| This study was supported by the Natural Science Foundation of Jiangsu Province (BK2009457).Cynanchum auriculatum has been used as a famous traditional Chinese medicine for the treatment of geriatric diseases and prolonging life. The pharmacological experiments indicated that C21 steroidal glycosides, the main active constituents in its root, exhibited broad-spectrum anti-tumor activity. And the inhibition activities of C21 steroidal glycosides in C. auriculatum against tumor cell lines have related with the structure of the sugar chain, that the steroidal aglycones and the compounds comprising 1 or 2 sugar residues have stronger activies than a large number of long chains of sugar compounds.Thus we select C21 steroidal glycosides in C. auriculatum to study the chemical degradation in different ways and the anti-rumor activity of C21 steroidal fraction degradation products so as to find constitutes that have stronger activities or a higher content.1. C21 steroidal glycosides of C. auriculatum degradation(l)The strong acid (hydrochloric acid)water solution was used to degrade the C21 steroidal glycosides of C. auriculatum. The effects of hydrochloric acid concentration, reaction temperature and reaction time'had been investigated using orthogonal design and the contents of kidjoranin3-0-β-digitoxopyranoside,caudatin,kidjoranin3-0-α-L-diginopyranosyl-(1→4)-(β-cym-aropyranoside and caudatin3-0-β-cymaropyranoside as markers were determined by the high performance of liquid chromatography. The optimal hydrolysis condition was established by orthognol experiment and multi-index test breakdown formula evaluation. The optimal conditions of degradation are as follows:refluxing for 6 hours of 0.5%dilute HC1 solution at 100℃.(2).The weak acid (acetic acid)water solution was used to degrade the C21 steroidal glycosides of C. auriculatum. The effects of acetic acid concentration, reaction temperature and reaction time had been investigated using orthogonal design and the contents of kidjoranin 3-0-(3-digitoxopyranoside,caudatin,kidjoranin3-0-α-L,-diginopyranosyl-(1→4)-β-cymaropyranos ide and caudatin 3-O-β-cymaropyranoside as response indexs were determined by the high performance of liquid chromatography. The optimal hydrolysis condition was established by orthognol experiment and multi-index test breakdown formula evaluation. The optimal conditions of degradation are as follows:refluxing for 6 hours of 5%dilute HAc solution at 100℃.2. Anti-tumor effect of the C21 steroidal glycosides degradation products and the acute toxicity experiments (1) The anti-tumor effect in vivo tests showed that the degradation products coming from hydrochloric acid solution and acetic acid solution can inhibit mice liver cancer H22 and Lewis lung cancer by intraperitoneal injection. The results showed that the tumor inhibition rates of acetic acid degradation products of total Baishouwu C21 steroidal glycosides is higher than the inhibition rate of hydrochloric acid degradation products, antitumor effects of both were higher than the effect of the total Baishouwu C21 steroidal glycosides. These prove that the degradation products of total Baishouwu C21 steroidal glycoside of can improve the inhibition of tumor activity.(2) The acute toxicity of the C21 steroidal glycosides and the degradation products coming from acetic acid solution showed that the LD50 were 984.0087 and 721.2675 mg/kg, and the process of degradation by acetic acid reduced the toxicity of the C21 steroidal glycosides in C. auriculatum.3. The isolation and identification of the C21 steroidal glycosides degradation productsThe chromatography, inculding silca gel column, sephadex LH-20 and ODS chromatography, and recrystallization were used to isolate the constituents from the degradation products coming from acetic acid solution. The structures of twelve chemical components were elucidated by the physic-chemical properties and the spectral methods including'H-NMR,13C-NMR ect.. They are caudatin-3-0-α-L-digin-(l→4)-P-D-cymaropyrannoside (B-1), kidjolanin (B-2), kidjoranin 3-0-α-L-digin-(l→4)-P-D-cymaropyrannoside (B-3), caudatin-3-O-β-D-cymaropyrannoside (B-4), caudatin-3-0-α-L,-diginopyranosyl-(1→4)-P-D-oleandropyranoside (B-5), caudatin-3-0-β-D-cymaropyranosyl-(l→4)-a-L-diginopyranosyl-(l→4)-β-D-cymaropyranoside (B-6), kidjolanin-3-O-β-D-cymaropyranosyl-(1→4)-a-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside (B-7), caudatin (B-8), kidjoranin-3-O-β-D-digitox (B-9), taraxasterol (B-10), acetatedaucosterol (B-11),β-Sitosterol (B-12). Compounds B-1 and B-9 were obtained from the degradation product.B-1 is a novel component.In conclusion, the content of the C21 steroidal aglycones and the compounds comprising 1 or 2 sugar residues was significantly increased by the degradation process of hydrochloric acid solution and acetic acid solution. The anti-tumor activity, can inhibit mice liver cancer H22 and Lewis lung cancer, was enhanced. But the acute toxicity was reduced. All the results of the study can offer credible foundation for the development of new anti-tumor drugs. |
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