Insights into Q-markers of honey-fried licorice in treating spleen deficiency based on substance and energy metabolism regulation.

BACKGROUND: Honey-fried Licorice (HFL) is a dosage form of Glycyrrhizae Radix et Rhizome processed with honey, which has been recorded to exhibit better efficacy in tonifying the spleen compared to the raw product. In contrast, different processing methods of Glycyrrhizae Radix et Rhizome exhibit different efficacies and applications, but their current quality control index components remain consistent. PURPOSE: Based on the discovery and research strategy of traditional Chinese medicine decoction piece quality marker (Q-marker), this study aimed to conduct a multidimensional integration of constituents absorbed into the body and metabolomics based on the tonifying spleen and stomach effects of HFL to effectively identify the Q-marker of HFL. METHODS: In this study, a spleen deficiency rat model was established using the "exhausted swimming + poor diet" method to investigate the pharmacodynamics of tonifying the spleen and stomach by HFL. The constituents absorbed into blood was conducted using UPLC-Q-TOF/MS, correlation analysis between metabolomics and constituents absorbed into blood recognized the Q-Marker of HFL. RESULTS: The pharmacodynamic data demonstrated that HFL exhibited a significant regulatory effect on the disordered levels of PP, trypsin, chymase, PL, α-Glu, MTL, GAS, VIP, IL-2, IFN-γ, and IgA in the spleen deficiency model. Furthermore, HFL was found to improve the pathological changes in the spleen and intestine in the spleen deficiency model, highlighting its significant "tonifying spleen and stomach" effect. In the serum containing HFL, a total of 17 constituents were identified as being absorbed into the blood. Among these, 11 were prototypical components, while 6 were metabolites. Metabolomics data revealed that 9 differentially expressed metabolic markers were observed. Furthermore, the analysis of endogenous metabolic markers indicated that 10 components exhibited significant correlations with these biomarkers. CONCLUSION: The effect of "tonifying spleen and stomach" of HFL is closely related to the regulation of the material and energy metabolism pathway. The Q-Marker of HFL is glycyrrhizic acid and 18ß-glycyrrhetinic acid as the main control standards and liquiritin, isoliquiritin, liquiritin, isoliquiritin, isolicorice flavonol, licorice chalcone C and Formononetin were used as auxiliary standards.

Hydroxy-Safflower Yellow A Mitigates Vascular Remodeling in Rat Pulmonary Arterial Hypertension.

Purpose: The underlying causes of pulmonary arterial hypertension (PAH) often remain obscure. Addressing PAH with effective treatments presents a formidable challenge. Studies have shown that Hydroxysafflor yellow A (HSYA) has a potential role in PAH, While the mechanism underlies its protective role is still unclear. The study was conducted to investigate the potential mechanisms of the protective effects of HSYA. Methods: Using databases such as PharmMapper and GeneCards, we identified active components of HSYA and associated PAH targets, pinpointed intersecting genes, and constructed a protein-protein interaction (PPI) network. Core targets were singled out using Cytoscape for the development of a model illustrating drug-component-target-disease interactions. Intersection targets underwent analysis for Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Selected components were then modeled for target interaction using Autodock and Pymol. In vivo validation in a monocrotaline-induced PAH (MCT-PAH) animal model was utilized to substantiate the predictions made by network pharmacology. Results: We associated HSYA with 113 targets, and PAH with 1737 targets, identifying 34 mutual targets for treatment by HSYA. HSYA predominantly affects 9 core targets. Molecular docking unveiled hydrogen bond interactions between HSYA and several PAH-related proteins such as ANXA5, EGFR, SRC, PPARG, PGR, and ESR1. Conclusion: Utilizing network pharmacology and molecular docking approaches, we investigated potential targets and relevant human disease pathways implicating HSYA in PAH therapy, such as the chemical carcinogenesis receptor activation pathway and the cancer pathway. Our findings were corroborated by the efficacious use of HSYA in an MCT-induced rat PAH model, confirming its therapeutic potential.

Safflower Yellow Inhibits Progression of Hepatocellular Carcinoma by Modulating Immunological Tolerance via FAK Pathway.

OBJECTIVE: To explore the anti-tumor effect of safflower yellow (SY) against hepatocellular carcinoma (HCC) and the underlying potential mechanism. METHODS: An in vitro model was established by mixing Luc-Hepa1-6 cells and CD3+CD8+ T cells, followed by adding programmed cell death protein 1 (PD-1) antibody (Anti-mPD-1) with or without SY. The apoptosis was detected by flow cytometry and the level of inflammatory cytokines was determined by enzyme-linked immunosorbent assay. The protein levels of programmed cell death 1 ligand 1 (PD-L1), chemokine ligand (CCL5), C-X-C motif chemokine ligand 10 (CXCL10) were measured by Western blot. An in situ animal model was established in mice followed by treatment with anti-mPD-1 with or without SY. Bioluminescence imaging was monitored with an AniView 100 imaging system. To establish the FAK-overexpressed Luc-Hepa1-6 cells, cells were transfected with adenovirus containing pcDNA3.1-FAK for 48 h. RESULTS: The fluorescence intensity, apoptotic rate, release of inflammatory cytokines, and CCL5/CXCL10 secretion were dramatically facilitated by anti-mPD-1 (P<0.01), accompanied by an inactivation of PD-1/PD-L1 axis, which were extremely further enhanced by SY (P<0.05 or P<0.01). Increased fluorescence intensity, elevated percentage of CD3+CD8+ T cells, facilitated release of inflammatory cytokines, inactivated PD-1/PD-L1 axis, and increased CCL5/CXCL10 secretion were observed in Anti-mPD-1 treated mice (P<0.01), which were markedly enhanced by SY (P<0.05 or P<0.01). Furthermore, the enhanced effects of SY on inhibiting tumor cell growth, facilitating apoptosis and inflammatory cytokine releasing, suppressing the PD-1/PD-L1 axis, and inducing the CCL5/CXCL10 secretion in Anti-mPD-1 treated mixture of Luc-Hepa1-6 cells and CD3+CD8+ T cells were abolished by FAK overexpression (P<0.01). CONCLUSION: SY inhibited the progression of HCC by mediating immunological tolerance through inhibiting FAK.

Hydroxysafflor Yellow A Promotes HaCaT Cell Proliferation and Migration by Regulating HBEGF/EGFR and PI3K/AKT Pathways and Circ_0084443.

OBJECTIVE: To investigate the effect and possible mechanism of hydroxysafflor yellow A (HSYA) on human immortalized keratinocyte cell proliferation and migration. METHODS: HaCaT cells were treated with HSYA. Cell proliferation was detected by the cell counting kit-8 assay, and cell migration was measured using wound healing assay and Transwell migration assay. The mRNA and protein expression levels of heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), EGF receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF-1α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA. The expression of circ_0084443 was detected by qRT-PCR. RESULTS: HSYA (800 µmol/L) significantly promoted HaCaT cell proliferation and migration (P<0.05 or P<0.01). It also increased the mRNA and protein expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and increased the phosphorylation levels of PI3K and AKT (P<0.05 or P<0.01). Furthermore, HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/mTOR signaling pathways (P<0.01). Circ_0084443 attenuated the mRNA expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α (P<0.05). HSYA inhibited the circ_0084443 expression, further antagonized the inhibition of circ_0084443 on HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and promoted the proliferation of circ_0084443-overexpressing HaCaT cells (P<0.05 or P<0.01). However, HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration (P>0.05). CONCLUSION: HSYA played an accelerative role in HaCaT cell proliferation and migration, which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways, and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.

Isoliquiritin treatment of osteoporosis by promoting osteogenic differentiation and autophagy of bone marrow mesenchymal stem cells.

Osteoporosis is a chronic progressive bone disease characterized by the decreased osteogenic ability of osteoblasts coupled with increased osteoclast activity. Natural products showing promising therapeutic potential for postmenopausal osteoporosis remain underexplored. In this study, we aimed to analyze the therapeutic effects of isoliquiritin (ISL) on osteoporosis in mice and its possible mechanism of action. An ovariectomy-induced osteoporosis mouse model and bone marrow mesenchymal stem cells (BMSCs) were used to analyze the effects of ISL on bone regeneration in vivo and in vitro, respectively. Mitogen-activated protein kinase (MAPK) and autophagy inhibitors were used, to investigate whether the MAPK signaling pathway and autophagy affect the osteogenic differentiation of BMSCs. ISL significantly improved bone formation and reduced bone resorption in mouse femurs without inducing any detectable toxicity in critical organs such as the liver, kidney, brain, heart, and spleen. In vitro experiments showed that ISL enhanced the proliferation and osteogenic differentiation of BMSCs and that its osteogenic effect was attenuated by p38/extracellular regulated protein kinase (ERK) and autophagy inhibitors. Further studies showed that the inhibition of phosphorylated p38/ERK blocked ISL autophagy in BMSCs. ISL promoted the osteogenic differentiation of BMSCs through the p38/ERK-autophagy pathway and was therapeutically effective in treating osteoporosis in ovariectomized mice without any observed toxicity to vital organs. These results strongly suggest the promising potential of ISL as a safe and efficacious candidate drug for the treatment of osteoporosis.

LC-MS/MS combined with blood-brain dual channel microdialysis for simultaneous determination of active components of astragali radix-safflower combination and neurotransmitters in rats with cerebral ischemia reperfusion injury: Application in pharmacokinetic and pharmacodynamic study.

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-ß-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-Emax 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-ß-glucoside, syringin and vernine in blood microdialysate and calycosin, syringin, vernine in brain microdialysate were narrow, which need be paid attention in clinical use.

[Research progress on mechanism of Carthamus tinctorius in ischemic stroke therapy].

Carthamus tinctorius is proved potent in treating ischemic stroke. Flavonoids, such as safflower yellow, hydroxysafflor yellow A(HSYA), nicotiflorin, safflower yellow B, and kaempferol-3-O-rutinoside, are the main substance basis of C. tinctorius in the treatment of ischemic stroke, and HSYA is the research hotspot. Current studies have shown that C. tinctorius can prevent and treat ischemic stroke by reducing inflammation, oxidative stress, and endoplasmic reticulum stress, inhibiting neuronal apoptosis and platelet aggregation, as well as increasing blood flow. C. tinctorius can regulate the pathways including nuclear factor(NF)-κB, mitogen-activated protein kinase(MAPK), signal transducer and activator of transcription protein 3(STAT3), and NF-κB/NLR family pyrin domain containing 3(NLRP3), and inhibit the activation of cyclooxygenase-2(COX-2)/prostaglandin D2/D prostanoid receptor pathway to alleviate the inflammatory development during ischemic stroke. Additionally, C. tinctorius can relieve oxidative stress injury by inhibiting oxidation and nitrification, regulating free radicals, and mediating nitric oxide(NO)/inducible nitric oxide synthase(iNOS) signals. Furthermore, mediating the activation of Janus kinase 2(JAK2)/STAT3/suppressor of cytokine signaling 3(SOCS3) signaling pathway and phosphoinositide 3-kinase(PI3 K)/protein kinase B(Akt)/glycogen synthase kinase-3ß(GSK3ß) signaling pathway and regulating the release of matrix metalloproteinase(MMP) inhibitor/MMP are main ways that C. tinctorius inhibits neuronal apoptosis. In addition, C. tinctorius exerts the therapeutic effect on ischemic stroke by regulating autophagy and endoplasmic reticulum stress. The present study reviewed the molecular mechanisms of C. tinctorius in the treatment of ischemic stroke to provide references for the clinical application of C. tinctorius.

Hydroxysafflor Yellow A Alleviates Ischemic Stroke in Rats via HIF-1[Formula: see text], BNIP3, and Notch1-Mediated Inhibition of Autophagy.

Stroke has become a major cause of death and disability worldwide. The cellular recycling pathway autophagy has been implicated in ischemia-induced neuronal changes, but whether autophagy plays a beneficial or detrimental role is controversial. Hydroxysafflor Yellow A (HSYA), a popular herbal medicine, is an extract of Carthamus tinctorius and is used to treat ischemic stroke (IS) in China. HSYA has been shown to prevent cardiovascular and cerebral ischemia/reperfusion injury in animal models. However, the specific active ingredients and molecular mechanisms of HSYA in IS remain unclear. Here, we investigated the effect of HSYA treatment on autophagy in a rat model of IS. IS was induced in rats by middle cerebral artery occlusion. Rats were treated once daily for 3 days with saline, HYSA, or the neuroprotective agent Edaravone. Neurobehavioral testing was performed on days 1, 2, and 3 post-surgery. Brains were removed on day 3 post-surgery for histological evaluation of infarct area, morphology, and for qRT-PCR and western blot analysis of the expression of the autophagy factor LC3 and the signaling molecules HIF-1[Formula: see text], BNIP3, and Notch1. Molecular docking studies were performed in silico to predict potential interactions between HSYA and LC3, HIF-1[Formula: see text], BNIP3, and Notch1 proteins. The result showed that HSYA treatment markedly alleviated IS-induced neurobehavioral deficits and reduced brain infarct area and tissue damage. HSYA also significantly reduced hippocampal expression levels of LC3, HIF-1[Formula: see text], BNIP3, and Notch1. The beneficial effect of HSYA was generally superior to that of Edaravone. Molecular modeling suggested that HSYA may bind strongly to HIF-1[Formula: see text], BNIP3, and Notch1 but weakly to LC3. In conclusion, HSYA inhibits post-IS autophagy induction in the brain, possibly by suppressing HIF-1[Formula: see text], BNIP3 and Notch1. HSYA may have utility as a post-IS neuroprotective agent.

Microwave-Assisted Extraction of Anticancer Flavonoid, 2',4'-Dihydroxy-6'-methoxy-3',5'-dimethyl Chalcone (DMC), Rich Extract from Syzygium nervosum Fruits.

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethyl chalcone (DMC) is a biological flavonoid that is present in the fruits of Syzygium nervosum (Ma-Kiang in Thai). Microwave-assisted extraction (MAE), which utilizes microwave radiation to heat the extraction solvent quickly and effectively, was used to recover DMC-rich extract from Syzygium nervosum fruit. To determine the DMC content, a highly accurate and precise HPLC technique was developed. The influences of MAE conditions, including the solid-liquid ratio, microwave power, and microwave duration on the content of DMC, were sequentially employed by a single factor investigation and response surface methodology (RSM) exploratory design. The predicted quadratic models were fitted due to their highly significant (p < 0.0001) and excellent determination coefficient (R2 = 0.9944). The optimal conditions for producing DMC-rich extract were a ratio of sample to solvent of 1:35 g/mL, a microwave power of 350 W, and a microwave time of 38 min. Under the optimal MAE setting, the DMC content reached 1409 ± 24 µg/g dry sample, which was greater than that of the conventional heat reflux extraction (HRE) (1337 ± 37 µg/g dry sample) and maceration (1225 ± 81 µg/g dry sample). The DMC-rich extract obtained from MAE showed stronger anticancer activities against A549 (human lung cancer cells) and HepG2 (human liver cancer cells) than the individual DMC substance, which makes MAE an effective method for extracting essential phytochemicals from plants in the nature.

A comparative study on the traditional versus modern yellow rice wine processing methods using Taohong Siwu Decoction for pharmaceutical production.

ETHNOPHARMACOLOGICAL RELEVANCE: Taohong Siwu Decoction (THSWD) is based on the "First Recipe of Gynecology." It is widely used in various blood stasis and deficiency syndromes, mainly in gynecological blood stasis, irregular menstruation, and dysmenorrhea. THSWD has great demand in traditional Chinese medicine (TCM), gynecology, orthopedics, and internal medicine. According to classical records, three medicinal materials, namely Rehmanniae radix, Angelica sinensis, and Carthamus tinctorius, used in THSWD need to be "washed with yellow rice wine." In the study of TCM prescriptions, the processing methods of medicinal materials not only needed to follow traditional records but also should consider modern technical conditions. Many medicinal materials in the repertoire of classical prescriptions involve yellow rice wine processing. Determining the processing method for medicinal materials is a key and difficult problem in the research and development of classical prescriptions. AIM OF THE STUDY: With THSWD as the representative, this study analyzed differences between no processing method, the modern processing method of "stir-frying the materials with yellow rice wine," and the traditional processing method of "washing with yellow rice wine." We focused on three aspects: composition, efficacy, and endogenous metabolism. This study aimed to provide a reference for research on the processing methods of medicinal materials used in classical prescriptions. MATERIALS AND METHODS: UPLC-Q-Orbitrap HRMS was used to quickly identify and classify the main chemical compounds of THSWD. A model of primary dysmenorrhea (PD) was established using estradiol benzoate combined with oxytocin. The latent period and writhing time; the levels of serum PGF2α, PGE2, ET-1, and ß-EP; and the pathological sections of the uterus were observed to determine their pharmacodynamic differences. GC-TOF/MS was used to analyze the differences in serum metabolites in rats. RESULTS: A total of 54 active compounds were identified, and the results showed that catalpol and rehmapicroside disappeared following yellow rice wine processing. Compared with materials processed by the traditional method, the relative contents of 15 components, such as 5-hydroxymethylfurfural and digitalis C, increased in materials processed by the modern method. However, the relative contents of 16 components, such as hydroxysafflor yellow A, verbascoside, and ferulic acid, decreased in the modern processing method. The modern and classic processing methods acted on PD through different metabolic pathways. THSWD obtained by classical processing methods mainly treated PD through anti-inflammatory and estrogen metabolism pathways, whereas THSWD obtained by modern processing methods mainly treated PD through anti-inflammatory metabolic pathways. CONCLUSION: The study revealed the differences in different yellow rice wine processing methods in terms of chemical composition of the THSWD obtained, as well as the mechanisms of action for the treatment of PD. This study provides a reference for the clinical application of THSWD and development of classical prescription preparations.

Hydroxysafflor yellow A can improve depressive behavior by inhibiting hippocampal inflammation and oxidative stress through regulating HPA axis.

Depression is characterized by indifferent and slow thinking, leading to highly unfavorable social and economic burden. Hydroxysafflor yellow A (HSYA) is a traditional Chinese medicine and has many pharmacological properties, such as anti-oxidative and anti-inflammatory activities. However, the underlying mechanism unraveling the effect of HSYA on depression is still unclear. Here, depression animal model was established. It was demonstrated that HSYA improved depressive behavior in rat model of depression, which increased horizontal movement, vertical movement, sucrose percent index and decreased immobility of depressed rats. Moreover, HSYA inhibited the activation of HPA signaling, inflammation and oxidative stress in brain of depressed rats. HSYA played an opposite effect on production of chronic unpredicted mild stress (CUMS)-induced pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß). CUMS increased MDA expression but decreased SOD and GSH-Px expression, which were reversed by HSYA treatment. Furthermore, HSYA exerted a suppressive role in TLR4/NF-jB signaling pathway in brain of depressed rats. In conclusion, these findings indicted that HSYA can improve depressive behavior through inhibiting HPA signaling, repressing hippocampal inflammation and oxidative stress, which will provide a new therapeutic method for treating depression.

Pharmacological Actions, Molecular Mechanisms, Pharmacokinetic Progressions, and Clinical Applications of Hydroxysafflor Yellow A in Antidiabetic Research.

Hydroxysafflor yellow A (HSYA), a nutraceutical compound derived from safflower (Carthamus tinctorius), has been shown as an effective therapeutic agent in cardiovascular diseases, cancer, and diabetes. Our previous study showed that the effect of HSYA on high-glucose-induced podocyte injury is related to its anti-inflammatory activities via macrophage polarization. Based on the information provided on PubMed, Scopus and Wanfang database, we currently aim to provide an updated overview of the role of HSYA in antidiabetic research from the following points: pharmacological actions, molecular mechanisms, pharmacokinetic progressions, and clinical applications. The pharmacokinetic research of HSYA has laid foundations for the clinical applications of HSYA injection in diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy. The application of HSYA as an antidiabetic oral medicament has been investigated based on its recent oral delivery system research. In vivo and in vitro pharmacological research indicated that the antidiabetic activities of HSYA were based mainly on its antioxidant and anti-inflammatory mechanisms via JNK/c-jun pathway, NOX4 pathway, and macrophage differentiation. Further anti-inflammatory exploration related to NF-κB signaling, MAPK pathway, and PI3K/Akt/mTOR pathway might deserve attention in the future. The anti-inflammatory activities of HSYA related to diabetes and diabetic complications will be a highlight in our following research.

[Comparison of effects of Xuebijing injection and its component hydroxysafflor yellow A on coagulation and survival rates of septic rats].

OBJECTIVE: To explore the effects of Xuebijing injection and its component hydroxysafflor yellow A on coagulation and survival rates of septic rats. METHODS: (1) Assessment of coagulation: 144 male Sprague-Dawley (SD) rats were divided into four groups by random number table: sham group, cecal ligation and puncture (CLP) induced sepsis model group (CLP group), CLP+Xuebijing group, and CLP+hydroxysafflor yellow A group, with 36 rats in each group. CLP was used for reproducing septic models. The cecum of the rats in the sham group was exposed by laparotomy and then returned to the abdominal cavity without CLP, while the other steps were the same as those in the CLP group. Rats in the CLP+Xuebijing group and CLP+hydroxysafflor yellow A group were injected with Xuebijing (4 mL/kg, twice a day) or hydroxysafflor yellow A solution (0.378 g/L, 298 µg each time, twice a day) through caudal vein after operation. Levels of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (Fib), and D-dimer in peripheral blood were measured by automatic coagulation analyzer at 6, 12, 24 hours after operation. The enzyme linked immunosorbent assay (ELISA) was applied to determine levels of tissue factor (TF), tissue factor pathway inhibitor (TFPI), and soluble thrombomodulin (sTM) in peripheral blood. (2) Analysis of survival rates: 120 rats were divided into four groups by random number table (the same groups with those in the section of assessment of coagulation), with 30 rats in each group. The Kaplan-Meier survival curve was plotted, and the cumulative survival rates were observed and recorded for 7 days after CLP surgery. RESULTS: (1) Results of coagulation assessment: compared with the sham group, septic rats in the CLP group showed significant dysfunction in coagulation early, as evidenced by prolonged PT at 6 hours after CLP (s: 8.9±0.2 vs. 8.4±0.4, P < 0.01), and significantly increased levels of Fib, D-dimer, TFPI and sTM [Fib (g/L): 2.8±0.3 vs. 2.3±0.1, D-dimer (ng/L): 1.8±0.2 vs. 1.5±0.1, TFPI (ng/L): 131.1±10.9 vs. 102.8±10.5, sTM (µg/L): 27.2±1.2 vs. 19.8±2.9, all P < 0.01]. The coagulation dysfunction became more and more serious at 12 hours after operation, and further deteriorated with time. The use of both Xuebijing and hydroxysafflor yellow A revealed significant improvement in coagulation of septic rats at 6 hours, as shown by shortened PT (s: 8.3±0.2, 8.3±0.1 vs. 8.9±0.2, both P < 0.01), and decreased Fib, D-dimer, TFPI and sTM as compared with those in the CLP group [Fib (g/L): 2.3±0.1, 2.3±0.2 vs. 2.8±0.3; D-dimer (ng/L): 1.5±0.1, 1.5±0.2 vs. 1.8±0.2; TFPI (ng/L): 109.5±10.2, 91.5±5.0 vs. 131.1±10.9; sTM (µg/L): 22.3±1.5, 21.1±1.8 vs. 27.2±1.2; all P < 0.01]. However, there was no significant difference in coagulation function between the two intervention groups. (2) Results of survival rates analysis: the rats in the sham group all survived 7 days after operation. The 7-day cumulative survival rate of the CLP group was only 36.67% (11/30). Compared with the CLP group, the cumulative survival rates were significantly increased in rats of the CLP+Xuebijing group and CLP+hydroxysafflor yellow A group [66.67% (20/30), 66.67% (20/30) vs. 36.67% (11/30), both P < 0.05], but no significant difference was found between the CLP+Xuebijing group and CLP+hydroxysafflor yellow A group. CONCLUSIONS: Both Xuebijing and its component hydroxysafflor yellow A appear to be capable of alleviating coagulation disorders and improving survival rates of septic rats effectively, and the effects show no significant difference between them.

Safflower yellow reduces DEN-induced hepatocellular carcinoma by enhancing liver immune infiltration through promotion of collagen degradation and modulation of gut microbiota.

Safflower yellow (SY) is the main active ingredient isolated from the traditional Chinese medicine Carthamus tinctorius, which is a valuable natural edible pigment that is widely used to treat cerebrovascular and cardiovascular diseases. However, the effect of SY on hepatocellular carcinoma (HCC) remains unclear. In this study, we showed that SY decreased the degree of injury and inhibited the release of inflammatory factors in the liver of a diethylnitrosamine (DEN)-induced HCC mouse model. Flow cytometry and immunoblotting showed that SY increased the infiltration of CD8+ T cells and Gr-1+ macrophages to improve the immune microenvironment by affecting the expression of collagen fibers. Further cellular experiments showed that SY degraded the collagens in the liver cells through the TGF-ß/Smad signalling pathway. SY also regulated the gut microbiota which may contribute to the immune microenvironment. In conclusion, SY exhibited a potent effect on the development of HCC by enhancing liver immune infiltration by promoting collagen degradation and modulating the gut microbiota. This study provides novel insights into the mechanism of SY as a candidate for the treatment of HCC in the future.

Hydroxysafflor yellow A, a natural compound from Carthamus tinctorius L with good effect of alleviating atherosclerosis.

BACKGROUND: Atherosclerosis is a chronic vascular inflammatory disease with complex pathogenesis. Its serious consequence is insufficient blood supply to heart and brain, which eventually leads to myocardial ischemia, infarction and stroke. Hydroxysafflor yellow A (HSYA), a single chalcone glycoside compound with a variety of pharmacological effects, which has shown a potential biological activity for prevention and treatment of atherosclerosis. PURPOSE: The main purpose of this review is to comprehensively elucidate the mechanism of HSYA on atherosclerosis and its risk factors (hyperlipidemia, hypertension and diabetes mellitus). METHOD: The literatures on HSYA in the treatment of atherosclerosis and its risk factors were searched in PubMed, Google Scholar, China National Knowledge Infrastructure, including in vitro (cell), in vivo (animal) and clinical (human) studies, and summarized reasonably. RESULTS: HSYA is a promising natural product for treating atherosclerosis. It can suppress foam cell formation, vascular endothelial cell dysfunction, vascular smooth muscle cell proliferation and migration, and platelet activation. The mechanisms are achieved by regulating the reverse cholesterol transport process, fatty acid synthesis, oxidative stress, PI3K/Akt/mTOR, NLRP3 inflammasome, TNFR1/NF-κB, NO-cGMP, Bax/Bcl-2, MAPKs, CDK/CyclinD and TLR4/Rac1/Akt signaling pathways. Besides, HSYA is devoted to lowering blood lipids, regulating ion channels, reducing vascular inflammation, and protecting pancreatic beta cells, which is conducive to reducing the harm of independent risk factors of atherosclerosis. CONCLUSIONS: HSYA exhibits the preventive and therapeutic effects on atherosclerosis and its risk factors in vivo and in vitro, which is relevant to multiple mechanisms. The clinical trials of HSYA need to be further investigated to provide a solid foundation for its clinical application.

Hydroxysafflor yellow A promotes osteogenesis and bone development via epigenetically regulating ß-catenin and prevents ovariectomy-induced bone loss.

In clinical treatment, there is increasingly prevalent that traditional Chinese medicine treats common bone diseases including osteoporosis. Hydroxysafflor yellow A (HSYA), one of the essential compounds of Safflower, has been used as the therapy for thrombus, myocardial ischemia, and inflammation, but its effect on osteogenesis through epigenetic control and ovariectomy-induced bone loss in vivo has not been explored. Therefore, the study aimed to explore the function and mechanism of HSYA on bone formation and development. We found HSYA could enhance the cell viability and promote osteogenesis of hBMSCs in vitro. Mechanistically, HSYA could increase the expression of ß-catenin leading to its accumulation in the nucleus and activation of downstream targets to promote osteogenesis. Besides, RNA-seq and quantitative RT-PCR and western blot showed KDM7A was significantly increased by HSYA. The occupancy of H3K27me2 on ß-catenin promoter was significantly decreased by HSYA, which could be reversed by silencing endogenous KDM7A. More importantly, HSYA promoted bone development in chick embryos and prevented ovariectomy (OVX)-induced bone loss in SD rats. Taken together, our study has shown convincing evidence that HSYA could promote osteogenesis and bone development via epigenetically regulating ß-catenin and prevent ovariectomy-induced bone loss.

Stability limits of a red Carthamin-cellulose complex as a potential food colourant.

This study covers aspects of stability and colouration of Carthamin- a unique red chalcone extracted from Carthamus tinctorius L. Due to its fast degradation in aqueous solutions even at room temperature, Carthamin has no significant use in the food industry. Therefore, obtaining Carthamin in a stable form is of high interest. Comparing UV-Vis spectra of Carthamin solutions and RGB-data of Carthamin-cellulose complex in the wet state showed a predominant formation of stable Carthamin conformation on the cellulose phase. It was determined that the wet Carthamin-cellulose complex acquires a stable and rich magenta colour in the pH range of 1-5. In aqueous suspensions with pH >6, the Carthamin-cellulose complex gets a purple colour, which is absolutely uncharacteristic for pure Carthamin in an aqueous solution. IR spectra indicate the fixation of Carthamin molecules on the cellulose, which presumably causes hindrance of free internal rotation of Carthamin molecules in the cellulose phase. The reduction of water activity in the cellulosic phase represents an additional stabilizing factor. As a result, the Carthamin-cellulose complex withstands heating up to 70 °C for 15 min in the pH range of 2-5, showing up to 90% of stability. These conditions are typical for the preparation of a wide range of food products. High stability in a food-like environment and magenta colour make the Carthamin-cellulose complex a prospective natural food dye.

Hydroxysafflor Yellow A Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Calcium Overload and Apoptosis.

Myocardial ischemia/reperfusion injury (MI/RI) is an urgent problem with a great impact on health globally. However, its pathological mechanisms have not been fully elucidated. Hydroxysafflor yellow A (HSYA) has a protective effect against MI/RI. This study is aimed at further clarifying the relationship between HSYA cardioprotection and calcium overload as well as the underlying mechanisms. We verified the protective effect of HSYA on neonatal rat primary cardiomyocytes (NPCMs) and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from hypoxia-reoxygenation (HR) injury. To explore the cardioprotective mechanism of HSYA, we employed calcium fluorescence, TUNEL assay, JC-1 staining, and western blotting. Finally, cardio-ECR and patch-clamp experiments were used to explain the regulation of L-type calcium channels (LTCC) in cardioprotection mediated by HSYA. The results showed that HSYA reduced the levels of myocardial enzymes and protected NPCMs from HR injury. HSYA also restored the contractile function of hiPSC-CMs and field potential signal abnormalities caused by HR and exerted a protective effect on cardiac function. Further, we demonstrated that HSYA protects cardiomyocytes from HR injury by decreasing mitochondrial membrane potential and inhibiting apoptosis and calcium overload. Patch-clamp results revealed that MI/RI caused a sharp increase in calcium currents, which was inhibited by pretreatment with HSYA. Furthermore, we found that HSYA restored contraction amplitude, beat rate, and field potential duration of hiPSC-CMs, which were disrupted by the LTCC agonist Bay-K8644. Patch-clamp experiments also showed that HSYA inhibits Bay-K8644-induced calcium current, with an effect similar to that of the LTCC inhibitor nisoldipine. Therefore, our data suggest that HSYA targets LTCC to inhibit calcium overload and apoptosis of cardiomyocytes, thereby exerting a cardioprotective effect and reducing MI/RI injury.

Rapid screening and identification of anticoagulation component from carthami flos by two-dimensional thrombin affinity chromatography combined with HPLC-MS/MS.

Carthami flos, commonly known as Honghua in China, is the dried floret of safflower and widely acknowledged as a blood stasis promoting herb. The study aimed at investigating the relationship between thrombin and carthami flos through a high-performance thrombin affinity chromatography combined with a high-performance liquid chromatography-tandem mass spectrometry system. First, thrombin was immobilized on the glutaraldehyde-modified amino silica gel to prepare the thrombin affinity stationary phase, which was packed into a small column (1.0 × 2.0 mm, id) for recognizing the anticoagulant active components of carthami flos. The target component was enriched and analyzed by the high-performance liquid chromatography-tandem mass spectrometry system. Finally, hydroxysafflor yellow A was screened out and identified as the active component. The anticoagulant effects of hydroxysafflor yellow A were analyzed by anticoagulant experiments in vitro, and the interaction of hydroxysafflor yellow A with thrombin was investigated by the molecular docking method. The results proved that hydroxysafflor yellow A (30 µg/mL, 0.05 mM) and carthami flos extract (30 µg/mL) could prolong activated partial thrombin time and thrombin time by 50 and 11%, respectively. Moreover, hydroxysafflor yellow A exhibits a good hydrogen bond field and stereo field matching with thrombin. Overall, it was concluded that hydroxysafflor yellow A might exert an anticoagulation effect by interacting with thrombin and thus could be potential anticoagulant drugs for the prevention and treatment of venous thrombosis.

Simultaneous determination of both kavalactone and flavokawain constituents by different single-marker methods in kava.

Kava, the rhizomes and roots of Piper methysticum Forst, is a popular edible medicinal herb traditionally used to prepare beverages for anxiety reduction. Since the German kava ban has been lifted by the court, the quality evaluation is particularly important for its application, especially the flavokawains which were believed to be responsible for hepatotoxicity. Now, by employing two different standard references and four different methods to calculate the relative correction factors, eight different quantitative analyses of multicomponents by single-marker methods have been developed for the simultaneous determination of eight major kavalactones and flavokawains in kava. The low standard method difference on quantitative measurement of the compounds among the external standard method and ours confirmed the reliability of the mentioned methods. A radar plot clearly illustrated that the contents of dihydrokavain and kavain were higher, whereas flavokawains A and B were lower in different kava samples. Only one of eight samples did not detect flavokawains that may be related to hepatotoxicity. In summary, by using different agents as an internal standard reference, the developed methods were believed as a powerful analytical tool not only for the qualitative and quantitative of kava constituents but also for the other multicomponents when authentic standard substances were unavailable.