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败酱草高速逆流色谱法分离纯化化合物并考察了化合物的体外抗肿瘤活性

败酱草高速逆流色谱法分离纯化化合物并考察了化合物的体外抗肿瘤活性
败酱草高速逆流色谱法分离纯化化合物并考察了化合物的体外抗肿瘤活性

Journal of Chromatography A,1115(2006)

103–111

Preparative isolation of four new and two known?avonoids from the

leaf of Patrinia villosa Juss.by counter-current chromatography and

evaluation of their anticancer activities in vitro

Jinyong Peng,Guorong Fan?,Yutian Wu

Shanghai Key Laboratory for Pharmaceutical Metabolite Research,School of Pharmacy,Second Military Medical University,

No.325Guohe Road,Shanghai200433,China

Received9December2005;received in revised form23February2006;accepted24February2006

Available online20March2006

Abstract

A preparative counter-current chromatography(CCC)was used to isolate and separate chemical constituents from the leaf of Patrinia villosa, a famous traditional Chinese medicinal herb.Six?avonoids including two known and four novel compounds were successfully simultaneous puri?ed by CCC with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water(10:13:13:10,v/v)by increasing the?ow rate of the mobile phase from1.0ml/min to2.0ml/min after110min to bring out the late eluters.The separation produced total of44.9mg fraction I with99.1%purity,35.5mg fraction II with98.8%purity,79.8mg fraction III with99.3%purity,45.8mg fraction IV with98.8%purity,39.8mg fraction V with98.6%purity and9.6mg fraction VI with97.5%purity from400mg crude extract in one sin-gle isolation procedure and less than10h,and the obtained fractions were all analyzed by high performance liquid chromatography(HPLC). Their chemical structures were elucidated as(2S)-5,7,2 ,6 -tetrahydroxy-6,8-di(?,?-dimethylallyl)?avanone(1),(2S)-5,7,2 ,6 -tetrahydroxy-6-lavandulylated?avanone(2),(2S)-5,7,2 ,6 -tetrahydroxy-4 -lavandulylated?avanone(3),(2S)-5,2 ,6 -trihydroxy-2 ,2 -dimethylpyrano[5 ,6 : 6,7]?avanone(4),(2S,3 S)-5,2 ,6 -trihydroxy-3 -?,?-dimethylallyl-2 ,2 -dimethyl-3 ,4 -dihydropyrano[5 ,6 :6,7]?avanone(5)and lico-agrochalcone B(6),respectively,by spectrum methods including UV,IR,high resolution(HR)-electrospray ionization(ESI)-MS,1-dimension (1D)and2-dimension(2D)NMR techniques.Among them,compounds2,3,4,and5were new compounds and discovered from nature for the ?rst time.The recoveries of the six compounds were91.2%,91.4%,92.1%,90.5%,90.3%and89.7%,respectively,in CCC step.Subsequently, their anticancer activities were also examined to inhibit human cancer cells’growth including A549,BEL-7402,SGC-7901,MCF-7,HT-29, K562and A498cell lines by MTT method in vitro.The results indicated that the compounds1,2and3exhibited high anticancer activities (IC50<7?g/ml),especially to K562cancer cell(IC50<3.1?g/ml),and the compounds4,5and6exhibited weaker inhibition effect(IC50<30?g/ml).

?2006Elsevier B.V.All rights reserved.

Keywords:Patrinia villosa Juss.;Preparative chromatography;Counter-current chromatography;Flavonoid

1.Introduction

Patrinia,a genus of about20species,is mainly distributed in central to east of Asia and northeast of North America,10 of which grows in China.Patrinia species have been used as medicinal plants for more than2000years from ShenNongBen-CaoJing,a famous ancient Chinese medicinal literary,and some of them still are used in folk medicine as anti-virus and anti-?Corresponding author.Tel.:+862125070388;fax:+862125070388.

E-mail address:Guorfan@https://www.wendangku.net/doc/e59533538.html,(G.Fan).bacteria[1,2],especially two species,P.scabiosaefolia Fisch and Patrinia villosa(BaiJiangCao in Chinese).

With regard to the chemical constituents of this genus, except for some iridoids[3,4],we have isolated and separated two C-glycosyl?avones(isovitexin and isoorientin)[5],a pep-tide derivative aurentiamide acetate[6]and several prenylated ?avonoids[7,8]from it.A literature search did not yield any more references to early report on study of chemicals from the leaf of the medicinal herb P.villosa.So,further chemical research and discovery from the leaf of P.villosa is warranted for exploiting new traditional Chinese medicine(TCM)products and pharmacological tests.

0021-9673/$–see front matter?2006Elsevier B.V.All rights reserved. doi:10.1016/j.chroma.2006.02.079

104J.Peng et al./J.Chromatogr.A 1115(2006)

103–111

Fig.1.The chemical structures of the six compounds.

Generally,some conventional methods including silica gel,polyamide and preparative RPLC are often used to isolate pure products from medicinal plants,but they are tedious,time consuming,requiring multiple chromatographic steps.Counter-current chromatography (CCC),a support free liquid–liquid partition chromatographic technique,eliminates irreversible adsorption of the sample onto solid support,has an excellent sample recovery.So,it has been successfully applied to isolate and purify chemicals from natural materials [9–12].

In the present paper,a preparative CCC was used as a tool to isolate and separate chemicals from the leaf of P .villosa and six compounds were simultaneously separated in only one CCC run.Their chemical structures were eluci-dated as (2S )-5,7,2 ,6 -tetrahydroxy-6,8-di (?,?-dimethylallyl)?avanone (1),(2S )-5,7,2 ,6 -tetrahydroxy-6-lavandulylated ?a-vanone (2)(2S )-5,7,2 ,6 -tetrahydroxy-4 -lavandulylated ?a-vanone (3),(2S )-5,2 ,6 -trihydroxy-2 ,2 -dimethylpyrano [5 ,6 :6,7]?avanone (4),(2S ,3 S )-5,2 ,6 -trihydroxy-3 -?,?-dimethylallyl-2 ,2 -dimethyl-3 ,4 -dihydropyrano [5 ,6 :6,7]?avanone (5)and licoagrochalcone B (6),respectively (shown in Fig.1).Among them,compounds 2,3,4,and 5were new compounds and discovered from nature for the ?rst time.Sub-sequently,the anticancer activities of the obtained compounds to inhibit human cancer cell lines’growth were evaluated by 3,-4,5-dimethyliazol-2,5-diphenyl tetrazolium bromide (MTT)method in vitro.

2.Experimental 2.1.Apparatus

Preparative CCC was carried out with a model TBE–300A counter-current chromatograph (Shenzhen,Tauto Biotech,China).The apparatus was equipped with a polytetra?uo-roethylene three preparative coils (diameter of tube,2.6mm,total volume,300ml)and a 20ml sample loop.The revolu-tion radius or the distance between the holder axis and cen-tral axis of the centrifuge (R )was 5cm,and the βvalue varied from 0.5at the internal terminal to 0.8at the exter-nal terminal (β=r /R where r is the distance from the coil to the holder shaft).The CCC system was equipped with a model S constant-?ow pump,a model UV-II detector oper-ating at 280nm,and a model N2010workstation (Zhejiang University,Hangzhou,China).The experimental temperature was adjusted by HX 1050constant temperature circulat-ing implement (Beijing Boyikang Lab Implement,Beijing,China).2.2.Reagents

n -Hexane,ethyl acetate,methanol,ethanol and acetic acid were analytical grade and purchased from WuLian Chemical Factory,Shanghai,China.While acetonitrile used for HPLC was HPLC grade (Merck,Germany).Reverse osmosis Milli-Q

J.Peng et al./J.Chromatogr.A1115(2006)103–111105

water(18M )(Millipore,USA)was used for all solutions and dilutions.

The leaf of P.villosa was purchased from a local drug store and identi?ed by Doctor Luping Qin(Department of Pharma-cognosy,College of Pharmacy,the Second Military Medical University,Shanghai,China).

2.3.Preparation of the crude extract for CCC isolation

The leaf of P.villosa was ground into powder,3.0kg of the powder was extracted by re?ux with3.0×104ml75%aqueous ethanol for two times.The mixture was?ltered,and2.1×104ml ?ltrate was collected.The extract was then concentrated to no ethanol by rotary vaporization at60?C under reduced pressure and600ml residue was obtained.Then the residue was re-dissolved in water(total volume1500ml),which was added into a glass column(6.0cm×60cm,contained2.0kg AB-8macro-porous resin,Nankai University,Tianjin,China).Five thousand millilitres water was?rst used to elute the resin until the elu-tion was nearly no color,and3000ml30%aqueous ethanol was used to elute the resin,too.Then6000ml85%aqueous ethanol was used to elute the target compounds,and20elution fractions (300ml for each)were collected and6(from5to10fractions) were united and evaporated to dryness according to HPLC anal-ysis,which was used for CCC isolation and separation.

2.4.Preparation of two-phase solvent system and sample solution

In the present paper,we selected several kinds of two-phase solvent systems.Each solvent system was thoroughly equili-brated in a separatory funnel at room temperature and the two phases were separated shortly before use.The sample solution was prepared by dissolving the crude extract in the solvent mix-ture of lower phase and upper phase(1:1,v/v)of the solvent system for isolation because the sample was not easily dissolved in either phase.

https://www.wendangku.net/doc/e59533538.html,C separation procedure

In each separation,the coil column was?rst entirely?lled with the upper phase(stationary phase),and then the apparatus was rotated at900rpm,while the lower phase(mobile phase) was pumped into the column at a?ow rate of1.0ml/min.After the mobile phase front emerged and hydrodynamic equilibrium was established in the column,approximately10ml of the sam-ple solution containing400mg of the crude extract was injected into the head of the column through the injection valve.After 110min,the?ow rate of the mobile phase was increased to 2.0ml/min.The ef?uent of the column was continuously mon-itored with a UV–vis detector at280nm and the separation temperature was controlled at35?C.Peak fractions were col-lected according to the elution pro?le.

2.6.HPLC analysis and identi?cation of CCC fractions

The analytical HPLC system used throughout this study con-sisted of515pump and2487detector(Waters),and a model N2000workstation(Zhejiang University,Hangzhou,China). The crude sample and peak fractions obtained by HSCCC were analyzed by HPLC.The column used was a reversed-phase Lichrospher C18(250mm×4.6mm I.D.,5?m)(Han-bang Science,Jiang Su Province,China)with a pre-column equipped with the same stationary phase,the mobile phase was CH3CN–MeOH–H2O–HAC(40:25:35:2,v/v/v).The?ow rate was0.8ml/min,and the ef?uent was monitored at280nm and the column temperature was set at30?C.

Identi?cation of CCC fractions was carried out by Shimadzu UV210A spectrometer(Japan),IR spectra(Hitachi275-50), MS(Finnigan MAT711),1-dimension(1D)and2-dimension (2D)NMR spectra(Varian Unity Inova–500).

2.7.Anticancer assay

MTT assay was performed as described[13].Brie?y,cells were seeded at a concentration of1.5×105cells/ml in a96well plates.After overnight incubation,serial concentrations of the compounds were added.Serial concentrations of test sample were prepared by dissolving the compound in dimethyl sulfox-ide(DMSO)followed by dilution with RPMI-1640medium to yield the?nal DMSO concentration in the assay well as0.2%. Each concentration was repeated three times.These cells were incubated in a humidi?ed atmosphere with5%CO2for3days. Then20?l MTT solution(4.2mg/ml)was added to each well and incubated at37?C for4h.The medium was removed and formazan was dissolved in DMSO and the optical density was measured at590nm using a Bio-assay reader(Bio Rad,USA). The growth inhibition was determined using:Growth inhibi-tion=(Control O.D.?Sample O.D.)/Control O.D.,and IC50, which is the drug concentration resulting in a50%inhibition of cell growth,is calculated from dose–inhibition curves.

3.Results and discussion

3.1.HPLC conditions

First,the crude extract used for further CCC isolation was analyzed by HPLC.So,a good HPLC condition was required. In our research,different mobile phases(methanol–water, acetonitrile–water,methanol–acetonitrile–water)with different concentration of acetic acid,different?ow rates,detection wavelength and column temperature were all tested.The result indicated that the mobile phase was composed of CH3CN–MeOH–H2O–HAC at the volume ratio of40:25: 35:2(v/v),and the?ow rate,column temperature and detection wavelength were set at0.8ml/min,30?C and 280nm,which were most suitable for our analysis.Under the above conditions,a satisfactory separation of the target compounds was obtained,and the HPLC chromatogram of the crude extract is given in Fig.2,which mainly contained six peaks,and peaks1,2,3,4,5,and6correspond to (2S)-5,7,2 ,6 -tetrahydroxy-6,8-di(?,?-dimethylallyl)?a-vanone,(2S)-5,7,2 ,6 -tetrahydroxy-6-lavandulylated?avanone (2S)-5,7,2 ,6 -tetrahydroxy-4 -lavandulylated?avanone), (2S)-5,2 ,6 -trihydroxy-2 ,2 -dimethylpyrano[5 ,6 :6,7]

106J.Peng et al./J.Chromatogr.A1115(2006)

103–111

Fig.2.HPLC chromatogram of the crude extract from the leaf of P.vil-losa after resin column chromatography.Column:reversed-phase Lichrospher C18(250mm×4.6mm I.D.,5?m);mobile phase:CH3CN–MeOH–H2O–HAC (40:25:35:2,v/v);?ow rate:0.8ml/min;UV wavelength:280nm;column temperature:30?C;peaks1,2,3,4,5,and6correspond to(2S)-5,7,2 ,6 -tetrahydroxy-6,8-di(?,?-dimethylallyl)?avanone,(2S)-5,7,2 ,6 -tetrahydroxy-6-lavandulylated?avanone,(2S)-5,7,2 ,6 -tetrahydroxy-4 -lavandulylated?a-vanone,(2S)-5,2 ,6 -trihydroxy-2 ,2 -dimethylpyrano[5 ,6 :6,7]?a-vanone,(2S,3 S)-5,2 ,6 -trihydroxy-3 -?,?-dimethylallyl-2 ,2 -dimethyl-3 ,4 -dihydro-pyrano[5 ,6 :6,7]?avanone and licoagrochalcone B,respec-tively.

?avanone,(2S,3 S)-5,2 ,6 -trihy-droxy-3 -?,?-dimethylallyl-2 ,2 -dimethyl-3 ,4 -dihydropyrano[5 ,6 :6,7]?avanone and licoagrochalcone B,and present the contents of12.2%, 9.6%,21.5%,12.5%,10.9%and2.8%,respectively.

https://www.wendangku.net/doc/e59533538.html,C isolation and separation

In CCC,the selection of two-phase solvent system is the most important for successful separation,and is also the most dif?-cult step;it is estimated that about90%of the entire work in CCC is spent on that.If only one component requires to be isolated from others,the standard CCC method,which uses a constant?ow-rate of the mobile phase,could be used.In order to isolate more different compounds,step-wise elution or stepwise increasing the?ow-rate of the mobile phase might be adopted[14–16].First,CCC experiments were carried out with the two-phase solvent system composed of n-hexane–methanol at a volume ratio of1:1(v/v),it was dif?cult to purify the tar-get compounds from the crude extract,because their retention time was short.Subsequently,a two-phase solvent system com-posed of n-hexane–ethyl acetate–methanol–water at a volume ratio of1:1:1:1(v/v)was tested.Although the peak reso-lution was improved,and peaks1,2and3could be isolated from others,it was dif?cult to separate other compounds.And then the two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water at a volume ratio of10:13:13:10was used,the peaks1,2,3and4were puri?ed and separated at the?ow rate of1.0ml/min in less than10h,but the other two compounds were retained in the column for a long time(15h) and more mobile phase was required.When the?ow rate was increased to2.0ml/min,peaks1and2were combined together, while other compounds were obtained from other constituents in less8h.Finally,the method with stepwise increasing the ?ow-rate of the mobile phase was attempted with this two-phase solvent system.That is,the?ow-rate of the mobile phase was kept at1.0ml/min before110min,and then increased to 2.0ml/min after that moment.

At the same time,the in?uence of the separation temperature and the revolution speed were also investigated.The tempera-ture has signi?cant effect on K values,the retention of stationary phase and the mutual solvency of the two phases,which mainly due to the effects of the thermodynamics and the re-partition of the two phases in CCC column,and high temperature led to resist the loss of stationary phase and increase the retention of the stationary phase[17].After tested at15?C,20?C,25?C,30?C, 35?C and40?C,it can be seen that good result can be obtained when the separation temperature was controlled at35?C.The revolution speed has a great in?uence to the retention of station-ary phase,high rotary speed can increase the retention of the stationary phase.In our experiment,the revolution speed was set at900rpm.

Under the above optimized separation conditions,the isola-tion of the target compounds was achieved with good resolution and the retention of the stationary phase was satisfactory(67%), and the CCC separation time was approximately400min(CCC chromatogram is shown in Fig.3).After the six compounds were eluted out,in order to save solvents and time,the remain-ing compounds in the column were removed by forcing out

the https://www.wendangku.net/doc/e59533538.html,C chromatogram of the crude extract from the leaf of P.villosa after cleaning-up by AB-8macroporous resin.Solvent system:n-hexane–ethyl acetate–methanol–water(10:13:13:10,v/v);stationary phase:upper phase; mobile phase:lower phase;revolution speed:900rpm;separation tempera-ture:35?C;sample size:400mg;retention of stationary phase:67%;sample loop:20ml;detection wavelength:280nm.I,II,III,IV,V and VI are collected fractions.The arrow indicates the?ow-rate of the mobile phase was increased stepwise from1.0to2.0ml/min after110min.

J.Peng et al./J.Chromatogr.A1115(2006)103–111

107

Fig.4.HPLC chromatography of the fractions obtained by CCC.HPLC analytical conditions and the peaks are the same shown in Fig.2.A:fraction I;B:fraction II;C:fraction III;D:fraction IV;E:fraction V;F:fraction VI.

stationary phase with pressurized nitrogen gas instead of elut-ing them with the mobile phase because the stationary phase was not to be reused.Fig.3shows the preparative CCC iso-lation of400mg of crude extract using the solvent system composed of n-hexane–ethyl acetate–methanol–water at a vol-ume ration of10:13:13:10(v/v)by increasing the?ow-rate of the mobile phase stepwise from1.0ml/min to2.0ml/min after110min.This separation yielded44.9mg compound1at 99.1%purity,35.5mg compound2at98.8%purity,79.8mg compound3at99.3%purity,45.8mg compound4at98.8% purity,39.8mg compound5at98.6%purity and9.6mg com-pound6at97.5%,respectively,from400mg of the crude extract in one-step separation run(HPLC chromatograms are shown in Fig.4).The recoveries of the six compounds were91.2%, 91.4%,92.1%,90.5%,90.3%and89.7%,respectively,only in CCC run.

3.3.Structural elucidation of the isolated compounds

Compounds1and6(fraction I and VI)were two known com-pounds and have been isolated by us from P.villosa[7].Their UV,IR,MS,1H NMR and13C NMR data are in agreement with(2S)-5,7,2 ,6 -tetrahydroxy-6,8-di(?,?-dimethylallyl)?a-vanone and licoagrochalcone B in the literatures[7,18].

Compound2was isolated as light yellow powder.Its molec-ular formula was determined as C25H28O6by high resolution (HR)-electrospray ionization(ESI)-MS m/z424.1562(calcd. 424.1234),ESI-MS m/z423[M-H]?and847[2M-H]?,13C and DEPT NMR experiments showed3×CH3,4×CH2,7×CH, 11×C.The IR spectrum(KBr)of2indicated the presence of hydroxyl(3434cm?1),conjugated carbonyl(1670cm?1), and aromatic(1600and1457cm?1)groups.The UV spectrum (MeOH,λmax)exhibited absorption bands at340,293,237nm. The dihydro?avanone of compound2was deduced from the NMR spectra which showed an oxymethine,a carbonyl group and a methylene atδC71.38,197.49and39.03,respectively,and an ABX system[δH2.47(dd,J=17.0,3.0Hz,H-3e),3.92(dd, J=17.0,14.0Hz,H-3a)and5.86(dd,J=14.0,3.0Hz,H-2),typ-ically assignable to two H-3and one H-2protons of a?avanone]. The1H NMR signals atδH6.39(d,2H,J=8.0Hz,H-3 ,5 )and 6.98(t,1H,J=8.0Hz,H-4 )were attributed for three adjacent protons on an aromatic ring.A lavandulyl group was deduced by the1H NMR signals at[δH1.47,1.51,1.58(s,3H×3),1.96(m, 2H),2.46(m,2H),2.45(m,1H),4.56(s,1H),4.53(s,1H),4.93 (brs,1H)]and13C NMR signals at[δC26.48(C-1 ),46.13(C-2 ),147.85(C-3 ),110.23(C-4 ),18.40(C-5 ),30.68(C-1 ), 123.32(C-2 ),130.24(C-3 ),17.31(C-4 )and25.20(C-5 )]. The point of attachment of the lavandulyl group was established unambiguously as C-6by HMBC experiment.The coupling con-stant(J2,3=14.0Hz)between protons in the2and3positions, which was indicative of axial–axial coupling,revealed the C-2 hydrogen is axial and ring B equatorial[19].The chiral centre of C-2was assigned as S-con?guration on the basis of its negative optical rotation,[?]D-150?(C.1.00MeOH)and equatorial B-ring[20].All1H and13C NMR assignments(shown in Table1) for compound2were performed by1H–1H COSY,HMQC and HMBC experiments(main HMBC and NOESY correlations are shown in Fig.5).Thus the compound2was determined to be (2S)-5,7,2 ,6 -tetrahydroxy-6-lavandulylated?avanone(shown in Fig.1).

108J.Peng et al./J.Chromatogr.A1115(2006)103–111

Table1

1H NMR(500MHz)and13C NMR(125MHz)data of compound2in[2H6]dimethyl sulfoxide(DMSO-d6)

PositionδCδH a,b PositionδCδH a,b 271.38 5.86dd(14.0,3.0)3 ,5 106.71 6.39d(8.0) 339.03 2.47dd(17.0,3.0)3.92dd(17.0,14.0)4 129.43 6.98t(8.0) 4197.49–1 26.48 2.46m 5161.2712.30brs2 46.13 2.45m 6106.19–3 147.85–

7164.684 110.23 4.56s4.53s 894.81 5.97s5 18.40 1.51s 9161.09–1 30.68 1.96m 10101.35–2 123.32 4.93brs

1 110.43–3 130.24–

2 ,6 157.12–4 17.31 1.47s

5 25.20 1.58s

a J in Hz.

b Signals were assigned by HMQC,HMBC and1H–1H COSY experiments.

Compound3was isolated as white powder,UVλMeOH

max :338,

290,232nm,IR(KBr)υmax cm?1:3410(OH),1686(C O), 1623,1469.ESI-MS:423[M-H]?,847[2M-H]?,HR-ESI-MS m/z424.1246for C25H28O6(calcd.424.1234).This formula can be validated through13C NMR,1H NMR and DEPT spectra.The 1H NMR signals at[δH5.84(dd,1H,J=14.0,3.0Hz,H-2),δ

2.42(dd,1H,J=17.0,

3.0Hz,H-3e)andδ3.94(dd,1H,J=1

4.0, 17.0Hz,H-3a)],and13C NMR signals at[δC71.50(C-2),39.65 (C-3)and197.74(C-4)]were characteristic of a dihydro?a-vanone for an ABX system.A lavandulyl group was deduced by the1H NMR signals at[δH1.52,1.60,1.65(s,3H×3),2.02(m, 2H),2.50(m,2H),2.47(m,1H),4.59(s,1H),4.53(s,1H),4.99 (brs,1H)]and13C NMR signals at[δC26.23(C-1 ),46.09(C-2 ),147.73(C-3 ),110.70(C-4 ),18.47(C-5 ),30.89(C-1 ), 123.58(C-2 ),130.45(C-3 ),17.57(C-4 )and2

5.44(C-5 )]. The1H NMR signals atδH5.89(2H,s,H-3 ,5 )and13C NMR signals at[δc157.96(C-2 ,6 ),10

6.4(C-3 ,5 )and158.79(C-4 )] indicated two hydroxyl group and two protons on B-ring,and the point of attachment of the lavandulyl group was established unambiguously as C-4 by the1H-13C long-range coupling and HMBC experiments.The1H NMR signals at?H5.68(1H,s, H-6)and5.87(1H,s,H-8)and13C NMR signals atδc94.03 (C-6)and94.18(C-8)indicated that there have substitutes on C-6and C-8.The absolute stereochemistry at C-2of compound 3was established as S likely to the compound2.All1H and13C NMR assignments(shown in Table2)for compound3were per-formed by1H–1H COSY,HMQC and HMBC experiments(main HMBC and NOESY correlations are shown in Fig.5).Thus the compound3was determined to be(2S)-5,7,2 ,6 -tetrahydroxy-4 -lavandulylated?avanone(shown in Fig.1).

Compound4was isolated as light yellow powder,mp: 167~168?C.The ESI-MS of4showed a molecular[M-H]

?Fig.5.The main HMBC and NOESY corrections of the four new compounds.

J.Peng et al./J.Chromatogr.A1115(2006)103–111109 Table2

1H NMR(500MHz)and13C NMR(125MHz)data of compound3in DMSO-d6

PositionδCδH a,b PositionδCδH a,b

271.50 5.84dd(14.0,3.0)3 ,5 106.40 5.89s 339.65 2.42dd(17.0,3.0)3.94dd(17.0,14.0)4 158.79–

4197.74–1 26.23 2.50m 5161.7712.56brs2 46.09 2.47m 694.03 5.68s3 147.73–

7164.894 110.70 4.59s4.53s 894.58 5.87s5 18.47 1.60s 9161.33–1 30.89 2.02m 10101.08–2 123.58 4.99brs

1 110.70–3 130.45–

2 ,6 157.96–4 17.57 1.52s

5 25.44 1.65s

a J in Hz.

b Signals were assigned by HMQC,HMBC and1H–1H COSY experiments.

ion at m/z353and the molecular formula of C20H18O6was

derived from the HR-ESI-MS(m/z354.1126calcd.354.1131).

This formula can also be validated through13C NMR,1H

NMR and DEPT spectra.The IR spectrum(KBr)of4indicated

the presence of hydroxyl(3412cm?1),conjugated carbonyl

(1668cm?1),and aromatic(1587and1465cm?1)groups.The

UV spectrum(MeOH,λmax)of4exhibited maxima at275and

310nm.The1H NMR spectrum of4(Table3)showed reso-

nances for an ABX system atδH2.48(dd,1H,J=3.0,17.0Hz),δH3.90(dd,1H,J=13.0,17.0Hz)andδH5.78(dd,1H,J=3.0, 13.0Hz),which is diagnostic for H-2and H-3of a?avanone

nucleus.The1H NMR signals atδH6.37(d,2H,J=8.0Hz,

H-3 ,5 )and6.99(t,1H,J=8.0Hz,H-4 )were attributed for

three adjacent protons on an aromatic ring,andδH12.28was

characteristic of5-OH.The dimethylchromene ring was elu-

cidated by the1H NMR signals at[δH1.36,1.39(s,each3H,

Me×2),5.94,6.53(d,each1H,J=10.0Hz,cis-ole?nic proton)]

and13C NMR signals[δc27.69,27.98(Me×2),77.88(C-2 ),

two cis-ole?nic carbons at126.37(C-3 )and115.02(C-4 )].

These data suggested that compound4has a?avanone skeleton

with one hydroxyl group and a dimethylpyran moiety,and these

inferences were con?rmed using the DEPT,1H–1H COSY and

HMQC NMR techniques.The presences of the substituents at

positions C-5(hydroxyl group),C-6and C-7(dimethylpyran)were deduced using the HMBC experiment(Fig.5).The cou-pling constant(J2,3=13.0Hz)between protons in the2and3 positions,which was indicative of axial–axial coupling,revealed the C-2hydrogen is axial and ring B equatorial.The chiral cen-tre of C-2was assigned as S-con?guration on the basis of its negative optical rotation,[?]D-100?(C.1.00MeOH)and equa-torial B-ring.All1H and13C NMR assignments for compound 4are shown in Table3.The chemical structure,main HMBC and NOESY correlations are shown in Figs.1and5.Thus the compound4was determined to be(2S)-5,2 ,6 -trihydroxy-2 ,2 -dimethylpyrano[5 ,6 :6,7]?avanone.

Compound5was isolated as white powder.Its molecu-lar formula was determined as C25H28O6by HR-ESI-MS m/z 424.1262(calcd.424.1234),ESI-MS m/z423[M-H]?and847 [2M-H]?,13C and DEPT NMR experiments showed4×CH3, 3×CH2,7×CH,11×C.The IR spectrum(KBr)of5indicated the presence of hydroxyl(3400cm?1),conjugated carbonyl (1665cm?1),and aromatic(1568and1443cm?1)groups.The UV spectrum(MeOH,λmax)exhibited absorption bands at330 and291nm.The dihydro?avanone of compound5was deduced from the NMR spectra which showed an oxymethine,a car-bonyl group and a methylene atδC71.94,197.84and39.04, respectively,and an ABX system[δH2.46(dd,J=17.0,3.0Hz, H-3e),3.89(dd,J=17.0,13.0Hz,H-3a)and5.80(dd,J=13.0,

Table3

1H NMR(500MHz)and13C NMR(125MHz)data of compound4in DMSO-d6

PositionδCδH a,b PositionδCδH a,b 272.00 5.78dd(3.0,13.0)1 109.96–

339.00 2.48dd(3.0,17.0)3.90dd(13.0,17.0)2 ,6 157.34–

4197.62–3 ,5 106.84 6.37d(8.0) 5159.5212.28brs4 129.89 6.99t(8.0) 6106.78–2 77.88–

7162.253 126.37 5.94d(10.0) 896.01 5.90s4 115.02 6.35d(10.0) 9157.86–2 -Me27.69 1.36s 10100.98–2 -Me27.98 1.39s

a J in Hz.

b Signals were assigned by HMQC,HMBC and1H–1H COSY experiments.

110J.Peng et al./J.Chromatogr.A1115(2006)103–111

Table4

1H NMR(500MHz)and13C NMR(125MHz)data of compound5in DMSO-d6

PositionδCδH a,b PositionδCδH a,b

271.94 5.80dd(14.0,3.0)3 ,5 106.90 6.39d(8.0)

339.04 2.46dd(17.0,3.0)4 129.92 6.98t(8.0)

3.89dd(17.0,1

4.0)

4197.84–2 79.15–

5160.9312.18brs3 40.16 2.18m

6101.92–4 21.53 2.20dd(16.0,12.0)

2.63dd(12.0,

3.0) 7161.432 -Me20.89 1.16s

895.65 5.89s2 -Me25.48 1.36s

9160.84–1 28.68 2.02ddd(16.5,12.2,7.5)

2.11ddd(12.2,7.8,

3.0) 10100.34–2 122.51 5.12brs

1 110.20–3 132.42–

2 ,6 157.35–4 17.56 1.51s

5 27.20 1.64s

a J in Hz.

b Signals were assigned by HMQC,HMBC and1H–1H COSY experiments.

3.0Hz,H-2),typically assignable to H-3and H-2protons of a?a-

vanone].The1H NMR signals atδH1.51,1.64(1H each,s,Me),δH5.12(1H,m,ole?nic proton,H-2 ),δH2.02,2.11(1H each, dd,J=8.0,8.0Hz,H-1 )and13C NMR signals at[δC17.56,

27.20(Me×2),28.68(C-1 ),122.51(C-2 ),132.42(C-3 )

indicated a dimethylallyl group was existed in the compound5

andδH12.18was characteristic of5-OH.The dihydro-dimethyl-

chromene ring was elucidated by the1H NMR signals[δH1.16,

1.36(3H each,s,Me×2),

2.18(1H,m,H-3 ),2.20,2.63(1H

each,dd,J=10.0,10.0Hz,H-4 )]and13C NMR signals[δc

20.89,25.48(Me×2),79.15(C-2 ),40.16(C-3 ),21.53(C-4 ).

Comparison with the dimethylchromene ring,no cis-ole?nic

protons and carbons were observed.These data suggested that

compound5has a?avanone skeleton with one hydroxyl group

and a dihydro-dimethylpyran moiety,and these inferences were

con?rmed using the DEPT,1H-1H COSY and HMQC NMR

techniques.The position of the substituents were deduced as

accruing at C-5and C-6,C-7(dihydro-dimethylpyran)using

the HMBC experiment.The absolute stereochemistry at C-2

of compound5was established as S based on NOESY tech-

nique.All1H and13C NMR assignments for compound5were

shown in Table4.The main HMBC and NOESY correlations

are shown in Fig.5.Thus the compound5was determined

to be(2S,3 S)-5,2 ,6 -trihydroxy-3 -?,?-dimethylallyl-2 ,2 -

dimethyl-3 ,4 -dihydropyrano[5 ,6 :6,7]?avanone(shown

in Fig.1).

3.4.Anticancer activity assay of the isolated compounds

In anticancer assay,seven kinds of typical human cancer

cell lines were selected to evaluate the compounds’inhibi-

tion activities to cancer cell growth including human lung

cancer cell A549,human liver cancer cell BEL-7402,human

esophageal cancer cell HT-29,human breast cancer sell MCF-7,

human gastric cancer cell SGC-7901,human leukemia can-

cer cell K562and human kidney cancer cell A498.The data Table5

The IC50values of the isolated compounds to inhibit human cancer cells growth by MTT method

Human cancer cells Compounds IC50(?g/ml)a

123456

A549 3.93 4.87 4.0215.219.6810.25 BEL-7402 4.60 3.64 2.8513.207.529.98 HT-29 4.08 5.28 4.2620.2214.3625.36 MCF-7 5.727.947.6235.4818.5328.54 SGC-7901 6.5910.209.2549.5230.2138.45 K562 1.64 2.42 3.0412.25 6.5812.23 A498 4.528.857.9246.2425.4635.21 a IC50was expressed as the drug concentration resulting in a50%inhibition of cell growth and calculated from dose–inhibition curves.

are reported Table5,and the results(IC50<7?g/ml)indicated that the compounds1,2and3exhibited high inhibition effect (IC50<10?g/ml)to tumor cells’growth in a dose-dependent manner,and when the concentration of the compounds was over15?g/ml,the cancer cells were totally inhibited,espe-cially to K562cancer cell(IC50<3.1?g/ml).The compounds 4,5and6could also inhibit the cancer cells’growth,but the inhibition effect was weaker than the compounds1,2and3 (IC50<30?g/ml).

4.Conclusion

Six?avanoids including four novel ones were successfully isolated and separated by high-speed counter-current chro-matography from the leaf of P.villosa.Our research demon-strated that CCC is a powerful technique to separate and isolate chemical constituents from medicinal plants,and the pharma-cology test showed that the compounds1,2and3exhibited high anticancer activities.In the light of these results,in vivo actions and clinical applications of the compounds are required further investigation.

J.Peng et al./J.Chromatogr.A1115(2006)103–111111

Acknowledgement

Financial support from Ministry of Science and Technology of China(863project)is gratefully acknowledged. References

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体外肿瘤药敏试验方法研究进展

体外肿瘤药敏试验方法研究进展 甘萍 治疗肿瘤的方法主要有3种,包括肿瘤化疗、放疗、手术。其中肿瘤化疗在消灭微小肿瘤转移灶和手术切除后的残留肿瘤细胞治疗中有着手术与放疗无法达到的显著优势,但肿瘤化疗中仍有许多问题:①肿瘤病人个体间对化疗药物敏感性差异大;②肿瘤对化疗药物有耐药性;③化疗药物对许多类型的肿瘤特别是实体瘤的有效率不高;④化疗药物的选择性差,毒性大,杀伤癌细胞的同时也杀伤正常细胞,给病人机体造成较大的损害。对于上述出现各种问题在临床上经验的化疗用药是无法保证肿瘤患者的个体有效性,因此个体化化疗在临床肿瘤化疗中就显得尤为重要。目前公认的较好的方法就是做肿瘤化疗药物敏感性试验(简称肿瘤药敏),该方法的特点是直接从患者体内获取新鲜的肿瘤组织进行首次培养,由于肿瘤细胞刚刚离体,生物学性状尚未发生大的变化,能较真实地反映整个肿瘤细胞群体的特性及不同供体的个体差异,能够比较确切地代表体内状态,为不同的病人准确筛选敏感的化疗药物,并确定其剂量,真正实现临床的个体化用药,以提高化疗的靶向性,减少化疗药物的不良反应,降低细胞耐药性,成为肿瘤治疗中迫切需要解决的问题。 目前,预测肿瘤药敏的方法已发展为体内和体外两大系列20多种药敏试验,并不断地朝着简单、快速、敏感、筛选作用方式不同的药物与临床有良好的相关性的方向迈进。而本文针对体外肿瘤药敏试验方法进行综述。 1 人体肿瘤细胞集落测定(HTCA) 它是利用肿瘤细胞悬液,置于双层琼脂中培养,通过选择性加入化疗药物培养后,计数细胞繁殖形成的集落数目,再评估肿瘤细胞对该药的敏感性。该法的优点:敏感、直接评价细胞增殖死亡。缺点:用于临床标本检测率低、周期长、集落计数繁琐。 2 放射性标记代谢物前体掺入法(3H-Tdr assay) 该法利用氚标记的胸腺嘧啶核苷和尿嘧啶作为核酸代谢物前体,测定一定时间内放射性标记的代谢物前体掺入的多少来判断药物对细胞增殖活性的抑制作用。该法的优点:操作简便、所需时间短、灵敏度高、重复性好,临床相关性与集落形成法相近,可适用于绝大多数恶性肿瘤。缺点:存在放射性污染,只能用于标记指数>5%的样品,显然无法测出药物对G0期细胞的杀灭作用,而且胸腺嘧啶池的大小也可影响结果,故标记指数的变化不一定系抗癌药所致。此外,同位素的放射性也限制了它的应用。 3 快速荧光分析法(FMCA) 它是采用一些特殊的荧光染料(荧光素二乙酸酯)对细胞的特定成分进行染

抗肿瘤药物的作用机制

抗肿瘤药物的作用机制 1.细胞生物学机制 几乎所有的肿瘤细胞都具有一个共同的特点,即与细胞增殖有关的基因被开启或激活,而与细胞分化有关的基因被关闭或抑制,从而使肿瘤细胞表现为不受机体约束的无限增殖状态。从细胞生物学角度,诱导肿瘤细胞分化,抑制肿瘤细胞增殖或者导致肿瘤细胞死亡的药物均可发挥抗肿瘤作用。 2.生化作用机制 (1)影响核酸生物合成:①阻止叶酸辅酶形成;②阻止嘌呤类核苷酸形成;③阻止嘧啶类核苷酸形成;④阻止核苷酸聚合;(2)破坏DNA结构和功能;(3)抑制转录过程阻止RNA 合成;(4)影响蛋白质合成与功能:影响纺锤丝形成;干扰核蛋白体功能;干扰氨基酸供应;(5)影响体内激素平衡。 烷化剂烷化剂可以进一步分为: 氮芥类:均有活跃的双氯乙基集团,比较重要的有氮芥、苯丁酸氮芥、环磷酰胺(CTX)、异环磷酰胺(IFO)等。其中环磷酰胺为潜伏化药物需要活化才能起作用。目前临床广泛用于治疗淋巴瘤、白血病、多发性骨髓瘤,对乳腺癌、肺癌等也有一定的疗效。 该药除具有骨髓抑制、脱发、消化道反应,还可以引起充血性膀胱炎,病人出现血尿,临床在使用此药时应鼓励病人多饮水,达到水化利尿,减少充血性膀胱炎的发生。还可以配合应用尿路保护剂美斯纳。 亚硝脲类:最早的结构是N-甲基亚硝脲(MNU)。以后,合成了加入氯乙集团的系列化合物,其中临床有效的有ACNU、BCNU、CCNU、甲基CCNU等,链氮霉素均曾进入临床,但目前已不用。其中ACNU、BCNU、CCNU、能通过血脑屏障,临床用于脑瘤及颅内转移瘤的治疗。主要不良反应是消化道反应及迟发性的骨髓抑制,应注意对血象`的观测,及时发现给予处理。 乙烯亚胺类:在研究氮芥作用的过程中,发现氮芥是以乙烯亚胺形式发挥烷化作用的,因此,合成了2,4,6-三乙烯亚胺三嗪化合物(TEM),并证明在临床具有抗肿瘤效应,但目前在临床应用的只有塞替派。此药用于治疗卵巢癌、乳腺癌、膀胱癌,不良反应主要为骨髓抑制,注意对血象定期监测。 甲烷磺酸酯类:为根据交叉键联系之复合成的系列化合物,目前临床常用的只有白消安(马利兰)。临床上主要用于慢性粒细胞白血病,主要不良反应是消化道反应及骨髓抑制,个别病人可引起纤维化为严重的不良反应。遇到这种情况应立即停药,更换其它药物。 其他:具有烷化作用的有达卡巴嗪(DTIC)、甲基苄肼(PCZ)六甲嘧胺(HHN)等。环氧化合物,由于严重不良反应目前已被淘汰。 抗代谢药物抗代谢类药物作用于核酸合成过程中不同的环节,按其作用可分为以下几类药物: 胸苷酸合成酶抑制剂:氟尿嘧啶(5-FU)、呋喃氟尿嘧啶(FT-207)、二喃氟啶(双呋啶FD-1)、优氟泰(UFT)、氟铁龙(5-DFUR)。 抗肿瘤作用主要由于其代谢活化物氟尿嘧啶脱氧核苷酸干扰了脱氧尿嘧啶苷酸向脱氧胸腺嘧啶核苷酸转变,因而影响了DNA的合成,经过四十年的临床应用,成为临床上常用的抗肿瘤药物,成为治疗肺癌、乳腺癌、消化道癌症的基本药物。 不良反应比较迟缓,用药6-7天出现消化道粘膜损伤,例如:口腔溃疡、食欲不振、恶心、呕吐、腹泻等,一周以后引起骨髓抑制。而连续96小时以上粘腺炎则成为其主要毒性反应。临床上如长时间连续点滴此类药物应做好病人的口腔护理,教会病人自己学会口腔清洁的方法,预防严重的粘膜炎发生。

抗肿瘤药物药效学实验方法及指导原则

抗肿瘤药物药效学实验方法及指导原则 一、基本原则 1. 抗肿瘤药物分类 (1) 细胞毒类药物(cytotoxic agent):包括干扰核酸和蛋白质合成、抑制拓扑异构酶及作用于微管系统的药物等; (2) 生物反应调节剂(biological response modifier); (3) 肿瘤耐药逆转剂(resistance reversal agent); (4) 肿瘤治疗增敏剂(oncotherapy sensitizer); (5) 肿瘤血管生成抑制剂(tumor angiogenesis inhibitor); (6)分化诱导剂(differentiation inducing agent); (7) 生长因子抑制剂(growth factor inhibitor); (8)反义寡核苷酸(antisense oligonucleotide) 。 2. 抗肿瘤药物药效学需研究内容 2.1 包括体外抗肿瘤试验,体内抗肿瘤试验。 2.2 评价药物的抗癌活性时,以体内试验结果为主,同时参考体外试验结果以做出正确的结论。 2.3 I类抗肿瘤新药应进行药物作用机制初步研究。 二、体外抗肿瘤活性试验 1. 试验目的 1.1 对候选化合物进行初步筛选; 1.2 了解候选化合物的抗瘤谱; 1.3 为随后进行的体内抗肿瘤试验提供参考,如剂量范围、肿瘤类别等。 2. 试验方法 选用10-15株人癌细胞株,根据试验目的选择相应细胞系及适量的细胞接种浓度,按常规细胞培养法进行培养;推荐使用四氮唑盐MTT还原法、XTT 还原法、磺酰罗丹明B(SR染色法、或51Cr释放试验、集落形成法等测定药物的抗癌作用。

抗肿瘤药物分类

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肿瘤细胞生物学特性

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抗肿瘤药物一医学的必看试题带详细解析答案

抗肿瘤药物一医学的必看试题带详细解析答案

278.抗肿瘤药物(一) 一、A型题:题干在前,选项在后。有A、B、 C、D、E五个备选答案其中只有一个为最佳答案。 1.属于植物来源的抗肿瘤药为 A.阿霉素 B.紫杉醇 C.米托蒽醌 D.来曲唑 E.昂丹司琼 正确答案:B 2.以下哪个不属于烷化剂类的抗肿瘤药 A.美法仑 B.白消安 C.塞替派 D.异环磷酰胺 E.巯嘌呤 正确答案:E 3.下列叙述不符合环磷酰胺的是 A.在体外无活性,进人体内经代谢而发挥作用,因此本身是前体药物 B.含一分子结晶水为固体,无水物为油状液体

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阿糖胞苷的抗肿瘤活性研究

摘要 随着近几年技术的发展,肿瘤化疗方面取得了很大的进步,肿瘤患者的寿命明显地延长了,尤其是那些患有白血病、恶性淋巴瘤等疾病的治疗和预防方面甚至有了很大的突破。本文从阿糖胞苷的来源及其抗肿瘤作用机制谈起,简单地介绍了阿糖胞苷的临床应用及其出现的不良反应和药动学特点,其次分析代谢为活性复合物-阿糖胞三磷酸核苷发挥作用,并分别针对细胞毒作用,大剂量给药-阿糖胞苷,诱导肿瘤细胞凋亡以及端粒酶作用等来阐述阿糖胞苷的抗肿瘤活性,并针对这些问题进行讨论,最后是全文的总结部分。 关键词肿瘤,药物,阿糖胞苷,研究

目录 摘要 (1) 引言 (3) 一、阿糖胞苷的简介 (3) (一)阿糖胞苷的来源 (3) (二)阿糖胞苷的抗肿瘤作用机制 (3) (三)阿糖胞苷的临床应用及其出现的不良反应[1] (4) (四)阿糖胞苷的药动学特点 (4) 二、阿糖胞苷的抗肿瘤活性研究 (4) (一)代谢为活性复合物-阿糖胞三磷酸核苷发挥作用 (5) 1、蛋白酪氨酸激酶(PTK)抑制剂 (5) 2、法尼基转移酶(FTase)抑制剂 (5) (二)细胞毒作用 (6) (三)大剂量给药-阿糖胞苷 (6) (四)诱导肿瘤细胞凋亡 (7) (五)端粒酶作用 (7) (六)其他 (8) 三、讨论 (8) 四、总结 (9) 参考文献 (10)

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