| purpose:Ilex pubescens Hook. Et Am. was widely used in south China as TCM for it clear effect on Coronary heart disease, angina and vasculitis etc., because of the effective part of Ilex pubescens mainly roots but stem and its poor ability to Renewable resources, the material found on the market most wild, partially traders use Ilex pubescens var. Clabra as Ilex pubescens. In 2012 Missouri Botanical Garden identified Ilex pubescens var. Glabra as synonym for Ilex pubescens Hook. Et Am., newest "Flora of China" not included in the species also, so it’s important to make clear the source of Ilex pubescens the establishment of precise, efficient and effective method of identifying imperative, method:In this study, ISSR molecular marker method was established to identify Ilex pubescens samples collected from five provinces in South China to distinguish Ilex pubescens Hook. Et Am. from Ilex pubescens var. Glabra on molecular level. Chemical samples from different parts and different harvest time was analyzed Using HPLC-DAD. Results:1. Protease K-MCTAB was Established for extracting cDNA from Ilex pubescens, by using buffer A which was CTAB free most phenols and partially water-soluble secondary metabolites was removed, protease K was added to enhance the yield of the nucleic acid while reducing impurity contamination. Results shows with method adopted optimized, cDNA yield extraction results at least 4 times than the DP3O5 kit with high quality and purity.2. Orthogonal design was used for Ilex pubescens ISSR reaction system optimization, the final result is optimized Mg2:2.5 mmol·L-1,dNTPs:0.15 mmol·L-1, Primer:0.4 μmol·L-1, DNA:70ng, Taq:0.5 U,11 primers were screened and annealing temperature for subsequent research.28 samples were amplified bands 2531, average 230.1 pre primer, which polymorphic bands 1870, accounting for 74%, average 170 pre primer.3. Similarity coefficient for 28 samples ranged from 0.6478 to 0.9870 between average similarities coefficients of 0.7693. Wherein A5 from Lechang, Guangdong Province, A27 from Meizhou, Guangdong Province under a minimum difference of 0.6478; A24 and A27 both from Meizhou, Guangdong Province, similarity coefficient up to 0.9870 the highest similarity. The presence of Ilex pubescens var. Glabra sample compared to other samples significantly different. All samples from the major genetic mainly in the 0.75-0.775 range, the genetic distance between Ilex pubescens var. Glabra group ranged from 0.725 to 0.75,distance within the group from 0.8 to 0.825. UPGMA cluster analysis showed that Ilex pubescens var. Glabra can be separate from Ilex pubescens at 0.34 level on distinct branch based on 11 ISSR primers. Also approved Ilex pubescens var. Glabra is different from Ilex pubescens at the molecular level.4. screening based on ISSR-PCR result of 28 gene pool, removal of occurrence probability<18% of the sequence established Ilex pubescens ISSR fingerprinting. With 5 parts of a random sample and 5 parts Ilex pubescens verification test random samples show bald Ilex pubescens amplification result Ilex pubescens ISSR fingerprint similarity between 0.8371-0.8962,5 bald Ilex pubescens sample amplification results Ilex pubescens ISSR fingerprint similarity between 0.6831-0.7411. Ilex pubescens ISSR fingerprinting proved able to distinguish Ilex pubescens from Ilex pubescens var. Glabra.5.sample test for different parts using HPLC-DAD show that:ilexsaponin A1 content root> stem> twigs, ilexsaponin B1 content twigs> roots> stems. Wherein the ratio ilexsaponin A1 content in root content of 45% -66%, the lowest twigs content, content ratio of 6% -27%, the content of the stem between the two,27% -39%. ilexsaponin B1 content content twigs largest proportion,56% -75%, the lowest stem content of 10%-15%, the content of the root between the two,15%-30%. although there are some individual differences Between different plants, but the same overall trend. Study of different harvest times revealed ilexsaponin A1, ilexsaponin B1 two goals over time there is a change in the composition of regular fluctuations, ilexsaponin A1 at May to July appears annual minimum content value, Sep. It may appear content maximum, ilexsaponin B1 of the year there will be twice the content of peak time in may to July, another time in November. DP002 and DP003 two samples ilexsaponin A1, ilexsaponin trends B1 content are basically the same, conclusion:Based on the results of ISSR molecular identification showed that 11 primers screened out from the present experiment able to distinguish from Ilex pubescens var. Glabra to Ilex pubescens clearly, prove the existence of differences between the two at the molecular level, combined with previous chemical research, it is necessary to consider further study on Ilex pubescens var. Glabra using as TCM. Alouth the main pharmacological components of Ilex pubescens is not clear, but studies have shown triterpenoid saponins its main component. This study selected ilexsaponin A1, ilexsaponin B1 as representative. The results showed that two compounds are quite different in different parts of the plant, its content changes throughout the year there are regular fluctuations, provide ideas and basis for the synthesis pathway for triterpenoids inside Ilex pubescens. |
