| Selaginella doederleinii belongs to the Selaginellaceae Genus plants of Pteridophyta.It can be used for heat-clearing and detoxication,dispelling wind and eliminating dampness,activating blood circulation to dissipate blood stasis.It is widely used to treat nasopharyngeal carcinoma,lung cancer,chorionic carcinoma and cervical cancer with high efficiency and low side effect.But there are few reports on its modern pharmaceutical research.Previous study reported that Selaginella doederleinii's main anti-cancer active ingredients were the bioflavonoids that concentrated on the ethyl acetate extracts.This paper establishes the content assay and fingerprinting analysis method of Selaginella doederleinii,preparation method and quality control standard of the total biflavonoids crude extract of Selaginella doederleinii.It lays the foundation for further development of APIs(Active Pharmaceutical Ingredients),and formulation based on total biflavone extract.The project includes the three parts outlined below:(1)Content assay and fingerprinting analysis of Selaginella doederleinii HieronA high-perofrmance liquid chromatographic(HPLC)method was developed for the content assay and fingerprinting analysis of the raw materials.The method was used for quality control of Selaginella doederleinii.Mehtod conditions: the column was Agilent C18 analytical column(4.6×250mm,5?m);the mobile phase was acetonitrile and 0.5% acetic acid in gradient elution mode;the flow rate was 1.0 mL/min;the UV absorption detector wavelength was 270 nm;and the run time was 55 minutes.14 peaks were identified for fingerprinting and 7 components were quantified.The method has excellent precision,recovery,and linearity.The linear ranges for amentoflavone,robustaflavone,2??,3??-dihydro-3?,3???-biapigenin,3?,3???-binaringenin,delicaflavone,heveaflavone,7,4?,7?,4??-tetra-O-methyl-amentoflavo-ne were 1.01-130?0.78-100?0.78-100?0.86-110?0.84-108?2.81-360?4.31 – 552 ?g/mL,respectively,with excellent linearity(r ? 0.999).The content of seven bioflavonoids in 12 batches of Selaginella doederleinii Hieron was determined by the established method.The content of heveaflavone was the highest,in the range of 0.191 ~ 0.376mg/g.The content of amentoflavone was the second,in the range of 0.098 ~ 0.213mg/g.The rest of the biflavone contents ranged 0.03 ~ 0.150 mg/g.Plants from different regions had different biflavone profiles.Using the 14 common characteristics peaks identified,the fingerprints of the samples were established and the fingerprints similarity of the 12 batches is greater than 0.9,indicating good consistency among the samples.According to the system cluster analysis results,12 samples from different habitats were classified into 5 groups,the clustering results were consistent with the analysis of similarity results,and the two methods were verified by each other.The analytical method established is simple and accurate,and can be used for the quality control of the medicinal materials.(2)Preparation of total biflavonoids crude extract of Selaginella doederleinii HieronSolvent extraction and the alkali-solution and acid-isolation extraction were compared.Solvent extraction method not only resulted in purer extract it also produced higher yield.Therefore,solvent extraction method was selected for further investigation.Solvent composition,solvent concentration,extraction temperature,extraction time and solid-liquid ratio were investigated by single factor test and orthogonal experiment design.Process evaluation criteria employed were extract yield,total content of flavonoids in the extracts and delicaflavone content.The optimal extraction process is adding 70% ethanol(45:1 mass ratio vs.the raw material),refluxing twice at 85 ?,1.5h for each time.Further purification of the crude extract was investigated.The crude extract of the total bioflavonoids was concentrated to the relative density of 1.10 ~1.15(60?).The purification was carried by extracting with equal volume of petroleum ether three times followed by with equal volume of ethyl acetate three times,collecting ethyl acetate layer,evaporating to dryness and freeze drying the extracts.It makes better sense to provide the percentage of the total bioflavonoids in the purified extract,which is about 4 times that of the original content(9.47%).(3)Quality control of total biflavonoids crude extract of Selaginella doederleinii HieronAccording to the quality standard of "Chinese Pharmacopoeia"(2015 Edition),the crude extract of total biflavonoids of Selaginella doederleinii Hieron was identified by thin layer chromatography.Other tests include water content,residue on ignition,heavy metal,arsenic content,as well as the content of the three major biflavonoids(including the main components of the amentoflavone,heveaflavone and the main active ingredient delicaflavone).Thin layer chromatogrphy results of the 5 batches of materials conformed the testing.The total biflavonoids content of the 5 batches was in the range of 365.8 ~ 390.5 mg/g.The content of amentoflavone was in the range of 35.3 ~ 43.7 mg/g,the content of delicaflavone was in the range of 15.9 ~ 20.4 mg/g and the content of heveaflavone was in the range of 39.0 ~ 47.2 mg/g.The methods established can be used for the quality control of the biflavonoids crude extract.In addition,the stability of the crude extract of Selaginella doederleinii was studied under different conditions(high temperature,high humidity and light)and under International Conference of Homonisation accelerated condition(40°C ± 2°C/75% ± 2%RH).The results showed that the crude extract of the total flavonoids produced by the optimized process was stable although it has a slight moisture absorption in the high temperature and humidity conditions.In this paper,we established the method of the determination and fingerprinting analysis of raw material of Selaginella doederleinii.The preparation method of crude extract of total biflavonoids was optimized.The quality control method of crude extract of Selaginella doederleinii was established. |
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