Rapid Discovery of Substances with Anticancer Potential from Marine Fungi Based on a One Strain-Many Compounds Strategy and UPLC-QTOF-MS.

The secondary metabolites of marine fungi with rich chemical diversity and biological activity are an important and exciting target for natural product research. This study aimed to investigate the fungal community in Quanzhou Bay, Fujian, and identified 28 strains of marine fungi. A total of 28 strains of marine fungi were screened for small-scale fermentation by the OSMAC (One Strain-Many Compounds) strategy, and 77 EtOAc crude extracts were obtained and assayed for cancer cell inhibition rate. A total of six strains of marine fungi (P-WZ-2, P-WZ-3-2, P-WZ-4, P-WZ-5, P56, and P341) with significant changes in cancer cell inhibition induced by the OSMAC strategy were analysed by UPLC-QTOF-MS. The ACD/MS Structure ID Suite software was used to predict the possible structures with inhibitory effects on cancer cells. A total of 23 compounds were identified, of which 10 compounds have been reported to have potential anticancer activity or cytotoxicity. In this study, the OSMAC strategy was combined with an untargeted metabolomics approach based on UPLC-QTOF-MS to efficiently analyse the effect of changes in culture conditions on anticancer potentials and to rapidly find active substances that inhibit cancer cell growth.

Total Synthesis and Target Identification of the Curcusone Diterpenes.

The curcusone natural products are complex diterpenes featuring a characteristic [6-7-5] tricyclic carbon skeleton similar to the daphnane and tigliane diterpenes. Among them, curcusones A-D demonstrated potent anticancer activity against a broad spectrum of human cancer cell lines. Prior to this study, no total synthesis of the curcusones was achieved and their anticancer mode of action remained unknown. Herein, we report our synthetic and chemoproteomics studies of the curcusone diterpenes which culminate in the first total synthesis of several curcusone natural products and identification of BRCA1-associated ATM activator 1 (BRAT1) as a cellular target. Our efficient synthesis is highly convergent, builds upon cheap and abundant starting materials, features a thermal [3,3]-sigmatropic rearrangement and a novel FeCl3-promoted cascade reaction to rapidly construct the critical cycloheptadienone core of the curcusones, and led us to complete the first total synthesis of curcusones A and B in only 9 steps, C and D in 10 steps, and dimericursone A in 12 steps. The chemical synthesis of dimericursone A from curcusones C and D provided direct evidence to support the proposed Diels-Alder dimerization and cheletropic elimination biosynthetic pathway. Using an alkyne-tagged probe molecule, BRAT1, an important but previously "undruggable" oncoprotein, was identified as a key cellular target via chemoproteomics. We further demonstrate for the first time that BRAT1 can be inhibited by curcusone D, resulting in impaired DNA damage response, reduced cancer cell migration, potentiated activity of the DNA damaging drug etoposide, and other phenotypes similar to BRAT1 knockdown.

Transcriptomic analyses reveal antiinflammatory mechanism of withanolides derived from the fruits of Physalis alkekengi L. var. franchetii.

In China, the fruits of Physalis alkekengi L. var. franchetii, which are conventionally utilized as edible berry, have attracted wide attention due to its significant biological activities. In the present study, phytochemical studies on the fruits of Physalis plants afforded six compounds, including two new withanolides (1-2) and four known agnologues (3-6). The inhibitory effects of these compounds on the formation of nitric oxide (NO) stimulated by lipopolysaccharide (LPS) in RAW264.7 macrophages were evaluated. Physapubescin M (1), with IC50 value of 1.58 µM, was selected for further study. The protein expression of COX-2 and iNOS, and LPS-induced production of cytokines (IL-6, IL-1ß and TNF-α) were reduced by physapubescin M (1) in a dose-dependent way. In addition, transcriptomic analyses were conducted to profile gene expression alterations in LPS-induced RAW264.7 cells upon treatment of physapubescin M (1) and the potential antiinflammatory mechanism of withnolides was mentioned. These results provide broad view to the underlying antiinflammatory mechanism of withnolides, and give a theoretical basis for the utilization of the fruits of P. alkekengi L. var. franchetii.

Physalis peruviana-Derived 4ß-Hydroxywithanolide E, a Novel Antagonist of Wnt Signaling, Inhibits Colorectal Cancer In Vitro and In Vivo.

Deregulation of the Wnt signaling pathway leads to colorectal cancer progression. Natural dietary compounds serve as promising candidates for development as chemopreventive agents by suppressing the Wnt/ß-catenin signaling pathway. Physalis peruviana-derived 4ßHWE showed a significant inhibitory activity with a calculated IC50 of 0.09 µΜ in a screening of novel inhibitors of Wnt signaling with the dual-luciferase reporter assay. This study investigated the anti-tumor effect of 4ßHWE and the potential Wnt signaling inhibitory mechanism. Both the western blot analysis and immunofluorescence assay showed that 4ßHWE promoted the phosphorylation and degradation of ß-catenin and the subsequent inhibition of its nuclear translocation to attenuate the endogenous Wnt target gene expression in colorectal cancer (CRC) cells. The cell viability assay indicated that 4ßHWE preferentially inhibited the proliferation of CRC compared with CCD-841-CoN, a normal human colonic epithelial cell line. 4ßHWE-mediated G0/G1 cell cycle arrest and apoptosis induction contributed to the suppression of the proliferation of CRC in the cell cycle and Annexin V-FITC/Propidium Iodide apoptosis analysis. Moreover, in vivo, 4ßHWE dramatically inhibited tumor growth in HCT116 xenografts by attenuating the Wnt/ß-catenin signaling pathway. In conclusion, our study suggested that 4ßHWE could be of potential use in anti-tumor agent development as a novel Wnt signaling inhibitor.

Dimericursones A and B: two unprecedented hexacyclic dimeric diterpenoids from the root barks of Jatropha curcas.

Dimericursones A and B (1 and 2), two unprecedented hexacyclic dimeric diterpenoids, were obtained from the root barks of Jatropha curcas. Their structures were elucidated by extensive spectroscopic analysis, electronic circular dichroism calculations, and single-crystal X-ray diffraction. Dimericursone B (2) showed significant inhibition on nitric oxide production of lipopolysaccharide-induced RAW264.7 macrophages with IC50 values of 5.65 µM.

The antigluconeogenic activity of cucurbitacins from Momordica charantia.

Five new cucurbitacins, kuguacins II-VI (1-5), along with five known analogues (6-10), were obtained from the fruit of Momordica charantia. Structures of the new compounds were elucidated as 5ß,19-epoxycucurbit-23-en-7-on-3ß,25-diol (1), 5ß,19-epoxycucurbit-7,23-dion-3ß,25-diol (2), 5ß,19-epoxycucurbit-6-en-19,23-dion-3ß,25-diol (3), 5ß,19-epoxy-23,24,25,26,27-pentanorcucurbit-6-en-7,19-dion-3ß,22-diol (4), and cucurbit-5-en-7,23-dion-3ß,19,25-triol (5) by extensive spectroscopic and single-crystal X-ray diffraction analyses. Some cucurbitane compounds from this species were screened for their potential antidiabetic properties in terms of antigluconeogenic activity. As a result, compounds 1, 10, 11, and 12 (at 25-100 µM) showed concentration-dependent inhibition on glucose production from liver cells. In addition, compounds 11 and 12 (at 100 µM) showed around 20-30 % inhibition on PEPCK activity.

Identification and antifeedant activities of limonoids from Azadirachta indica.

Four new limonoids, azadiraindins A-D (1-4, resp.), together with seven known analogs, were isolated from the MeOH extract of Azadirachta indica. The structures of 1-4 were elucidated by NMR and MS spectroscopic analyses, and the relative configuration of 1 was determined by single-crystal X-ray crystallography. The compounds isolated in comparatively large amount were evaluated for their antifeedant activities against Plutella xylostella; the antifeedant rate of 10 was 90.6% and the corrected mortality of 8 was 79.2%.

Three new limonoids from Azadirachta indica.

Three new limonoids, azadiraindins E-G (1-3, respectively), together with six known analogs, were isolated from the fresh fruit coats of Azadirachta indica. The structures of these compounds were elucidated by spectroscopic methods (IR, MS, HR-ESI-MS, 1D NMR, and 2D NMR).

Hepatoprotective effects of triterpenoids from Ganoderma cochlear.

Two novel trinorlanostanes, cochlates A and B (1 and 2), with a 3,4-seco-9,10-seco-9,19-cyclo skeleton, as well as six new triterpenoids, fornicatins D-F (3-5) and ganodercochlearins A-C (6-8), together with five known triterpenoids (9-13), were obtained from the fruiting bodies of Ganoderma cochlear. The structural elucidation was achieved by interpretation of spectroscopic data, and compounds 2 and 7a were further characterized by X-ray crystallographic analysis. Fornicatins A, D, and F (10, 3, and 5) and fredelin (13) lowered the ALT and AST levels in HepG2 cells treated with H2O2, suggesting that they could display in vivo hepatoprotective activities.

Three new tetranorditerpenes from aerial parts of acerola cherry (Malpighia emarginata).

Acerola cherry is a world famous fruit which contains abundant antioxidants such as vitamin C, anthocyanins, flavonoids, and phenolics. However, studies concerning bioactivity components from aerial parts of acerola (Malpighia emarginata) are scarce. In view of this, we have examined the constituents of aerial parts of acerola, and three new tetranorditerpenes acerolanins A-C (1-3) with a rare 2H-benz[e]inden-2-one substructure were isolated. Their structures were determined on the basis of spectral studies and acerolanin C was confirmed by X-ray crystallographic analysis. Furthermore, three new compounds have been studied for their cytotoxic activity.

Determination of arbutin in vitro and in vivo by LC-MS/MS: Pre-clinical evaluation of natural product arbutin for its early medicinal properties.

ETHNOPHARMACOLOGICAL RELEVANCE: Arbutin is a naturally occurring glucoside extracted from plants, known for its antioxidant and tyrosinase inhibiting properties. It is widely used in cosmetic and pharmaceutical industries. With in-depth study of arbutin, its application in disease treatment is expanding, presenting promising development prospects. However, reports on the metabolic stability, plasma protein binding rate, and pharmacokinetic properties of arbutin are scarce. AIM OF THE STUDY: The aim of this study is to enrich the data of metabolic stability and pharmacokinetics of arbutin through the early pre-clinical evaluation, thereby providing some experimental basis for advancing arbutin into clinical research. MATERIALS AND METHODS: We developed an efficient and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for determining arbutin in plasma. We investigated the metabolic and pharmacokinetic properties of arbutin through in vitro metabolism assay, cytochrome enzymes P450 (CYP450) inhibition studies, plasma protein binding rate analysis, Caco-2 cell permeability tests, and rat pharmacokinetics to understand its in vivo performance. RESULTS: In vitro studies show that arbutin is stable, albeit with some species differences. It exhibits low plasma protein binding (35.35 ± 11.03% âˆ¼ 40.25 ± 2.47%), low lipophilicity, low permeability, short half-life (0.42 ± 0.30 h) and high oral bioavailability (65 ± 11.6%). Arbutin is primarily found in the liver and kidneys and is eliminated in the urine. It does not significantly inhibit CYP450 up to 10 µM, suggesting a low potential for drug interactions. Futhermore, preliminary toxicological experiments indicate arbutin's safety, supporting its potential as a therapeutic agent. CONCLUSION: This study provides a comprehensive analysis the drug metabolism and pharmacokinetics (DMPK) of arbutin, enriching our understanding of its metabolism stability and pharmacokinetics properties, It establishes a foundation for further structural optimization, pharmacological studies, and the clinical development of arbutin.

Isolation and bioactivity evaluation of terpenoids from the medicinal fungus Ganoderma sinense.

A new pentanorlanostane, ganosineniol A (1), eight new lanostane triterpenoids, ganosinoside A (2), ganoderic acid Jc (3), ganoderic acid Jd (4), ganodermatetraol (5), ganolucidic acid γa (6), ganolucidate F (7), ganoderiol J ( 8), and methyl lucidenate Ha ( 9), and a new sesquiterpenoid, ganosinensine (10), together with eleven known triterpenoids (11- 21), were isolated from the fruiting bodies of the fungus Ganoderma sinense. Chemical structures were determined based on spectroscopic evidence, including 1D, 2D NMR, and mass spectral data. Furthermore, all isolates were tested for cytotoxic activity and induction ability of hPXR-mediated CYP3A4 expression. Among them, ganoderic acid Jc (3) displayed selective inhibitory activity against HL-60 cells (IC50 = 8.30 µM), and ganoderiol E (11) exhibited selective cytotoxic activity against MCF-7 cells (IC50 = 6.35 µM). Meanwhile, compounds 5, 7, and ganolucidic acids B and C (19, 20) showed induction ability of hPXR-mediated CYP3A4 expression.

Ganoderma Lucidum Triterpenoids Improve Maternal Separation-Induced Anxiety- and Depression-like Behaviors in Mice by Mitigating Inflammation in the Periphery and Brain.

Although early life stress (ELS) can increase susceptibility to adulthood psychiatric disorders and produce a greater inflammatory response in a stressful event, targeted preventive and therapeutic drugs still remain scarce. Ganoderma lucidum triterpenoids (GLTs) can exert anti-inflammatory effects in the periphery and central nervous systems. This study employed a combined model of "childhood maternal separation + adulthood sub-stress" to explore whether GLTs may alleviate anxiety- and depression-like behaviors in male and female mice by mitigating inflammation. Male and female pups were separated from their mothers for four hours per day from postnatal day 1 (PND 1) to PND 21; starting from PND 56, GLTs were administered intraperitoneally once daily for three weeks and followed by three days of sub-stress. Results showed that maternal separation increased the anxiety- and depression-like behaviors in both male and female mice, which disappeared after the preemptive GLTs treatment (40 mg/kg) before adulthood sub-stress. Maternal separation up-regulated the pro-inflammatory markers in the periphery and brain, and activated microglia in the prefrontal cortex and hippocampus. All the abnormalities were reversed by GLTs administration, with no adverse effects on immune organ indices, liver, and renal function. Our findings suggest that GLTs can be a promising candidate in treating ELS-induced psychiatric disorders.

The synthesis and cytotoxic activity of derivatives of 4ß-hydroxywithanolide E.

4ß-Hydroxywithanolide E, which can be obtained in large amounts from the Physalis genus, possessed anti-proliferative effects on a variety of human cancer cell lines. For discussing its anti-tumor structure-activity relationship, a series of 4ß-hydroxywithanolide E derivatives (1-17) were synthesized and evaluated for their antitumor activity in vitro towards acute promyelocytic leukemia NB4 cell line by the Alarma blue assay. Cytotoxicity data revealed that the enone structure and C-4 hydroxyl substituents of ring A, together with the side chain (C-20-C-28) play an important effect on the cytotoxicity.

New lathyrane diterpenoids with anti-inflammatory activity isolated from the roots of Jatropha curcas L.

ETHNOPHARMACOLOGICAL RELEVANCE: Jatropha curcas L. (Euphorbiaceae), as a drought resistant shrub mainly cultivated in tropical and subtropical areas worldwide, is widely used as traditional medicine to cure arthritis, dysentery, abscess and pneumonia in Asian, African and South American folklores. The methanolic extracts of the roots have been revealed the anti-inflammatory activity in vivo and vitro. AIM OF STUDY: This research aimed to provide promising anti-inflammatory candidates from the roots of J. curcas. In addition, RNA-Seq was conducted to give targeted genes involved in the anti-inflammatory action. MATERIALS AND METHODS: The diterpenoids were isolated from the CH2Cl2 fraction of the methanolic extract from the roots of J. curcas by column chromatography (CC): silica gel, Sephadex LH-20, ODS, semi-preparative reversed-phase high-performance liquid chromatography (HPLC). The structures were identified based on HR-ESI-MS and 1D, 2D-NMR spectroscopic analysis. Their anti-inflammatory effects were tested on lipopolysaccharide (LPS, 500 ng/mL)-stimulated murine RAW264.7 macrophages. Furthermore, we conducted transcriptome-wide RNA sequencing to profile gene expression alterations in LPS-induced RAW264.7 cells upon treatment with jatrocurcasenone I (4) and analyzed the underlying genes targeted by this compound. RESULTS: Six diterpenoids were obtained from J. curcas, and four of them were identified to be new lathyrane diterpenoids: jatrocurcasenones F-I (1-4). Compounds 3 and 4 exhibited potent inhibitory activities against LPS-induced nitric oxide (NO) production in RAW264.7 cells with IC50 values of 11.28 µM and 7.71 µM, respectively. Western blotting analysis showed that the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were suppressed with the supplementation of 3 and 4. The results of RNA-seq showed that 4 (20 µM) exhibited regulation on the 587 differentially expressed genes (DEGs) induced by LPS (500 ng/mL). Transcriptome-wide RNA sequencing indicated that the protective activity of 4 supplementation was most likely driven by modulating expression levels of IL-1α, IL-1ß, IL-1f6, IL-6, IL-1rn, IL-27, Ccl2, Ccl5, Ccl7, Ccl9, Ccl22, Cxcl10, Tnfsf12, Tnfsf15, Lta, Trim25, Bcl2a1a, Dusp1, Dusp2, Ptgs2, Edn1 and Nr4a1. CONCLUSIONS: This study offered four new lathyrane diterpenoids, of them, jatrocurcasenone I (4) showed significant anti-inflammatory activity. RNA-Seq suggested that jatrocurcasenone I (4) could be a candidate drug for the prevention inflammation-mediated diseases by modulating 24 candidate DEGs.

Hydroxy-octadecenoic acids instead of phorbol esters are responsible for the Jatropha curcas kernel cake's toxicity.

The toxic kernel cake of Jatropha curcas (KCakeJ) is an emerging health and environmental concern. Although phorbol esters are widely recognized as the major toxin of KCakeJ, convincing evidence is absent. Here, we show that rather than phorbol esters an isomeric mixture of 11-hydroxy-9E-octadecenoic acid, 12-hydroxy-10E-octadecenoic acid and 12-hydroxy-10Z-octadecenoic acid (hydroxy-octadecenoic acids, molecular formula C18H34O3) is the major toxic component. The toxicities of hydroxy-octadecenoic acids on experimental animals, e.g. acute lethality, causing inflammation, pulmonary hemorrhage and thrombi, allergies, diarrhea and abortion, are consistent with those on human/animals caused by Jatropha seed and/or KCakeJ. The hydroxyl group and the double bond are essential for hydroxy-octadecenoic acids' toxicity. The main pathway of the toxicity mechanism includes down-regulating UCP3 gene expression, promoting ROS production, thus activating CD62P expression (platelet activation) and mast cell degranulation. The identification of the major toxin of KCakeJ lays a foundation for establishing an environmentally friendly Jatropha biofuel industry.

New cucurbitane triterpenoids and steroidal glycoside from Momordica charantia.

Three new cucurbitane triterpenoids 1-3 and one new steroidal glycoside 4, were isolated together with ten known compounds from Momordica charantia. The structures of new compounds were determined to be 19(R)-n-butanoxy-5 beta,19-epoxycucurbita-6,23-diene-3beta,25-diol 3-O-beta-glucopyranoside (1), 23-O-beta-allopyranosylecucurbita-5,24-dien-7 alpha,3beta,22(R),23(S)-tetraol 3-O-beta-allopyranoside. (2), 23(R),24(S),25-trihydroxycucurbit-5-ene 3-O-{[beta-glucopyranosyl(1-->6)]-O-beta-glucopyranosyl}-25-O-beta-glucopyranoside (3), and 24(R)-stigmastan-3beta,5 alpha,6 beta-triol-25-ene 3-O-beta-glucopyranoside (4), respectively. Their structures were elucidated by the combination of mass spectrometry (MS), one and two-dimensional NMR experiments and chemical reactions.

Three new withanolides from the calyces of Nicandra physaloides.

Chemical investigation on ethyl acetate extract of the calyces of Nicandra physaloides resulted in the isolation of three new withanolides named as nicphysatone A (1), nicphysatone B (2), nicphysatone C (3), together with five known withanolides, nic 17 (4), nic 7 (5), nic 2 (6), withahisolide G (7) and nicaphysalin B (8). The structures were determined by comprehensive spectroscopic experiments. The discovery enriched the diversity of natural withanolides and could serve as scaffolds for the synthesis of more potent modified withanolides.

Two new farnesyl phenolic compounds with anti-inflammatory activities from Ganoderma duripora.

Ganoderma fungi have long been used as a famous traditional medicine and food in country of East Asia. In this work, two new farnesyl phenolic compounds, ganoduriporols A and B (1 and 2), were isolated from the fruiting bodies of Ganoderma duripora, and their structures were elucidated using various spectroscopic methods. Anti-inflammatory activities were assayed and evaluated for the two compounds. Ganoduriporols A and B exhibited dose-dependent anti-inflammatory effects in RAW 264.7 cells. Furthermore, ganoduriporol A was demonstrated to inhibit the production of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and prostaglandin E2 (PGE2) through the suppression of COX-2, MAPK and NF-κB signaling pathway in LPS-induced macrophage cells. These results suggested that these two new farnesyl phenolic compounds and the fruiting body of G. duripora could serve as anti-inflammatory agents for medicinal use or functional food.

Withanolides from aerial parts of Nicandra physalodes.

Twenty withanolides, including previously unknown nicanlodes A-M, were isolated from aerial parts of Nicandra physalodes. Their structural elucidations were unambiguously achieved through interpretation of extensive spectroscopic data (NMR and HRMS) and by comparison with literature data. Nicanlodes A and B have an unusual aromatic amine moiety. The isolated compounds were evaluated for their cytotoxicity against five human cancer cell lines.