Quantitative estimation of Carthamin and Carthamidin from the Florets C. tinctorius L., (Safflower Florets)

Natural colorants derived fom plant materials have gained increasing popularity due to their non toxic nature. pigment extraction from the florets is normally done by Soxhlet extraction, maceration, and hydro distillation are conventional methods that have been widely used in industry and laboratory .phytochemical analysis of safflower florets revealed the plant presence of high amount of Carthamin and carthamidin.

Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius / 中草药·英文版

OBJECTIVE@#Flavonoids are the bioactive compounds in safflower (Carthamus tinctorius), in which chalcone synthase (CHS) is the first limiting enzyme. However, it is unclear that which chalcone synthase genes (CHSs) are participated in flavonoids biosynthesis in C. tinctorius. In this study, the CHSs in the molecular characterization and enzyme activities were investigated.@*METHODS@#Putative chalcone biosynthase genes were screened by the full-length transcriptome sequences data in C. tinctorius. Chalcone biosynthase genes in C. tinctorius (CtCHSs) were cloned from cDNA of flowers of C. tinctorius. The cloned gene sequences were analyzed by bioinformatics, and their expression patterns were analyzed by real-time PCR (RT-PCR). The protein of CtCHS in the development of flowers was detected by polyclonal antibody Western blot. A recombinant vector of CtCHS was constructed. The CtCHS recombinant protein was induced and purified to detect the enzyme reaction (catalyzing the reaction of p-coumaryl-CoA and malonyl-CoA to produce naringin chalcone). The reaction product was detected by HPLC and LC-MS.@*RESULTS@#Two full-length CtCHS genes were successfully cloned from the flowers of safflower (CtCHS1 and CtCHS3), with gene lengths of 1525 bp and 1358 bp, respectively. RT-PCR analysis showed that both genes were highly expressed in the flowers, but the expression of CtCHS1 was higher than that of CtCHS3 at each developmental stage of the flowers. WB analysis showed that only CtCHS1 protein could be detected at each developmental stage of the flowers. HPLC and LC-MS analyses showed that CtCHS1 could catalyze the conversion of p-coumaryl-CoA and malonyl-CoA substrates to naringin chalcone.@*CONCLUSION@#CtCHS1 is involved in the biosynthesis of naringin chalcone in safflower.

Characterization and function of short-chain dehydrogenases/reductases in hydroxysafflower yellow A biosynthesis pathway / 药学实践杂志

Objective To explore the function of short-chain dehydrogenases/reductases (SDRs) in safflower flavonoid, especially hydroxysafflower yellow A (HSYA) biosynthesis. Methods SDRs involved in HSYA biosynthesis pathway were screened based on safflower transcriptome database and metabolome database. The expression pattern was analyzed by qRT-PCR. The overexpression vector was constructed by seamless cloning technology, then genetically transformed to the Yunnan Weishan safflower strain by Agrobacterium gv3101. The transgenic T2 generation plants were positively verified, and the gene expression of corolla SDRs was analyzed. The content of secondary metabolites was assayed by UPLC-Q-TOF/MS. Results Three SDRs genes named CtSDR1, CtSDR2 and CtSDR3 involved in HSYA biosynthesis pathway were screened. Their expression in safflower from high to low was corolla > leaf > stem > root. The expression level in corolla increased gradually with corolla development. qRT-PCR analysis of corolla with positive verification of genome insertion sequence showed that the transcription level of CtSDR3 in corolla of T2 positive plants increased by 2~3 times compared with the blank control group, and the content of secondary metabolite HSYA increased by 7.1%~16.6% (P< 0.05). Conclusion CtSDR3 may be involved in the biosynthesis of flavonoids, especially HSYA, in safflower. It provides the support data for explaining the function of CtSDR3 in HSYA biosynthesis pathway.

Quantitative analysis of effects of salvianic acid a combined with hydroxy safflower yellow a on rat endothelial cells after hypoxic injury using the combination index method

Abstract Cerebrovascular disease is the second most serious disease in the world. It has the features of high morbidity, high mortality and recurrence rate. Numerous research on the compatibility of Chinese medicine with effective ingredients of cerebral ischemia has been made during the past decades. The purpose of this study is to quantitatively analyze the combined pharmacological effect of effective ingredients in Danshen and Honghua (Dan Hong) on rat microvascular endothelial cells after gradually oxygen-glucose deprivation. The experimental concentration range for the compatibility of two effective ingredients were determined in the preliminary experiments by Cell Counting kit-8 (CCK-8) method. Drugs were added to rat brain microvascular endothelial cells at a non-toxic dose level. After that, the cells were cultured for 12 h, and placed in a hypoxic environment. Finally, the cell survival rate was used as a measure of drug effect. In order to determine synergism or antagonism, the combination index (CI)-isobologram method was performed to analyze the data from the experiments. Based on this theory, the potencies of each drug and the shapes of their does-effect curves are both taken into account. The results show that the synergism or the antagonism between two effective ingredients compatibility change with different proportion and dosage. Furthermore, it can be seen from the results of these experiments that when these drugs are used in combination, the dosage required to achieve the same therapeutic effects is greatly reduced compared with the case of single one. It is worth mentioning that our experiments also prove that the median-effect equation and the CI method can be applied in the field of traditional Chinese medicine.

Effects of components in stasis-resolving and collateral-dredging Chinese herbal medicines on angiogenesis and inflammatory response of human umbilical vein endothelial cells induced by VEGF / 中国中药杂志

The present study investigated the mechanism of components in stasis-resolving and collateral-dredging Chinese herbal medicines, including scutellarin(Scu), paeonol(Pae), and hydroxy safflower yellow A(HSYA), in the treatment of psoriasis by regulating angiogenesis and inflammation. The human umbilical vein endothelial cells(HUVECs) cultured in vitro were divided into a normal group, a model group, a VEGFR tyrosine kinase inhibitor Ⅱ(VRI) group, and Scu, Pae, and HSYA groups with low, me-dium, and high doses. Cell viability was detected by the CCK-8 assay. Cell migration was detected by wound healing assay. Tube formation assay was used to measure the tube formation ability. Western blot was used to detect the protein expression of the VEGFR2/Akt/ERK1/2 signaling pathway. The secretion levels of inflammatory cytokines IFN-γ, IL-1β, IL-6, and TNF-α were detected by ELISA. The results showed that compared with the model group, all the Scu, Pae, and HSYA groups could reduce cell viability, inhibit cell migration and tube formation(P<0.05, P<0.01), and down-regulated the protein expression of VEGFR2, p-VEGFR2, Akt, p-Akt, ERK1/2, and p-ERK1/2. Scu and Pae could down-regulate VEGFR2 expression(P<0.05, P<0.01), while other groups only showed a downward trend. Scu and Pae significantly reduced IFN-γ and IL-6 levels(P<0.01), and HSYA significantly reduced the levels of IFN-γ, IL-1β, and IL-6(P<0.01). Scu, Pae, and HSYA had no significant effect on TNF-α. The results suggested that Scu, Pae, and HSYA may exert a therapeutic role in psoriasis-related angiogenesis and inflammation by inhibiting VEGFR2/Akt/ERK1/2 signaling pathway and inhibiting the secretion of IFN-γ, IL-1β, and IL-6.

Method validation of hydroxysafflor yellow A in “Carthamus tinctorius” injection by High-performance liquid chromatography / Монголын Анагаах Ухаан

Introduction@#Carthamus tinctorius L. widely accepted as Safflower or false saffron, belongs to the Compositae or Asteraceae family. Hydroxysafflor yellow A is the main active chemical compound present in florets of Carthamus tinctorius L. A joint research team of the “Tsombo Pharm” Co., LTD and the Drug research Institute is conducting an experiment to produce a solution of “Carthamus tinctorius” injection prepared by Carthamus tinctorius L. @*Goal @#The aim of this study was to develop the validation method of hydroxysafflor yellow A in “Carthamus tinctorius” injection.@*Material and Methods @#As a test sample “Carthamus tinctorius” injection was produced by “Tsombo pharma” Co., LTD. The standard Hydroxysafflor yellow A was supplied from Sigma-Aldrich Co., Ltd. The reagent were high-performance liquid chromatography grade acetonitrile, phosphoric acid, methanol and purified water. </br> Shimadzu HPLC (CMB-20 A, UV detector Shimadzu SPD-20A was used as the analytical instrument and the analysis conditions were as follows Table 1. @*Results@#A Shimpack С18 column was used with methanol:acetonitrile:0.7% phosphoric acid as the mobile phase under the condition of gradient elution. The hydroxysafflor yellow A were analyzed by using a timed wavelength measure according to their maximum absorption wavelength. Accuracy and precision were assessed by analyzing five sets of samples, independently prepared at low (50%) middle (100%) and high (150%) concentrations. The intraday and interday precisions of the investigated compound were less than 1.59 % and the average recoveries ranged from 81.9% to 101.5%. </br> There were good linear correlations between the concentrations of the hydroxysafflor yellow A and its chromatographic peak areas (R2 = 0.998), the proposed method was successfully applied to determine the hydroxysafflor yellow A in “Carthamus tinctorius” injection. @*Conclusions@#The results indicated that the proposed method is simple, stable, and accurate and could be readily utilized as a quality control method for manufacturing process of “Carthamus tinctorius” injection.

Tissue culture of safflower and analysis of secondary metabolites in suspension cells / 中国中药杂志

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.

Improvement Effects of Safflower Yellow on Lung Function of Chronic Obstructive Pulmonary Disease Model Rats / 中国药房

OBJECTIVE：To study the improvement effects of saf flower yellow on lung function in chronic obstructive pulmonary disease （COPD）model rats. METHODS ：SD rats were randomly divided into control group ，model group ，positive control group （dexamethasone，0.09 mg/kg），safflower yellow low-dose ，medium-dose and high-dose groups （5，10，20 mg/kg）， with 10 rats in control group and 11 rats in other groups. Except for control group ，other groups were given lipopolysaccharide combined with fumigation to induce COPD model. After modeling ，control group and model group were given constant volume of normal saline intragastrically ，and administration groups were given relevant medicine intragastrically ，once a day ，for consecutive 12 weeks. After last medication ，the levels of TNF-α，IL-6 and IL- 8 were detected by ELISA. The levels of blood gas indexes （PaO2，SaO2 and PaCO 2）in whole blood were detected by blood gas analyzer. The levels of lung function indexes （FVC，FEV1， FEV1/FVC，PEF and MMEF ）were detected by lung function analyzer. The expression of TLR 4，NF-κB and I κB-α protein were detected by Western blot. HE staining was used to observe the pathological changes of lung tissue. RESULTS ：Compared with control group ，the serum levels of TNF-α，IL-6 and IL- 8，the level of PaCO 2 in whole blood as well as the protein expression of TLR4 and NF-κB in lung tissue were increased significantly in model group（P＜0.01）；the levels of PaO 2 and SaO 2 in whole blood，the levels of lung function as FVC ，FEV1，FEV1/ FVC，PEF and MMEF as well as the protein expression of IκBα in lung tissue were decreased significantly（P＜0.01）； there were obvious degeneration and necrosis in the epithelial cells of lung tissue ，and obvious inflammatory infiltration in the interstitial cells. Compared with model group ，the levels o f inflammatory factors in serum ，blood gas indexes in whole blood and lung function indexes as well as the expression of related protein in lung tissue （except for IκBα in low-dose group）were reversed significantly in safflower yellow groups （P＜0.05 or P＜ 0.01）；the necrosis ，exfoliation and inflammatory infiltration of epithelial cells in lung tissue were improved in varying degrees. CONCLUSIONS：Safflower yellow can significantly improve the lung function of COPD model rats ，and its mechanism may be related to inhibiting the expression of inflammatory factors and regulating the expression of TLR 4/NF-κB pathway-related proteins.

Screening of type II 5α-reductase inhibitors from traditional Chinese medicine based on molecular docking and molecular dynamics simulation technology / 中草药

Objective: To screen the potential type II 5α-reductase inhibitors from active ingredients of traditional Chinese medicine (TCM) based on molecular docking and molecular dynamics (MD) simulation technology. Methods: The molecular docking was used to screen 26 monomer compositions from TCM. Based on the docking results, MD was performed to evaluate the binding strength of compounds with protein. The binding free energy of the system was calculated using the MM/PBSA method. The in vitro micro-reaction system was used to investigate biological activity. Results: The binding energies of 26 monomer compositions from TCM to type II 5-alpha Reductase were different. Among them, ligustroflavone, safflower yellow and hinokiflavone have low binding energies to type II 5-alpha reductase, and their binding abilities were strong. The molecular dynamics simulation results are consistent with the docking results (binding capacity: ligustroflavone-protein > safflower yellow-protein > hinokiflavone-protein). The three components ligustroflavone, safflower yellow and hinokiflavone have a certain inhibitory activity on type II 5α-reductase with the IC50 value of (42.12 ± 3.83), (69.06 ± 6.35), and (191.28 ± 5.90) μmol/L, respectively. Conclusion: Among the screened 26 monomer compositions, ligustroflavone, safflower yellow and hinokiflavone have the potential to be used in the study of treatment and prevention of androgen-dependent diseases, which provides a reference for further exploration and discovery of type II 5α-reductase inhibitors.

Bioinformatics analysis of safflower WD40 transcription factor family genes / 中国中药杂志

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.

Occurrence regularity and integrated control of leaf miner in safflower / 中国中药杂志

Leaf miner is one of the major pests on safflower, which causes yield loss and poor quality seriously. "Weihonghua", "nine safflower varieties" and "three chemical insecticides" 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.

Virtual screening of components of safflower against myocardial ischemia based on molecular docking technology / 中国中药杂志

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.

The mechanism of methyl jasmonate-induced accumulation of hydroxysafflor yellow A in safflower of different colors / 药学学报

To explore the mechanism hydroxysafflor yellow A (HSYA) biosynthesis and regulation, the effect of methyl jasmonate (MeJA) treatment on gene expression related to the biosynthesis of hydroxysafflor yellow A (HSYA) was analyzed, and expression differences in genes involved in HSYA biosynthesis in safflower of different colors was quantified. MeJA at concentrations of 0, 50, 100, and 200 μmol·L-1 was sprayed onto safflower florets to determine the optimal concentration of MeJA. Safflower was treated with 100 μmol·L-1 MeJA and florets were harvested 0, 3, 6, 12 and 24 h after treatment. The content of MeJA was determined by high performance liquid chromatography (HPLC). RNA was extracted from safflower florets treated with 100 μmol·L-1 MeJA for 6 h. The transcription of key genes involved in the biosynthesis of HSYA was quantified by qRT-PCR and differentially expressed genes were identified. The content of HSYA increased after treatment with MeJA, with 100 μmol·L-1 MeJA treatment for 6 h having the greatest effect on HSYA accumulation. qRT-PCR results showed that MeJA could significantly increase the transcription of HSYA biosynthesis genes including PAL2, PAL4, 4CL2, 4CL4, 4CL5, CHS3, CHS4 and CHI2. The content of HSYA differed between safflowers of different colors with a trend of red>orange-yellow>yellow>white. The results of qRT-PCR showed that the expression of CHS1 and CHI2 in red, orange and yellow safflower was significantly higher than that in white safflower. These results indicate that MeJA promotes the accumulation of HSYA by up-regulating the expression of genes involved in the biosynthesis of HSYA such as PAL2, PAL4, 4CL2, 4CL4, 4CL5, CHS3, CHS4 and CHI2, and the variation of HSYA content in safflower of different colors was related to a difference in the level of expression of CHS1 and CHI2.

Determination of hydroxylsafflower yellow A in safflower W/O cream by RP-HPLC / 中国药科大学学报

@#A reversed phase HPLC method for determination of hydroxylsafflower yellow A in safflower W/O cream was established. The column was Zorbax Eclipse C18 column(4. 6 mm×250 mm, 5 μm), and the mobile phase was composed of methanol, acetonitrile and 0. 02% phosphoric acid solution(26 ∶2〓 ∶72). The flow rate of mobile phase was set at 1. 0 mL/min, and the column temperature was kept at 55 °C. The detection wavelength was 403 nm. Safflower W/O cream was successively demulsified with methanol at high temperature and followed by the addition of purified water for the extraction. The results showed that the excipients did not interfere with the chromatographic peak of hydroxylsafflower yellow A. Hydroxylsafflower yellow A presented a good linear relationship in the range of 1. 236- 12. 36 μg/mL(y=156. 17x+1. 198 3, r=0. 999 5), and the detection limit was 23. 6 ng/mL with the quantitative limit of 118 ng/mL. The percentage of extracting recovery was in the range of 99. 7% to 103. 3%. The precision RSD was 0. 12%(n=6), and the sample stability was acceptable when being stored at room temperature for 24 h. The developed method in this study was simple, rapid, accurate and reproducible, and can be used for the determination of hydroxylsafflower yellow A in safflower W/O cream.

The characteristics and functions of CtXTH1: the gene booststhe corolla elongation in safflower / 药学学报

In this study, 13 xyloglucan endotransglycosylases/hydrolases (XTHs) and 8 expansin (EXPs) were screened from safflower floret transcriptome database. Through correlation analysis between the safflower gene expression profile chip and the corolla development, only 4 XTHs (CtXTH1-4) and 1 EXP (CtEXP1) have positive relevance with corolla elongation (r≥0.60) and were therefore validated by qRT-PCR. The full length of these genes were cloned by RACE. According to the bioinformatic analysis, CtXTH1 correlated with the development of the floret, and the expression pattern analysis indicated that CtXTH1 had accumulated in the floret. The recombinant vector (pMT39-CtXTH1) was constructed for gene transformation. Overexpression of CtXTH1 significantly increased the corolla length (about 5.34% to 10.25%) and corolla weight (about 30.00% to 36.02%) in transgenic safflower. The overexpression lines also showed an increasing tendency in the weight of seeds, average number of corollas per cone and average number of seeds in each cone. Meanwhile, overexpression of CtXTH1 had no significant effect on flavonoids. According to the corolla microstructure, the OVX-line tubular part of floret exhibited a looser and irregular character. These data suggested that CtXTH1 can potentially increase relaxation of the tissues and boost corolla elongation. Our study provides a valuable clue for plant breeding in the future.

Determination of common dyes in dyed safflower by near infrared spectroscopy / 中国中药杂志

Because the red and bright color of corolla is the main indicator for the quality assessment of good safflower,the dyed safflower is sometimes found at the herbal market,what is influence on this herb quality and efficacy. A total of 127 safflower samples was therefore collected from different cultivating areas and herbal markets in China to develop a rapid method to identify the dyed safflower. Near-infrared spectroscopy(NIRS) combined with characteristic identification,high performance liquid chromatography(HPLC),principal component analysis(PCA) and partial least squares regression analysis(PLS) were employed to differentiate safflower from dyed safflower samples,and further quantify the levels of the 6 dyes,i.e. tartrazine,carmine,sunset yellow,azorubine,acid red 73 and orange Ⅱ in the dyed safflower. The results indicated that the 50 safflower samples and 77 dyed safflower samples were located at different regions in PCA cluster diagram by NIR spectra. Tartrazine,carmineand and sunset yellow were found in the 77 dyed safflower samples with the amounts of 0. 60-3. 66,0. 11-1. 37,0. 10-0. 71 mg·g-1,respectively. It indicated that the three dyes were the common and main dyes in the dyed safflower. However,azorubine,acid red 73 and orange Ⅱ were not detected in all herb samples. A total of 62 dyed safflower samples were chosen as calibration samples to develop the model for estimating the amount of dyes in dyed safflower. The estimating accuracy was verified by another 15 dyed safflower samples. The values of tartrazine,carmine and sunset yellow in dyed safflower samples were compared between the NIRS and HPLC methods. Each value of mean absolute difference(MAD) was less than 5%. The correlation coefficients of tartrazine,carmineand and sunset yellow were 0. 970,0. 975,0. 971,respectively. It indicated the data quantified by NIRS and HPLC were consistence. It is concluded that NIRS can not only differentiate safflower from dyed safflower,but also quantify the amount of the dyes. NIRS is suitable for rapidly identify the quality of safflower.

Study on safflower yellow for injection based on cell degranulation and acute anaphylactoid model / 中国中药杂志

This paper was aimed to establish screening methods of anaphylactoid reaction caused by safflower yellow for injection based on RBL-2 H3 cell degranulation model and mice model for acute anaphylactoid reaction,and evaluate the hypersensitivity caused by safflower yellow for injection from different batches. An in vitro cell model was used to keep the cells stimulated for an hour with different batches of safflower yellow for injection as the drug group,serum-free MEM medium as negative control group and 30 mg·L-1 C48/80 as positive control group respectively. The supernatant was then absorbed,and neutral red staining technique was used to detect the effect of safflower yellow injection on the degranulation of RBL-2 H3 cells with the positive cell rate of degranulation as the indicator.An in vivo model was established to validate the experimental results,and mice model for acute anaphylactoid reaction and ELISA method were adopted to detect the plasma histamine content,and screen the hypersensitivity caused by safflower yellow for injection at the animal level by using plasma histamine content as a test index. The results of the neutral red staining experiments showed that the positive control C48/80 could cause cell degranulation,and most of the cells were deeply stained. There was significant difference in positive cell rate between different batches of safflower yellow and positive control group. In the mice model for acute anaphylactoid reaction,it was found that the positive control C48/80 significantly increased the histamine content in the plasma of mice,while the safflower yellow in each batch did not cause a significant increase in plasma histamine( P<0. 000 1). The mechanism of anaphylactoid reaction is relatively complicated. This study was mainly based on the release of histamine and other active substances by degranulation of mast cells. No significant degranulation reaction of RBL-2 H3 cells induced by safflower yellow for injection was detected,nor was the plasma histamine level significantly increased in mice from the in vitro and in vivo aspects.

Protection of diabetic retinal ganglion cells by safflower yellow pigment regulated p38MAPK signaling pathway / 中草药

Objective: To investigate the protective effect of safflower yellow pigment (SYP) on diabetic (DM) retinal ganglion cells (RGC) by regulating p38 mitogen-activated protein kinase (MAPK) signaling pathway. Methods: Selected 50 rats and 40 of them were used to establish DM rat model by single intraperitoneal injection of streptozotocin. They were randomly divided into model group and safflower yellow pigment low, medium and high dose groups, and the remaining 10 rats was control group. Safflower yellow pigment low, medium and high dose groups were intraperitoneally injected with 20, 40, and 80 mg/kg of SYP. Model group and control group were intraperitoneally injected with the same amount of physiological saline solution for six weeks. The general state of the rats was observed, and the morphological changes of RGC were observed by hematoxylin-eosin (HE) staining, and the number of RGC cells was counted. RGC apoptosis rate was detected by in situ apoptosis (TUNEL) method. Real-time quantitative polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expressions of p38 mitogen-activated protein kinase (MAPK), phosphorylated p38MAPK (p-p38MAPK) and cysteine-containing aspartate proteolytic enzyme-3 (Caspasae-3), vascular endothelial growth factor polyclonal antibody (VEGF), and protein expression ratio of p-p38/p38MAPK was compared. Results: All the rats survived during the experiment. There were no abnormalities in the rats in the control group. The blood glucose in the model group was higher, the diet and urine volume were larger, and the body weight was reduced. The safflower yellow pigment low, medium and high dose groups were better than the model group. Pathological observation showed that there were no abnormalities in the cells of the retina of the control group, while the RGC cells in the model group were disordered arranged and the nucleus were sparse, and the number of cells in the bipolar cell layer and the photoreceptor layer was reduced, and the arrangement was sparse. The abnormalities of RGC cells and outer bipolar cells in the safflower yellow pigment low, medium and high dose groups were significantly lower than those in the model group, and the high dose groups was the most obvious. There was significant difference in the number of RGC between groups (P < 0.05). Compared with the control group, the number of RGC in the model group was significantly decreased (P < 0.05), the apoptotic rate was significantly increased (P < 0.05), the levels of Caspase-3 and VEGF in the retina were significantly increased (P < 0.05), and the values of p-p38MAPK/p38MAPK were significantly increased (P < 0.05). Compared with the model group, the number of RGC was increased significantly (P < 0.05), the apoptotic rate of RGC was decreased significantly (P < 0.05), the expression levels of Caspase-3, VEGF and protein in retina were decreased significantly (P < 0.05), and the value of p-p38MAPK/p38MAPK was decreased significantly (P < 0.05), and the differences among the dose groups were significant (P < 0.05). Conclusion: Safflower yellow pigment can protect RGC of DM rats by inhibiting p38MAPK signaling pathway, and reduce RGC apoptosis. The 80 mg/kg of SYP has the best protective effect.

Preparation and quality evaluation of hydroxysafflor yellow A solid lipid nanoparticles / 国际中医中药杂志

Objective@#To optimize the formulation and preparation technology of hydroxy safflower yellow A solid lipid nanoparticles and evaluate its quality.@*Metheds@#Hydroxy safflower yellow A solid lipid nanoparticles were prepared by the method of hot melt emulsification ultrasonic-low temperature using hydroxypropyl methylcellulose as a lipid material and glyceryl monostearate as an emulsifier. Using entrapment efficiency as indexes, the amount of hydroxypropyl methylcellulose, purified water, glyceryl monostearate, and Tween 80 aqueous solution (1%) as factors, orthogonal test was applied to optimize the formulation and preparation technology. Dialysis method was used to measure encapsulation efficiency. The morphology and uniformity of the nanoparticles were observed by transmission scanning electron microscopy. The particle size, polydispersion index and Zeta potential were determined by nano-particle size analyzer. And hydroxy safflower yellow A solid lipid nanoparticles sustained releasing characteristics was evaluated by the percentage of cumulative drug release.@*Results@#The optimal process of prepared hydroxy safflower yellow A solid lipid nanoparticles was 5 mg of hydroxypropyl methylcellulose, 0.2 ml of purified water, 100 mg of glyceryl monostearate, and 1.8 ml of Tween 80 aqueous solution (1%). The size of the prepared nanoparticles was uniform and spherical. And the average particle size were (99.85 ± 3.04) nm, polydispersion index were (0.390 ± 0.021), Zeta potential were (-27.63 ± 2.12) mV, and the encapsulation efficiency of the hydroxy safflower yellow A solid lipid nanoparticles were (63.35 ± 2.65)%. The release rate of the nanoparticles was (44.35 ± 0.49)%.@*Conclusions@#The prepared hydroxy safflower yellow A solid lipid nanoparticles have good uniformity and good sustained release properties.

Preparation and quality evaluation of hydroxysafflor yellow A solid lipid nanoparticles / 国际中医中药杂志

Objective To optimize the formulation and preparation technology of hydroxy safflower yellow A solid lipid nanoparticles and evaluate its quality. Metheds Hydroxy safflower yellow A solid lipid nanoparticles were prepared by the method of hot melt emulsification ultrasonic-low temperature using hydroxypropyl methylcellulose as a lipid material and glyceryl monostearate as an emulsifier. Using entrapment efficiency as indexes, the amount of hydroxypropyl methylcellulose, purified water, glyceryl monostearate, and Tween 80 aqueous solution (1%) as factors, orthogonal test was applied to optimize the formulation and preparation technology. Dialysis method was used to measure encapsulation efficiency. The morphology and uniformity of the nanoparticles were observed by transmission scanning electron microscopy. The particle size, polydispersion index and Zeta potential were determined by nano-particle size analyzer. And hydroxy safflower yellow A solid lipid nanoparticles sustained releasing characteristics was evaluated by the percentage of cumulative drug release. Results The optimal process of prepared hydroxy safflower yellow A solid lipid nanoparticles was 5 mg of hydroxypropyl methylcellulose, 0.2 ml of purified water, 100 mg of glyceryl monostearate, and 1.8 ml of Tween 80 aqueous solution (1%). The size of the prepared nanoparticles was uniform and spherical. And the average particle size were (99.85 ± 3.04) nm, polydispersion index were (0.390 ± 0.021), Zeta potential were (-27.63 ± 2.12) mV, and the encapsulation efficiency of the hydroxy safflower yellow A solid lipid nanoparticles were (63.35 ± 2.65)%. The release rate of the nanoparticles was (44.35 ± 0.49)%. Conclusions The prepared hydroxy safflower yellow A solid lipid nanoparticles have good uniformity and good sustained release properties.