In vivo pharmacokinetics of and tissue distribution study of physalin B after intravenous administration in rats by liquid chromatography with tandem mass spectrometry.

A rapid and sensitive liquid chromatography tandem mass spectrometry quantitative analysis method was established for the pharmacokinetics and tissue distribution study of physalin B in rat. Physalin B and physalin H (internal standard, IS) were separated on an Agilent Eclips XDB C8 column. MS detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode with a positive eletrospray ionization source. The assay was validated in the concentration ranges of 22.6-22600 ng/mL for heart and lung and 4.52-4520 ng/mL for other tissues. The intra- and inter-day precisions (RSD) were ≤9.23 and ≤12.51%, respectively, with accuracy (%) in the range of 88.07-113.2%. A pharmacokinetic study showed that physalin B has a long dwell time with a half-life of 321.2 ± 29.5 min and clearance of 175.4 ± 25.7 mL/min/kg after intravenous administration. Additionally, physalin B showed a wide tissue distribution with a special higher penetration in lung. The data presented in this study could provide useful information for the further study of physalin B. Copyright © 2016 John Wiley & Sons, Ltd.

Isolation, pharmacological activity and structure determination of physalin B and 5ß,6ß-epoxyphysalin B isolated from Congolese Physalis angulata L.

Physalis angulata L., an annual herb from the Solanaceae family, is widely used in popular medicine in tropical countries to treat a variety of diseases. Two products, (X) and (Y), were isolated from a crude CH2Cl2 extract of dried Congolese Physalis angulata L. plants and crystallized from acetone for structure elucidation. Compound (X) corresponds to a physalin B dimer acetone solvate hydrate (2C28H30O9·C3H6O·0.22H2O), while compound (Y) crystallizes as a mixed crystal containing two physalin B molecules which overlap with 5ß,6ß-epoxyphysalin B, also known as physalin F, and one acetone molecule in the asymmetric unit (1.332C28H30O9·0.668C28H30O10·C3H6O). Antiplasmodial activity, cytotoxic activity and selectivity indices were determined for crude extracts and the two isolated products (X) and (Y).

Physalin B inhibits cell proliferation and induces apoptosis in undifferentiated human gastric cancer HGC-27 cells.

BACKGROUND: Physalin B (PB) from Physalis angulata L. (Solanaceae) is a naturally occurring secosteroid with multiple biological activities, including anti-inflammatory and anticancer activity. However, PB's effects and mechanisms in human gastric cancer (GC) cells are not well characterized. METHODS: The undifferentiated GC cell line HGC-27 and semi-differentiated GC cell line SGC-7901 were treated with PB. Cell counting kit-8 (CCK-8) and colony formation assays were performed to evaluate cell viability. Apoptosis and the cell cycle were assessed by Annexin V/PI and PI/RNase DNA staining assays, respectively, and Western blotting was used to evaluate the expression of a protein. RESULTS: PB significantly inhibited the proliferation of HGC-27 cells in a dose- and time-dependent manner. Moreover, PB induced G0/G1 cycle arrest and caspase-dependent apoptosis of HGC-27 cells. Cleaved caspases 8, 3, and 7, poly(ADP)-ribose polymerase (PARP), and the cyclin-dependent kinase (CDK) inhibitor p-Chk2 was induced by PB in HGC-27 cells, while the cell cycle-related proteins cyclin D1, cyclin D3, CDK4, CDK6, cyclin E, and phosphorylated retinoblastoma tumor suppressor protein (p-Rb) were downregulated in a dose-dependent manner. CONCLUSIONS: PB inhibits proliferation via cyclin-dependent kinase and induces caspase-dependent apoptosis in HGC-27 cells, suggesting that PB might be a novel and effective agent for undifferentiated GC therapy.

Physalin B ameliorates inflammatory responses in lipopolysaccharide-induced acute lung injury mice by inhibiting NF-κB and NLRP3 via the activation of the PI3K/Akt pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Physalin B (PB) is an active constituent of Physalis alkekengi L. var. Franchetii, which is a traditional medicine for clearing heat and detoxification, resolving phlegm, and diuresis. It has been commonly applied to treat sore throat, phlegm-heat, cough, dysuria, pemphigus, and eczema. AIM OF STUDY: Physalin B has shown efficacy as an anti-acute lung injury (ALI) agent previously; however, its mechanisms of action remain unclear. In the present study, we established a lipopolysaccharide-induced septic ALI model using BALB/c mice to further confirm the therapeutic potential of PB and to assess the underlying molecular mechanisms. MATERIALS AND METHODS: We used 75% ethanol and macroporous resin for extraction, separation, and enrichment of PB. The LPS-induced ALI mouse model was used to determine anti-inflammatory effects of PB. The severity of acute lung injury was evaluated by hematoxylin and eosin staining, wet/dry lung ratio, and myeloperoxidase (MPO) activity in lung tissue. An automatic analyzer was used to measure the arterial blood gas index. Protein levels of pro-inflammatory cytokines in serum, bronchoalveolar lavage fluid (BALF), and lung tissue was measured using an ELISA. Quantitative RT-PCR was used to measure changes in RNA levels of pro-inflammatory cytokines in the lungs. A fluorometric assay kit was used for determination of apoptosis-related factors to assess anti-apoptotic effects of PB. Western blotting was used to assess levels of key pathway proteins and apoptosis-related proteins. Connections between the pathways were tested through inhibitor experiments. RESULTS: Pretreatment with PB (15 mg kg-1 d-1, i.g.) significantly reduced lung wet/dry weight ratios and MPO activity in blood and BALF of ALI mice, and it alleviated LPS-induced inflammatory cell infiltration in lung tissue. The levels of pro-inflammatory factors TNF-α, IL-6, and IL-1ß and their mRNA levels in blood, BALF, and lung tissue were reduced following PB pretreatment. PB pretreatment also downregulated the apoptotic factors caspase-3, caspase-9, and apoptotic protein Bax, and it upregulated apoptotic protein Bcl-2. The NF-κB and NLRP3 pathways were inhibited through activation of the PI3K/Akt pathway due to PB pretreatment, whereas administration of PI3K inhibitors increased activation of these pathways. CONCLUSIONS: Taken together, our results suggest that the anti-ALI properties of PB are closely associated with the inactivation of NF-κB and NLRP3 by altering the PI3K/Akt pathway. Furthermore, our findings provide a novel strategy for application of PB as a potential agent for treating patients with ALI. To the best of our knowledge, this is the first study to elucidate the underlying mechanism of action of PB against ALI.

Physalin B ameliorates nonalcoholic steatohepatitis by stimulating autophagy and NRF2 activation mediated improvement in oxidative stress.

Non-alcoholic steatohepatitis (NASH) is the progressive stage of non-alcoholic fatty liver disease that may ultimately lead to cirrhosis and liver cancer, and there are few therapeutic options for its treatment. Physalin B (PB), a withanolide isolated from Physalis species (Solanaceae), exhibits a broad spectrum of biological activities, however, the potential role of PB in NASH has not been evaluated. The present study investigated the protective effects of PB against NASH and further elucidated the mechanisms of PB in hepatic autophagy and oxidative stress in vitro and in vivo. We conducted a series of experiments using methionine-choline deficient (MCD) diet induced NASH mice and cultured L02 cells. Serum markers of liver injury, morphology, and the histology of liver tissues were investigated. Western blot assays and quantitative real-time PCR were used to investigate the hepatoprotective effect of PB. PB significantly ameliorated hepatic injury, including hepatic index, transaminase activities, histology, and inflammation in MCD-induced mice. Moreover, PB markedly increased the expression of P62 and the ratio of LC3Ⅱ/Ⅰ in vitro and in vivo. Furthermore, PB promoted the interaction between endogenous KEAP1 and P62, reduced the interaction between KEAP1 and NRF2, activated the nuclear translocation of NRF2 and NRF2 target gene expression, and ultimately attenuated oxidative stress. In addition, knockdown of P62 blocked PB-mediated activation of NRF2 in L02 cells. These results clearly indicated that PB ameliorated NASH by stimulating autophagy and P62-KEAP1-NRF2 antioxidative signaling, suggesting that PB is expected to become a novel therapeutic drug for NASH.

Physalin B reduces Aß 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-regulated ß-amyloid (Aß) 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 in ß-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 Aß secretion, which was caused by down-regulation of ß-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 Aß secretion 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 Aßsecretion by activating the expression of FoxO1 and inhibiting the phosphorylation of STAT3.

Physalin B attenuates liver fibrosis via suppressing LAP2α-HDAC1-mediated deacetylation of the transcription factor GLI1 and hepatic stellate cell activation.

BACKGROUND AND PURPOSE: Liver fibrosis is one of the leading causes of morbidity and mortality worldwide but lacks any acceptable therapy. The transcription factor glioma-associated oncogene homologue 1 (GLI1) is a potentially important therapeutic target in liver fibrosis. This study investigates the anti-fibrotic activities and potential mechanisms of the phytochemical, physalin B. EXPERIMENTAL APPROACH: Two mouse models (CCl4 challenge and bile duct ligation) were used to assess antifibrotic effects of physalin B in vivo. Mouse primary hepatic stellate cells (pHSCs) and human HSC line LX-2 also served as in vitro liver fibrosis models. Liver fibrogenic genes, GLI1 and GLI1 downstream genes were examined using Western blot and quantitative real-time PCR (qRT-PCR). GLI1 acetylation and LAP2α-HDAC1 interaction were analysed by co-immunoprecipitation. KEY RESULTS: In vivo, physalin B administration attenuated hepatic histopathological injury and collagen accumulation and decreased expression of fibrogenic genes. Physalin B dose-dependently suppressed fibrotic marker expression in LX-2 cells and mouse pHSCs. Mechanistic studies showed that physalin B inhibited GLI activity by non-canonical Hedgehog signalling. Physalin B blocked formation of lamina-associated polypeptide 2α (LAP2α)/histone deacetylase 1 (HDAC1) complexes, thus inhibiting HDAC1-mediated GLI1 deacetylation. Physalin B up-regulated acetylation of GLI1, down-regulated expression of GLI1 and subsequently inhibited HSC activation. CONCLUSION AND IMPLICATIONS: Physalin B exerted potent antifibrotic effects in vitro and in vivo by disrupting LAP2α/HDAC1 complexes, increasing GLI1 acetylation and inactivating GLI1. This indicates that the phytochemical physalin B may be a potential therapeutic candidate for the treatment of liver fibrosis.

Anti-colitic effects of Physalin B on dextran sodium sulfate-induced BALB/c mice by suppressing multiple inflammatory signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Physalin B is one of the main active withanolide existed in Physalis alkekengi L. var. franchetii (Mast.) Makino, a famous traditional Chinese food and herbal medicine, which has been widely used as heat-clearing and toxin-resolving medicine for the treatment of various inflammatory disease, such as cough, excessive phlegm, pharyngitis, sore throat, pemphigus, eczema, and jaundice. AIM OF THE STUDY: We aimed to confirm the therapeutic effects of Physalin B on ulcerative colitis (UC) and enrich the further application of its traditional anti-inflammatory effect. MATERIALS AND METHODS: The anti-UC effects of Physalin B were evaluated in Balb/c mice with dextran sulfate sodium (DSS) induction. The body weight, colon length, disease activity index (DAI) and pathological changes of colon tissue were measured. Cytokine levels were detected by ELISA. NF-κB pathway and protein levels of related pathways, such as signal transducer and activator of transcription 3 (STAT3), ß-arrestin1 and NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome were detected by western blot. RESULTS: The dose of Physalin B that is not cytotoxic could dramatically reduce the levels of TNF-α, IL-6 and IL-1ß on LPS-stimulated RAW 264.7 cells. Meanwhile, Physalin B dramatically improved clinical signs and symptoms, alleviated body weight loss and colon length shortening in DSS-induced UC mice. Meanwhile, Physalin B also dramatically relieved the pathological damage, reduced in the activity of myeloperoxidase (MPO) and reestablished the balance of pro-inflammatory cytokines. Physalin B could suppress DSS-induced activation of NF-κB. Moreover, Physalin B also markedly suppressed the activation of STAT3, ß-arrestin1 and NLRP3 inflammasome. CONCLUSION: This study preliminary confirmed the therapeutic effect of Physalin B on experimental acute UC mice and provided robust evidence support for the anti-inflammatory effect of Physalin B, suggesting that Physalin B might be a potential agent for the therapeutic efficacy on UC.

Physalin B induces cell cycle arrest and triggers apoptosis in breast cancer cells through modulating p53-dependent apoptotic pathway.

Physalin B (PB), one of the major active steroidal constituents of Cape gooseberry (Physalis alkekengi L.), possesses a wide spectrum of biological activities. Although the anticancer activity of PB was reported in previous studies, the underlying mechanisms are still not well stated. In this study, the anticancer effect and the underlying mechanisms of PB were investigated in breast cancer cells. PB significantly reduced the viability of three human breast cancer cell lines, MCF-7, MDA-MB-231 and T-47D, in a concentration- and time-dependent manner. PB induced cell cycle arrest at G2/M phase and promoted cleavage of PARP (poly (ADP-ribose) polymerase), caspases 3, caspase 7 and caspase 9 to stimulate cell apoptosis. Further studies showed that PB induced breast cancer cells apoptosis in a p53-dependent manner in MCF-7 cells. PB also suppressed the phosphorylation of Akt (protein kinase B) and PI3K (phosphoinositide 3-kinase), and increased the phosphorylation of GSK-3ß (glycogen synthase kinase 3ß). Taken together, our results indicated that PB might serve as a potential therapeutic agent for breast cancer.

Physalis alkekengi L. var. franchetii (Mast.) Makino: An ethnomedical, phytochemical and pharmacological review.

ETHNOPHARMACOLOGICAL RELEVANCE: The calyxes and fruits of Physalis alkekengi L. var. franchetii (Mast.) Makino (Physalis Calyx seu Fructus), have been widely used in traditional and indigenous Chinese medicines for the therapy of cough, excessive phlegm, pharyngitis, sore throat, dysuria, pemphigus, eczema, and jaundice with a long history. AIM OF THE REVIEW: The present review aims to achieve a comprehensive and up-to-date investigation in ethnomedical uses, phytochemistry, pharmacology, and toxicity of P. alkekengi var. franchetii, particularly its calyxes and fruits. Through analysis of these findings, evidences supporting their applications in ethnomedicines are illustrated. Possible perspectives and opportunities for the future research are analyzed to highlight the gaps in our knowledge that deserves further investigation. MATERIAL AND METHODS: Information on P. alkekengi var. franchetii was collected via electronic search of major scientific databases (e.g. Web of Science, SciFinder, Google Scholar, Pubmed, Elsevier, SpringerLink, Wiley online and China Knowledge Resource Integrated) for publications on this medicinal plant. Information was also obtained from local classic herbal literature on ethnopharmacology. RESULTS: About 124 chemical ingredients have been characterized from different parts of this plant. Steroids (particularly physalins) and flavonoids are the major characteristic and bioactive constituents. The crude extracts and the isolated compounds have demonstrated various in vitro and in vivo pharmacological functions, such as anti-inflammation, inhibition of tumor cell proliferation, antimicrobial activity, diuretic effect, anti-diabetes, anti-asthma, immunomodulation, and anti-oxidation. CONCLUSIONS: P. alkekengi var. franchetii is an important medicinal plant for the ethnomedical therapy of microbial infection, inflammation, and respiratory diseases (e.g. cough, excessive phlegm, pharyngitis). Phytochemical and pharmacological investigations of this plant definitely increased in the past half century. The chemical profiles, including ingredients and structures, have been adequately verified. Modern pharmacological studies supported its uses in the traditional and folk medicines, however, the molecular mechanisms of purified compounds remained unclear and were worth of further exploration. Therefore, the researchers should be paid more attention to a better utilization of this plant.

Six new physalins from Physalis alkekengi var. franchetii and their cytotoxicity and antibacterial activity.

Six new physalin steroids, 7ß-methoxylisophysalin B (1), 7ß-methoxylphysalin C (2), physalin V (3), physalin VI (4), physalin VII (5), isophysalin I (6), together with 20 known physalins (7-26) were isolated from calyces of Physalis alkekengi var. franchetii. Structures of the new compounds were revealed through 1D and 2D NMR and mass spectroscopic methods. Compounds 1-26 were evaluated for cytotoxicity against human HL-60, SMMC-7721, A-549, MCF-7 and SW-480, and the results indicated that compounds 8, 11, and 14 displayed potent cytotoxicities (IC50<5µM) in vitro. Further antibacterial assay indicated that compounds 8, 14, and 19 showed high antibacterial activities against Bacillus subtilis and Escherichia coli.