红花(Carthamus tinctorius L.)种子醇溶蛋白遗传多样性分析

利用A—PAGE（Acid—polyacrylamide gel electrophoresis）技术对来源于32个国家的53份红花材料进行了种子醇溶蛋白检测。结果表明，这些红花材料具有丰富的醇溶蛋白等位变异，共分离出15条迁移率不同的谱带。其中，每份材料可电泳出5-12条谱带，平均8．5条，所有材料有1条共有带。红花种子醇溶蛋白可作为评价红花遗传多样性的工具之一。不同材料的遗传相似系数（GS）变异范围为0．375—1．000，平均值为0．752。聚类分析结果表明，在GS值为0．752的水平上供试材料聚为6大类，其亲缘关系远近与地理来源关系不大。其中，来自中国的7份材料分别被聚在了3个大类中，表明中国红花种子醇溶蛋白遗传多样性比较丰富。

SRAP Analysis on Genetic Relationship of Carthamus tinctorius L Varieties in Xinjiang

[Objective] This study aimed to analyze the genetic relationship of the Carthamus tinctorius L（safflower） varieties in Xinjiang, and thus to provide a scientific basis for its protection and development. [Method] The genomic DNA of five safflower varieties from Xinjiang and one from Yunnan were analyzed and compared by SRAP molecular marker. [Result] Twelve pairs of SRAP primers were selected to amplify the genomic DNA of the six materials. Among the 171 clear DNA bands finally obtained, 93 were polymorphic, accounting for 54.4% of total. Genetic similarity coefficient of the six safflower varieties ranged from 0.60 to 0.92. [Conclusion]SRAP molecular marker is suitable for safflower varieties research and to guide the molecular breeding.

Progress of safflower (Carthamus tinctorius L.) regeneration through tissue culture

Background: Safflower regeneration through tissue culture has long been limited to low frequency and lack of an efficient protocol that suitable for most safflower cultivars. In past decades, researches had been carried out to investigate safflower regeneration through tissue culture and great progress had been made. Objective: To investigate factors that affect safflower regeneration through tissue culture principally. Methods: This article summarized available literatures about advancements in safflower regeneration, especially discussed factors affecting safflower tissue culture in detail. Results: Safflower regeneration was fairly hard than other congeneric plants, such as chrysanthemum. The genotype, seedling age, type of explants, medium components, plant growth regulators and other additives all had specific influences on safflower tissue culture. More deepgoing researches need to be undertaken to establish an effective safflower regeneration system.

Color spaces of safflower (Carthamus tinctorius L.) for quality assessment

Objective:In this study,safflower(Carthamus tinctorius L.)was taken as a representative example to examine the application of color characteristics to evaluate quality.Methods:A computer vision system was established for the objective and nondestructive assessment of color using image processing algorithms.Color parameters were investigated based on the RGB,L*a*b and HSV color spaces.The content of hydroxysafflor yellow A(HSYA),a major bioactive constituent of safflower,was determined by high-performance liquid chromatography.The relationship between HSYA content and color values was investigated by Pearson correlation analysis.A multiple linear regression model was established to predict the HSYA content from color values.Results:The red color and lightness of safflower were found to be significantly related to HSYA content.The prediction equation obtained by multiple regression was reliable with an R2 value of 0.805(P<.01).Conclusion:The results suggest that the computer vision technique could be used as a promising and non-destructive technology for color measurement and quality evaluation of CHM.

Safflower (Carthamus Tinctorius L.) a Potential Source of Drugs against Cryptococcal Infections, Malaria and Leishmaniasis

In this research we present that Carthamus Tinctorius L.(gen.Asteraceae,otherwise known as Safflower)(Fig.1)may contain agents active in Cryptococcal infections,malaria and Leishmaniasis,as treatment options are becoming scarce due to drug resistance development.Phytochemistry and pharmacological activities(antimicrobial,antimalarial,antileishmanial)of C.tinctorius L.were analyzed.The composition of volatile oil of safflower dried flowers was analyzed by gas chromatography-mass spectrophotometry with flame ionization detector(GC-FID)and in vitro sensitivity assays were performed to assess biological activity.8 known and 3 unknown compounds were detected in the extract(Fig.1).Then the Safflower ointment was manufactured and its acute toxicity study on rats was tested.The volatile oil of C.tinctorius L exhibited activity against Cryptococcus neoformans,Plasmodium falciparum and Leishmania donovani.Safflower volatile oil has anticryptococcal,antimalarial and antileishmanial effects.The prepared ointment had an excellent acute toxicity safety profile.

Assay for the developmental toxicity of safflower (Carthamus tinctorius L.) to zebrafish embryos/larvae

Objective:To explore the developmental toxicity and the potential toxicological mechanism of safflower (Carthamus tinctorius L.) on zebrafish embryos/larvae.Methods:Mortality,malformations and increased apoptosis induced by safflower were assessed in zebraflsh embryos from 6 to 96 hours post-fertilization.Enzymes and genes in the anti-oxidative and apoptotic pathways were also assayed.Results:The lethal concentration 50 of safflower to zebrafish embryos was 345.6 mg/L.Hatching inhibition,abnormal spontaneous movement,depressed heart rate,pericardial edema,yolk sac edema,abnormal head-trunk angle,inhibition of melanin release,enlarged yolk,and short body length were observed in safflower-treated zebrafish.Additional apoptotic cells mainly appeared around the heart.Safflower exposure changed the activities of defense enzymes (SOD T,CAT↑,MDA↑,GPX without trend),increased MDA content,decreased caspase-3 activity,and altered mRNA levels of related genes (ogg1 ↓,p53↓,Cu/Zn-sod↑,Mn-sod↓,cat↓,gpx↑).Conclusion:Safflower exhibits developmental toxicity for zebrafish embryos/larvae.The developing heart was speculated as the target organ of toxicity.Oxidative stress and increased apoptosis have roles in the developmental toxicity of safflower.This article provides a novel method to research the teratogenicity and possible mechanisms of toxicity of traditional Chinese medicines that are prohibited or contraindicated in pregnant women.

Influence of Meta-Topolin on Efficient Plant Regeneration via Micropropagation and Organogenesis of Safflower (<i>Carthamus tinctorius</i>L.) cv. NARI-H-15

The effect of meta-Topolin (mT) was assessed to develop a reliable protocol for efficient plant regeneration of safflower (Carthamus tinctorius L.) cv. NARI-H-15. For micropropagation, 7 - 9 days old shoot-tip explants cultured on MS basal medium supplemented with 3.0 mg/L meta-Topolin (mT) + 0.5 mg/L CPPU showed 97.7% adventitious shoot formation (42.4 shootlets) than node after 45 days of culture. For organogenesis, the seedling explants of immature leaf cultured on 1.5 mg/L CPPU or 1.5 mg/L NAA fortified medium produced high amount of callus than cotyledon and stem calli after 60 days of culture. However, MS basal medium fortified with 4.0 mg/L mT + 1.5 mg/L CPPU was found beneficial to stimulate 100% organogenic response (74.7 shootlets) from immature leaf calli than cotyledon and stem derived calli after 45 days of culture. The healthy plantlets obtained from micropropagation and organogenesis process cultured on 1/4 MS basal salts, 1.5% sucrose (w/v) and 0.8% agar (w/v) medium supplemented with NAA (1.5 mg/L) and mT (0.1 mg/L) produced maximum of 96% (12.8 rootlets) and 84% (7.3 rootlets) adventitious rooting, respectively than mT and CPPU tested medium. However, maximum of 67% and 42% survival rate was noticed when in vitro raised plants from micropropagation and organogenesis were hardened in pots containing soil mix and maintained under green house condition. This optimized regeneration protocol might be helpful in regeneration of new genotypes and cultivars of safflower to improve agronomic traits through in vitro selection process and Agrobacterium-mediated genetic transformation system.

Molecular mechanism of Salvia miltiorrhiza Bunge and Carthamus tinctorius L. in the treatment of myocardial ischemia based on network pharmacology: a comparative study

Based on network pharmacology and molecular docking technology,the similarities and differences of anti-myocardial ischemia mechanism between Salvia miltiorrhiza Bunge(SM)and Carthamus tinctorius L.(CT)were studied.Firstly,based on the traditional Chinese medicine systems pharmacology(TCMSP),the related compounds of SM and CT were obtained,and the potential targets of these compounds were collected by the target fishing method.Genecards database was used to obtain targets related to myocardial ischemia.The cross targets of CT,SM,and myocardial ischemia were then selected,and the protein-protein interaction(PPI)network was constructed based on the STRING database.The cross targets were imported into the Metascape database for Gene Oncology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.Cytoscape software was used to build the topological network diagram of the drug-compound-target path.Finally,the binding ability of the active ingredient and the key target was verified by molecular docking.65 active ingredients and 38 potential targets were screened from SM,and 22 active ingredients and 58 potential targets were excavated from CT.Important targets common to SM and CT were TNF,IL6,VEGFA,AKT1,etc.The common enrichment pathways involved are fluid shear stress and atherosclerosis,IL-17 signaling pathway,pathways in cancer,and toxoplasmosis.The findings suggested that the two traditional Chinese medicines exerted the effect of myocardial ischemia through the characteristics of multiple targets,multiple pathways,and multiple compounds.

红花RP-HPLC指纹图谱的建立及其质量研究

目的 建立红花RP HPLC指纹图谱分析法 ,研究不同产地红花药材的质量。方法 采用梯度洗脱的方法进行色谱分离 ,使用“计算机辅助相似度评价软件”进行数据处理 ,对不同产地红花药材指纹图谱的相似度进行比较分析。结果 不同产地红花药材指纹图谱相似度较好 ,但仍有少数产地的药材指纹图谱中有明显差别。结论 采用RP HPLC方法控制药材的指纹图谱 ,方法重现性好 ,用于红花的质量评价切实可行。不同产地红花药材化学组成相似 ,其相对比例较稳定。

UV,HPLC测定红花中黄色素、多糖和腺苷的含量

目的：为红花药材的质量评价提供依据。方法：采用ＵＶ，ＨＰＬＣ分析不同产地红花的化学成分含量（黄色素、多糖、腺苷）。结果：不同产地红花黄色素含量在２４．９０％～４０．３４％的范围内，多糖含量在５．６２％～１０．２２％的范围内，腺苷含量在３８．７～３９２．７μｇ·ｇ－１的范围内。结论：不同产地红花化学成分含量存在差异（Ｐ＜０．０１），巍山红花黄色素含量最高，新乡红花多糖含量最高；吉木萨尔红花腺苷含量最高。

红花的HPLC指纹图谱研究

目的建立评价红花质量优劣的指纹图谱分析方法。方法采用高效液相色谱分析,色谱柱Zorbax SB-C18柱(4.6 mm ×250 mm,5 μm),以乙腈-0.5%磷酸水溶液为流动相,采用梯度洗脱的方法进行色谱分离,流速为0.8 mL·min-1,检测波长275 nm,枉温为30 0℃测定了23批不同产地的红花药材的HPLC指纹图谱,并使用“计算机辅助相似度评价软件”和相似度分析对数据进行处理,对不同批药材指纹图谱的相似度进行比较分析。结果根据相似度分析处理结果,将红花药材分为2类,合格药材与不合格药材结论本方法简便、快捷、可靠,为提高红花质量控制标准提供参考。

中国红花遗传多样性的AFLP分子标记(英文)

目的考察红花的种内变异，为进一步进行红花种质资源选育和筛选品质相关基因奠定基础。方法采用扩增片段长度多态性（AFLP）技术，研究中国红花28个不同栽培居群在DNA水平的多态性。采用UPGMA构建系统树图进行聚类分析和计算遗传距离。结果筛选得到的12对引物的扩增片段具有丰富的多态性。各居群间的遗传相似系数在0．48-0．96。聚类分析显示它们分为3大主要类群和一个中间类群。结论红花种内存在一定的遗传多样性。基于AFLP条带统计的聚类结果和表型特征并不完全一致，可能是基因型和环境共同作用于表型的结果。

HPLC法测定红花中红花黄色素A的含量

目的:建立了高效液相色谱法测定红花药材中红花黄色素A的含量,考察不同产地红花中红花黄色素A的含量。方法:以香草酸为内标物,色谱柱为ODS2(200 mm×4.6 mm,5μm),甲醇-0.2%乙酸水溶液(10:90)为流动相,流量为1.0 mL·min-1,柱温为25℃,检测波长为222 nm。结果:线性范围9.4-150.4μg·mL-1,r=0.9999,平均回收率(n=3)为97.6%,RSD为1.7%。结论:本法简便、准确、重现性好,可作为红花药材质量控制的有效方法。

油用型红花(Carthamus tinctoris L.)种子醇溶蛋白遗传多样性分析

利用A-PAGE（Acid-polyacrylamide gel electrophoresis）技术对来源于不同国家的79份油用型红花材料醇溶蛋白位点进行检测。结果表明，油用型红花醇溶蛋白位点存在丰富的变异类型，共分离出15条迁移率不同的谱带，每份材料具有3—12条不等，平均8．5条。材料间平均遗传相似系数（GS）为0．5303，变幅为0．1333—1．000，且各洲间遗传多样性大于洲内遗传多样性。在GS值为0．512的水平上，供试材料聚为六大类，聚类结果表明，红花醇溶蛋白图谱类型与其地理分布有一定的相关，但不显著。

RP-HPLC法测定红花中黄酮醇的含量

用ＲＰＨＰＬＣ法分离并测定了红花中３种黄酮醇类化合物芦丁（Ｉ）、槲皮素（Ｉ）和山萘酚（ＩＩ）。以大豆甙元为内标物，分析柱ＹＷＧＣ１８（２５ｃｍ×４６ｍｍＩＤ，１０μｍ），甲醇—水—磷酸（４８５∶５１５∶０２５，ｐＨ３５）为流动相，流速１０ｍｌ·ｍｉｎ－１，柱温为室温，检测波长３６０ｎｍ，线性范围０１１～０８０μｇ，相关系数γ＝０９９９５～０９９９８，回收率９７８％～９８９％。本法简便、快速、灵敏。

注射用丹红(粉针)-中间体-药材HPLC指纹图谱相关性研究

探讨中药指纹图谱相关性研究方法,运用Agilent 1100 DAD-HPLC高效液相色谱仪,采用色谱条件:大连依利特SinoChrom ODS-BP(250 mm×4.6 mm,5μm)色谱柱,甲醇-0.1%磷酸水梯度洗脱,柱温30℃,流速1.0 mL.min-1,检测波长280 nm,进样量20μL。建立了的相同条件下丹红注射剂、中间体和药材的HPLC指纹图谱。所建立的指纹图谱个峰分离度较好,指纹信息完整,符合指纹图谱技术规范。进一步利用相似度分析软件进行了指纹图谱相关性分析,揭示了丹红注射剂与中间体和药材具有较好的相关性。共有峰的相对保留时间及相对峰面积参数,可作为中药注射剂质量控制的重要依据。

红花HPLC的指纹图谱研究

目的:利用高效液相色谱建立红花的色谱指纹图谱。方法:采用反相C18柱,乙腈-0.5磷酸水溶液梯度洗脱;检测波长275nm;流速1.0mL/min进行试验。结果:从10个省出产的红花获得了34个色谱共有峰,基本特征一致。羟基红花黄色素A为指标成分,新疆红花的含量为最高。结论:该法为中药样品的鉴定提供了较全面的信息,红花样品指纹图谱及指标成分含量测定可用于全面控制红花药材的质量。

HPLC同时测定红花药材中4种化学成分含量

目的建立HPLC同时测定红花药材中羟基红花黄色素A、芦丁、槲皮素、山柰素4种化学成分含量的方法。方法采用Agilent ZORBAX SB-C18色谱柱(4.6 mm×250 mm,5μm),DAD检测器,以0.5%磷酸溶液(A)-甲醇(B)进行梯度洗脱,柱温为30℃,流速为0.8 m L/min,进样量为10μL,检测波长为360 nm。结果羟基红花黄色素A在0.077 6~0.776 0μg(r=0.999 9)、芦丁在0.046 0~0.460 0μg(r=0.999 6)、槲皮素在0.006 4~0.064 0μg(r=0.999 9)、山柰素在0.012 8~0.128 0μg(r=0.999 7)范围内呈良好线性关系。羟基红花黄色素A、芦丁、槲皮素、山柰素的平均回收率分别为99.68%、99.78%、102.03%、97.03%,RSD(n=6)分别为2.29%、1.52%、1.02%、2.05%。结论该法简便、准确,灵敏度高,稳定性和重复性好,可用于红花药材的质量控制。

红花cDNA-AFLP反应体系的优化研究

目的为构建红花的遗传连锁图谱和功能基因的研究,探讨影响红花cDNA扩增片段长度多态性(cDNA amplified fragment length polymorphism,cDNA-AFLP)的各种因素,建立并优化红花cDNA-AFLP反应体系。方法以红花新鲜花瓣为材料,针对其内含物特殊性对Trizol法加以改进提取RNA,采用无RNase H活性的鼠源反转录酶(M-MLV RTase)结合置换合成法反转录双链cDNA;两步法酶切与连接,优化酶切时间;连接产物和预扩产物分别设置不同稀释倍数扩增并对选扩反应体系略微调整后,用6%的聚丙烯酰胺凝胶(PAGE)电泳分离,银染检测。结果改进Trizol法得到的红花总RNA样品较为完整,纯度较高,可直接用于双链的合成;经无RNase H活性反转录酶结合置换合成法得到高质量的cDNA模板。建立优化后的红花cDNA-AFLP体系为:250ng cDNA 37℃6 h经限制性内切酶MseI和EcoRI完全酶切,16℃连接12 h;连接产物最佳稀释倍数为10倍;预扩产物稀释为150倍;PAGE电泳得到清晰、稳定、分辨率较高的多态性条带。结论本研究建立的反应体系适用于红花功能基因的cDNA-AFLP研究。

红花miR397a基因表达及对靶基因LAC2的调控作用

【目的】研究红花miR397a前体基因及成熟基因在红花不同组织中的表达水平,并对miR397a基因序列、靶基因及基因功能进行分析,为深入研究红花抗逆机制提供参考。【方法】提取红花籽粒、花瓣、茎、根、叶片等5个组织材料的总RNA,使用荧光定量PCR鉴定miR397a在不同组织中的表达水平;利用生物信息学方法分析红花miR397a基因序列,用原生质体转化方法验证红花miR397a调控的靶基因LAC2,并利用转基因拟南芥表型研究miR397a基因的功能。【结果】miR397a前体基因和成熟基因均能在红花不同组织中表达,且均以叶片组织中的表达量最高,分别是籽粒组织的2.5与1.9倍,差异达极显著水平;miR397a序列在不同物种间高度保守,序列中仅存在1-2个碱基的错配或缺失;miR397a对LAC2基因有调控作用,miR397a的过表达可引起LAC2表达水平降低,红花miR397a表达引起植物对NaCl的敏感性增加。【结论】红花miR397a在叶片组织中的表达量最高,基因序列保守,且红花miR397a表达可增强植物对NaCl的敏感性。