| DiWu is the dried rhizome of Anemone flaccida Fr.Schmilt,belonging to the Anemone genus in the Ranunculaceae family.Its medicinal material characteristic is warm,taste slight bitter and has the effect of dispersing,dispelling rheumatoid,helping bones and muscles,treating cold arthralgia and falling injury.In the present study,its pharmacological active ingredient,triterpenoid saponins,has the exact effect in anticancer,anti-inflammatory,antimicrobial properties and immune regulation.At present,they are mainly in wild,distributed in the provinces of the middle and lower reaches of the Yangtze River.With the increasing demand for treatment and slowly growing,Diwu is in the endangered edge.The yield of wild resources cannot meet the demand of the market.So the commercial medicinal materials appear mixed varieties,and the quality is also a problem,which probably leads some risk in the application.In this paper,the resources and quality of diwu were systematically studied from three aspects: the suitability producing area,phylogenetic study and the rapid identification of the original plants of Anemone,the fingerprint of its active components and the dynamic analysis of metabolomics,and it provides a more accurate and effective method and basis for the identification and quality evaluation in order to solve the problem of shortage of DiWu resources.Based on Global Geographic Information System for Medicinal Plant,it makes a first survey and analysis on the distribution of the resources and growth conditions of Anemone flaccida: it is mainly distributed in 32 countries,including the United States,Russia,Canada,China and Australia.Its ecological suitability in China mainly includes 10 provinces,such as Shanxi,Guizhou,Gansu,Sichuan and Hubei.In the main distribution area,the lowest mean temperature in cold season is-26.2°C,while the highest in the hottest season is 30.8?.In the main cultivation area,the mean temperature in hottest season is 8.8 ?-30.8 ?,while-26.2 ?-22.6? in the coldest season.The annual average temperature is 20.2?-27.5?.The annual average precipitation is 163-2364 mm.The annual average relative humidity is 26.3-75.2%.The average annual sunshine is 89.2-204.5 W/m2,which is mainly suitable for growth in leached soil,epigastric soil,thin layer soil,dark color soil,highly active strong acid soil,artificial soil and other environments,providing scientific basis for industrial structure adjustment and rational development of diwu medicinal materials.By selecting the four hot candidate sequences of ITS2,rbc L,mat K and psb K-psb I for PCR amplification,sequencing and sequence comparison of 52 samples from 15 species in Anemone,and using the maximum likelihood and Bayesian inference to rebuild their phylogenetic relationship,it is shown that Anemone flaccida can clearly identify with other group by ITS2 barcoding and Anemone flaccida var.hofengensis exists in different classification.Combined with high resolution melting(Bar-HRM)analysis technology,it gets 26 different melting curve peak and Tm value,sets up melting curve in DiWu and provides a new way identification of traditional Chinese medicine and important basis for safe circulation.At the same time,using the second generation of high-throughput sequencing technology,Anemone flaccida chloroplast genome has been sequence was sequenced,assembled and annotation.The result is that the codon preferences are greatly influenced by the selection pressure.Then UUU,UUA and UCU 15 codes were selected as optimal codon,which is helpful to system evolution and genetic engineering research.Using UPLC-QTF-MS /MS,a three-step strategy was developed to identify or characterize oleanane-type triterpenoid saponins in DiWu.The first step is to investigate the collision-induced dissociation(CID)MS/MS fragmentation pattern of oleanane-type triterpenoid saponins using five standards.The neutral loss of 176 Da,162 Da,146 Da,and 132 Da indicated the presence of a glucuronyl(Glu A)group,a glucosyl(Glc)group,a rhamnosyl(Rha)group and an arabinose(Ara)or a xylose(Xyl)group,respectively.Second,obtaining the exact mass and molecular formula of each potential triterpenoid saponin by comparing the fragmentation pathway with that of the five standards.The third step is determining the species and conjugation positions of sugar moiety based on the MS/MS product ions.The targeted profile facilitated the qualitative distribution and relative quantitative analysis of oleanane-type triterpenoid saponins in a single injection.Finally,34 oleanane-type triterpenoid saponins were characterized in the DiWu extracts.In the electrospray ionized positive ion mode,each saponin was separated at baseline within 32 min.The methodology showed that the results of this method were reproducible,repeatable and stable.And the use of curcumin as an internal reference could effectively eliminate the matrix effect,providing a scientific basis for the quality control and pharmacokinetic study of DiWu.The same way was used to analyze the dynamic accumulation changes of total saponins and 34 saponins in 90 samples of wild and introduced DiWu at different harvest periods during March 2017 to June 2017.It was obvious that the contents of total saponin in wild DiWu were significantly higher than those in introduced DiWu for any corresponding period.27 oleanane-type saponinswere present in both wild and introduced DiWu.It is reasonable to assume that the difference in growth environment had a significant effect on the expression of relative genes controlling these enzymes in wild and introduced DiWu,which leads to differences in saponin type and content.The relative quantitative analysis with 34 oleanane-type triterpenoid saponins was conducted to evaluate wild and introduced DiWu samples at different harvest periods and find out the best harvest period and the new quality control index in DiWu.The result shows that the total saponins content paralleled changes with the harvesting periods in the two different growth environments,which all began to decline after an initial increase.In the fourth harvesting period(April 11,2017),the total saponin content first maximized and then fell to the lowest value in the tenth period(June 10,2017).Furthermore,dynamic changes in the contents of triterpenoid saponins throughout various harvesting periods were clearly explained and mid-April was identified as the appropriate harvest time.Moreover,results indicate that the highest contents of six main saponins(Anhuienoside E,glycoside St-I4 a,Hemsgiganoside B,Flaccidoside ?,Hederasaponin B and a unknown saponin)were the most abundant during the fourth harvesting period,so the content of these five components can be considered as an indicator in the DiWu quality assessment. |
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