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Objective To explore the effect of circadian rhythm genes on flavonoids biosynthesis in safflower and its molecular mechanism. Methods Based on the transcriptome and metabolomic database of safflower corolla, we screened the circadian rhythm genes that correlate with biosynthesis of flavonoids in safflower. qPCR was used to quantify the expressions of circadian rhythm genes in different flowering stages at different time points in a single day. LC-MS was performed to determine the accumulation of flavonoids. The correlation between them was analyzed as well. Yeast Two-Hybrid experiment was used to verify the interactive proteins of these genes. Results Seven circadian rhythm genes PRR1, PRR2, ELF3, FT, PHYB, GI and ZTL were obtained. PRR1 gene was positively correlated with flavonoids accumulation (r≥0.7). The full length of PRR1 is 3 201 bp, encoding 421 amino acids, which is highly homologous with rice OsPRR73 gene and named as CtPRR1 (GenBank accession number: MW492035). CtPRR1 was mainly expressed in flowers, and the expression level increased in the daytime and declined in the evening gradually. Correspondingly, the content of flavonoids showed an opposite variation. Both of them displayed a circadian rhythm with a negative correlation (r≥−0.7). In addition, 2 heat shock proteins along with 3 AP2 transcription factors interacting with CtPRR1 protein were obtained via Yeast Two-Hybrid experiment. Conclusion CtPRR1 negatively regulated the safflower flavonoids accumulation in a circadian rhythm way, which may be affected by these interacting proteins.
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Objective To investigate the long-term protective effects of the nicotiflorin on ischemic stroke rats. Methods Ischemic stroke model in rats was established for this study. The effects of nicotiflorin on long-term survival rate, nervous system function, body weight and brain neurons in rats were observed. Results The nicotiflorin had significantly improved the long-term survival rate of cerebral ischemia rats, which also promoted weight gain, alleviated pathological damage of brain tissue, maintained morphology of brain neurons and function of nervous system. Conclusion The nicotiflorin has obvious long-term protective effect on ischemic stroke rats and the mechanism may be related to the protection of the structure and function of brain neurons.
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Objective To explore the function of short-chain dehydrogenases/reductases (SDRs) in safflower flavonoid, especially hydroxysafflower yellow A (HSYA) biosynthesis. Methods SDRs involved in HSYA biosynthesis pathway were screened based on safflower transcriptome database and metabolome database. The expression pattern was analyzed by qRT-PCR. The overexpression vector was constructed by seamless cloning technology, then genetically transformed to the Yunnan Weishan safflower strain by Agrobacterium gv3101. The transgenic T2 generation plants were positively verified, and the gene expression of corolla SDRs was analyzed. The content of secondary metabolites was assayed by UPLC-Q-TOF/MS. Results Three SDRs genes named CtSDR1, CtSDR2 and CtSDR3 involved in HSYA biosynthesis pathway were screened. Their expression in safflower from high to low was corolla > leaf > stem > root. The expression level in corolla increased gradually with corolla development. qRT-PCR analysis of corolla with positive verification of genome insertion sequence showed that the transcription level of CtSDR3 in corolla of T2 positive plants increased by 2~3 times compared with the blank control group, and the content of secondary metabolite HSYA increased by 7.1%~16.6% (P< 0.05). Conclusion CtSDR3 may be involved in the biosynthesis of flavonoids, especially HSYA, in safflower. It provides the support data for explaining the function of CtSDR3 in HSYA biosynthesis pathway.
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Flavonoids are secondary metabolites that widely existing in traditional Chinese medicine. They have many biological activities and pharmacological effects. In recent years, studies have found that flavonoids can play an effective protective role in the treatments of ischemic stroke and ischemic heart disease through various ways. The mechanisms of their protective effects have been systematically explained in detail. For example, flavonoids can affect pathophysiological mechanisms such as antioxidant, inhibition of inflammatory response, apoptosis and autophagy pathway. This paper reviews the protective effect and mechanisms of the treatments of flavonoids on cardiovascular and cerebrovascular ischemic diseases by referring to relevant literatures. The purpose is to provide reference for future research and development of drugs for the treatments of cardiovascular and cerebrovascular diseases.
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Objective To investigate the protective effects of the total bakkenolides from P.tricholobus on improving hypoxia tolerance in mice. Methods Mice normobaric pressure hypoxia model and oxygen glucose deprivation model in PC12 cells were established, and the effects of PTB on survival time, serum lactate dehydrogenase (LDH) activity and malondialdehyde (MDA) content, brain and heart superoxide dismutase (SOD) and reduced glutathione (GSH) activities, brain tissue pathological changes and cell survival were observed. Results The total bakkenolides from P.tricholobus had prolonged the survival time of mice in confined spaces, increased the activity of SOD and GSH, reduced the production of lipid peroxidation, decreased the degree of anaerobic glycolysis, protected the structure and function of neural cells, and improved the survival rate of OGD-treated cells. Conclusion The total bakkenolides from P.tricholobus could promote the hypoxia tolerance in mice which might be related to scavenging oxygen free radicals, inhibiting lipid peroxidation reaction and protecting the structures and functions of nerve cells.
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Objective To study the protective effects of the total bakkenolides from P .tricholobus on high altitude hy-poxia .Method Normobaric hypoxia model and acute hypobaric hypoxia model in mice ,hypobaric hypoxia model in rats were established for this study .Survival time and survival rate of mice were recorded .The level of blood sugar and glycogen ,adeno-sine triphosphate (ATP) ,lactic acid (LD) ,lactic dehydrogenase (LDH) were detected in different organs of rats .Results The total bakkenolides significantly prolonged the survival time of mice in normobaric hypoxia model and reduced the death rate of mice in acute hypobaric hypoxia model .The total bakkenolides suppressed blood sugar level in rats and increased the glyco-gen level in rat liver ,skeletal muscle and myocardium .It also elevated the ATP content in rat brain ,liver ,skeletal muscle and myocardium .Meanwhile ,the content of LD in plasma ,skeletal muscle ,myocardium and LDH level in myocardium were re-duced .Conclusion The total bakkenolides from P .tricholobus have protective effect on normobaric hypoxia model and acute hypobaric hypoxia model in mice as well as hypobaric hypoxia model in rats .Its anti-hypoxia efficacy at high altitude may relate to the increased blood sugar ,glycogen ,and ATP level and reduced LD ,LDH level in major organs .
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Objective To summarize the clinical outcomes of pectoralis major myocutaneous flap for repairing large defects in oral and maxillofacial area after resection of malignant tumor. Methods The clinical data of 27 patients underwent resection of malignant tumor in oral and maxillofacial area and reconstructed with pectoralis major myocutaneous flap were collected in our hospital from August 1998 to January 2015. The pectoralis major myocutaneous flaps were harvested with sizes ranging from 6 cm × 4 cm to 11 cm × 9 cm. The major myocutaneous flaps were used to reconstruct the defects of oral mucosa in 26 cases, and flap was used to reconstruct the defect of facial skin in 1 case. Seventeen major myocutaneous flaps reached the neck via the subclavicular tunnel, the other 10 were transferred over the clavicle. Results After surgery, 20 flaps (74.1%) were survived completely, 6 were partial necrosis (22.2%) and one was total necrosis (3.7%). Thirteen cases showed postoperative complications (48.1%), in which 10 cases were wound infection (37.0%), including 8 patients with infection at the recipient site and 2 patients with infection at the donor site. The wound infection was found in all of 7 patients with flap necrosis. The other complications included wound dehiscence in 1 patient (3.7%), neck hematoma in 1 patient (3.7%), and lung infection in 1 patient (3.7%). Conclusion In order to avoid the flap necrosis and reduce wound infection at the recipient site, the major myocutaneous flap should be designed based on the characteristics of blood supply, and the vascular pedicle should be protected carefully in the operation.
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Objective To study the liver protection of total celosins (CES) in different liver damage models .Method A-cute liver damage mouse model ,chronic liver damage rat model and immune liver damage mouse model were established for this study .Liver function tests ,liver lipid peroxidation indicators and liver pathology were observed .Results CES of each dose group can lower serum ALT and AST in different degrees ,can significantly reduce the content of MDA in liver homogenate , while can increase SOD activity .Pathological examination showed that animals administered significantly reduced the degree of liver cell damage .Conclusion CES have protective effects on acute liver damage induced by CCl 4 ,chronic liver damage induced by CCl4 and immunological liver damage induced by concanavalin A .Its hepatoprotective effect may be related to its antioxidant effects .
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The safflower floret is a traditional Chinese medicine used to promote blood circulation and remove obstruction in the channels. The spines on its bracts are considered a handicap when manual harvest is involved. In this study, cDNA-SRAP was used to systematically investigate which genes are associated with the spines. Sixty pairs of possible primer combinations were used on two cDNA pools representing spininess and spinelessness. Six transcript-derived fragments were identified, of which two with low recombination were sequenced successfully and named as GPY-1 and GPY-2. By using the RACE method, the full-length cDNA of GPY-2 is cloned and named as CTL-spn. The full-length cDNA of CTL-spn was 1 679 bp long with a 1 524 bp ORF encoding a 508 aminoacid protein. The deduced amino acid sequence of the CTL-spn gene shared a high homology (97%) with other known ATP synthase CF1 alpha subunits. Semiquantitative RT-PCR analysis revealed that the mRNA of GPY-1 and GPY-2 accumulated in only spiny lines. Considering the important role of ATP synthase CF1 alpha subunit in plants, it may directly take part in the formation process of spininess and enhancing resistance reaction of spiny safflower. Also, our results provide the important insights for breeding spineless cultivars of safflower.
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Objective To investigate the genetype distribution of mycoplasma pneumoniae(MP) by denaturing high-performance liquid chromatography(DHPLC).Methods A total of 300 cases nasopharyngeal aspirate were collected from our hospital.The MP genes of standard strains and clinical specimens isolates were amplified by PCR followed by DHPLC and genetype determination.Results A total of 110 cases were positive after 24 hours fermentation from 300 cases with pharyngeal swab.By the specific primers of MP-129,MP-FH standard strain and specimens,2 280 bp and 2 580 bp gene fragments were made out respectively.One hundred and ten strains of clinical isolates were detected by DHPLC.One hundred and seven strains of P1-Ⅰ were 1b subtype,3 were type P1-Ⅱ which were all 2a subtype.Conclusion The genetype of MP infection in children from our hospital is P1-Ⅰ,1b subtype by using DHPLC technology.
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Aim To investigate the intraspecific variation of Carthamus tinctorius L. (safflower) and establish foundation for further breeding of safflower germplasm resource and screening the quality correlation genes. Methods Amplified fragment length polymorphism (AFLP) was carried out to analyze genetic variation of 28 safflower populations collected in China. Unweighed pair-group method of with arithmetical averages (UPGMA) cluster analysis was used to construct a dendrogram and to estimate the genetic distances among the populations. Results All populations could be uniquely distinguished using 12 selected primer combinations. Similarity coefficients ranged from 0. 48 to 0. 96 among the populations.Dendrogram revealed distinct segregation of all the cultivars into three main groups and one midst group.Conclusion Limited genetic diversity exists within the tested 28 collections at intra specific level and AFLP-based phylogeny was not absolutely consistent with that based on morphological characters may be due to the interaction effect between genotype and environment.
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Objective:To analyze the chemical constituents of the ether extracts from the fruits of 3 species of Chaenomeles . Methods: The chemical constituents were analyzed by GC MS. Results: Sixty one constituents were firstly isolated and identified from the fruits of 3 species of Chaenomeles . Forty compounds in Chaenomeles lagenaria , thirty four compounds in Xuan mugua ( C.lagenaria from Xuanzhou) and 23 compounds in C.sinensis were isolated and identified respectively. There were 10 compounds in all 3 species of Chaenomeles , but their contents were very much different. They were acetic acid, benzaldehyde, n caproic acid, 14 methyl pentadecanoic methyl ester, glycerin, benzoic acid, 6,9 octadecadienoic methyl ester, squalane, stearic acid and oleic eicosyl ester. Conclusion: The composition and content vary with different species and district of Chaenomeles . The result may be useful for the evaluation of Chaenomeles fruits quality. [
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Objective:To study the chemical constituents of the methylene chloride of Rubus chingii Hu..Methods:Chromatography on silica gel column,Sephadex LH-20 column,and recrystallization technique were used to isolate and purify the compounds.Spectroscopy methods including EI-MS,ES-MS,1H-NMR,13CNMR,HMQC,and HMBC were used to elucidate the structures of compounds.Results:Ten compounds were obtained and 9 compounds were identified as:hexacosanol(Ⅰ),?-sitosterol(Ⅱ),4-hydro-3-methlbenzal acid(Ⅲ),4-hydrobenzal dehyde(Ⅳ),oleanolic acid(Ⅴ),stigmast-5-en-3-ol,oleate(Ⅵ),1H-2-indenone,2,4,5,6,7,7a-hexahydro-3-(1-methylethyl)-7a-methyl(Ⅷ),4-hydroxy-3-methoxybenzoic acide(Ⅸ),and liballinol(Ⅹ).Conclusion:Compounds Ⅰ,Ⅲ,Ⅳ,Ⅵ,Ⅷ,and Ⅹhave been obtained from Rubus chingii for the first time.
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Objective:To study the chemical constituents in the root of Petasites tricholobus Franch.and evaluate their anti-inflammatory activity.Methods: The primary extraction was done with 95% ethanol and subsequently with other agents including ethyl acetate.The compounds extracted with ethyl acetate were isolated by silica gel column chromatography and purified by means of recrystallization.The structures of the compounds were elucidated by employing chemical and spectral methods.The anti-inflammatory activities of the compounds were evaluated through their inhibitory effect on the trachea contraction of guinea pig induced by histamine in vitro.Results: Six compounds were isolated from the root of Petasites tricholobus Franch.,namely,homofukinolide(1),bakkenolide-B(2),stigmasterol(3),and ?-sitosterol(4),bakkenolide-D(5),and ?-sitosterol-3-O-?-D-glucopyranoside(6).Bakkenolide-B and bakkenolide-D demonstrated inhibitory effect on the trachea contraction induced by histamine in vitro.Conclusion: Homofukinolide,?-sitosterol,and ?-sitosterol3-O-?-D-glucopyranoside are isolated for the first time from Petasites tricholobus Franch..Bakkenolide-B and-D have obvious anti-histamine activities.
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Objective To construct for the heredity linkage map and to study the functional gene research of Carthamus tinctorius,the factors affecting cDNA amplified fragment length polymorphism(cDNA-AFLP) of C.tinctorius were investigated with developing and optimizing the cDNA-AFLP reaction system.Methods Improved Trizol method was used to extract RNA from compounds in new petals specific to safflower.With the help of M-MLV RTase without RNase activity combined with replacement synthesis method,double-stranded cDNA was synthesized from total RNA;cDNA was digested by restriction enzyme MseI/EcoRI and ligated by two steps.Then the products were provided for pre-amplification and selected amplification of different concentration gradients.After tiny modifications of system concentration,finally PAGE electrophoresis and silver-staining were performed.Results High purity and integrated total RNA for later cDNA synthesis were obtained and high quality cDNA was synthesized with the help of M-MLV RTase without RNase activity combined with replacement synthesis method.The cDNA-AFLP reaction system in C.tinctorius was as follows: 250 ng integrity cDNA was digested thoroughly at 37 ℃ for 6 h,and ligated 12 h at 16 ℃.Furthermore,the sample dilution multiplication was 10 fold for pre-amplification and 150 fold for selected amplification under the proper system concentration.According to the above reaction system,the polymorphous strips with high resolution power in PAGE electrophoresis were clear and stable.Conclusion The cDNA-AFLP reaction system established and optimized in this experiment is suitable for the functional gene analysis of C.tinctorius.
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Objective:To study the high-performance liquid chromatographic(HPLC) fingerprints of different germplasms of Flos Carthami. Methods: HPLC was applied in chromatographic separation of 36 different germplasms of Flos Carthami collected from 11 provinces of China.The chromatography condition was: Agilent Zorbax C_(18) column(4.6 mm?250 mm,5 mm);mobile phase: CAN,0.5% H_3PO_4(from 1090 to 8020);running time: 60 min;flow speed: 1.0 ml/min;detection wavelength: 265 nm;and temperature: 20℃.The data were processed by Chromatographic Fingerprint Station designed by Department of Pharmaceutical Analysis,School of Pharmacy,Second Military Medical University.Different germplasms of Flos Carthami were subjected to cluster analysis and their similarity was also evaluated.Results: The different germplasms of Flos Carthami in this study were grouped into 2 chemical types.Samples from northwest of China were of high similarity;samples from the middle China were of high similarity to those from South China.The similarity of some germplasms of Flos Carthami was significantly different.Conclusion: The chemical components of different germplasms of Flos Carthami are obviously different from each other;selection of germplasm is very important in quality control of Flos Carthami.