| Peganum harmala L.(Zygophyllaceae)is a desert perennial herb that is widely distributed throughout Asia.In traditional Chinese medicine,the whole plant has been used to treat apoplexy,rheumatism,lumbago,and other diseases.The seeds of this plant,which possess hypothermic and hallucinogenic properties,were used in folk medicine as a stimulating agent of the central nervous system.The seeds were also frequently prescribed for the treatment of alimentary tract cancers and malaria.Previous phytochemical investigations revealed that the seeds contained abundant alkaloids,mostly β-carboline and quinazoline alkaloids,which play crucial roles in the pharmacological and therapeutic effects of the seeds,such as anti-tumour,anti-inflammatory,antibacterial,cholinesterase inhibitory,and anti-HIV activities.However,few studies have focused on the non-alkaloids constituents of these seeds.The continuing search for anti-proliferative bioactive natural products from higher plants has led to extensive research on the seeds of P.harmala.In this study,the 70%ethanol extract of the seeds of P harmala,was suspended in water and acidified to pH 1.0 with 10%hydrochloric acid.The non-alkaloid fraction(236.4 g)was obtained by extraction with the same volume of CHCl3 for four times.Fourty-seven compounds were isolated and purified from the non-alkaloid fraction of the seeds of P harmala by various chromatographic means such as silica gel column chromatography(CC),ODS MPLC,Sephadex LH-20 CC and prepared reverse phase HPLC.The 47 compounds were identified through chemical and spectroscopic methods.They were:(1)Triterpenoids:3β-acetoxy-27-(4-hydroxy-3-methoxy-E-cinnamoyloxy)lup-en-28-oic acid methyl ester(1*),3β-acetoxy-27-hydroxylup-20(29)-en-28-oic acid(2*),3-oxo-27-transcaffeoyloxylup-20(29)-en-28-oic acid methyl ester(3*),3α-acetoxy-14α-hydroxylup-20(29)-en-11-oxo-28-oic acid methyl ester(4*),3a-acetoxy-11α,14α-dihydroxylup-20(29)-en-16-oxo-28-oic acid methyl ester(5*),3-oxo-27-hydroxylup-20(29)-en-28-oic acid methyl ester(6),3-oxo-27-hydroxylup-20(29)-en-28-oic acid(7),methyl 1up-20(29)-en-3-oxo-28-oate(8),betulonic acid(9),lupeol(10),3a-acetoxy-27-hydroxylup-20(29)-en-28-oic acid methyl ester(11),3β-acetoxy-27-hydroxylup-20(29)-en-28-oic acid methyl ester(12),3a,27-dihydroxylup-20(29)-en-28-oic acid methyl ester(13),betulin(14),betulinic acid(15),3α-acetoxy-27-hydroxyolean-12-en-11-oxo-28-oic acid methyl ester(16*),3α-hydroxy-27-(4-hydroxy-3-methoxy-E-cinnamoyloxy)olean-12-en-28-oic acid methyl ester(17*),3α-acetoxy-27-hydroxyolean-12-en-28-oic acid methyl ester(18),3β-acetoxy-27-hydroxyolean-12-en-28-oic acid methyl ester(19),3α-acetoxy-27-hydroxyolean-12-en-28-oic acid(20*),3α-acetoxyoleanolic acid(21),3α,28-dihydroxy-12-en-olean(22),3β,28-dihydrox y-12-en-olean(23),3-oxo-27-hydroxy olean-12-en-28-oic methyl ester(24),3α-acetoxy-9α,11α,26-trihydroxy-olean-12-en-28-oic acid methyl ester(25),urs-12-ene-28-carboxy-3α-tetradecanoate(26),3α-acetoxy-12,27-dihydroxyolean-12-en-11-oxo-28-oic acid methyl ester(27),29-hydroxy-3-oxotirucalla-7,24diene-21-methyl ester(28),3β-acetyl-20S,24S-epoxy-25-hydroxydammarane(29).(2)Phenolics:4-hydroxybenzylcyanide(30),p-methoxybenzeneacetonitrile(31),syringylethanone(32),7-hydroxy-6-methoxy-3-(3-methyl-2-butenyl)coumarin(33),p-methoxybenzoic acid(34),N-(2-phenylethyl)acetamide(35).(3)Other types:cyclohexenone(36),dehydrovomifoliol(37),(E,4R)-4-hydroxy-4,5,5-trimethyl-3-(3-oxobut-1-enyl)cyclohex-2-enone(38),loliolide(39),cyclo(L)-Pro-(L)-Leu(40),cyclo-(S)-Pro-(R)-Phe(41),cyclo(D)-Pro-(D)-Val(42),cyclo(D)-Pro-(D)-Leu(43),proline(44),3,4-dimethyl-5-hydroxy-5-pentylfuran-2(5H)-one(45),pinoresinol(46),3’-O-demethylatedepiturpin(47).The 47 compounds,include 29 triterpenoids(1-29),6 phenolics(30-35),and 12 other type compounds(36-47),and compounds 1-5,16,17 and 20 are new compounds.The antitumor activity of the isolated triterpenoids were assayed in vitro.Compounds 2,12,and 21 exhibited the most significant anti-proliferative activities against HeLa cells(human cervical cancer cells),HepG2 cells(human hcc cells)and SGC-7901 cells(human gastric cancer cells)with the IC50 of 8.29-17.58 μM.Compounds 6,11,20 and 26 also exhibited anti-proliferative activities(IC50 of 15.09-37.75 μM).The colony formation assay was performed to evaluate the effects of compounds 2,12,and 20 on the proliferation of HeLa cells.The results indicated that they could decrease the cell colony formation ability in a concentration-dependent manner.Increasing in the concentration of compounds 2,12,and 20,the colonies were significantly reduced.By using the Hoechst 33258 staining fluorescence microscope analysis experiment,the results showed that compounds 2,12,and 20 could inhibit the viability of HeLa cells and induce morphological characteristics of apoptosis.To further confirm whether apoptosis was triggered in cells,the expression levels of apoptosis-associated proteins were analysed by Western blot analysis after HeLa cells were treated with compounds 2,12,and 20.Compounds 2,12,and 20 increased the expression of cleaved-PARP-1 and BAX,and reduced the expression of BCL-2,in a dose-dependent manner.These data indicated that the HeLa cells were undergoing apoptosis.This study provides chemical basis for the further utilization of seeds of P.harmala. |
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