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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.
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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.
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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.
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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.
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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.
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Objective: To clarify the structure features of polysaccharides from Chrysanthemum morifolium (PCM) and to study their activities against tumor cells and NF-κB. Methods: Six homogeneous neutral polysaccharides were obtained from three kinds of C. morifolium (Hangju, Huaiju, and Boju) flowers by successive hot water extraction, followed by ethanol precipitation, ion-exchange chromatography, and gel permeation chromatography. Their primary structures were characterized by HPGPC, IR, GC, and GC-MS analyses. Their bioactivities were examined by MTT assay using PANC-1 and LO2 cells. In addition, NF-κB signaling activation in PANC-1 and LO2 cells treated by polysaccharides were also measured. Results: The weight-average molecular mass of the six PCM, CMTA0S1, CMTA0S3, CMJA0S1, CMJA0S2, CMBA0S1, and CMBA0S3 was 7.523 × 104, 7.80 × 103, 7.80 × 104, 1.04 × 104, 5.79 × 104, and 1.35 × 104, respectively. CMTA0S1, CMJA0S1, and CMBA0S1 mainly contained galactose (Gal), arabinose (Ara) and glucose (Glc) residues in molar ratio of 1.23:1.00:0.20, 2.18:1.00:0.53, and 3.30:1.00:0.75, while CMTA0S3, CMJA0S2, and CMBA0S3 mainly contained Gal, Ara, Glc, and mannose (Man) residues in molar ratio of 0.73:1.00:0.40:0.21, 1.39:1.00:0.84: 0.55, and 1.19:1.00:0.48:0.19. Methylation analysis indicated that six PCM primarily consisted of T-arabinofuranosyl, 1, 5-arabinofuranosyl, 1, 4-galactopyranosyl, 1, 3, 6-galactopyranosyl, and 1, 4-glucopyranosyl residues. The biological activity study suggested that all the PCM could inhibit the growth of PANC-1 cells. Among them the inhibitory rates of CMTA0S3 and CMJA0S2 were at most to 70% with concentration-effect relationship. The NF-κB inhibition test indicated that only the crude polysaccharide CMBA had strong immunosuppressive activity, and homogeneous polysaccharides CMTA0S1 and CMJA0S1 showed potential immunostimulation. Conclusion: The six homogeneous polysaccharides share similar structures and inhibition on PANC-1 cells growth. Meanwhile they also may regulate the NF-κB activation.
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Objective: The NaCl stress conditions were simulated to study the effect of the endophytic fungi C1, C4 on antisalty characteristic of Chrysanthemum morifolium in the adverse circumstance. Methods: Endophytic Botrytis sp. (C1) and Chaetomium globosum (C4) were inoculated to the C. morifolium plantlets which were planted in the pots in order to research the effects of salt stress on physiological indicators of C. morifolium. Results: With the increase of NaCl concentration, the water content of root and leaf decreased in every group. The loss of root and leaf's water in fungi-treated group was smaller than that in the control group. SOD activities in every group increased with the increase of NaCl concentration, and achieved the peak value at 20 g/L NaCl. The SOD activity in fungi-treated group was higher than that in the control group. Soluble protein of fungi-treated group was higher than that in the control group, and C4 group surpassed C1 group. POD activity increased firstly and then decreased, and compared to the control group, the POD activities in C4 and C1 groups increased by 25.50% and 1.35%, respectively at 15 g/L NaCl. PAL activity of C4 treated group was seven folds compared to the control group at 15 g/L NaCl. Conclusion: Endophytic fungi could enhance the salt-tolerant ability of C. morifolium, and the effect in C4 group was better than C1 group.
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Objective: Interspecific hybridization was made between Chrysanthemum morifolium 'Hongxinju' (♀), C. morifolium 'Xinbaiju' (♀) and C. indicum (♂), C. nankingense (♂), and F1 hybrids were identificated in order to discover inheritance of main characters in Chrysanthemum morifolium. Methods: Interspecific hybridization of C. morifolium were made by bud pollination. Morphological, cytological, and RAPD analyses were used for the identification of interspecific hybrids. Results: The hybrids showed interspecific morphological traits of their parents or unique morphological traits their parents did not possess. RAPD results indicated that the hybrids produced specific bands of male parent or new bands absent in both parents. Chromosome number of F1 hybrids was the average of their parents. Conclusion: Bud pollination could improve heterozggosity of interspecific hybridization of C. morifolium 'Hongxinju' and 'Xinbaiju'. Hybrids could be aneuploid chromosomes and show the significant segregation of character.
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Object To determine the amount of acacetin-7-O-β-D-glucoside and apigenin-7-O-β-D-glucoside in Chrysanthemum morifolium Ramat. from different localities. Methods By the use of HPLC. Results The contents of acacetin-7-O-β-D-glucoside in Boju1, Chuju2, Gongju3 and Hangju4 were 0.082 6%～0.124 9%, 0.015 7% and 0.033 5% respectively; the contents of apigenin-7-O-β-D-glucoside in Chuju, Boju, Gongju and Hangju were 0.012 6%, 0.027 4%, 0.117 7% and 0.587 7% respectively. Conclusion The contents of the two flavone glycoside in C. morifolium from different localities were markedly different.
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Objective To study and compare the metabolism of Chrysanthemum morifolium extracts(CME) in vitro intestinal flora from human,rat,Beagle dog,and rabbit.Methods Intestinal flora of human,rat,Beagle dog,and rabbit and CME were anaerobically cultured at 37 ℃ in vitro. After being extracted by acetic ether,the main metabolites of CME were separated and quantified by HPLC.Results In vitro,CME was easily metabolized by the intestinal flora and the main metabolites in incubation medium were determined in high concentration after 0.5 h.By LC-MS,luteolin and apigenin were identified,respectively;both metabolites were degraded with prolongation of incubation time and the concentrations of luteolin and apigenin were low after 24 h;moreover,CME was quickly metabolized by human,rat,Beagle dog intestinal flora,and gently by rabbit intesinal flora.Conclusion In vitro,CME is easily metabolized by intestinal flora human,rat,Beagle dog,and rabbit and the main metabolites are luteolin and apigenin.
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Objective To compare the botanical characters, yield, and active components in flowers of seven various cultivars from Chrysanthemum morifolium in planting base in Sheyang County, Northern Jiangsu Province and provide the theoretical and practical basis for plangting and expanding reproduction of improved cultivars of C. morifolium. Methods Based on the field experiment, comparison, and analysis of the botanical characters, yield, and active components (total flavonoid and chlorogenic acid) in flowers of the seven cultivars from C. morifolium, such as “Hongxinbaiju”, “Changbanbaiju”, and “Hubaiju” etc. Results The botanical characters of flowers are vary different among the seven cultivars from medicinal C. morifolium in the planting base; the amount of single capitulum is the main factor influencing on the yield of C. morifolium, the yield of “Hongxinbaiju” is the highest; the contents of total flavonoid and chlorogenic acid of “Changbanbaiju” are the highest in the seven cultivars. Conclusion From all the aspects, in the planting base of Northern Jiangsu Province, “Hongxinbaiju” and “Changbanbaiju” can be extended as major cultivars with good characters of superior quality and high yield, “Hubaiju” and “Huangjuhua” also can be cultivated at present.
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extraction hours. No significant difference of flavonoids content was observed among the different collecting time of C. morifolium. Conclusion The optimum extraction conditions were 75% ethanol, refluence for 1 5 h at 100 ℃ water bath for three times. The flavonoids would not be bound up with the harvest time.
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Objective To investigate the genetic diversity of Chrysanthemum morifolium and provide the evidence for evaluation and exploitation of C.morifolium germplasm.Methods The genetic diversities of 31 germplasm from different habitats were investigated with the technique of inter-simple sequence repeat(ISSR),which were 29 of C.morifolium,1 of C.indicum,and 1 of C.nankingense.Results Twenty-two primers were selected to produce highly reproducible ISSR bands.Among 182 amplified bands,149 showed polymorphism,the percentage of polymorphic bands(PPB) reached to 81.87%. Observed effective number of alleles(Ne),Nei's gene diversity index(He),and Shannon information index (Ⅰ) were 1.348 1,0.219 1,and 0.345 1,respectively.Conclusion ISSR Method is suitable for identification and genetic diversity analysis of C.morifolium.
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Objective To establish and optimize ISSR-PCR systems of Chrysanthemum morifolium and lay the foundation for its genetic diversity research. Methods Based on the analysis of variance, an orthogonal design was used to optimize the ISSR-PCR amplification system on C. morifolium by four factors (Taq polymerase, Mg2+, dNTP, and primer) at three concentration levels, respectively. Results A suitable ISSR reaction system was constructed with the 20 ?L reaction system containing 1.00 U Taq polymerase, 2.00 mmol/L Mg2+, 0.20 mmol/L dNTP, and 0.50 ?mol/L primer. Conclusion ISSR-PCR is significantly influenced by the concentration of Taq polymerase, Mg2+, and dNTP. This ISSR-PCR system could provide clear bands, reliable reaction system, and abundant polymorphisms . It is proved to be suitable for the study of the genetic diversity of C. morifolium
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Objective To seek the optimization hormone combination for the callus induction and differentiation from cotyledon and hypocotyl explants of Chrysanthemum morifolium through tissue culture. Methods Cotyledon and hypocotyl explants were cultured on various medium with different hormone combinations. Results Callus could be inducted on every media designed in this experiment,but the effects to the induction and differentiation culture of buds were different.The results showed that the medium MS+IAA 0.3 mg/L+6-BA 12 mg/L was suitable to the induction of callus from cotyledon and hypocotyl.The optimal hormone combination was MS+IAA 0.3 mg/L+6-BA 4 mg/L for the initiation of adventitious bud of cotyledon.The shoot regeneration percentage reached 67.5%.The optimal medium for the initiation of adventitious bud of hypocotyl was MS+KT 2 mg/L+NAA 0.2 mg/L.The shoot regeneration percentage reached 62.5%.Rooting was induced on 1/2 MS+NAA 0.1 mg/L,and the root inducting ratio reached 100% in 7 d. Conclusion A rapid plantlet regeneration system for C. morifolium is established from cotyledon and hypocotyl explants.Bud induction frequency is higher and the shoots in vitro grow vigorously.
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Objective Taking the total amino-acid, total flavones, chlorogenic acid, and volatile oil in Chrysanthemum morifolium as index to investigate the internal quality which relates to collection periods and processing methvods in order to compare the quality of species from oringinal habitats. Methods The contents of colorogenic acid in C. morifolium was analyzed by HPLC; the content of total flavones and amino-acid in C. morifolium were measured by spectrophotometery; and the volatile oil obtained by steam distilation extraction was weighted. Results The every indexes of viviparious chrysanthemum except chlorogenic acid was the best among various flowering periods so the viviparious chrysanthemum can be used as the first-class tea. The common tea produced by half-booming and full-booming flowers with higher yield and appropriate index. The machine processing is fast and suitable for the production to a large-scale. Conclusion The quality of C. morifolium planted in Ruicheng, Shanxi Province is equal to that in Tongxiang, Zhejiang Province, which depends on the collecting periods and processing methods, so does the volatile oil rather than the evironment of the habitat.
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Objective: To determine luteolin 7 O ? D glucoside in Chrysanthemum morirolium Ramat. Methods: The content of luteolin 7 O ? D glucoside was determined by HPLC on Symmetry Shield TM RP S column with MeoH H 2O(49∶51) as a mobile phase and detection wavelength at 348nm. Results: The liuear relationship of this method was well and the average recovery of the added sample is 99.74%. Conclusion: The analytical time is short, separating degree is good and results are accurate when luteolin 7 O ? D glucoside is determined by this method.
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AIM: To analyze the liposoluble constituents from two Varieties of Chrysanthemum morifolium Ramat(Changhong and Lengyan) cultivated in Kaifeng. METHODS: Changhong and Lengyan were extracted by Soxhlet extraction,and GC-MS was used to analyze their components. RESULTS: Forty compounds were identified from Changhong,amounting to 90.09% of the total constituents.Thirty-five compounds were identified from Lengyan,amounting to 91.57% of the total constituents. CONCLUSION: There appears to be considerable difference in triterpenoids and sterols between the samples of Changong and Lengyan.
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AIM: To investigate the vasorelaxant effect and mechanism of EtOAc extract from Chrysanthemum morifolium Ramat (CME). METHODS: The effects of CME on the contraction of rat thoracic a orta were examined. RESULTS: CME caused concentration-dependent relaxation of aorta rings precontricted with phenylephrine and K+. The effect in endothelium-intac t aorta was more effective than that in endothelium-deduced aorta. NG-nitro-L- arginine methylester, methylene blue and glibenclamide attenuated the effect of C ME significantly. However, indomethacin, propranolol, tetraethylammonium, BaCl 2, 4-aminopyridine and 5-hydroxydecanoate did not affect CME effect. The effect of SKF-525A combined with L-NAME had no obvious difference with that of L-NAME o n CME-induced relaxation. NOS activity in aorta was increased markedly by CME in vitro. CME did not reduced the contraction elicited by PE in Ca 2+-f ree medium, but reduced the contraction induced by PE in K+-free solution or C a 2+ free following input Ca 2+. CONCLUSION: CME induces both endothelium-dependent and independe nt relaxation. NO and cGMP are likely involved in the endothelium-dependent rela xation, inhibition of voltage-dependent or receptor-operate Ca 2+ channel a nd activation of ATP-sensitive K+ channel contribute in part to the endotheliu m-independent relaxation by CME.
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AIM: To explore the influence of Chrysanthemum morifolium Ramat on TNF-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in human colon cancer cell line DLD-1 and its possible mechanism. METHODS: Adenovirus-mediated TRAIL gene AD/hTERT-gTRAIL was applied either alone or by combination with Chrysanthemum morifolium Ramat in human colon DLD-1 cell line. Cell growth and apoptosis were measured by inverted microscope, MTT method and flow cytometry. The expression of TRAIL mRNA, TRAIL-Rs mRNA and TRAIL protein expression after exposure to Chrysanthemum morifolium Ramat were measured by semi-quantitive RT-PCR and FACS, respectively. RESULTS: The suppression percentages and apoptotic rate of DLD-1 by Ad/hTERT-gTRAIL alone were 31.4% and 13.5%, respectively. Combination of TRAIL gene transfection with Chrysanthemum morifolium Ramat, the suppression and the apoptosis rate raised to 93.1% and 45.4%, respectively (P