Green synthesis and biological evaluation of glaucanic acid and dihydrocompactin acid by endophytic fungus Penicillium polonicum from Zingiber officinale.

Fungal endophytes are valued for biosynthesizing chemically diverse metabolic cascade with interesting biological activities. In the current investigation, two compounds were isolated from Penicillium polonicum, an endophyte of Zingiber officinale. The active moieties, glaucanic acid (1) and dihydrocompactin acid (2) were isolated from the ethyl acetate extract of P. polonicum and characterized by NMR and mass spectroscopy. Further, bioactive potential of the isolated compounds was evaluated by antimicrobial, antioxidant and cytotoxicity assays. Compounds 1 and 2 displayed antifungal activity against phytopathogen Colletotrichum gloeosporioides with more than 50% reduction in its growth. Both the compounds exhibited antioxidant activity against free radicals (DPPH and ABTS) and cytotoxicity activity against cancer cell lines respectively. The compounds, glaucanic acid and dihydrocompactin acid are being reported for the first time from an endophytic fungus. This is the first report on the biological activities of Dihydrocompactin acid produced by endophytic fungal strain.

LCMS-DNP based dereplication of Araucaria cunninghamii Mudie gum-resin: identification of new cytotoxic labdane diterpene.

As a part of natural defense, plants initiate the secretion of gum containing numerous pharmacologically active essential metabolites. A fraction of such gum-resin from Araucaria cunninghamii Mudie, when screened against human cancer cell lines, was found to be active. Further, it was subjected to an LCMS-DNP (Dictionary of Natural Products) based dereplication study followed by a detailed phytochemical investigation to obtain pure metabolites. Also, the gum resin of A. cunninghamii was found to be a rich source of abietanes and labdanes. The LCMS-DNP-based dereplication study identified many known metabolites, which were isolated for the first time from this plant as well as a new labdane diterpenoid (9). The compounds were characterized via spectroscopic techniques, which were subsequently compared with the already existing literature data. The metabolites were screened against seven human cancer cell lines. The anticancer activity was further supported by molecular docking studies.

2-Pyridin-4-yl-methylene-beta-boswellic Acid-A Potential Candidate for Targeting O6-Methylguanine-DNA Methyltransferase Epi-transcriptional Reprogramming in KRAS G13D-Microsatellite Stable, G12V-Microsatellite Instable Mutant Colon Cancer.

PMBA (2-Pyridin-4-yl-methylene-beta-boswellic acid), screened from among the 21 novel series of semisynthetic analogues of ß-boswellic acid, is being presented as a lead compound for integrative management of KRAS mutant colorectal cancer (CRC), upon testing and analysis for its anticancerous activity on a panel of NCI-60 cancer cell lines and in vivo models of the disease. PMBA (1.7-29 µM) exhibited potent proliferation inhibition on the cell lines and showed sensitivity in microsatellite instability and microsatellite stable (GSE39582 and GSE92921) subsets of KRAS gene (Kirsten rat sarcoma viral oncogene homolog)-mutated colon cell lines, as revealed via flow cytometry analysis. A considerable decrease in mitogen-activated protein kinase pathway downstream effectors was observed in the treated cell lines via the western blot and STRING (Search tool for the retrieval of interacting genes/proteins) analysis. PMBA was further found to target KRAS at its guanosine diphosphate site. Treatment of the cell lines with PMBA showed significant reduction in MGMT promoter methylation but restored MGMT (O6-methylguanine-DNA methyltransferase) messenger ribonucleic acid expression via significant demethylation of the hypermethylated CpG (Cytosine phosphate guanine) sites in the MGMT promoter. A significant decrease in dimethylated H3K9 (Dimethylation of lysine 9 on histone 3) levels in the MGMT promoter in DNA hypo- and hypermethylated HCT-116G13D and SW-620G12V cells was observed after treatment. In the MNU (N-methyl-N-nitrosourea)-induced CRC in vivo model, PMBA instillation restricted and repressed polyp formation, suppressed tumor proliferation marker Ki67 (Marker of proliferation), ablated KRAS-associated cytokine signaling, and decreased mortality. Clinical trial data for the parent molecule revealed its effectiveness against the disease, oral bioavailability, and system tolerance. Comprehensively, PMBA represents a new class of KRAS inhibitors having a therapeutic window in the scope of a drug candidate. The findings suggest that the PMBA analogue could inhibit the growth of human CRC in vivo through downregulation of cancer-associated biomarkers as well as reactivate expression of the MGMT gene associated with increased H3K9 acetylation and H3K4 methylation with facilitated transcriptional activation, which might be important in silencing of genes associated with upregulation in the activity of KRAS.

Tandem MS-Based Metabolite Profiling of 19,20-Epoxycytochalasin C Reveals the Importance of a Hydroxy Group at the C7 Position for Biological Activity.

Seven cytochalasins, 19,20-epoxycytochalasin N, cytochalasin P1, deacetyl 19,20-epoxycytochalasin C, 19,20-epoxycytochalasin D, 19,20-epoxycytochalasin C, cytochalasin D, and cytochalasin C, were isolated from a fungal (Rosellinia sanctae-cruciana) crude extract. A cytotoxicity assay (sulforhodamine B) was performed on a series of cancer cell lines: HT-29, A-549, PC-3, HCT-116, SW-620, and MCF-7. Simultaneously, the liquid chromatography-mass spectrometry (LC-MS)/MS profile of 19,20-epoxycytochalasin C-treated cell lines revealed that 19,20-epoxycytochalasin C (m/z 524.25) oxidized to a metabolite of m/z 522.25 Da (-2 Da (-2H) from 19,20-epoxycytochalasin C). Further chemical oxidation of 19,20-epoxycytochalasin C using the Dess-Martin reagent produced an identical metabolite. It has been noticed that the parent molecule (19,20-epoxycytochalasin C) showed an IC50 of 650 nM (on HT-29), whereas for the oxidized metabolite (m/z 522.24) of 19,20-epoxycytochalasin C, the IC50 was >10 µM. It is clear that the parent molecule had 16 times higher cytotoxic potential as compared to the oxidized metabolite. The spectroscopic investigation indicated that the oxidation of the hydroxyl (-OH) group occurred at the C7 position in 19,20-epoxycyctochalsin C and led to the inactivation of 19,20-epoxycytochalasin C. Further, cell cycle analysis and histopathological evidence support the findings, and CDK2 could be a possible target of 19,20-epoxycyctochalasin C.

14-Residue peptaibol velutibol A from Trichoderma velutinum: its structural and cytotoxic evaluation.

Velutibol A (1), a new 14-residue peptaibol was isolated from the Himalayan cold habitat fungus Trichoderma velutinum. The structural characterization was carried out by 1D and 2D NMR studies, and tandem mass studies, and Marfey's method aided in determining the stereochemistry of the amino acids. The CD analysis revealed folding of the peptide in a 310-helical conformation. The intramolecular H-bonding was determined by an NMR-VT experiment. Cytotoxic evaluation was carried out against a panel of cancer cell lines. The cell cycle assay was carried out on human myeloid leukaemia (HL-60) cells and revealed the formation of apoptotic bodies and DNA damage in a dose-dependent manner. Three other peptaibols namely velutibol B (2), velutibol C (3), and velutibol D (4) were also isolated in trace amounts from the psychotropic fungus and characterized through tandem mass spectroscopy and Marfey's analysis.

Valproic acid induces three novel cytotoxic secondary metabolites in Diaporthe sp., an endophytic fungus from Datura inoxia Mill.

Addition of the valproic acid (histone deacetylases inhibitor) to a culture of an endophytic fungus Diaporthe sp. harbored from Datura inoxia significantly altered its secondary metabolic profile and resulted in the isolation of three novel compounds, identified as xylarolide A (1), diportharine A (2) and xylarolide B (3) along with one known compound xylarolide (4). The structures of all the compounds (1-4) were determined by detailed analysis of 1D and 2D NMR spectroscopic data. The relative configurations of compounds 1-3 were determined with the help of NOESY data and comparison of optical rotations with similar compounds with established stereochemistry. All the isolated compounds were screened for antibacterial, antioxidant and cytotoxic activities. Xylarolide A (1) and xylarolide (4) displayed significant growth inhibition of MIAPaCa-2 with an IC50 of 20 and 32 µM respectively and against PC-3 with an IC50 of 14 and 18 µM respectively. Moreover, compound 1 displayed significant DPPH scavenging activity with EC50 of 10.3 µM using ascorbic acid as a positive control.

Lipovelutibols A-D: Cytotoxic Lipopeptaibols from the Himalayan Cold Habitat Fungus Trichoderma velutinum.

Four novel lipovelutibols A (1), B (2), C (3), and D (4) containing six amino acid residues with leucinol at the C-terminus and a fatty acyl moiety (n-octanoyl) at its N-terminus were isolated from the psychrotrophic fungus Trichoderma velutinum collected from the Himalayan cold habitat. The structures (1-4) were determined by NMR and MS/MS, and the stereochemistry of amino acids by Marfey's method. Lipopeptaibols 2 and 4 were found to contain d-isovaline, a nonproteinogenic amino acid, but lacked α-aminoisobutyric acid, characteristic of peptaibols. Cytotoxic activity of 2 and 4 was observed against HL-60, LS180, MDA-MB-231, and A549 cancer cell lines.

Isolation and characterization of bioactive metabolites from Xylaria psidii, an endophytic fungus of the medicinal plant Aegle marmelos and their role in mitochondrial dependent apoptosis against pancreatic cancer cells.

BACKGROUND: The genus Xylaria has been reported as a rich source of biologically active secondary metabolites. In the present study, an endophytic fungus Xylaria psidii has been isolated from the leaf sample of Aegle marmelos (L.) Corr., characterized on the basis of its morphological features and sequence data for the ITS region (KU291350) of the nuclear ribosomal DNA. Biological screening of ethyl acetate extract of Xylaria psidii displayed a potential therapeutic effect on pancreatic cancer cells. HYPOTHESIS: This study was designed systematically to explore Xylaria psidii, an endophytic fungus for the identification of biologically active secondary metabolites against pancreatic cancer cells. METHODS: While exploring the bioactive secondary metabolites, a sensitive and reliable LC-MS based dereplication approach was applied to identify four compounds A-D from fungal extract. Further bioactivity guided isolation of fungal extract yielded two major metabolites 1 and 2. The structures of 1 and 2 have been determined by detailed spectroscopic analysis including MS, NMR, IR and UV data and similarity with published data. Xylarione A (1) is new whereas (-) 5-methylmellein (2) is reported for the first time from X. psidii. Both the isolated compounds were screened for their effect on the viability and proliferation against a panel of cancer cell lines (MCF-7, MIA-Pa-Ca-2, NCI-H226, HepG2 and DU145) of different tissue origin. RESULTS: Compounds 1 and 2 exhibited cytotoxicity against pancreatic cancer (MIA-Pa-Ca-2) cells with IC50 values of 16.0 and 19.0 µm, respectively. The cell cycle distribution in MIA-Pa-Ca-2 cells, confirmed a cell cycle arrest at the sub-G1 phase. Cell death induced by 1 and 2 displayed features characteristic of apoptosis. Flow cytometry based analysis of 1 and 2 using Rhodamine-123 displayed substantial loss of mitochondrial membrane potential in a concentration dependent manner by both the compounds. CONCLUSION: Results conclude that the isolated compounds 1 and 2 are responsible for the activity shown by crude ethyl acetate extract and may act as potential leads for medicinal chemists for designing more potent analogs.

Terpenoid and flavonoid spectrum of in vitro cultures of Glycyrrhiza glabra revealed high chemical heterogeneity: platform to understand biosynthesis.

Simultaneous qualitative and quantitative assessment of eight flavonoids and two terpenoids were performed in fourteen in vitro raised morphogenic cultures of Glycyrrhiza glabra. Our study revealed that the spectrum and production of ten compounds, under investigation, were higher in organized tissue than the undifferentiated mass, however, aerial portions of the in vitro raised plants (leaf and stem) were found to be devoid of therapeutically relevant triterpenoid, glycyrrhizin. A correlation was observed between cell maturation, morphological differentiation and glycyrrhizin accumulation. Mature stolons (4 months) were characterized by the maximum accumulation of glycyrrhizin (8.60 µg/mg) in in vitro plantlets. The cytotoxic effect of the extracts evaluated against a panel of human cancer cell lines (in vitro) indicated that the pancreatic cell line (MIA-PaCa-2) were sensitive to all the fourteen extracts investigated. To the best of our knowledge this is the first comprehensive report relating plant growth regulators to metabolite spectrum and cytotoxic assessment in in vitro raised G. glabra cultures. Overall, our findings demonstrated that the metabolite spectrum of in vitro raised morphogenetic lines, under different stages of maturation, might offer a platform to understand the regulatory aspects of the concerned metabolite pathway and their consequent role in differentiation.

A therapeutically relevant, 3,3'-diindolylmethane derivative NGD16 attenuates angiogenesis by targeting glucose regulated protein, 78kDa (GRP78).

Angiogenesis remain a critical procedure for tumor progression and malignancy. Anticancer agents targeting angiogenic cascades have been proved to be an effective strategy in the field of cancer therapeutics. The current study aims to explore the mechanistic prevention of angiogenesis and cancer cell proliferation by 1,1'-ß-d-glucopyranosyl-3,3'-bis(5-bromoindolyl)-octyl methane (NGD16), a novel N-glycosylated derivative of 3,3'-diindolylmethane (DIM). NGD16 suppressed the viability of prostate cancer (PC-3), pancreatic adenocarcinoma (MiaPaca-2), colorectal cancer (COLO-205) and human umbilical vein endothelial cells (HUVECs) effectively with IC50 values 0.8 µM, 2.8 µM, 5.3 µM and 2.5 µM respectively. Abrogation of angiogenesis by NGD16 was promising in in vivo mouse Matrigel plug assay as well as in ex vivo sprouting of rat thoracic aorta. At the molecular level, NGD16 inhibited the expression of glucose regulated protein, 78 kDa (GRP78), vascular endothelial growth factor receptor-2 (VEGFR2) and matrix metalloproteinase-9 (MMP-9) expression, the main mediators of angiogenesis and neovessel formation. Overexpression of GRP78 upregulated the expression of MMP-9 and VEGFR2 in PC-3 and HUVECs. Antibody blocking of GRP78 further potentiated NGD16 in attenuating angiogenesis through inhibition of MMP-9. NGD16 depicted its promising biodistribution profile in a pharmacokinetic study with 46.9% intraperitoneal bioavailability. Our findings suggest NGD16 is a potent inhibitor of neo-angiogenesis with a desirable pharmacokinetic profile, which can be taken forward in its development as an anticancer drug.

Dysoxylum binectariferum bark as a new source of anticancer drug camptothecin: bioactivity-guided isolation and LCMS-based quantification.

Camptothecin (CPT, 1) is a potent anticancer natural product which led to the discovery of two clinically used anticancer drugs topotecan and irinotecan. These two drugs are semisynthetic analogs of CPT, and thus the commercial production of CPT as a raw material from various plant sources and tissue culture methods is highly demanding. In the present study, the Dysoxylum binectariferum bark, was identified as an alternative source of CPT, through bioassay-guided isolation. The barks showed presence of CPT (1) and its 9-methoxy analog 2, whereas CPT alkaloids were not present in seeds and leaves. This is the first report on isolation of CPT alkaloids from Meliaceae family. An efficient chromatography-free protocol for enrichment and isolation of CPT from D. binectariferum has been established, which was able to enrich CPT up to 21% in the crude extract. The LCMS (MRM)-based quantification method revealed the presence of 0.105% of CPT in dry barks of D. binectariferum. The discovery of CPT from D. binectariferum bark will certainly create a global interest in cultivation of this plant as a new crop for commercial production of CPT. Isolation of anticancer drug CPT from this plant, indicates that along with rohitukine, CPT and 9-methoxy CPT also contributes significantly to the cytotoxicity of D. binectariferum.

Biodegradable polymeric system for cisplatin delivery: development, in vitro characterization and investigation of toxicity profile.

Cisplatin is one of the most potent anticancer agent used in the treatment of various solid tumors, however, its clinical use is limited due to severe adverse effects including nephrotoxicity. In this investigation cisplatin loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles were developed and characterized for various in vitro characteristics including size distribution, zeta potential, drug loading and release profile. PLGA nanoparticles were successfully developed as investigated using scanning electron microscopy and exhibited average particles size and zeta potential as 284.8 nm and -15.8 mV, respectively. Fourier transform infrared spectroscopy and differential scanning calorimetry indicated an absence of any polymer-drug interactions. Cisplatin nanoparticles exhibited in vitro anticancer activity against A549 cells comparable to that of cisplatin solution. The biodistribution study in mice indicated that the kidney cisplatin level was significantly (p<0.01) lower with cisplatin nanoparticles than cisplatin solution. Following two cycles of cisplatin treatment, a week apart, blood urea nitrogen level was found to be higher in case of cisplatin solution as compared to cisplatin nanoparticles. Further, there was a significant (p<0.01) increase in plasma creatinine level in case of cisplatin solution as compared to cisplatin nanoparticles. Histopathological examination of kidney from cisplatin nanoparticles treated group revealed no kidney damage, however, a sign of nephrotoxicity was observed in the case of cisplatin solution. The results suggest that PLGA nanoparticle based formulation could be a potential option for cisplatin delivery.

Capsaicin production by Alternaria alternata, an endophytic fungus from Capsicum annum; LC-ESI-MS/MS analysis.

Alternaria alternata, an endophytic fungus capable of producing capsaicin (1) was isolated from Capsicum annum. The endophyte was found to produce capsaicin upto three generations. Upscaling of the fermentation broth led to the isolation of one known and one compound characterized as 2,4-di-tert-butyl phenol (2) and alternariol-10-methyl ether (3) respectively. Compound 1 and 3 were identified and quantified using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) system through multiple reaction monitoring (MRM). Furthermore, compound 3 displayed a range of cytotoxicity against a panel of human cancer cell lines and was found to induce apoptosis evidenced by Hoechst staining and loss of mitochondrial-membrane potential in HL-60 cells.

Quantification of the Polyisoprenylated Benzophenones Garcinol and Isogarcinol Using Multiple Reaction Monitoring LC/Electrospray Ionization-MS/MS Analysis of Ultrasound-Assisted Extracts of Garcinia indica Fruits.

This paper describes a method that includes an optimized extraction process and identification and quantification of two anticancer compounds (garcinol and isogarcinol) by LC/electrospray ionization (ESI)-MS/MS in the multiple reaction monitoring (MRM) mode. The study aimed to develop a fast, accurate, and sensitive method for the quantification of garcinol and isogarcinol in different extracts of Garcinia indica fruits. The compounds were detected using LC/ESI-MS/MS in the positive-ion mode and quantified in the MRM mode using a transition mass of m/z 603.3/411 taken as the quantifier and 603.3/343.2 as the qualifier for garcinol and isogarcinol. Five point calibration curves were linear in the range of 2 to 10 ng/mL for garcinol and 0.5 to 6 ng/mL for isogarcinol, with a correlation coefficient of ≥0.990 for both. LOQ for garcinol and isogarcinol was 0.06 and 0.05 ng/mL, respectively, while LOD was 0.021 and 0.017 ng/mL respectively. Our work demonstrated optimization of extraction procedure, fast and highly sensitive quantification (pg level LOQ), and validation of the developed method for the investigated compounds in fruit extracts of G. indica.