| Spatholobi caulis is derived from the vine stem of Spatholobus suberectus Dunn,which belongs to the Leguminosae.It has the effects of nourishing blood and activating blood,regulating menstruation and analgesia,and relieving muscles and activating collaterals.There are three types of Chinese herbal medicines in the"XueTeng" category included in the Chinese Pharmacopoeia(2015 edition):"DaXueTeng","JiXueTeng" and "Dian JiXueTeng".Among them,"JiXueTeng" has the most original species,up to 26 species,belonging to 6 families and 12 genera.Spatholobi caulis has been used for thousands of years in China,but its original is complex,the kinship is not clear,mixed with a variety of adulterants,leading to drug confusion,seriously affecting the safety and effectiveness of clinical medication.At present,the commercial "JiXueTeng" in the market are mainly derived from wild resources.Due to years of harvesting,the reserves have been very low.According to the herb,most species of the Millettia which is from the Leguminosae can be used as the original of "JiXueTeng".Therefore,it is clear that the original relationship of spatholobi caulis and its adulterants provides a favorable scientific basis for the accuracy of the use of spatholobi caulis and the safety of clinical drug use in the Chinese herbal medicine market in the future,and lays a scientific foundation for expanding the search for its alternative resources.ObjectiveThe original of spatholobi caulis is complex,the kinship is not clear,mixed with a variety of adulterants,leading to drug confusion,seriously affecting the safety and effectiveness of clinical medication.In this experiment,the reserves and distribution of medicinal resources of the Millettia.were investigated through the research of herbal medicine and the collection of specimens from the CVH library.Collection of experimental samples of spatholobi caulis and its adulterants through field investigations.And combined with the DNA barcode to use PCR amplification technology to accurately identify spatholobi caulis and its adulterants.Finally,Comparing the anti-blood deficiency effects of spatholobi caulis and Millettia.nitida Benth.var.hirsutissima,provide a certain experimental basis for the follow-up research of themMethods1.Find out the medicinal resources,efficacy and national medicines of the Millettia by referring to domestic and foreign references and herbal books,and through the Chinese Virtual Herbarium(CVH)(http://www.cvh.ac.cn/)collection specimens summarized the spatial distribution of medicinal plants of Millettia.It provides a reference for the full development and utilization of experiments and provides an experimental basis for subsequent research2.In this study,by participating in the census work of Jiangxi Pilot,the fourth national census of traditional Chinese medicine resources,we went to Longnan,Yongfeng,Fengcheng and other places to collect samples of the Medicinal plants of the Millettia.During the collection process,the information such as the date,location,latitude,longitude,and altitude of the samples were recorded,and accurately identified in combination with the "Chinese Flora" search form.The collected samples were taken from fresh plant young leaves in a timely manner and stored in silica gel for storage3.A total of 72 samples of spatholobi caulis and its adulterants were collected,the sample DNA was extracted,the ITS2,matK,psbA-trnH,ITS,and rbcL sequences were amplified and sequenced,and the amplification success rate and sequencing success rate of each sequence were calculated.The alignment of all sequences were determined by MEGA 7.0 software,and the interspecies and intraspecific genetic distance of them were analyzed to evaluate the Barcoding gap,based on the Kimura-2-Parameter(K2P)two-parameter model.Phylosuite software was used to construct the ITS2,matK and psbA-trnH and multi-gene(I-M-P)phylogenetic tree.4.In this experiment,a blood-deficiency model of mice was established,the expression of EPO in the serum was measured by ELISA,the G6PDH content was measured,and the percentage of bone marrow hematopoietic tissue and the changes in spleen tissue were observed by HE-staining to compare spatholobi caulis and Millettia.nitida Benth.var.hirsutissima Anti-blood deficiency effect.Result1.From the specimens collected in the CVH library,specimens of 26 Medicinal plants of the Millettia were sorted out.It was found that Millettia dielsiana,Millettia reticulata,and Millettia pachycarpa are abundant in China.Guangxi,Guangdong,Hunan,Guizhou,Yunnan and Jiangxi provinces are rich in resources.A total of 88 species of four types of the Millettia were collected in the field.2.DNA barcode sequences were used to evaluate for ITS2,matK,psbA-trnH,ITS,and rbcL.Among them,ITS2 had the highest amplification success rate and sequencing success rate,both at 100%.matK and psbA-trnH sequencing success rates were 94.4%and 91.7%,while rbcL and ITS are only 72.2%and 61.1%.A total of 72 ITS2 sequences,68 matK sequences,and 66 psbA-trnH sequences were obtained,and the variation site information of ITS2,matK,and psbA-trnH DNA barcode sequences of the samples of spatholobi caulis and its adulterants were analyzed and compared by MEGA7.0 software.Compared with other barcode sequences,ITS2 has obvious Barcoding gap,and there is less overlap between species.phylogenetic trees show that ITS2,matK and psbA-trnH single gene sequences can significantly cluster spatholobi caulis and its adulterants into different branches.The I-M-P phylogenetic tree can also cluster spatholobi caulis and its adulterants obviously into different branches,but the genetic distance between matK species is smaller.Therefore,ITS2 and psbA-trnH were more suitable for the identification of spatholobi caulis and its adulterants than matK.especially ITS2 had better results,followed by psbA-trnH.3.The blood deficiency model of mice was established by acetylphenyl hydrazine combined with cyclophosphamide.The content of EPO expression and G6PDH in serum was detected by ELISA,and the content of EPO and G6PDH in the serum in the model group was significantly higher than that in the blank group(p<0.01),which indicated that the model was established successfully.the percentage of bone marrow hematopoietic tissue area of femoral bone marrow HE-stained sections was calculated by image analysis software,and the hematopoietic tissue percentage of the model group was significantly lower than that of the blank group(p<0.01),and there was no significant difference between the high,medium and low dose of spatholobi caulis and the high,medium and low dose(p<0.01)of Millettia nitida Benth.var.hirsutissima in the model group.The HE staining method was used to observe the pathological sections of spleen tissue,found that the drug group had a significant recovery effectConclusionSpatholobi caulis is a traditional Chinese medicine used for thousands of years,but its original is complex,the kinship is not clear,mixed with a variety of adulterants(xi supplies),leading to drug confusion,seriously affecting the safety and effectiveness of clinical medication.according to the herb,most species of the Millettia which is from the Leguminosae can be used as the original of "JiXueTeng".In this experiment,the efficacy,national medicinal status,reserves and distribution of the medicinal plants were investigated.The molecular identification technology of DNA barcode was used to identify spatholobi caulis and its adulterants successfully,and an identification method based on ITS2 and supplemented by the psbA-trnH sequence was obtained for fast and accurate identification of them,to provide the basis for the safety and accuracy of rational use of clinical drugs.By comparing the anti-blood deficiency effect of spatholobi caulis and Millettia.nitida Benth.var.hirsutissima,it lays a certain experimental foundation for the further study of the mechanism of them. |
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