Correlations between content of linarin in Chrysanthemum indicum and climatic factors in habitats / 中国中药杂志

Chrysanthemi Indici Flos(CIF), the capitulum of Chrysanthemum indicum, is widely used in proprietary Chinese medicine and daily chemical products. At present, CIF is mainly produced from wild resources and rarely cultivated. This study aims to reveal the correlations between linarin content in CIF and climatic factors in different habitats, and provide a theoretical basis for suitable zoning and rational production of medicinal materials. The content of linarin in CIF was determined by HPLC. Grey relational analysis and Pearson correlation analysis were carried out for linarin content with climatic factors. The results showed that the content of linarin in CIF was significantly different among different habitats. The grey relational degrees of climatic factors with linarin content was in an order of average annual precipitation>annual average sunshine hours>annual average temperature>longitude>annual frost-free period>latitude>altitude. Longitude, annual average temperature and average annual precipitation had significantly positive correlations with the content of linarin in CIF, whereas latitude and altitude showed negative correlations with it. The annual frost-free period and annual average sunshine hours had no significant correlation with the content of linarin in CIF. The content of linarin in CIF varied significantly in different habitats. High longitude, low latitude, low altitude, high annual average temperature and high annual average precipitation could be used as indicators for the habitats of high-quality Ch. indicum. This study provides a reference for selecting suitable producing areas of Ch. indicum and establishing artificial cultivation system.

Comprehensive antioxidant and anti-inflammatory activity of alcohol extracts from Chrysanthemum indicum in different areas based on entropy weight and TOPSIS methodology / 中国中药杂志

The study is aiming at investigating the application of entropy weight TOPSIS method in the comparison of the scavenging effect of DPPH, ABTS and hydroxyl radical and the inhibition effect of xanthine oxidase(XOD) and lipoxygenase(LOX) of Chrysanthemum indicum. The DPPH, ABTS, salicylic acid and spectrophotometry were used to determine the scavenging effect of DPPH, ABTS and hydroxyl radical and the inhibition effect of xanthine oxidase(XOD) and lipoxygenase(LOX) of Ch. indicum from 31 different areas in vitro. Take the half inhibition rate of as the evaluation index, two principal components were extracted by the principal component analysis, and their cumulative contribution rate reached at 92.4%. The different areas of Ch. indicum could be divided into Dabei Mountain and Qinling-Taihang Mountain by use principal component to analysis. The entropy weight TOPSIS method was used to objectively assign weights to five indexes, calculate the weight of each index and set up the best and worst scheme of the evaluation object, and the relative proximity(C_i) was used as the measure to construct the multi-index comprehensive evaluation model of Ch. indicum. And then sort with the relative proximity value. The results showed that the relative proximity was between 0.098 and 0.983 which represents there were significant differences in the scavenging effect of DPPH, ABTS and hydroxyl radical and the inhibition effect of xanthine oxidase(XOD) and lipoxygenase(LOX) between extracts of Ch. indicum from different areas. The Ch. indicum from Dabie Mountain area have a relatively high relative degree of measurement and high-quality ranking. Taken together, the quality of Ch. indicum.from the Dabie Mountain area is better. The index weight coefficient and the classification result of producing area are basically consistent with the result of principal component analysis. The results show that the TOPSIS method based on entropy weight method can be used to evaluate the comprehensive quality of Ch. indicum.

Effects of application of N, P, and K on traits and active ingredients of Chrysanthemum indicum / 中草药

Objective To explore the effect of N, P, and K fertilizers on the growth of Chrysanthemum indicum and accumulation of active components. Methods This research used different levels and ratios of nitrogen, phosphorus and potassium through orthogonal experiment; The flower of C. indicum traits were observed in the harvest period; The content of the dried flower active ingredient was measured using HPLC. Results The result showed the noticeable differences among the 14 groups in the number of capitulum, the diameter of capitulum and tubiform floret, yield per plant, total flavonoid content and linarin content as well as fertilizer efficiency of different mineral elements. Appropriate increase of N, K concentration and low P concentration was beneficial to the growth and development of C. indicum and the output of dried flowers. Low concentration of N, P, K promoted the cotent of linarin and total flavonoids; The content of linarin treated with different combination all accorded with the standard of Chinese Phamaeopoeia. Conclusion Compared comprehensively, it was concluded that the optimal proportion of nitrogen, phosphorus and potassium in the growth of chrysanthemum inflorescence15:0.1:3. Flavonoid and linarin content reached the highest point without fertilization.

Cloning and prokaryotic expression of CHI in Chrysanthemum morifolium cv.'Hangju' / 中国中药杂志

Three Chrysanthemum-chalcone-isomerase genes( CmCHI) were successfully cloned by PCR from the database of Chrysanthemum transcriptome and named CmCHI1,CmCHI2 and CmCHI3,respectively. Bioinformatics analysis showed that the base numbers of CmCHI1-3 open reading frame were 708,633 and 681 bp,encoding 235,210 and 226 amino acids,respectively. Three fusion proteins of about 30 kDa were successfully induced by prokaryotic expression technology,and the corresponding recombinant fusion proteins were isolated and purified by Ni-NTA resin column. Clustering analysis showed that the 3 CmCHI were homologous with Compositae plants,and CmCHI1 and CmCHI3 belonged to type Ⅰ CHI. CmCHI2 belongs to type Ⅳ CHI. Using β-actin as an internal reference gene,RT-qPCR was used to detect and analyze the expression of CmCHI1-3 genes in Hangju. The results showed that the expression levels of CmCHI1 and CmCHI3 were higher,while the expression levels of CmCHI2 were lower. It was concluded that CmCHI1 and CmCHI3 were the main chalcone isomerase genes involved in the synthesis of flavonoids in Hangju,and CmCHI2 was a helper gene. Flooding treatment significantly promoted the expression of CmCHI1 and CmCHI3 genes,but had no regulatory effect on CmCHI2. The above results provided a basis for further study of the molecular regulation mechanism of CHI gene in the metabolism of flavonoids in Hangju,which laid a foundation for improving the content of flavonoids in Hangju and finally improving the medicinal quality of Hangju.

Study on wild tending of Chrysanthemum indicum / 中国中药杂志

To provide theoretical basis for the formation of Chrysanthemum indicum resource and quality,the differences in biology,ecology,yield and quality of Ch. indicum population in the wild tending,the cultivated and the wild were analyzed and compared in this study. The results showed that in the aspect of biology,there was no significant difference between the condition of wild tending and cultivated Ch. indicum at the height of plant,both were significantly higher than wild Ch. indicum. In the aspect of ecology,in the condition of wild tending is less likely to occur water logging than cultivated Ch. indicum. In the condition of wild tending is less likely to occur plant diseases and insect pests than Ch. indicum in the condition of wild. In the aspect of production,the average output of the sample of the condition of wild tending was significantly higher than that the condition of artificial cultivation and wild quadrat. There was no significant difference between the three kinds of condition on the traits of capitulum dichasium,the content of buddleoside of the wild tending Ch. indicum was significantly higher than that of 0. 80% required by the Chinese Pharmacopoeia( 2015 edition). Therefore,the artificial supplement and the appropriate artificial management of wild tending is a mode of production to increase the population density of Ch. indicum and obtain high quality and high yield medicinal materials.

Effect of short-day treatment on expression of CO gene in Chrysanthemum indicum / 中国中药杂志

This experiment studied the expression pattern of key gene CO in the photoperiod of Chrysanthemum indicum. The CDS sequence of the Ch. indicum CO gene was cloned by RT-PCR. The open reading frame was 1 380 bp in length and encoded 459 amino acids. The bioinformatics analysis results showed that the Ch. indicum CO had higher homology with Ch. lavandulifolium and Artemisia annua,and the CO was more conservative in the same family. The molecular weight of the predicted protein encode by CO is 52. 04 k Da,the p I is 4. 81,the α-helix structure accounted for 17. 65%,the random coil accounted for 76. 69%,the extension chain accounted for 5. 66%,there are no β-fold and signal peptide. The experimental results showed that short-day treatment could increase the expression level of CO gene in Ch. indicum and induce its flowering. The results of qRT-PCR showed that the relative expression of CO gene in different tissues and different treatment periods of Ch. indicum was significantly different. In this paper,we studied the effect of short-day treatment on the expression of key genes in the flowering cycle of Ch. indicum,providing a basis for photoperiod regulation and harvesting period of Ch.indicum.

Identification of Chrysanthemum indicum and its adulterants based on ITS2 barcode / 中国中药杂志

DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum based on ITS2 sequences. The total DNA was extracted from 22 collected samples,and the ITS2 sequence was amplified by PCR and sequenced,and the information of ITS2 sequence was obtained. Another 14 items of the same family or the same genus were downloaded from Gen Bank.We aligned all 36 sequences,calculated the intraspecific and interspecific distances,and constructed Neighbor Joining( NJ) phylogenetic tree,using MEGA 7. 0. The difference of the secondary structure between the ITS2 sequences was compared. The results showed that the genetic distance of Ch. indicum and Ch. morifolium was overlapped,but the maximum intraspecific distance was far less than the minimum interspecific distance between and among Ch. indicum and other species,with an obvious barcoding gap. The NJ tree showed that Ch. indicum and Ch. morifolium shared a clade,and most of Ch. morifolium with some Ch. indicum were shared a subclade,while Inula lineariifolia,Sinosenecio oldhamianus and Senecio scandens belonged to one clade separately. ITS2 secondary structures for I. lineariifolia,S. oldhamianus and S. scandens were significantly different enough to identify completely but Ch. indicum and Ch. morifolium shared two secondary structures of A and B. It was proved that Ch. indicum was one of the evolutionary sources of Ch.morifolium. Therefore ITS2 sequence as DNA barcode can identify Ch. indicum and its adulterants accurately and quickly. The study provides an important basis for Ch. indicum for the identification of germplasm resources and the safety of clinical medication.

Prokaryotic expression and in vitro enzyme activity analysis of flavonol synthase in Chrysanthemum morifolium cv. 'Hangju' / 中国中药杂志

We cloned flavonol synthase gene (named as CmFLS) by RACE from Chrysanthemum morifolium cv. 'Hangju' based on transcriptome database. Sequencing results showed that 1 235 bp sequence was acquired with the largest open reading frame (ORF) of 1 008 bp, which encoded 335 amino acids. The predicted CmFLS encoded protein had an isoelectric point (pI) of 5.41. The phylogenetic tree analysis indicated that CmFLS was highly homologous to other FLSs, which identified from the species of Compositae. The recombinant fusion protein, with a molecular mass of 43 kDa, was successfully expressed by prokaryotic expression system. Meanwhile, Ni-NTA resin was used to purify the recombinant fusion protein, and the Ni-Native Buffer containing 250 mmol·L⁻¹ imidazole was most favorable for elution. The purified recombinant fusion protein was subjected to in vitro catalytic reaction, and then the products were extracted and analyzed by HPLC. The results showed that the recombinant fusion protein CmFLS was able to catalyze the production of quercetin by dihydroquercetin under specific buffer and reaction conditions, which indicated that the functional protein encoded by CmFLS had dioxygenase activity in the biosynthetic pathway of flavonoids biosynthesis in Ch. morifolium cv. 'Hangju'. The above results laid the foundation for further studying on CmFLS, and provided new ideas for the regulation of flavonoids metabolism from the molecular level and the catalytic synthesis of flavonols in vitro.

Cloning and expression analysis of and quantification of downstream products in under flooding stress / 中国中药杂志

To investigate the effects of the expression of flavonoid 3' hydroxylase gene ( and active ingredients in under flooding stress, we cloned F3'H from Hangju (temporarily named ) and conducted bioinformatics analysis. During the flower bud differentiation stage, we flooded the and then used the Real-time PCR to detect the relative expression of ; Finally, active ingredients of the inflorescence were measured by HPLC.The sequencing results showed that 1 562 bp sequence was acquired with the largest open reading frame of 1 527 bp, which encoded 508 amino acids. The phylogenetic tree found that was highly homologous to other species of Compositae. Real-time PCR results showed that had a significant response to flooding stress and had the highest expression level after flooding for 24 h, which was about 9 times as that of the control group. The results of HPLC showed that luteolin and luteoloside, the downstream products catalyzed by the F3'H, were significantly higher than those in the control group. It was also found that the contents of chlorogenic acid and 3,5- acid were also significantly higher than those of the control group. Therefore, regulates the synthesis of downstream products by regulating the expression of in the flavonoid synthesis pathway under flooding stress, thereby responding to flooding stress. The flooding stress during flower bud differentiation can significantly enhance the accumulation of active ingredients.

Comparative study on content of heavy metals in Chrysanthemum indicum and soil in different areas / 中国中药杂志

ICP-MS was used to determine the contents of five heavy metals such as lead (Pb), cadmium (Cd), mercury (Hg), arsenic (As) and copper (Cu) in Chrysanthemum indicum and soil samples. Pearson correlation and path analysis were used to analyze the effects of latitude and longitude and altitude on the content of heavy metal elements in Ch. indicum. To explore the relationship between geographical location of heavy metal content in Ch. indicum and the content of heavy metal elements in soil. The results showed that the Ch. indicum had high enrichment of Cd and had a synergistic effect on the absorption of five heavy metal elements. Elevation directly affected the accumulation of Pb and Cd in Ch. indicum and was a positive effect. The direct positive effect of latitude acts on the accumulation of Hg and Cu in Ch. indicum. The effect of longitude on the Cd content was the negative effect of longitude. The geographical location had different effects on the accumulation of heavy metal elements in Ch. indicum. There was a certain interaction between direct and indirect effects. Ch. indicum had different absorption and enrichment characteristics for different heavy metal species. When controlling the heavy metal content and medicinal material quality of Ch. indicum, we must fully consider the influence of geographical location on the quality of medicinal materials, select suitable geographic regions to carry out acquisitions, and choose a good ecological environment. Regional establishment of Chinese herbal medicine planting bases, or adopting certain agricultural measures to reduce the absorption of heavy metal elements in Ch. indicum herbs, while conducting research on chemical composition, strengthening the research on the effective forms of heavy metals, and paying attention to the safety of medicinal herbs.