| Objective Huaier is mainly used for Supporting the Healthy Energy,Blood-Activating and Mass-Eliminating as a medicinal fungi.It is suitable for patients with primary liver cancer who are not suitable for surgery and chemotherapy.And it can also be used in the treatment of lung cancer,gastric cancer and breast cancer on the basis of standard anti-cancer treatment of chemicals.In this paper,the anti-tumor effect of the extracts of Huaier in vitro was studied.The main effective parts were flavonoids,triterpenes and polysaccharides.On the basis of this,the preparation technology and in vitro antitumor efficacy of freeze-dried powder in the effective part of Huaier were studied.Then the quality standard of the preparation and the preliminary stability were studied.The experimental data were accumulated for the development of freeze-dried powder.Methods Preformulation study:The proliferation inhibition test was performed by Huaier extracts on 6 kinds of tumor cells to screen out tumor cells with good efficacy.Then HCT-116 colon cancer cells and BGC-823 gastric cancer cell proliferation inhibition test to selected effective parts of Huaier.Establishment of methodology:The method of establishing total flavonoids with rutin as index;Oleanolic acid was used as the index to establish the total three terpene methodology;Taking glucose as index,the law of total polysaccharide was established.Purification process:The alcoholic extracts were initially purified by different polar organic solvents.And then the total flavonoids and total triterpenes of Huaier were purified by macroporous resin.On this basis,using MTT method to study proliferation on HCT-116 colon cancer cells and BGC-823 gastric cancer cells with extracts of unpurified and purified total flavonoids,total triterpenes.To determine whether it is necessary to purify,and to screen out the best ingredient on inhibition of tumor proliferation,the optimal time and concentration,laying the foundation for the formulation of prescription research.Preparation process:Using the single factor test to investigate the type and dosage of protective agent and the drug concentration of freeze-dried powder.Measuring the temperature of separator and the sample by the freeze dryer,and drawing the cooling curve to determine the eutectic point of the sample.By using single factor and orthogonal test,taking the appearance,water content,rehydration time and freeze drying rate as the main indexes on the quality of freeze-dried powder,then determining the optimum freeze-drying process parameters.In vitro efficacy study:Studying the effect of freeze-dried powder on the active fraction of Huaier on proliferation of HCT-116 cells by in vitro cell proliferation inhibition experiment.Seting the 5-Fu group and 5-Fu combined with freeze-dried powder to study whether the freeze-dried powder could reduce the dosage of 5-Fu without reducing the curative effect.Experimental study of cell migration:the movement,migration and invasion of colon cancer cell line HCT-116 were detected by scratch test.Quality standard of preparation:According to "China Pharmacopoeia"(2015 Edition),the standard research and evaluation were carried out from the aspects of character,TLC identification,water content,rehydration time,content uniformity,microbial limit test and so on.And using the contents of total flavonoids,total three terpene and total polysaccharides as the main indexes of the quality of freeze-dried powder.Stability study:According to items and methods provided by the clinical quality standards and the "Chinese Pharmacopoeia"(2015 Edition),the samples were placed in 92.5%high humidity,60 ℃ high temperature and 4500±500 lx strong illumination conditions,respectively,and the influencing factors were investigated.Results Preformulation study:Through six kinds of tumor cell screening test,it was confirmed that extracts of Huaier had stronger inhibitory effect on proliferation of HCT-116 colon cancer cells and BGC-823 gastric cancer cells.On this basis,the effective part of Huaier was further screened as the alcohol and water extract.Establishment of methodology:The methodology of total flavonoids,total triterpenes and total polysaccharides were established,the total flavonoids was with the indicator of rutin:the standard curve was Y=0.01147X+0.01567,R2=0.9990,it had good linear relationship within the scope of 11.84～71.04 μg/mL.The total three terpene was with the indicator of oleanolic acid:the standard curve was Y=0.05564X-0.03366,R2=0.99933,it had good linear relationship within the scope of 5.550～19.425μg/mL.The total polysaccharide was with the indicator of glucose:the standard curve was Y=0.0548X-0.01716,R2=0.99927,it had good linear relationship within the scope of 5.595～14.920 μg/mL.The three ways had good precision,repeatability,stability,standard recovery met the demand,method were feasible.Purification process:The total flavonoids,total triterpenes and total polysaccharides that were not purified had stronger inhibitory effect on cell proliferation of HCT-116 colon cancer cells and BGC-823 gastric cancer cells,and were not purified in the later stage.The alcohol and water extracts of Huaier had stronger inhibitory effect on the cell proliferation of HCT-116 colon cancer cells and BGC-823 gastric cancer cells than other parts,to further determine the effective parts for the alcohol and water extracts of Huaier.Molding process:The Lyophilized formulation formula was as follows:The concentration of Lyophilized liquid was 4%,the best protectant was 10%mannitol,the best solubilizer was 2%poloxamer 188,the lowest eutectic point of the sample is:-5～-1℃;the best freeze-drying process is:-56℃ that was pre-freezing 3 h,-15～-10℃ sublimation drying 12 h,45～50℃,analytical drying for 9 h.In vitro efficacy study:The freeze-dried powder preparation prepared in this study had obvious inhibitory effect on HCT-116 colon cancer cells.IC50 value of 24 h and 48 h were 767.80,614.09 μg/mL,respectively;freeze-dried powder combined with 5-Fu can significantly reduce the amount of 5-Fu.Experimental study on cell migration:The freeze-dried powder prepared in this study can reduce the migration ability of colon cancer HCT-116 cells,and the effect of combined application with 5-Fu is better.Quality standard of preparation:Huaier freeze-dried powder is light yellow,loose cake-like,uniform and full.The average loading capacity is 1.375 g/bottle,the redissolve time,content uniformity,microbial limit test are meet the requirements;TLC identification:corresponding position was the same spots with Huaier test solution and control drug solution,While the negative control solution sample without interference;Determination of freeze-dried powder content:The content of total flavonoids in the samples was not lower than 34.74 mg/g,the transfer rate was 60.84%;The content of total triterpenoids was not lower 15.81 mg/g and the transfer rate was 68.72%.The content of total polysaccharide was not less than 66.80 mg/g,the transfer rate was 95.00%.Stability study:The freeze-dried powder prepared in this paper has good stability in high temperature test,strong light test and high humidity test condition.Conclusion In this study,the active parts of Huaier(total flavonoids,total triterpenes and total polysaccharides)were prepared into freeze-dried powder preparations.The research of the preformulation,extraction process,purification process,preparation molding process,quality standard evaluation and in vitro efficacy study and other content were finished.Screening the effective parts of Huaier by in vitro tumor cell proliferation inhibition test.The preparation process of effective parts of Huaier freeze-dried powder was studied by combining the advantages of freeze-dried powder preparation.The formulation and freeze drying parameters of preparation process were optimized.The cell viability test and cell migration test were carried out.Using modern analytical techniques,the character,TLC identification Check items(quantity difference,rehydration time,water content and so on),content determination and other aspects of freeze-dried powder had a more systematic study,developed a more perfect quality standards.Where in the check indicators in line with the Flushing agent inspection requirements in "China Pharmacopoeia"(2015 Edition),the method of determination of total components and single component content is feasible and can effectively control the quality of freeze-dried powder of effective parts of Huaier.According to "China Pharmacopoeia"(2015 Edition),taking quality standard for freeze-dried powder of active constituents of Huaier as the index,studying the stability of freeze-dried powder by using natural storage test and accelerated test methods.The inspection indicators were in line with the requirements,showed that the freeze-dried powder formula was reasonable,the preparation process was feasible,the quality and safety were controllable and the stability was good. |
PDF Full Text Request