Hydroxysafflor Yellow Research Articles

Integrating molecular characterization and metabolites profile revealed CtCHI1\u2019s significant role in Carthamus tinctorius L.

BackgroundAs a traditional Chinese herb, safflower (Carthamus tinctorius L.) is valued for its florets to prevent cardiovascular and cerebrovascular diseases. Basing on previous chemical analysis, the main active compounds are flavonoids in its florets. Although flavonoid biosynthetic pathway has been well-documented in many model species, unique biosynthetic pathway remains to be explored in safflower. Of note, as an important class of transitional enzymes, chalcone isomerase (CHI) has not been characterized in safflower.ResultsAccording to our previous research, CHIs were identified in a safflower transcriptome library built by our lab. To characterize CHI in safflower, a CHI gene named CtCHI1 was identified. A multiple sequences alignment and phylogenetic tree demonstrate that CtCHI1 shares 92% amino acid identity and close relationship with CHI to Saussurea medusa. Additionally, subcellular localization analysis indicated CtCHI1-GFP fusion protein was mainly in the cell nucleus. Further, we purified CtCHI1 protein from E. coli which can effectively catalyze isomerization of 2′,4′,4,6′-tetrahydroxychalcone into naringenin in vitro. Via genetic engineer technology, we successfully obtained transgenic tobacco and safflower lines. In transgenic tobacco, overexpression of CtCHI1 significantly inhibited main secondary metabolites accumulation, including quercetin (~ 79.63% for ovx-5 line) and anthocyanins (~ 64.55% for ovx-15 line). As shown in transgenic safflower, overexpression of CtCHI1 resulted in upstream genes CtPAL3 and CtC4H1 increasing dramatically (up to ~ 3.9fold) while Ct4CL3, CtF3H and CtDFR2 were inhibited. Also, comparing the whole metabolomics database by PCA and PLS-DA between transgenic and control group, 788 potential differential metabolites were marked and most of them displayed up-regulated trends. In parallel, some isolated secondary metabolites, such as hydroxysafflor yellow A (HSYA), rutin, kaempferol-3-O-β-rutinoside and dihydrokaempferol, accumulated in transgenic safflower plants.ConclusionsIn this study, we found that CtCHI1 is an active, functional, catalytic protein. Moreover, CtCHI1 can negatively and competitively regulate anthocyanins and quercetin pathway branches in tobacco. By contrast, CtCHI1 can positively regulate flavonol and chalcone metabolic flow in safflower. This research provides some clues to understand CHI’s differential biochemical functional characterization involving in flavonoid pathway. More molecular mechanisms of CHI remain to be explored in the near future.

Inhibitory Effect of Hydroxysafflor Yellow B on the Proliferation of Human Breast Cancer MCF-7 Cells.

A recent patent has been issued for hydroxysafflor yellow A (HSYA) as a drug to prevent blood circulation disorders. Hydroxysafflor yellow B (HSYB), an isomer of HSYA with antioxidative effects, has been isolated from the florets of Carthamus tinctorius. The effects of HSYB on the proliferation of cancer cells and its mechanism of action have not been investigated. The aims of this study were to investigate the anti-cancer effects and the molecular mechanism of HSYB for breast cancer MCF-7 cells. MTT assays and colony formation assays were used to assess the survival and proliferation of MCF-7 cells, respectively. Hoechst 33258 and flow cytometry were used to measure cell apoptosis and flow cytometry to determine effects on the cell cycle. Western blots were used to measure protein levels. Treatment with HSYB reduced survival and proliferation of human breast cancer MCF-7 cells in a dose-dependent manner. Furthermore, HSYB arrested the MCF-7 cell cycle at the S phase and downregulated cyclin D1, cyclin E, and CDK2. Compared with a control group, HSYB suppressed the protein levels of p-PI3K, PI3K, AKT, and p-AKT in MCF-7 cells. In addition, HSYB decreased the levels of Bcl- 2, increased the levels of Bax, cleaved caspase-3 and caspase-9, and subsequently induced MCF-7 cell apoptosis. These data demonstrate that HSYB arrests the MCF-7 cell cycle at the S phase and induces cell apoptosis. Patent US20170246228 indicates that HSYB can be potentially used for the prevention and treatment of human breast cancer.

Hydroxysafflor Yellow A mitigated myocardial ischemia/reperfusion injury by inhibiting theactivation of the JAK2/STAT1 pathway.

Hydroxysafflor Yellow A (HSYA) may reduce ischemia/reperfusion (I/R) injury. However, the underlying molecular mechanisms remain unclear. The present study explored the effect and the mechanisms of HSYA on myocardial injury in vivo and in vitro. Myocardial infarct size was assessed by Evans blue/2,3,5-triphenyltetrazoliumchloride staining. Levels of cardiac troponin I (cTnI), interleukin-6 (IL-6), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured using commercial kits. Alteration of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation was determined by fluorescent signals. Apoptosis was detected by terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling staining, flow cytometry assay and caspase-3 activity. Expression levels of the apoptosis-associated proteins were detected by reverse transcription quantitative polymerase chain reaction and western blot analysis. In vivo, animals treated with HSYA presented less severe myocardial injury and decreased janus kinase 2 (JAK2)/signal transducer and activator of transcription 1 (STAT1) activity, improved antioxidant capacity and decreased apoptosis. In vitro, compared with the hypoxia (H)/reoxygenation (R) + HSYA group, AG490 and S1491 treatment decreased the releases of cTnI, IL-6 and LDH and enhanced the resistance to oxidative stress by maintaining MMP and decreasing ROS generation. In addition, AG490 and S1491 were also identified to alleviate the H/R-induced apoptosis by inhibiting caspase 3 activity and modulating the expression levels of cleaved caspase-3, tumor necrosis factor receptor superfamily member 6 (Fas), Fas ligand, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein. These data suggested that inactivation of the JAK2/STAT1 pathway strengthened the HSYA-induced protective effect in H/R-induced myocardial injury. In conclusion, the treatment of HSYA was effective in decreasing IR-induced myocardial injury, and this may be largely dependent on the JAK2/STAT1 pathway. Therefore, the present study provided a potential strategy to prevent myocardial I/R injury.

Amelioration of dry eye syndrome in db/db mice with diabetes mellitus by treatment with Tibetan Medicine Formula Jikan Mingmu Drops

Ethnopharmacological relevanceJikan Mingmu Drops (JMD), a traditional Tibetan medicine containing six herbs, has been used to treat dry eye syndrome (DES) in individuals with diabetes mellitus. Aim of studyHowever, the activity of JMD ameliorates DES with diabetes mellitus has not been previously examined. The aim of the study is to investigate the molecular mechanism of JMD on db/db mice. Materials and methodsThe main chemical constituents of JMD were analyzed by high-performance liquid chromatography and gas chromatography–mass spectrometry. DES was then induced in db/db mice by applying 0.2% benzalkonium chloride to the ocular surface for 7 days. Eye drops containing JMD (0.25, 0.5, or 1 g/mL) or vehicle subsequently were administered three times daily for another 7 days, and the therapeutic effects were evaluated by phenol red thread tear and sodium fluorescein tests. Conjunctival specimens were subjected to hematoxylin and eosin staining and periodic acid–Schiff staining to examine pathological changes and number of goblet cells. ELISA was performed to assess the levels of various inflammatory cytokines. ResultsJMD contains hydroxysafflor yellow A, magnoflorine, jatrorrhizine hydrochloride, palmatine hydrochloride, berberine hydrochloride, gallic acid, ellagic acid, tauroursodeoxycholic acid, camphor, isoborneol, borneol, trans-cinnamic acid, and muscone. JMD treatment significantly increased the tear volume, decreased the corneal fluorescein staining score, restored the morphology and structure of conjunctival epithelial cells, and markedly downregulated the levels of interleukin (IL)-6, IL-17α, IL-1β, tumor necrosis factor-α, and vascular endothelial growth factor in the conjunctiva. Further data showed that these protective effects were accompanied by inhibition of inflammation in a dose-dependent manner. ConclusionsAmelioration of DES in db/db mice with diabetes mellitus by treatment with Tibetan medicine formula JMD maybe related to its anti-inflammatory effects.

Validated LC-MS/MS method for simultaneous quantification of seven components of Naodesheng in rat serum after oral administration and its application to a pharmacokinetic study

A simple, precise and reliable LC-MS/MS method was developed and validated for simultaneous quantification of vitexin, notoginsenoside R1, hydroxysafflor yellow A, ginsenoside Rd, puerarin, daidzein and senkyunolide I as components of Naodesheng (NDS) in rat serum. The Linearity ranges in rat serum were 0.045–4.5 μg/mL for vitexin, 0.0476–4.76 μg/mL for notoginsenoside R1, 0.0422–4.22 μg/mL for hydroxysafflor yellow A, 0.0426–4.26 μg/mL for ginsenoside Rd, 0.0436–4.36 μg/mL for puerarin, 0.026–2.6 μg/mL for daidzein, and 0.05–5 μg/mL for senkyunolide I, with the correlation coefficients greater than 0.99. The established method was validated in terms of intra- and inter-day precision and accuracy, recovery, matrix effect and stability. Furthermore, the method was successfully applied for pharmacokinetic study of these seven components in rat serum after oral administration of NDS.

Simultaneous determination of major components of Huangqi-Honghua extract in rat plasma using LC-MS/MS and application to a pharmacokinetic study.

A sensitive and reliable LC-MS/MS method was developed and validated for simultaneous quantification of the major components of Huangqi-Honghua extact in rat plasma, including hydroxysafflor yellow A (HSYA), astragaloside IV (ASIV), calycosin-7-O-β-d-glucoside (CAG), calycosin, calycosin-3'-O-glucuronide (C-3'-G) and calycosin-3'-O-sulfate (C-3'-S). After extraction by protein precipitation with acetonitrile and methanol from plasma, the analytes were separated on a Hypersil BDS C18 column by gradient elution with acetonitrile and 5 mM ammonium acetate. The detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization source switched between negative and positive modes. HSYA was monitored in negative ionization mode from 0 to 4.9 min, and ASIV, CAG, calycosin, C-3'-G and C-3'-S were determined in positive ionization mode from 4.9 to 10 min. The lower limits of quantification of the analytes were 6.25 ng/mL for HSYA, 0.781 ng/mL for CAG and 1.56 ng/mL for ASIV and calycosin. The intra- and inter-assay precision (RSD) values were within 13.43%, and accuracy (RE) ranged from -8.75 to 9.92%. The validated method was then applied to the pharmacokinetic study of HSYA, ASIV, CAG, calycosin, C-3'-G and C-3'-S in rat after an oral administration of Huangqi-Honghua extract.

A NMR-Based Metabonomics Approach to Determine Protective Effect of a Combination of Multiple Components Derived from Naodesheng on Ischemic Stroke Rats.

Naodesheng (NDS) is a widely used traditional Chinese medicine (TCM) prescription for the treatment of ischemic stroke. A combination of 10 components is derived from NDS. They are: Notoginsenoside R1, ginsenoside Rg1, ginsenoside b1, ginsenoside Rd, hydroxysafflor yellow A, senkyunolide I, puerarin, daidzein, vitexin, and ferulic acid. This study aimed to investigate the protective effect of the ten-component combination derived from NDS (TCNDS) on ischemic stroke rats with a middle cerebral artery occlusion (MCAO) model by integrating an NMR-based metabonomics approach with biochemical assessment. Our results showed that TCNDS could improve neurobehavioral function, decrease the cerebral infarct area, and ameliorate pathological features in MCAO model rats. In addition, TCNDS was found to decrease plasma lactate dehydrogenase (LDH) and malondialdehyde (MDA) production and increase plasma superoxide dismutase (SOD) production. Furthermore, 1H-NMR metabonomic analysis indicated that TCNDS could regulate the disturbed metabolites in the plasma, urine, and brain tissue of MCAO rats, and the possible mechanisms were involved oxidative stress, energy metabolism, lipid metabolism, amino acid metabolism, and inflammation. Correlation analysis were then performed to further confirm the metabolites involved in oxidative stress. Correlation analysis showed that six plasma metabolites had high correlations with plasma LDH, MDA, and SOD. This study provides evidence that an NMR-based metabonomics approach integrated with biochemical assessment can help to better understand the underlying mechanisms as well as the holistic effect of multiple compounds from TCM.

Hydroxysafflor yellow A inhibited lipopolysaccharide-induced non-small cell lung cancer cell proliferation, migration, and invasion by suppressing the PI3K/AKT/mTOR and ERK/MAPK signaling pathways.

BackgroundChronic inflammation plays a significant role in the occurrence and development of non‐small cell lung cancer (NSCLC). Hydroxysafflor yellow A (HSYA), a chemical compound of the yellow color pigments extracted from the safflower, has been widely used in clinical treatment with positive antioxidation, anti‐inflammation, and antitumor effects. However, the role and underlying mechanisms of HYSA on development and progress in inflammation‐mediated NSCLC are unknown.MethodsCell counting kit‐8, colony formation, EdU, cell apoptosis, wound healing, Transwell migration and invasion, and enzyme‐linked immunosorbent assays; flow cytometry; and Western blotting were conducted using human NSCLC cell lines A549 and H1299.ResultsLipopolysaccharide (LPS) significantly promoted the proliferation and enhanced colony formation of A549 and H1299 cells, while HYSA notably reversed the effects of LPS. HYSA induced apoptosis of LPS‐mediated A549 and H1299 cells in a dose dependent manner; and remarkably suppressed migration, invasion, and epithelial–mesenchymal transition (EMT), significantly regulated production of LPS‐induced inflammation cytokines, and downregulated protein expression of PI3K/Akt/mTOR and ERK/MAPK signaling pathways in LPS‐induced A549 and H1299 cells. Furthermore, PI3K (LY294002) and ERK (SCH772984) inhibitors remarkably inhibited proliferation, migration, invasion, and EMT, and induced apoptosis in LPS‐mediated A549 and H1299 cells. These effects were even more obvious in the presence of HYSA and LY294002 or SCH772984 compared to those of either agent alone.ConclusionHYSA suppressed LPS‐mediated proliferation, migration, invasion, and EMT in A549 and H1299 cells by inhibiting the PI3K/Akt/mTOR and ERK/MAPK signaling pathways, indicating that HYSA may be a potential candidate to treat inflammation‐mediated NSCLC.

Hydroxysafflor yellow A (HSYA) alleviates apoptosis and autophagy of neural stem cells induced by heat stress via p38 MAPK/MK2/Hsp27-78 signaling pathway

This study aimed to explore mechanisms of the effects of hydroxysafflor yellow A (HSYA) on neural stem cells (NSCs) after heat stress (HS). Rat NSCs cells were cultured at 42 °C to impose heat stress. Cell counting kit-8 and Edu assay were used to analyze NSC proliferation. Annexin V/PI apoptosis kit was used to detect NSC apoptosis. Expression and phosphorylation of autophagy and apoptosis-associated proteins were determined by western blotting. We showed that HSYA significantly promoted proliferation and attenuated apoptosis of NSCs after heat stress. HSYA also increased Bcl-2 expression but decreased the expression of Bax and cleaved caspase-3 in NSCs induced by heat stress. In addition, HSYA decreased p38 and Hsp27-78 phosphorylation and MK-2 expression after heat stress, which was consistent with NSCs treated with SB203850 treatment or p38 knockdown. Furthermore, we demonstrated that heat stress increased LC3-II expression and mTOR phosphorylation, and decreased the expression of p62 in NSCs, while HSYA, SB203850 treatment or p38 knockdown reversed these alterations. In conclusion, HSYA significantly reversed the apoptosis and autophagy of NSCs induced by heat stress (P < 0.05), via downregulating MK2 expression and p38 and Hsp27-78 phosphorylation.

Hydroxysafflor Yellow A Alleviates Ovalbumin-Induced Asthma in a Guinea Pig Model by Attenuateing the Expression of Inflammatory Cytokines and Signal Transduction.

Hydroxysafflor yellow A (HSYA) is an effective ingredient of the Chinese herb Carthamus tinctorius L. In this study, we aimed to evaluate the effects of HSYA on ovalbumin (OVA)-induced asthma in guinea pigs, and to elucidate the underlying mechanisms. We established a guinea pig asthma model by intraperitoneal injection and atomized administration OVA. Guinea pigs were injected intraperitoneally with HSYA (50, 75, 112.5 mg/kg) once daily from days 2 to 22 before OVA administration. We examined biomarkers including lung function, pulmonary histopathology, immunoglobulin E (IgE), Th1/Th2 relative inflammatory mediators, and related pathways. Pathological changes in lung tissues were detected by hematoxylin and eosin and periodic acid-Schiff staining. Phosphorylation levels of JNK mitogen-activated protein kinase (MAPK), p38 MAPK, ERK MAPK, and inhibitor of nuclear factor κBα (IκBα) were detected by western blot. plasma levels of total IgE, platelet-activating factor (PAF), and interleukin (IL)-3 were detected by enzyme-linked immunosorbent assay (ELISA). Expression levels of tumor necrosis factor (TNF)-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-13, and interferon (IFN)-γ were detected by ELISA and real-time quantitative polymerase chain reaction. HSYA significantly reduced airway resistance, improved dynamic lung compliance, and attenuated the pathologic changes. HSYA also inhibited the phosphorylation of JNK MAPK, p38 MAPK, ERK MAPK, and IκBα, and inhibited the OVA-induced elevations of IgE, PAF, IL-1β, IL-6, IL-4, IL-5, and IL-13 and the decreases in TNF-α, IFN-γ, IL-2, and IL-3. These findings suggest that HSYA has a protective effect on OVA-induced asthma through inhibiting the Th1/Th2 cell imbalance and inhibiting activation of the MAPK signaling pathway.

CtACO1 Overexpression Resulted in the Alteration of the Flavonoids Profile of Safflower.

Background: Flavonoids with various structures play a vital role in plant acclimatization to varying environments as well as in plant growth, development, and reproduction. Exogenous applications of ethylene and 1-aminocyclopropane carboxylic acid (ACC), could affect the accumulation of flavonoids. Very few attempts have been made to investigate the effect of 1-aminocyclopropane carboxylic acid oxidase (ACO), a unique enzyme that catalyzes ACC to ethylene, on genes and metabolites in the flavonoid biosynthetic pathway. In this study, two ACOs in safflower (CtACOs) were cloned, and then transgenic safflower with overexpressed CtACO1 was generated through the Agrobacterium-mediated floral dipping method. Results: CtACO1 and CtACO2 were both characterized by the 2-oxoglutarate binding domain RxS and the ferrous iron binding site HxDxnH as ACOs from other plants. However, the transcript levels of CtACO1 in flowers at stages I, II, III, and IV were all higher than those of CtACO2. At the cellular level, by using electroporation transformation, CtACO1 was found to be localized at the cytomembrane in onion epidermal cells. CtACO1 overexpression had varying effects on genes involved in the ethylene and flavonoid biosynthetic pathways. The metabolites analysis showed that CtACO1 overexpression lines had a higher accumulation of quercetin and its glycosylated derivatives (quercetin 3-β-d-glucoside and rutin). In contrast, the accumulation of quinochalcones (hydroxysafflor yellow A and carthamin), kaempferol glycosylated derivatives (kaempferol-3-O-β-rutinoside and kaempferol-3-O-β-d-glucoside), apigenin, and luteolin in CtACO1 overexpression lines were decreased. Conclusion: This study confirmed the feasibility of applying the floral dipping method to safflower and showed a novel regulatory effect of CtACO1 in the flavonoid biosynthetic pathway. It provides hypothetical and practical groundwork for further research on regulating the overall metabolic flux of flavonoids in safflower, particularly hydroxysafflor yellow A and other quinochalcones, by using appropriate genetic engineering strategies.

Proteomic Analysis of Hydroxysafflor Yellow A Against Cerebral Ischemia/Reperfusion Injury in Rats.

Hydroxysafflor yellow A (HSYA), an active component from Chinese medicinal herb, has been applied to the prevention and treatment of cerebral ischemia/reperfusion injury (CIRI). To clarify the comprehensive mechanisms HSYA for stroke, we used label-free quantitative proteomic analysis to investigate the modulated proteins of rats subjected to CIRI and their alteration by HSYA. Neurological examination, infarct assessment, and biochemical assay were performed to validate the effects of HSYA, and the results indicated that HSYA played a significant role in brain protection. A total of 13 proteins were identified as overlapped proteins by label-free quantitative proteomic analysis. Gene Ontology and pathway analysis showed that these differentially expressed proteins were mainly enriched in the hypoxia-inducible factor 1 (HIF-1) signaling pathway. Furthermore, networks were constructed with respect to protein function interactions. The results suggested that seven proteins were identified as hub proteins between model and sham groups, while 25 proteins were identified as hub proteins between HSYA and model groups. In addition, the expressions of three overlapping proteins were validated by Western blot, and their levels were consistent with the results of label-free analysis. In conclusion, Eftud2, mTOR, Rab11, Ppp2r5e, and HIF-1 signaling pathways have been detected as key hub proteins and pathways in HSYA against CIRI through proteomic analysis. Our research has provided convincing explanations for the mechanism of HSYA against CIRI and the identified key proteins and pathways might provide novel therapeutics for CIRI.

Hydroxysafflor yellow A attenuates high glucose-induced human umbilical vein endothelial cell dysfunction.

High glucose (HG) induces vascular injury in diabetes. Hydroxysafflor yellow A (HSYA) has been used to ameliorate ischemic cardiovascular diseases in China for many years. In the present study, we assessed whether HSYA has a potential protective role in HG-induced human umbilical vein endothelial cell (HUVEC) injury. Cell viability was determined with an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Cell apoptosis was detected by fluorescein isothiocyanate/propidium iodide staining assay. The endothelial cell permeability was measured with a permeability assay. Cell adhesion molecule (CAM) expression, vascular endothelial growth factor, and basic fibroblast growth factor levels were detected with an enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) formation was measured with a DCF-DA assay. Protein expression of NADPH oxidase 4 (NOX4) was measured by Western blotting. Our data indicated that HG increases HUVEC apoptosis, vascular permeability, monocyte adhesion, the level of CAMs, the formation of ROS, and NOX4 expression. Our data revealed that HG increases vascular injury, which is attenuated by HSYA. Because vascular inflammation has a key role in the development of diabetes mellitus, our results implied that HSYA is considered as a potential agent for diabetic vascular injury treatment.

Neuroprotection of hydroxysafflor yellow A in experimental cerebral ischemia/reperfusion injury via metabolic inhibition of phenylalanine and mitochondrial biogenesis

Stroke is the second most frequent cause of mortality, resulting in a huge societal burden worldwide. Timely reperfusion is the most effective therapy; however, it is difficult to prevent ischemia/reperfusion (I/R) injury. In traditional Chinese medicine, hydroxysafflor yellow A (HSYA) has been widely used for the treatment of cerebrovascular disease and as a protective therapy against I/R injury. Evidence has demonstrated that HSYA could reduce the levels of reactive oxygen species and suppress cellular apoptosis; however, whether HSYA alters the metabolic profile as its underlying mechanism for neuroprotection remains unknown. In the present study, using a metabolomic screening, phenylalanine was identified to significantly increase in an experimental model of mouse cerebral I/R injury. Notably, western blotting and qPCR analysis were conducted to test the expression level of apoptosis-associated factors, and HSYA was identified to be able to protect neuronal cells by reducing phenylalanine level associated with I/R injury. Additionally, these findings were confirmed in primary mouse neurons and PC12 cells exposed to oxygen and glucose deprivation/reoxygenation (OGD/R) stress. Of note, HSYA was observed to regulate the mRNA expression of key metabolic enzymes, phenylalanine hydroxylase, tyrosine aminotransferase and aspartate aminotransferase, which are responsible for phenylalanine metabolism. Furthermore, by performing mitochondrial labeling and JC-1 fluorescence assay, HSYA was identified to promote mitochondrial function and biogenesis suppressed by OGD/R. The findings of the present study demonstrated that I/R injury could increase the levels of phenylalanine, and HSYA may inhibit phenylalanine synthesis to enhance mitochondrial function and biogenesis for neuroprotection. The present study proposed a novel metabolite biomarker for cerebral I/R injury and the evaluated the efficacy of HSYA as a potential therapeutic treatment I/R injury.

Application of multiple chemical and biological approaches for quality assessment of Carthamus tinctorius L. (safflower) by determining both the primary and secondary metabolites

BackgroundThe florets of Carthamus tinctorius L. (safflower) serve as the source of a reputable herbal medicine targeting gynecological diseases. Conventional investigations regarding the quality control of safflower, however, mainly focused on the secondary metabolites with primary metabolites ignored. PurposeTo holistically evaluate the quality difference of safflower samples collected from five different producing regions by multiple chemical and biological approaches with both the primary and secondary metabolites considered. MethodsA precursor ions list-triggered data-dependent MS2 approach was established by ultra-high performance liquid chromatography/Q-Orbitrap mass spectrometry (UHPLC/Q-Orbitrap MS) to comprehensively identify the secondary metabolites from safflower. Primary metabolites were identified by various 1D and 2D nuclear magnetic resonance (NMR) experiments. Similarity evaluation and quantitative assays of all the characterized primary metabolites and a quinochalcone C-glycoside (QCG) marker, hydroxysafflor yellow A (HSYA), were performed by quantitative 1H NMR (qNMR) using an external standard method. Multiple in vitro models with respect to the antioxidant, anti-platelet aggregation, and antioxidant stress injury effects, were assayed to determine the efficacy differences. ResultsTotally thirteen primary metabolites (including one nucleoside, two sugars, five organic alkali/acids, and five amino acids) and 135 secondary metabolites (97 QCGs and 38 flavonoids) could be identified or tentatively characterized from safflower. Good chemical consistency was observed between the commercial safflower samples and a standard safflower sample, with similarity varying in the range of 0.95‒0.99. The results from qNMR-oriented quantitative experiments (thirteen primary metabolites and HSYA) and biological assays indicated the quality of safflower samples from Xinjiang (XJ-2 and XJ-4), Hunan (HuN-1 and HuN-2), and Sichuan (SC), was comparable to the standard safflower sample. ConclusionThe integration of multiple chemical (using two analytical platforms, UHPLC/Q-Orbitrap MS and NMR) and biological (four in vitro models) approaches by determining both the primary and secondary metabolites demonstrated a powerful strategy that could facilitate the holistic quality evaluation of traditional Chinese medicine.

Hydroxysafflor yellow A (HSYA) targets the platelet-activating factor (PAF) receptor and inhibits human bronchial smooth muscle activation induced by PAF.

Hydroxysafflor yellow A (HSYA) is the main active ingredient of edible plant safflower. HSYA has demonstrated anti-inflammatory effects. The inflammatory response is the key mechanism responsible for asthma, and the pro-inflammatory platelet-activating factor (PAF) is known to play a role in the pathology of bronchial asthma. In this study, we stimulated human bronchial smooth muscle cells (HBSMCs) with PAF and examined the effects of HSYA on the resulting asthma-related process. PAF stimulation induced HBSMC activation, induced proliferation, increased expression of the pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor-α, and activated asthma-related signaling pathways. All these effects were significantly inhibited by treatment with HSYA (9, 27, 81 μmol L-1). The effects of HSYA were prevented by the addition of a PAF receptor (PAFR) antagonist or by PAFR gene silencing with small interfering RNA. These results suggest that HSYA may inhibit PAF-induced activation of HBSMCs by targeting the PAFR. Overall, these findings provide evidence that HSYA can be applied as a potential therapeutic agent in the treatment of bronchial asthma.

Protective Effect of Hydroxysafflor Yellow A on Inflammatory Injury in Chronic Obstructive Pulmonary Disease Rats.

To investigate the attenuating effect of Hydroxysafflor yellow A (HSYA) on inflammatory injury in chronic obstructive pulmonary disease (COPD). Rats were randomly assigned to 7 groups according to body weight including normal control group, HSYA blank group (76.8 mg/kg), COPD group, COPD+HSYA (30, 48, 76.8 mg/kg) groups and COPD+dexamethasone (2 mg/kg), 10 in each group. Passive cigarette smoke and intratracheal instillation of lipopolysaccharides were used to establish a COPD model in rats. Hematoxylin and eosin staining of lung tissue sections was used, real-time polymerase chain reaction (PCR) was used to assay mRNA levels of some cytokines in lung tissues, the cytokines in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA), Western blot analysis was used to determine phosphorylated p38 mitogen-activated protein kinase (MAPK) levels in lung tissues, and nuclear factor-κB (NF-κB) p65 protein levels in lung tissues were detected by immunohistochemistry. Lung alveolar septa destruction, alveolus fusion, inflammatory cell infiltration, and bronchiole exudation were observed. These pathological changes were alleviated in the COPD+HSYA group. The mRNA expression of inflammatory factors were significantly increased in lung tissues from COPD rats (all P<0.01) and were inhibited by HSYA. Levels of inflammatory cytokines in BALF of COPD rats were significantly increased (all P<0.01) which were inhibited by HSYA (all P<0.01, 48, 76.8 mg/kg). The levels of p38 MAPK phosphorylation and p65 in lung tissues of COPD rats were significantly increased (all P<0.01) and were suppressed by HSYA (all P<0.01, 48, 76.8 mg/kg). HSYA could alleviate inflammatory cell infiltration and other pathological changes in the lungs of COPD rats. HSYA inhibited inflammatory cytokine expression, and increase phosphorylation of p38 MAPK and NF-κB p65 in the lungs of COPD rats. The protective mechanism of HSYA to inhibit COPD inflammation might be by attenuating NF-κB and p38MAPK signal transduction.

Biological activity and hydroxy safflor yellow A content of intermediates in preparation of Safflower Injection

In order to investigate the effect of various production processes on the quality of Safflower Injection, the biological activities of the intermediates were evaluated by measuring activated partial thromboplastin time (APTT) and adenosine diphosphate (ADP) induced platelet aggregation in vitro. Intermediates were produced by key processes, such as extraction, concentration, twice alcohol precipitation, water sedimentation and two sterilizations during the production of Safflower Injection. The content of main chemical components in intermediates was determined by HPLC. The results showed that with the advance of the preparation process of Safflower Injection, the inhibition of ADP-induced platelet aggregation rate of each intermediate decreased gradually, and the trend of extending APTT activity decreased first and then increased. Meanwhile, the content of hydroxy safflor yellow A (HSYA) was gradually lowered, the content of p-hydroxy-cinnamic acid was increased, and new chemical component p-hydroxybenzaldehyde was produced. In conclusion, sterilization played a key role in the biological activity and HSYA content of Safflower injection.

Hydroxysafflor yellow A sensitizes ovarian cancer cells to chemotherapeutic agent by decreasing WSB1 expression

IntroductionCarthamus tinctorius L. (safflower) is a traditional Chinese medicine, the active ingredient of which is hydroxysafflor yellow A (HSYA).22HSYA: hydroxysafflor yellow A HSYA has been shown to have the potential to inhibit tumor growth. However, the molecular mechanisms whereby HSYA exerts its antitumor functions remain largely unclear. In this study, we investigated the antitumor mechanisms of HSYA in ovarian cancer cell line Skov3. MethodsThe cell proliferation assay was conducted using a Cell Proliferation Assay kit. The cell viability assay was performed using the CellTiter-Blue Cell Viability kit. Microarray was conducted to identify the global gene expression change of ovarian cancer cells caused by HSYA treatment. Small interfering RNA (SiRNA)33SiRNA: Small interfering RNA transfection was conducted to knock down WD repeat and SOCS box-containing protein 1 (WSB1)44WSB1: WD repeat and SOCS box-containing protein 1. WSB1 expression was detected by quantitative reverse transcription-quantitative polymerase chain reaction (qRT-PCR)55qRT-PCR: quantitative reverse transcription-quantitative polymerase chain reaction. The protein expression of extracellular signal-related kinase (Erk)66Erk: extracellular signal-related kinase1/2 and phosphorylation-Erk1/2 was detected by western blot. ResultsHSYA inhibited Skov3 cell proliferation in a dose-dependent manner (P < 0.05). When cells were cultured with HSYA and doxorubicin, cell viability was further reduced (P < 0.05). HSYA could decrease the expression of WSB1. Through knocking down of WSB1, ovarian cancer cell proliferation was inhibited and further reduced by treating with doxorubicin (P < 0.05), the expression of Erk1/2 and Erk phosphorylation were downregulated. ConclusionHSYA may inhibit ovarian cancer cell line Skov3 proliferation and sensitize Skov3 cells to chemotherapeutic agents through the reduction of WSB1 expression.

Comparative Study on Excretive Characterization of Main Components in Herb Pair Notoginseng-Safflower and Single Herbs by LC⁻MS/MS.

The herbal medicine combination of notoginseng-safflower has been commonly used clinically for the prevention and treatment of cardiovascular diseases. A reliable liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of six bioactive components (hydroxysafflor yellow A, notoginsenoide R1, ginsenoside Rb1, Re, Rd, and Rg1) in rat urine and feces after oral administration of notoginseng total saponins (NS), safflower total flavonoids (SF), and the combination of NS and SF (CNS). The chromatographic separation was achieved on a Waters HSS T3 column under gradient elution with acetonitrile and water containing formic acid as the mobile phase. The calibration curves were linear, with correlation coefficient (r) > 0.99 for six components. The intra- and interday precision (RSD) and accuracy (RE) of QC samples were within −14.9% and 14.9%, respectively. The method was successfully applied to study of the urinary and fecal excretion of six bioactive constituents following oral administration of NS, SF, and CNS in rats. Compared to the single herb, the cumulative excretion ratios of six constituents were decreased in the herbal combination. The study indicated that the combination of notoginseng and safflower could reduce the renal and fecal excretion of the major bioactive constituents and promote their absorption in rats.