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Background@#In 2021, 5981 of cancer new cases was registered in Mongolian population. Among those cases, liver cancer was commonly registered with a prevalence of 32.7%. Studies on anticancer agents with no-adverse effects and good-preventive efficacy against cancer have been attracted more attention from the researchers in the field of pharmaceutical sciences. Scutellaria baicalensis Georgi, Saussurrea amara.L, Chiazospermum erectum Berh, and Carthamus tinctorius.L are well recognized as effective agent against liver diseases. Using these raw materials, researchers have been invented a traditional prescription and named as Hepaclin-4. In this study, we aimed to investigate the qualitative study of raw materials and some biologically active sub- stances in the compounds.@*Purpose@#To study the qualitative study of raw materials for Hepaclin-4 prescription@*Materials and methods@#Some qualitative properties of raw materials for Hepaclin-4 prescription, including appearance, minerals, some organic compounds, total ash, water-soluble substances and fungi, were investigated according to Mongolian pharmacopeia and total flavonoid was detected by thin layer chromatography.@*Results@#No changes were observed on the appearance of raw materials, and minerals and organic compounds weren’t detected in the prescription. No contamination with fungi and insects were identified. The moist in the raw materials were 5.9 to 8.1%, total ash was 4.7 to 13.3% and the water-soluble substances were detected 33.8 to 42.9%. Number of aerobic bacteria, fungi and E.coli, Salmonella species were detected in normal range, indicating that the prescription was matched with the requirement of pharmacopeia. According to the thin layer chromatography study of the raw materials, a yellow spot on the chromatogram were identified and same as quercetin (Rf=0.9-0.98) and rutin ((Rf=0.18-0.23)) as standard compounds, which indicated that the spot which indicated that the spot was flavonoids in the prescription.@*Conclusions@#These results showed that the appearance, moist, minerals, organic compound, water-soluble substances, ash and biologically active substances of the raw materials for Hepaclin-4 prescription was corresponded with the requirements of pharmacopeia, and flavonoid was detected in raw materials of Hepaclin-4.
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OBJECTIVE@#Flavonoids are the bioactive compounds in safflower (Carthamus tinctorius), in which chalcone synthase (CHS) is the first limiting enzyme. However, it is unclear that which chalcone synthase genes (CHSs) are participated in flavonoids biosynthesis in C. tinctorius. In this study, the CHSs in the molecular characterization and enzyme activities were investigated.@*METHODS@#Putative chalcone biosynthase genes were screened by the full-length transcriptome sequences data in C. tinctorius. Chalcone biosynthase genes in C. tinctorius (CtCHSs) were cloned from cDNA of flowers of C. tinctorius. The cloned gene sequences were analyzed by bioinformatics, and their expression patterns were analyzed by real-time PCR (RT-PCR). The protein of CtCHS in the development of flowers was detected by polyclonal antibody Western blot. A recombinant vector of CtCHS was constructed. The CtCHS recombinant protein was induced and purified to detect the enzyme reaction (catalyzing the reaction of p-coumaryl-CoA and malonyl-CoA to produce naringin chalcone). The reaction product was detected by HPLC and LC-MS.@*RESULTS@#Two full-length CtCHS genes were successfully cloned from the flowers of safflower (CtCHS1 and CtCHS3), with gene lengths of 1525 bp and 1358 bp, respectively. RT-PCR analysis showed that both genes were highly expressed in the flowers, but the expression of CtCHS1 was higher than that of CtCHS3 at each developmental stage of the flowers. WB analysis showed that only CtCHS1 protein could be detected at each developmental stage of the flowers. HPLC and LC-MS analyses showed that CtCHS1 could catalyze the conversion of p-coumaryl-CoA and malonyl-CoA substrates to naringin chalcone.@*CONCLUSION@#CtCHS1 is involved in the biosynthesis of naringin chalcone in safflower.
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In assessing the quality of seed lots, the vigor tests are complementary to the germination test and they identify differences in the degree of deterioration of the lots. For safflower, there is little information regarding these tests. In this way, the intention of this study was to adapt the accelerated aging test methodology to assess the physiological potential of safflower seeds (Carthamus tinctorius). For this purpose, 12 seed lots were evaluated for thousand-seed weight, germination, first germination count, seedling emergence test (emergence percentage, emergence speed index, relative emergence frequency and the initial, final and mean times) and accelerated aging. For the accelerated aging test, the traditional and saline methods were used. For this, the samples were conditioned in periods of 0, 8, 16, 24, 32 and 48 hours at 42 °C. Afterwards, they were submitted to the germination test, with evaluation of normal seedlings on the 3rd day. The 12 lots were evaluated within each period, in independent experiments. The data were submitted to analysis of variance and the means were compared using the Scott-Knott clustering method at 5% probability. In the traditional accelerated aging test the periods of 16, 24, 32 and 48 hours were more efficient in differentiating the lots in vigor levels, as they stratified the lots in three classes and the time of 8 hour classified the lots in two levels of vigor. In the accelerated saline aging method the time 32 hours were more efficient since it ranked seed lots at three levels of vigor and the periods of 8, 16 and 24 hour stratified the lots in two levels. In results obtained by the principal component analysis it was verified. The variables traditional accelerated aging for 24 and 32 hours correlated with emergence in the field. Therefore, the traditional accelerated aging test at 42 °C for 24 hours are promising for evaluating the physiological quality of safflower seeds.
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Plant Physiological Phenomena , Carthamus tinctorius/physiologyABSTRACT
UDP-glucose: flavonoid 3-O-glucosyltransferase (UF3GT) uses flavones, dihydroflavonol or anthocyanin as the acceptor and uridine 5′-diphosphate-sugar as the donor to catalyze the production of flavonoid 3-O-glycoside compounds. Based on sequence homology and transcriptome data, we screened and cloned a UF3GT gene named CtUF3GT (GenBank No. OM948976) from safflower. Biological information analysis demonstrate that CtUF3GT has highly conserved PSPG motif. The open reading frame of CtUF3GT is 1 446 bp, encoding 481 amino acids, with a presumed molecular weight of 52.36 kD and a theoretical isoelectric point of 5.33. Multiple sequence alignment indicate that CtUF3GT has a high homology with UF3GT from Asteraceae, and phylogenetic analysis showed that CtUF3GT clusters with functional identified UF3GTs from other species. The purified recombinant protein glucosylated kaempferol and quercetin to biosynthesis of kaempferol 3-O-glucoside and quercetin 3-O-glucoside, respectively. And CtUF3GT prefered to use kaempferol as substrate. qRT-PCR analysis showed that the UF3GT gene was most highly expressed in flowers, followed by leaves, with very low expression in bracts and stems, and no expression in roots. The expression of UF3GT gene showed a trend of increasing and then decreasing at different stages of flower development. The expression of CtUF3GT gene in safflower with different flower color was highly significant (P < 0.01) at S1, S2, S5, S6 and S7 stages of flower development, in which the expression of CtUF3GT in white safflower was 5.3 and 3.1 times higher than that in red safflower at S6 and S7 stages. This study lays the foundation for further exploring the role of CtUF3GT in the mechanism of safflower flavonoid secondary metabolite biosynthesis and accumulation.
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ObjectiveTo study on the suitable cryopreservation conditions of Carthamus tinctorius seeds. MethodThe germination rate，relative conductivity，soluble sugar，soluble protein， and related enzyme activities of C. tinctorius seeds， as well as the hydroxysafflor yellow A （HSYA） content in Carthami Flos after storage and breeding for four months were detected under different temperature conditions （long-term storage，medium-term storage，short-term storage，room temperature，and ultra-low temperature refrigerator），different water content （8.1%，6.6%，5.2%，and 3.9%），and different storage time （2，4，6，8， 10 months）. SPSS 20.0 was used for statistical analysis. ResultDuring the storage for 10 months，the changing trend of the germination rate of C. tinctorius seeds revealed that it was more suitable to store seeds with low water content at a lower temperature. The differences in germination rate of seeds caused by storage temperature，seeds water content， and storage time were statistically significant. After storage for 10 months，the germination rate was significantly correlated with other detection indexes. ConclusionThe proper water content of C. tinctorius seeds in long-term and medium-term storage is 5.2% or 6.6%，and that in short-term and ultra-low temperature refrigerator is 3.9% or 5.2%. As revealed by the comparison results， the optimal storage conditions for C. tinctorius seeds were long-term storage and water content of 5.2%， which resulted in the highest germination rate and content of soluble sugar and soluble protein and the lowest relative conductivity after storage for 10 months. Additionally， the content of hydroxy safflor yellow A （HSYA） in Carthami Flos obtained after breeding and regeneration for four months was higher than that obtained after room temperature storage.
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ABSTRACT: The safflower (Carthamus tinctoriusL.) has an uneven flowering and fruiting, which can cause problems in seed production and harvesting in regions with hot and humid climates. However, little is known about the optimal safflower harvest time. Therefore, this study evaluated the optimumtiming for seed harvest of three safflower genotypes (2106, S-325, and 7329).The experiment was a randomized complete block design with six replications. The harvest started 16 days after flowering (DAF) and ended at 52 DAF. Ten harvests were made in total. Seed water content, seeds fresh and dry matter, seed germination, and first germination counts were evaluated.Genotypes 2106 and 7329 had germination rates of 79% and 91%, respectively, at 34 and 38 DAF, while genotype S-325 had 90% germination at 37 DAF. Harvesting at 52 DAF combined with a rainy season impaired the germination of safflower seeds. The harvest time most suitable for safflower occurred between 34 and 42DAF, when the seeds have the seed water content between 26% and 33%.
RESUMO: O cártamo (Carthamus tinctoriusL.) apresenta flores e frutos irregulares, o que pode causar problemas na produção e colheita de sementes em regiões com clima quente e úmido. No entanto, pouco se sabe sobre o tempo ideal de colheita de cártamo. Portanto, o objetivo desteestudo foi avaliar o momento ideal para a colheita de sementes de três genótipos de cártamo(2106, S-325 e 7329). O experimento foi delineado em blocos casualizados, com seis repetições. A colheita começou 16 dias após o florescimento (DAF) e terminou aos 52 DAF. Dez colheitas foram feitas no total. Foram avaliados o teor de água das sementes, matéria fresca e seca das sementes, germinação das sementes e primeira contagem de germinação. Os genótipos 2106 e 7329 tiveram taxas de germinação de 79% e 91%, respectivamente, aos 34 e 38 dias após a floração (DAF), enquanto o genótipo S-325 teve 90% de germinação aos 37 DAF. A colheita aos 52 DAF combinada com uma estação chuvosa prejudicou a germinação das sementes de cártamo. A época de colheita mais adequada para o cártamo ocorreu entre 34 e 42 DAF, quando as sementes apresentam teor de água entre 26% e 33%.
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Objective: In order to obtain new glycosyltransferases with highly efficient catalysis, the glycosyltransferases from Carthamus tinctorius which contains diverse types of glycosides were mined. Methods: A new glycosyltransferase gene (UGT88B2) with full length was obtained by PCR and further transformed into Escherichia coli for heterologous expression. The catalytic activity of recombinant UGT88B2 was determined by HPLC-MSn. The structures of representative catalytic products were elucidated by MS and NMR. Results: UGT88B2 exhibited catalytic promiscuity and various patterns in glycosylation of flavonoids with high efficiency. Conclusion: A new glycosyltransferase named UGT88B2 was successfully mined and can be employed as enzymatic tools in glycosylation of flavonoids.
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Objective: To prepare the self-microemulsion gel drug delivery system of Carthamus tinctorius extract based on Mentha haplocalyx oil as oil phase. Methods: M. haplocalyx oil was used as the oil phase, and C. tinctorius extract was used as the water phase. The prescription of self-microemulsion were optimized by pseudo-ternary phase diagram, the process and prescription of gel were screened by single-factor method and the appearance, viscosity and pH value were evaluated. Result: The optimal formulation of CTE-SMEDDS-BGs was as following: F68 was the emulsifier, anhydrous ethanol was the co-emulsifier, the Km ratio was 1:1, and the total amount of emulsifier and co-emulsifier to M. plocalyx oil was 8:2, carbopol-980 was 2%, glycerin was 6%, and C. tinctorius extract was 5 mL. The CTE-SMEDDS-BGs was obtained by adding the CTE-SMEDDS into swelling gel matrix and triethylamine was used to adjust the pH to 6.0. The characteristics of appearance were yellow translucent, moderate viscosity, uniform and delicate, non-greasy, and easy to spread on the skin, the viscosity was 4.98 × 104 mPa•s (RSD was 1.53%), pH was 6.04 (RSD was 0.44%). Conclusion: The CTE- SMEDDS-BGs with M. plocalyx oil as oil phase is simple and stable, and meets the requirements of gel topical preparations.
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OBJECTIVE：To investigate the correlation of storage life and effective composition content with color value of Carthamus tinctorius ，and to provide reference for the quality evaluation of C. tinctorius with different years of storage. METHODS：Using 24 batches of C. tinctorius from same place of production with different years of storage （0，1，2 years，8 batches each type ）as samples ，the contents of hydroxysafflor yellow A （HSYA）and kaempferol were determined by HPLC. Color value [lightness value （L*），red-green value （a*），yellow-blue value （b*）] were determined by spectrophotometer. SPSS 19.0 statistical software was used to analyze the correlation of storage life and effective composition content with color value. RESULTS：Kaempferol content was still high after 1 year or 2 years of storage （0.161％，0.061％，respectively）. However ，the content of HSYA decreased with the prolongation of the storage life （the average content of HSYA were 2.46％，1.58％，and 1.51％ after storage 0，1 and 2 years，respectively），and the color of the drug became darker （a* value decreased ）. Results of correlation analysis showed that the content of HSYA was positively associated with color value L*，a*（r＝0.430，0.781，P＜0.05 or P＜0.01）；the content of HSAY was negatively associated with storage life （r＝－0.777，P＜0.01）. There was no correlation between the remaining variables （P＞0.05）. CONCLUSIONS ：The longer the storage life ，the darker the color and the lower the content of HSYA ，so it is not suitable for over year and multiyear preservation.
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We determined a component-target-disease network for Carthamus tinctorius L. and the key compounds, identified by topological analysis, were related to vasculitis, coronary heart and cerebrovascular disease. Based on these compounds, the chromatographic fingerprint of Carthamus tinctorius L. was established. Firstly, 132 compounds were obtained from TCMID and TCMSP databases. Their targets were predicted in the PharmMapp and HemMapper databases. CardioGenBase, Therapeutic Target Database and DisGeNET databases were used to collect targets of vasculitis, coronary heart disease and cerebrovascular disease. The corresponding relationships between component and target protein were established by mapping. Finally, the "component-target-disease" network was built with Cytoscape software. The core network and key nodes were analyzed with the Cytohubba plug-in. The results showed that the 24 key compounds were alpha-tocopherol, adenosine, quinone chalcone pigments such as hydroxysafflor yellow A, safflower yellow, quercetin, kaempferol and other flavonoids, organic acids such as stearic acid, linolenic acid, coumaric acid and cinnamic acid. This resulting chromatographic fingerprint of Carthamus tinctorius L. showed good consistency, and the core chemical compounds obtained by topological analysis of the network of "component-target-disease", could be used as quality control markers. Our research provides a new approach for the identification of quality control indicators in Chinese medicinal materials.
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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.
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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 , GeneticsABSTRACT
Objective To study the alkaloids from leaves of Carthamus tinctorius. Methods The alkaloids were isolated and purified by silica gel, MCI, Sephadex LH-20 column chromatographies, and semi-preparative HPLC, and their structures were elucidated by physical and spectroscopic analysis. Results A new β-carboline alkaloid, 4,9-dimethoxy-1-ethyl-β-carboline (1) along with one known analogue 4-methoxy-1-ethyl-β-carboline (2), were isolated from the leaves of C. tinctorius. Compounds 1 and 2 showed the cytotoxicity against HepG2 cell lines with IC50 values of (15.2 ± 0.58) μmol/L and (17.4 ± 0.33) μmol/L, respectively. Conclusion Compounds 1 and 2 are firstly obtained from Carthamus genus, and compound 1 is a new compound named carthine A. Both compounds 1 and 2 exhibited cytotoxicity against HepG2 cell lines.
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Objective: To clone a coding region sequence of AP2/ERF transcription factor family from Carthamus tinctorius, and construct a plant expression vector. Methods: A gene (CtERF1) of AP2/ERF family transcription factor was cloned by RT-PCR based on the sequence of C. tinctorius transcription sequencing, the phylogenetic tree was constructed by ClustalW 1.83 software, Spe I and Xba I restriction sites were introduced to construct over-expression vector pBASTA-CtERF1 containing 35S promoter. Results: CtERF1 gene had a functional domain of a typical AP2/ERF gene encoding 297 amino acids, and contained an AP2 region speculated to be located in cytoplasm and nucleus, which was ERF subprotein. Systematic evolution analysis showed that CtERF1 gene had some homology with other plant species, among which the relationship with Populus deltoides and Panax japonicus were the closest. The pBASTA-CtERF1 plant expression vector was constructed successfully by molecular biology. Conclusion: A CtERF1 gene of C. tinctorius AP2/ERF transcription factor family was cloned and the plant expression vector pBASTA-CtERF1 was constructed successfully.
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Objective: To clone CtWD40 transcription factors (TFs) and analyze its expression level in different tissues of Carthamus tinctorius and relationship with the content of hydroxysafflor yellow A (HSYA). Methods: CtWD40 gene was obtained by cloning the WD40 candidate gene from transcriptome of C. tinctorius as reference. Its conserved domain, three-dimensional structure and phylogeny analysis were analyzed by bioinformatics methods. The CtWD40 expression pattern was also analyzed by qRT-PCR method, in the same time, HSYA content in different petals were analyzed by HPLC. Results: Gene sequence of CtWD40 was obtained and eight conserved WD domains were found in CtWD40 gene sequences. Phylogenetic analysis revealed that CtWD40 had a closed homology with WD40 from composite plants. Conclusion: The expression of CtWD40 gene was first increased and then decreased in petal tissues of different flowering stages.Pearson coefficient revealed significant correlation between CtWD40 expression and HSYA content in C. tinctorius petal.
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Objective: To optimize the preparation technology of phospholipid complex of Carthamus tinctorius (safflower) extract and investigate its permeability. Methods: On the basis of single factor experiment, the preparation process was optimized by using the response surface analysis method, taking the compound rate of phospholipid complex of safflower extract as the index. It was characterized by UV-vis absorption spectrum and infrared spectrum. The modified Franz diffusion cell was used to evaluate the membrane permeability of safflower extract and phospholipid complex of safflower extract with different drug-lipid ratios in vitro. Results: The optimum preparation technology of phospholipid complex of safflower extract was as follows: methanol was used as compound solvent, the concentration of safflower extract was 5.0 mg/mL, and the mass ratio of phospholipid to phospholipid was 1∶1, the reaction time was 1.5 h, and the reaction temperature was 55 ℃. The results of transmembrane experiment showed that the 24-hour cumulative permeability (Q24) of safflower extract phospholipid complex with drug-fat ratio of 2, 1, and 0.5 was (15.07 ± 1.24), (15.61 ± 0.92), (21.94 ± 1.54), and (21.05 ± 1.39) μg/cm2, respectively. Conclusion: The optimized preparation process is reasonable and feasible, and the phospholipid complex of safflower extract can obviously improve its membrane permeability.
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Objective: To establish a Logistic model for quality assessment of Carthamus tinctorius based on content determination and bioactivity. Methods: A method to determinate hydroxy safflor yellow A by using ultra-high performance liquid chromatography (UPLC) was proposed. The activity of anticoagulant was reflected by thrombin time (TT) and the anti-oxidant activity was expressed by scavenging ability of hydroxyl radical and DPPH radical. Later, a Logistic model grade evaluation of C. tinctorius was constructed based on correlation analysis between content and biological activity Logistic.Results: The constructed Logistic model had outstanding stability and high prediction accuracy of grades and it divides 19 batches of C. tinctorius into four grades. Conclusion: The Logistic model based on content determination and biological activity is applicable to assess quality of C. tinctorius slices and provides some reference for quality control.
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OBJECTIVE： To study the mechanism of Prunus persica-Carthamus tinctorius couplet medicine in the treatment of osteonecrosis of the femoral head （ONFH）. METHODS： The network pharmacology was adopted. The active components of P. persica -C. tinctorius couplet medicine and ONFH target were screened through TCM systematic pharmacological analysis platform target （TCMSP）， DRAR-CPI， hnuman gene database （GeneCards） and online medelian inheritance in man （OMIM） using oral availability of compounds （OB）＞30％ and drug like （DL）＞0.18 as standard. Network topology attribute analysis software Cytoscape 3.6.0 was utilized to construct the active components-ONFH targets network. Target protein interaction network was established on the basis of STRING database， and top 5 target proteins in the list of connectivity were screened， and molecular docking server was used to predict the combination activity of active components from P. persica -C. tinctorius couplet medicine. The biological processes of target gene ontology （GO） and metabolic pathways in Kyoto encyclopedia of genes and genomes （KEGG） were enriched and analyzed by DAVID. RESULTS： A total of 44 active components were screened from P. persica -C. tinctorius couplet medicine， including baicalin， quercetin， etc.， and 78 targets related to ONFH including VEGF， VEGI， CRP， etc. Through analysis of molecular docking server， binding activity of active components of P. persica -C. tinctorius couplet medicine to target protein was strong. GO and KEGG pathway enrichment analysis showed that biological process of P. persica -C. tinctorius couplet medicine for ONFH was related with negative regulation of apoptosis process and positive regulation of nuclear factor-κB transcription factor， mainly through regulating secretory glycoprotein signaling pathway， melanogenesis signaling pathway， VEGF signaling pathway， signaling pathway of basal cell carcinoma， adenosine-activated protein kinase signaling pathway. CONCLUSIONS： This study preliminarily validates the major targets and pathways of P. persica -C. tinctorius couplet medicine for ONFH， which lay a foundation for further study on their pharmacological action.
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The specific PCR primer was designed base on ITS2 sequence in GenBank, and we developed a SYBRGreen real-time fluorescence quantitative PCR system for identification of Crocus sativus and Carthamus tinctorius source. Compared with Chinese herbal medicine DNA barcode technique, this method showed characteristics of shorter time, higher specificity and sensitivity. Using this method to detect 15 samples, 4 were C. sativus, 8 were C. tinctorius, and the other 3 samples were none of them. The result was in accordance with Chinese herbal medicine DNA barcode. This study lays the foundation for identification of related Chinese medical materials.
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Carthamus tinctorius , Crocus , Real-Time Polymerase Chain ReactionABSTRACT
The expression of fibroblast growth factor 9 (FGF9) recombinant fusion protein in Carthamus tinctorius was used to identify its effect on hair regrowth and wound repair system in mice, providing a basis for C. tinctorius as a plant bioreactor, and establishing a foundation for commercial applications of FGF9 fusion protein in hair regrowth and wound repair. The identified pOTBar-oleosin-rhFGF9 plasmid was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method, and the oleosin-rhFGF9 gene was transformed into safflower leaves by A. tumefaciens mediated method. Transgenic safflower seedlings were then obtained by tissue culture. After basta screening, transgenic T₃ safflower seeds were obtained by grafting method, PCR verification and propagation. The expression of oleosin-rhFGF9 was detected by Western blot, and the content of oleosin-rhFGF9 fusion protein was 0.09% by using ELISA quantitative method. It was observed that 60 μg·L⁻¹ transgenic safflower oil had better effect on promoting NIH/3T3 cells proliferation in a certain dose-dependent manner. Sixty C57BL/6 mice were used to establish alopecia model and wound model respectively, and then were randomly divided into control group (treated with PBS or saline), negative group (treated with wild type safflower seed oil bodies, 60 g·L⁻¹), positive group (treated with FGF9, 0.054 g·L⁻¹), low dose group (treated with transgenic safflower oil bodies, 10 g·L⁻¹) and high dose group (treated with transgenic safflower oil bodies, 60 g·L⁻¹). The skin of all above-mentioned mice models were coated with soft adhesive manner every other day, 100 μL/time. After 15 days, the mice skin was cut and embedded for histological analysis. The hair regrowth experimental results showed that the hair of mice grew well, and the mice in high dose group had bushy hair, with significant effect on regeneration hair number as compared with the positive group. The healing was obvious in wound experiment, with significant healing effect in positive group, high dose group and low dose group as compared to blank control group. Furthermore, high dose group remarkably showed a better and higher healing effect than the positive group at day 5. Oleosin-rhFGF9 was successfully transformed into safflower, and T₃ transgenic safflower oil bodies expressed oleosin-rhFGF9 fusion protein were obtained, with the role of promoting hair regeneration and wound repair in mice.
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Objective To explore the protective effects of Salvia miltiorrhiza and Carthamus tinctorius active ingredients in different compatibility on cerebral ischemia-reperfusion injury in rats. Methods Adult male Sprague-Dawley (SD) rats were selected to establish a cerebral ischemia-reperfusion (I/R) injury model by middle cerebral artery occlusion (MCAO) method. The rats were randomly divided into sham operation group, model group, and positive control group (Danhong Injection 2 mL/kg); And orthogonal test method L9(34) was adopted to compose nine compatibility groups from main active ingredients of S. miltiorrhiza and C. tinctorius (trashinol, salvianolic acid A, salvianolic acid B, and HYSA). Rats were tail iv administrated once daily for continuous 3 d. Score neurological deficit was evaluated after 3 d; qRT-PCR was used to detect mRNA expression of glucose regulated protein 78 (GRP-78), nuclear factor-κB (NF-κB), C/EBP homologous protein (CHOP), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6); HE staining was used to detect the pathological changes of cerebral cortex, and the expression of NF-κB p65 protein in cerebral cortex was detected by immunohistochemistry. Results Compared to the model group, Danhong Injection group and orthogonal compatibility nine groups significantly reduced the neurological deficit scores of rats with cerebral ischemia-reperfusion; increased the mRNA expression of GRP-78 in the cerebral cortex; decrease the mRNA expression of NF-κB, CHOP, TNF-α, and IL-6, and decrease the protein expression of NF-κB p65 in the cerebral cortex. The results also showed that the protective effects of the 4th group (danshensu 30 mg/kg, salvianolic acid A 2.5 mg/kg, salvianolic acid B 16 mg/kg, and HYSA 8 mg/kg), 6th group (trashinol 30 mg/kg, salvianolic acid A 10 mg/kg, salvianolic acid B 8 mg/kg, and HYSA 4 mg/kg) in the 9th group were more significant for cerebral ischemia reperfusion injury in rats. Conclusion The active components of S. miltiorrhiza and C. tinctorius can play a good protective role in endoplasmic reticulum stress and inflammation in rats with cerebral ischemia reperfusion injury.