Investigation into safflower injection as a prophylactic treatment for retinal vein occlusion in a rabbit model.

The study aimed to assess the effect and mechanism of safflower injection in preventing retinal vein thrombosis in rabbits. Twenty healthy adult pigmented rabbits were randomly assigned to either the experimental group, receiving safflower injection, or the control group, receiving normal saline. After two weeks of treatment, blood samples were collected to analyze platelet adhesion and aggregation rates. Photodynamic therapy was applied to induce occlusion in the target retinal vein. Fundus photography and fluorescein angiography were recorded using a dynamic microscopic monitoring system, and laser speckle imaging was employed to assess blood flow in the affected vein. The experimental group exhibited significantly lower rates of platelet adhesion and aggregation compared to the control group. Following the induction of retinal vein occlusion, the experimental group showed a lower complete occlusion rate of the target retinal vein. Although initial blood flow in the target vein was similar between groups, the blood flow at 1, 3, and 5 min post-occlusion was significantly higher in the experimental group. Safflower injection delayed retinal vein thrombosis formation, preserved blood flow in the affected retinal area, and reduced platelet adhesion and aggregation. These effects facilitated vascular reperfusion within a limited timeframe.

Uncovering the Role of Hydroxycinnamoyl Transferase in Boosting Chlorogenic Acid Accumulation in Carthamus tinctorius Cells under Methyl Jasmonate Elicitation.

Chlorogenic acids (CGAs) are bioactive compounds widely used in the food, pharmaceutical, and cosmetic industries. Carthamus tinctorius is an important economic crop, and its suspension cells are rich in CGAs. However, little is known about the biosynthesis and regulation of CGAs in Carthamus tinctorius cells. This study first elucidated the regulatory mechanism of CGA biosynthesis in methyl jasmonate (MeJA)-treated Carthamus tinctorius cells and the role of the MeJA-responsive hydroxycinnamoyl transferase (HCT) gene in enhancing their CGA accumulation. Firstly, temporal changes in intracellular metabolites showed that MeJA increased the intracellular CGA content up to 1.61-fold to 100.23 mg·g-1. Meanwhile, 31 primary metabolites showed significant differences, with 6 precursors related to increasing CGA biosynthesis. Secondly, the transcriptome data revealed 3637 new genes previously unannotated in the Carthamus tinctorius genome and 3653 differentially expressed genes. The genes involved in the plant signaling pathway and the biosynthesis of CGAs and their precursors showed a general up-regulation, especially the HCT gene family, which ultimately promoted CGA biosynthesis. Thirdly, the expression of a newly annotated and MeJA-responsive HCT gene (CtHCT, CtNewGene_3476) was demonstrated to be positively correlated with CGA accumulation in the cells, and transient overexpression of CtHCT enhanced CGA accumulation in tobacco. Finally, in vitro catalysis kinetics and molecular docking simulations revealed the ability and mechanism of the CtHCT protein to bind to various substrates and catalyze the formation of four hydroxycinnamic esters, including CGAs. These findings strengthened our understanding of the regulatory mechanism of CGA biosynthesis, thereby providing theoretical support for the efficient production of CGAs.

Investigating the therapeutic effects and mechanisms of Carthamus tinctorius L.-derived nanovesicles in atherosclerosis treatment.

BACKGROUND: Carthamus tinctorius L., a traditional herbal medicine used for atherosclerosis (AS), lacks a clear understanding of its therapeutic mechanisms. This study aimed to investigate the therapeutic effects and mechanisms of Carthamus tinctorius L.-derived nanovesicles (CDNVs) in AS treatment. METHODS: CDNVs were isolated and characterized using improved isolation methods. Transmission electron microscopy, nanoparticle tracking analysis, and protein analysis confirmed their morphology, size, and protein composition. Small RNA sequencing was performed to identify the miRNA profile of CDNVs, and bioinformatics analysis was used to determine their potential biological roles. In vivo biodistribution and toxicity studies were conducted in mice to assess the stability and safety of orally administered CDNVs. The anti-atherosclerotic effects of CDNVs were evaluated in ApoE-/- mice through plaque burden analysis. The protective effects of CDNVs on ox-LDL-treated endothelial cells were assessed through proliferation, apoptosis, reactive oxygen species activation, and monocyte adhesion assays. miRNA and mRNA sequencing of CDNV-treated endothelial cells were performed to explore their regulatory effects and potential target genes. RESULTS: CDNVs were successfully isolated and purified from Carthamus tinctorius L. tissue lysates. They exhibited a saucer-shaped or cup-shaped morphology, with an average particle size of 142.6 ± 0.7 nm, and expressed EV markers CD63 and TSG101. CDNVs contained proteins, small RNAs, and metabolites, including the therapeutic compound HSYA. Small RNA sequencing identified 95 miRNAs, with 10 common miRNAs accounting for 72.63% of the total miRNAs. These miRNAs targeted genes involved in cell adhesion, apoptosis, and cell proliferation, suggesting their relevance in cardiovascular disease. Orally administered CDNVs were stable in the gastrointestinal tract, absorbed into the bloodstream, and accumulated in the liver, lungs, heart, and aorta. They significantly reduced the burden of atherosclerotic plaques in ApoE-/- mice and exhibited superior effects compared to HSYA. In vitro studies demonstrated that CDNVs were taken up by HUVECs, promoted proliferation, attenuated ox-LDL-induced apoptosis and ROS activation, and reduced monocyte adhesion. CDNV treatment resulted in significant changes in miRNA and mRNA expression profiles of HUVECs, with enrichment in inflammation-related genes. CXCL12 was identified as a potential direct target of miR166a-3p. CONCLUSION: CDNVs isolated from Carthamus tinctorius L. tissue lysates represent a promising oral therapeutic option for cardiovascular diseases. The delivery of miRNAs by CDNVs regulates inflammation-related genes, including CXCL12, in HUVECs, suggesting their potential role in modulating endothelial inflammation. These findings provide valuable insights into the therapeutic potential of CDNVs and their miRNAs in cardiovascular disease.

Growth performance, meat quality and hematological parameters of broiler chickens fed safflower seed.

The aim of the current study was to determine the effects of dietary supplementation of safflower seed (SS) on the growth performance and hematological parameters of broiler birds along with the physicochemical, textural and sensory attributes of chicken meat. A total of 200 male chickens (7-days-old) were distributed into 5 groups (40 chickens in each) with 5 replicates of 8 chicks in a 42-day experiment. Each group was allocated to one of 5 dietary treatments, i.e., 0, 2.5, 5, 7.5, and 10% SS. The experimental diets were formulated for starter (7 to 21 days) and finisher (22 to 42 days) phases. Inclusion of SS in the diet improved growth performances in treatment groups between 7 and 42 days. The highest and lowest body weights were observed at the 5% SS and 0% SS levels, respectively. The physicochemical attributes of breast and thigh meat were found (P > 0.05) except for crude fat. The crude fat was significantly (P < 0.05) increased with increasing levels of SS in the diet. The inclusion of SS in the diet did not negatively impact the textural properties, i.e., hardness, cohesiveness, springiness, gumminess, chewiness, and shear force of breast and thigh meat. There was no significant difference in the sensory parameters of cooked chicken meat with increasing levels of SS in the diet. The results demonstrated a significant (P < 0.01) improvement in hematological parameters in the blood samples of broiler chickens fed diet supplemented with various levels of SS for five weeks. These findings suggest that, SS may be used as an oil seed for broiler chicken feed.

Synergistic effects of vermicompost and mycorrhizal inoculation on arsenic tolerance and phytostabilization in safflower (Carthamus tinctorius L.).

Arsenic (As) is a hazardous metalloid, and mycorrhizal inoculation and vermicompost amendment can influence As bioremediation. However, the studies concerning the sole and joint effects of arbuscular mycorrhizal fungi (AMF) and vermicompost on the phytoremediation efficacy are limited. In the present study at first, the impact of various levels of vermicompost (0, 2, 4, and 8% w/w) was investigated on As mobility in soil and safflower (Carthamus tinctorius L.) plants grown in soils of spiked with 0, 40, and 80 mg kg-1 As. Results revealed that with increasing dose of vermicompost, bioavailable As in soil decreased which resulted in a lower bioaccumulation factor and translocation factor (TF) and led to a significant increase of tolerance index (TI) and total chlorophyll content in plants. The highest effect on TI and total As accumulation per plant was obtained in the dosage of 8% vermicompost. Therefore, in the second experiment, the sole and joint effects of 8% vermicompost and inoculation with Rhizophagus intraradices were assessed on the tolerance and accumulation of As in safflower. The addition of vermicompost aggravated mycorrhizal colonization but did not significantly influence mycorrhizal dependency under As stress. The joint effects of AMF and vermicompost improved the dry weight of roots and shoots, increased P concentration and P:As ratio in shoots, reduced malondialdehyde content, and moderated ascorbate peroxidase activity in leaves of As-stressed plants. Interestingly, co-application of AMF and vermicompost more than their sole usage decreased As concentration in shoots and TF and more strongly increased total As accumulation per plant. These findings suggest that mycorrhizal inoculation and vermicompost have a synergistic effect on As tolerance and phytostabilization efficacy of safflower plants, and their combined application may be a new option to remediate As-contaminated soils.

Highly Promiscuous Flavonoid Di-O-glycosyltransferases from Carthamus tinctorius L.

Safflower (Carthamus tinctorius L.) has been recognized for its medicinal value, but there have been limited studies on the glycosyltransferases involved in the biosynthesis of flavonoid glycosides from safflower. In this research, we identified two highly efficient flavonoid O-glycosyltransferases, CtOGT1 and CtOGT2, from safflower performing local BLAST alignment. By constructing a prokaryotic expression vector, we conducted in vitro enzymatic reactions and discovered that these enzymes were capable of catalyzing two-step O-glycosylation using substrates such as kaempferol, quercetin, and eriodictyol. Moreover, they exhibited efficient catalytic activity towards various compounds, including flavones (apigenin, scutellarein), dihydrochalcone (phloretin), isoflavones (genistein, daidzein), flavanones (naringenin, glycyrrhizin), and flavanonols (dihydrokaempferol), leading to the formation of O-glycosides. The broad substrate specificity of these enzymes is noteworthy. This study provides valuable insights into the biosynthetic pathways of flavonoid glycosides in safflower. The discovery of CtOGT1 and CtOGT2 enhances our understanding of the enzymatic processes involved in synthesizing flavonoid glycosides in safflower, contributing to the overall comprehension of secondary metabolite biosynthesis in this plant species.

Antibacterial, ROS scavenging and angiogenesis promotingϵ-Polylysine/gelatin based hydrogel containing CTLP to regulate macrophages for pressure ulcer healing.

Pressure ulcers (PUs) have emerged as a substantial burden on individuals and society. The introduction of innovative dressings that facilitate the healing of pressure ulcer wounds represents a cost-effective alternative for treatment. In this study, the emphasis is on the preparation of Carthamus tinctorius L. polysaccharide (CTLP) as hydrogel microspheres (MPs), which are then encapsulated within a hydrogel matrix crosslinked with phenylboronic acid gelatin (Gelatin-PBA) andϵ-polylysine-grafted catechol (ϵ-PL-Cat) to enable sustained release for promoting pressure ulcer healing. The presented Gelatin-PBA/ϵ-PL-Cat (GPL)/CTLP-MPs hydrogel demonstrated outstanding self-healing properties. In addition,in vitroexperiments revealed that the hydrogel exhibited remarkable antibacterial activity, excellent biocompatibility. And it showed the capacity to promote vascular formation, effectively scavenge reactive oxygen species, and facilitate macrophage polarization from the M1 to M2 phenotype.In vivowound healing of mice PUs indicated that the prepared GPL/CTLP-MPs hydrogel effectively accelerated the formation of granulation tissue and facilitated the healing of the wounds. In summary,in vivoandin vitroexperiments consistently highlight the therapeutic potential of GPL/CTLP-MPs hydrogel in facilitating the healing process of PUs.

The mechanism of Semen Persicae-Flos Carthami in treating hypertrophic scar: A study based on network pharmacological analysis and in vitro experiments.

Traditional medicine believes that hypertrophic scar (HS) falls into the category of "blood stasis". Chinese herbs for promoting blood circulation and removing blood stasis, activating meridians, and relieving pain are usually selected to treat HS by traditional Chinese medicine (TCM). Both Semen Persicae (SP) and Flos Carthami (FC) are confirmed to be effective for HS. Clinically, SP and FC are often used in combination with each other. However, the pharmacodynamic mechanism and molecular target of SP-FC in the treatment of HS are still unclear. Therefore, this study is intended to explore the mechanism and target of SP-FC in the treatment of HS through network pharmacology combined with in vitro cell and molecular biology experiments. Target genes of SP-FC were obtained from the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP), and targets of HS-related diseases were searched from databases such as Disgenet and GeneCards. Based on the targets searched and obtained, a Venn diagram was plotted to acquire common targets of SP-FC-HS. Next, STRING 11.0 was employed for protein-protein interaction (PPI) network analysis of common targets; and cytoscape 3.9.0 for connection relationship analysis of PPI and plotting of a "drug-component-target" network diagram. Besides, a modified explant culture method was applied to separate primary hypertrophic scar fibroblasts (HSFs); MTT assay to detect cell viability of HSFs after treatment by SP-FC for 24 h; Annexin V-FITC/PI double staining combined with flow cytometry to test apoptosis; western blot to check the protein expression level of p53; and real-time fluorescence quantitative PCR to determine mRNA level of p53. In the analysis of network pharmacology, 269 pharmacological targets of SP, 449 pharmacological targets of FC, and 2569 targets of HS-related diseases were screened from the databases. After plotting the Venn diagram, 116 common targets of SP-FC-HS were acquired. In vitro experiments showed that the expression of p53 in HSFs was decreased. SP-FC significantly reduces the viability of HSFs, increases p53 levels in HSFs, and promotes apoptosis. SP-FC can reduce scar formation by promoting p53 expression.

Uncovering Mechanism and Efficacy of Salvia Miltiorrhiza-Safflower in Cerebral Ischemia-Reperfusion Injury.

Cerebral ischemia (CI) is the main cause of stroke morbidity and disability. This study aims to identify the early molecular regulation responsible for the therapeutic effectiveness of the Herb pair Danshen-Honghua (DH) for CI. The major targets of DH were identified by searching the public database of traditional Chinese medicine (TCM). In addition, GeneCards, Disgenet, and GeneMap databases in OMIM were used to determine the disease targets of CI. A total of 88 common targets of DH and CI were selected, a protein-protein interaction (PPI) network was established by Cytoscape, and 19 core targets were screened. These genes were primarily enriched in biological processes including wound healing, reaction to oxidative stress, and response to peptides, lipid and atherosclerosis, Age-rage signaling pathway, and TNF signaling pathway by KEGG and GO enrichments. The effective components of DH had stable binding to these key targets by molecular docking. Finally, it was verified that the mechanism of DH on CI treatment may be related to the activation of the TNF-α/JNK signaling pathway by establishing the middle cerebral artery occlusion (MCAO) rat model.

Elevated atmospheric CO2 concentration mitigates salt damages to safflower: Evidence from physiological and biochemical examinations.

The physiological and biochemical responses of salt-stressed safflower to elevated CO2 remain inadequately known. This study investigated the interactive effects of high CO2 concentration (700 ± 50 vs. 400 ± 50 µmol mol-1) and salinity stress levels (0.4, 6, and 12 dS m-1, NaCl) on growth and physiological properties of four safflower (Carthamus tinctorius L.) genotypes, under open chamber conditions. Results showed that the effects of CO2 on biomass of shoot and grains depend on salt stress and plant genotype. Elevated CO2 conditions increased shoot dry weight under moderate salinity stress and decreased it under severe stress. The increased CO2 concentration also increased the safflower genotypes' relative water content and their K+/Na + concentrations. Also enriched CO2 increased total carotenoid levels in safflower genotypes and improved membrane stability index by reducing H2O2 levels. In addition, increased CO2 level led to an increase in seed oil content, under both saline and non-saline conditions. This effect was particularly pronounced under severe saline conditions. Under conditions of high CO2 and salinity, the Koseh genotype exhibited higher grain weight and seed oil content than other genotypes. This advantage is due to the higher relative water content, maximum quantum efficiency of photosystem II (Fv/Fm), and K+/Na+, as well as the lower Na+ and H2O2 concentrations. Results indicate that the high CO2 level mitigated the destructive effect of salinity on safflower growth by reducing Na + uptake and increasing the Fv/Fm, total soluble carbohydrates, and membrane stability index. This finding can be used in safflower breeding programs to develop cultivars that can thrive in arid regions with changing climatic conditions.

Metagenomic sequencing revealed the regulative effect of Danshen and Honghua herb pair on the gut microbiota in rats with myocardial ischemia injury.

In recent years, more and more evidence has shown that the disorder of gut microbiota (GM) is closely correlated with myocardial ischemia (MI). Even though the Danshen and Honghua herb pair (DHHP) is widely used in treating cardiovascular disease in China and exhibits obvious clinical efficacy on MI, the anti-MI mechanism of DHHP remains and needs to be explored in depth. Thus, in this study, we investigated whether the amelioration effect and molecular mechanism of DHHP on MI were related to regulating GM through pharmacodynamics evaluation and metagenomic sequencing. Histopathological testing results showed that DHHP treatment could alleviate the pathological changes of myocardial tissue in the acute MI (AMI) rats induced by isoproterenol (ISO), especially structural disorder, irregular distribution, and enlargement of the myocardial space. These pathological changes were all alleviated to some extent by DHHP treatment. Biochemical analysis results suggested that compared with the control group, the serum levels of AST, CTn-I, CK-MB, and TNF-α in model group rats were notably decreased, and the CAT and SOD levels in serum were markedly increased. These abnormal trends were significantly reversed by DHHP treatment. Furthermore, metagenomic sequencing analysis results indicated that DHHP could improve disorders in the composition and function of GM in AMI rats, mainly reflected in increasing diversity and richness, and obviously enhancing the abundance of Bacteroides fluxus, B. uniformis, B. stercoris, Roseburia hominis, Schaedlerella arabinosiphila, and R. intestinalis, and reducing the abundance of Enterococcus avium and E. canintestini, which were associated with purine metabolism, tyrosine metabolism, cyanoamino acid metabolism, and glutathione metabolism. In conclusion, DHHP may attenuate ISO-induced MI by regulating the structure, composition, and function of GM, thus contributing to further our understanding of the anti-MI mechanisms of DHHP and providing new therapeutic ideas and diagnostic targets for the clinical studies of MI.

The potential cutaneous benefits of Carthamus tinctorius oleosomes.

Safflower (Carthamus tinctorius) oleosomes are unique organelles that house triglycerides and fatty acids and demonstrate a natural resilience to environmental stresses. There is recent growing interest in safflower oleosomes due to their potential applications in dermatology, especially as a carrier technology to improve drug penetration through the skin. This paper explores various aspects of safflower oleosomes, including their production, safety, absorption, and applications in photoprotection and epidermal remodeling. Oleosomes have shown encouraging results in targeted drug delivery in in vitro and in vivo animal models; however, human clinical research is required to determine their efficacy and safety in dermatology. Oleosomes are comprise a novel biotechnology that has the potential to transform sustainable and natural treatments in dermatology by utilizing their unique structure. Safflower oleosomes are stable lipid molecules that can deliver small and large molecules with high efficacy. This review will examine the current research findings and prospective future applications of oleosomes.

Carthamus Tinctorius L. (Safflower) extracts inhibit expression of metastatic genes of MDA-MB-231 breast cancer cells.

Breast cancer is the most common type of cancer in women and the second  cause of cancer-related death after lung cancer. Although the common methods used in the treatment of breast cancer are chemotherapy, radiotherapy and surgery, the search for alternative treatments continues.  The leading  alternative treatments are medicinal plants which actually inspire the production of many cancer drugs. In this study, the proliferative and metastatic effects of Carthamus tinctorius L., known for its many therapeutic properties, on metastatic breast cancer were investigated. Here, intending to evaluate the the content and actions of different extracts of safflower leaves extracts were prepared by extracting in water, alcohol and oil and analysed by FTIR. Their antioxidant effect was tested and then the extracts were applied to metastatic breast cancer cells. FTIR spectrums of all three extracts have revealed the presence of organic compounds.  It is found that all extracts but mostly the oil extract has antioxidant property. MTT assay, wound healing assay and gene expression analysis were performed to assess the antiproliferative and anti metastatic effects of the extracts on breast cancer cells. It is found that, there is no significant antiproliferative effect of extracts on MDA-MB-231 cells except the alcohol extract. However, all safflower extracts, especially the oil extract, significantly reduced the metastatic potential of breast cancer cells. It is concluded that safflower contents are potent chemicals which inhibit the cellular mechanisms underlying the spreading of cancer cells and further analysis may lead to new initiatives in drug design research.

Identification of genes associated with fatty acid biosynthesis based on 214 safflower core germplasm.

BACKGROUND: Safflower (Carthamus tinctorius L.) is an oilseed crop with substantial medicinal and economic value. However, the methods for constructing safflower core germplasm resources are limited, and the molecular mechanisms of lipid biosynthesis in safflower seeds are not well understood. RESULTS: In this study, 11 oil-related quantitative traits and 50 pairs of InDel markers were used to assess the diversity of a collection of 605 safflower germplasms. The original safflower germplasm exhibited rich phenotypic diversity, with high variation for most of the phenotypic traits under investigation. Similarly, high genetic diversity was evaluated in the original germplasm, in which the mean Shannon's information index (I), observed heterozygosity (H0), and expected heterozygosity (He) were 0.553, 0.182, and 0.374, respectively. Four subgroups with strong genetic structures were identified and a core germplasm of 214 cultivars was constructed, which is well represented in the original germplasm. Meanwhile, differential expression analysis of the transcriptomes of high and low linoleic acid safflower varieties at two stages of seed development identified a total of 47 genes associated with lipid biosynthesis. High expression of the genes KAS II and SAD enhanced the synthesis and accumulation of oleic acid, while FAD genes like FAD2 (Chr8G0104100), FAD3, FAD7 and FAD8 promoted the consumption of oleic acid conversion. The coordinated regulation of these multiple genes ensures the high accumulation of oleic acid in safflower seed oil. CONCLUSIONS: Based on these findings, a core germplasm of 214 cultivars was constructed and 47 candidate genes related to unsaturated fatty acid biosynthesis and lipid accumulation were identified. These results not only provide guidance for further studies to elucidate the molecular basis of oil lipid accumulation in safflower seeds, but also contribute to safflower cultivar improvements.

Safflower (Carthamus tinctorius Linn.) Inhibits Cell Proliferation and Induces Apoptotic in Breast Cancer Cell Lines T47D.

<b>Background and Objective:</b> Safflower (<i>Carthamus tinctorius</i> Linn.) is one of the medicinal plants that contain secondary metabolites that have the potential to as anti-cancer by inducing apoptosis. This study aims to determine the content of secondary metabolite compounds and the induction activity of apoptosis from ethanol extract of safflower in the T47D breast cancer cell line. <b>Materials and Methods:</b> Safflower was extracted using 96% ethanol and assayed for phytochemical screening, cytotoxic tests by cell counting kit-8 to determine inhibitory concentration and apoptosis induction activity by flow cytometry to determine the ability of samples induce the programmed cell cancer in death. The data collected was analyzed with the PRISM GraphPad version. <b>Results:</b> The ethanol extract of safflower contains flavonoid compounds, alkaloids, saponins, tannins and terpenoids. The results of the anticancer activity test showed an IC₅₀ value of 479 µg mL¹ and the best percentage of apoptosis at a concentration of 200 µg mL¹ was 16.61% at the beginning of apoptosis and 10.52% at the end of apoptosis. <b>Conclusion:</b> The safflower can be developed as a breast anticancer agent that works through the induction of apoptosis to improve the effectiveness of breast cancer treatment.

Optimized Separation of Carthamin from Safflower by Macroporous Adsorption Resins and Its Protective Effects on PC12 Cells Injured by OGD/R via Nrf2 Signaling Pathway.

The growing demand for safe natural products has reignited people's interest in natural food pigments. Here, we proposed the use of macroporous adsorption resins (MARs) to separate and purify carthamin from safflower. The optimal parameters for carthamin purification with HPD400 MAR were determined as follows: a mass ratio of crude carthamin in sample solution to wet resin of 0.3, a crude carthamin solution concentration of 0.125 g·mL-1, a pH of 6.00, a sample volume flow rate of 0.5 mL·min-1, an ethanol volume fraction of 58%, an elution volume of 4 BV, and an elution volume flow rate of 1.0 mL·min-1. Under the above purification conditions, the recovery rate of carthamin was above 96%. Carthamin dramatically improved the survival rate of PC12 cells damaged by oxygen-glucose deprivation/reoxygenation and protected them from oxidative stress by inhibiting the generation of reactive oxygen species and increasing the total antioxidant capacity and glutathione (GSH) levels. Carthamin promoted extracellularly regulated protein kinase phosphorylation into the nucleus, permitting Nrf2 nuclear translocation and upregulating the gene expression of the rate-limiting enzymes glutamate-cysteine ligase catalytic subunit and glutamate-cysteine ligase regulatory subunit of GSH synthesis to obliterate free radicals and exert antioxidant effects. This study revealed the purification method of carthamin and its antioxidant protective effects, providing important insights into the application of carthamin in functional foods.

A Muti-Substrate Flavonol O-glucosyltransferases from Safflower.

To explore the complete biosynthesis process of flavonoid glycosides in safflower, specifically the key glycosyltransferase that might be involved, as well as to develop an efficient biocatalyst to synthesize flavonoid glycosides, a glycosyltransferase CtUGT4, with flavonoid-O-glycosyltransferase activity, was identified in safflower. The fusion protein of CtUGT4 was heterologously expressed in Escherichia coli, and the target protein was purified. The recombinant protein can catalyze quercetin to form quercetin-7-O-glucoside, and kaempferol to form kaempferol-3-O in vitro, and a series of flavones, flavonols, dihydroflavones, chalcones, and chalcone glycosides were used as substrates to generate new products. CtUGT4 was expressed in the tobacco transient expression system, and the enzyme activity results showed that it could catalyze kaempferol to kaempferol-3-O-glucoside, and quercetin to quercetin-3-O-glucoside. After overexpressing CtUGT4 in safflower, the content of quercetin-3-O-rutinoside in the safflower florets increased significantly, and the content of quercetin-3-O-glucoside also tended to increase, which preliminarily confirmed the function of CtUGT4 flavonoid-O-glycosyltransferase. This work demonstrated the flavonoid-O-glycosyltransferase function of safflower CtUGT4 and showed differences in the affinity for different flavonoid substrates and the regioselectivity of catalytic sites in safflower, both in vivo and in vitro, providing clues for further research regarding the function of UGT genes, as well as new ideas for the cultivation engineering of the directional improvement of effective metabolites in safflower.

[Effect of sowing dates on physiological characteristics, yield, and quality of Carthamus tinctorius].

A field experiment was conducted to measure the physiological characteristics, yield, active ingredient content, and other indicators of Carthamus tinctorius leaves undergoing 13 sowing date treatments. The principal component analysis(PCA) and redundancy analysis were used to analyze the correlation between these indicators to explore the effect of sowing date on the yield and active ingredient content of C. tinctorius in Liupanshan of Ningxia. The results illustrated that the early sowing in autumn and spring had significant effects on leaf photosynthetic parameters, SPAD value, antioxidant enzyme activity, nitrogen metabolism enzyme activity, filament yield, grain yield, and hydroxy safflower yellow A(HYSA) of C. tinctorius. Sowing in mid-November and late March had the best effect. Leaf transpiration rate, stomatal conductance, nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase increased by 44.9%, 52.4%, 15.9%, 60.8%, 10.3%, and 38.3%, respectively. The activities of superoxide dismutase, peroxidase, and catalase decreased by 10.8%, 4.1%, and 20.9%, respectively. The improvement of photosynthetic physiological characteristics promoted the dry matter accumulation and reproductive growth of C. tinctorius. The yield of filaments and seeds increased by 15.5% and 11.7%, and the yield of HYSA and kaempferol increased by 17.9% and 20.0%. In short, the suitable sowing date can promote the growth and development of C. tinctorius in Liupanshan of Ningxia, and significantly improve the yield and quality, which is conducive to the high quality and efficient production of C. tinctorius.

Anti-osteoporosis effects of mammalian lignans and their precursors from flaxseed and safflower seed using zebrafish model.

Secoisolariciresinol diglucoside (SDG) and tracheloside (TCL) are the main lignan components of flaxseed cake and safflower seed cake, which are by-products of oil extraction. Both SDG and TCL are metabolized into mammalian lignan enterolactone (EL) with the involvement of intestinal bacteria. In this research, we evaluated the anti-osteoporosis effects of SDG and the in vivo metabolites EL and enterodiol (ED) prepared in our previous work, as well as the newly isolated chemical constituents from safflower seed, including TCL, the lactone ring opening product of TCL (OTCL) and two alkaloids on the alloxan-induced zebrafish model. All the compounds showed significant anti-osteoporosis effects at 80 µM, with p < 0.05 for EL and p < 0.001 for other compounds compared with the model. SDG and TCL showed the most significant and concentration-dependent effects, with p < 0.001 compared with model at 20 µM. The alkaloids, N-coumaroylserotonin glucoside and N-feruloylserotonin glucoside, also showed anti-osteoporosis at 20 µM with p < 0.01, whereas EL, ED, and OTCL showed no significant effects. Quantitative real-time polymerase chain reaction revealed that SDG and TCL upregulated the expression of osteogenic genes Runx2, SP7, OPG, Col1a1a, Alp, ON, OPN, and OCN in alloxan-treated zebrafish. The in vivo metabolite of lignans, EL, showed significant anti-inflammatory effect (p < 0.01) at 20 µM, which might also help to combat osteoporosis and other complications caused by excessive immune response in the body. The results provided scientific data for using the oil extraction by-products as sources of anti-osteoporosis compounds. PRACTICAL APPLICATION: This study found that lignans in flaxseed cake and safflower seed cake exhibited anti-osteoporosis effects by upregulating the expression of osteogenic genes, making the oil extraction by-products sources of anti-osteoporosis compounds.

Identification and Characterization of CtUGT3 as the Key Player of Astragalin Biosynthesis in Carthamus tinctorius L.

Safflower (Carthamus tinctorius L.) is a multipurpose economic crop that is distributed worldwide. Flavonoid glycosides are the main bioactive components in safflower, but only a few UDP-glycosyltransferases (UGT) have been identified. Three differentially expressed UGT genes related with the accumulation of 9 flavonoid O-glycosides were screened from metabolomics and transcriptome analysis. Safflower corolla protoplasts were used to confirm the glycosylation ability of UGT candidates in vivo for the first time. The astragalin content was significantly increased only when CtUGT3 was overexpressed. CtUGT3 also showed flavonoid 3-OH and 7-OH glycosylation activities in vitro. Molecular modeling and site-directed mutagenesis revealed that G15, T136, S276, and E384 were critical catalytic residues for the glycosylation ability of CtUGT3. These results demonstrate that CtUGT3 has a flavonoid 3-OH glycosylation function and is involved in the biosynthesis of astragalin in safflower. This study provides a reference for flavonoid biosynthesis genes research in nonmodel plants.