| Objective:Pulsatilla chinensis (Bge.) Regel is the dired root from a ranunculaceae plant, The main chemical constituents is pentacyclic triterpenes, Our preliminary studies have demonstrated that the alkali hydrolysate of Pulsatilla chinensis(Bge.)Regel total saponins activity increased apparently. In view of the great change in chemical constituents after alkali hydrolysation and their possible impact on the activity. We plan to develop the research from the following aspects to elucidate the active components of alkali hydrolysation of Pulsatilla chinensis (Bge.) Regel total saponins and further development for new drug To investigate the chemical constituents of alkali hydrolysation of Pulsatilla chinensis (Bge.) Regel total saponins, identify and analysis the main effective component; (2) To confirm the antitumor activity of the main components in vitro and in vivo; (3) To evaluate the druggability of the main components by study the pharmacokinetic parameters, distribution, excretion, metabolite in vitro and in vivo.The ultimate aim of this study is to lay the foundations for the development of antitumor active fraction from alkali hydrolysation of Pulsatilla chinensis (Bge.) Regel total saponins new drug of class V (TCM)and futher development new drug of class I (TCM)or drug candidate from Pulsatilla chinensis (Bge.) RegelMethod:1. Prepare the alkali hydrolysation of Pulsatilla chinensis (Bge.) Regel total saponins by means of organic solvent extraction, alkali hydrolysation, macroporous adsorption resin, separate the main component by ODS MPLC method, identify and analysis the main component by UHPLC-Q-TOF-MS, Extracting the relative peak area by accurate mass use Peakview 1.2 software and find the component which possess the highest content.2. To confirm the antitumor activity of the main components (B3) in vitro and in vivo. (1) In vitro:To study the proliferation inhibition activity of B3 in 11 Tumor cells by MTT; (2)To establish the KM H22 mouse liver cancer model study the effection of B3 on tumor growth, body weight and immune organs(thymus and lineal index).3.To establish the determination of B3 in plasma by LC-MS/MS method in accordance with SFDA guidelines for non-clinical pharmacokinetic and US FDA The guiding principle for quantitative analysis of biological samples.4. To determine the plasma concentration of B3 after intragastric and intravenous different doses of B3, establish the drug concentration time curve, analysis the Pharmacokinetic parameters by Winnolin 6.1, acquire the pharmacokinetic characteristics and bioavailability of B3 after intragastric and intravenous.5. To establish preliminarily of the determination of B3 in tissue by LC-MS/MS method, investigate the distribution of B3 in the tissues, primarily clarify the distribution of B3 in different time and accumulates.6. To establish preliminarily of the determination of B3 in bile, urine and feces by LC-MS/MS method, study the excretion of B3 after intravenous.7. To establish an UHPLC-TOF/MS/MS method to identify the metablites of B3 in rat plasma, urine and feces samples, understand the B3 in vivo biotransformation of B3, and clarify its mechanism in vivo.8. To establish an UHPLC-Q-trap-MS method to identify the metablites of B3 in rat intestinal flora in vitro, understand the biotransformation in vitro.Result:1. Six compounds were isolated from alkali hydrolysation of Pulsatilla chinensis (Bge.) Regel total saponins, by use UHPLC-Q-TOF-MS and Extracting the relative peak area by Accurate mass use Peakview 1.2 software, confirm that 3-O-a-L-rhamnose-(1'2) [(3-D-glucose-(1'4)]-a-L-arabinose-hederagenin (B3) were the main component, approximately 2.5 g B3 were isolated at a time,25 g B3 were isolated by column chromatography separation repeatedly.2. The in vitro and in vivo anti-tumor experiment results show:By MTT method, the cell growth inhibition effect showed good concentration dependence, the IC50 were 1.94-9.47 μg/mL. The antitumor activity in vivo study show that B3 (2.5,5,7.5 mg/kg, ip) and (100,200 mg/kg, ig) significantly lower the tumor weight of tumor-bearing mice, the anti-hepatoma activity were 28.34%,47.59%,58.82%and, 69.13%,57.94% respectively. B3 show strong antitumor activity in vitro and in vivo. B3 had no significant effect on tumor bearing mice red blood cell, hemoglobin, platelet, which no statistical significance compared with the model group, B3 had no effect on the hematopoietic function of bone marrow.3. A LC-MS/MS method were established for the determination of B3 in plasma. total run time was 2.5 min, samples pretreated were simply, concentration range of B3 were 2.0-2000 ng/mL, can meet the needs of bioanalytical of B3, lay the foundations for the Pharmacokinetic parameters, distribution, excretion, metabolite study.4. Pharmacokinetic and bioavailability result:(1) The result of pharmacokinetic by intragastric administration show:The rats intragastric administration 60mg/kg, 120 mg/kg,240 mg/kg dose of B3, was absorbed rapidly, stomach and digestive tract was the main absorb site,and eliminate slowly.The AUClast ratio of three dose were (1:0.47:1.41) and (1:2:4), no linear correlation. t1/2 were irregular with dose. The absorbtion of B3 were nonlinear pharmacokinetics. The absorbtion of B3 may be saturation. The apparent volume of distribution of the three dose were high,may be B3 easy to accumulate in the body; (1) The result of pharmacokinetic by intravenous administration show:The rats intravenous administration 0.625 mg/kg, 1.25 mg/kg, 2.5 mg/kg dose of B3, was absorbed rapidly,,and eliminate slowly.The AUClast ratio of three dose were (1:3.5:11.3) and (1:2:4), No linear correlation.t1/2 were Irregular with dose. The absorbtion of B3 were nonlinear pharmacokinetics. The absorbtion of B3 may be saturation. The apparent volume of distribution of the three dose were high, may be B3 easy to accumulate in the body; (3) Bioavailability:The bioavailability of B3 were 0.22%,it was very low.5. The concentration B3 were higher in lung, liver, spleen and kidney after intravenous administration, prompt that B3 have an affinity with above-mentioned tissues. In different segment of intestine (duodenum, jejunum, ileum and colon), the concentration of B3 was higher, prompt that B3 may be has a better effect on Colon cancer.6. After intravenous administration of B3, the cumulative excretion rate of B3 in bile, urine feces and urine were 0.11%、9.37% and 0.42%, prompt that feces excretion B3 was the main method. The total cumulative excretion rate of B3 was lower than 10%.7. A total of 18 metabolites were identified in rat plasma, urine and feces samples based on MS and MS/MS data by using ESI-Q-TOF-MS, and eight of them (M11-M18) were reported for the first time, extensive metabolite were occurred in vivo.8. B3 (M0, Parent) and senven metabolites were identified by UHPLC-Q-trap-MS, B3 can be metabolite by rat intestinal flora in vitro.Conclusion:1. This phenomenon is identical with past research, the antitumor activity of alkali hydrolysation of Pulsatilla chinensis (Bge.) Regel total saponins increased apparently, may be relevant with the saponins glycosidation in C-28 transformed to 28-COOH. Six compounds were isolated from alkali hydrolysation of Pulsatilla chinensis (Bge.) Regel total saponins, which were all 28-COOH saponins. By use UHPLC-Q-TOF-MS and extracting the relative peak area by accurate mass use Peakview 1.2 software, confirmed that B3 the main component (highest content and yield).2. B3 were not only the main component in alkali hydrolysation of Pulsatilla chinensis (Bge.) Regel total saponins, but also the mainly antitumor substance. The sudy in vitro show that B3 have a strong tumor cell growth inhibition effect with low effect and poisonous in equal dose. The sudy in vivo show that B3 can significantly lower the tumor weight of tumor-bearing mice, showed good concentration dependence and tumor growth, body weight and immune organs (thymus and lineal index), but a bit weaker with CTX.3.(1) The result of pharmacokinetic by intragastric administration show that, like other saponins, the intragastric bioavailability of B3was only 0.22%.The Tmax and Cmax of different rats in the same dose were very different, the intragastric absorbtion of B3 was irregully, prompt that we must use some dosage can increase the absorbtion or use non-intragastric administration. In addition, the effect pattern of B3 may be not only the parent but also the metabolites. The apparent volume of distribution of intragastric administration and intravenous administration were high, may be B3 easy to accumulate in the some tissue, prompt that B3 have an affinity with above-mentioned tissues. B3 may have a good effection on some solid tumours, B3 had no effect on the hematopoietic function of bone marrow.4. The total cumulative excretion rate of B3 was low, B3 may be experience extentive biotransformation. he cumulative excretion rate of B3 in bile, urine feces and urine were 0.11%、9.37% and 0.42%, prompt that feces excretion B3 was the main method. The total cumulative excretion rate of B3 was lower than 10%, A large number of B3 whereabouts unknown, need further study.5. B3 may experienced extentive biotransformation in vivo. A total of 18 metabolites were identified in rat plasma, urine and feces samples based on MS and MS/MS data by using UHPLC-TOF/MS/MS, and eight of them (Ml 1-M18) were reported for the first time, from the abundances of the metabolites in rat plasma, we speculated that isomeric of B3 (Ml) and (M7) aglycon of B3was the main metabolite in the circulation besides B3 parent.these metabolites were the effective compound. The proposed metabolite pathway were speculated, it’s also very useful for other pentacyclic triterpene.6. B3 can be metabolite by rat intestinal flora in vitro. B3 was a pentacyclic triterpene, the characteristic of these compounds was strong active and poor Bioavailability, the intestinal flora may play an important role in active, metabolic is an important key. From the study in vitro we can concluded that the 5 in 7 metablites formed in rat intestinal flora in vitro were find in vivo metabolites, similarities and difference were exist in vitro and in vivo.All told, the sources of raw materials B3 was adequately, antitumor activity is quite clear, the pharmacokinetic and distribution, excretion, metabolic in vitro and in vivo was clarified. It was worthy of being a new drug of class I (TCM)or drug candidate to further development. |
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