Optimization of biotransformation processes of Camarosporium laburnicola to improve production yields of potent telomerase activators.

BACKGROUND: Telomerase activators are promising agents for the healthy aging process and the treatment/prevention of short telomere-related and age-related diseases. The discovery of new telomerase activators and later optimizing their activities through chemical and biological transformations are crucial for the pharmaceutical sector. In our previous studies, several potent telomerase activators were discovered via fungal biotransformation, which in turn necessitated optimization of their production. It is practical to improve the production processes by implementing the design of experiment (DoE) strategy, leading to increased yield and productivity. In this study, we focused on optimizing biotransformation conditions utilizing Camarosporium laburnicola, a recently discovered filamentous fungus, to afford the target telomerase activators (E-CG-01, E-AG-01, and E-AG-02). RESULTS: DoE approaches were used to optimize the microbial biotransformation processes of C. laburnicola. Nine parameters were screened by Plackett-Burman Design, and three significant parameters (biotransformation time, temperature, shaking speed) were optimized using Central Composite Design. After conducting validation experiments, we were able to further enhance the production yield of target metabolites through scale-up studies in shake flasks (55.3-fold for E-AG-01, 13-fold for E-AG-02, and 1.96-fold for E-CG-01). CONCLUSION: Following a process optimization study using C. laburnicola, a significant increase was achieved in the production yields. Thus, the present study demonstrates a promising methodology to increase the production yield of potent telomerase activators. Furthermore, C. laburnicola is identified as a potential biocatalyst for further industrial utilization.

Potent telomerase activators from a novel sapogenin via biotransformation utilizing Camarosporium laburnicola, an endophytic fungus.

BACKGROUND: Cycloartane-type triterpenoids possess important biological activities, including immunostimulant, wound healing, and telomerase activation. Biotransformation is one of the derivatization strategies of natural products to improve their bioactivities. Endophytic fungi have attracted attention in biotransformation studies because of their ability to perform modifications in complex structures with a high degree of stereospecificity. RESULTS: This study focuses on biotransformation studies on cyclocephagenol (1), a novel cycloartane-type sapogenin from Astragalus species, and its 12-hydroxy derivatives (2 and 3) to obtain new telomerase activators. Since the hTERT protein levels of cyclocephagenol (1) and its 12-hydroxy derivatives (2 and 3) on HEKn cells were found to be notable, biotransformation studies were carried out on cyclocephagenol and its 12-hydroxy derivatives using Camarosporium laburnicola, an endophytic fungus isolated from Astragalus angustifolius. Later, immunoblotting and PCR-based ELISA assay were used to screen starting compounds and biotransformation products for their effects on hTERT protein levels and telomerase activation. All compounds showed improved telomerase activation compared to the control group. CONCLUSIONS: As a result of biotransformation studies, seven new metabolites were obtained and characterized, verifying the potential of C. laburnicola as a biocatalyst. Additionally, the bioactivity results showed that this endophytic biocatalyst is unique in transforming the metabolites of its host to afford potent telomerase activators.

Astragalus Saponins, Astragaloside VII and Newly Synthesized Derivatives, Induce Dendritic Cell Maturation and T Cell Activation.

Astragaloside VII (AST VII), a triterpenic saponin isolated from Astragalus species, shows promise as a vaccine adjuvant, as it supported a balanced Th1/Th2 immune response in previous in vivo studies. However, the underlying mechanisms of its adjuvant activity have not been defined. Here, we investigated the impact of AST VII and its newly synthesized semi-synthetic analogs on human whole blood cells, as well as on mouse bone marrow-derived dendritic cells (BMDCs). Cells were stimulated with AST VII and its derivatives in the presence or absence of LPS or PMA/ionomycin and the secretion of cytokines and the expression of activation markers were analyzed using ELISA and flow cytometry, respectively. AST VII and its analogs increased the production of IL-1ß in PMA/ionomycin-stimulated human whole blood cells. In LPS-treated mouse BMDCs, AST VII increased the production of IL-1ß and IL-12, and the expression of MHC II, CD86, and CD80. In mixed leukocyte reaction, AST VII and derivatives increased the expression of the activation marker CD44 on mouse CD4+ and CD8+ T cells. In conclusion, AST VII and its derivatives strengthen pro-inflammatory responses and support dendritic cell maturation and T cell activation in vitro. Our results provide insights into the mechanisms of the adjuvant activities of AST VII and its analogs, which will be instrumental to improve their utility as a vaccine adjuvant.

Neuroprotective metabolites via fungal biotransformation of a novel sapogenin, cyclocephagenol.

Cyclocephagenol (1), a novel cycloartane-type sapogenin with tetrahydropyran unit, is only encountered in Astragalus species. This rare sapogenin has never been a topic of biological activity or modification studies. The objectives of this study were; (i) to perform microbial transformation studies on cyclocephagenol (1) using Astragalus endophyte, Alternaria eureka 1E1BL1, followed by isolation and structural characterization of the metabolites; (ii) to investigate neuroprotective activities of the metabolites; (iii) to understand structure-activity relationships towards neuroprotection. The microbial transformation of cyclocephagenol (1) using Alternaria eureka resulted in the production of twenty-one (2-22) previously undescribed metabolites. Oxidation, monooxygenation, dehydration, methyl migration, epoxidation, and ring expansion reactions were observed on the triterpenoid skeleton. Structures of the compounds were established by 1D-, 2D-NMR, and HR-MS analyses. The neuroprotective activities of metabolites and parent compound (1) were evaluated against H2O2-induced cell injury. The structure-activity relationship (SAR) was established, and the results revealed that 1 and several other metabolites had potent neuroprotective activity. Further studies revealed that selected compounds reduced the amount of ROS and preserved the integrity of the mitochondrial membrane. This is the first report of microbial transformation of cyclocephagenol (1).

Non-apoptotic cell death induction via sapogenin based supramolecular particles.

The discovery of novel chemotherapeutics that act through different mechanisms is critical for dealing with tumor heterogeneity and therapeutic resistance. We previously reported a saponin analog (AG-08) that induces non-canonical necrotic cell death and is auspicious for cancer therapy. Here, we describe that the key element in triggering this unique cell death mechanism of AG-08 is its ability to form supramolecular particles. These self-assembled particles are internalized via a different endocytosis pathway than those previously described. Microarray analysis suggested that AG-08 supramolecular structures affect several cell signaling pathways, including unfolded protein response, immune response, and oxidative stress. Finally, through investigation of its 18 analogs, we further determined the structural features required for the formation of particulate structures and the stimulation of the unprecedented cell death mechanism of AG-08. The unique results of AG-08 indicated that supramolecular assemblies of small molecules are promising for the field of anticancer drug development, although they have widely been accepted as nuisance in drug discovery studies.

The role of cycloastragenol at the intersection of NRF2/ARE, telomerase, and proteasome activity.

Aging is well-characterized by the gradual decline of cellular functionality. As redox balance, proteostasis, and telomerase systems have been found to be associated with aging and age-related diseases, targeting these systems with small compounds has been considered a promising therapeutic approach. Cycloastragenol (CA), a small molecule telomerase activator obtained from Astragalus species, has been reported to positively affect several age-related pathophysiologies, but the mechanisms underlying CA activity have yet to be reported. Here, we presented that CA increased NRF2 nuclear localization and activity leading to upregulation of cytoprotective enzymes and attenuation of oxidative stress-induced ROS levels. Furthermore, CA-mediated induction of telomerase activity was found to be regulated by NRF2. CA not only increased the expression of hTERT but also its nuclear localization via upregulating the Hsp90-chaperon complex. In addition to modulating nuclear hTERT levels at unstressed conditions, CA alleviated oxidative stress-induced mitochondrial hTERT levels while increasing nuclear hTERT levels. Concomitantly, H2O2-induced mitochondrial ROS level was found to be significantly decreased by CA administration. Our data also revealed that CA strongly enhanced proteasome activity and assembly. More importantly, the proteasome activator effect of CA is dependent on the induction of telomerase activity, which is mediated by NRF2 system. In conclusion, our results not only revealed the cross-talk among NRF2, telomerase, and proteasome systems but also that CA functions at the intersection of these three major aging-related cellular pathways.

Undescribed polyether ionophores from Streptomyces cacaoi and their antibacterial and antiproliferative activities.

Polyether ionophores represent a large group of naturally occurring compounds mainly produced by Streptomyces species. With previously proven varieties of bioactivity including antibacterial, antifungal, antiparasitic, antiviral and anti-tumor effects, the discovery of undescribed polyethers leading to development of efficient therapeutics has become important. As part of our research on polyether-rich Streptomyces cacaoi, we previously performed modification studies on fermentation conditions to induce synthesis of specialized metabolites. Here, we report four undescribed and nine known polyether compounds from S. cacaoi grown in optimized conditions. Antimicrobial activity assays revealed that four compounds, including the undescribed (6), showed strong inhibitory effects over both Bacillus subtilis and methicillin-resistant Staphylococcus aureus (MRSA) growth. Additionally, K41-A and its C15-demethoxy derivative exhibited significant cytotoxicity. These results signified that selectivity of C15-demethoxy K41-A towards cancer cells was higher than K41-A, which prompted us to conduct mechanistic experiments. These studies showed that this uninvestigated compound acts as a multitarget compound by inhibiting autophagic flux, inducing reactive oxygen species formation, abolishing proteasome activity, and stimulating ER stress. Consequently, the optimized fermentation conditions of S. cacaoi led to the isolation of undescribed and known polyethers displaying promising activities.

New Cardenolides from Biotransformation of Gitoxigenin by the Endophytic Fungus Alternaria eureka 1E1BL1: Characterization and Cytotoxic Activities.

Microbial biotransformation is an important tool in drug discovery and for metabolism studies. To expand our bioactive natural product library via modification and to identify possible mammalian metabolites, a cytotoxic cardenolide (gitoxigenin) was biotransformed using the endophytic fungus Alternaria eureka 1E1BL1. Initially, oleandrin was isolated from the dried leaves of Nerium oleander L. and subjected to an acid-catalysed hydrolysis to obtain the substrate gitoxigenin (yield; ~25%). After 21 days of incubation, five new cardenolides 1, 3, 4, 6, and 8 and three previously- identified compounds 2, 5 and 7 were isolated using chromatographic methods. Structural elucidations were accomplished through 1D/2D NMR, HR-ESI-MS and FT-IR analysis. A. eureka catalyzed oxygenation, oxidation, epimerization and dimethyl acetal formation reactions on the substrate. Cytotoxicity of the metabolites were evaluated using MTT cell viability method, whereas doxorubicin and oleandrin were used as positive controls. Biotransformation products displayed less cytotoxicity than the substrate. The new metabolite 8 exhibited the highest activity with IC50 values of 8.25, 1.95 and 3.4 µM against A549, PANC-1 and MIA PaCa-2 cells, respectively, without causing toxicity on healthy cell lines (MRC-5 and HEK-293) up to concentration of 10 µM. Our results suggest that A. eureka is an effective biocatalyst for modifying cardenolide-type secondary metabolites.

Telomerase activators from 20(27)-octanor-cycloastragenol via biotransformation by the fungal endophytes.

Cycloastragenol [20(R),24(S)-epoxy-3ß,6α,16ß,25-tetrahydroxycycloartane] (CA), the principle sapogenol of many cycloartane-type glycosides found in Astragalus genus, is currently the only natural product in the anti-aging market as telomerase activator. Here, we report biotransformation of 20(27)-octanor-cycloastragenol (1), a thermal degradation product of CA, using Astragalus species originated endophytic fungi, viz. Penicillium roseopurpureum, Alternaria eureka, Neosartorya hiratsukae and Camarosporium laburnicola. Fifteen new biotransformation products (2-16) were isolated, and their structures were established by NMR and HRESIMS. Endophytic fungi were found to be capable of performing hydroxylation, oxidation, ring cleavage-methyl migration, dehydrogenation and Baeyer-Villiger type oxidation reactions on the starting compound (1), which would be difficult to achieve by conventional synthetic methods. In addition, the ability of the metabolites to increase telomerase activation in Hekn cells was evaluated, which showed from 1.08 to 12.4-fold activation compared to the control cells treated with DMSO. Among the compounds tested, 10, 11 and 12 were found to be the most potent in terms of telomerase activation with 12.40-, 7.89- and 5.43-fold increase, respectively (at 0.1, 2 and 10 nM concentrations, respectively).

Evaluation of adjuvant activity of Astragaloside VII and its combination with different immunostimulating agents in Newcastle Disease vaccine.

Astragaloside VII (AST-VII), a major cycloartane saponin isolated from Turkish Astragalus species, turned out to be one of the most active metabolites demonstrating Th1/Th2 balanced immune response. As Quillaja saponins are extensively used in adjuvant systems, this study made an attempt to improve AST-VII based adjuvant systems by using different immunostimulatory/delivery agents (monophosphoryllipid A (MPL), Astragalus polysaccharide (APS) and squalene) and to induce cellular and humoral immune response against a viral vaccine. For this purpose, Newcastle Disease vaccine (NDV) was chosen as a model vaccine. Swiss albino mice were immunized subcutaneously with LaSota vaccines in the presence/absence of AST-VII or developed adjuvant systems. AST-VII administration both in live/inactivated LaSota vaccines induced neutralizing and NDV specific IgG, IgG1 and IgG2b antibodies response as well as IL-2 and IL-4 production. APS based delivery systems enhanced the production of neutralizing antibody and the minor augmentation of IFN-γ and IL-2 levels. Squalene emulsion (SE) alone or combined with AST-VII were effective in NDV restimulated splenocyte proliferation. As a conclusion, AST-VII and AST-VII containing adjuvant systems demonstrated Th1/Th2 balanced antibody and cellular immune responses in NDV vaccines. Thus, these systems could be developed as vaccine adjuvants in viral vaccines as alternative to saponin-based adjuvants.

Identification of a Noncanonical Necrotic Cell Death Triggered via Enhanced Proteolysis by a Novel Sapogenol Derivative.

Small molecules which activate distinct cell death pathways have promising high potential for anticancer drug research. Especially, regulated necrosis draws attention as an alternative cell death mechanism to overcome the drug resistance. Here, we report that a new semisynthetic saponin analogue (AG-08) triggers necrotic cell death with unprecedented pathways. AG-08-mediated necrosis depends on enhanced global proteolysis involving calpains, cathepsins, and caspases. Moreover, AG-08 generates several alterations in lysosomal function and physiology including membrane permeabilization, redistribution toward the perinuclear area, and lastly excessive tubulation. As a consequence of lysosomal impairment, the autophagic process was abolished via AG-08 treatment. Collectively, in addition to its ability to induce necrotic cell death, which makes AG-08 a promising candidate to cope with drug resistance, its unique activity mechanisms including autophagy/lysosome impairment and enhancement of proteolysis leading a strong death capacity emphasizes its potential for anticancer drug research.

Adjuvant potency of Astragaloside VII embedded cholesterol nanoparticles for H3N2 influenza vaccine.

Adjuvants are substances that increase the immune response to a given antigen. In the development of novel vaccine adjuvants/systems, saponins are one of the most attractive molecules due to their altered immunomodulatory activities. In this study, we tried to develop PEG (polyethylene glycol)/cholesterol-based lipid nanoparticles (LNPs) to deliver the Astragaloside VII (AST-VII) and potentiate adjuvant properties of AST-VII for the influenza vaccine. In the formation of PEG/cholesterol/AST-VII-based LNPs (PEG300: Chol-AST-VII LNPs), 3 different primary solvents (acetone, ethanol, and chloroform) were evaluated, employing their effects on hydrodynamic particle size, distribution, surface chemistry, and colloidal stability. Prepared nanoparticles were simply admixtured with inactivated influenza antigen (H3N2) and applied to PMA (phorbol 12-myristate 13-acetate)-ionomycin treated human whole blood to evaluate their cytokine release profile. PEG300: Chol-AST-VII LNPs (80.2 ± 7.7 nm) were obtained using chloroform as a desolvation agent. Co-treatment of PMA-ionomycin with AST-VII and PEG300: Chol-AST-VII LNPs significantly increased the levels of IL-2 and IFN-g, compared to PMA-ionomycin alone. In the presence of H3N2, AST-VII was able to augment IL-17A, while PEG300: Chol-AST-VII LNPs stimulated the production of IFN-g. Hemolysis was only observed in PEG300: Chol-AST-VII LNPs (250 µg/mL) treatment. AST-VII and AST-VII-integrated LNPs could be used as efficacious adjuvants for an inactivated H3N2 vaccine in vitro, and cytokine response through Th1/Th17 route was reported.

Investigations on the anti-ulcerogenic activity of Sideritis caesarea H. Duman, Aytaç & Baser.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Sideritis caesarea H. Duman, Aytaç & Baser are used for complaints such as stomach-aches, and intestinal spasms as traditional medicine in Kayseri, Turkey. AIM OF STUDY: To investigate the anti-ulcerogenic activity by using bioassay guided fractionation technique (BAGF) and to identify the compound(s) that are responsible for anti-ulcerogenic activity through ethanol-induced anti-ulcerogenic activity model in vivo. MATERIALS AND METHODS: Liquid-liquid partition and then different chromatographic techniques were utilized for the BAGF of the ethanol (80%) extract of the aerial parts of Sideritis caesarea. Ethanol-induced gastric ulcer method on rats was employed for the determination of the anti-ulcerogenic activity, and the ulcer index was also calculated for anti-ulcerogenic activity detection. RESULTS: The ethanol (80%) extract of S. caesarea showed statistically potent anti-ulcerogenic activity (95.9% ulcer inhibition, p < 0.001). Among the liquid-liquid fractions, strongest anti-ulcerogenic activity was observed with the ethyl acetate fraction (91.4% inhibition, p < 0.001) and therefore BAGF studies were proceeded with the ethyl acetate fraction. Two anti-ulcerogenic flavonoids {4'-O-methylhypolaetin-7-O-[6‴-O-acetyl-ß-D-allopyranosyl-(1→2)]-6″-O-acetyl-ß-D-glucopyranoside and isoscutellarein-7-O-[6‴-O-acetyl-ß-D-allopyranosyl-(1→2)]-6″-O-acetyl-ß-D-glucopyranoside} were isolated from this fraction together with a sesquiterpene glycoside [(2E,6E)-2,6,10-trimethyl-2,6,11-dodecatriene-1,10-diol-1-O-ß-D-glucopyranoside] and two additional flavonoids {4'-O-methylhypolaetin-7-O-[6‴-O-acetyl-ß-D-allopyranosyl-(1→2)]-ß-D-glucopyranoside and isoscutellarein-7-O-[6‴-O-acetyl-ß-D-allopyranosyl-(1→2)]-ß-D-glucopyranoside}. CONCLUSIONS: Traditional use of S. caesarea in the treatment of stomach-aches was supported by this study and four flavonoids were isolated by using BAGF method and two of them were determined to have significant anti-ulcerogenic activity. Additionally, (2E,6E)-2,6,10-trimethyl-2,6,11-dodecatriene-1,10-diol-1-O-ß-D-glucopyranoside was obtained from a Sideritis genus for the first time.

Evaluation of the potential aphrodisiac activity of sesquiterpenoids from roots of Ferula huber-morathii Pesmen in male rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Several species of Ferula L. genus have been used in traditional Turkish medicine as aphrodisiac to treat male sexual dysfunction. Especially, roots and oleo gum resin of F. elaeochytris Korovin, F. communis L., F. assa-foetida L. and F. gummosa Boiss. were claimed to be used for aphrodisiac activity, menstrual regulation and treatment of gastric pain in Anatolia. Ferula L. is represented by 23 taxa in Turkey, 13 of which are endemic species. F. huber-morathii Pesmen (FHM), an endemic plant, is popularly known as ''helizan, çagsir''. AIM OF THE STUDY: This study aimed to isolate sesquiterpenoids from the roots of Ferula huber-morathii (FHM) and to confirm their aphrodisiac potential in male rats. MATERIAL AND METHODS: In a preliminary experiment, the effects of aqueous (H2O) and chloroform (CHCl3) extracts of FHM were tested for their potential aphrodisiac activities in male rats. Then, sesquiterpene derivatives were isolated from the active chloroform extract of FHM roots (FHM-R) and characterized (TLC, 1D, 2D NMR, HR-MS and CD). Moreover, some of the isolates with adequate quantities were evaluated for their possible aphrodisiac effects on male rats. Single doses (10 mg/kg BW) of sildenafil citrate (SC, positive control), gummosin, mogoltavidin, deacetylkellerin, ferukrin acetate with kellerin, elaeochytrin-A and ferutinin were administered orally by gavages to male Wistar albino rats. Mount latency (ML), mount frequency (MF), intromission latency (IL), intromission frequency (IF), ejaculation latency (EL) and postejaculatory interval (PEI) were studied. In addition, copulatory efficiency (CE) and intercopulatory efficiency (ICE) were calculated. RESULTS: The preliminary experiment revealed that the chloroform extract was the main source of the active compounds as it showed the higher aphrodisiac activity while the aqueous extract was found to be inactive. Eleven sesquiterpene derivatives, viz. gummosin, mogoltavidin, farnesiferol A, deacetylkellerin, ferukrin acetate, kellerin, teuclatriol, feruhermonin C, ferutinin, elaeochytrin A and teferidin, were isolated from the FHM-CHCl3 extract. Oral administration of deacetylkellerin, elaeochytrin-A and ferutinin significantly increased MF and IF. The ML and IL were significantly reduced, and ejaculation latencies were prolonged. Administration of these sesquiterpenoids also reduced the PEI. The present results revealed that ferutinin was the most effective aphrodisiac compound compared to other sesquiterpenoids. The results of 10 mg/kg of ferutinin are comparable to SC, the positive control. The results revealed that gummosin, mogoltavidin and ferukrin acetate with kellerin did not significantly alter the aphrodisiac parameters. CONCLUSIONS: This study has established that the CHCl3 extract of FHM root contains sesquiterpene derivatives, especially coumarin ethers and benzoic esters. Findings of the present study demonstrate that the chloroform extract and some of the sesquiterpene derivatives significantly stimulates sexual behavior in male rats, thus suggesting that F. huber-morathii possesses an aphrodisiac activity.

Microbial Transformation of Cycloastragenol and Astragenol by Endophytic Fungi Isolated from Astragalus Species.

Biotransformation of Astragalus sapogenins (cycloastragenol (1) and astragenol (2)) by Astragalus species originated endophytic fungi resulted in the production of five new metabolites (3, 7, 10, 12, 14) together with 10 known compounds. The structures of the new compounds were established by NMR spectroscopic and HRMS analysis. Oxygenation, oxidation, epoxidation, dehydrogenation, and ring cleavage reactions were observed on the cycloartane (9,19-cyclolanostane) nucleus. The ability of the compounds to increase telomerase activity in neonatal cells was also evaluated. After prescreening studies to define potent telomerase activators, four compounds were selected for subsequent bioassays. These were performed using very low doses ranging from 0.1 to 30 nM compared to the control cells treated with DMSO. The positive control cycloastragenol and 8 were found to be the most active compounds, with 5.2- (2 nM) and 5.1- (0.5 nM) fold activations versus DMSO, respectively. At the lowest dose of 0.1 nM, compounds 4 and 13 provided 3.5- and 3.8-fold activations, respectively, while cycloastragenol showed a limited activation (1.5-fold).

Development of adjuvant nanocarrier systems for seasonal influenza A (H3N2) vaccine based on Astragaloside VII and gum tragacanth (APS).

Adjuvants are chemical/biological substances that are used in vaccines to increase the immunogenicity of antigens. A few adjuvants have been developed for use in human vaccines because of their limitations including lack of efficacy, unacceptable local or systemic toxicity, the difficulty of manufacturing, poor stability, and high cost. For that reasons, novel adjuvants/adjuvant systems are under search. Astragaloside VII (AST-VII), isolated from Astragalus trojanus, exhibited significant cellular and humoral immune responses. The polysaccharides (APS) obtained from the roots of Astragalus species have been used in traditional Chinese medicine and possess strong immunomodulatory properties. In the present study, the immunomodulatory effects of a newly developed nanocarrier system (APNS: APS containing carrier) and its AST-VII containing formulation (ANS: AST-VII + APNS), on seasonal influenza A (H3N2) vaccine were investigated. Inactivated H3N2 alone or its combinations with test compounds/formulations were intramuscularly injected into Swiss albino mice. Four weeks after immunization, the immune responses were evaluated in terms of antibody and cytokine responses as well as splenocyte proliferation. APNS demonstrated Th2 mediated response by increasing IgG1 antibody titers, whereas ANS showed response towards Th1/Th2 balance and Th17 by producing of IFN-γ, IL-17A and IgG2a. Based on these results, we propose that APNS and ANS are good candidates to be utilized in seasonal influenza A vaccines as adjuvants/carrier systems.

Phytochemical screening and evaluation of the antimicrobial and antioxidant activities of Ferula caspica M. Bieb. extracts.

Chloroform, ethyl acetate and methanol extracts from the aerial parts of Ferula caspica M. Bieb. were tested for their antioxidant capacities by CUPRAC, ABTS, FRAP, Folin-Ciocalteu and aluminum chloride methods and for antimicrobial activities by the broth microdilution method. Chloroform and ethyl acetate extracts showed the highest antioxidant capacity and antimicrobial activity. Three known sesquiterpene derivatives; 1-(2',4'-dihydroxyphenyl)-3,7,11-trimethyl-3-vinyl-6(E),10-dodecadien-1-one (1), 2,3-dihydro-7-hydroxy-2,3-dimethyl-2-[4',8'-dimethyl-3',7'-nonadienyl]-furo[3,2,c]coumarin (2), 2,3-dihydro-7-hydroxy-2,3-dimethyl-3-[4',8'-dimethyl-3',7'-nonadienyl]-furo[3,2,c]coumarin(3); phenylpropanoid; laserine/2-epilaserine (4/5) and steroid mixtures; stigmasterol and ß-sitosterol (6/7) were isolated from chloroform extract; three known flavonoids; kaempferol-3-O-ß-glucopyranoside (8), kaempferol-3-O-α-rhamnopyranoside (9), quercetin-3-O-ß-glucopyranoside (10), and one benzoic acid derivative; 2,4-dihydroxybenzoic acid (11) were isolated from the ethyl acetate extract. The structures were elucidated by spectroscopic methods.

Polyethers isolated from the marine actinobacterium Streptomyces cacaoi inhibit autophagy and induce apoptosis in cancer cells.

Polyether compounds, a large group of biologically active metabolites produced by Streptomyces species have been reported to show a variety of bioactivity such as antibacterial, antifungal, antiparasitic, antiviral, and tumour cell cytotoxicity. Since some of these compounds target cancer stem cells and multi-drug resistant cancer cells, this family of compounds have become of high interest. In this study, three polyether-type metabolites (1-3), one of which was a new natural product (3), were isolated from the marine derived Streptomyces cacaoi via antimicrobial activity-guided fractionation studies. As several polyether compounds with structural similarity such as monensin have been linked with autophagy and cell death, we first assessed the cytotoxicity of these three compounds. Compounds 2 and 3, but not 1, were found to be cytotoxic in several cell lines with a higher potency towards cancer cells. Furthermore, 2 and 3 caused accumulation of both autophagy flux markers LC3-II and p62 along with cleavage of caspase-3, caspase-9 and poly (ADP-ribose) polymerase 1 (PARP-1). Interestingly, prolonged treatment of the compounds caused a dramatic downregulation of the proteins related to autophagasome formation in a dose dependent manner. Our findings provide insights on the molecular mechanisms of the polyether-type polyketides, and signify their potency as chemotherapeutic agents through inhibiting autophagy and inducing apoptosis.

Biotransformation of Neoruscogenin by the Endophytic Fungus Alternaria eureka.

Biotransformation of neoruscogenin (NR, 1, spirosta-5,25(27)-diene-1ß,3ß-diol), the major bioactive sapogenin of Ruscus preparations, was carried out with the endophytic fungus Alternaria eureka. Fourteen new biotransformation products (2-15) were isolated, and their structures were elucidated by NMR and HRESIMS data analyses. A. eureka affected mainly oxygenation, oxidation, and epoxidation reactions on the B and C rings of the sapogenin to afford compounds 8-15. In addition to these, cleavage of the spiroketal system as in compounds 2-7 and subsequent transformations provided unusual metabolites. This is the first study reporting conversion of the spirostanol skeleton to cholestane-type metabolites 2-5. Additionally, the cleavage of the C-22/C-26 oxygen bridge yielding a furostanol-type steroidal framework and subsequent formation of the epoxy bridge between C-18 and C-22 in 7 was encountered for the first time in steroid chemistry.

Biotransformation of cyclocanthogenol by the endophytic fungus Alternaria eureka 1E1BL1.

The microbial transformation of cyclocanthogenol (CCG), Astragalus sp. originated sapogenin, by the endophytic fungus Alternaria eureka 1E1BL1 isolated from Astragalus angustifolius was investigated. Hydroxylation, oxidation, epoxidation, O-methylation, ring-expansion and methyl migration reactions were observed on the triterpenoid skeleton. As a result, eight metabolites were isolated and the structures of the previously undescribed compounds were established by 1-D, 2-D NMR and HR-MS analyses.