Menthol based hydrophobic deep eutectic solvent for extraction and purification of ergosterol using response surface methodology.

Vitamin D deficiency has been linked to various diseases, but could be rectified via fortified food stuffs or supplementation. In this study 39 different hydrophobic deep eutectic solvents were evaluated for green extraction of ergosterol from mushroom. Extraction parameters (e.g. time, solvent volume) were optimized using response surface methodology (RSM) and a maximum extraction yield of 6995.00 µg ergosterol/g dry weight mushroom was attained with menthol: pyruvic acid. The extracted ergosterol was purified using a novel methodology and the extraction solvent was reused for six cycles, while retaining extraction efficiency (up to 28%). The ergosterol was exposed to ultra-violet radiation for conversion to ergocalciferol (vitamin D2) resulting in a yield of ergocalciferol that was equivalent to 2142.01 µg/g dry weight mushroom.

Supercritical and ultrasound-assisted extracts from Pleurotus pulmonarius mushroom: chemical profiles, antioxidative, and enzyme-inhibitory properties.

BACKGROUND: This study provides an insight into the impact of ultrasound-assisted extraction with water as solvent (UAEW) and extraction by supercritical carbon dioxide (SC-CO2 ) with 5% EtOH on antioxidant and enzyme inhibitory activity in regard to the chemical profile of the edible and medicinal mushroom, Pleurotus pulmonarius. RESULTS: Extraction efficiency was between 0.36% and 63.32%, depending on the extraction technique. The main compounds in the extracts were fatty acids. Supercritical CO2 extraction with co-solvent was the most suitable method for obtaining extracts that were rich in ergosterol content, reaching a value of 40.1 mg g-1 . The UAEW of crude mushroom powder ensured the highest yield, as well as the extracts with best antioxidative activity. The measurements of enzyme inhibitory activity revealed that all types of investigated extracts exhibited only tyrosinase and amylase inhibition at a significant level. CONCLUSION: Based on our results, the extraction methods significantly affected the chemical profile and bioactivity of P. pulmonarius. © 2020 Society of Chemical Industry.

Ergosterol Peroxide from the Egyptian Red Lingzhi or Reishi Mushroom, Ganoderma resinaceum (Agaricomycetes), Showed Preferred Inhibition of MCF-7 over MDA-MB-231 Breast Cancer Cell Lines.

Ergosterol peroxide and ganoderic acid AMI were isolated for the first time from the mycelium of the Egyptian Ganoderma resinaceum mushroom. The structure of these two metabolites was established by detailed analysis of 1D and 2D NMR. The isolated compounds were tested for their antitumor in vitro activities in MCF-7 and MDA-MB-231 breast cancer cell lines. Ergosterol peroxide showed preferred inhibition of MCF-7 (ER +ve) cell lines relative to MDA-MB-231 (ER -ve) cell lines with an IC50 of 1.18 µM and 12.82 µM respectively. Our data suggest that ergosterol peroxide targets estrogen receptors.

Isolation and Characterization of Chemical Constituents from the Poroid Medicinal Mushroom Porodaedalea chrysoloma (Agaricomycetes) and Their Antioxidant Activity.

The chemical analysis of the methanol extract of Porodaedalea chrysoloma (Fr.) Fiasson & Niemela afforded the isolation of five compounds (1-5). The first two are phenolic derivatives: methyl (E)-3-(4-methoxycar-bonylphenoxy)-acrylate (1) is a new natural product, while methyl 3-(4-methoxycarbonylphenoxy)-propionate (2) was isolated from a natural source for the first time. The triterpene steroids ergone (3), 3ß-hydroxyergosta-7,22-diene (4), and ergosterol (5) have not been previously identified in this species. The structures of the compounds were determined on the basis of NMR and MS spectroscopic analysis. The isolated fungal metabolites 1-5 were evaluated for their antioxidant activity. Compounds 1, 2, and 4 proved to possess considerable antioxidant effect in the ORAC assay with 2.21 ± 0.34, 1.58 ± 0.18, and 5.02 ± 0.47 mmol TE/g, respectively.

Anti-diabetic effects of Grifola frondosa bioactive compound and its related molecular signaling pathways in palmitate-induced C2C12 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Grifola frondosa (GF), a high value medicinal mushroom, is popularly consumed as traditional medicines and health foods in China and Japan. It is a herbal medicine traditionally used for treating inflammation, cancer and diabetes. AIM OF THE STUDY: This study aimed to examine the anti-diabetic effects of a GF bioactive compound ergosterol peroxide (EPO), and its mechanism(s) of action in palmitate (PA)-induced C2C12 cells. MATERIALS AND METHODS: EPO was isolated and purified from GF fruiting bodies, and used to test for anti-diabetic activity in PA-induced murine C2C12 skeletal muscle cells through measuring glucose uptake, intracellular ROS production, and expressions of MAPKs, IRS-1, PI3K, Akt and GLUT-4 proteins. RESULTS: EPO significantly up-regulated glucose absorption and increased cell growth. At 5 µM, EPO significantly enhanced glucose uptake and decreased ROS formation, as well as up-regulated the expression of IRS-1, p-IRS-1, PI3K, Akt, p-Akt, and GLUT-4 proteins in PA-induced cells, while their p-JNK and p-p38 expression were down-regulated. GLUT-4 siRNA treatment effectively down-regulated the EPO-induced absorption of glucose and inhibited the expression of GLUT-4. CONCLUSION: These results suggest that the anti-diabetic effect of GF was from its bioactive compound EPO through the inhibition of ROS production, up-regulation of glucose absorption, and modulation of PI3K/Akt, MAPKs and GLUT-4 signaling transduction pathways.

Ergostane-type steroids from the Cameroonian 'white tiama' Entandrophragma angolense.

Two new ergostane-type steroids named tiamenones A and B (1-2) were isolated from the bark of Entandrophragma angolense (Meliaceae) along with ten known compounds identified as 20S-hydroxy-4,6,24(28)-ergostatrien-3-one (3), 3ß,7α,20ß-trihydroxyergosta-5,24(28)-diene (4), 3ß,5α-dihydroxyergosta-7,22-diene (5), stigmasterol, ß-sitosterol, ß-amyrin, oleanolic acid, asperphenamate, sucrose and daucosterol. The structures of the isolated compounds have been established using NMR spectroscopic and mass spectrometric analyses. The assignment of relative and absolute configurations of compound 1 was achieved by a NOESY experiment and comparison of its NMR data with those of known compound reported in literature. Compounds 1-3, ß-amyrin and asperphenamate were evaluated for their antibacterial potencies against five bacterial model strains viz. Escherichia coli DSMZ 1058, Pseudomonas agarici DSMZ 11810, Bacillus subtilis DSMZ 704, Micrococcus luteus DMSZ 1605 and Staphylococcus warneri DSMZ 20036 and their cytotoxicity on two cell lines KB3-1 and HT-29. No potencies were exhibited by the tested compounds even at the highest concentration of 0.5 mg/mL. Compounds 1-3 were found to be potential HIV-1 inhibitors based on their molecular docking analyses.

Characterization of the bioactivities of an ethanol extract and some of its constituents from the New Zealand native mushroom Hericium novae-zealandiae.

In this study, we investigated the potential bioactivities of an ethanol extract of Hericium novae-zealandiae and four of its constituents, namely hericenone C, hericene B, ergosterol and ergosterol peroxide. The proliferation of three prostate cancer cell lines, namely DU145, LNCaP and PC3, was evaluated after treatment with the extract and constituents. It was found that both the ethanol extract and ergosterol peroxide possess anti-proliferative activities to the three prostate cancer cell lines. Ergosterol peroxide was considered likely to be one of the major compounds responsible for the anti-proliferative effect of the ethanol extract. Subsequently, the results of RT-qPCR assay showed two possible mechanisms for these anti-proliferative activities. One is apoptosis, supported by the up-regulation of CASP3, CASP8, CASP9, and an increase in the ratio of Bax/Bcl2. The other is anti-inflammation, indicated by the down-regulation of IL6 and up-regulation of IL24. The ethanol extract also exhibited antioxidant and AChE inhibitory (though weak) activities. However, none of the four compounds were found to account for these latter two activities. This is the first report of the bioactivities, and the corresponding active ingredients of lipophilic constituents from H. novae-zealandiae.

Cytotoxic ergosterols from cultures of the basidiomycete Psathyrella candolleana.

Three newly isolated ergosterols, psathergosterols A-C (1-3), together with two known ones (4 and 5), have been isolated from cultures of the basdiomycete Psathyrella candolleana. Their structures with the absolute configuration were elucidated by means of spectroscopic methods and the single crystal X-ray diffraction. Compounds 2-4 exhibited certain cytotoxicities to five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480).

Anti-Inflammatory Effects of 5α,8α-Epidioxycholest-6-en-3ß-ol, a Steroidal Endoperoxide Isolated from Aplysia depilans, Based on Bioguided Fractionation and NMR Analysis.

Sea hares of Aplysia genus are recognized as a source of a diverse range of metabolites. 5α,8α-Endoperoxides belong to a group of oxidized sterols commonly found in marine organisms and display several bioactivities, including antimicrobial, anti-tumor, and immunomodulatory properties. Herein we report the isolation of 5α,8α-epidioxycholest-6-en-3ß-ol (EnP(5,8)) from Aplysia depilans Gmelin, based on bioguided fractionation and nuclear magnetic resonance (NMR) analysis, as well as the first disclosure of its anti-inflammatory properties. EnP(5,8) revealed capacity to decrease cellular nitric oxide (NO) levels in RAW 264.7 macrophages treated with lipopolysaccharide (LPS) by downregulation of the Nos2 (inducible nitric oxide synthase, iNOS) gene. Moreover, EnP(5,8) also inhibited the LPS-induced expression of cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) at the mRNA and protein levels. Mild selective inhibition of COX-2 enzyme activity was also evidenced. Our findings provide evidence of EnP(5,8) as a potential lead drug molecule for the development of new anti-inflammatory agents.

Isolation, characterization and DFT studies of epoxy ring containing new withanolides from Withania coagulans Dunal.

A new withanolide named as withacoagulin J (1) along with a known withanolide H (2) from Withania coagulans Dunal are reported in this paper. The isolated compounds were elucidated by using 1D-NMR (1H NMR, 13C NMR) and 2D-NMR including homonuclear (COSY, NOESY) and heteronuclear (HSQC, HMBC); along with Mass spectrometry, UV-Visible and IR spectroscopic techniques. The molecular formula based on Fast-Atom Bombardment Mass Spectrometry [FAB-MS (M + 1)] for 1 and 2 were deduced as C28H37O5 and C28H39O6 with m/z values 453.2624 and 471.6041, respectively. The quantum mechanical studies of both compounds are based on DFT calculations. The DFT studies show band gaps of 4.86 and 4.83 eV for 1 and 2, respectively. The band gaps of 1 and 2 reflect high stability and resistivity towards oxidation-reduction reactions. The energies of HOMO and LUMO for compound 1 are -6.11 and -1.25 eV and for compound 2: -6.47 and -1.64 eV respectively. Theoretical and experimental FTIR data closely match for both the compounds which support the high accuracy of the computational protocol selection. Other parameters such as bond lengths, bond angles and dihedral angles for both compounds are also studied.

A natural food ingredient based on ergosterol: optimization of the extraction from Agaricus blazei, evaluation of bioactive properties and incorporation in yogurts.

In recent years, mycosterols have emerged as potential functional ingredients for the development of sterol-enriched food products and dietary supplements. Agaricus blazei is a mushroom rich in bioactive compounds. For commercial purposes, their fruiting bodies must obey rigid morphological criteria. Those not conforming to these criteria are usually discarded, although this does not mean impairment of their content in bioactives. The aim of the present work was to propose the use of commercially discarded A. blazei fruiting bodies for obtaining an extract rich in ergosterol as a fortifier ingredient for yogurts. For extraction, the Soxhlet technology was used and the highest ergosterol yield (around 12%) was achieved in the 5th cycle, yielding 58.53 ± 1.72 µg of ergosterol per 100 g of mushroom (dry weight). The ergosterol rich extract presented notable antioxidant and antimicrobial properties, besides showing no hepatotoxicity. When added to the yogurts it significantly enhanced their antioxidant properties. Furthermore, it did not significantly alter the nutritional or the individual fatty acid profiles of the final dairy products. Thus, A. blazei fruiting bodies that do not conform to the commercial requirements of the market and are normally discarded could be exploited for obtaining a natural high added-value food additive, following the circular bioeconomy concept.

Apoptosis induction by Pleurotus sajor-caju (Fr.) Singer extracts on colorectal cancer cell lines.

Pleurotus sajor-caju (PSC) is an edible mushroom used in food supplements, presenting antitumor properties through induction of cell death pathways. The PSC potential against colorectal cancer was analyzed by exposing HCT116wt cells to different PSC extracts. The PSC n-hexane extract (PSC-hex) showed the highest cytotoxicity effect (IC50 value 0.05 mg/mL). The observed cytotoxicity was then associated to apoptosis-promoting and cell cycle-arrest pathways. PSC-hex was able to induce apoptosis related to breakdown of mitochondrial membrane potential and ROS generation. The absence of cytotoxicity in HTC116-p53 and HTC116-Bax cells, alongside with an increase in p53, Bax and Caspase-3 expression, and decrease in Bcl-2 expression, supports that the pro-apoptotic effect is probably induced through a p53 associated pathway. PSC-hex induced cell cycle arrest at G2/M in HCT116wt without cytotoxicity in HTC116-p21 cells. These findings suggest that a p21/p53 cell cycle regulation pathway is probably disrupted by compounds present on PSC-hex. Identification of the major components was then performed with ergosta-5,7,22-trien-3ß-ol representing 30.6% of total weight. In silico docking studies of ergosta-5,7,22-trien-3ß against Bcl-2 were performed and results show a credible interaction with the Bcl-2 hydrophobic cleft. The results show that PSC-hex can be used as supplementary food for adjuvant therapy in colorectal carcinoma.

In Vitro and In Vivo Activity of Fomitopsis Pinicola (Sw. Ex Fr.) Karst Chloroform (Fpkc) Extract Against S180 Tumor Cells.

BACKGROUND/AIMS: Non-toxic fomitopsis is has been traditionally used in folk medicine in many countries for its anti-inflammatory and anti-vascular disease activities. The present study investigates the antitumor effect of Fomitopsis pinicola (Sw. Ex Fr.) Karst chloroform extract (FPKc) on S180 tumor cells in vitro and in vivo and we determined the underlying mechanisms. METHODS: HPLC was employed to analyze the constituents of FPKc. In-vitro 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to quantify the growth inhibition of FPKc; Propidium iodide (PI) exclusion assay and scanning electron microscopy (SEM) were used to observe the damage on the cell membrane and the changes of the cell morphology; Staining with Hoechst 33342/propidium iodide (HO/PI) and the application of the Annexin V-FITC/PI analysis permitted to observe the cell death triggered by FPKc; DNA damage and cell cycle arrest were detected by flow cytometry; Rhodamine 123 (RH123) and Cytochrome C were used as dyes to investigate the alterations of the mitochondria. In-vivo tumor inhibition and mice survival time were determined. RESULTS: The results of the HPLC assay indicated that FPKc might contain DA (dehydroeburiconic acid), PA (pachymic acid), and ES (ergosterol), at percentages of 0.25%, 17.8%, and 10.5%, respectively. Concerning the study of the biological function, the results showed that FPKc exhibited preferential and significant suppression of proliferation on specific cell lines including S180, HL-60, U937, K562, SMMC-7721, and Eca-109 cells, which induced plasma membrane and cell morphology damages, triggering S180 tumor-cells late apoptosis and leading to DNA damage and S phase arrest. Mitochondria were suspected to play a vital role in these changes. In vivo data indicated that FPKc inhibited the solid tumor growth and prolonged the survival time of tumor-bearing mice. Moreover, FPKc provoked only little damage on normal cells in vitro and also on normal tissues in vivo. CONCLUSION: FPKc inhibited S180 tumor cells growth and prolonged the lifespan of mice. In vitro, we found that FPKc induced S180 tumor cells apoptosis and cell cycle arrest, possibly via the mitochondrial pathway.

Anti-Inflammatory Activity and Bioactive Constituents of Cultivated Fruiting Bodies of Xylaria nigripes (Ascomycetes), a Chinese Medicinal Fungus.

Xylaria nigripes, also known as Wu Ling Shen, is popular for treating insomnia and trauma in traditional Chinese medicine. This study aimed to examine the anti-inflammatory activity and bioactive constituents of cultivated X. nigripes fruiting bodies in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Results showed that among the different extracts, the hexane fraction exhibited the best protection against cell toxicity induced by 1 µg/mL LPS and the strongest inhibitory effect on nitric oxide (NO) production. This fraction led to the isolation of 2 bioactive compounds (namely, XN-CP1 and XN-CP2), which were confirmed to be ergostarien-3ß-ol and ergosterol peroxide, respectively. Although both XN-CP1 and XN-CP2 showed good inhibitory effects on NO, tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and prostaglandin E2 production in LPS-stimulated macrophages, XN-CP2 was shown to have a stronger anti-inflammatory activity; this was further supported by its strong suppressive effects on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and nuclear factor (NF)-κB activation. These results conclude that ergosterol peroxide (XN-CP2) could be the main bioactive compound contributing to the potent anti-inflammatory activity of X. nigripes, and its mechanism of action is mediated through inhibition of iNOS and COX-2 expression via the NF-κB signaling pathway.

In Vitro Expression of Toll-Like Receptors and Proinflammatory Molecules Induced by Ergosta- 7,22-Dien-3-One Isolated from a Wild Mexican Strain of Ganoderma oerstedii (Agaricomycetes).

Compounds showing pharmacological activity on the immune system are of interest because of their therapeutic potential in the treatment of many diseases. However, data from primary human immune cells and in vivo studies are limited. The aim of this study was to analyze the ability to induce the expression of Toll-like receptors (TLRs) and proinflammatory molecules on cells involved in the immune system using the compound ergosta-7,22-dien-3- one, isolated from a wild Mexican strain of Ganoderma oerstedii. According to our study, ergosta-7,22-dien-3-one did not present any cytotoxic effect on HeLa or J774A.1 cells, and it was able to stimulate nitric oxide production, induce the expression of genes, and induce the production of TLRs, cytokines, chemokines, and cellular adhesion molecules in J774A.1 cells, based on reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. Here we report a new pro-inflammatory property of ergosta-7,22-dien-3-one, which should be considered as a possible adjuvant property in view of its biological activity.

Ergosterol Peroxide: A Mushroom-Derived Compound with Promising Biological Activities-A Review.

Ergosterol peroxide (EP; 5α,8α-epidioxy-22E-ergosta-6,22-dien-3ß-ol) is a C28-sterol and a component of many medicinal mushrooms. Since its discovery nearly a century ago, many sources and biological effects of this compound have been described. Effects range from antimicrobial to cytotoxic to immunosuppressive and other activities. This review covers biological investigations of EP, its activities, and possible mechanisms of action. EP is a promising candidate for drug development and contributes to the health-promoting effects of medicinal mushrooms.

Fluconazole treatment enhances extracellular release of red pigments in the fungus Monascus purpureus.

Traditional methods for the production of food grade pigments from the fungus Monascus spp. mostly rely on submerged fermentation. However, the cell-bound nature and intracellular accumulation of pigments in Monascus spp. is a major hurdle in pigment production by submerged fermentation. The present study focused on the investigation of the effect of the antifungal agent fluconazole on red pigment production from Monascus purpureus (NMCC-PF01). At the optimized concentration of fluconazole (30 µg ml-1), pigment production was found to be enhanced by 88% after 96 h and it remained constant even after further incubation up to 168 h. Ergosterol, a sterol specific to fungi, was also extracted and estimated as a function of fungal growth. The concentration of ergosterol in fluconazole-treated fermentation broth was reduced by 49% as compared to control broth. Thus it could be responsible for facilitating the release of intracellular and cell-bound pigments. Nevertheless, the role of cell transporters in transporting out the red pigments cannot be ignored and deserves further attention. Qualitative analysis of red pigment by thin layer chromatography, UV spectroscopy and mass spectrometric analysis (ESIMS) has confirmed the presence of the well-known pigment rubropunctamine. In addition, this fermentation process produces citrinin-free pigments. This novel approach will be useful to facilitate increased pigment production by the release of intracellular or cell-bound Monascus pigments.

The structure of antrocarine E, an ergostane isolated from Antrocaryon klaineanum Pierre (Anacardiaceae).

Eight compounds were isolated from the stem bark of Antrocaryon klaineanum, and their structures determined by chemical and spectroscopic methods. Among these compounds, the ergostane-type antrocarine E (1a) is a new compound, although the structure had already been published by mismatching the spectroscopic data with those of 2. In this paper, we are reporting the valid spectroscopic values for antrocarine E and X-ray diffraction results.

Metabolite Profiling in Withania somnifera Roots Hydroalcoholic Extract Using LC/MS, GC/MS and NMR Spectroscopy.

Ashwagandha (Withania somnifera) is a very well-known herbal medicine and it was well studied for its active metabolites throughout the World. Although, nearly 40 withanolides were isolated from W. somnifera root extract, still there is remaining unidentified metabolites due to very low abundance and geographical variation. Advanced separation technology with online identification by mass and nuclear magnetic resonance (NMR) are nowadays used to find out the new compounds in the crude herbal extract. This article described the metabolite profiling of ashwagandha root hydroalcoholic extract using ultra-performance liquid chromatography coupled with a positive ion electrospray ionization tandem mass spectrometry through gas chromatography mass spectrometry (GC/MS) and NMR spectroscopy. A total of 43 possible withanolides was identified and proposed their structures based on the mass of molecular and fragment ions. GC/MS and NMR analysis indicated the presence of several known withanolides including withaferin A, withanolide D, withanoside IV or VI, withanolide sulfoxide, etc. To the best of our knowledge, dihydrowithanolide D at m/z 473 (tR 7.86 min) and ixocarpalactone A at m/z 505 (tR 8.43 min) were first time identified in the ashwagandha root hydroalcoholic extract. The current study that described the identification of withanolides with summarized literature review might be helpful for designing the experiment to identify of the new chemical constituents in Withania species.

A new compound from Stereum insigne CGMCC5.57.

Studies of the EtOAc extract of the culture broth and methanol extract of the mycelium of Stereum insigne CGMCC5.57 led to the isolation of one new dihydrobenzofuran (1) and six known compounds (2-7). The structures of compounds were elucidated mainly by HRESIMS experiments, and 1D, 2D NMR spectroscopy analysis. This is the first report about the chemical constitutes of the fungus S. insigne.