Optimal harvest period and quality control markers of cultivated Flos Chrysanthemi Indici using untargeted/targeted metabolomics, chemometric analysis and in vivo study.

ETHNOPHARMACOLOGICAL RELEVANCE: Flos Chrysanthemi Indici (FCI), the flower of Chrysanthemum Indicum L., is a popular traditional Chinese medicine (TCM) for treatment of inflammatory diseases in China. FCI is also a functional food, and is widely used as herbal tea for clearing heat and detoxicating. AIM OF THE STUDY: To explore quality control markers of FCI based on the optimal harvest period. MATERIALS AND METHODS: First, UPLC-Q-TOF/MS based untargeted metabolomics was applied to explore the chemical profiles of FCIs collected at bud stages (BS), initial stages (IS), full bloom stages (FS) and eventual stages (ES) from eight cultivated regions in China. Subsequently, lipopolysaccharide (LPS)-induced RAW264.7 cell inflammatory model and carrageenan-induced rat paw edema model were used to confirm the anti-inflammatory effect of FCIs collected at IS/FS. Then, UPLC-PDA targeted metabolomics was used to quantitatively analyze 9 constituents with anti-inflammatory activity (7 flavonoids and 2 phenolic acids) changed significantly (VIP > 4) during flowering stages. Finally, ROC curves combined with PCA analysis based on the variation of 9 active constituents in FCIs from different flowering stages were applied to screen the quality markers of FCI. RESULTS: FCIs at IS/FS had almost same chemical characteristics, but quite different from those at BS and ES. A total of 32 constituents in FCIs including flavonoids and phenolic acids were changed during flowering development. Most of the varied constituents had the highest or higher contents at IS/FS compared with those at ES, indicating that the optimal harvest period of FCI should be at IS/FS. FCI extract could effectively suppress nitric oxide (NO) production in LPS-induced RAW264.7 cells and regulate the abnormal levels of cytokines and PGE2 in carrageenan-induced paw edema model rat. The results of quantitatively analysis revealed that the variation trends of phenolic acids and flavonoids in FCIs were different during flowering development, but most of them had higher contents at IS/FS than those at ES in all FCIs collected from eight cultivated regions, except one sample from Anhui. Finally, linarin, luteolin, apigenin and 3,5-dicaffeoylquinic acid were selected as the Q-markers based on the contribution of their AUC values in ROC and clustering of PCA analysis. CONCLUSIONS: Our study demonstrates the optimal harvest period of FCI and specifies the multi-constituents Q-markers of FCI based on the influence of growth progression on the active constituents using untargeted/targeted metabolomics. The findings not only greatly increase the utilization rate of FCI resources and improve quality control of FCI products, but also offer new strategy to identify the Q-markers of FCI.

The haplotype-resolved genome of diploid Chrysanthemum indicum unveils new acacetin synthases genes and their evolutionary history.

Acacetin, a flavonoid compound, possesses a wide range of pharmacological effects, including antimicrobial, immune regulation, and anticancer effects. Some key steps in its biosynthetic pathway were largely unknown in flowering plants. Here, we present the first haplotype-resolved genome of Chrysanthemum indicum, whose dried flowers contain abundant flavonoids and have been utilized as traditional Chinese medicine. Various phylogenetic analyses revealed almost equal proportion of three tree topologies among three Chrysanthemum species (C. indicum, C. nankingense, and C. lavandulifolium), indicating that frequent gene flow among Chrysanthemum species or incomplete lineage sorting due to rapid speciation might contribute to conflict topologies. The expanded gene families in C. indicum were associated with oxidative functions. Through comprehensive candidate gene screening, we identified five flavonoid O-methyltransferase (FOMT) candidates, which were highly expressed in flowers and whose expressional levels were significantly correlated with the content of acacetin. Further experiments validated two FOMTs (CI02A009970 and CI03A006662) were capable of catalyzing the conversion of apigenin into acacetin, and these two genes are possibly responsible acacetin accumulation in disc florets and young leaves, respectively. Furthermore, combined analyses of ancestral chromosome reconstruction and phylogenetic trees revealed the distinct evolutionary fates of the two validated FOMT genes. Our study provides new insights into the biosynthetic pathway of flavonoid compounds in the Asteraceae family and offers a model for tracing the origin and evolutionary routes of single genes. These findings will facilitate in vitro biosynthetic production of flavonoid compounds through cellular and metabolic engineering and expedite molecular breeding of C. indicum cultivars.

The CmMYB3 transcription factors isolated from the Chrysanthemum morifolium regulate flavonol biosynthesis in Arabidopsis thaliana.

KEY MESSAGE: Chrysanthemum morifolium MYB3 factors are transcriptional activators for the regulation of flavonol biosynthesis. Flavonol was not only the critical secondary metabolite participating in the growth and development of plants but also the main active ingredient in medicinal chrysanthemum. However, few pieces of research revealed the transcriptional regulation of flavonol biosynthesis in Chrysanthemum morifolium. Here, we isolated two CmMYB3 transcription factors (CmMYB3a and CmMYB3b) from the capitulum of Chrysanthemum morifolium cv 'Hangju'. According to the sequence characteristics, the CmMYB3a and CmMYB3b belonged to the R2R3-MYB subgroup 7, whose members were often reported to regulate flavonol biosynthesis positively. CmMYB3a and CmMYB3b factors were identified to localize in the nucleus by subcellular localization assay. Besides, both of them have obvious transcriptional self-activation activity in their C-terminal. After the overexpression of CmMYB3 genes in Nicotiana benthamiana and Arabidopsis thaliana, the flavonol contents in plants were increased, and the expression of AtCHS, AtCHI, AtF3H, and AtFLS genes in A. thaliana was also improved. Interestingly, the CmMYB3a factor had stronger functions in improving flavonol contents and related gene expression levels than CmMYB3b. The interaction analysis between transcription factors and promoters suggested that CmMYB3 could bind and activate the promoters of CmCHI and CmFLS genes in C. morifolium, and CmMYB3a also functioned more powerfully. Overall, these results indicated that CmMYB3a and CmMYB3b work as transcriptional activators in controlling flavonol biosynthesis.

Comparison of metabolome characteristics and screening of chemical markers in Chrysanthemum indicum from different habitats.

Chrysanthemum indicum is a polymorphic species with many ecological, geographical or eco-geographic populations, but there are few studies on the metabolic characteristics of different populations. This study conducted widely targeted metabolomics studies on Ch. indicum from seven typical producing areas. As a result, a total of 802 metabolites were detected and identified, among which the top three categories of metabolites were flavonoids, organic acids and amino acids and derivatives. Through multivariate statistical analysis, the seven samples from different habitats could be divided into four categories, and the significantly changed metabolites between different categories were mainly concentrated in the flavonoid synthesis pathway. Through a variety of cluster analysis, it was observed that the Ch. nankingense (Nakai) Tzvel (Chinese name Juhuanao) had the largest separation degree from other samples and were clustered into a single category. Furthermore, the corresponding candidate chemical markers were screened in this study to distinguish the Juhuanao. Correlation analysis showed that climatic factors were not the main reason for the differences in the metabolic characteristics of Ch. indicum in different populations, which indicated that Ch. indicum is indeed a species with rich variation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-022-01137-z.

[Correlations between content of linarin in Chrysanthemum indicum and climatic factors in habitats].

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].

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.

[Cloning and prokaryotic expression of CHI in Chrysanthemum morifolium cv.'Hangju'].

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 ß-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.

Widely Targeted Metabolomics Analysis Reveals the Effect of Flooding Stress on the Synthesis of Flavonoids in Chrysanthemum morifolium.

Chrysanthemum morifolium. cv "Hangju" is an important medicinal material with many functions in China. Flavonoids as the main secondary metabolites are a major class of medicinal components in "Hangju" and its composition and content can change significantly after flooding. This study mimicked the flooding stress of "Hangju" during flower bud differentiation and detected its metabolites in different growth stages. From widely targeted metabolomics data, 661 metabolites were detected, of which 46 differential metabolites exist simultaneously in the different growth stages of "Hangju". The top three types of the 46 differential metabolites were flavone C-glycosides, flavonol and flavone. Our results demonstrated that the accumulation of flavonoids in different growth stages of "Hangju" was different; however, quercetin, eriodictyol and most of the flavone C-glycosides were significantly enhanced in the two stages after flooding stress. The expression of key enzyme genes in the flavonoid synthesis pathway were determined using RT-qPCR, which verified the consistency of the expression levels of CHI, F3H, DFR and ANS with the content of the corresponding flavonoids. A regulatory network of flavonoid biosynthesis was established to illustrate that flooding stress can change the accumulation of flavonoids by affecting the expression of the corresponding key enzymes in the flavonoid synthesis pathway.

[Study on wild tending of Chrysanthemum indicum].

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].

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 ß-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].

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'].

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.

[Comparative study on content of heavy metals in Chrysanthemum indicum and soil in different areas].

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.

[Cloning and expression analysis of F3'H and quantification of downstream products in Chrysanthemum morifolium under flooding stress].

To investigate the effects of the expression of flavonoid 3' hydroxylase gene (F3'H) and active ingredients in Chrysanthemum morifolium under flooding stress, we cloned F3'H from Hangju (temporarily named CmF3'H) and conducted bioinformatics analysis. During the flower bud differentiation stage, we flooded the Ch. morifolium and then used the Real-time PCR to detect the relative expression of CmF3'H; 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 CmF3'H was highly homologous to other species of Compositae. Real-time PCR results showed that CmF3'H 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-O-di-caffeoylquinic acid were also significantly higher than those of the control group. Therefore, Ch. morifolium regulates the synthesis of downstream products by regulating the expression of CmF3'H 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.