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Introduction: Traditional Chinese medicinal plant, safflower, shows effective for treating pulmonary arterial hypertension (PAH), yet the underlying mechanisms remain largely unexplored. This study is aimed at exploring the potential molecular mechanisms of safflower in the treatment of PAH., Methods: Network pharmacology approach and molecular docking were applied to identify the core active compounds, therapeutic targets, and potential signaling pathways of safflower against PAH. Meanwhile, high-performance liquid chromatography (HPLC) assay was performed to determine the core compounds from safflower. Further, the mechanism of action of safflower on PAH was verified by in vivo and in vitro experiments., Results: A total of 15 active compounds and 177 targets were screened from safflower against PAH. Enrichment analysis indicated that these therapeutic targets were mainly involved in multiple key pathways, such as TNF signaling pathway and Th17 cell differentiation. Notably, molecular docking revealed that quercetin (core compound in safflower) displayed highest binding capacity with NLRP3. In vivo, safflower exerted therapeutic effects on PAH by inhibiting right ventricular hypertrophy, inflammatory factor release, and pulmonary vascular remodeling. Mechanistically, it significantly reduced the expression of proangiogenesis-related factors (MMP-2, MMP-9, Collagen 1, and Collagen 3) and NLRP3 inflammasome components (NLRP3, ASC, and Caspase-1) in PAH model. Similarly, these results were observed in vitro. Besides, we further confirmed that NLRP3 inhibitor had the same therapeutic effect as safflower in vitro., Conclusion: Our findings suggest that safflower mitigates PAH primarily by inhibiting NLRP3 inflammasome activation. This provides novel insights into the potential use of safflower as an alternative therapeutic approach for PAH., (© 2024 The Author(s). The Clinical Respiratory Journal published by John Wiley & Sons Ltd.)

Carthamus tinctorius L. (Safflower) is extensively used as a functional food and herbal medicine, with its application closely associated with hydroxysafflor yellow A (HSYA). However, the low oral bioavailability of HSYA in safflower extract (SFE) limits its health benefits and application. Our study found that co-administration of 250, 330, and 400 mg/kg peach kernel oil (PKO) increased the oral bioavailability of HSYA in SFE by 1.99-, 2.11-, and 2.49-fold, respectively. The enhanced bioavailability is attributed to improved lipid solubility and intestinal permeability of HSYA in SFE due to PKO. PKO is believed to modify membrane fluidity and tight junctions, increase paracellular penetration, and inhibit the expression and function of P-glycoprotein, enhancing the transcellular transport of substrates. These mechanisms suggest that PKO is an effective absorption enhancer. Our findings provide valuable insights for developing functional foods with improved bioavailability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Crop simulation models are valuable tools for decision making regarding evaluation and crop improvement under different field conditions. CSM-CROPGRO model integrates genotype, environment and crop management portfolios to simulate growth, development and yield. Modeling the safflower response to varied climate regimes are needed to strengthen its productivity dynamics. The main objective of the study was to evaluate the performance of DSSAT-CSM-CROPGRO-Safflower (Version 4.8.2) under diverse climatic conditions. The model was calibrated using the field observations for phenology, biomass and safflower grain yield (SGY) of the year 2016-17. Estimation of genetic coefficients was performed using GLUE (Genetic Likelihood Uncertainty Estimation) program. Simulated results for days to flowering, maturity, biomass at flowering and maturity and SGY were predicted reasonably with good statistical indices. Model evaluation results elucidate phenological events with low root mean square error (6.32 and 6.52) and high d-index (0.95 and 0.96) for days to flowering and maturity respectively for all genotypes and climate conditions. Fair prediction of safflower biomass at flowering and maturity showed low RMSE (887.3 and 564.3 kg ha -1 ) and high d-index (0.67 and 0.93) for the studied genotypes across the environments. RMSE for validated safflower grain yield (101.8 kg ha -1 ) and d-index (0.95) depicted that model outperformed for all genotypes and growing conditions. Longer appropriate growing conditions at NARC-Islamabad took optimal duration to assimilate photosynthetic products lead to higher grain yield. Safflower resilience to different environments showed that it can be used as an alternate crop for different agroecological regions. Furthermore, CROPGRO-Safflower model can be used as tool to further evaluate inclusion of safflower in the existing cropping systems of studied regions., (© 2024. The Author(s) under exclusive licence to International Society of Biometeorology.)

Modifying biochar with nano-nutrients is one of the most effective methods in improving the efficiency of biochar in reducing the adverse effects of environmental stresses such as salinity on plant growth and productivity. The possible effects of solid biochar, nano-silicon dioxide enriched biochar, nano-calcium carbonate enriched biochar, and combined application of these enriched biochars on physiological performance of safflower ( Carthamus tinctorius L.) were evaluated under different levels of salt stress (non-saline, 6 and 12 dSm -1 ). Salt stress increased sodium content, reactive oxygen species generation, and antioxidant enzymes activity, but decreased potassium, calcium, magnesium, iron, zinc, silicon, photosynthetic pigments, leaf water content, and seed yield (by about 36%) of safflower plants. The addition of biochar forms to the saline soil improved growth (up to 24.6%) and seed yield (up to 37%) of safflower by reducing sodium accumulation (by about 32%) and ROS generation and enhancing nutrient uptake, photosynthetic pigments, and water contents of leaves. The combined forms of enriched biochars were the best treatment on reducing salt stress effects on safflower plants. Therefore, application of enriched biochars has a high potential to reduce the harmful effects of salt stress on plants.

Domesticated safflower (Carthamus tinctorius L.) is a widely cultivated edible oil crop. However, despite its economic importance, the genetic basis underlying key traits such as oil content, resistance to biotic and abiotic stresses, and flowering time remains poorly understood. Here, we present the genome assembly for C. tinctorius variety Jihong01, which was obtained by integrating Oxford Nanopore Technologies (ONT) and BGI-SEQ500 sequencing results. The assembled genome was 1,061.1 Mb, and consisted of 32,379 protein-coding genes, 97.71% of which were functionally annotated. Safflower had a recent whole genome duplication (WGD) event in evolution history and diverged from sunflower approximately 37.3 million years ago. Through comparative genomic analysis at five seed development stages, we unveiled the pivotal roles of fatty acid desaturase 2 (FAD2) and fatty acid desaturase 6 (FAD6) in linoleic acid (LA) biosynthesis. Similarly, the differential gene expression analysis further reinforced the significance of these genes in regulating LA accumulation. Moreover, our investigation of seed fatty acid composition at different seed developmental stages unveiled the crucial roles of FAD2 and FAD6 in LA biosynthesis. These findings offer important insights into enhancing breeding programs for the improvement of quality traits and provide reference resource for further research on the natural properties of safflower., (© 2024. The Author(s).)

Flavonol synthase gene ( FLS ) is a member of the 2-oxoglutarate-dependent dioxygenase ( 2-ODD ) superfamily and plays an important role in plant flavonoids biosynthetic pathways. Safflower ( Carthamus tinctorius L.), a key source of traditional Chinese medicine, is widely cultivated in China. Although the flavonoid biosynthetic pathway has been studied in several model species, it still remains to be explored in safflower. In this study, we aimed to elucidate the role of CtFLS1 gene in flavonoid biosynthesis and drought stress responses. The bioinformatics analysis on the CtFLS1 gene showed that it contains two FLS-specific motifs (PxxxIRxxxEQP and SxxTxLVP), suggesting its independent evolution. Further, the expression level of CtFLS1 in safflower showed a positive correlation with the accumulation level of total flavonoid content in four different flowering stages. In addition, CtFLS1 -overexpression (OE) Arabidopsis plants significantly induced the expression levels of key genes involved in flavonol pathway. On the contrary, the expression of anthocyanin pathway-related genes and MYB transcription factors showed down-regulation. Furthermore, CtFLS1 -OE plants promoted seed germination, as well as resistance to osmotic pressure and drought, and reduced sensitivity to ABA compared to mutant and wild-type plants. Moreover, CtFLS1 and CtANS1 were both subcellularly located at the cell membrane and nucleus; the yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assay showed that they interacted with each other at the cell membrane. Altogether, these findings suggest the positive role of CtFLS1 in alleviating drought stress by stimulating flavonols and anthocyanin accumulation in safflower.

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., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Objectives: Safflower is a traditional Chinese medicine for the treatment of gynecological diseases and its flavonoids have potential anti-inflammatory effects. The purpose is to explore the possible effects of safflower total flavonoids (STF) on lipopolysaccharide (LPS)-induced inflammatory damage of Ishikawa cells., Method: In this study, LPS-induced endometrial carcinoma Ishikawa cells were used to establish an inflammatory injury model. The effective concentration of STF was screened by CCK-8 and enzyme-linked immunosorbent assay. The apoptosis of damaged Ishikawa cells was detected by flow cytometry. The contents of caspase11 and caspase 3 in Ishikawa cells were observed by fluorescence imaging. Western blot and RT-qPCR were used to detect the expression of related proteins and mRNA in damaged Ishikawa cells, and the possible mechanism of safflower flavonoids against LPS-induced endometrial carcinoma Ishikawa cells was analyzed by cell transcriptomics., Key Findings: The STF-reduced tumor necrosis factor α, interleukin-1β, and interleukin-6 expression level; the expression level of the proteins ASK1, Caspase3, and Caspase11 was also significantly decreased, and the proteins ERα, p-PI3K, and p-AKT were significantly increased. The transcriptome results showed that the PI3K-Akt signal pathway may be the main signal pathway for the STF., Conclusion: The STF could regulate the PI3K/AKT signal pathway to treat the inflammatory injury of Ishikawa cells., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 Ct UGT4, with flavonoid- O -glycosyltransferase activity, was identified in safflower. The fusion protein of Ct UGT4 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. Ct UGT4 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 Ct UGT4 flavonoid- O -glycosyltransferase. This work demonstrated the flavonoid- O -glycosyltransferase function of safflower Ct UGT4 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.

Oilseeds with high productivity and tolerance to various environmental stresses are in high demand in the food and industrial sectors. Safflower, grown under residual moisture in the semi-arid tropics, is adapted to moisture stress at certain levels. However, a substantial reduction in soil moisture has a significant impact on its productivity. Therefore, assessing genetic variation for water use efficiency traits like transpiration efficiency (TE), water uptake, and canopy temperature depression (CTD) is essential for enhancing crop adaptation to drought. The response of safflower genotypes ( n = 12) to progressive soil moisture depletion was studied in terms of water uptake, TE, and CTD under a series of pot and field experiments. The normalised transpiration rate (NTR) in relation to the fraction of transpirable soil water (FTSW) varied significantly among genotypes. The genotypes A-1, Bhima, GMU-2347, and CO-1 had higher NTR-FTSW threshold values of 0.79 (R 2 = 0.92), 0.74 (R 2 = 0.96), 0.71 (R 2 = 0.96), and 0.71 (R 2 = 0.91), respectively, whereas GMU-2644 had the lowest 0.38 (R 2 = 0.93). TE was high in genotype GMU-2347, indicating that it could produce maximum biomass per unit of water transpired. At both the vegetative and reproductive stages, significant positive relationships between TE, SPAD chlorophyll metre reading (SCMR) ( p < 0.01) and CTD ( p < 0.01) were observed under field conditions by linear regression. The genotypes with high FTSW-NTR thresholds, high SCMR, and low CTD may be useful clues in identifying a genotype's ability to adapt to moisture stress. The findings showed that the safflower genotypes A-1, Bhima, GMU-2347, and CO-1 exhibited an early decline and regulated water uptake by conserving it for later growth stages under progressive soil water depletion., Competing Interests: Ratnakumar Pasala is an Academic Editor for PeerJ., (© 2023 Manikanta et al.)

&lt;b&gt;Background and Objective:&lt;/b&gt; Safflower (&lt;i&gt;Carthamus tinctorius&lt;/i&gt; 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. &lt;b&gt;Materials and Methods:&lt;/b&gt; 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. &lt;b&gt;Results:&lt;/b&gt; The ethanol extract of safflower contains flavonoid compounds, alkaloids, saponins, tannins and terpenoids. The results of the anticancer activity test showed an IC&lt;sub&gt;50&lt;/sub&gt; value of 479 μg mL&lt;sup&gt;1&lt;/sup&gt; and the best percentage of apoptosis at a concentration of 200 μg mL&lt;sup&gt;1&lt;/sup&gt; was 16.61% at the beginning of apoptosis and 10.52% at the end of apoptosis. &lt;b&gt;Conclusion:&lt;/b&gt; 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.

The cytochrome P450 superfamily of monooxygenases plays a major role in the evolution and diversification of plant natural products. The function of cytochrome P450s in physiological adaptability, secondary metabolism, and xenobiotic detoxification has been studied extensively in numerous plant species. However, their underlying regulatory mechanism in safflower still remained unclear. In this study, we aimed to elucidate the functional role of a putative CtCYP82G24-encoding gene in safflower, which suggests crucial insights into the regulation of methyl jasmonate-induced flavonoid accumulation in transgenic plants. The results showed that methyl jasmonate (MeJA) was associated with a progressive upregulation of CtCYP82G24 expression in safflower among other treatment conditions including light, dark, and polyethylene glycol (PEG). In addition, transgenic plants overexpressing CtCYP82G24 demonstrated increased expression level of other key flavonoid biosynthetic genes, such as AtDFR, AtANS, and AtFLS, and higher content of flavonoid and anthocyanin accumulation when compared with wild-type and mutant plants. Under exogenous MeJA treatment, the CtCYP82G24 transgenic overexpressed lines showed a significant spike in flavonoid and anthocyanin content compared with wild-type and mutant plants. Moreover, the virus-induced gene silencing (VIGS) assay of CtCYP82G24 in safflower leaves exhibited decreased flavonoid and anthocyanin accumulation and reduced expression of key flavonoid biosynthetic genes, suggesting a possible coordination between transcriptional regulation of CtCYP82G24 and flavonoid accumulation. Together, our findings confirmed the likely role of CtCYP82G24 during MeJA-induced flavonoid accumulation in safflower., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Safflower (Carthamus tinctorius L.) has been explored as a source of natural antioxidant. However, quercetin 7-O-beta-D-glucopyranoside and luteolin 7-O-beta-D-glucopyranoside, as its bioactive compounds, possessed poor aqueous solubility, limiting its efficacy. Here, we developed solid lipid nanoparticles (SLNs) decorated with hydroxypropyl beta-cyclodextrin (HPβCD) incorporated into dry floating gel in situ systems to control the release of both compounds. Using Geleol® as a lipid matrix, SLNs were <200 nm in size with >80 % of encapsulation efficiency. Importantly, following the decoration using HPβCD, the stability of SLNs in gastric environment was significantly improved. Furthermore, the solubility of both compounds was also enhanced. The incorporation of SLNs into gellan gum-based floating gel in situ provided desired flow and floating properties, with <30 s gelation time. The floating gel in situ system could control the release of bioactive compounds in FaSSGF (Fasted-State Simulated Gastric Fluid). Furthermore, to assess the effect of food intake on release behavior, we found that the formulation could show a sustained release pattern in FeSSGF (Fed-State Simulated Gastric Fluid) for 24 h after being released in FaSGGF for 2 h. This indicated that this combination approach could be a promising oral delivery for bioactive compounds in safflower., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The regulatory mechanism of the MBW (MYB-bHLH-WD40) complex in safflower (Carthamus tinctorius) remains unclear. In the present study, we show that the separate overexpression of the genes CtbHLH41, CtMYB63, and CtWD40-6 in Arabidopsis thaliana increased anthocyanin and procyanidin contents in the transgenic plants and partially rescued the trichome reduction phenotype of the corresponding bhlh41, myb63, and wd40-6 single mutants. Overexpression of CtbHLH41, CtMYB63, or CtWD40-6 in safflower significantly increased the content of the natural pigment hydroxysafflor yellow A (HYSA) and negatively regulated safflower petal size. Yeast-two-hybrid, functional, and genetic assays demonstrated that the safflower E3 ligase CtBB1 (BIG BROTHER 1) can ubiquitinate CtbHLH41, marking it for degradation through the 26S proteasome and negatively regulating flavonoid accumulation. CtMYB63/CtWD40-6 enhanced the transcriptional activity of CtbHLH41 on the CtDFR (dihydroflavonol 4-reductase) promoter. We propose that the MBW-CtBB1 regulatory module may play an important role in coordinating HYSA accumulation with other response mechanisms., (© 2023 Institute of Botany, Chinese Academy of Sciences.)

Flavonoids are the most abundant class of secondary metabolites that are ubiquitously involved in plant development and resistance to biotic and abiotic stresses. Flavonoid biosynthesis involves multiple channels of orchestrated molecular regulatory factors. Methyl jasmonate (MeJA) has been demonstrated to enhance flavonoid accumulation in numerous plant species; however, the underlying molecular mechanism of MeJA-induced flavonoid biosynthesis in safflower is still not evident. In the present study, we revealed the underlying molecular basis of a putative F3'5'H gene from safflower imparting MeJA-induced flavonoid accumulation in transgenic plants. The constitutive expression of the CtF3'5'H1 gene was validated at different flowering stages, indicating their diverse transcriptional regulation through flower development in safflower. Similarly, the CtF3'5'H1 -overexpressed Arabidopsis plants exhibit a higher expression level, with significantly increased anthocyanins and flavonoid content, but less proanthocyanidins than wild-type plants. In addition, transgenic plants treated with exogenous MeJA revealed the up-regulation of CtF3'5'H1 expression over different time points with significantly enhanced anthocyanin and flavonoid content as confirmed by HPLC analysis. Moreover, CtF3'5'H1 - overexpressed Arabidopsis plants under methyl violet and UV-B irradiation also indicated significant increase in the expression level of CtF3'5'H1 with improved anthocyanin and flavonoid content, respectively. Noticeably, the virus-induced gene silencing (VIGS) assay of CtF3'5'H1 in safflower leaves also confirmed reduced anthocyanin accumulation. However, the CtF3'5'H1 suppression in safflower leaves under MeJA elicitation demonstrated significant increase in total flavonoid content. Together, our findings confirmed that CtF3'5'H1 is likely mediating methyl jasmonate-induced flavonoid biosynthesis in transgenic plants via enhanced anthocyanin accumulation.

The flower of the safflower (Carthamus tinctorius L.) is a traditional Chinese medicine that can improve cerebral blood flow due to its enrichment in flavonoids. Light is one of the main environmental factors that affects safflower growth and flavonoid synthesis. Elongated hypocotyl 5 (HY5) plays an important role in plants' light signal transduction. However, no study of HY5 in safflower has been conducted. In this study, a 462-bp sequence of CtHY5 was successfully cloned. The expression pattern of CtHY5 in different safflower tissues and the expression patterns of CtHY5 and CtCHS1 in full-blooming flowers that were treated under different light intensities were studied. The subcellular localization and the overexpression of CtHY5 were carried out as well. CtHY5 has a DNA-binding region belonging to the basic leucine zipper transcription factor family. CtHY5 was specifically expressed in flowers. The expression level of CtHY5 first increased and then decreased with increasing light intensity, which was similar to the expression pattern of CtCHS1. The subcellular localization study was implemented in safflower protoplasts and the YFP fluorescence was observed in nucleus. The overexpression analysis initially verified the promotion effect of CtHY5 to the expression of CtCHS1 and the content of flavonoids., (© 2022 International Union of Biochemistry and Molecular Biology, Inc.)

The quality of traditional Chinese medicine (TCM) guarantees its clinical efficacy. Although advanced analytical techniques and methods can accurately determine the content of chemical components in TCM, it is difficult to accurately determine its clinical efficacy. In addition, the current analytical methods and technologies are complex and have difficulty meeting the requirements of a rapid, accurate and convenient determination of TCM quality. In this study, we first propose the concept of "indistinct" evaluation of the quality of TCM, that is, combining biological potency with character evaluation, quantifying the character evaluation, and preparing the safflower quality grade evaluation card based on the character analysis, which provides research ideas and methods for the rapid and accurate evaluation of the quality of TCM. We determined the biological potency of different batches of safflower based on the in vitro antiplatelet aggregation model and divided the safflower samples into two grades based on the biological potency. We further collected the color information of different grades of safflower samples, quantified the color information of different grades of safflower, drew a quality grade evaluation card for the rapid judgment of safflower quality grade and verified its accuracy by pharmacodynamic evaluation. To further analyze the differences in the material basis of different grades of safflower, the LC-MS method was used to simultaneously determine the contents of 19 chemical components, such as myricetin, in different grades of safflower samples to analyze the differences in the material basis of different grades of safflower. The result shows that the different grades of safflower exhibited significant differences in color. The pharmacodynamic results show that the quality evaluation card prepared based on color information can accurately evaluate quality, and the effect of first-class safflower is significantly better than that of second-class safflower. The chemical analysis results of different grades of safflower show that there are also significant differences between them, among which hypericin, 6-hydroxyapin-6-O-glucose-7-O-glucuronide, 6-hydroxykaempferol-3,6-O-diglucoside-7-O-glucuronic acid glycoside, 6-hydroxykaempferol-3,6,7-tri-O-glucoside and hydroxysafflower yellow A exhibit significant differences, which may be the main differentiating components of different grades of safflower. This study preliminarily confirmed that the "indistinct" evaluation of the quality of TCM based on character analysis is accurate and scientific, and the quality evaluation card prepared can accurately judge the quality of TCM, providing a reference for the rapid application of TCM character evaluation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

C4H (cinnamate 4-hydroxylase) is a pivotal gene in the phenylpropanoid pathway, which is involved in the regulation of flavonoids and lignin biosynthesis of plants. However, the molecular mechanism of C4H-induced antioxidant activity in safflower still remains to be elucidated. In this study, a CtC4H1 gene was identified from safflower with combined analysis of transcriptome and functional characterization, regulating flavonoid biosynthesis and antioxidant defense system under drought stress in Arabidopsis. The expression level of CtC4H1 was shown to be differentially regulated in response to abiotic stresses; however, a significant increase was observed under drought exposure. The interaction between CtC4H1 and CtPAL1 was detected using a yeast two-hybrid assay and then verified using a bimolecular fluorescence complementation (BiFC) analysis. Phenotypic and statistical analysis of CtC4H1 overexpressed Arabidopsis demonstrated slightly wider leaves, long and early stem development as well as an increased level of total metabolite and anthocyanin contents. These findings imply that CtC4H1 may regulate plant development and defense systems in transgenic plants via specialized metabolism. Furthermore, transgenic Arabidopsis lines overexpressing CtC4H1 exhibited increased antioxidant activity as confirmed using a visible phenotype and different physiological indicators. In addition, the low accumulation of reactive oxygen species (ROS) in transgenic Arabidopsis exposed to drought conditions has confirmed the reduction of oxidative damage by stimulating the antioxidant defensive system, resulting in osmotic balance. Together, these findings have provided crucial insights into the functional role of CtC4H1 in regulating flavonoid biosynthesis and antioxidant defense system in safflower.

Safflower ( Carthamus tinctorius . L) possesses anti-tumor, anti-thrombotic, anti-oxidative, immunoregulatory, and cardio-cerebral protective effects. It is used clinically for the treatment of cardio-cerebrovascular disease in China. This study aimed to investigate the effects and mechanisms of action of safflower extract on myocardial ischemia-reperfusion (MIR) injury in a left anterior descending (LAD)-ligated model based on integrative pharmacology study and ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometer (UPLC-QTOF-MS/MS). Safflower (62.5, 125, 250 mg/kg) was administered immediately before reperfusion. Triphenyl tetrazolium chloride (TTC)/Evans blue, echocardiography, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, lactate dehydrogenase (LDH) ability, and superoxide dismutase (SOD) levels were determined after 24 h of reperfusion. Chemical components were obtained using UPLC-QTOF-MS/MS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to analyze mRNA and protein levels, respectively. Safflower dose-dependently reduced myocardial infarct size, improved cardiac function, decreased LDH levels, and increased SOD levels in C57/BL6 mice. A total of 11 key components and 31 hub targets were filtered based on the network analysis. Comprehensive analysis indicated that safflower alleviated inflammatory effects by downregulating the expression of NFκB1, IL-6, IL-1β, IL-18, TNFα, and MCP-1 and upregulating NFκBia, and markedly increased the expression of phosphorylated PI3K, AKT, PKC, and ERK/2, HIF1α, VEGFA, and BCL2, and decreased the level of BAX and phosphorylated p65. Safflower shows a significant cardioprotective effect by activating multiple inflammation-related signaling pathways, including the NFκB, HIF-1α, MAPK, TNF, and PI3K/AKT signaling pathways. These findings provide valuable insights into the clinical applications of safflower.

Polysaccharides are the main bioactive components in safflower. In this study, safflower polysaccharides (SPs) were extracted by ultrasonic assisted extraction, and four purified safflower polysaccharide fractions (named SSP1, SSP2, SSP3, and SSP4, respectively) were obtained. The physicochemical properties and in vitro physiological activities of the four fractions were investigated. The molecular weights (M W ) of the SSPs were 38.03 kDa, 43.17 kDa, 54.49 kDa, and 76.92 kDa, respectively. Glucuronic acid, galactose acid, glucose, galactose, and arabinose were the main monosaccharides. The Fourier transform infrared spectroscopy (FT-IR) indicated that the polysaccharides had α - and β -glycosidic bonds. Nuclear magnetic resonance (NMR) analysis showed that SSP1 had 6 different types of glycosidic bonds, while SSP3 had 8 different types. SSP3 exhibited relatively higher ABTS + scavenging activity, Fe +3 -reduction activity, and antiproliferative activity. The results will offer a theoretical framework for the use of SPs in the industry of functional foods and medications., Competing Interests: The authors declare no conflict of interest.

In the present study, we used exogenous naringenin (0.5 mM) pretreatment before the stress (25 mM NaCl) on the growth and tolerance of safflower seedlings under non-salinity conditions and salinity conditions. Our results showed that salinity treatment significantly declined the biomass, leaf relative water content, chlorophyll content, K + content, and K + /Na + ratio by 28%, 28%, 12%, 36%, and 56%, respectively, as compared to untreated control. The results obtained in the present study showed the beneficial effects of the pretreatment of naringenin in safflower seedlings under non-salinity conditions concerning increasing plant biomass, total phenolic compound, radical scavenging activity (RSA), soluble sugar content, proline, glutathione, enzymatic antioxidants, and K + content. Nevertheless, naringenin pretreated plants showed a clear increment in the values of biomass, RSA, total phenolic compound, and catalase enzyme activity parameters under salinity stress. Salinity stress caused ionic phytotoxicity and oxidative stress by enhancing Na +  content, H 2 O 2 accumulation, malondialdehyde (MDA), and antioxidants. However, naringenin alleviated salt-induced oxidative stress by decreasing H 2 O 2 and MDA content in the leaves and improving the catalase activity in treated plants. Generally, it could be concluded pretreatment of naringenin before stress could partly diminish NaCl-caused oxidative stress in safflower seedlings, probably due to improvement in enzymatic and non-enzymatic antioxidants and reduced cell membrane damage.

Key Message: The nuclear Factor YB of Carthamus tinctorius L. increased the content of unsaturated fatty acids by regulating the expression of genes involved in fatty acid synthesis and oil accumulation. Safflower (Carthamus tinctorius L.) seed oil is rich in linoleic acid and is widely used in food and medicine. Therefore, key genes regulating oil synthesis were mined through genetic engineering to provide genetic resources for improving oil content. Based on the conserved domain of the NF-YB, we screened and identified 14 CtNF-YB transcription factors in the safflower genome and divided them into three subfamilies through phylogenetic analysis. Regulatory motif analysis of the CtNF-YB promoter revealed specific cis-regulatory elements related to abiotic stress, growth, and development. Expression analysis of CtNF-YB family genes showed that non-Leafy Cotyledon 1(non-LEC1) genes were highly expressed in roots, leaves, and flowers; Leafy Cotyledon 1(LEC1) genes were highly expressed during early seed development; and Dr1-like genes were highly expressed in roots, stems, and leaves. CtNF-YB12 was identified as a LEC1 transcription factor based on phylogeny and BLAST alignment. Heterologous CtNF-YB12 expression in Arabidopsis thaliana increased seed pod length and seed size. Moreover, CtNF-YB12 overexpression increased the oil content of seeds, upregulated genes involved in fatty acid biosynthesis and glycolysis, and altered the content of unsaturated fatty acids, including oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3), as well as of sucrose, fructose, and glucose. CtNF-YB12 may increase the oil content by regulating key enzyme genes of oil synthesis, so it can be used as a reliable target., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Notoginseng and safflower are commonly used traditional Chinese medicines for benefiting qi and activating blood circulation. A previous study by our group showed that the compatibility of the effective components of total saponins of notoginseng (NS) and total flavonoids of safflower (SF), named NS-SF, had a preventive effect on isoproterenol (ISO)-induced myocardial infarction (MI) in rats. However, the therapeutic effect on MI and the synergistic mechanism of NS-SF are still unclear. Therefore, integrated metabolomics, combined with immunohistochemistry and other pharmacological methods, was used to systematically research the therapeutic effect of NS-SF on MI rats and the synergistic mechanism of NS and SF. Compared to NS and SF, the results demonstrated that NS-SF exhibited a significantly better role in ameliorating myocardial damage, apoptosis, easing oxidative stress and anti-inflammation. NS-SF showed a more significant regulatory effect on metabolites involved in sphingolipid metabolism, glycine, serine, and threonine metabolism, primary bile acid biosynthesis, aminoacyl-tRNA biosynthesis, and tricarboxylic acid cycle, such as sphingosine, lysophosphatidylcholine (18:0), lysophosphatidylethanolamine (22:5/0:0), chenodeoxycholic acid, L-valine, glycine, and succinate, than NS or SF alone, indicating that NS and SF produced a synergistic effect on the treatment of MI. This study will provide a theoretical basis for the clinical development of NS-SF.

Flavonoids with significant therapeutic properties play an essential role in plant growth, development, and adaptation to various environments. The biosynthetic pathway of flavonoids has long been studied in plants; however, its regulatory mechanism in safflower largely remains unclear. Here, we carried out comprehensive genome-wide identification and functional characterization of a putative cytochrome P45081E8 gene encoding an isoflavone 2'-hydroxylase from safflower. A total of 15 CtCYP81E genes were identified from the safflower genome. Phylogenetic classification and conserved topology of CtCYP81E gene structures, protein motifs, and cis-elements elucidated crucial insights into plant growth, development, and stress responses. The diverse expression pattern of CtCYP81E genes in four different flowering stages suggested important clues into the regulation of secondary metabolites. Similarly, the variable expression of CtCYP81E8 during multiple flowering stages further highlighted a strong relationship with metabolite accumulation. Furthermore, the orchestrated link between transcriptional regulation of CtCYP81E8 and flavonoid accumulation was further validated in the yellow- and red-type safflower. The spatiotemporal expression of CtCYP81E8 under methyl jasmonate, polyethylene glycol, light, and dark conditions further highlighted its likely significance in abiotic stress adaption. Moreover, the over-expressed transgenic Arabidopsis lines showed enhanced transcript abundance in OE-13 line with approximately eight-fold increased expression. The upregulation of AtCHS , AtF3'H , and AtDFR genes and the detection of several types of flavonoids in the OE-13 transgenic line also provides crucial insights into the potential role of CtCYP81E8 during flavonoid accumulation. Together, our findings shed light on the fundamental role of CtCYP81E8 encoding a putative isoflavone 2'-hydroxylase via constitutive expression during flavonoid biosynthesis.

Background: Astragali Radix-Safflower combination (ARSC) is widely utilized in clinic to treat cerebral ischemia/reperfusion injury (CI/RI). Whereas, there is no in-depth research of the pharmacokinetics (PK) and pharmacodynamics (PD) analysis of ARSC after intragastric administration in rats with CI/RI., Purpose: The purpose of this research is to investigate the PK characteristics of eight active ingredients (astragaloside IV, calycosin, calycosin-7-O-β-glucoside, formononetin, ononin, hydroxysafflor yellow A, syringin and vernine) of ARSC, and the regulation of neurotransmitters disorders, revealing the pharmacodynamic substance basis and the mechanism of ARSC in treating CI/RI from the molecular level., Methods: We established a new method which based on blood-brain dual channel microdialysis (MD) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to continuously gather, and determine the components of ARSC and neurotransmitters related to CI/RI in vivo. The collected data were analyzed by sigmoid-E max function. The neurotransmitters primarily regulated in CI/RI rat were discussed by principal component analysis and the compound most associated with total pharmacodynamics was chosen by partial least squares regression., Results: The validated LC-MS/MS method had specificity and selectivity to simultaneously analyze the concentration of eight active components of ARSC extract and five neurotransmitters of CI/RI rats. The recovery rates of brain MD probe and blood MD probe were stable within six hours. The MD probes recovery rates decreased with the increase of flow rates, but the solution concentration had little effect on the probes recovery rates. It was feasible to correct the recovery rates of probes in vivo by using reverse dialysis method. All eight active ingredients of ARSC could pass across the blood brain barrier after CI/RI. ARSC regulated the release of glutamate (Glu), γ-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and aspartic acid (Asp). Notably, astragaloside IV and hydroxysafflor yellow A might have better regulatory effect on neurotransmitters in comparison with other six measured components of ARSC, and Glu was the neurotransmitter mainly regulated in CI/RI rats., Conclusion: The ARSC was able to treat CI/RI through ameliorating neurotransmitters disorders. There was a hysteresis between the peaked drug concentration and maximum therapeutic effect of ARSC. The drug effective concentrations range of ASIV, calycosin, calycosin-7-O-β-glucoside, syringin and vernine in blood microdialysate and calycosin, syringin, vernine in brain microdialysate were narrow, which need be paid attention in clinical use., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2022. Published by Elsevier GmbH.)

Safflower (Carthamus tinctorius L.) is cultivated in various countries for the flavonoid compounds it contains. These flavonoids have been used in many industries as drugs and/or dyes. Over 60 flavonoids have been isolated from safflower. These flavonoids can be divided into two groups: special and common, both of which are active pharmaceutical ingredients efficacious in the treatment of cardiovascular and cerebrovascular diseases. Gene functions have been studied to figure out the biosynthesis of flavonoids in safflower. However, there is no comprehensive summary of the flavonoids in safflower. Research was recognised through systematic searches of ScienceDirect, PubMed, Web of Science, and CNKI databases by searching terms of "Carthamus tinctorius L.", "safflower", "flavonoid", "pharmacology", and "gene". More than 200 research reports were included after eligibility checks. This study summarizes the application of flavonoids in medicine and other industries. Comprehensively collects the chemical structure information of the two groups of flavonoids, and organic acids, alkaloids, spermidine, polyacetylene, and polysaccharides. The mechanism of two groups of flavonoids in treatment of cardiovascular and cerebrovascular diseases was describe in detail, and pharmacological mechanisms of protecting liver, lung and bone, and anti-cancer and anti-inflammatory were also summarised. Besides, the study updated the latest information on the molecular biology of safflower flavonoids. It is found that two groups of flavonoids in safflower have obvious differences in application, chemical structure, pharmacological mechanism, and biosynthetic pathway. It is hoped that this summative research will provide a new insight to flavonoids research in safflower., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Masson SAS.)

Cadmium (Cd) pollution in agricultural soils induces oxidative stress in plants that in turn is the foremost limiting factor for agricultural productivity. In past few decades, plant-metal-microbe interaction is of great interest as an emerging environmentally friendly technology that can be exploited to alleviate metal stress in plants. Considering these, in the present study an endophytic bacterium strain EPS has been isolated from the roots of common bean. The present strain was identified as Stenotrophomonas maltophilia based on 16S rRNA gene sequence. The strain showed Cd tolerance and Cd-adsorption potentials. The inoculation of strain EPS in safflower seeds significantly enhanced the antioxidant defense of plants under Cd-stress conditions through increasing the levels of antioxidant molecules like phenolics, flavonoids and carotenoids as well as improving the activities of the antioxidative enzymes including guaiacol peroxidase (POX), ascorbate peroxidase (APX) and superoxide dismutase (SOD). The output of this study is that strain EPS inoculation mitigates Cd-induced oxidative stress and consequently it may be beneficial, especially in Cd-contaminated crop fields., (© 2022. The Author(s).)

Background: Safflower ( Carthamus tinctorius L.), well known for its flower, is widely used as a dye and traditional Chinese medicine. Flavonoids, especially flavonoid glycosides, are the main pigments and active components. However, their biosynthesis is largely unknown. Interestingly, the colour of flowers in safflower changed from yellow to red during flower development, while much of the gene and chemical bases during colour transition are unclear., Methods: In this research, widely targeted metabolomics and transcriptomics were used to elucidate the changes in flavonoid biosynthesis from the gene and chemical points of view in flowers of safflower during colour transition. The screening of differential metabolites depended on fold change and variable importance in project (VIP) value. Differential expressed genes (DEGs) were screened by DESeq2 method. RT-PCR was used to analyse relative expressions of DEGs., Results: A total of 212 flavonoid metabolites, including hydroxysafflor yellow A, carthamin and anthocyanins, were detected and showed a large difference. The candidate genes of glycosyltransferases and flavonoid hydroxylase that might participate in flavonoid glycoside biosynthesis were screened. Ten candidate genes were screened. Through integrated metabolomics and transcriptome analysis, a uridine diphosphate glucose glycosyltransferase gene, CtUGT9 showed a significant correlation with flavonoid glycosides in safflower. In addition, expression analysis showed that CtUGT9 was mainly expressed in the middle development of flowers and was significantly upregulated under MeJA treatment. Our results indicated that CtUGT9 might play an important role in flavonoid glycoside biosynthesis during colour-transition in safflower., Competing Interests: The authors declare that they have no competing interests., (© 2022 Ren et al.)

Main Conclusion: We performed genome-wide and heterologous expression analysis of the safflower cysteine protease family and found that inhibition of CtCP1 expression enhanced plant cold resistance. Cysteine protease (CP) is mainly involved in plant senescence and stress responses. However, the molecular mechanism of endogenous cysteine protease inhibition in plant stress tolerance is yet unknown. Here, we report the discovery and functional characterization of a candidate CP1 gene from safflower. The conserved structural topology of CtCPs revealed important insights into their possible roles in plant growth and stress responses. The qRT-PCR results implied that most of CtCP genes were highly expressed at fading stage suggesting that they are most likely involved in senescence process. The CtCP1 expression was significantly induced at different time points under cold, NaCl, H 2 O 2 and PEG stress, respectively. The in-vitro activity of heterologously expressed CtCP1 protein showed highest protease activity for casein and azocasein substrates. The expression and phenotypic data together with antioxidant activity and physiological indicators revealed that transgenic plants inhibited by CtCP1-anti showed higher tolerance to low temperature than WT and CtCP1-OE plants. Our findings demonstrated the discovery of a new Cysteine protease 1 gene that exerted a detrimental effect on transgenic Arabidopsis under low-temperature stress., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Seed ageing has an important effect on germination and productivity. During natural ageing, seed vigour decreases rapidly but, to date, the molecular mechanisms underlying this decrease have not been fully elucidated. Using omics, some of the details regarding seed vigour decline during natural ageing might be elucidated through integrated analysis. Safflower seed germination and physio-biochemical changes during natural ageing (stored for 4, 16 and 28 months) were determined. Proteome and lipidome profiling during natural seed ageing was performed, and the differentially expressed proteins and lipid metabolite species analysed. The surface and internal structures of cotyledons were observed. An integrating analysis of the proteome and lipidome was also carried out. Natural seed ageing significantly decreased safflower seed germination and vigour. 4,184 proteins and 1,193 lipids were quantified, both of which show huge differences among the different naturally aged seeds. The surface of the cotyledons collapsed and cracked, and the oil bodies become looser during natural ageing. The total content of DAG and PA increased, while the content of TAG and PL (PC, PE, PS, PI and PL) significantly decreased during seeds ageing. Two lipase genes (HH-026818-RA and HH-025320) likely participated in this degradation of lipids. We conclude that the enzymes that participate in glycerolipid metabolism and fatty acid degradation probably lead to the degradation of oil bodies (TAG) and membrane lipids (PC, PE, PS, PI, PG) and, ultimately, destroy the structure, causing a decline in seed vigour during natural seed ageing., (© 2021 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

Carthamin, a dimeric quinochalcone that is sparingly soluble in water, is obtained from the yellow-orange corolla of fully blooming safflower (Carthamus tinctorius L.) florets. Carthamin is a natural red colorant, which has been used worldwide for more than 4500 years and is the major component of Japanese 'beni' used for dyeing textiles, in cosmetics and as a food colorant. The biosynthetic pathway of carthamin has long remained uncertain. Previously, carthamin was proposed to be derived from precarthamin (PC), a water-soluble quinochalcone, via a single enzymatic process. In this study, we identified the genes coding for the enzyme responsible for the formation of carthamin from PC, termed 'carthamin synthase' (CarS), using enzyme purification and transcriptome analysis. The CarS proteins were purified from the cream-colored corolla of safflower and identified as peroxidase homologs (CtPOD1, CtPOD2 and CtPOD3). The purified enzyme catalyzed the oxidative decarboxylation of PC to produce carthamin using O2, instead of H2O2, as an electron acceptor. In addition, CarS catalyzed the decomposition of carthamin. However, this enzymatic decomposition of carthamin could be circumvented by adsorption of the pigment to cellulose. These CtPOD isozymes were not only expressed in the corolla of the carthamin-producing orange safflower cultivars but were also abundantly expressed in tissues and organs that did not produce carthamin and PC. One CtPOD isozyme, CtPOD2, was localized in the extracellular space. Based on the results obtained, a model for the stable red pigmentation of safflower florets during flower senescence and the traditional 'beni' manufacturing process is proposed., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Safflower (Carthamus tinctorius) petals have been used for centuries as a spice, in tea blends and in traditional Asian medicine. Aqueous extracts of Safflower petals have been used as a colouring food over the last 30 years due to their bright colour. Publications in the past raised concerns about fertility impairing, maternal toxicity, fetotoxic and teratogenic properties in rodents. As the tested extracts were poorly characterized and the studies were not performed according to guidelines, a need for further evaluation was seen. In silico predictions for the main pigments provided negative results for bacterial mutagenicity. Further, in vitro genotoxicity and in vivo reproductive toxicity studies of a well-characterized aqueous safflower concentrate generated more relevant data for risk assessment of its use in food. In vitro AMES tests and a mouse lymphoma cell assay were negative. An OECD guideline 421 screening study was performed in rats with oral daily doses of up to 1000 mg/kg bodyweight, applied via gavage to simulate a bolus effect. The highest dose reflected a toxicological limit test. The study did not give indications of general toxicity, did not show any effect on fertility and reproduction nor any effect on prenatal development and, also in contrast to previous results, treatment did not affect estradiol and FSH levels. Furthermore, pups raised until PND 14-16, developed normally with no adverse effects observed. With the established NOAEL of at least 1000 mg/kg/d, a considerable margin of exposure is achieved when compared with human intake estimates., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Safflower (Carthamus tinctorius L.), a member of the Asteraceae, is a popular crop due to its high linoleic acid (LA) and flavonoid (such as hydroxysafflor yellow A) contents. Here, we report the first high-quality genome assembly (contig N50 of 21.23 Mb) for the 12 pseudochromosomes of safflower using single-molecule real-time sequencing, Hi-C mapping technologies and a genetic linkage map. Phyloge nomic analysis showed that safflower diverged from artichoke (Cynara cardunculus) and sunflower (Helianthus annuus) approximately 30.7 and 60.5 million years ago, respectively. Comparative genomic analyses revealed that uniquely expanded gene families in safflower were enriched for those predicted to be involved in lipid metabolism and transport and abscisic acid signalling. Notably, the fatty acid desaturase 2 (FAD2) and chalcone synthase (CHS) families, which function in the LA and flavonoid biosynthesis pathways, respectively, were expanded via tandem duplications in safflower. CarFAD2-12 was specifically expressed in seeds and was vital for high-LA content in seeds, while tandemly duplicated CarFAD2 genes were up-regulated in ovaries compared to CarFAD2-12, which indicates regulatory divergence of FAD2 in seeds and ovaries. CarCHS1, CarCHS4 and tandem-duplicated CarCHS5˜CarCHS6, which were up-regulated compared to other CarCHS members at early stages, contribute to the accumulation of major flavonoids in flowers. In addition, our data reveal multiple alternative splicing events in gene families related to fatty acid and flavonoid biosynthesis. Together, these results provide a high-quality reference genome and evolutionary insights into the molecular basis of fatty acid and flavonoid biosynthesis in safflower., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Safflower(Carthamus tinctorius), a valuable traditional Chinese medicinal plant, has attracted much attention in recent years. This study established a stable tissue culture system of safflower and analyzed the chromatogram of its secondary metabolites, providing high-quality experimental materials for further research on natural products in safflower. The calluses were established from the safflower seeds germinated in a sterile environment, and then they were differentiated into the aseptic seedlings, or cultured to obtain suspension cells in liquid medium. The ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF-MS), Progenesis QI, and principal component analysis(PCA) were used to detect and analyze the secondary metabolites in the suspension cells before and after induction with different elicitors(methyl jasmonate, silver nitrate, salicylic acid and yeast extract). A total of 23 secondary metabolites including flavonoids, phenylpropanoids, alkaloids, fatty acids and aromatic glycosides were detected in safflower suspension cells. In response to the four elicitors, 11 compounds showed increased or decreased relative content. The results indicate that different elicitors have various effects on the accumulation of secondary metabolites in safflower suspension cells, and yeast extract shows more obvious positive induction. Therefore, different elicitors may play a role in the expression of related genes in the biosynthetic pathway of specific secondary metabolites. The results facilitate the discovery of targeted elicitors and the large-scale production of valuable secondary metabolites in the future.

Application of biochar-based metal oxide nanocomposites can acquire new composites and combine the benefits of biochar with nanomaterials. For the first time, this research was conducted to evaluate the possible effects of solid biochar (25 g biochar kg -1 soil) and biochar-based nanocomposites (BNCs) of magnesium oxide (25 g BNC-MgO kg -1 soil), manganese oxide (25 g BNC-MnO biochar kg -1 soil) and combined use of these nanocomposites (12.5 g BNC-MgO + 12.5 g BNC-MnO kg -1 soil) on salt (non-saline, 6 and 12 dSm -1 NaCl salinities) tolerance of safflower plants (Carthamus tinctorius L.). Salinity reduced potassium, magnesium and manganese contents in root and leaf tissues, chlorophyll content index, photosynthetic pigments, maximum quantum yield of photosystem II (Fv/Fm) and relative photosynthetic electron transport rate (RETR), leaf water content and plant biomass, but increased the sodium content, reactive oxygen species generation (ROS), oxidative stress and antioxidants and ROS detoxification potential of safflower roots and leaves. Application of biochar and BNCs increased the contents of potassium, manganese and magnesium in plant tissues, photosynthetic pigments, Fv/Fm and RETR, leaf water content and reduced sodium accumulation, ROS generation and oxidative stress under saline conditions, leading to a higher plant biomass in comparison with control. The BNC-MgO + BNC-MnO was the superior treatment on reducing salt toxicity. This treatment reduced oxidative stress by enhancing photosynthetic pigments, Fv/Fm and RETR of safflower under salt stress. These results revealed that BNCs have a great potential for improving salt tolerance of plants through increasing RETR and decreasing sodium accumulation and ROS generation.

Background: The investigation of molecular mechanisms involved in lipid metabolism plays a critical role for the genetic engineering of safflower (Carthamus tinctorius L.) to increase the oil accumulation level or to change the oil composition. Although transcript sequences are currently available for the leaves and flowers of safflower, a wide range scan of temporal transcripts at different stages of seed development has not been conducted for safflower., Results: In this study, temporal transcriptome sequencing was executed at 10, 14, 18, and 22 days after flowering (DAF) to uncover the molecular networks concerned in the biosynthesis of unsaturated fatty acids (USFAs). The results revealed that the biosynthesis of fatty acids is a dominant cellular process from 10 to 14 DAF, while degradation mainly happens after 18 DAF. Significant expression changes of two genes, stearoyl-[acyl-carrier-protein] 9-desaturase gene (SAD) from 10 to 14 DAF and oleate desaturase (FAD2-1) from 14 to 18 DAF, were detected at the transcriptomic levels, and the temporal expression patterns revealed by the transcriptomic analysis were confirmed using quantitative real-time PCR experiments. In addition, 13 candidate transcription factors (TFs) involved in regulating the expression level of the FAD2-1 gene were identified., Conclusions: These results create a link between fatty acid biosynthesis and gene expression at different developmental stages of the seeds, provide insight into the underlying lipid metabolism, and meanwhile lay an important foundation for the genetic engineering of safflower varieties. We have identified novel candidate genes, including TFs, that are worthy of further exploration.

Water deficit is one of the most limiting factors for plant growth and production. Polyamines are osmo-active compounds and have important roles in plant resistance to water limitation. A pot experiment was undertaken in a greenhouse as factorial based on complete randomized block design with three replications to assess the physiological and biochemical responses of safflower to different levels of water supply (100% and 40% field capacity) and spermine (0, 40 and 60 µM). Ascorbate peroxidase and peroxidase activities (POX), malondialdehyde (MDA), hydrogen peroxide (H 2 O 2 ), anthocyanins, soluble protein, soluble sugars and proline contents in shoots increased, while total phenols, flavonoids, and photosynthetic pigments significantly decreased due to water deficit. Foliar spray of spermine mitigated the adverse effects of water deficit by increasing the catalase, superoxide dismutase, POX activities, soluble proteins and photosynthetic pigments, and by decreasing MDA and H 2 O 2 contents. Spermine could, therefore, play an important role in protecting photosynthetic system and cellular membranes during drought stress in safflower., (© 2020. Akadémiai Kiadó Zrt.)

The quality of safflower (Carthamus tinctorius L.) in the market is uneven due to the problems of dyeing and adulteration of safflower, and there is no perfect standard for the classification of quality grade of safflower at present. In this study, computer vision (CV) and near-infrared (NIR) were combined to realize the rapid and nondestructive analysis of safflower. First, the partial least squares discrimination analysis (PLS-DA) model was used to qualitatively identify the dyed safflower from 150 samples. Then the partial least squares (PLS) model was used for quantitative analysis of the hydroxy safflower yellow pigment A (HSYA) and water extract of undyed safflower. Herein, the discrimination rate of PLS-DA model reached 100%, and the residual predictive deviation (RPD) values of the PLS models for HSYA and water extract were 2.5046 and 5.6195, respectively. It indicated that the rapid analysis method combining CV and NIR was reliable, and its results can provide important reference for the formulation of safflower quality classification standards in the market., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Safflower has both medicinal and edible values but research on its nutrient composition is still lacking. This study was established for the quantitative determination of 28 nucleosides, nucleobases, and amino acids based on the ultra-performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry. Analysis of 30 batches of safflower from different producing areas indicated that the contents of l-proline, l-asparagine, l(+)-arginine, l-serine, l-histidine, uracil, guanosine, and uridine was high in safflower. Principle component analysis and cluster analysis found that samples from different regions could be distinguished well, and samples from the same area could be clustered into one class, different geographical environments may cause the differences of nucleosides, nucleobases, and amino acids in safflower. The analysis of principal component analysis, cluster analysis, and counter propagation artificial neural network show similar results. Then the content of nucleosides, nucleobases, and essential amino acids were compared, and found that the content in safflower from Gansu was higher than those from other regions, and there was a little difference between the samples from Xinjiang, Sichuan, and Yunnan. This research revealed the composition of nucleosides, nucleobases, and amino acids in safflower, and provided a theoretical basis for utilization of safflower., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Safflower (Carthamus tinctorius L.) is an important cash crop, of which the dried tube flower is not only an important raw material for dyes and cosmetics but also an important herb widely used in traditional Chinese medicine. The pigment and bioactive compounds are composed of flavonoids (mainly quinone chalcones), and studies have reported that MeJA can promote the biosynthesis of quinone chalcones, but the mechanism underlying the effect of MeJA in safflower remains unclear. Here, we attempt to use metabolomics and transcriptome technologies to analyse the molecular mechanism of flavonoid biosynthesis under MeJA treatment in safflower., Results: Based on a UHPLC-ESI-MS/MS detection platform and a self-built database (including hydroxysafflor yellow A, HSYA), a total of 209 flavonoid metabolites were detected, and 35 metabolites were significantly different after treatment with MeJA. Among them, 24 metabolites were upregulated upon MeJA treatment, especially HSYA. Eleven metabolites were downregulated after MeJA treatment. Integrated metabolomics and transcriptome analysis showed that MeJA might upregulate the expression of upstream genes in the flavonoid biosynthesis pathway (such as CHSs, CHIs and HCTs) and downregulate the expression of downstream genes (such as F3Ms, ANRs and ANSs), thus promoting the biosynthesis of quinone chalcones, such as HSYA. The transcription expressions of these genes were validated by real-time PCR. In addition, the promoters of two genes (CtCHI and CtHCT) that were significantly upregulated under MeJA treatment were cloned and analysed. 7 and 3 MeJA response elements were found in the promoters, respectively., Conclusions: MeJA might upregulate the expression of the upstream genes in the flavonoid biosynthesis pathway and downregulate the expression of the downstream genes, thus promoting the biosynthesis of quinone chalcones. Our results provide insights and basic data for the molecular mechanism analysis of flavonoid synthesis in safflower under MeJA treatment.

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.

Leaf miner is one of the major pests on safflower, which causes yield loss and poor quality seriously. &quot;Weihonghua&quot;, &quot;nine safflower varieties&quot; and &quot;three chemical insecticides&quot; as materials that used to evaluate variety and regularity of leaf miner, safflower resistant level, and different proportions insecticides in field efficiency test. The results showed that Liriomyza sativae and L. huidobrensis accounted for 80%, the peak period of two pests was all in July; but Phytomyza horticola is relative less, its peak period occured in June. Three were great difference of resistance to leaf miner among safflower varieties, FQ12 and YJ65 expressed higher resistibility to leaf miner by ratio method. With abamectin 2% emulsifiable concentrate diluted for 2 000 times, or the mixture three insecticides(bifenthrin 20% water emulsions, thiamethoxam 25% water dispersible granule, abamectin 2% emulsifiable concentrate=1∶1∶1) diluted for 3 000 times, which were sprayed on leaves at squaring stage and lethal rate was 96% after 48 h in the study. Through comparative study on the variety and regularity of leaf miner, screen for resistant varieties to leaf miner and for high efficiency pesticide. The study provides theoretical basis and reference for integrated pest management of leaf miner.

The screening of active components of traditional Chinese medicine has always been the focus and difficulty in modern research of Chinese medicine preparations. With the continuous development of life science, omics and computer technology, the virtual screening technology for active components of traditional Chinese medicine has gradually come into people's vision. Molecular docking technology is an important method for screening active components of traditional Chinese medicine. It not only has a short cycle and strong operability, but also avoids the disadvantage of poor stability in pharmacological experiments. Safflower extract can effectively alleviate the symptoms of myocardial ischemia, but its active components are not clear. In this study, with use of the molecular docking technology, the active components in safflower against myocardial ischemic were virtually screened based on the screening method of active components. Forty-six chemical components and 5 target proteins which showed high correlation with myocardial ischemia were obtained from the existing database and related literature reports. With the molecules of three commercially available drugs diltiazem, trimetazidine and verapamil as positive control molecules, the compomnents were docked with 5 target proteins. Active components were screened according to docking scores and interactions between molecules and targets, and then the active ingredients can be inferred. Fourteen chemical components were screened to have the most potential anti-myocardial ischemic activity, and all of them were flavonoids. Therefore, it can be inferred that the flavonoid components are the most potential anti-myocardial ischemic components in safflower. The screening of active anti-myocardial ischemia components in safflower was completed in this study, laying the foundation for subsequent researches.

Ethnopharmacological Relevance: Safflower (Carthamus tinctorius) has many applications in folk medicine. Its oil is used traditionally to treat obesity and other metabolic disorders. The anti-hypercholesterolemic and antioxidant effects of this plant have been well documented, but the anti-inflammatory effects and its role on fatty acid oxidation and homeostasis of trace elements are not fully understood., Objective: The aim of this study was to evaluate the protective effects of different doses of oil and extract of safflower seed against fructose induced metabolic syndrome by investigating the homeostasis of trace elements, TNF-α, and fatty acids metabolism., Methods: Eighty adult male Sprague-Dawley rats were randomly divided into ten groups and treated daily for 16 weeks. At the end of the study, plasma levels of liver enzymes, lipid profiles, blood glucose, insulin and TNF-α were measured. The levels of antioxidant enzymes and lipid peroxidation were also measured along with the expression of CD36, fatty acyl-CoA synthetase (FAS), and Carnitine palmitoyl transferase I (CPT-1) beta genes in the liver., Results: The antioxidant enzymes activity significantly decreased and lipid peroxidation, lipid profiles, liver enzymes, and TNF-α significantly increased in fructose-induced metabolic syndrome compared to the control groups, as well as the level of some trace elements significantly changed (p < 0.05). Treatment with oil and safflower seed extract in a dose dependent manner could improve biochemical parameters in groups of metabolic syndrome treated with oil and extract compared to metabolic syndrome (p < 0.05). The results also showed that the expression of above mentioned genes significantly increased in groups of metabolic syndrome treated with oil and extract compared to control and metabolic syndrome groups (p < 0.05)., Conclusion: It can be concluded that safflower seed extract and its oil can improve fructose-induced metabolic syndrome through antioxidant and anti-inflammatory effects, adjustment of homeostasis of trace elements, and enhancing the beta-oxidation capacity of the liver by increasing the expression of CD36, FAS, and CPT-1beta genes., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A water-soluble polysaccharide (SPAW) was purified from Safflower and it was identified to be (1→3)-linked β-d-Glucan. The therapeutic effect and underlying mechanism of SPAW on steroid-induced avascular necrosis of the femoral head (SANFH) in a rabbit model was performed here. The abnormal histopathologic changes and apoptosis of femoral head in model group were significantly reverted after SPAW (25, 100 and 200 mg/kg) administration for 60 days, as evidenced by the a decline of empty lacunae rate, the average bone marrow fat cell size and the proportion of apoptotic cells. Furthermore, administration of SPAW significantly decreased the Bax and caspase-3 protein expression, but increased the protein expression of Bcl-2 when compared these in model rabbits. Meanwhile, increased hydroxyproline (HOP) and decreased serum hexosamine (HOM) concentration in rabbit serum were turned to the opposite way. The present study suggested that SPAW may provide an alternative treatment for the treatment of SANFH., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

An evaluation of the ultrasonic extraction process and the antioxidant activities of hydroxysafflor yellow A (HSYA) and anhydrosafflor yellow B (AHSYB) from safflower are presented herein. Using response surface methodology (RSM), based on a four-factor-three-level Box-Behnken design (BBD), the extraction parameters, namely, temperature, extraction time, solvent-to-material ratio, and extraction power, were optimized for maximizing the yields of HSYA and AHSYB. The maximum yield was obtained at a temperature of 66 °C with an extraction time of 36 min, solvent-to-material ratio of 16 mL/g, and the extraction power of 150 W, which was adjusted according to the actual conditions. The HSYA and AHSYB contents were determined using high performance liquid chromatography (HPLC). The yield and the comprehensive evaluation value of HSYA and AHSYB were calculated. The antioxidant activities of the extracts were determined using a ferric reducing antioxidant power (FRAP) kit and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. The results suggested that the safflower extracts possessed obvious ferric reducing and DPPH radical scavenging activities. The antioxidant activity increased with increasing concentration. The results suggested that optimizing the conditions of ultrasonic extraction using RSM can significantly increase the yields of HSYA and AHSYB from safflower. The safflower extracts showed better antioxidant activity. This study can encourage future research on cardiovascular and cerebrovascular diseases.

Safflower (Carthamus tinctorius L.) is one of the most important oilseed crops for its seed oil rich in unsaturated fatty acids. Precise utilization of diverse genetic resources is fundamental in breeding programs to improve high yield genotypes with desirable traits. In this study, for the first time we report successful application of DArTseq technology; an efficient genotyping-by-sequencing (NGS); to analysis genetic diversity and population structure of 89 safflower accessions from worldwide origins. Totally, 19,639 DArTseq markers (10,130 SilicoDArTs and 9509 SNPs) generated through DArTseq genotyping. After filtering the data, 3431 polymorphic DArTseq markers (1136 SilicoDArTs and 2295 SNPs) used for genetic diversity, population structure and linkage disequilibrium analysis in safflower genotypes. All the SilicoDArT and SNP markers showed high reproducibility and call rate. Polymorphism information content (PIC) values ranged from 0.1 to 0.5, while ≥ 0.50% of SilicoDArTs and ≥ 0.64% SNPs showed PIC values more than median. Genotypes grouping using DArTseq markers resulted in three distinct clusters. Results showed weak correlation between safflower diversity pattern and origins. Analysis of molecular variance revealed that the majority of genetic variation was attributed to the differences among varieties within cluster populations and there was no significant molecular variance between origins. However, safflower of accessions belonged to Iran, Turkey, Pakistan and India indeed appear to be genetically similar and grouped close in referred cluster, while the accessions from Near East (Afghanistan, China) being distinct. Our results were in agreement with hypothesis that safflower domesticated in somewhere west of Fertile Crescent and then expanded through Africa and Europe. Present study using a panel of globally diverse safflower accessions and large number of DArTseq markers set the stage for future analysis of safflower domestication using large germplasm from proposed domestication centers. Also, studied germplasm in this study can be used as a valuable source for future genomic studies in safflower for mapping desirable traits through genome-wide association mapping studies.

Productivity of safflower (Carthamus tinctorius L.) is markedly reduced by salt stress. This study is based on analysis of proteins profile of safflower plants treated with 200 mM NaCl, with exogenously applied salicylic acid (SA) and penconazole (PEN), as growth regulators. Plants were investigated through a gel-based proteomic approach, which resulted in the identification of 17 salt-responsive proteins related to different metabolic modifications. Of these, seven different proteins were up or down regulated by both SA and PEN, suggesting the synergistic and antagonistic effects of SA and PEN. The classification of differentially expressed proteins showed that salt-responsive proteins were mainly involved in photosynthesis, ion homeostasis, and oxidative stress response, as well as nitrogen, protein, and carbohydrate metabolism. The identified stress-responsive proteins in this study could pave the way to develop salt tolerance in safflower, thus sustaining its productivity under salinity. In addition, SA and PEN may be considered as a foliar application to ameliorate salinity effects, due to their low price and availability., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In the present study, deep eutectic solvents (DESs) were prepared and firstly applied to extract flavonoids from Safflower using ultrasonic-assisted extraction (UAE), and DES2-UAE was selected as a green and efficient extraction method. The effects of extraction parameters on extraction efficiency (EE) of flavonoids were investigated by single-factor experiments and optimized using a Box-Behnken design (BBD). The optimal results were 56.37 mL/g liquid-solid ratio, 55.85 min extraction time, 41.44 °C extraction temperature and 188.55 W ultrasonic power, respectively. Under optimal extraction conditions, the highest EE of flavonoids (55.41 mg/g) was obtained. The verification experimental results were in good correlation with the predicted results. The adsorption and desorption experiments of flavonoids on five types of macroporous resins (NKA-2, AB-8, HPD-100, D-101 and S-8) were carried out, NKA-2 and S-8 showed higher adsorption/desorption capacities, successively followed by AB-8, D-101 and HPD-100. The adsorption behaviors were better explained by Langmuir isotherm model according to correlation coefficient on the basis of static adsorption test at different temperature. In conclusion, the developed DES2-UAE combined with macroporous resin enrichment can be an alternative method for the green and efficient extraction of bioactive flavonoids from plant materials., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The aim of this study was to determine the effect of different extraction solvents (petroleum benzene, hexane, diethyl ether and acetone) and extraction methods (hot and cold) on oil yield of safflower seeds and its fatty acid compositions. Oil contents of safflower seeds extracted by hot extraction system were changed between 37.40% (acetone) and 39.53% (petroleum benzene), while that of cold extraction was varied between 39.96% (petroleum benzene) and 39.40% (diethyl ether). Regarding the extraction solvents, the highest oil yield (39.53%) was obtained with petroleum benzene, while the minimum value (37.40%) was found with acetone under hot extraction condition. The main fatty acids observed in all extracted oil samples were linoleic, oleic and palmitic acids. Oleic acid contents of safflower oils extracted by hot extraction system was ranged between 41.20% (acetone) and 42.54% (hexane), its content in oils obtained by cold extraction method was varied between 40.58% (acetone) and 42.10% (hexane and diethyl ether). Linoleic content of safflower oil extracted by hot extraction system was found between 48.23% (acetone) and 49.62% (hexane), while that oil extracted by cold method range from 48.07 (hexane) to 49.09% (acetone). The fatty acid composition of safflower seeds oil showed significant (p < 0.05) differences depending on solvent type and extraction method. The results of this study provide relevant information that can be used to improve organic solvent extraction processes of vegetable oil.