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.

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.

Extraction,Purification,and Anti-Inflammatory Activity of Steroid Fraction from Physalis Alkekengi L.Var.Franchetii(Mast.)Makino

Objective: As a traditional medicinal plant listed in the Chinese Pharmacopeia, Physalis alkekengi L. var. franchetii(Mast.) Makino(PAF) has a long medicinal history and high economic value. PAF has immunomodulatory properties and can be used to treat acute lung injury and eczema.The aim of this study is to solve the problems of extraction and purification of active components from PAF. Materials and Methods: The solvent to be used for extraction and its concentration, the solid-to-liquid ratio, and extraction duration were investigated using a single-factor experiment.An orthogonal design(L_(9)[3^(4)]) was used to determine the optimum extraction conditions. After optimization, the sample’s concentrations and flow velocity, the eluents and their velocity, adsorption time, and the removed water volume were measured. The content of the five steroids in the sample was determined by high-performance liquid chromatography(HPLC). We also investigated the anti-inflammatory property of PAF calyxes before and after purification. Results: The optimum extraction and purification processes were determined by single-factor analysis.AB-8 was identified as the best macroporous adsorption resin for enrichment. After optimization, the average total steroid content was 71.83%,and the average recovery was 90% after purification. Among the five steroid components detected by HPLC, physalin F showed the highest content. Furthermore, the sample obtained after purification could significantly inhibit paw edema by egg whites induced. Conclusions: An environmentally-sustainable, efficient, and stable process was first optimized for enriching and purifying total steroids from PAF. The process has the potential for further development and utilization in the pharmaceutical industry.