Linarin ameliorates innate inflammatory response in an experimental dry eye model via modulation of the NLRP3 inflammasome

Objective To investigate the effect and mechanism of linarin(LA) in an experimental dry eye model.Methods LA or vehicle was applied in two dry eye models: an in vitro hyperosmotic stress model and an in vivo desiccating stress(DS) murine model. The viability of human corneal epithelial cells(HCECs) was measured using a cell counting kit(CCK-8).Tear secretion was assessed using the phenol red cotton test. The tear break-up time(TBUT) was recorded using 0.1% liquid fluorescein sodium. Corneal epithelial permeability was evaluated through Oregon green dextran(OGD) staining.Conjunctival goblet cells were counted using periodic acid-Schiff(PAS) staining. Terminal deoxynucleotidyl transfer d UTP nickend labeling(TUNEL) staining was used to quantify apoptotic cells in both models. The expression of Ki-67 was measured in HCECs in the cell model while that of matrix metalloproteinase(MMP)-3 and-9 was measured in the murine model through immunofluorescence staining. Real-time quantitative PCR(RTqPCR) was performed to assess the expression of MMP-3 and MMP-9 in the corneal epithelium and NLRP3, ASC, Caspase-1,interleukin(IL)-1β, IL-18, and tumor necrosis factor(TNF)-α in the conjunctiva. The protein expression levels of NLRP3, ASC,Caspase-1, IL-1β, and IL-18 in the conjunctiva were assessed via Western blot.Results In the in vitro model, treatment of HCECs with LA showed no toxicity, increased proliferation, and reduced apoptosis. In the murine model, compared to the control, LA significantly increased tear production and TBUT, improved OGD staining, and increased the number of goblet cells. Topical treatment of LA to mice provided decreased expression of MMP-3, MMP-9, TNF-α, and apoptotic corneal epithelium. Topical administration of LA also suppressed the NLRP3 inflammasome in the dry eye disease(DED) murine model by decreasing the expression of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the conjunctiva.Conclusion Our findings support the safety and efficacy of LA in the treatment of DED. LA alleviated corneal epithelial damage and suppressed NLRP3 inflammasome-mediated immunity in the conjunctiva in a murine model of DED.

Investigation of Linarinic acid and one of its derivatives against cerebral ischemia in mice

The study aims to investigate the effects of(-)-Linarinic acid(LA) and one of its derivatives(LAd) on brain injury induced by ischemia. Malonaldehyde(MDA) is determined as an index for lipid peroxidation both in vitro and vivo. Mice were pre-treated with LA and LAd for 3 d.Thereafter, they were induced to have incomplete cerebral ischemia with both bilateral carotid artery occlusion and hypotension(BCAOH). In the first part of the in vivo experiment, mice were divided into four groups: sham(control), ischemia, ischemia + LA(200 mg/kg, i.g.) and ischemia + LAd(200 mg/kg, i.g.). In the second part, the dose-response of LAd was investigated at 100, 200 and 400 mg/kg i.g., respectively. A modified neurological severity score was developed for evaluating behavioral deficits of the mice with ischemia. Brains of the mice were excised in order to determinate MDA after ischemia for 6 h. Survival time, survival rate, neurological injury score and MDA level in brains were observed. Results were: 1) The data in vitro showed that both LA and LAd could inhibit the generation of MDA. IC50 values obtained by Probit analysis were 2.9 mM for LAd and 4.88 mM for LA;2) BCAOH could significantly shorten the survival span, reduce the survival rate and cause neurological deficits,which were associated with high level of lipid hydroperoxide production in cerebral tissues;3) LAd decreased lipid peroxidation and improved the neurological outcome more than LA.It is concluded that LAd offers a better neuroprotection than LA against brain damage caused by cerebral ischemia.

Anti-diabetic effects of linarin from Chrysanthemi Indici Flos via AMPK activation

Objective:The purpose of this study is to investigate the anti-diabetic effects of linarin,a flavonoid extracted from Chrysanthemi Indici Flos(CIF),and its potential mechanisms.Methods:The effects of linarin on cell viability and glucose consumption in Hep G2 cells were measured.Meanwhile,monosodium glutamate(MSG) mouse model was constructed to monitor the changes of insulin tolerance,glucose tolerance,triglyceride and cholesterol.The protein expression levels of pAMPK,p-ACC,PEPCK and p-GS were detected by Western blot.Results:Linarin could increase the relative glucose consumption of Hep G2 cells,improve insulin tolerance and glucose tolerance,and decrease the levels of triglyceride and cholesterol of MSG mice.Simultaneously,the expression levels of p-AMPK and p-ACC in Hep G2 cells and the liver tissue of MSG mice were increased,while the expression levels of PEPCK and p-GS were decreased after treatment with linarin.Conclusion:Insulin resistance could be ameliorated by linarin in type 2 diabetes,and its mechanism may be related to AMPK signaling pathway.

蒙花苷通过抑制TLR4/NF-κB通路抑制小鼠脊髓损伤后小胶质细胞活化介导的神经炎症和神经元凋亡

目的探讨蒙花苷(LIN)在脊髓损伤(SCI)后对激活小胶质细胞介导的炎症和神经元凋亡的神经保护作用和潜在机制。方法将50只8~10周龄C57BL/6J小鼠随机分为假手术组(Sham组)、脊髓损伤模型组(SCI组)和LIN药物治疗组(12.5、25、50 mg/kg),10只/组。使用巴索小鼠评分量表(BMS)、斜板实验和脚印分析评估SCI小鼠的运动功能恢复情况;通过HE染色和LFB染色分别评估脊髓组织损伤面积和髓鞘化面积。采用Nissl染色、免疫荧光及Western blotting观察损伤脊髓组织前角运动神经元的保留情况。体外实验通过LPS诱导BV2细胞建立体外炎症模型,同时给予蒙花苷进行干预,将BV2细胞分为对照组(Con-B)、脂多糖组(LPS-B)及蒙花苷组(LIN-B)。通过免疫荧光、Western blotting、RT-qPCR及ELISA检测体内外小胶质细胞活化及炎症因子的释放情况,进一步采用Western blotting验证信号通路的改变。构建体外BV2细胞和HT22细胞共培养模型,分为Con-H组、LPS-H组以及LIN-H组。采用Western blotting检测激活小胶质细胞介导的HT22细胞凋亡情况。结果行为学检测发现,LIN组小鼠BMS评分显著提高,承受的斜板高度更高,后肢行走协调且足迹清晰(P<0.05)。组织学染色结果显示,LIN组小鼠脊髓组织损伤面积减少,髓鞘化面积增加,并且小鼠脊髓前角运动神经元的数量增加(P<0.05)。体内外实验证实,LIN显著抑制小胶质细胞的活化和炎症因子(iNOS、COX-2、TNF-α、IL-6、IL-1β)的释放,并减少了体内神经元的凋亡(P<0.05)。Western blotting显示LIN抑制小胶质细胞活化可能与抑制TLR4/NF-κB信号通路有关。体外共培养实验结果显示,LIN的干预能够减少炎症介导的神经元凋亡数量(P<0.05)。结论LIN可能通过抑制TLR4/NF-κB信号通路抑制小胶质细胞的激活,减轻神经炎症,改善SCI后的神经元凋亡,提高SCI小鼠运动能力,发挥其神经保护作用。

维吾尔族习用药神香草特征图谱与含量测定

目的建立神香草高效液相色谱(HPLC)特征图谱,地奥司明、蒙花苷的含量测定方法。方法分析采用C18色谱柱(4.6 mm×250 mm,5μm);乙腈-0.1%磷酸为流动相,梯度洗脱;流速1.0 mL·min^(-1);特征图谱检测波长328 nm,含量测定检测波长333 nm。结果神香草HPLC特征图谱中有18个主要色谱峰。地奥司明在10.773~430.92 ng、蒙花苷在0.3247~324.73 ng范围内线性关系良好,平均回收率(n=9)分别为97.1%(RSD=2.2%)、100.2%(RSD=0.7%)。结论该方法准确可靠,可用于神香草的质量控制。

HPLC法测定喉痛灵片的蒙花苷含量

本文研究用HPLC法测定喉痛灵片中蒙花苷的含量。用TYPE MGII C_(18)(5μm)的色谱柱为固定相,乙腈-水(25∶75)溶剂作流动相,流速为1.0 mL/min,测定的波长为326 nm,柱温为接近室温。实验结果显示,蒙花苷在2.538~101.528μg/mL之间区域内浓度呈良好稳定的正线性关系,线性范围宽,相关系数r=0.9999;加样回收率为103.1%~109.6%,平均值为104.0%,RSD=1.6%。本研究的测定方法分析时间短,样品前处理简单方便,专属性强,重现性好,实验数据良好,质量可控,值得推广应用。

Preparation and pharmacokinetics in vivo of linarin solid dispersion and liposome

Objective:The current investigation aimed to determine the appropriate dosage form by comparing solid dispersion and liposome to achieve the purpose of improving the solubility and bioavailability of linarin.Methods:Linarin solid dispersion(LSD)and linarin liposome(LL)were developed via the solvent method and the thin film hydration method respectively.The Transwell chamber model of Caco-2 cells was established to evaluate the absorption of drug.The pharmacokinetics of linarin,LSD and LL in rats after ig administration were carried out by high performance liquid chromatography(HPLC)method.Results:The solubility of LSD and LL was severally 3.29 times and 3.09 times than that of linarin.The permeation coefficients of LSD and LL were greater than 10^(-6),indicating that the absorption of LSD and LL were both better than linarin.The bioavailability of the LSD was 3.363 times higher than that of linarin,and the bioavailability of LL was 0.9886 times higher than that of linarin.Conclusion:The linarin was more suitable for making solid dispersion to enhance its solubility and bioavailability.

野菊花药用活性成分及调控措施研究进展

野菊花(Chrysanthemum indicum L.)具有清热解毒、预防感冒、抗高血压和抗肿瘤等多种药理作用,是多种中成药的主要组成成分。综述野菊花的药用活性成分黄酮类、萜类、挥发油类及有机酸药理作用的相关研究,同时对黄酮类化合物中重要组成成分蒙花苷含量的调控措施,包括海拔、土壤养分等环境因子的调控效应进行了论述,以期为野菊花资源的进一步研究和开发提供参考。

蒙花苷体内外抗肺纤维化作用及其机制研究

目的 考察蒙花苷的体内外抗纤维化作用,并初步探讨其作用机制。方法 将C57BL/6J小鼠随机分为正常组(羧甲基纤维素钠)、模型组(羧甲基纤维素钠)、阳性对照组(吡非尼酮,200 mg/kg)和蒙花苷低、高剂量组(12.5、25 mg/kg),每组8只。除正常组外,其余各组小鼠均制备肺纤维化模型。造模结束后,灌胃给药,每天1次,连续14 d。观察小鼠一般情况,测定其肺指数,检测其血清中肿瘤坏死因子α(TNF-α)、转化生长因子β1(TGF-β1)水平以及肺组织中白细胞介素6(IL-6)水平;采用苏木素-伊红(HE)染色及Masson染色观察其肺组织病理学变化,并参照Ashcroft评分标准进行肺纤维化评分;检测其肺组织中α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(CollagenⅠ)、磷酸化胞外信号调节激酶(p-ERK1/2)、TGF-β1的表达水平。使用TGF-β1刺激人胚肺成纤维细胞HFL1建立体外肺纤维化模型,实验设置正常组、模型组和蒙花苷低、中、高浓度组(3.7、7.4、14.8 mg/L),培养48 h后,检测细胞中α-SMA、CollagenⅠ、p-ERK1/2蛋白表达水平。结果 在体内实验中,与正常组比较,模型组小鼠肺指数和TNF-α、TGF-β1、IL-6水平均显著升高(P<0.01);肺泡中出现大量炎症浸染及细胞纤维化等病变,且有大量胶原蛋白沉积,HE染色和Masson染色评分均显著升高(P<0.01);肺组织中α-SMA、CollagenⅠ、p-ERK1/2及TGF-β1蛋白表达均显著上调(P<0.01)。与模型组比较,蒙花苷高剂量组小鼠上述指标均显著改善(P<0.05或P<0.01),蒙花苷低剂量组大部分指标(除肺指数外)均显著改善(P<0.05或P<0.01)。在体外实验中,与空白组比较,模型组细胞出现密度变大、明显增殖等变化,细胞中α-SMA、CollagenⅠ和p-ERK1/2蛋白表达均显著上调(P<0.05或P<0.01)。与模型组比较,蒙花苷各浓度组细胞密度变小,形态逐渐恢复正常;蒙花苷高浓度组细胞中上述蛋白及蒙花苷中浓度组细胞中p-ERK1/2蛋白表达均显著下调(P<0.05或P<0.01)。结论 蒙花苷可能通过调控ERK及炎症相关通路,减轻炎症反应,从而发挥抗肺纤维化作用。

妇科九味洗剂高效液相色谱法特征指纹图谱及成分定量研究

目的建立妇科九味洗剂的高效液相色谱法(HPLC)特征指纹图谱及多指标成分测定方法。方法该研究于2020年5—8月在芜湖市中医医院制剂室实验室中进行。采用HPLC法,色谱柱Agilent Zorbax SB-C_(18)(4.6 mm×150 mm,5µm),流动相甲醇-0.4%甲酸不同比例梯度洗脱,波长334 nm,柱温30℃;13批妇科九味洗剂样品的特征图谱运用“中药色谱指纹图谱相似度评价系统”(2012版)评价其相似度;同时测定咖啡酸、木犀草苷和蒙花苷的含量。结果13批样品共标定16个共有峰,相似度均在0.91以上;通过对照品比对,识别出咖啡酸、木犀草苷和蒙花苷成分。3个成分分别在5.82~58.20 ng(r=0.9995)、9.44~94.40 ng(r=0.9995)、20.08~200.80 ng(r=0.9996)范围内呈现良好的线性关系。结论所建立的方法简单,准确可靠,可用于妇科九味洗剂的质量控制。

UPLC-MS/MS法同时测定珍菊降压片中5种成分的含量

文中建立一种UPLC-MS/MS法同时测定珍菊降压片中可乐定、蒙花苷、绿原酸、氢氯噻嗪、芦丁含量。采用ACQUITY UPLC?BEH柱(C182.1 mm×50 mm1.7μm),以0.1%甲酸水溶液-甲醇为流动相梯度洗脱,流速:0.3 mL·min^(-1),柱温35℃,进样量2μL。质谱采用ESI(电喷雾离子源),以正负离子多反应监测模式定量分析。实验结果显示,可乐定、蒙花苷、绿原酸、氢氯噻嗪、芦丁的线性范围为0.02648~0.8472μg·m L^(-1)、0.1011~3.235μg·m L^(-1)、0.08055~2.578μg·mL^(-1)、0.02600~0.8320μg·mL^(-1)、0.1033~3.306μg·mL^(-1)(r^(2)为0.99167~0.99907),平均加标回收率分别为95.1%、93.2%、91.7%、94.6%、92.8%,RSD为2.81%、2.02%、3.41%、3.15%、1.79%(n=6)。结果表明该方法操作简单、便捷、快速,可一次性实现多个不同成分的检测,结果准确可靠。

分离鉴定黄连花薹的差异性成分并测定其中4个成分的含量

目的采用薄层鉴别法与高效液相色谱法对比寻找出黄连药材与黄连花薹中含量最大的差异性成分,并对其进行鉴定;同时建立一测多评法测定黄连花薹中4个化学成分的含量。方法采用聚酰胺柱层析等分离纯化手段对黄连花薹与黄连药材差异性成分进行提取、分离、纯化,并采用液质联用以及核磁鉴定的方法对差异性成分进行确证;采用资生堂C18色谱柱(250 mm×4.6 mm,5μm),检测波长为340 nm,柱温为40℃,流速为1.0 mL·min^(-1),进样量为10μL,流动相为乙腈(A)-0.1%磷酸(每1000 mL加入20μL三乙胺)(B),梯度洗脱。以盐酸小檗碱为内标物,计算其他3个成分的相对校正因子,建立用一测多评法和常规外标法测定黄连花薹中4个化学成分的含量,并对两种方法的含量测定结果进行比较。结果分离鉴定出黄连花薹与黄连药材的差异性成分蒙花苷,同时在各自的线性范围(r>0.9999)内,芦丁、绿原酸、蒙花苷的相对校正因子分别为2.5704、2.4532、1.7700。6批黄连花薹一测多评法测定结果与外标法测定结果无显著差异,但大大提高了检测的效益,节约了检测成本。结论分离鉴定出黄连花薹与黄连药材的差异性成分蒙花苷,同时建立的一测多评法测定结果准确,可用于黄连花薹的定量分析及质量控制。

基于正交试验设计的大黄甘草洗液提取工艺研究

目的对大黄甘草洗液的提取工艺进行优化,为中试放大生产提供质量控制依据。方法利用高效液相色谱法测定蒙花苷的含量,采用正交试验考察加水倍量、浸泡时间、提取时间及提取次数等因素对提取效果的影响。结果大黄甘草洗液的最佳提取工艺为药材加14倍量水浸泡1小时,提取2次,每次2小时。结论提取工艺优化后稳定可靠,可为该药的量产提供保证。

基于Box-Behnken结合总评“归一值”优化贝加尔唐松草黄酮成分的提取工艺及其抗氧化和抑菌活性研究

为优化贝加尔唐松草黄酮成分的提取工艺,考察总黄酮的抗氧化和抑菌活性,采用热回流提取技术,在单因素实验基础上,以贝加尔唐松草中总黄酮得率、蒙花苷得率及其二者的总评“归一值(OD)”为综合评价指标进行Box-Behnken响应面试验,优化贝加尔唐松草黄酮成分的最佳提取工艺;以总黄酮对1,1-二苯基-2-三硝基苯肼(DPPH)自由基、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS^(+)·)和羟自由基(·OH)的清除率为指标,评价其体外抗氧化活性,并用抑菌圈法评价其对6种供试菌的抑菌活性。贝加尔唐松草黄酮成分的提取工艺参数为:乙醇浓度58%,料液比1:28(g:m L),提取时间34 min,提取温度70℃,在此条件下,蒙花苷得率为5.23±0.04 mg·g^(-1),总黄酮得率为40.78±0.12 mg·g^(-1);贝加尔唐松草总黄酮清除DPPH、ABTS^(+)·和·OH 3种自由基的半抑制浓度(IC_(50))值分别为0.341 mg·mL^(-1)、0.115 mg·mL^(-1)和0.481 mg·mL^(-1),对6种供试菌的最低抑菌浓度(MIC)在12.5~50.0μg·mL^(-1);在实验浓度范围类,总黄酮对3种自由基的清除率和抑菌活性与其浓度均呈一定的关系。在Box-Behnken响应面优化的参数下,贝加尔唐松草总黄酮和蒙花苷的提取工艺稳定、提取得率高。并且发现总黄酮表现出了良好的清除自由基能力和抑菌活性,具有良好的应用潜力。本研究为贝加尔唐松草活性成分的深入研究和资源的综合利用奠定了良好的基础。

HPLC测定野菊花中蒙花苷与木犀草素的含量

目的：建立野菊花中蒙花苷与木犀草素的含量测定方法。方法：运用高效液相色谱法测定，色谱柱为Thermo Hypersil BDSC18（4．6mm×250mm，5μm），流动相为甲醇-四氢呋喃-水（50：4：46），流速0．6mL·min^-1；检测波长350nm。结果：蒙花苷与木犀草素分别在0．0152～0．152mg·mL^-1和0．0018～0．018mg·mL^-1线性关系良好，r分别为0．9998，0．9994。平均回收率分别为98．9％，97．2％，RSD均小于3．0％。结论：本法方便，准确，可以用于野菊花药材的质量控制。

高效液相色谱法测定密蒙花中蒙花苷的含量

目的 :建立高效液相色谱法测定密蒙花中蒙花苷的含量。方法 :采用RP -C1 8色谱柱 ,甲醇 -水 -醋酸 (4 5∶5 4 5∶0 5 )为流动相 ,流速 0 8mL·min- 1 ,32 6nm为检测波长 ,室温下对密蒙花中的蒙花苷进行含量测定。结果 :蒙花苷在 0 4～ 2 9μg范围内 ,面积与其浓度线性关系良好 (r=0 9991) ;样品平均回收率为 97 0 2 % ,RSD为 1 5 % (n =4)。结论 :该法简单、快速、结果准确可靠。

薄荷非挥发性成分研究

目的：为促进薄荷的综合利用，对其非挥发性成分进行系统研究。方法：采用硅胶柱色谱、重结晶和光谱法分离鉴定化合物。结果：从薄荷中分离并鉴定了8个化合物，分别是刺槐素（Ⅰ）、椴树素（Ⅱ）、蒙花苷（Ⅲ）、正丁基-β-D-吡喃果糖苷（Ⅳ）、熊果酸（Ⅴ）、齐墩果酸（Ⅵ）、β-甾醇（Ⅶ）和胡萝卜苷（Ⅷ）。结论：化合物Ⅰ-Ⅴ均为首次从该植物中分离得到。

墨旱莲的化学成分研究

目的研究墨旱莲的化学成分。方法应用溶剂法进行提取,硅胶、Sephadex LH-20等手段反复柱色谱分离、纯化,通过熔点、NMR等手段确定结构。结果从其乙醇提取物的石油醚、醋酸乙酯以及正丁醇萃取部分共得到10个化合物,分别为2,2′,5″,2″-三噻吩-5-羧酸(Ⅰ)、β-谷甾醇(Ⅱ)、芹菜素(Ⅲ)、槲皮素(Ⅳ)、木犀草素(Ⅴ)、蟛蜞菊内酯(Ⅵ)、去甲蟛蜞菊内酯(Ⅶ)、旱莲苷C(Ⅷ)、木犀草苷(Ⅸ)、蒙花苷(Ⅹ)。结论化合物Ⅰ为一新天然产物,Ⅹ为首次从墨旱莲中分离得到。

高效液相色谱法测定玉叶解毒颗粒中蒙花苷的含量

目的:建立 HPLC 法测定玉叶解毒颗粒中蒙花苷的含量。方法:以 HiQ-sil C_(18)V(250mm×4.6mm,5μm)为分析柱,甲醇-0.5%冰醋酸溶液(45∶55)为流动相,检测波长:326nm,流速:1.0mL·min^(-1)。结果:蒙花苷在0.1-0.6μg范围内呈良好的线性关系(r=0.9991),平均回收率为96.6%,RSD 为1.6%(n=5)。结论:该方法简便、准确,可作为玉叶解毒颗粒的含量测定方法。

RP-HPLC法同时测定夏桑菊颗粒中绿原酸、异迷迭香酸苷、迷迭香酸和蒙花苷

目的建立RP—HPLC法同时测定夏桑菊颗粒（夏枯草、桑叶、野菊花）中绿原酸、异迷迭香酸苷、迷迭香酸和蒙花苷4种活性成分的方法。方法采用AgilentEclipseXDB—C18色谱柱（4．6mm×250mm，5μm）；流动相为乙腈-1．0％醋酸水溶液，采用梯度洗脱，检测波长为320nm，体积流量1．0mL／min，柱温30℃，进样体积10μL。结果绿原酸、异迷迭香酸苷、迷迭香酸和蒙花苷的线性范围分别为0．0389～0．7771μg、0．0067～4．200μg、0．0480～0．9600Ixg和0．0386～0．7714μg（0．9992〈r〈0．9999），平均回收率在98．35％和98．62％之间。结论该法检测效率高，专属性强，重复性好，可用于夏桑菊颗粒中绿原酸、异迷迭香酸苷、迷迭香酸和蒙花苷的定量测定，可作为该制剂的质量控制方法之一。