Studies Of Chemical Constituents And Bioactivities Of Cynanchum Auriculatum Royle Ex Wight

Cynanchum auriculatum Royle ex Wight,widely distributes in east of China and also found in India.Root tuber of it has been used for more than 1,000 years in traditional Chinese medicine,with a famous name Baishouwu.It is sweet and a little bitter in taste and slightly warm in nature.This herbal medicine is very effective in tonification and functions in replenishing the liver and kidney,enriching vital essence and blood,astringing primordial energy and clearing away toxins(Kaibao Materia Medica,Song Dynasty,China).It has long been used for many symptoms induced by Yin deficiency of liver and kidney such as dizziness,insomnia and forgetfulness,premature grey hair,aching and weakness of the loins and knees,weakness of muscles and bones,oppression in the chest and heart pain,dyspepsia and so on.Previous studies reported the isolation of steroids,acetophenones and triterpenes from the titled plant with significant bioactivities.The acetophenones isolated from the plant can protect live cells from the chronic damage of carbon tetrachloride(CCl₄).The steroidal glycosides from the plant showed cytotoxic activity.This dissertation describes the chemical and pharmacological studies on this plant,especially the bio-assay guided isolation of steroidal glycosides and acetophenones,as well as their anti-tumor,neuroprotective and gastroprotective activities.The chemical investigation of 95%ethanol extract of this plant resulted in the isolation of 90 compounds.Structures of them were established,on the basis of spectral data including UV, IR,ESI-MS,1D-NMR,2D-NMR,physical and chemical properties,as follows:*SL-EYNPX-1:Auriculosideâ… (penupogenin 3-O-β-D-digitoxopyranosyl-(1→4)-β-D-cy maropyranoside)*SL-EYNPX-2:Auriculosideâ…¡(kidjolanin 3-O-β-D-digitoxopyranosyl-(1→4)-β-D-digit oxopyranoside)*SL-EYNPX-3:Auriculosideâ…¢(gagaminine 3-O-β-D-digitoxopyranosyl-(1→4)-β-D-cy maropyranoside)*SL-EYNPX-4:Auriculosideâ…£(kidjolanin 3-O-β-D-cymaropyranosyl-(1→4)-β-D-cyma ropyranoside)*SL-EYNPX-5:Auriculosideâ…¤(kidjolanin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cy maropyranoside)*SL-EYNPX-6:Auriculosideâ…¥(kidjolanin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-ol eandropyranoside)*SL-EYNPX-7:Auriculosideâ…¦(kidjolanin 3-O-α-L-cymaropyranosyl-(1→4)-β-D-dig itoxopyranosyl-(1→4)-β-D-cymaropyranoside)*SL-EYNPX-8:Auriculosideâ…§(kidjolanin 3-O-β-D-cymaropyranosyl-(1→4)-α-L-di ginopyranosyl-(1→4)-β-D-cymaropyranoside)*SL-EYNPX-9:Auriculosideâ…¨(kidjolanin 3-O-β-D-glucopyranosyl-(1→4)-β-D-cymar opyranosyl-(1→4)-β-D-cymaropyranoside)*SL-EYNPX-10:Aurieulosideâ…©(kidjolanin 3-O-β-D-glucopyranosyl-(1→4)-6-deoxy 3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside)*SL-EYNPX-11:Auriculosideâ…ª(caudatin 3-O-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside)*SL-EYNPX-12:Auriculosideâ…«(kidjolanin 3-O-6-deoxy-3-O-methyl-β-D-allopyrano syl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside)*SL-EYNPX-13:Auriculosideâ…©â…¢(metaplexigenin 3-O-6-deoxy-3-O-methyl-β-D-allo pyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside)*SL-EYNPX-14:Auriculosideâ…©â…£(caudatin 3-O-6-deoxy-3-O-methyl-β-D-allopyrano syl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside)*SL-EYNPX-15:Auriculosideâ…©â…¤(dihydrosarcostin 3-O-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyra noside)*SL-EYNPX-16:Auriculosideâ…©â…¥(20-(S)-O-acetyl-dihydrosarcostin 3-O-β-D-glucopy ranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4) -β-D-cymaropyranoside)*SL-EYNPX-17:Auriculosideâ…©â…¦(dihydrosarcostin 3-O-6-deoxy-3-O-methyl-β-D-al lopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside)*SL-EYNPX-18:Auriculosideâ…©â…§(caudatin 3-O-α-L-diginopyranosyl-(1→4)-β-D-ol eandropyranoside) SL-EYNPX-19:Caudatin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-20:Caudatin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-21:Caudatin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-22:Caudatin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-23:Caudatin 3-O-β-D-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-24:Caudatin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-25:Caudatin 3-O-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-26:Caudatin 3-O-α-L-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1 4)-β-D-cymaropyranosideSL-EYNPX-27:Caudatin 3-O-β-D-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-28:Caudatin 3-O-β-D-glucose-α-L-cymaropyranosyl-(1→4)-β-D-oleandrop yranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-29:Caudatin 3-O-α-L-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-30:Caudatin 3-O-β-D-glucose-β-D-cymaropyranosyl-β-D-oleandropyranosy 1-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-31:Caudatin 3-O-β-D-glucose-(1→4)-α-L-oleandropyranosyl-(1→4)-β-D-c ymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cy maropyranosideSL-EYNPX-32:Caudatin 3-O-α-L-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside SL-EYNPX-33:Caudatin 3-O-β-D-glucose-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-c ymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-34:Caudatin 3-O-β-D-glucose-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-ol eandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-35:Caudatin 3-O-β-D-glucose-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-c ymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cy maropyranosideSL-EYNPX-36:Caudatin 3-O-β-D-glucose-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-o leandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-c ymaropyranosideSL-EYNPX-37:Caudatin 3-O-α-L-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-38:Wilfoside C1N(Caudatin 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cymar opyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside)SL-EYNPX-39:Wilfoside C3N(Caudatin 3-O-β-D-cymaropyranosyl-(1→4)-α-L-digino pyranosyl-(1→4)-β-D-cymaropyranoside)SL-EYNPX-40:Wilfoside C1G(Caudatin 3-O-β-D-glucopyranosyl-(1→4)-α-L-cymar opyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropy ranoside)SL-EYNPX-41:Wilfoside C2G(Caudatin 3-O-β-D-glucopyranosyl-(1→4)-α-L-cymaro pyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-digitoxopyr anoside)SL-EYNPX-42:Cynanchogenin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyrano syl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-43:Cynanchogenin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranos yl-(1→4)-β-D-cymaropyranosideSL-EYNPX-44:Cynanchogenin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyrano syl-(1→4)-β-D-cymaropyranosideSL-EYNPX-45:Cynanchogenin 3-O-β-D-cymaropyranosyl-(1→4)-β-D-oleandropyranos yl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-46:Cynanchogenin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyrano syl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosi deSL-EYNPX-47:Cynanchogenin 3-O-β-D-cymaropyranosyl-(1→4)-β-D-oleandropyranos yl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosi deSL-EYNPX-48:Cynanchogenin 3-O-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosi deSL-EYNPX-49:Cynanchogenin 3-O-α-L-cymaropyranosyl-(1→4)-β-D-oleandropyranos yl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-50:Gagaminine 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-( 1→4)-β-D-cymaropyranosideSL-EYNPX-51:Gagaminine 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-52:Gagaminine 3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl -(1→4)-β-D-cymaropyranosideSL-EYNPX-53:Gagaminine 3-O-α-L-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-( 1→4)-β-D-cymaropyranosideSL-EYNPX-54:Sarcostin 3-O-β-D-cymaropyranosideSL-EYNPX-55:Sarcostin 3-O-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-56:Sarcostin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-57:Kidjolanin 3-O-α-L-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-58:Wilfoside K1N(kidjolanin 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cym aropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside)SL-EYNPX-59:Taiwanoside D(kdjoranin 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cyma ropyranosyl-(1→4)-α-L-diginopyranoside) SL-EYNPX-60:Taiwanoside A(metaplexgenin 3-O-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside)SL-EYNPX-61:Taiwanoside B(metaplexgenin 3-O-α-L-cymaropyranosyl-(1→4)-β-D-c ymaropyranosyl-(1→4)-α-L-diginopyranoside)SL-EYNPX-62:Deacylmetaplexigenin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandro pyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosideSL-EYNPX-63:Wilfoside M1N(deacylmetaplexigenin 3-O-α-L-cymaropyranosyl-(1 4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside)SL-EYNPX-64:Taiwanoside E(deacylmetaplexigenin 3-O-α-L-cymaropyranosyl-(1→4) -β-D-cymaropyranosyl-(1→4)-α-L-diginopyranoside)SL-EYNPX-65:CaudatinSL-EYNPX-66:KidjolaninSL-EYNPX-67:GagaminineSL-EYNPX-68:Deacylmetaplexigenin*SL-EYNPX-69:Cynandione ESL-EYNPX-70:Cynandione ASL-EYNPX-71:Cynandione BSL-EYNPX-72:Bungeiside DSL-EYNPX-73:AcetoveratroneSL-EYNPX-74:Cynanchone ASL-EYNPX-75:CynantetroneSL-EYNPX-76:Bungeiside A(2-O-glucosyl-5-hydroxyacetophenone)SL-EYNPX-77:Bungeiside B(2-O-glucosyl-4-hydroxyacetophenone)SL-EYNPX-78:Cynanoneside ASL-EYNPX-79:Bungeiside CSL-EYNPX-80:Resacetophenone(2,4-dihydroxyacetophenone) SL-EYNPX-81:Quinacetophenone(2,5-dihydroxyacetophenone)SL-EYNPX-82:Acetovanillone(4-hydroxy-3-methoxyacetophenone)SL-EYNPX-83:Acetylpyrocatechol(3,4-dihydroxyacetophenone)SL-EYNPX-84:Piceol(4-hydroxyacetophenone)SL-EYNPX-85:20-hydroxyecdysoneSL-EYNPX-86:Ursolic acidSL-EYNPX-87:β-amyrin acetateSL-EYNPX-88:Oleanolic acid-3-acetateSL-EYNPX-89:Azelaic acidSL-EYNPX-90:Diallyl carbonateAmong all the compounds mentioned above,SL-EYNPX-1～SL-EYNPX-18 and SL-EYNPX-69 were new compounds,and 61 compounds,SL-EYNPX-19～SL-EYNPX-64 and SL-EYNPX-70～SL-EYNPX-84,were isolated from the titled plant for the first time.Anti-tumor studies demonstrated that the ethanol extract of C.auriculatum as well as the CHCl₃ fraction(total steroidal glycoside) have significant anti-tumor activity,which were evaluated both in vitro and in vivo.This work also confirmed that the total steroidal glycoside is the effectve fraction for C.auriculatum's anti-tumor activity.Further screening of single steroids indicated that SL-EYNPX-65(caudatin) and its glycosides showed significant cytotoxicity against several human tumor cell lines in vitro which allowed a potent anti-tumor perspective.The qualitative structure-activity relationship(QSAR) of steroidal glycosides' anti-tumor activity were investigated.The results suggested that the long sugar chain moiety at C-3 blocks the activity of caudatin.The ikemaoic acid moiety at C-12 is also important for cytotoxicity of caudatin.The cinnamic acid moiety at C-12 decreases the toxicity of aglycones and glycosides.The dimeric acetophenone,SL-EYNPX-69(cynandione A) was found to alleviate L-glutamate-induced cell injury with an estimated 50%effective concentration(EC₅₀) of 0.018μg/ml and 0.027μg/ml on PC 12 and neuron cells,respectively.The mechanism study indicated that the protective effect of cynandione A in hippocampal neurons against L-glutamate toxicity is mediated by the Ras/MAPK signaling pathway.Cynandione A induces a transient activation of ERK1 and ERK2.Futhermore,the climbing scores of Aβ₄₂ transgenic Drosophila exposing to the compound cynandione A increased sigificantly compared with the Aβ₄₂ transgenic Drosophila exposing to the phosphate buffered saline(p＜0.05).It indicated that cynandione A had an interesting anti-Alzheimer's disease potential,which could possibly lead to an effective new anti-Alzheimer's disease drug.The ethanol extracts and CHCl₃ fraction of C.aurieulatum showed significant gastroprotective effect on ethanol-, indomethacin-induced gastric lesions and histamine-induced gastric acid secretion in rats.The pH value of total steroidal glycosides treated group increased nearly to the normal level, showing that it had significant gastric acid-secretion inhibition effect after histamine-induced gastric lesion.