a molecular formula C19H20O7determined from the HREIMS ([M]+ion at m/z360.1210,calc.360.1209).The IR spectrum of3 showed three particularly broad and inten bands at1772,1727 and1698cmÀ1which are characteristic of d-lactone,c-lactone
H.Liu et al./Phytochemistry71(2010)1174–11801177
and carbonyl group,respectively.A hydroxyl group at3424cmÀ1 and another band at876cmÀ1suggested the prence of a furan moiety.The1H and13C NMR spectroscopic data(Tables1and2) of3cloly rembled tho of1with characteristic signals for a b-substituted furan ring,d-lactone and a ketone carbonyl,showing typical resonances for a norclerodane diterpene skeleton.The main differences were the conformation of the A/B rings and the appear-ance of an hydroxyl group and a c-lactone bridged at C-2and C-4. Evidence for a carbon signal at C-5downfield shifted from d C49.6 (d)in1to d C73.6(s)in3indicated that an hydroxyl group was at-tached to C-5,which was further confirmed by HMBC correlations of the proton resonances at d H1.77(H-3b),2.10(H-1a),2.30(H-10 or H-3a),and2.74(H-4)with the carbon signal at d C73.6.The c-lactone ring bridged at C-2and C-4was deduced from the carbon resonance at C-2downfield shifted from d C65.0in1to d C75.9in3 due to esterification,and was also confirmed by the HMBC correla-tion of the proton signal at d H4.75(H-2)with the carbon resonance at d C175.7(C-18).The position of a ketone carbonyl at C-6was determined by HMBC correlations of the protons at d H2.30(H-10),2.67(H-7b or H-8),2.74(H-4),and3.21(H-7a)with the car-bon signal at d C205.6.
The relative configuration of3was established by a NOESY experiment.Starting from a conventional
b-orientation of Me-19 in norclerodane diterpene,NOESY correlations of Me-19(d H1.09) with H-1b(d H1.59),H-8(d H2.67)and H-7b(d H2.67),H-10(d H 2.30)with H-12(d H5.55)and OH-5(d H6.61)suggested that the OH-5had an a-orientation and that the A/B ring and B/d-lactone were cis-fud.Furthermore,the structure and relative configura-tion of3was confirmed by single crystal X-ray diffraction analysis (Fig.2).The X-ray crystallography affirmed the relative configura-tion of3and the result was consistent with the data from the NOESY experiment.Thus,structure3was therefore concluded to be15,16-epoxy-6-keto-5a-hydroxyl-8b,5-norclerodane-13(16), 14(15)-dien-17,12b;18,2a-diolide and given the trivial name of diosbulbin M.
Compound4was obtained as colourless needles,and its molec-ular formula C25H30O12was established by positive-ion HRMALDI-FTMS([M+Na]+m/z545.1663,calc.545.1635).The IR spectrum showed3415cmÀ1(hydroxyl group),1719cmÀ1(carbonyl group) and874cmÀ1(furan)absorption peaks.The1H and13C NMR spec-troscopic data(Tables1and2)established the prence of25car-bons assigned to a hexosyl moiety and a norclerodane diterpenoidoid aglycone containing a b-substituted furan moiety, two carbonyl carbons(d C177.9and174.4),a tetrasubstituted ole-fin(d C118.8and145.2),one methyl group(d H0.99/d C17.4),two quaternary carbons,four methines,and four methylenes.The agly-cone of4was the same as that of3,except for an additional enol and a diffe
rent position of the hydroxyl group.The position of the hydroxyl group was determined as C-8by the evidence of the carbon signal downfield shifted from d C45.8(d)in3to d C 75.0(s)in4,and further identified by HMBC correlations of the proton resonances at d H0.99(Me-19),3.24(H-7a),2.41(H-7b) and2.43(H-11a)with the carbon signal at d C75.0(C-8)(Fig.3). The HMBC correlations of the proton resonances at d H4.11(H-4), 3.24(H-7a)and2.41(H-7b)with the carbon signal at d C118.8 (C-5)and145.2(C-6)indicated that the olefin was at C-5–C-6. The identification of the glucosyl moiety was confirmed by TLC and GC analys.The absolute configuration of gluco was deter-mined to be D by GC–MS analysis of chiral derivatives of the sugar in an acidic hydrolysate.The configuration of the anomeric proton of D-glucopyranosyl unit was determined as b,bad on the1H NMR coupling constant(3J
1;2
=7.8>7Hz).In the HMBC spectrum, the anomeric proton at d H4.75(H-10)of the gluco showed a long-range correlation with C-6(d C145.2).The relative configura-tion at position8was confirmed by a NOESY experiment in which the correlation of H-10(d H3.10)with H-12(d H5.69)indicated that the B/C(d-lactone)ring is cis-fud.The key HMBC and NOE corre-lations of4are shown in Fig.3.Therefore,compound4was char-acterized as15,16-epoxy-8b-hydroxyl-norclerodane-5(6),13(16),
14(15)-trien-17,12b;18,2a-diolide-6-O-b-D-glucopyranoside and it was designated diosbulbinoside G.
The13C NMR spectroscopic data for compounds6–8,which were not reported before,were assigned(e Table2).
3.Concluding remarks
In conclusion,the structures of four norclerodane diterpenoids (1–4)along with four others(5–8)isolated from D.bulbifera were identified,including an unusual5a,10a-cis-clerodane(3)deter-mined by X-ray diffraction analysis.While norclerodane diterpe-noids have usually been described as the primary and characteristic constituents of this species,the purified compounds studied here did not show remarkable anti-tumor activities in vitro against human hepatocellular carcinoma cells(Bel-7402)at a con-centration of10l M nor anti-bacterial activities against four fungal species at a concentration of200l g/mL.Since this herb has been in u for the treatment of cancer in TCM by oral administration,the isolated compounds may need to be activated through metabolism by gutflora or by microsome P450enzymes in liver to display their bioactivity.So further in vivo pharmacological studies on the purified norclerodane diterpenoids should be carried out.
4.Experimental
4.1.General experimental procedures
Melting points were determined on a Buchi Melting Point B-540 apparatus(Buchi Labortechnik AG in Flawil,Switzerland)and are uncorrected.Optical rotations were taken in MeOH on a KRÜSS P8000-T polarimeter(A.KRÜSS Optronic GmbH,Germany).IR spectra were measured as afilm on a KBr pellet using a Thermo Nicolet380FT-IR spectrometer(Thermo Electron Corporation, USA).The1D and2D NMR spectra were acquired on Bruker AV400or AV500spectrometers.The HRESIMS and HRMALDI-FTMS were obtained on an FTMS-7(Bruker Daltonic)and an Ionspec4.7T HisRes MALDI-FTMS(Ionspec,Irvine,CA,USA)spectrometers, respectively.HREIMS spectra were measured on a Waters GCT CA176spectrometer(Waters Corporation,USA).Open column chromatography(CC)was carried out using silica gel(200–300 and300–400mesh,Qingdao Marine Chemical Co.,China),and Sephadex LH-20(GE Healthcare,Sweden)as stationary phas. TLC was performed on HSGF254(0.2mm,Qingdao Marine Chemical Co.,China)plates.
4.2.Plant material
Dried rhizomes of D.bulbifera were collected from Qingyang, Anhui Province,China,in September200
ps动作
6,and were identified by Professor Shou-Jin Liu(Anhui College of Traditional Chine Medicine,Anhui,China).A voucher specimen(LH0609-1)was deposited in the herbarium of the Institute of Traditional Chine Medicine,Shanghai University of Traditional Chine Medicine. 4.3.Extraction and isolation
Dried rhizomes of D.bulbifera(30kg)were crushed,extracted three times(solvent:material ratio=6:1and each time for4h) with EtOH–H2O(4:1,v/v,3Â180L)at85°C,and concentrated in vacuo to yield a black liquid.The EtOH extract was partitioned be-tween petroleum ether and H2O(1:1)to afford a petroleum ether (60–90°C)fraction(160g),with the former then again extracted
1178H.Liu et al./Phytochemistry71(2010)1174–1180
