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<0.05or 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/m TOR 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_0084443on HBEGF,EGFR,PI3K,AKT,m TOR 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.

Protective Effect of Hydroxysafflor Yellow A on Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Rats

Objective： To observe the effect of hydroxysafflor yellow A （HSYA）, an active ingredient of a traditional Chinese herbal medicine Carthamus tinctorius L., on lung inflammation and pulmonary fibrosis induced by bleomycin （BLM） in rats. Methods： Animals were divided into 6 groups including normal group, model group, three HSYA groups and dexamethasone （DXM） group. Three doses of HSYA （35.6, 53.3, and 80.0 mg？kg–1？day–1） were intraperitoneally （i.p.） injected in rats for 3 weeks after BLM administration and DXM was used as the positive control （n=8 or 12）. Arterial blood gas was assayed and morphological changes were observed. Lung mRNA expressions of tumor necrosis factor （TNF）-α, interleukin （IL）-1β, IL-6 and some cytokines in lung tissue were detected by real-time polymerase chain reaction. Nuclear factor-κB p65 or α-smooth muscle actin （α-SMA） protein distribution in rat lung tissue was observed by immunohistochemistry. Results： On the 7th day after BLM administration, lung tissue showed serious inflammation. Treatment with HSYA or DXM ameliorated lung inflammation. After treatment with HSYA or DXM, oxygen partial pressure （PaO2） increased （HSYA 80.0 mg？kg–1, P〈0.01） and CO2 partial pressure （PaCO2） decreased （HSYA 53.3, 80.0 mg？kg–1, P〈0.05）. Moreover, the mRNA expression of TNF-α, IL-1β, and IL-6; and the number of NF-κB p65 positive cells was lower in HSYA 53.3 and 80.0 mg？kg–1 groups than those in the model group （all P〈0.05）. Twenty-one days after BLM administration, HSYA or DXM treatment ameliorated fibrosis, increased PaO2 （HSYA 53.3, 80.0 mg？kg–1, P〈0.01）, and decreased PaCO2 （53.3 and 80.0 mg？kg–1, P〈0.05）. Further, the mRNA expression of TGF-β1, α-SMA, and collagen Ⅰ as well as the number of α-SMA positive cells increased in the model group and HSYA can attenuate these changes （53.3, 80.0 mg？kg–1, P〈0.05）. Hematoxylin and eosin and Masson＇s trichrome staining indicated that the fibrosis and collagen deposition were ameliorated in HSYA groups （53.3, 80.0 mg？kg–1, P〈0.05）. Conclusion： HSYA could alleviate acute lung inflammation and chronic pulmonary fibrosis induced by BLM in rats.

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

Objective: To investigate the attenuating effect of Hydroxysafflor yellow A(HSYA) on inflammatory injury in chronic obstructive pulmonary disease(COPD). Methods: 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 instil ation 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 m RNA 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. Results: Lung alveolar septa destruction, alveolus fusion, inflammatory cel infiltration, and bronchiole exudation were observed. These pathological changes were al eviated in the COPD+HSYA group. The m RNA 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(al P<0.01) and were suppressed by HSYA(all P<0.01, 48, 76.8 mg/kg). Conclusions: HSYA could alleviate inflammatory cell infiltration and other pathological changes in the lungs of COPD rats. HSYA could inhibit 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 p38 MAPK signal transduction.

丹参通络解毒汤中羟基红花黄色素A和丹酚酸B的含量测定

目的运用高效液相色谱法(HPLC)同时测定丹参通络解毒汤中羟基红花黄色素A(HSYA)和丹酚酸B(Sal B)含量。方法采用Agilent ZORBAX SB-C18色谱柱,流动相乙腈-0.1%磷酸梯度洗脱,流速1.0 mL·min-1,检测波长280 nm。结果 HSYA在21.2~63.3μg/mL呈良好的线性关系(r=0.999 6),平均回收率为99.23%,RSD为1.15%。Sal B在16.8~50.4μg/mL呈良好的线性关系(r=0.999 5),平均回收率为99.01%,RSD为1.12%。结论此方法简便、准确、重复性好,可作为丹参通络解毒汤的质量控制的检测方法。

RP-HPLC法同时测定丹红粉针中丹参素、原儿茶醛、红花黄色素A和丹酚酸B的含量

目的:建立 RP-HPLC 法测定丹红粉针中丹参素、原儿茶醛、红花黄色素 A 和丹酚酸 B 等4种有效成分的含量。方法:采用 Apollo-C_(18)(250 mm×4.6 mm,5μm)色谱柱;以1%冰醋酸-乙腈为流动相梯度洗脱;流速1.0 mL·min^(-1);柱温25℃;检测波长280 nm。结果:丹参素、原儿茶醛、红花黄色素 A 和丹酚酸 B 浓度分别在1.12～11.2,0.256～2.56,3.46～34.6,36.2～362μg·mL^(-1)范围内与峰面积线性关系良好(r≥0.9995),方法平均回收率分别为99.5%、101.0%、98.8%和100.8%(RSD<3.0%,n=9)。结论:本方法简便、准确,重复性好,可用于同时测定丹红注射剂中丹参素、原儿茶醛、红花黄色素 A 和丹酚酸 B 的含量。

HPLC波长切换法同时测定金嗓散结胶囊中的(R,S)-告依春、羟基红花黄色素A、丹酚酸B、木蝴蝶苷B和木蝴蝶苷A

目的:建立金嗓散结胶囊中(R,S)-告依春、羟基红花黄色素A、丹酚酸B、木蝴蝶苷B和木蝴蝶苷A的同时测定方法。方法:采用HPLC法,应用Agilent HC-C18(250 mm×4.6 mm,5μm)色谱柱;以甲醇-乙腈(3∶1)与0.4%磷酸溶液为流动相,进行梯度洗脱;流速为1.0 m L·min^(-1);供试品采用50%甲醇超声提取。结果:(R,S)告依春、羟基红花黄色素A、丹酚酸B、木蝴蝶苷B和木蝴蝶苷A的线性范围分别为4.150~83.00、5.120~102.4、11.07~221.4、4.590~91.80、8.140~162.8μg·m L^(-1),相关系数分别为0.999 2、0.999 9、0.999 4、0.999 7、0.999 5;5种成分分离良好,阴性对测定无干扰,平均加样回收率及相应的RSD分别为99.06%(1.27%)、99.54%(1.15%)、98.19%(0.89%)、97.88%(1.71%)、97.22%(0.99%)。结论:所建立的方法可用于金嗓散结胶囊的质量控制。

HPLC同时测定心可宁胶囊中羟基红花黄色素A和丹酚酸B的含量

目的建立同时测定心可宁胶囊中羟基红花黄色素A和丹酚酸B含量的方法。方法采用高效液相色谱法(HPLC),色谱柱为Agilent Zorbax Extend C_(18)柱(250 mm×4.6 mm,5μm),柱温30℃,流动相为甲醇-0.5%磷酸(35:65),流速1.0 ml/min,检测波长403 nm(羟基红花黄色素A)、286 nm(丹酚酸B),进样量10μl。结果羟基红花黄色素A在0.1184～0.8880μg的范围内呈良好的线性关系(r=0.9994),丹酚酸B在0.8390～6.2925μg的范围内呈良好的线性关系(r=0.9998);羟基红花黄色素A平均加样回收率为99.17%(n=6),RSD为0.87%,丹酚酸B平均加样回收率为99.41%,RSD为0.87%。结论该方法操作简便、准确、重复性好,可用于心可宁胶囊的质量控制。

大孔树脂柱色谱法制备红花黄色素和羟基红花黄色素A

目的 建立简便有效的制备红花有效部位红花黄色素 (safflor yellow,SY)及有效成分羟基红花黄色素 A(hydroxysafflor yellow A,HSYA)的方法。方法 室温水提减压浓缩所得红花水提液上 D- 4 0 2 0型非极性大孔树脂柱 ,水洗脱糖类等杂质 ,以 30 %和 10 %乙醇洗脱分别得 SY和 HSYA;采用分光光度法和 HPL C法分析水洗脱除糖过程中 SY和 HSYA的损失率及醇洗脱 SY和 HSYA的回收率。结果 聚酰胺薄膜色谱结果显示 SY和 HSYA洗脱液分别可见 4～ 5个和 1个黄色荧光斑点。 HPL C分析结果表明 ,两洗脱液中所含 SY和 HSYA纯度分别为92 .4 %和 83.0 %。在水洗除杂质和稀醇洗脱过程中 ,损失率和回收率分别为 SY:7.5 %和 80 .6 % ,HSYA:1.6 4 %和77.3%。结论 本法适于大规模制备 SY和 HSYA。

羟基红花黄色素A对实验性脑缺血的保护作用

Aim To investigate the protective effect of hydroxysafflor yellow A（HSYA）,a soluble element extracted from Carthamus tinctorius L.,on focal cerebral ischemia in rats.Methods Focal cerebral ischemia in male Wistar-Kyoto（WKY） rats were induced by permanent middle cerebral artery occlusion（MCAO）.Three doses of 1.5,3.0 and 6.0 mg·kg-1 of HSYA were administrated to three groups of rats,separately,via sublingular vein injection 30 min after the onset of ischemia.24 h after ischemia in rats,neurological deficit scores were evaluated and the infarction area of brain was assessed by quantitative image analysis.The in vitro neuroprotective effect of HSYA was tested in cultured fetal cortical neurons exposed to glutamate and sodium cyanide（NaCN）.Results HSYA at doses of 3.0 and 6.0(mg·kg-1) exerted significant neuroprotective effects on rats with focal cerebral ischemic injury as expressed by neurological deficit scores and reduced the infarct area as compared with saline group,and the potency of HSYA at dose of 6.0 mg·kg-1 was similar to that of 0.2 mg·kg-1 of nimodipine.In vitro studies,HSYA significantly inhibited neurons damage induced by exposure to glutamate and NaCN in cultured fetal cortical cells.Conclusion HSYA has potential neuroprotective action against focal cerebral ischemia in rats and cultured rat fetal cortical neurons as well.

红花药材黄色素A含量的RP-HPLC测定及其资源的质量评价

目的：为红花药材质量评价体系的建立、红花优良品种的筛选提供依据。方法：选择有代表性的红花品种22个，在不同生态地区和不同年份进行栽培试验，并采用RP-HPLE测定各品种中主要活性成分黄色素A含量，建立药材质量评价标准，比较品种间差异，考察其遗传稳定性。结果：红花不同品种黄色素A的含量在0．70％-1．85％，品种间存在非常显著差异（P〈0．01），其中，鱼台红花、合肥红花、芮城红花含量居前3位。结论：红花药材中的主要活性成分黄色素A为质量评价的重要指标，鱼台红花、合肥红花、芮城红花为红花优良种质资源。

红花黄色素A在小鼠体内的分布

目的 对红花黄色素A在小鼠体内的代谢、分布等药动学指标进行考察。方法 建立生物样品中红花黄色素A的HPLC测定法 ,并测定了小鼠给药后的组织中药物浓度。结果 小鼠尾静脉注射 (31 8mg·kg- 1 )红花黄色素A后 ,药物在小鼠体内AUC的大小顺序为血、肾、肝、肺、心、脾 ,脑中未检测到。

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。

红花黄色素粉针剂中羟基红花黄色素A血浆蛋白结合率的研究

目的测定红花黄色素粉针剂中羟基红花黄色素A的血浆蛋白结合率。方法采用超滤离心法除掉血浆蛋白,并用高效液相色谱法对低、中、高剂量静脉注射后的羟基红花黄色素A血药浓度进行测定。结果低、中、高剂量羟基红花黄色素A的血浆蛋白结合率分别为77.22%、78.31%、78.10%。结论羟基红花黄色素A血浆蛋白结合率较高,在静脉给予5～20 mg/kg范围内血浆蛋白结合率不具有质量浓度依赖性。

红花查尔酮异构酶基因的克隆分析表达及HSYA动态累积关系

目的：克隆红花中查耳酮异构酶（Chalcone-flavonone isomerase,CHI）基因,进行生物信息学分析、开花1~7 d CHI表达量及羟基红花黄色素A（hydroxysafflor yellow A,HSYA）含量分析,为红花中CHI功能验证、黄酮类成分合成及调控机制研究提供基础。方法：克隆CHI序列,通过生物信息学预测CHI特性,使用Real-time PCR检测红花开花1~7 d CHI的相对表达量,HPLC检测HSYA动态累积的百分含量,并进行关联分析。结果：克隆获得红花CHI序列全长,检测得到红花开花1~7 d CHI的相对表达量及HSYA动态累积的百分含量。CHI表达量及HSYA动态累积百分含量呈现相似的变化趋势：均在1~4 d逐渐升高,在第4天达到最高峰,第5~7天急剧降低。结论：此研究可为红花中CHI功能验证、黄酮类成分合成及调控机制研究提供基础。

红花中羟基红花黄色素A的提取纯化工艺研究

目的:优化红花中羟基红花黄色素A(HSYA)的提取纯化工艺。方法:以羟基红花黄色素A的提取率为考核指标,采用正交试验设计对红花提取工艺参数进行考察,进而考察大孔树脂对羟基红花黄色素A的纯化。结果:红花提取工艺条件是20倍量水浸泡40min;再采用HPD100大孔树脂对羟基红花黄色素A纯化,得到纯度较高的HSYA。结论:优选羟基红花黄色素A的提取纯化工艺操作简单、科学合理,可用于其单体及制剂的制备。

新疆裕民华卫红花GAP基地红花采收时间的研究

[目的]确定新疆裕民华卫红花GAP基地红花的最佳采收时间。[方法]采用HPLC法对2008～2010年红花采收期内5个不同时段采收的红花中羟基红花黄色素A、山奈素含量进行测定,并对指纹图谱进行分析。[结果]采收时间对红花中羟基红花黄色素A、山奈素含量及指纹图谱无明显影响。[结论]红花在采收期1 d内任一时间进行采收,对红花质量无明显影响,可以在红花GAP基地生产中进行推广应用。

羟基红花黄色素A的提取及含量测定

目的:对羟基红花黄色素A进行提取工艺改进。方法:采用ZTC-Ⅱ型澄清剂甲、D-4020型大孔吸附树脂和Sephadex LH-20柱层析法,从红花中提取分离羟基红花黄色素A单体,采用高效液相色谱法(HPLC)测定羟基红花黄色素A的含量。结果:采用HPLC法测定羟基红花黄色素A的平均含量为97.3%。结论:此工艺具有成本低、纯度较高的优点,可以作为羟基红花黄色素A的提取工艺使用。

红花中羟基红花黄色素A的提取动力学研究

目的确定羟基红花黄色素A的最佳提取工艺,并确立能够表征羟基红花黄色素A提取过程的动力学方程。方法以高效液相法测定不同温度、不同时间提取液中羟基红花黄色素A的含量,以Fick第一扩散定律为基础,建立浸提动力学方程,计算提取的速率常数、活化能等函数值。结果最佳提取工艺为加适量水,在90℃温浸提取60 min,拟合的动力学方程为lnk=-3 263.3(1/T)+6.524 4,该模型能较好地描述红花中羟基红花黄色素A提取的动态过程,活化能为27.13 k J·mol-1。结论红花中羟基红花黄色素A提取过程的动力学符合一级动力学方程特征。

RP-HPLC法测定红花中羟基红花黄色素A的含量

建立了红花中的主要功效成分羟基红花黄色素A含量测定的RP-HPLC方法．色谱柱为Betabasic 18柱（250mm×4．6mm，5μm），流动相为甲醇和0．5％乙酸水溶液，梯度洗脱，流速1mL／min，柱温30℃，检测波长为402nm，线性范围：0．0432～0．864μg，检测限2．1ng（按信噪比S／N=3计），该方法快速、准确。

红花羟基黄色素A动态积累规律及热稳定性研究

研究红花花丝羟基黄素色A（HSYA）动态积累规律和HSYA的热稳定性,为红花生产和应用提供了技术支持和理论支撑。以‘延津’红花和‘原阳’红花为研究材料,采用高效液相色谱技术分析红花花丝不同发育时期的HSYA含量以及不同温度和时间对HSYA制剂和花丝HSYA含量的影响。结果表明：HSYA在花蕾形成时期就开始产生并积累,在开花后第3天,其含量到达平台期。HSYA制剂含量随着保存温度（50~100℃）增加而呈直线下降;在保存温度一定的条件下,随着保存时间（30~120 min）的增加也呈现直线下降的趋势。红花花丝在50℃烘干条件下,以烘干时间为240 min时花丝HSYA含量最高;在70℃烘干条件下,以烘干时间为90 min时花丝HSYA含量最高。红花花丝在100~140℃的烘干条件下,HSYA含量呈直线下降;在烘干温度一定的条件下,HSYA含量随着烘干时间（30~240 min）的增加均呈现直线下降的趋势。因此,红花采摘以花后第3天为最好,花丝烘干可以采用40~50℃烘干,而红花制剂在生产过程中尽量避免采用高温浓缩技术。