Study On The Evolution Of Chromosomes And The Extraction Of Flavonoids From Chrysosplenium L.

The species of Chrysosplenium L.is a member of the Saxifragaceae family,which was formally established by Linnaeus in 1753.There are more than 80 species in the world,most species are distribted in China.There are more than 50 species including varieties,which are widely distributed in southwest,central and Northeast China.However,the study on the evolution of chromosome number in China is rarely reported.Based on the previous work of our laboratory,we supplemented the chromosome data of 12 species of Chrysosplenium in China,and analyzed the chromosome phylogeny of 24 species of them by using the mat K sequence in chloroplast genome.On the one hand,this study can provide cytological evidence for the revision of this genus classification system and the identification of new species.On the other hand,it provide more information for the global chromosome evolution of Chrysosplenium.Chrysosplenium has high medicinal value because of its rich flavonoid compounds.They are widely used in traditional Chinese medicine.However,due to the short plant,special habitat,difficulty in artificial cultivation,and most are endangered species,it is difficult to collect materials.At present,there are little researches on the extraction of total flavonoids about them.In order to fill this gap,we have studied the seed germination and the production technology of the total flavonoid compound extraction of Chrysosplenium sinicum and Chrysosplenium macrophyllum,which laid a preliminary foundation for the development of germplasm resources and medicinal value of Chrysosplenium.The results are as follows:1.In this study,the karyotypes of 12 species of Chrysosplenium in China were studied,and a karyotype production method suitable for them was established and applied for patent.The karyotype photos obtained are clear and fill in the blank of chromosome data of some species.The results showed that the number of Chrysosplenium chromosomes was 2n=8,16,18,24,32,36,42,46,48,which indicated that polyploidy and multiple chromosome bases existed.In addition,we found that the chromosome numbers of the same species in different cohabitation groups were different.For example,in C.henryi,the morphological characteristics of three populations are slightly different,but the chromosome number is not the same.Analysis of the causes,there may be variations and chromosome polyploidy.2.The chloroplast mat K sequence of 24 species Chrysosplenium were used for phylogenetic analysis.The evolutionary tree constructed by the maximum likelihood method and the Bayesian method shows the same topological structure.Except for C.microspermum,other species can be divided into two categories and several sub categories,which are consistent with the classification results based on morphological characteristics.Based on Bayesian tree and 8 kinds of chromosome cardinality data,we use mesquite 3.51 software to calculate the chromosome cardinality of this genus.It conclude that the chromosome cardinality of this genus is n=12(94.24%),and make a preliminary guess on the evolution process of its cardinality.However,because only one gene sequence is selected and the chromosome data is not comprehensive enough,the specific evolution process of cardinality in the evolution process needs more targeted and detailed research.3.C.sinicum,which is widely distributed in altitude and has a large amount of seed acquisition,was selected as an example to explore the effects of light,low temperature and Gibberellin Treatment on its seed germination rate.The results showed that:(1)The seed of C.sinicum has dormancy;(2)Under light,the germination rate,germination index and germination potential were higher than under full dark;(3)Low temperature treatment at 5°C can increase its germination potential and germination index,and it is positively correlated with treated time of low temperature,but has no significant effect on germination rate;(4)The germination rate reached the highest(97.78±1.57)%after soaking seeds for 12 hours with GA₃concentration of 150mg/L.Seed soaking time was negatively correlated with seed activity.4.C.macrophyllum,the largest plant of this genus and widely distributed in China,was selected as the representative.Using the single factor experiment combined with Box-Behnken central composite experiment,and the content of total flavonoids as the evaluation index,we optimized reflux extraction of total flavonoids of C.macrophyllum.The results showed that the optimal extraction conditions were as follows:ethanol concentration was 80%,extraction temperature was 80?,liquid-to-solid ratio was 1:30(g:m L)and extraction time was 50 min.Under these conditions,the extraction yield of total flavonoids was(5.529±0.121)mg/g.The results are basically consistent with the theoretical prediction value of Design-Expert 12.0,which shows that the extraction process is feasible.