Genetic diversity analysis and fingerprints of Chrysanthemum×morifolium based on SSR molecular markers / 生物工程学报

The present study aims to explore the genetic diversity of germplasm resources of Chrysanthemum×morifolium (hereinafter, C.×morifolium) at the molecular level and to establish a fingerprint database of C.×morifolium varieties. We employed 12 pairs of primers with high levels of polymorphism, clear bands, and high degrees of reproducibility to analyze the SSR molecular markers and genetic diversity of 91 C.×morifolium materials and 14 chrysanthemum- related materials. With regard to constructing the fingerprints of the tested materials, we chose 9 pairs of core primers. The findings revealed that 12 primer pairs detected 104 alleles in 105 samples, ranging from 2 to 26. The average number of observed alleles (Na) per site was 9.25. The average number of effective alleles (Ne) per site was 2.745 6, with its range being 1.276 0 to 4.742 5. Shannon genetic diversity index (I) values ranged between 0.513 3 and 2.239 9 (M=1.209 0). Nei's gene diversity index (H) ranged between 0.216 3 and 0.789 1 (M=0.578 0). The observed heterozygosity (Ho) ranged between 0.223 3 and 0.895 2 (M=0.557 5). The expected heterozygosity (He) ranged between 0.217 4 and 0.793 3 (M=0.580 8). The polymorphism information content (PIC) ranged between 0.211 5 and 0.774 0 (M=0.532 9). The genetic similarity (GS) ranged between 0.228 5 and 1.000 0 (M=0.608 3). Cluster analysis revealed that when the genetic distance (GD) equals to 0.30, the tested materials can be classified into 2 groups. When the GD equals to 0.27, the first group can be divided into 6 subgroups; accordingly, 105 tested materials can be divided into 7 subgroups. The cophenetic correlation test was carried out based on the cluster analysis, and the corresponding results showed that the cluster map correlated with the genetic similarity coefficient (r=0.952 73). According to the results of Structure population analysis, we obtained the optimal population number, with the true number of populations (K) being 3 and the population being divided concerning Q≥0.5. Three subgroups, i.e., Q1, Q2 and Q3, included 34, 33 and 28 germplasms, respectively, and the remaining 10 germplasms were identified as the mixed population. During the experiment, 9 pairs of core primers were screened among the total of 12 for a complete differentiation regarding 105 tested materials, and the fingerprints of 91 C.×morifolium materials and 14 chrysanthemum-related materials were further constructed. Overall, there were significant genetic differences and rich genetic diversity among C.×morifolium materials, which would shed light on the garden application and variety selection fields of C.×morifolium. The fingerprint database of 105 C.×morifolium varieties and chrysanthemum-related species may provide technical support for future research regarding the identification and screening system of C.×morifolium varieties.

A new polyacetylene from the stems and leaves of Chrysanthemum morifolium Ramat / 药学学报

Eight polyacetylenes were isolated from the extract of the stems and leaves of Chrysanthemum morifolium by various chromatographic methods. Their structures were determined as 2E,4E,12Z-tetradecatriene-1-pyrrolidine-1-oxo-8,10-diynoic (1), tetradeca-2E,4E,12E-trien-8,10-diynoic acid pyrrolidide (2), tetradeca-2E,4E-dien-8,10-diynoic acid pyrrolidide (3), tetradeca-2E,4E,10Z-trien-8-ynoic acid pyrrolidide (4), 2E,4E,12E-tetradecatriene-8,10-diynoic acid isobutylamide (5), 2E,4E-undecyldiene-8,10-diynoic acid isobutylamide (6), 2E,4E,10E-N-isobutyl-2,4,10-tetradecatrien-8-ynoic acid amide (7), and undeca-2E,4E-diene-8,10-diynoic acid phenylethylamide (8) by spectroscopic methods, including UV, IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR spectra. Among them, compound 1 is a new polyacetylene, and compounds 2-8 were isolated from this plant for the first time. Compounds 5-8 inhibited the proliferation of A549 cell significantly at certain concentration, showing potent antitumor activity.

Content characteristics of mineral elements and its correlation with soil nutrients and effective components in Chrysanthemum morifolium from different producing areas in Macheng / 中国中药杂志

In this study, 24 copies of samples of Chrysanthemum morifolium and soil from two main production towns in Macheng city were collected, and the contents of 13 mineral elements, 5 effective components and 14 soil nutrient factors in Ch. morifolium were determined. The enrichment characteristics of available soil nutrients by mineral elements were analyzed and the dominant factors affecting the effective components of Ch. morifolium were screened. The results showed that the content of mineral elements and soil nutrients and effective components are very different, and variation of soil fertility was much greater than that of inorganic elements in chrysanthemum plants. In general, the level of element content in Ch. morifolium from different producing areas is K>N>P>Mg>Ca>Fe>Mn>Zn>Cu>Ni>Cr>Pb>Cd. The content of K, N and Mg is higher than that of common crops, and the content of Cu, Cd and Pb in Ch. morifolium from various producing areas does not exceed the relevant standards. The N, P and K enrichment capacity in soil was stronger than that of other elements, and the Ca enrichment ability was the worst. The content of AvCu in the soil was positively correlated with the contents of N, Mg, K, Fe and Cu elements in Ch. morifolium. The contents of chlorogenic acid, luteolin, 3,5-O-dicaffeoylquinic acid reached the pharmacopoeia standard. The percentage of chlorogenic acid and 3,5-O-dicaffeoylquinic acid in Ch. morifolium that from Huangtugang town in the active components were generally higher than that from Futianhe town, and the diffe-rences of luteolin contents in the two producing areas were relatively small. The correlation and regression analysis showed that the contents of Cu, Zn and Cr in Ch. morifolium were positively correlated with the active components, while the contents of Fe, Mn and Ni were negatively correlated with the contents of AvP, AvK, TK, AvMn and AvCu in soil. In general, Zn and Ca fertilizer should be added to the ecological planting of Ch. morifolium, K fertilizer should be added, and N and P fertilizer should be applied appropriately.

Analysis and evaluation of mineral elements of Chrysanthemum morifolium for medicinal and tea use of different germplasm resources / 中国中药杂志

In this study, 23 germplasm resources of Chrysanthemum morifolium used in medicine and tea were collected from Dabie Mountains and its surrounding producing areas, and the contents of 13 mineral elements were determined and compared. The thermal maps of correlation analysis, principal component analysis and cluster analysis were used for comprehensive evaluation. The results showed that the average content of each element in Ch. morifolium of different germplasm resources was: K>N>P>Mg>Ca>Fe>Mn>Zn>Cu>Ni>Cr>Pb>Cd, and the leaves were: K>N>Ca>Mg>P>Fe>Mn>Zn>Cr>Cu>Ni>Pb>Cd. There are rich contents of N, P, K, Ca, Mg and Fe in Ch. morifolium flowers and their leaves, among them, K element has the largest change range, while N, Ca, Fe, Mg and Zn elements have a larger change range. The absorption and accumulation of each element in the leaves of different germplasm resources varied greatly. The correlation analysis shows that there is a strong positive correlation between Ca element, Mg, Mn and Cd element.Principal component analysis in Ch. morifolium flowers characteristic elements for Mn, Cr, Cu, P, K, can be used as a Ch. morifolium resources to identify the characteristics of the elements, choose top five principal component(F1-F5) comprehensive evalua-tion of medicinal Ch. morifolium, scored in the top five varieties for Hangiu-Fuhuangju, Hangju-Xiaoyangju, Hangju-Sheyangju, Hangju-Dayanghua, Hangju-Subeiju,indicates that in terms of mineral elements, the five medicinal Ch. morifolium resources quality is better. The PCA score chart can divide 23 Ch. morifolium resources into 4 groups, and the cluster analysis heat map divides 23 Ch. morifolium resources into 5 groups. All the Ch. morifolium resources of the same type can be well clustered together, indicating that the difference in mineral element content of Ch. morifolium germplasm resources is closely related to genetic factors.

Fusarium Wilt Changes Microbial Community Structure in Rhizosphere Soil of Chrysanthemum morifolium / 中国实验方剂学杂志

Objective:To explore the differences in rhizosphere microbial community structure between <italic>Fusarium</italic> wilt-infected and healthy <italic>Chrysanthemum morifolium </italic>plants<italic>.</italic> Method:The rhizosphere soils of diseased and healthy<italic> C. morifolium </italic>plants were sampled and subjected to high-throughput 16S ribosomal DNA （rDNA） and internal transcribed spacer （ITS） sequencing， to identify the microbial community structure including bacteria and fungi. Result:<italic>Fusarium</italic> wilt reduced the bacterial abundance and diversity but had no significant effect on fungal alpha-diversity.The proportions of Acidobacteria， Gemmatimonadetes， and Nitrospirae in rhizosphere soil of healthy <italic>C.morifolium</italic> plants were higher than those of diseased plants， while the proportions of Proteobacteria and Bacteroidetes were lower（<italic>P</italic><0.05）. <italic>Fusarium</italic> fungi accounted for 27.49%， 14.53%， and 11.94% in diseased plants whereas 0.47%， 1.01%， and 0.67% in healthy plants.Pathogenic bacteria <italic>Pectobacterium</italic> and <italic>Dickeya</italic> were enriched in rhizosphere soil of diseased plants. The abundances of nitrifying， detoxifying， and photosynthetic bacteria in rhizosphere soil of healthy plants were higher than those of diseased plants. Conclusion:<italic>Fusarium</italic> wilt reduces the bacterial richness and diversity and triggers the enrichment of massive <italic>Fusarium</italic> fungi， <italic>Pectobacterium</italic>， and <italic>Dickeya</italic>. The proportion of beneficial bacteria in rhizosphere soil of healthy plants is significantly higher than that of diseased plants.

Status and prospects of resource utilization of non-medicinal parts of medicinal Chrysanthemum morifolium / 中草药

Chrysanthemum morifolium is a traditional Chinese medicinal material in China. The yield of non-medicinal parts is much higher than the inflorescence, and the yield of stems and leaves of C. morifolium is 3.5 times of medicinal parts. For a long time, the non-medicinal parts of C. morifolium have not been fully used, resulting in great waste of resources and environmental pollution. Therefore, the in-depth research and development of non-medicinal parts of C. morifolium deserve attention. Research shows that the non-medicinal parts of C. morifolium is rich in volatile oil, flavonoids, phenolic acids, polysaccharides and other components, which have antibacterial, anti-inflammatory, antioxidant, anti-convulsion and improvement of intestinal disorders. This article summarizes the research situation of chemical components, pharmacological effects, and resource utilization status of stems, leaves, roots and other non-medicinal parts produced during the cultivation and production of medicinal C. morifolium, in order to provide the scientific basis and reference for the development, utilization and industrialization of the non-medicinal parts of medicinal C. morifolium.

Effects of planting period on growth, yield and medicinal quality of Chrysanthemum morifolium / 中国中药杂志

In this paper, Chrysanthemum morifolium was used as the experimental object, eight different planting periods were set up in field plot experiment from April to August, which were 04-15, 05-19, 05-30, 06-09, 06-19, 07-20, 07-31, 08-15. The effects of different treatments on the occurrence of root rot, agronnmic traits, mineral element absorption and content of effective components of Ch. morifolium in Macheng country of Hubei province were studied. The results showed that delaying the planting time could effectively reduce the diseases occurrence of root rot of Ch. morifolium. With the advance of transplanting period, the plant height, the weight of one hundred flowers and the number of flowers of Ch. morifolium showed a trend of gradual decrease, while the number of primary branches and the thickness of main stem and the primary branch increased first and then decreased. The yield of Ch. morifolium per plant and per mu increased with the advance of the planting period, and the yield per mu increased during the planting period on June 19, which was 91.96% higher than that on April 15. And with the delay of the planting period,the absorption and accumulation of potassium(K) elements was promotes. The content of active ingredients such as chlorogenic acid, rutin, luteolin, and 3,5-O dicoffeoacy lquinic acid in the Ch. morifolium increased significantly and then gradually decreased with the delay of the planting period, which indicates that late planting can significantly improve the quality of Ch. morifolium. Considering factors such as the occurrence of root rot disease, yield and active ingredient content, combined with climatic conditions in the Dabie Mountains in eastern Hubei, the optimum planting period of Ch. morifolium was determined from mid-late June to early July.

Cloning and expression of key enzyme genes for chlorogenic acid synthesis in Chrysanthemum morifolium / 中草药

To investigate the effects of the expression of coumaroylquinate 3’-monooxygenase (C3’H) and shikimate O-hydroxycinnamoyltransferase (HCT) in the chlorogenic acid-producing pathway and active ingredients in Chrysanthemum morifolium under flooding stress, we cloned two C3’H genes which were CmC3’H1 and CmC3’H2 and two HCT genes which were CmHCT1 and CmHCT2 by the RT-PCR from Hangju and conducted bioinformatics analysis. During the flower bud differentiation stage, we flooded the C. morifolium and then used β-actin as the reference gene to detect the relative expression of the four genes by the qRT-PCR. Finally, the content of downstream products and other indicators of these four genes in C. morifolium were measured by HPLC. We obtained the four genes’ complete open reading frame and predicted the relative molecular mass of the amino acid sequence and the theoretical isoelectric point (pI). And the protein tertiary structure models were constructed. The qRT-PCR results showed that flooding the C. morifolium for 3 days during the flower bud differentiation stage resulted in significant expression changes of the four genes at different growth stages. The results of HPLC showed that chlorogenic acid, the downstream product catalyzed by the C3’H and the HCT, was significantly higher than that in the control group. It was also found that the content of luteoloside and 3,5-O-di-caffeoylquinic acid was also significantly higher than those of the control group. Therefore, C. morifolium regulates the synthesis of downstream products by regulating the expression of the four genes under flooding stress, thereby responding to flooding stress. And the flooding stress during flower bud differentiation can significantly enhance the accumulation of active ingredients of C. morifolium.

Extracts from stems and leaves of Chrysanthemum morifolium ameliorate gut dysfunction by a fecal metabolomic approach / 中草药

Objective: To holistically evaluate the protective effects of water and ethanol extracts of stems and leaves of Chrysanthemum morifolium and its underlying mechanism. Methods: Gut dysfunction model was established by injection of liposaccharide in rabbits, and administrated with water and ethanol extracts of stems and leaves of C. morifolium to investigate the treatment. Feces of rabbits in each group were collected and analyzed by 1H-NMR complemented with multivariate statistical methods to evaluate the metabolic alteration. Results: Compared with the control group, the levels of lactate and formate in liposaccharide intoxicated model group were significantly increased, and the concentrations of propionate, glutamine, glutamate, aspartate were notably decreased. Both the water and ethanol extracts of stems and leaves of C. morifolium ameliorated gut dysfunction of rabbits in a similar manner, increased the decreased levels of aspartate, adenine, phenylalanine, tyrosine induced by liposaccharide, and reduced the elevated content of formate. Conclusion: Pathway analysis revealed that both the water and ethanol extracts of stems and leaves of C. morifolium could regulate the disturbed phenylalanine, tyrosine and tryptophan biosynthesis; disordered alanine, aspartate and glutamate metabolism and imbalanced glycine, serine and threonine amino acid metabolism, exerting a holistic protective effect on gut disorder. Thus, this study lays a scientific foundation for the resource utilization of stems and leaves of C. morifolium after the harvest of the inflorescence.

Research progress on chemical constituents and pharmacological effects of Chrysanthemum morifolium and predictive analysis on quality markers / 中草药

As a kind of Chinese medicinal material, Chrysanthemum morifolium is numerous and widely distributed, which has high medicinal value. C. morifolium has a good effect in medicinal and health care, and it is also the first batch of being used for both medicine and food issued by Ministry of Health of China. In recent years, C. morifolium has been widely used in medicine and health foods. Flavonoids, volatile oils, and phenylpropanoids are its main effective components. Based on the review of its chemical composition and pharmacological effects, combined with the definition of Q-marker, this study processed predictive analysis on Q-marker of C. morifolium at aspects of chemical composition, clinical efficacy, pharmacokinetics, traditional medicinal properties, traditional pharmacodynamics, different storage conditions, and different processing methods, which can establish scientific quality standards of C. morifolium.

Qualitative and quantitative analyses of Chrysanthemum morifolium ‘Fubaiju’ by LC-MSn and HPLC-UV / 中国药科大学学报

@#A qualitative analytical method of liquid chromatography coupled with mass spectrometry(LC-MSn)was developed for the identification of main constituents in Chrysanthemum morifolium ‘Fubaiju’. High-performance liquid chromatography(HPLC)was developed for the quantification of five active components, including chlorogenic acid(1), luteolin-7-O-β-D-glucopyranoside(2), luteolin-7-O-β-D-glucopyranuronide(3), 3, 5-Di-caffeoylquinic acid(4), and apigenin-7-O-β-D-glucopyranoside(5). A total of 22 compounds, including 13 flavonoids and 9 phenolic acids, were identified based on their retention behaviors, UV profiles and MS fragment information. Furthermore, a validation method with good linearity(r> 0. 999 9), precision, stability, repeatability and recovery was successfully applied for simultaneous determination of five major components in 10 batches of C. morifolium ‘Fubaiju’ by HPLC-UV method. The established method was proved to be a validation strategy for the quality evaluation of C. morifolium ‘Fubaiju’.

Study on Quality Control Components in Water Extract of Chrysanthemum morifolium and Network Pharmacology Effects / 中国药房

OBJECTIVE： To screen the quality control components of Chrysanthemum morifolium based multiple component metabolism， and study its network pharmacology effect. METHODS： The water extract of C. morifolium was prepared. A total of one rats were selected， water extract of C. morifolium was perfused in jejunum segment after abdominal anesthesia； plasma sample 1 was collected by double perfusion collection. Other 3 rats were given water extract of C. morifolium intragastrically， and plasma sample 2 was collected by abdominal aorta blood collection. UPLC-MS/MS was used to analyze water extract of C. morifolium and plasma sample component， and prototype blood-entry component in water extract of C. morifolium was identified after metabolism. TCMSP and Swiss Target Prediction database were used to screen the core target of prototype blood-entry component. DAVID database was used to enrich the related pathways of core target. The quality control components were screened according to topological parameters. Cytoscape software was used to analyze pharmacological effect of quality control components of C. morifolium. RESULTS： After UPLC-MS/MS analysis， 27 compounds were identified in water extract of C. morifolium， among which there were 12 prototype blood-entry components. After network pharmacology analysis， 7 quality control components were identified， i.e. cosmosiin， apigenin-7-O-glucuronide， luteolin， tilianin， apigenin， hesperetin， acacetin. It was possible to treat cancer， cardiovascular and cerebrovascular diseases， and neurological diseases by acting on metabolic pathway， cancer related pathway， signal transduction related pathway， adipocyte lipolysis regulatory pathway， etc. CONCLUSIONS： The study screen the possible quality control components of water extract of C. morifolium. The theoretical pharmacological effect of it can be clarified through network pharmacology， which can provide a new idea for the utilization of C. morifolium.

Identification of Chrysanthemum indicum in different geographical populations and Ch. morifolium based on DNA barcodes of psbA-trnH,matK and trnL / 中国中药杂志

DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum and Ch. morifolium based on psbA-trn H,mat K and trn L sequences. The total DNA was extracted from 21 samples collected,and the psbA-trn H,mat K,trn L sequences were amplified by PCR and sequenced. The information of these sequences were obtained. We aligned all 63 sequences,calculated the intraspecific and interspecific distances,analysed the SNPs distribution of psbA-trn H+mat K+trn L combination sequences and constructed the Neighbor-joining( NJ) Tree,using MEGA 7. 0. The results showed that the genetic distances of Ch. indicum,Ch. indicum( Juhuanao)and Ch. morifolium were overlapped. The SNPs analysis of psbA-trn H+mat K+trn L combination sequences showed that there were 19 nucleotide polymorphism loci( SNPs) and nine parsim-informative sites in the combination sequences. In addition,Ch. indicum showed more obvious sequence polymorphism than those of Ch. indicum( Juhuanao) and Ch. morifolium. The psbA-trn H sequences showed obvious length variation.The NJ Tree showed that Ch. morifolium numbered C2-C5 were clustered into a single subbranch with a bootstrap value of 62%,and Ch.morifolium could be distinguished from Ch. indicum and Ch. indicum( Juhuanao). Moreover,Ch. indicum numbered Z9 and Z10 collected from Gansu province were singly clustered into one branch with a bootstrap value of 77%. It was also found that the changes of psbA-trn H and trn L sequences information of Ch. indicum samples from the northwest were obviously related to the geography and environment. Moreover,Ch.indicum and Ch. indicum( Juhuanao) had obvious differentiation,were also regarded as the evolutionary sources of Ch. morifolium. Therefore,psbA-trn H+mat K+trn L combination sequences as DNA barcode can identify Ch. indicum and Ch. morifolium accurately and rapidly,which provides an important basis for germplasm resources identification and species identification.

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.

Caffeoyl quinic acids in Chrysanthemum morifolium improve LPS-induced HUVEC vascular endothelial cell injury by regulating ERK/MAPK signaling pathway / 药学学报

To explore the effect of total extract of Chrysanthemum morifolium on lipopolysaccharide (LPS)-induced acute lung injury in mice, we studied the effects of three caffeoyl quinic acids isolated from Chrysanthemum morifolium on vascular endothelial cell injury and their mechanisms of action. All animal experiments were carried out strictly in accordance with the National Animal Welfare Ethics and Protection Regulations. A mouse model of acute lung injury was established by intranasal instillation of LPS. After 6 days of oral administration of chrysanthemum extract, the lung wet weight/dry weight ratio, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were measured in mouse bronchoalveolar lavage fluid. Human umbilical vein endothelial cells (HUVEC) were serum starved for 12 h and treated with 2.5 μg·mL-1 LPS for 24 h to establish the in vitro model of vascular endothelial cell injury. After 24 h of treatment of caffeoyl quinic acids from Chrysanthemum morifolium, the levels of TNF-α, IL-6, IL-1β, vascular cell adhesion molecule-1 (VCAM-1) and endothelin-1 (ET-1) were measured by ELISA in the cell culture supernatant, the malondialdehyde (MDA) level was detected by TBA method, the superoxide dismutase (SOD) level was determined by hydroxylamine method, and the nitric oxide (NO) level was assayed by a one-step method. The levels of p-MEK1/2, MEK1/2, p-ERK1/2, ERK1/2, p-JNK, JNK, p-P38 and P38 of mitogen-activated protein kinase (MAPK) signaling pathway were detected by Western blot. The total extract of Chrysanthemum morifolium can significantly reduce the wet weight/dry weight ratio of lung in mice and the levels of TNF-α, IL-6 and IL-1β in alveolar lavage fluid. The caffeoyl quinic acids from Chrysanthemum morifolium significantly increased the levels of SOD and NO, decreased the levels of TNF-α, IL-6, IL-1β, VCAM-1, ET-1 and MDA, and significantly reduced the levels of p-MEK1/2, p-ERK1/2. In conclusion, total extracts of Chrysanthemum morifolium exhibit certain protective effect on mice with acute lung injury, and three caffeoyl quinic acids from Chrysanthemum morifolium may improve LPS-induced vascular endothelial cell injury by inhibiting inflammatory cytokines and oxidative stress, and regulating inter-cellular adhesion molecule and vasomotor factors through ERK/MAPK signaling pathway.

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.

Enzymatic characteristics of peroxidase from Chrysanthemum morifolium cv. Bo-ju / 中国中药杂志

The enzymatic browning is one of the main reasons for affecting the quality of medicinal flowers. In the process of chrysanthemum harvesting and processing, improper treatment will lead to the browning and severely impact the appearance and quality of chrysanthemum. Peroxidase enzyme is one of the oxidoreductases that cause enzymatic browning of fresh chrysanthemum. The enzymatic characteristics of peroxidase (POD) in chrysanthemum were studied in this paper. In this experiment, the effects of different reaction substrates and their concentrations, PH value of buffer and reaction temperatures on the activity of POD enzyme were investigated. The results showed that the optimal substrate of POD was guaiacol, and the optimal concentration of POD was 50 mmol·L⁻¹. The optimal pH value and reaction temperature were 4.4 and 30-35 °C, respectively. Michaelis-Menten equation was obtained to express the kinetics of enzyme-catalyzed reaction of POD, Km=0.193 mol·L⁻¹, Vmax=0.329 D·min⁻¹. In addition, the results of POD enzyme thermal stability test showed that the POD enzyme activity was inhibited when being treated at 80 °C for 4 min or at 100 °C for 2 min. The above results were of practical significance to reveal the enzymatic browning mechanism, control the enzymatic browning and improve the quality of chrysanthemum, and can also provide the basis for the harvesting and processing of medicinal materials containing polyphenols.

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.

Review of the Changes of the Species and Processing Methods of Chrysanthemum morifolium / 中国药学杂志

Chrysanthemum morifolium has a long history of culture and use in China. Due to different germplasm resources, producing areas, and processing methods, many cultivated varieties have formed now. The varieties and processing methods of C. morifolium are affected by economic interests and processing cost, which change gradually. On the basis of spot investigation and related literature study, the changes of the varieties and processing methods of C. morifolium were summarized in this paper. It will provide theoretical evidence for the culture, processing, quality evaluation, and clinical application of C. morifolium.

Flavonoids from the flowers of Chrysanthemum morifolium Ramat / 中国药学杂志

OBJECTIVE: To study the chemical constituents in the flowers of Chrysanthemum morifolium Ramat. METHODS: The compounds were isolated with Diaion HP-20, Toyopearl HW-40, Sephadex LH-20, silica gel column chromatography and preparative HPLC. The structures of the compounds were identified by physiochemical properties and spectral analysis. RESULTS: Twenty compounds were obtained, and their structures were identified as luteolin (1), apigenin (2), acacetin (3), diosmetin (4), acacetin 7-O-β-D-glucoside (5), acacetin 7-O-β-D-glucoside (6), acacetin7-O-(6″-O-acetyl)-β-D-glucoside (7), eriodictyol (8), naringenin (9), artemetin (10), 5-hydroxy-6, 7, 3', 4'-tetramethoxyflavone (11), 5, 7-dihydroxy-3', 4'-dimethoxyflavone (12), 4'-methoxyctricin (13), 3', 5'-dimethoxy-4', 5, 7-trihydroxyflavone (14), 5, 6-dihydroxy-3, 7, 3', 4'-tetramethoxyflavone (15), luteolin 7-O-β-D-glucuronide methyle ester (16), dihydroquercetin-7-β-D-glucoside (17), quercetin 3-O-β-D-glucoside(18), quercetin 3-O-β-D-glucoside (19), and acacetin 7-O-β-(6″-(E)-crotonylglucopyranoside) (20). CONCLUSION: Compounds 9-20 were isolated for the first time from this plant.