Carthami Flos, as a traditional blood-activating and stasis-resolving drug, possesses anti-tumor, anti-inflammatory, and immunomodulatory pharmacological activities. Flavonoid glycosides are the main bioactive components in Carthamus tinctorius. Glycosyltransferase deserves to be studied in depth as a downstream modification enzyme in the biosynthesis of active glycoside compounds. This study reported a flavonoid glycosyltransferase CtUGT49 from C. tinctorius based on the transcriptome data, followed by bioinformatic analysis and the investigation of enzymatic properties. The open reading frame(ORF) of the gene was 1 416 bp, encoding 471 amino acid residues with the molecular weight of about 52 kDa. Phylogenetic analysis showed that CtUGT49 belonged to the UGT73 family. According to in vitro enzymatic results, CtUGT49 could catalyze naringenin chalcone to the prunin and choerospondin, and catalyze phloretin to phlorizin and trilobatin, exhibiting good substrate versatility. After the recombinant protein CtUGT49 was obtained by hetero-logous expression and purification, the enzymatic properties of CtUGT49 catalyzing the formation of prunin from naringenin chalcone were investigated. The results showed that the optimal pH value for CtUGT49 catalysis was 7.0, the optimal temperature was 37 ℃, and the highest substrate conversion rate was achieved after 8 h of reaction. The results of enzymatic kinetic parameters showed that the K_m value was 209.90 μmol·L~(-1) and k_(cat) was 48.36 s~(-1) calculated with the method of Michaelis-Menten plot. The discovery of the novel glycosyltransferase CtUGT49 is important for enriching the library of glycosylation tool enzymes and provides a basis for analyzing the glycosylation process of flavonoid glycosides in C. tinctorius.
Carthamus tinctorius/chemistry* ; Phylogeny ; Flavonoids/analysis* ; Glycosides/analysis* ; Glycosyltransferases/genetics* ; Anti-Inflammatory Agents ; Chalcones

The NAC(NAM/ATAF/CUC) transcription factors are members of the largest transcriptional gene family in plants and play an essential role in the response of plants to drought stress. To identify the number and function of the NAC gene family in Carthamus tinctorius, the present study adopted bioinformatics methods to identify NAC gene family members based on the whole genome data of C. tinctorius, and analyzed their physicochemical properties, chromosomal location, phylogenetic relationship, gene structure, conserved domain, and conserved motif. Meanwhile, the real-time fluorescence-based quantitative RT-PCR(qRT-PCR) was used to analyze the transcription level of four NAC genes under drought stress in different time. The results showed that C. tinctorius contained 87 NAC genes unevenly distributed on 11 chromosomes, while no NAC gene was found on chromosome 12. The encoded proteins were 103-974 amino acids and the number of CDS ranged from 3 to 9. According to the phylogenetic relationships, 87 NAC genes were clustered into17 subfamilies. The analysis of conserved domains and motifs revealed that most of the genes contained five conserved subdomains, A-E and motif2 was the most conserved among NAC genes. The expression pattern analysis showed that the transcription levels of four NAC genes related to drought resistance were all up-regulated after drought stress treatment for different time, suggesting that these four NAC genes may be related to drought resistance of C. tinctorius. This study is expected to provide a theoretical basis for further functional analysis of NAC transcription factors in C. tinctorius and references for the cultivation of drought-tolerant C. tinctorius varieties.
Droughts ; Carthamus tinctorius/genetics* ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism* ; Phylogeny ; Transcription Factors/metabolism* ; Stress, Physiological/genetics* ; Multigene Family

The WD40 transcription factor family is a gene superfamily widely found in eukaryotes, which is closely related to plant growth and development regulation. It has been reported that the WD40 transcription factor was involved in the synthesis of anthocyanins, which is one of the vital components of safflower flavonoid compounds. In this study, 40 CtWD40 members in the safflower genome were identified though bioinformatics tools and gene expression analysis methods. According to the WD40 protein sequence and phylogenetic characteristics of Arabidopsis and other plants, the safflower CtWD40 family was classified into 7 subfamilies. Conservative motif analysis was used to reveal the specific conserved motifs and gene structures of each subfamily member, and there exist a certain degree of similarities in the conserved motifs and gene structure between the closely related family members. Subsequently, the search for cis-acting elements of gene promoters found CtWD40-specific promoter elements, revealing the metabolic pathways which may involve. Next, enrichment of function analysis was employed to analyze the functional categories and cellular localization of the CtWD40 protein. Furthermore, the interactions between CtWD40 proteins predicted its potential regulatory function. Finally, 19 members of the safflower CtWD40 subfamily were analyzed by qRT-PCR, the result showed the expression patterns of these members were different in diverse tissue and flowering period. This study provides a basis for the functional and expression research of the CtWD40 genes.
Carthamus tinctorius ; Computational Biology ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Genome, Plant ; Multigene Family ; Phylogeny ; Plant Proteins ; genetics ; Transcription Factors ; genetics

To clone bHLH( basic helix-loop-helix) gene from Carthamus tinctorius,analyze the expression level in different plant tissues and construct the plant expression vector. The bHLH1 gene was cloned by RT-PCR techniques,and the protein characteristics were analyzed by bioinformatics,and phylogenetic tree was constructed. The expression of bHLH1 gene in different tissues and the roots after inoculated by Fusarium oxysporum were analyzed using real time-PCR,and the plant expression vector p BASTA-bHLH1 was constructed. The obtained ORF sequence of bHLH1 gene was 897 bp,encoded a protein of 298 amino acids. Sequence alignment and phylogenetic tree analyses showed that C. tinctorius bHLH1 had a certain homology with other species of amino acids,and was the most similar to the amino acid sequence of tobacco. Real-time PCR results showed significant differences,CtbHLH1 gene in red flower petals in different tissues and different flowering period had remarkable difference in expression level,its high amount expressed in petals,flowers third day after blossom expressed the highest quantity,at the end of the flowering the expression quantity is low. In addition,it is expressed in the root,and the expression in the stem and leaves is extremely low. The bHLH1 gene of C. tinctorius is successfully cloned,and the expression is analyzed. The plant expression vector p BASTA-bHLH is constructed.
Amino Acid Sequence ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Carthamus tinctorius ; genetics ; Cloning, Molecular ; Flowers ; genetics ; Gene Expression Regulation, Plant ; Genetic Vectors ; Phylogeny ; Plant Proteins ; genetics

OBJECTIVETo explore the clinical efficacy and the effect mechanism of hooking therapy and safflower injection in the treatment of lumbar disc herniation (LDH).

METHODSSixty patients of LDH were randomized into an observation group and a control group at ratio of 1 to 1, 30 cases in each one. In the control group, safflower injection was applied to three points of lumbar region, once a day. Seven treatments made one session, and totally 2 sessions were required. In the observation group, under local anesthesia, the big hook needle was used to stimulate the three points of lumbar region first, followed by the safflower injection, once every 6 to 7 days. If the efficacy was not satisfactory enough, the second treatment was given. The visual analogue scale (VAS) and the modified Japanese Orthopaedic Association (M-JOA) scores were observed before and after treatment in the patients of the two groups and the clinical efficacy was evaluated. The levels of peripheral interleukin-6 (IL-6) and nitric oxide (NO) were determined.

RESULTSVAS and M-JOA scores were all improved apparently after treatment as compared with those before treatment in the patients of the two groups (both P < 0.01). The improvements in the observation group were much more obvious than those in the control group (both P < 0.01). In the observation group, the curative and remarkably effective rate was 76.7% (23/30) and the total effective rate was 96.7% (29/ 30), better than 43.3% (13/30) and 70.0% (21/30) in the control group separately (both P < 0.05). After treatment, the levels of IL-6 and NO were all reduced as compared with those before treatment in the two groups; (both P < 0.01). The above indices were changed more obviously in the observation group as compared with those in the control group (both P < 0.01).

CONCLUSIONThe combined therapy of hooking therapy and safflower injection apparently relieves pain and clinical symptoms of LDH. The effect mechanism is relevant with reducing the levels of IL-6 and NO in the peripheral blood.

Acupuncture Points ; Acupuncture Therapy ; Adult ; Carthamus tinctorius ; chemistry ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Interleukin-6 ; genetics ; metabolism ; Intervertebral Disc Displacement ; drug therapy ; genetics ; metabolism ; therapy ; Male ; Middle Aged ; Nitric Oxide ; genetics ; metabolism ; Treatment Outcome ; Young Adult

Flavonol synthase (FLS) is one of the key enzymes in flavonoids metabolic pathways. In this study, middle sequence was obtained from Carthamus tinctorius transcriptome sequencing results. Full-length cDNAs of FLS was cloned from petals of C. tinctorius to FLS by using RT-PCR and RACE technology. Its full-length cDNA was 1,201 bp, with an open reading frame of 1,101 bp and 336 encoded amino acids. The phylogenetic analysis showed that, FLS gene encoded amino acids in C. tinctorius were highly homologous with amino acids in congeneric Compositae species, especially Rudbeckia laciniata. The pBASTA-FLS plant expression vector was successfully built by the molecular biology method, which lays a foundation for further studying biology functions of the gene and biosynthesis mechanism of flavonoids.
Amino Acid Sequence ; Base Sequence ; Carthamus tinctorius ; classification ; enzymology ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; metabolism ; Molecular Sequence Data ; Open Reading Frames ; Oxidoreductases ; genetics ; metabolism ; Phylogeny ; Plant Proteins ; genetics ; metabolism

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.
Adenosine Triphosphate ; Amino Acid Sequence ; Carthamus tinctorius ; enzymology ; genetics ; Chloroplast Proton-Translocating ATPases ; genetics ; DNA Primers ; DNA, Complementary ; Plant Proteins ; genetics

OBJECTIVETo study the effect of safflower injection on the proliferation and apoptosis of human leukemia cell line HEL and the relevant molecular mechanisms.

METHODSHEL cells were treated with different concentrations of safflower injection. HEL cells without safflower injection treatment were used as the control group. MTT method was used to detect the inhibitory rate of the HEL cells at 24, 48 and 72 hours after various concentrations of safflower injection treatment (10, 20, 30, 40 and 50 mg/mL). The cell cycle and apoptosis were detected using flow cytometry and the HOXB3-mRNA expression was measured by RT-PCR at 48 hours after safflower injection treatment (10, 20 and 30 mg/mL).

RESULTSCompared with the control group, various concentrations of safflower injection inhibited HEL cell proliferation in a dose-dependent manner (P<0.05). At 48 hours after various concentrations of safflower injection treatment, the number of treated cells in the G2/M phase increased, but that in the S phase decreased, and the apoptosis rate was significantly higher than that in the control group, with a dose-dependent manner (P<0.05). The expression of HOXB3-mRNA in safflower injection-treated cells decreased in a dose-dependent manner compared with the control group (P<0.05).

CONCLUSIONSSafflower injection can inhibit proliferation and induce apoptosis of HEL cells in vitro, and its underlying mechanisms may involve down-regulation of the HOXB3-mRNA expression.

Apoptosis ; drug effects ; Carthamus tinctorius ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Homeodomain Proteins ; genetics ; Humans ; Injections ; Leukemia ; drug therapy ; metabolism ; pathology

OBJECTIVETo investigate the expression of acidic fibroblast growth factor (aFGF) in transgenic safflower and lay the foundation for the use of the plant bioreactor large-scale production aFGF.

METHODThe haFGF gene was transformed into plant preference of the aFGF sequence as a basis for design of primers, plant preferences aFGF gene sequences was amplified by PCR. The vegetable body expression vector was constructed by using digested connection method and then transferred to Agrobacterium tumefaciens EHA105 by the freeze-thaw method. It transferred to safflowers by agrobacterium-mediated transformation method, and identified by PCR, southern blot and RT-PCR.

RESULTThe full-length aFGF gene sequences were amplified through PCR and constructed into plant expression vector with soybean oleosin and promoter, and transformed into safflower. Three independently transformed safflower plant units with point insertion were successfully obtained, which showed the same size of aFGF expression at the transcriptional level.

CONCLUSIONThe plant oil body expression vectors were successfully constructed, and the optimal condition for genetic transformation was selected. The transgenic safflower plants were obtained.

Agrobacterium tumefaciens ; genetics ; Carthamus tinctorius ; genetics ; Fibroblast Growth Factor 1 ; genetics ; Gene Expression Regulation, Plant ; Genetic Vectors ; Humans ; Membrane Proteins ; genetics ; Plant Proteins ; genetics ; Plants, Genetically Modified ; Polymerase Chain Reaction ; Transformation, Genetic

Carthamus tinctorius L. is a traditional Chinese medicine with the effect of promoting blood circulation and removing blood stasis. HSYA (hydroxysafflor yellow A) is the main effective component of Carthamus tinctorius L. In order to study the inhibitory effects of HSYA against PMN (polymorphonuclear) activation induced by LPS (lipopolysaccharide), rabbit PMN adhesion potency which was activated by LPS through colorimetry method was observed. Cellular free calcium concentration was determined by fluorescence spectrophotometry. RT-PCR was applied to study the effect of HSYA on PMN TNF-alpha and IL-6 mRNA expression; The inhibition of HSYA on NF-kappaB activation was monitored with immunofluorescence. The results showed that after treated with HSYA, the increase of adhesion potency (HSYA dose 1.01 x 10(-4) mol x L(-1)), free calcium concentration (HSYA dose 3.1 x 10(-5) mol x L(-1)), TNF-alpha and IL-6 mRNA expression elevation (HSYA dose 5.2 x 10(-1) mol x L(-1)) induced by LPS were inhibited. HSYA can inhibit NF-kappaB p65 subgroup nuclear translocation (HSYA dose 5.2 x 10(-5) mol x L(-1)). It is suggested that HSYA is effective in PMN activation induced by LPS.
Animals ; Calcium ; metabolism ; Carthamus tinctorius ; chemistry ; Cell Adhesion ; drug effects ; Chalcone ; analogs & derivatives ; isolation & purification ; pharmacology ; Interleukin-6 ; genetics ; metabolism ; Lipopolysaccharides ; toxicity ; Male ; Neutrophil Activation ; drug effects ; Neutrophils ; cytology ; metabolism ; Plants, Medicinal ; chemistry ; Quinones ; isolation & purification ; pharmacology ; RNA, Messenger ; metabolism ; Rabbits ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism