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.

Biogenesis-Guided Synthesis and Structural Revision of Sarocladione Enabled by Ruthenium-Catalyzed Endoperoxide Fragmentation.

Sarocladione is the first 5,10:8,9-diseco-steroid with a 14-membered macrocyclic diketone framework to have been isolated from a natural source. Herein we report a biomimetic synthesis of sarocladione in only two or seven steps from inexpensive, commercially available ergosterol. The key feature of this synthesis was a novel ruthenium-catalyzed endoperoxide fragmentation, which transformed various saturated endoperoxides into olefinic diketones by cleavage of two C-C bonds. This synthesis allowed us to unambiguously determine the structure of sarocladione and provided experimental support for its revised biosynthetic origin. This work also vividly demonstrates that consideration of the biogenesis is a powerful tool for elucidating the structures of natural products.

Biomimetic Synthesis of the CDE Ring Moiety of Physalins, Complex 13,14-Secosteroids.

Physalins are a structurally complex family of 13,14-secosteroids isolated from the genus Physalis. We disclose a two-step construction of the CDE ring moiety of the physalins from a steroidal compound bearing 14-OH, 18-COOMe, and 17, 20-α-epoxide based on our biosynthetic proposal. C13-C14 bond cleavage by an alkoxy radical at C-14 and spontaneous epoxide ring opening gave a compound having a cyclononene and γ-lactone. Diastereoselective dihydroxylation of the resulting alkene with OsO4 provided the CDE ring moiety of physalin.

Synthesis of DFGH-Ring Derivatives of Physalins via One-Pot Construction of GH-Ring and Evaluation of Their NF-κB-Inhibitory Activity.

We designed and synthesized a series of derivatives containing the right-side DFGH-ring structure of physalin-type natural products, decorated with a hydrophobic substituent. The synthetic scheme utilizes a highly efficient, one-pot protocol for simultaneous construction of the GH-ring system, promoted by HF/pyridine. Among the compounds synthesized, 5d inhibited TNF-α-stimulated NF-κB activation with similar potency to physalin B.

1H NMR-Based Isolation of Anti-Inflammatory 9,11-Secosteroids from the Octocoral Sinularia leptoclados.

Octocoral Sinularia leptoclados has been identified as a source of bioactive 9,11-secosteroids. This study adopted a targeted isolation approach to the discovery and analysis of five 9,11-secosteroids, including two novel compounds named sinleptosterols A (1) and B (2) as well as five known analogues (8αH-3ß,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9-one (3), 8ßH-3ß,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9-one (4), leptosterol A (5), (24S)-3ß,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9-one (6), and 3ß,11-dihydroxy-9,11-secogorgost-5-en-9-one (7)) in terms of 1H-NMR patterns and potency against neutrophilic inflammation. The structure of secosteroids 1 and 2 was deduced from general spectroscopic analysis and an examination of NMR spectra. Among the above-mentioned isolates, compound 4 had the most pronounced effect in inhibiting elastase release and superoxide anion generation, with the IC50 values of 2.96 and 1.63 µM, respectively.

Ubiquitin-Proteasome Modulating Dolabellanes and Secosteroids from Soft Coral Clavularia flava.

We performed a high-content screening (HCS) assay aiming to discover bioactive molecules with proteasome inhibitory activity. By structural elucidation, we identified six compounds purified from soft coral Clavularia flava, which potentiates proteasome inhibition. Chemical structure elucidation revealed they are dolabellane- and secosteroid-based compounds including a new dolabellane, clavinflol C (1), three known dolabellanes, stolonidiol (2), stolonidiol-17-acetate (3), and clavinflol B (4) as well as two new secosteroids, 3,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9,23-dione (5) and 3,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9,23-dione (6). All six compounds show less cytotoxicity than those of known proteasome inhibitors, bortezomib and MG132. In summary, the high-content measurements of control inhibitors, bortezomib and MG132, manifest the highest ratio >2 in high-content measurement. Of the isolated compounds, 2 and 5 showed higher activity, followed by 3 and 6, and then 1 and 4 exhibited moderate inhibition.

A new 22,26-seco physalin steroid from Physalis angulata.

A new 22,26-seco physalin, physalin XI (1) together with 5 known compounds, were isolated from the dichloromethane extract of Physalis angulata L. The structure of isolated compounds was elucidated by spectroscopic analysis. The effects of isolated compounds on in vitro cytotoxicity were investigated. Compound 1 was assessed for its cytotoxicity against cancer cell lines (HepG2, HeLa, HuCCA-1, T47-D and A-549) and a normal cell line (MRC-5), and the result showed that it has no activity. Compounds 2 and 4 are highly toxic to H69AR and MDA-MB-23 cell lines. This property appears to be related to the presence of their conjugated double bond or epoxy groups and is a more reliable indication of toxicity than substitution on C(5)-C(6).

Sarocladione, a unique 5,10:8,9-diseco-steroid from the deep-sea-derived fungus Sarocladium kiliense.

A novel ergostane, sarocladione (1), was isolated from the deep-sea-derived fungus Sarocladium kiliense, along with 20 known compounds. The structure of 1 was determined mainly by a detailed analysis of its experimental and calculated NMR spectroscopic data. It is worth noting that 1 was the first steroid bearing a 5,10:8,9-diseco moiety. All 21 compounds were tested for in vitro antitumor activities against five cancer cell lines. ß-Sitostenone (7) and 4,6-dihydroxyeudesmane (20) showed significant effects on HeLa-S3 cells with the IC50 values of 9.2 µM and 9.3 µM, respectively.

Physalin D regulates macrophage M1/M2 polarization via the STAT1/6 pathway.

The in vitro and in vivo effects of physalin D on macrophage M1/M2 polarization were investigated. In silico analysis was first performed for biological function prediction of different physalins. The results suggest physalins have similar predicted biological functions due to their similarities in chemical structures. The cytotoxicity of physalins was then analyzed based on cell apoptosis rate and cell viability evaluation. Physalin D was chosen for further study due to its minimal cytotoxicity. Bone marrow macrophages were isolated and induced with lipopolysaccharide/interferon (IFN)-γ for M1 polarization and interleukin (IL)-4/IL-13 for M2 polarization. The results showed that physalin D can repolarize M1 phenotype cells toward M2 phenotype. In addition, physalin D is protective in M2 macrophages to maintain the M2 phenotype in the presence of IFN-γ. On the molecular level, we found that physalin D suppressed the signal transducers and activators of transcription (STAT)1 activation and blocked STAT1 nuclear translocation. Conversely, physalin D can also activate STAT6 and enhance STAT6 nuclear translocation for M2 polarization. Taken together, these results suggested that physalin D regulates macrophage M1/M2 polarization via the STAT1/6 pathway.

Aspersecosteroids A and B, Two 11(9 → 10)- abeo-5,10-Secosteroids with a Dioxatetraheterocyclic Ring System from Aspergillus flocculosus 16D-1.

Two 11(9 → 10)- abeo-5,10-secosteroids possessing an unprecedented dioxatetraheterocyclic ring system, aspersecosteroids A (1) and B (2), and a new ergosteroid, asperflosterol (3), were isolated from the sponge-derived fungus Aspergillus flocculosus 16D-1. Their structures were determined by spectroscopic analysis, X-ray diffraction, and computational methods. Compounds 1-3 demonstrated inhibitory effects on key pro-inflammatory cytokine production in THP-1 cells. A biogenetic pathway with oxidative cleavage, Wagner-Meerwein rearrangement, and sequential acetalization as key steps is proposed for 1 and 2.

YAP-dependent ubiquitination and degradation of ß-catenin mediates inhibition of Wnt signalling induced by Physalin F in colorectal cancer.

Aberrant activation of Wnt/ß-catenin signalling is critical in the progression of human cancers, especially colorectal cancer (CRC). Therefore, inhibition of Wnt/ß-catenin signalling is a significant potential target for CRC therapy. Here, we identified for the first time that Physalin F (PF), a steroid derivative isolated from Physalis angulate, acts as an antagonist of Wnt/ß-catenin signalling. In vitro, PF decreased Wnt3a-induced TOPFlash reporter activity in HEK293T cells and promoted the formation of the ß-catenin destruction complex. Importantly, PF also inhibited Wnt/ß-catenin signalling and accelerated the degradation of ß-catenin in CRC cells. However, PF did not affect the stabilization of Axin or the interaction of ß-catenin with E-cadherin. Interestingly, we further found that PF promoted YAP binding to the ß-catenin destruction complex, which facilitated the ubiquitination and degradation of ß-catenin. Silencing and pharmacological inhibition of YAP reversed the formation of the ß-catenin destruction complex induced by PF, implying that YAP binding to the ß-catenin destruction complex was responsible for PF-mediated inhibition of Wnt/ß-catenin signalling. Furthermore, PF observably inhibited tumour growth by down-regulating ß-catenin in tumour-bearing mice. Collectively, our findings indicated that PF inhibited Wnt/ß-catenin signalling by accelerating the ubiquitination and degradation of ß-catenin in a YAP-dependent manner and therefore PF could be a novel potential candidate for CRC therapy.

Cynatratoside-C from Cynanchum atratum displays anti-inflammatory effect via suppressing TLR4 mediated NF-κB and MAPK signaling pathways in LPS-induced mastitis in mice.

The present study was conducted to isolate anti-inflammatory compound from Cynanchum atratum and investigate the molecular mechanisms of active compound against lipopolysaccharide (LPS)-induced mastitis in mice. Bioassay-guided fractionations and isolation (via ex vivo tests) of compounds with anti-inflammatory activity were performed on roots of C. atratum yielding a pure bioactive compound: Cynatratoside-C, identified by comparing spectral data (EI-MS, 1H NMR and 13C NMR) with literature values. Ex vivo tests showed that Cynatratoside-C inhibited the expression of TLR4 and pro-inflammatory cytokine (TNF-α, IL-6 and IL-1ß) production in LPS-stimulated primary mouse mammary epithelial cells. In vivo results indicated that Cynatratoside-C markedly attenuated LPS-induced mammary histopathologic changes and mammary oxidative stress (MDA, SOD, GPx) activity. Besides, Cynatratoside-C blocked the expression of Toll-like receptor 4 (TLR4) and then suppressed the phosphorylation of nuclear transcription factor-kappa B (NF-κB) p65 and degradation inhibitor of NF-κBα (IκBα). Further study showed that Cynatratoside-C could suppress the phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) in mitogen-activated protein kinase (MAPK) signal pathway. In conclusion, our results suggest that Cynatratoside-C played an anti-inflammatory role in LPS-induced mastitis by regulating TLR4 and the NF-κB and MAPK signaling pathways in mammary gland tissues. Cynatratoside-C may be a promising potential therapeutic reagent for the treatment of mastitis.

High performance thin-layer chromatography-mass spectrometry enables reliable analysis of physalins in different plant parts of Physalis alkekengi L.

We developed first HPTLC and HPTLC-MS/MS methods which enable characterization of structurally similar and complex biologically active compounds - physalins - from crude extracts of Chinese lantern (Physalis alkekengi L.). Separation on HPTLC silica gel plates developed with ethyl acetate-toluene-formic acid (7:3:0.2, v/v) enabled densitometric screening of physalins in absorption and, after post-chromatographic derivatization with sulfuric acid reagent, also in fluorescence mode. Compared to existing (U)HPLC methods, in this case TLC provides an alternative selectivity, better sensitivity and higher resolution, which was exemplified by the separation of physalin L standard and its impurity, identified as 2,3,25,27-tetrahydrophysalin A. Strong ion suppression caused by the developing solvent additive - formic acid - was efficiently solved by two successive plate pre-developments with methanol-formic acid (9:1, v/v) and methanol. This significantly improved the sensitivity of HPTLC-MS/MS method, but also required a slightly modified developing solvent ethyl acetate-toluene-formic acid (6:4:0.2, v/v). Simultaneous hyphenation of HPTLC with a triple quadrupole and an ion trap mass analyzer enabled a reliable and straightforward non-targeted characterization of physalins from the same chromatographic zone (band) and determination of physalin types. The performance of developed HPTLC-densitometric and HPTLC-MS/MS methods was demonstrated by the analysis of physalins from the aqueous extracts, prepared by an optimized fast and simple extraction method under reflux. Variations in physalin profiles and abundances in different parts of P. alkekengi L. harvested at different stages of maturity were observed. This indicates that not all parts of the plant, or plant as a whole, are appropriate for specific medicinal applications. Husks are proposed as the most suitable plant part for P. alkekengi L. quality control, because they exhibited the most obvious MS2 fingerprints of physalins with minimal interferences.

Synthesis of cytotoxically active derivatives based on alkylated 2,3-seco-triterpenoids.

Extremely low content of biologically active triterpenoids with the fragmented or contracted ring A extractable from plants is the main disadvantage of their use in drug discovery and practical pharmacology. Development of new methods for synthesis of these compounds and their structural analogs from bioavailable triterpene precursors gives an opportunity to obtain promising agents for pharmacology with excellent yields. A new approach to synthesis of alkylated A-seco-triterpenoids, including the Beckmann fragmentation of 3-methyl-substituted allobetulin or betulinic acid methyl ester with 2-hydroxyimino group in the ring A was proposed. These compounds were used to prepare a series of 2,3-seco- and five-membered ring A lupane and oleanane derivatives, cytotoxicity of which was screened in vitro against the cancer (HEp-2, HCT 116, A549, RD TE32, MS) and non-cancerous (HEK 293) cell lines. Methyl 3-bromomethyl-1-cyano-3-oxo-2,3-seco-2-norlup-20(29)-en-30-al-28-oate was selected as the most active compound (IC50 3.4-10.4 µM for HEp-2, HCT 116, RD TE32, MS cells) capable of triggering caspase-8-mediated apoptosis in HCT 116 cells accompanied by typical apoptotic chromatin condensation, without any loss of mitochondrial membrane permeability.

Isolation and quantitative analysis of physalin D in the fruit and calyx of Physalis alkekengi L.

Physalin D was isolated from the methanol extract of Physalis alkekengi L. fruits by combination of different chromatographic methods (CPC, TLC, HPLC). The structure was elucidated based on 1H and 13C NMR spectral analysis with the aid of 2D-correlation spectroscopy (1H, 1H-COSY, HSQC and HMBC) and comparison with literature data. The quantity of physalin D in mature and immature fruits and calyces was determined by RP-HPLC-UV method. Among the studied samples, immature calyx showed the highest content of physalin D (0.7880 ± 0.0612%), while mature calyx contained 4 times less amount (0.2028 ± 0.016%). The physalin D content of the fruit was much lower; immature fruits contained 0.0992 ± 0.0083% physalin D and mature fruits 0.0259 ± 0.0021%. The antiproliferative activity of the CHCl3 extract and its fractions was tested on three cancer cell lines (HeLa, MCF-7 and A431). The antiproliferative activity of physalin D is discussed with regard the published data.

LC-MS/MS method for simultaneous determination of flavonoids and physalins in rat plasma: Application to pharmacokinetic study after oral administration of Physalis alkekengi var. franchetii (Chinese lantern) extract.

A rapid and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of luteolin, luteolin-7-O-ß-D-glucopyranoside, physalin A, physalin D and physalin L in rat plasma. Scutellarein and dexamethasone were used as the internal standards (IS). Plasma samples were prepared by liquid-liquid extraction with ethyl acetate. The five constituents were separated on an Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm). A gradient elution procedure was used with acetonitrile (A)-0.1% aqueous formic acid (B). Mass spectrometric detection was performed in negative ion multiple reaction monitoring mode with an electrospray ionization (ESI) source. This method showed good linearity (r2 > 0.997) over a concentration range of 2.0-500 ng/mL with a lower limit of quantification of 2.0 ng/mL for all five compounds. The inter- and intra-day accuracy ranged from 91.7 to 104%, and precisions (RSD) were <6.46% for all analytes. The extraction recoveries of all analytes were >85%. This validated method was successfully applied for the first time to the pharmacokinetic study of five ingredients after oral administration of 70% ethanol extract of Chinese lantern in rats.

Anti-inflammatory effects of physalin E from Physalis angulata on lipopolysaccharide-stimulated RAW 264.7 cells through inhibition of NF-κB pathway.

Physalin E is a naturally occurring seco-steroid isolated from the stems and aerial parts of Physalis angulata L. (Solanaceae). This study was aimed to explore the anti-inflammatory effects of physalin E on RAW 264.7 mouse macrophages stimulated by lipopolysaccharide (LPS) and the potential underlying mechanisms. The results showed that physalin E significantly inhibited LPS-induced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) expression and secretion in a dose-dependent manner. Unlike dexamethasone, these effects could not be blocked by miferstone (RU486). Meanwhile, physalin E reduced the degradation of I-kappa B protein in the cytoplasm and downregulated the nuclear factor-κB (NF-κB) p65 protein in the nuclear, which resulted in the inhibition of the NF-κB nuclear translocation. In conclusion, physalin E exerts its anti-inflammatory activities in LPS-induced macrophages. Physalin E can inhibit the production of inflammatory cytokines by targeting the NF-κB signaling pathway.

New Anti-Inflammatory 9,11-Secosterols with a Rare Tricyclo[5,2,1,1]decane Ring from a Formosan Gorgonian Pinnigorgia sp.

Pinnigorgiols D (1) and E (2), two new 9,11-secosterols with a rearranged carbon skeleton, were isolated from a Taiwan gorgonian Pinnigorgia sp. The structures of these two compounds were elucidated on the basis of spectroscopic methods and were proven to possess a tricyclo[5,2,1,1]decane ring. The new secosterols 1 and 2 displayed significant inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils.

Synthesis of Strophasterol†A Guided by a Proposed Biosynthesis and Innate Reactivity.

The synthesis of strophasterol A, a moderator of endoplasmatic reticulum (ER) stress in Alzheimer's disease, and the first member of a structurally unprecedented class of secosterols, was achieved through the implementation of a key step of its proposed biosynthesis and two C-H oxidations. Analysis of the innate reactivity of the intermediates enabled the identification of a novel way to prepare an α-chloro-γ-hydroxy-δ-keto enone, as well as its vinylogous α-ketol rearrangement to a δ-keto carboxylic acid.

New anti-inflammatory sterols from a gorgonian Pinnigorgia sp.

Chemical investigation on the EtOAc-soluble fraction from the MeOH/DCM extract of a gorgonian Pinnigorgia sp. afforded two new sterols, 11-acetoxy-24S-methyl-3ß,5α,6α-trihydroxy-9,11-secocholest-7-en-9-one (1) and 5ß,6ß-epoxy-(22E,24R)-ergosta-8,22-diene-3ß,7ß-diol (2). The structures of sterols 1 and 2 were elucidated on the basis of spectroscopic analysis and by comparison of their spectroscopic data with those of related analogues. Both 1 and 2 were shown to significantly inhibit the accumulation of the pro-inflammatory iNOS and COX-2 protein in LPS-stimulated RAW264.7 macrophage cells.