Study Of Saponin Fraction From Gleditsia Sinensis On Anti-hepatoma Mechanisms And Its Microsphere Formulation

Objective:Base on the literature research and pharmacological experiment,we foun d that Gleditsia sinensis has obvious anti-tumor effect,and Saponins are the main active ingredient^[1-6].On the basis of the previous research,th e separating and purifying method and pharmacological method were us ed in this study to prepare SFGS.We explore the anti-hepatoma effect of SFGS by pharmacological experiment,then clarify the possible mecha nism of SFGS enhance sorafenib cytotoxicity.Because SFGS can stimul ate the mucosa as its pungent smell,we choose Chitosan nanoparticles to carry SFGS in clinical application.then evaluate characteristics and eva luate the pharmacodynamics of it.Methods:Experiment 1 Isolation and purification of saponins from SFGS and the study on the activity of anti-liver cancer1.The technologies of extraction Saponins from SFGS were studied.The different factors were investigated for extraction of Saponins.2.The total extract was prepared from 50 g fructus gleditsiae.We separa ted and purified the effective fraction in the total extract by Macroreticu lar Adsorbents along with pharmacological experiment.3.We prepared the total extract from 29 kg fructus gleditsiae according t o the optimal extraction method followed by separation and purification with macroporous resin.The effective fraction was separated and purifie d by silica gel column.Then the effective fraction was screened from t hose extracts.4.The effective fraction from silica gel column was separated and purifi ed by MCI column.And then,the effective fraction was screened from it.5.The effective fraction of MCI column were analysised and identified b y Mass spectrometry.And the content of Saponins were determined by U V-spectrophotometry.Experiment 2 Investigate the mechanism of inhibition effect of sorafenib combined with the SFGS on hepatoma1.Pharmacological experiments and Western Blot assay were used to inv estigate whether combinations of sorafenib and SFGS had synergetic int eraction.2.Pharmacological experiments and Western Blot assay were used to inv estigate whether SFGS could activate endoplasmic reticulum stress?ERS?of liver cancer cells,and whether ERS could take part in the anti-tum or function of the SFGS.3.The Western Blot assay was used to examine the proteins expression of the Wnt/?-Catenin signal pathways under SFGS treatment.Use XAV939 to treat liver cancer cells,found out whether the inhibition of Wnt/?-Catenin signaling pathways induce apoptosis of liver cancer cells.4.Pharmacological experiments and Western Blot assay were used to inv estigate whether Wnt/?-Catenin signaling pathways could mediate by ER S.5.The tumor model was established to investigate the inhibition effect of sorafenib combined with the SFGS.Experiment 3 Study of the preparation of CS-SFGS nanometer microsph eres and its anti-tumor effect in vitro.1.Select the preparation method of CS-SFGS nanometer microspheres,an d evaluate the effect of different single factor.2.Prepare the CS-SFGS nanometer microspheres,and detect its characteri stics.3.The tumor model was established to investigate the anti-tumor effect o f CS-SFGS nanometer microspheres.Result:Experiment 1 The separation and purification of SFGS and anti-hepatom a research1.The results showed that 75%ethanol was the preferably solvent and t he alcohol reflux extraction was the optimal method.2.Triterpenoid saponin exist in three fractions:30%ethanol elution part and 70%ethanol elution part.70%ethanol elution part is the best activ e fractions for anti-tumor.3.The methylene chloride-methanol?2:1?elution parts of silica gel col umn is the best active fractions for anti-tumor.4.The 60%ethanol elution parts of MCI column is the best active fracti ons for anti-tumor,5.17 constituents were identified by Mass spectrometry.The content of S aponins in SFGS was 77.4±2.1%.Experiment 2 Investigate the mechanism of inhibition effect of sorafenib combined with the SFGS on hepatoma1.The combination of sorafenib and SFGS showed synergetic effect in a nti-tumor.2.SFGS could induce ERS in liver cancer cells.And ERS could take pa rt in the anti-tumor function of the SFGS.3.SFGS could inhibit Wnt/?-Catenin signaling pathways.And the inhibi tion of Wnt/?-Catenin signaling pathways could induce apoptosis of live r cancer cells.4.Wnt/?-Catenin signaling pathways could mediate by ERS.5.In vitro,the combination of sorafenib and SFGS have a better anti-tum or pharmacological action than Sorafenib alone.Experiment 3 Study of the preparation of CS-SFGS nanometer microsph eres and its anti-tumor effect in vitro.1.CS-SFGS nanometer microspheres were prepared by ionic cross-linking method.Optimal prescription:chitosan?2.0 mg/mL?;Tripolyphosphate sod ium?1.5 mg/mL?;Material input ratio?4:1?.2.To determine the characteristics of CS-SFGS nanometer microspheres:Good sphericity and equally distributed;PDI?0.183?;Partical size?184.7nm?;Zeta potential?26.3mV?;Drug-loading content?14.64%?;Encapsulatio n rate?84.17%?;Total release quantity in 24 h?91.3±5.4%,n=3?.3.In vitro,the CS-SFGS nanometer microspheres have a better anti-tumor pharmacological action than Sorafenib alone.Conclusion:SFGS could obviously inhance the anti-tumor effect of sorafenib which was a evident for SFGS as a good radiosensitizer for sorafenib.So we could reduce the side effects or resistance of sorafenib through combinat ion with SFGS.The results showed that SFGS was a natural ERS acti vator.SFGS could inhibit Wnt/?-Catenin signaling pathways through ac tivating ERS.And the inhibition of Wnt/?-Catenin signaling pathways co uld induce apoptosis of liver cancer cells.The anti-tumor effect of CS-S FGS nanometer microspheres on sorafenib was better than on SFGS.This study provided a new perspective for anti-tumor effect and clinical rese arch of Gleditsia sinensis in the future.