Three new physalins from Physalis alkekengi var. franchetii

Three new physalin steroids,physalin Ⅲ(1),physalin IV(2),3-O-methylphysalin X(3),together with five known physalins(4-8)were isolated from the 80%EtOH extract of calyces of Physalis alkekengi var.franchetii.The structures of the new compounds were revealed through 1D and 2D NMR and mass spectroscopic studies.

Extraction and Crystal Structure of Physalin B

The title compound of physalin B（C28H32O9）, a main active physalin of Physalis angulata L, was isolated from the whole plant of Physalis angulata L, and characterized by X-ray diffraction analysis. It crystallizes in the monoclinic system, space group P21 with C28H32O9, a = 12.4996（2）, b = 14.35620（10）, c = 14.75190（10）, V = 2607.97（5） 3, Z = 4, Dc = 1.382 mg/cm3, Mr = 542.56, F（000） = 1152, and μ = 0.870 mm-1. The final R = 0.0389 and wR = 0.1037 for 47670 observed reflections（I 〉 2σ（I））. The rigid molecule consists of eight fused rings involving two lactones. There are two C28H32O9 molecules in an symmetric unit, and the title compound is stacked into a 3D layer structure through hydrogen bonds. In the 5~20 μmol/L range, physalin B can significantly inhibit the secretion of inflammatory cytokines TNF-α and IL-6 on RAW264.7 cells. The results suggest that physalin B has anti-inflammatory activity in vitro.

Physalin B reduce secretion of Aβ by inhibiting phosphorylation of STAT3 via down-regulated expression of β-secretase and γ-secretase

OBJECTIVE Extracellular amyloid-β(Aβ) plaques are one of the major pathological hallmarks of Alzheimer disease(AD). Therefore, decreasing Aβ levels is one strategyfor preventing the etiology of AD. Aβ peptides are generated from the cleavage of amyloid precursor protein(APP) by the β-secretase(BACE1) and γ-secretase(PS1). Inhibition of these secretases represents an obvious logical strategy to inhibit the generation of Aβ. In addition, signal transducer and activator of transcription 3(STAT3) is known to regulate many genes, and to significantly affect Aβ generation by controlling BACE1 and PS1 expression. Physalin B(PB), one of the major active steroidal constituents of solanaceaephysalis plants, possesses a wide variety of biological activities. PB down-regulates BACE1 and PS1 expression while it is unclear whether PB can regulate Aβ in N2 a/APPswe cells, and if so, whether it is by inhibiting the phosphorylation of STAT3. METHODS N2 a/APPswe cells were treated with PB in different concentrations for 24 h.(1) We used CCK8 method to detect the effects of different concentrations of PB on cell viability, and selected the best concentration for drug treatment to the cells.(2)The contents of Aβ40 and Aβ42 were determined by ELISA.(3) Western blotting was used to detect the expression levels of p-STAT3 and APP metabolism-related proteins, including APP, CTFα, CTFβ, BACE1,PS1, ADAM10 and so on.(4) RT-PCR was performed to detected the m RNA expression of BACE1 and PS1.(5) β-secretase activity Fluorometric assay kit was used to analyzed β-secretase activity.(6) In order to further explore the underlying mechanisms, N2 a/APPswe cells were pre-treated with 100 μmol·L-1 S3 I-201(a STAT3 inhibitor,can effectively prevent STAT3 phosphorylation) for 30 min and then treated with 3 μmol·L-1 PB incubated for 24 h. Then we evaluated the level of expression of STAT3 and p-STAT3 by Western blotting. RESULTS(1) CCK8 experiment results illustrated that PB did not show cytotoxicity at the applied concentration when cells were treated with PB(0, 0.3, 1 and 3 μmol·L-1). So, we used these concentrations in the following experiment.(2) ELISA results showed, compared to the control group, the contents of Aβ40 and Aβ42 decreased with the increasing of PB concentration.(3)According to Western blotting results, PB significant down-regulated the expression of BACE1, PS1, APP, CTFβ and p-STAT3 compared to the control group.(4) RT-PCR results indicated that PB can reduced the m RNA expression of BACE1 and PS1 effectively.(5) It turns out that PB can significantly inhibit BACE1 activity tested by BACE1 activity Fluorometric assay kit.(6) S3 I-201 had a similar manner to significantly inhibited STAT3 phosphorylation with PB through Western blotting. Moreover, co-treated with S3 I-201 and PB inhibited STAT3 phosphorylation much more than treatment with S3 I-201 alone. CONCLUSION These findings indicate that PB can effectively inhibit the expression of BACE1 and PS1 to reduce Aβ secretion by inhibiting the phosphorylation of STAT3.

UPLC-MS/MS法测定苦蘵宿萼中physalin H和physalin D的含量

建立了同时测定苦蘵中2种酸浆苦味素类化合物含量的方法.应用超高效液相色谱(UPLC)与Qtrap质谱(MS)联用同时测定苦蘵中酸浆苦味素H(physalin H)和酸浆苦味素D(physalin D)2个成分的含量.采用Waters Acquity BEH C18(100 mm×2.1 mm,1.7μm)色谱柱,以水(0.1%甲酸)-乙腈(0.1%甲酸)为流动相,梯度洗脱,柱温35℃,流速0.3 mL·min^(-1),使用ESI+离子源,多反应监测(MRM)模式扫描,对physalin H和physalin D进行定量.结果表明physalin H和physalin D在5~10000 ng·mL^(-1)范围内具有良好的线性关系,相关系数(r)分别为0.9997和0.9995;平均加样回收率(n=5)分别为71.1%和71.0%,RSD值分别为1.36%和1.47%.该方法准确可靠,可用于苦蘵药材中physalin H和physalin D的含量测定,为有效控制苦蘵药材的质量奠定了基础.

HPLC-CAD法测定锦灯笼中3个酸浆苦素类成分的含量

目的:建立HPLC-CAD法同时测定锦灯笼中physalin F、physalin H、physalin B三个成分含量的方法。方法:采用Thermo Hypersil GOLD C18色谱柱(250 mm×4.6 mm,5μm),以乙腈(A)-水(B)为流动相,梯度洗脱(0—8 min,32%~38%A;8—20 min,38%~45%A;20—21.1 min,45%~55%A;20.1—23.5 min,55%A),流速1.0 mL·min-1,柱温30℃;电雾式检测器(CAD)参数:采集频率10 Hz,滤波为5 s,雾化温度35℃。结果:Physalin F、physalin H、physalin B分别在2.081-33.300μg·mL^(-1)(r=0.9996)、1.550-24.800μg·mL^(-1)(r=0.9996)、2.338-37.400μg·mL^(-1)(r=0.9991)范围内线性关系良好,平均加样回收率分别为98.69%、105.71%、98.50%,RSD值分别为2.72%、2.18%、2.27%。结论:此方法准确、简便、可靠,可用于测定锦灯笼中酸浆苦素类成分的含量。

Physalin B reduces Aβ secretion through down-regulation of BACE1 expression by activating FoxO1 and inhibiting STAT3 phosphorylation

Physalin B(PB),one of the major active steroidal constituents of Solanaceae Physalis plants,has a wide variety of biological activities.We found that PB significantly down-regulatedβ-amyloid(Aβ)secretion in N2a/APPsw cells.However,the underlying mechanisms are not well understood.In the current study,we investigated the changes in key enzymes involved inβ-amyloid precursor protein(APP)metabolism and other APP metabolites by treating N2a/APPsw cells with PB at different concentrations.The results indicated that PB reduced Aβ secretion,which was caused by down-regulation of β-secretase(BACE1)expression,as indicated at both the protein and mRNA levels.Further research revealed that PB regulated BACE1 expression by inducing the activation of forkhead box O1(FoxO1)and inhibiting the phosphorylation of signal transducer and activator of transcription 3(STAT3).In addition,the effect of PB on BACE1 expression and Aβsecretion was reversed by treatment with FoxO1 siRNA and STAT3 antagonist S3I-201.In conclusion,these data demonstrated that PB can effectively down-regulate the expression of BACE1 to reduce Aβsecretion by activating the expression of FoxO1 and inhibiting the phosphorylation of STAT3.

酸浆宿萼中酸浆苦素的提取工艺及降糖活性

该试验采用低共熔溶剂-超声波辅助提取法对酸浆宿萼中的酸浆苦素进行提取并进行降糖活性研究。该试验合成5种不同的低共熔溶剂,在单因素试验的基础上,采用四因素三水平的响应面法优化酸浆宿萼中的酸浆苦素的提取工艺条件,通过对α-葡萄糖苷酶、α-淀粉酶抑制能力测定评价酸浆苦素体外降糖活性。结果表明:氯化胆碱∶葡萄糖=2∶1(摩尔比)合成的低共熔溶剂酸浆苦素提取量最高,且明显高于75%乙醇组;最佳提取工艺为料液比1∶10(g/mL)、超声功率320 W、超声时间30 min、含水量15%,在该条件下,酸浆苦素提取量为(8.96±0.15)mg/g,酸浆苦素有降糖活性。

苦蘵化学成分及抗炎活性研究

研究苦蘵的化学成分及抗炎活性。采用硅胶、Sephadex LH-20和ODS等柱色谱分离技术对苦蘵乙酸乙酯部位进行分离纯化,通过波谱学技术鉴定化合物结构,并采用LPS诱导的RAW264.7释放NO的细胞模型,评价化合物的体外抗炎活性。从苦蘵中分得13个化合物:5,4′-二羟基-3,3′,7-三甲氧基-黄酮(1)、3β-hydroxy-5α,8α-epidioxyergosta-6,22-diene(2)、(22R)-5β,6β∶14α,17∶14β,26-triepoxy-2α-ethoxy-13,20,22-trihydroxy-1,15-dioxo-16α,24-cyclo-13,14-secoergosta-18,27-dioic acid 18→20,27→22-dilactone(3)、alkesterol B(4)、withaminilide F(5)、酸浆苦素B(6)、酸浆苦素D(7)、酸浆苦素U(8)、withaphysalin R(9)、rel-(18R,22R)-5β,6β∶18β,20-diepoxy-3β,18α-dimethoxy-4β-hydroxy-1-oxowith-24-enolide(10)、withaphysalin P(11)、β-谷甾醇(12)、棕榈酸(13)。化合物1和2首次从酸浆属中分离得到,化合物3~5、10、11首次从该植物中得到。体外抗炎结果表明,化合物5、6、10、11抗炎效果显著。

锦灯笼化学成分及生物活性研究进展

锦灯笼为茄科植物酸浆Physalis alkekengi L.var.franchetii(Mast.)Makino的干燥宿萼或带果实的宿萼。秋季果实成熟、宿萼呈红色或橙红色时采收。迄今为止已从锦灯笼中分离鉴别出189种化合物,包括酸浆苦素类、黄酮类、苯丙素类、生物碱类、萜类等化学成分,现代药理学研究表明锦灯笼具有抗炎、抗菌、抗氧化、抗肿瘤等多种生物活性,本文主要对锦灯笼化学成分及生物活性研究进展进行综述,为锦灯笼的开发和利用提供理论依据。

酸浆化学成分及酸浆苦素G的结构订正

目的：对吉林产酸浆的化学成分进行研究。方法：运用层析手段和波谱方法分离并鉴定了8个化合物。结果：对酸浆苦素G（physalin G，1）的结构进行了订正并对其核磁数据进行了正确归属，其余化合物分别为酸浆苦素A（physalin A，2），酸浆苦素B（physalin B，3），酸浆苦素D（physalin D，4），酸浆苦素P（physalin P，5），金圣草黄素（chrysoeriol，6），马尾藻甾醇（saringosteml，7），丁香酸（syringicacid，8）。结论：化合物1的6位构型订正为尺，化合物6～8为首次从该属中分离得到。

中药锦灯笼的研究进展

目的对茄科植物锦灯笼的化学成分和药理作用进行阐述,为该植物的进一步研究与开发奠定基础。方法结合近年来的40篇相关文献,对从锦灯笼中分离得到的化学成分和药理活性进行综述。结果与结论锦灯笼果实和宿萼中含有多种化合物,如甾体、甾醇、氨基酸和生物碱等,对该植物的镇痛、强心、抗菌、利尿及抗癌等作用进行总结,为深入研究锦灯笼提供参考。

RP-HPLC测定酸浆药材中3种有效成分的含量

目的:采用 RP-HPLC 建立同时测定酸浆药材中酸浆苦味素 A、酸浆苦味素 D 及木犀草素含量的方法。方法:分析柱采用 Diamonsil C_(18)(200 mm×4.6 mm,5 μm)色谱柱,流动相为甲醇-0.2%醋酸(45:55,v/v);流速:1.0 mL·min^(-1);检测波长:220 nm。结果:酸浆苦味素 A、酸浆苦味素 D 及木犀草素浓度分别在0.15～1.20 mg·mL^(-1)(r=0.9995),0.10～0.81 mg·mL^(-1)(r=0.9996),0.12～0.96 mg·mL^(-1)(r=0.9997)范围内与峰面积呈良好的线性关系,酸浆苦味素 A 平均回收率分别为95.9%,97.1%,103%;RSD 分别为2.0%,0.7%,1.4%。酸浆苦味素 D 的平均回收率分别为97.3%,101%,97.1%;RSD 分别为1.0%,1.9%,1.3%。木犀草素的平均回收率分别为97.8%,97.8%,97.7%;RSD 分别为0.4%,2.9%,0.5%。结论:采用 RP-HPLC 同时测定酸浆药材中酸浆苦味素 A、酸浆苦味素 D 及木犀草素3种成分的含量,该法简单、可靠,可作为控制酸浆药材质量的参考标准。

酸浆宿萼的化学成分

目的研究茄科植物酸浆宿萼(Physalis alkekengiL.var.franchetii(Mast.)Makino)的化学成分。方法用体积分数为80%的乙醇溶液对酸浆宿萼进行加热回流提取,回收乙醇,浓缩后用水混悬,依次用环己烷、乙酸乙酯萃取,取乙酸乙酯层经硅胶柱色谱,CHCl3MeOH混合溶剂做梯度洗脱;CHCl3MeOH体积比为100∶2、100∶6,洗脱部分以及萃取后的水层再次经硅胶柱色谱、Sephadex LH 20柱色谱、反相ODS开放柱色谱及RP HPLC等,共得到7个化合物;利用其理化性质和波谱学分析数据,鉴定化学结构。结果分离得到了7个化合物中4个为酸浆苦素类化合物,分别鉴定为酸浆苦素D(Ⅰ)、酸浆苦素L(Ⅱ)、酸浆苦素O(Ⅲ)、4,7二脱氢新酸浆苦素B(Ⅳ);3个黄酮类化合物,分别鉴定为木犀草素(Ⅴ)、商陆素(Ⅵ)、木犀草素7,4′二OβD葡萄糖苷(Ⅶ)。结论商陆素(Ⅵ)、木犀草素7,4′二OβD葡萄糖苷(Ⅶ)为首次从该植物中分离得到的2个已知化合物。

酸浆苦素B的体外抗肿瘤活性研究

【目的】研究苦蘵中提取的酸浆苦素B(physalin B,Ph B)的体外抗肿瘤活性。【方法】采用四甲基偶氮唑蓝(MTT)比色法和细胞形态观察法研究Ph B对2种肿瘤细胞株Hep G2和SGC7901增殖的抑制作用及形态的影响,并与抗癌药物羟基喜树碱(hydroxycamptothecine,HCTP)作对照比较;采用4’6-二脒基-2-苯基吲哚(DAPI)染色法观察Ph B诱导Hep G2和SGC7901细胞凋亡。【结果】MTT实验结果显示Ph B对Hep G2和SGC7901的生长均有明显的抑制作用,并具有一定的时间剂量依赖关系,且高浓度时作用优于阳性对照药HCTP,倒置相差显微镜结果也显示其显著改变了细胞形态,并能够证实MTT实验结果的可靠性;DAPI染色结果表明Ph B可促进Hep G2和SGC7901细胞凋亡。【结论】苦蘵提取物Ph B对肿瘤细胞Hep G2和SGC7901具有一定的增殖抑制作用。

贮藏条件对锦灯笼药材的质量影响

目的:测定不同贮藏条件下锦灯笼药材有效成分的含量变化,研究贮存条件对锦灯笼药材的质量影响。方法:采用HPLC法测定锦灯笼药材在室温、加热、冷冻、暴晒等条件下存放一定时间后酸浆苦素成分的含量变化。结果:不同贮藏条件下,酸浆宿萼中酸浆苦素类成分含量变化显著。结论:贮藏条件对锦灯笼药材的质量有很大影响应,应严格控制。

锦灯笼的化学成分研究

从锦灯笼[Physalis alkekengi L.var.franchetii(Mast.)Makino]中提取物中分离出12个化合物,通过波谱方法和理化性质分别鉴定为:酸浆苦素B(1),酸浆苦素A(2),4,7-didehydroneophyalin B(3),(Z)-9,10,11-三羟基-12-十八碳烯酸(4),酸浆苦素F(5)山奈酚-3-O-β-D-葡萄糖苷(6),3′,4′-二甲基槲皮素(7),山奈酚-3,7-二氧-α-L-双鼠李糖苷(8),十五碳酸(9),二十四碳酸(10),3,4-二羟基苯乙醇(11),齐墩果酸(12)。化合物6-11为首次从该属植物中分出,化合物5为首次从该种植物中分出。

酸浆宿萼中酸浆苦素抑菌稳定性的研究

考察温度、pH值、可见光和紫外线对酸浆宿萼中提取的酸浆苦素抑菌特性的影响.通过对几种常见腐败菌和致病菌的抑菌性测定,结果表明：酸浆苦素对于革兰氏阳性菌的抑菌效果较明显,对部分霉菌有抑制效果,对酵母菌无抑菌效果.对于金黄色葡萄球菌的最小抑菌浓度（minimum inhibitory concentration,MIC）为31.3 μg/mL,对于霉菌中纯黄丝衣霉的MIC为62.5 μg/mL.pH值对酸浆苦素抑菌性影响较大,其次是温度,可见光和紫外线对其抑菌效果影响不明显.在20～35℃时抑菌活性最强,高温对其抑菌活力影响不大.酸浆苦素抑菌性在酸性、中性、弱碱性溶液中均较为稳定,而强碱性环境会迅速破坏其抑菌性能.

锦灯笼药材最佳采收期研究

目的：研究锦灯笼药用部位有效成分在不同生长期的动态累积变化情况,确定最佳采收期。方法：采用高效液相法,考察有效成分在药用部位的动态累积变化。结果：锦灯笼宿萼的有效成分随生长时间显著变化,酸浆苦素A、酸浆苦素O、酸浆苦素L含量在7月初开始逐渐上升,至8月初达到最大值,然后缓慢下降,月初含量骤降;而酸浆苦素B,酸浆苦素D的含量,在7~9月期间无明显变化,霜降含量迅速下降。黄酮类成分含量在7~8月很低,至8月下旬迅速上升,八月末达到最大值,然后开始下降。结论：依据药用部位的上述两种有效成分的含量变化情况,建议东北地区锦灯笼最佳采收期在八月下旬。

酸浆苦素B、E抗炎、抗菌作用实验研究

目的观察酸浆苦素(physalin)B和E的抗炎、抗菌作用。方法采用二甲苯致小鼠耳肿胀的试验方法,考察酸浆苦素B和E对小鼠的耳缘肿胀率的影响;采用体外抑菌试验,观察酸浆苦素B、E对乙型溶血性链球菌、肺炎链球菌、金黄色葡萄球菌、甲型溶血性链球菌、卡塔球菌的抑制作用。结果酸浆苦素B对二甲苯致小鼠耳肿胀具有极显著的抑制作用,酸浆苦素E对二甲苯致小鼠耳肿胀具有明显的抑制作用。酸浆苦素B、E具有良好的抑菌作用,酸浆苦素B对乙型溶血性链球菌、肺炎链球菌、金黄色葡萄球菌、甲型溶血性链球菌、卡塔球菌的最低抑菌浓度分别为6.25、25、200、200、400μg/m L。酸浆苦素E对乙型溶血性链球菌、肺炎链球菌、金黄色葡萄球菌、甲型溶血性链球菌、卡塔球菌的最低抑菌浓度分别为50、50、400、400、800μg/m L,酸浆苦素B较之酸浆苦素E抗菌活性更强。结论酸浆苦素B和E具有一定的抗炎、抗菌作用,酸浆苦素B活性较酸浆苦素E强。

酸浆绿茶体外抗氧化活性研究

对酸浆绿茶体外抗氧化活性进行研究,结果表明,酸浆绿茶具有一定的还原力;对DPPH.自由基、.OH自由基、O2-.自由基具有一定的清除能力,对脂质过氧化物有抑制作用,其IC50值分别为0.45g·L-1、0.99g·L-1、15.43g·L-1、0.83g·L-1;对DPPH.自由基的清除能力较强,对O2-.自由基的清除能力较弱。