Studies On Identification?Quality Analysis And Antitumor Activity Of Selaginellae Braunii Herba

Selaginella braunii Baker is distributed in the south of the Yangtze River.The dried whole plant is used as the folk medicine,which had the function of clear heat and detoxify,clear lung to relieve cough and stop bleeding.So far,there is only one report about its chemical constituents.No systematic pharmacognosy identification,content determination of active ingredients,HPLC fingerprint and pharmacological effects have been reported.It possesses similar appearances with other plant medicines from the same genus,leading to confusing and error-prone application.The macroscopic characteristics,microscopic characteristics,TLC and HPLC fingerprint of Selaginellae braunii herba collected in Western Hubei Province were compared with the 6 other medicines from the same genus,namely,the dried whole plants of S.delicatula(Desv.)Alston,S.uncinata(Desv.)Spring,S.moellendorffii Hieron.,S.doederleinii Hieron.,S.remotifolia Spring and S.sinensis(Desv.)Spring.and the exclusive identification methods were established.According to the characteristics of this drug(main stem accounts for a large proportion),the main stem and branches and leaves of the samples were separated,the contents of total flavonoids and amentoflavone of the dried whole plant,main stem,branches and leaves of it were determined.Inhibitory activities of different extracts from the above-mentioned parts of the herb on human hepatoma cell HepG2 were tested and compared with the same genus of other herbal medicines(S.delicatula(Desv.)Alston,S.uncinata(Desv.)Spring,S.doederleinii Hieron.).Pharmacognostical identification results showed that the main characteristics of this crude drug were as follows: the upper part of the dried herb are curled,adventitious roots are very short,forked at the apex,the main stem is nearly subquadrangular or even cylindrical(rare),on the middle and lower part of the main stem,no branches can be found.The main microscopic characteristics show that The xylem is slightly "? " shaped,but the protruding parts at both ends of the "? " are sometimes not obvious.Meanwhile,the xylem of the tender stems is slightly "? " shaped.In TLC,the chromatogram of the sample showed six well-separated spots and two main spots with characteristics under the same experimental conditions.The above characteristics can effectively distinguish Selaginellae braunii herba from the same genus so that its accurate application could be improved.The method for the determination of the total flavonoids in Selaginellae braunii herba was established through experiments,and then determined the different batches of the dried whole plant,main stem,branches and leaves of this crude drug.The results showed that the total flavonoids content of the samples were above 2.0% except for individual samples.Comparing the total flavonoids contents of the samples collected in the same place in July and October 2018,we can find that the total flavonoids contents have little change during the growth period,and preliminarily confirm that the harvest time is from July to October.The contents of the total flavonoids in the branches and leaves of the different batches of the medicinal materials were higher than those in the main stems,and the ratio was 3.81%-53.03%.It was preliminarily considered that it was more reasonable to use the branches and leaves as the medicinal material.The common pattern of HPLC fingerprint profiles of Selaginellae braunii herba was established and the 14 common peaks were identified.Comparing the chromatograms of Selaginellae braunii herba and the 6 medicines from the same genus under the same experimental conditions,the results showed that the number of peaks,the time of peaks and the number of common peaks were different among different medicines,which could effectively distinguish Selaginellae braunii herba from the same genus.Peak 1,5 and 6 were identified as amentoflavone,robustaflavone and hinokiflavone separately,the contents of amentoflavone in the different batches of the drug?s dried whole plants,main stems,branches and leaves were determined.The content of amentoflavone of the dried whole plant varying greatly with different harvesting sites and dates,and in the samples collected in October was significantly higher than that in July.This content in branches and leaves was 74.8% higher than that in the main stem,which further confirmed that the quality of the medicine with branches and leaves was better.According to the related research reports on anti-tumor activities of Selaginella medicinal plants,human hepatocellular carcinoma HepG2 cells was selected as the research object,using the MTT method to detect their anti-tumor activity.The results showed that the ethyl acetate and methanol fractions of Selaginellae braunii herba showed better anti-tumor activity,the former had the strongest activity,and the inhibition activity was time and dose dependent.The activities of extracting parts of the branches and leaves was stronger than that of the main stem,which further proved the quality characteristics of branches and leaves.The effects of different doses of ethyl acetate extracts from the herbal medicine with the best activity on the tumor cells were observed by inverted microscopy,Hoechst 33258 fluorescence microscopy and flow cytometry Annexin V-FITC/PI double staining.The results showed that with the increase of dosage,the morphology of tumor cells changed significantly,and the apoptotic rate also increased.It is confirmed that Selaginellae braunii herba has a good inhibitory effect on human hepatocellular carcinoma HepG2 cells.In this study,we established specific and quality control methods for identification of Selaginellae braunii herba,and verified its anti-tumor activity.It provides an important experimental basis for the identification,quality evaluation and application development of it.