Anti-tumor Principles Of Asparagus Filicinus: Structure, Bioactivity And Analysis

Xiao-Bai-Bu,the dried root of Asparagus filicinus Buch.-Ham(Asparagaceae), is used for the treatment of cough,tracheitis and pneumonia in traditional Chinese medicine.It was reported that Xiao-Bai-Bu has significant anti-tumor activity.Our previous research shows that the crude saponins portion(AAPS)of Xiao-Bai-Bu exhibited growth inhibition activity toward several tumor cell lines.In order to elucidate the anti-tumor principles of Xiao-Bai-Bu and provide scientific foundation for the development of AAPS,the chemical constituents of AAPS was investigated first of all.And then,the analytical method was established for the quality analysis of AAPS and crude herbal medicine.The primary pharmacokinetic of AAPS was also investigated.The investigation on the chemical constituents of AAPS led to the isolation and identification of 16 compounds,including 12 steroidal saponins and 4 ecdysones,and their structures were elucidated by IR,MS,NMR spectroscopic study and chemical analysis.They are filiasparosides A～D(ZS-1～ZS-4),aspafiliosides E～G (ZS-5～ZS-7),aspafiliosides A(ZS-8)and B(ZS-9),asparosides B(ZS-10)and A (ZS-12),aspafilioside C(ZS-11),ecdysone(ZY-1),20-hydroxyecdysone(ZY-2), turkesterone(ZY-3),and 20-hydroxyecdysone-20,22-monocetonide(ZY-4). Filiasparosides A～D and aspafiliosides E～G are new compounds.11 compounds were evaluated for in vitro cytotoxicity against human lung carcinoma(A-549)and breast adenocarcinoma(MCF-7)cell lines,and 7 compounds(filiasparosides A～D and aspafiliosides A,B and E)were found to show varying degrees of cytotoxic activity against the two cell lines,except that ZS-8 had no effect on the MCF-7 cell line(EC₅₀ >20μg/ml).The most potent compound,ZS-3,exhibited significant cytotoxicity against A549 and MCF-7 cell lines with EC₅₀values of 2.28 and 3.04ug/ml, respectively.ZS-4 was as potent against the A549 cell line(EC₅₀2.38μg/ml)but less potent against the MCF-7 cell line(EC₅₀10.29μg/ml).So,we can conclude that steroidal saponins are the main bioactive components in AAPS.A high-performance liquid chromatography coupled with evaporative light scattering detector(HPLC-ELSD)method was developed for the quality evaluation of AAPS,through a simultaneous determination of one ecdysone and six steroidal saponins,including aspafilioside A(ZS-8),B(ZS-9),C(ZS-11),E(ZS-5),G(ZS-7), filiasparoside A(ZS-1),and 20-hydroxyecdysone(ZY-2).This method was successfully applied to validate several different preparative methods of AAPS,and the ADS-7 macroporous resin preparative method that previously established in our lab was proved to be the most effective one.13 samples of A.filicinus from different localities in China were also analyzed by this developed method,and it was found that the contents of the 7 compounds differed greatly in the samples.In vivo pharmacological experiment of AAPS showed no anti-tumor function on tumorous mice.In order to find the exact reason for the ineffectivity,we investigate the water-solubility,stability in gastrointestinal tract,absorption of small intestine, and the pharmacokinetic characteristic of AAPS.Solutol HS-15 was selected as an effective surfactant to enhance the water-solubility of AAPS,and the contents of bioactive compounds were significant increased in the condition of presence of Solutol HS-15.Study of the stability of AAPS in artificial gastrointestinal juice found that most components of AAPS were stable except ZS-7.Some components of AAPS could be changed to ZS-7 under acidic condition and resulted into the increase of ZS-7 content.Everted gut model was used to evaluate the absorption characteristic of AAPS.At the 200mg/ml concentration of AAPS,the uptakes of active steroidal saponins in intestine were very low and could not be detected by the developed HPLC-ELSD method.So,it could be concluded that the low bioavailability of active components is the main reason for the ineffectivity of AAPS in vivo by oral administration.The pharmacokinetic behavior of AAPS after tail venous(iv)route of administration was investigated.The content of ZS-11 was higher than ZS-7 in AAPS, but the concentration of ZS-7 was obviously higher than ZS-11 in the plasma sample after iv administration(AAPS 200mg/kg).The half-time ofβ-phase of ZS-7(T_1/2β= 9.86h)is longer than ZS-11(T_1/2β=1.51h).From above observations,it could be concluded that ZS-11 was metabolized and transformed to ZS-7 quickly in vivo,and that resulted into the higher concentration in plasma and longer eliminate phase of ZS-7.At the dose of 100 and 200mg/kg,AAPS had severe haemolytic phenomenon and it was not feasible to be used by iv administration.The study on metabolites of AAPS in vivo indicated that the metabolites were mainly founded in feces.The quantitative analysis of the feces sample gathered after oral administration(2g/kg)were carried out and the result showed that the cumulate amount of two active compounds(ZS-8 and ZS-9)in feces were two and nearly four times of dosages respectively,and the cumulate excreted rates of the other two active compounds(ZS-1 and ZS-5)were 72%and 44%.This result suggested that the abundant non-active components in AAPS such as ZS-11 were transformed to ZS-8 and ZS-9 after in vivo metabolized.Based on the above conclusions,the anti-tumor foundation of A.filicinus were elucidated by phytohemical research.A HPLC-ELSD method was developed for the quantitative analysis of the main and active compounds in AAPS and the crude A. filicinus.The pharmacokinetics research of AAPS proved that the low bioavailability of the active compounds is the main reason for non-activity in vivo by oral administration.