Inhibitory Effects of Saponins From Anemarrhena asphodeloides Bunge on the Growth of Vascular Smooth Muscle Cells

Objective To investigate the effects of saponins from Anemarrhena asphodeloides Bunge （SAaB） （Botanical Name： Anemarrhena Asphodeloidis Rhizoma） on the growth of vascular smooth muscle cells （VSMCs）. Methods Cell proliferation was measured by a newly developed cell proliferation reagent, WST-1. Cell apoptosis was assayed by flow cytometry through detecting annexin V. Nitric oxide production was evaluated using confocal laser scanning microscopy with diaminofluorescein diacetate （DAF-2, DA）. Cell aldose reductase （AR） activity, as well as the effect of Epalrestat and interleukin-1β were also explored. Results WST assay showed that cell proliferation induced by serum was significantly inhibited by SAaB （P〈0.01）. Flow cytometry analysis revealed that SAaB could enhance apoptotic rate of VSMCs （P〈0.01）. Nitric oxide production was significantly enhanced after administration of SAaB and interleukin-Iβ Moreover, AR activity of VSMCs was also remarkably inhibited by both SAaB and Epalrestat （P〈 0.01）. Conclusion SAaB can inhibit proliferation and enhance apoptosis of VSMCs. It may protect vascular cells by inhibiting VSMC proliferation and augmenting apoptotic rate of VSMCs via NO-dependent pathway.

Two new saponins from Anemarrhena asphodeloides Bge.

Two new saponins 3-O-β-d-glucopyranosyl (1 → 2)-β-d-mannopyranosyl sarsasapogenin, named timosaponin A IV(1) and (5β, 25S)-26-O-β-d-glucopyranosyl-furost-20(22)-en-3,26-diol-3-O-β-d-glucopyranosyl (1 → 4) glucopyranosyl (1 → 2)-β-d-galacopyranoside, named timosaponin B IV(2), were isolated by silica gel column chromatography and preparative HPLC from Anemarrhena asphodeloides Bge. Their structures were elucidated by chemical characters and spectroscopic analysis.

Antiviral activity of mangiferin from the rhizome of Anemarrhena asphodeloides against herpes simplex virus type 1

Objective:To evaluate the antiviral activity of pure compounds against herpes simplex virus type 1(HSV-1)from the rhizome of Anemarrhena asphodeloides.Methods:Bioassay-guided isolation was conducted to separate the active compound and its chemical structure was elucidated by spectral analysis.In vitro antiviral efficacy of active compound was detected by Cell Counting Kit-8 assay,plaque reduction assay,and fluorescence observation.RT-PCR was used to determine the viral load and the cytokine-related gene expression after HSV-1 infection.In vivo study was also conducted to further determine antiviral efficacy of an active compound against HSV-1.Results:An active compound was isolated and elucidated as mangiferin.Mangiferin significantly inhibited the replication of HSV-1 in Vero cells with a half maximal inhibitory concentration(IC_(50))of 64.0 mg/L.Time-of-addition and time-of-removal assays demonstrated that mangiferin could effectively inhibit the replication of HSV-1 in the early stage(8 h).UL12,UL42,and UL54 gene expression levels of HSV-1 in the 64 mg/L mangiferin-treated group were markedly reduced as compared with the HSV-1 group(P<0.01).Fluorescence observation showed that mangiferin attenuated the mitochondrial damage maintainingΔΨm induced by HSV-1 in Vero cells.The expression of inflammatory factors TNF-α,IL-1β,and IL-6 was remarkably increased in the virus-infected group as compared with that in the normal group(P<0.05),the levels of these inflammatory factors dropped after treatment with mangiferin.Mangiferin significantly decreased the viral load and attenuated the HSV-1-induced up-regulation of TNF-α,IL-1β,and IL-6.The relative protection rate of HSV-1-infected mice could reach up to55.5%when the concentration of mangiferin was 4 g/kg.Conclusions:Mangiferin exhibits promising antiviral activity against HSV-1 in vitro and in vivo and could be a potential antiviral agent for HSV-1.

Simultaneous determination of steroidal saponins in Anemarrhena asphodeloides Bge. by ultra high-performance liquid chromatography/ quadrupole time-of-flight mass spectrometry

The objective of the research is to develop a quantitative method by ultra high-performance liquid chromatography/ quadrupole time-of-flight mass spectrometry （UHPLC/Q-TOF MS） for the analysis of seven major steroidal saponins （timosaponin N, timosaponin El, timosaponin BII, timosaponin B, anemarrhenasaponin I, anemarrhenasaponin A2, and timosaponin AIII） in Anemarrhena asphodeloides Bge. The complete separation of these seven steroidal saponins was achieved within 18 min with an ACQUITY UPLC HSS T3 column using an acetonitrile-water （contain 0.1% formic acid） gradient system. The limits of quantitation （LOQ）, 0.18-0.75 ng/pL for seven steroidal saponins, were determined experimentally. The limits of detection （LOD） were found to be 0.05-0.22 ng/μL for these saponins. The correlation coefficients （r2） for calibration curves varied from 0.9902 to 0.9979. This method showed good repeatability for the quantification of these saponins in rhizomes ofA. asphodeloides, with intra-day and inter-day variations of less than 5.0% for seven steroidal saponins. The recoveries of seven steroidal saponins were from 97.13% to 101.98%. The validated method was successfully applied to quantifying seven steroidal saponins in various sources ofA. asphodeloides （different collecting places or processing methods） and Zhimu concentrate-granules （ZMCG）.

中药知母组培快繁体系的建立

为了建立知母高效组织培养及快速繁殖体系,将知母的叶片、根茎和幼嫩花葶分别接种于9种组合培养基上,筛选最佳的外植体材料;以MS培养基为基本培养基,通过不同种类激素和浓度的配比试验,筛选出知母离体组培快繁不同阶段的培养基组合;测定不同种类激素和浓度组合培养基下炼苗移栽成功的知母组培苗芒果苷含量。结果表明,幼嫩花葶为诱导愈伤组织最佳外植体,诱导知母幼嫩花葶产生愈伤组织的培养基组合为MS+1.0 mg/L KT+0.5 mg/L NAA,出愈率为79.40%;诱导愈伤组织产生不定芽的培养基组合为MS+0.5 mg/L 6-BA+0.5 mg/L NAA,生芽率为100%;诱导不定芽生根培养的培养基组合为MS+0.5 mg/L NAA,生根率为100%,且在此培养基组合下炼苗移栽的知母组培苗芒果苷含量最高。

金花清感颗粒治疗新型冠状病毒感染的理论探析

金花清感颗粒研发于甲型H1N1流感病毒疫情盛行时期,其组方以经典名方银翘散和麻杏石甘汤为主体传承创新而成,主治病机为风温初起、卫气同病,其中知母为组方的点睛之笔,起到坚阴护卫之效。金花清感颗粒在新型冠状病毒感染疫情防治中均展现出很高的临床疗效,其在新型冠状病毒感染防治中主要针对的证型为:燥伏太阴、伏燥兼湿、卫气同病,进一步强调了伏燥病机在新型冠状病毒感染发病中的重要性,为今后的防治提供了新思路。

Two new steroidal saponins isolated from Anemarrhena asphodeloides

Two new steroidal saponins,named timosaponin P(1) and timosaponin Q(2),were isolated from the rhizome parts of Anemarrhena asphodeloides Bunge using various chromatographic methods.Their structures and absolute configurations were elucidated by a combination of spectroscopic and spectrometric data,including 1D,2D NMR,HR-ESI-MS and ECD calculations,and this is the first time the absolute configuration of C-23 of steroidal saponin was confirmed by ECD calculations.

Screening potential α-glucosidase inhibitors from Anemarrhena asphodeloides using response surface methodology coupled with grey relational analysis

Objective: To screen potential α-glucosidase inhibitors from Anemarrhena asphodeloides.Methods: Response surface methodology employing Box-Behnken design was used to optimize conditions for the extraction of α-glucosidase inhibitory active compounds from A. asphodeloides. The powders(20.0 g) of A. asphodeloides were extracted under the optimized conditions. The extract was applied to a D-101 macroporous resin column. It was eluted with ethanol and water to give six fractions. Compounds from the active fraction were identified by UPLC-Q-TOF-MS. The structure-activity relationship was discussed based on grey relational analysis.Results: The optimum extraction conditions were as follows: ethanol concentration, 100%; extraction temperature, 51 ℃; and solvent to solid ratio, 23 mL/g. It indicated that the active compounds were concentrated into 80% ethanol fraction. Twenty five steroid saponins from 80%ethanol fraction were identified by UPLC-Q-TOF-MS. Peaks 19 and 23 were tentatively identified as new structures. The predicted α-glucosidase inhibitory activities of the compounds were 7 > 2 > 1 > 22 > 23 > 3 > 9 > 21 > 24 > 4 > 13 > 8 > 14 > 16 > 17 > 25 > 6 > 19.Conclusion: The fraction eluted by 80% ethanol showed the best inhibitory activity. After analyzing the data of UPLC-Q-TOF-MS, 25 steroid saponins were tentatively identified in this fraction.

Exploring the catalytic function and active sites of a novel C-glycosyltransferase from Anemarrhena asphodeloides

Anemarrhena asphodeloides is an immensely popular medicinal herb in China,which contains an abundant of mangiferin.As an important bioactive xanthone C-glycoside,mangiferin possesses a variety of pharmacological activities and is derived from the cyclization reaction of a benzophenone C-glycoside(maclurin).Biosyntheti-cally,C-glycosyltransferases are critical for the formation of benzophenone C-glycosides.However,the benzo-phenone C-glycosyltransferases from Anemarrhena asphodeloides have not been discovered.Herein,a promiscuous C-glycosyltransferase(AaCGT)was identified from Anemarrhena asphodeloides.It was able to catalyze efficiently mono-C-glycosylation of benzophenone,together with di-C-glycosylation of dihydrochalcone.It also exhibited the weak O-glycosylation or potent S-glycosylation capacities toward 12 other types of flavonoid scaffolds and a simple aromatic compound with–SH group.Homology modeling and mutagenesis experiments revealed that the glycosylation reaction of AaCGT was initiated by the conserved residue H23 as the catalytic base.Three critical residues H356,W359 and D380 were involved in the recognition of sugar donor through hydrogen-bonding interactions.In particular,the double mutant of F94W/L378M led to an unexpected enzy-matic conversion of mono-C-to di-C-glycosylation.This study highlights the important value of AaCGT as a potential biocatalyst for efficiently synthesizing high-value C-glycosides.

在体单向肠灌流法研究知母提取物肠吸收特性

目的:研究知母提取物7个活性成分在大鼠肠道的吸收特性,揭示知母提取物活性成分的肠吸收机制。方法:选用大鼠单向肠灌流模型进行知母7种活性成分的肠吸收实验,采用固相萃取法处理肠灌流样品,通过LCMS/MS定量方法测定知母多种活性成分的吸收变化。结果:从Ka来看,芒果苷、新芒果苷、知母皂苷BⅡ、宝藿苷Ⅰ以及菝葜皂苷元的Ka存在显著性差异(P<0.05),提示这些物质主要吸收部位在空肠。知母提取物中主要有效成分P_(eff)值均>1.0×10^(-3)cm/min,提示各成分在大鼠十二指肠段和空肠的吸收均较好。结论:芒果苷、新芒果苷、知母皂苷BⅡ、宝藿苷Ⅰ以及菝葜皂苷元的主要吸收部位在空肠。

知母多糖对链脲佐菌素诱导糖尿病大鼠的降血糖作用

目的:研究分子量在30~100 k Da知母多糖对链脲佐菌素(STZ)致糖尿病SD大鼠的降血糖效果及其机制。方法:采用STZ造模实验的糖尿病SD大鼠,分为正常对照组、高血糖模型组、二甲双胍组、知母多糖组,连续给药4周后,分别测定糖尿病大鼠体重、空腹血糖、总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)、空腹胰岛素(FINS)等指标,并进行胰腺组织的HE染色观察。结果:与模型组相比,分子量在30~100 k Da知母多糖组的血糖值、TC、LDL-C和胰岛素抵抗指数均极显著降低(p<0.01),TG和血清胰岛素显著降低(p<0.05),血糖抑制率达到62.4%,体重和HDL-C极显著升高(p<0.01);HE染色观察后发现知母多糖可减轻STZ对糖尿病大鼠胰岛细胞的选择性损伤,对被特异性破坏后胰岛细胞有修复作用。结论:分子量在30~100 k Da知母多糖对糖尿病SD大鼠的有明显的降血糖,降血脂效果,该作用机制可能与改善胰岛素抵抗和修复受损胰岛细胞组织有关。

西陵知母中甾体皂苷的分离与鉴定

目的：从新鲜西陵知母（ＡｎｅｍａｒｒｈｅｎａａｓｐｈｏｄｅｌｏｉｄｅｓＢｕｎｇｅ．）根茎中分离出有生理活性的化学成分—甾体皂苷。方法：用柱色谱法分离纯化，通过理化方法及光谱分析（ＵＶ，ＩＲ，１ＨＮＭＲ，１３ＣＮＭＲ，ＥＩＭＳ，ＥＳＩＭＳ，ＨＭＢＣ，ＨＭＱＣ）鉴定化学结构。结果：从新鲜西陵知母根茎的乙醇提取物中分离得到西陵知母皂苷Ａ与Ｂ，其化学结构分别为萨尔萨皂苷元３ＯβＤ葡糖基（１→２）ＯβＤ半乳糖苷和萨尔萨皂苷元３ＯβＤ葡糖基（１→３）ＯβＤ葡糖基（１→２）ＯβＤ半乳糖苷。结论：西陵皂苷Ｂ为一新化合物。

知母中呋甾皂甙的研究

自知母ＡｎｅｍａｒｒｈｅｎａａｓｐｈｏｄｅｌｏｉｄｅｓＢｇｅ．根茎的乙醇提取物中，经硅胶柱层析和制备型ＨＰＬＣ分得四种呋甾皂甙。用化学反应和波谱（ＩＲ，ＦＡＢ－ＭＳ，ＥＩ－ＭＳ，１ＨＮＭＲ，１３ＣＮＭＲ，ＤＥＰＴ，一维多重接力ＣｏＳＹ，二维接力ＨＯＨＡＨＡ，１Ｈ－１ＨＣＯＳＹ，１Ｈ－１３ＣＣＯＳＹ和ＮＯＥ差谱）解析，确定其结构为知母皂贰Ｂ（ａｎｅｍａｒｓａｐｏｎｉｎＢ，Ｉ），（２５Ｓ）－２６－Ｏ－β－Ｄ－吡喃葡萄糖基－５β－呋甾－２０（２２）－烯－３β，２６－二醇－３－Ｏ－β－Ｄ－吡喃葡萄糖基（１→２）－β－Ｄ－吡喃哺葡萄糖甙（Ⅱ），（２５Ｓ）－２６－Ｏ－β－Ｄ－吡喃葡萄糖基－２２－羟基－５β－呋甾－３β，２６－二醉－３－Ｏ－β－Ｄ－吡喃葡萄糖基（１→２）－β－Ｄ－吡喃半乳糖甙（Ⅲ），（２５Ｓ）－２６－Ｏ－β－Ｄ－吡喃葡萄糖基－２２－甲氧基－５β－呋甾－３β，２６－二醇－３－Ｏ－β－Ｄ－吡喃葡萄糖基（１→２）－β－Ｄ－吡喃半乳糖甙（Ⅳ）。Ⅱ为一新的呋甾皂甙，命名为知母皂甙Ｃ；化合物Ⅳ为首次从知母中分离得到，命名为知母皂甙Ｅ。初步的药理实验显示，化合物１～Ⅳ均有一定的清除羟自由基的作用。

心血管相关基因芯片的制备及其在知母皂苷作用机理研究中的应用

目的 研究知母皂苷活血化瘀作用的分子机制。方法 制备了包含 87个心血管疾病相关基因探针的寡核苷酸芯片。将 80mg·L- 1 知母皂苷作用 8h及未作用的培养人血管内皮细胞提取总RNA ,通过逆转录分别标记Cy 5和Cy 3荧光染料 ,然后与芯片杂交 ,检测知母皂苷对血管内皮细胞中心血管疾病相关基因表达的影响。 结果知母皂苷作用于血管内皮细胞后 ,血管紧张素酶原 (AGT)基因、肾上腺素α2A受体 (ADRA2R)基因和内皮素转换酶 1(ECE 1)基因的表达均有不同程度的下调。结论 知母皂苷有调控血管内皮细胞功能的作用 ,可能是其治疗心血管疾病的机制之一。

西陵知母中新芒果甙的分离与结构鉴定

西陵知母中新芒果甙的分离与结构鉴定洪永福韩公羽郭学敏（第二军医大学药学院中西药研究室，上海２００４３３）知母（ＡｎｅｍａｒｈｅｎａａｓｐｈｏｄｅｌｏｉｄｅｓＢｇｅ．）为常用中药，以根茎入药。文献［１］记载，我国河北易县西陵出产的为最佳品，以“西陵知母...

知母各化学拆分组分的抗炎及免疫调节活性

目的:研究知母及其各化学拆分组分的抗炎免疫活性及其物质基础的对应关系。方法:C57小鼠除正常对照组外均采用2,4二硝基氟苯诱发超敏反应模型,观察小鼠免疫器官指数、耳肿胀度、耳组织IL-4及IFN-γ含量,体外实验观察刀豆蛋白(Con A)诱导的小鼠脾淋巴细胞增殖力、脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7炎症因子分泌量。结果:知母煎液组、皂苷组及多糖组小鼠耳肿胀度及免疫器官指数显著降低(P<0.05),免疫细胞增殖能力显著降低(P<0.01),小鼠巨噬细胞炎症因子NO、TNF-α分泌显著减少(P<0.05)。结论:知母具有显著的抗炎及免疫调节活性,其物质基础为皂苷组分和多糖组分。

知母中的两种新呋甾皂苷

目的研究知母根茎的化学成分。方法采用水煎提取、大孔吸附树脂SP825柱色谱、反相C18柱色谱等进行分离,并通过化学手段和光谱分析(FAB-MS,1HNMR,13CNMR,1H-1HCOSY)鉴定其化学结构。结果从知母根茎中分离得到6种甾体皂苷,分别鉴定为:(25S)-26-O-β-D-吡喃葡糖基-22-羟基-5β-呋甾-2β,3β,26-三醇-3-O-β-D-吡喃葡糖基-(1→2)-β-D-吡喃半乳糖苷(知母皂苷N,1),知母皂苷E1(2),(25S)-26-O-β-D-吡喃葡糖基-22-甲氧基-5β-呋甾-2β,3β,26-三醇-3-O-β-D-吡喃葡糖基-(1→2)-β-D-吡喃半乳糖苷(知母皂苷O,3),知母皂苷E2(4),(25R)-26-O-β-D-吡喃葡糖基-22-羟基-5α-呋甾-2α,3β,26-三醇-3-O-β-D-吡喃葡糖基-(1→2)-[β-D-吡喃木糖基-(1→3)]-β-D-吡喃葡糖基-(1→4)-β-D-吡喃半乳糖苷(purpureagitosid,5),marcogenin-3-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranoside(6)。结论化合物1和3为新化合物,命名为知母皂苷N和知母皂苷O,化合物5为首次从知母中分离得到。

知母的皂苷成分

目的：研究知母的化学成分，寻找新的活性物质。方法：利用大孔树脂柱色谱、硅胶柱色谱及高效液相色谱等手段进行分离，根据化合物的理化性质及光谱（ＩＲ，ＦＡＢＭＳ，ＥＳＩＭＳ，１ＨＮＭＲ，１３ＣＮＭＲ，ＤＥＰＴ，ＨＭＱＣ，ＨＭＢＣ，ＮＯＥＳＹ和ＲＯＥＳＹ）数据鉴定结构。结果：从中药知母分离得到两种甾体皂苷，确定其结构是：（５β，２５Ｓ）螺甾烷３β，１５α，２３α三醇３ＯβＤ吡喃葡萄糖基（１→２）βＤ吡喃半乳糖苷（１）和（５β，２５Ｓ）螺甾烷３β，２３α二醇３ＯβＤ吡喃葡萄糖基（１→２）βＤ吡喃半乳糖苷（２）。结论：１和２均是新化合物，分别命名为知母皂苷Ｆ和知母皂苷Ｇ。

知母提取物对马铃薯晚疫病菌的抑制作用及防病效果

本研究测试了知母提取物对马铃薯晚疫病菌菌丝生长、游动孢子释放、休止孢萌发和孢子囊直接萌发的抑制作用。结果表明,知母的水提取物对菌丝生长、游动孢子释放和休止孢萌发的抑制中浓度分别为1.91 mg/mL、1.23 mg/mL和1.04 mg/mL;对孢子囊直接萌发也具一定的抑制作用,在10.00 mg/mL浓度下,抑制率达72.89%。离体叶片测试发现,浓度高于0.5%的知母提取物对马铃薯晚疫病具有显著的防治效果。盆栽植株测试结果表明,1%的知母提取物对马铃薯晚疫病的保护效果达70%以上,防效优于离体叶片试验结果,且持效期可达5 d。2%和1%的知母提取物对马铃薯晚疫病的治疗效果不明显。

UPLC-LTQ-Orbitrap XL分析知母-黄柏药对化学成分

目的:分析知母-黄柏药对的化学成分。方法:采用超高效液相-线性离子阱-轨道阱质谱联用仪(UPLCLTQ-Orbitrap XL),在正、负两种离子模式下,利用质谱的高分辨率以及成分的二级碎片信息,鉴别药对中的成分。结果:共鉴别了55个化学成分,其中从知母中鉴别了18个化学成分,黄柏中鉴别了37个化学成分,3个化学成分为首次在该药对中报道。结论:UPLC-LTQ-Orbitrap XL能快速、精确的对知母-黄柏药对的化学成分定性分析,为知母-黄柏药对药效物质基础研究提供依据。