Optimization of liquid fermentation conditions for Coprinus comatus to enhance antioxidant activity.

Coprinus comatus is an edible and medicinal fungus. In this study, the antioxidant activity of the fermentation product of C. comatus was investigated through optimization of fermentation process. The results indicated that the fermentation product of C. comatus had obvious scavenging ability for 2,2'-Azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) free radical. The EC50 of the n-butanol extract from the fermentation product on ABTS·+ was 0.65 ± 0.02 mg/mL. On this basis, the liquid fermentation conditions of C. comatus were optimized through single factor and response surface optimization experiments according to the scavenging ability of ABTS·+ to improve the antioxidant capacity of the fermentation product. The results showed that when the 14% of C. comatus was fermented in a culture medium with a C/N ratio of 48:1 for 6 days, the ABTS·+ scavenging ability was the strongest, and the EC50 of n-butanol extract was 0.57 ± 0.01 mg/mL, which was 12.31% higher than the initial activity. This study laid the foundation for the development of C. comatus.

Ethanol extract of the mushroom Coprinus comatus exhibits antidiabetic and antioxidant activities in streptozotocin-induced diabetic rats.

CONTEXT: Edible mushrooms have a long history of use in traditional Chinese or Japanese medicine. Coprinus comatus (O.F. Müll.) Pers. (Agaricaceae) contains antioxidant and antidiabetic agents. OBJECTIVE: To identify the benefits of ethanol extracts of the C. comatus fruit body in streptozotocin-induced hyperglycaemic rats by evaluating their blood glucose, glycosylated haemoglobin (HbA1c), insulin, glucagon-like peptide-1 (GLP-1), dipeptidyl peptidase-4 (DPP-4), and glutathione (GSH) levels, with and without extract administration. MATERIALS AND METHODS: Wistar rats were either left untreated or were administered 45 mg/kg body weight (BW) streptozotocin; 45 mg/kg BW metformin; or 250, 500, or 750 mg/kg BW extract for 14 days. The blood glucose, GLP-1, DPP-4, GSH, insulin, and HbA1c levels were determined. Data were analysed using analysis of variance and Duncan's multiple range tests. RESULTS: Preliminary data showed that administration of C. comatus ethanol extract dose of 250, 500, and 750 mg orally has no toxicity effects after 24 h administration. The ethanolic extract of fruiting body of C. comatus considerably reduced the rat's fasting blood glucose levels 26.69%, and DPP-4 6.97% at dose of 750 mg. The extract reduced HbA1c 4-4.30%, increased GLP-1 71.09%, GSH 11.19% at dose of 500 mg, and increased insulin levels 13.83%. Extracts contain bioactive compounds such as flavonoid, alkaloid, terpenoids, vitamins C and E, rutin, and saponin. CONCLUSIONS: The C. comatus extract can be used as herbal medicine that reduces diabetic symptoms. Further investigation on C. comatus extracts should be conducted with gas chromatography-mass spectrometry to characterise the bioactive compounds.

The two faces of Coprinus comatus-Functional properties and potential hazards.

Mushrooms have been used for centuries not only as food but also in traditional medicine as a source of components with pro-health activity. One of them is Coprinus comatus (O.F.Müll.) Pers. also called shaggy mane, chicken drumstick mushroom, or lawyer's wig. In Asian countries, C. comatus (CC) is approved as edible mushroom and often cultivated for consumption, whereas in many other countries, although it is widespread, it is unrecognized and not used. In this review, for the first time, we discussed about the composition related to functional properties as well as the potential risks associated with consumption of CC by reviewing scientific literature. The information has been collected in order to get to know this species thoroughly. Various studies show many of the physiological activities, such as antioxidant, anticancer, antiandrogenic, hepatoprotective, acetylcholinesterase inhibitory, antiinflammatory, antidiabetic, antiobesity, antibacterial, antifungal, antinematode, and antiviral. Besides positive physiological properties, CC has also negative features, for example, skin reactions in patients with dermatitis and atopic predisposition, risk of confusion with poisonous mushrooms, quick autolysis after collection, and contamination of toxic elements.

Biosynthesis of lagopodins in mushroom involves a complex network of oxidation reactions.

Use of the ku70-deficient strain of Coprinopsis cinerea enabled confirmation within the native context of the central role the sesquiterpene synthase Cop6 plays in lagopodin biosynthesis. Furthermore, yeast in vivo bioconversion and in vitro assays of two cytochrome P450 monooxygenases Cox1 and Cox2 allowed elucidation of the network of oxidation steps that build structural complexity onto the α-cuprenene framework during the biosynthesis of lagopodins. Three new compounds were identified as intermediates formed by the redox enzymes.

Spirobenzofuran (SBF): An isolate from Acremonium sp. HKI 0230 and Coprinus echinosporus suppresses inflammation in RAW 264.7 cells.

Inflammation is an aggregate of different pathologic responses in body that leads to life threatening conditions if not combated at early stages. A variety of chemical medications from low quality to high quality are available in market for treatment of inflammation. However the side effects posed by these medications cannot be ignored. Here in our study we have shown for the first time, the anti-inflammatory effects of SBF compound that is obtained from wild mushroom species that are Acremonium sp. HKI 0230 and Coprinus echinosporus. We employed Nitric oxide determination, cell viability assay, RT-PCR and western blot analysis to check the anti-inflammatory effects of SBF. The antioxidant activity of this compound has been studied in detail in past, but our results have shown that SBF potently suppressed the Nitric oxide production (NO) without any cytotoxicity to the model cell line; RAW 264.7 cells. It also inhibited the production of major proinflammatory mediators and cytokines i.e. iNOS, COX-2, IL-1ß, IL-6 and TNF-α. SBF elicited its anti-inflammatory effects via the canonical NF-κB and MAPK pathway. Taken together, our results have shown that SBF exhibits excellent anti-inflammatory activity in vitro and further experimentations may warrant its application as a commercial herbal remedy for inflammation related anomalies.

Coprinuslactone protects the edible mushroom Coprinus comatus against biofilm infections by blocking both quorum-sensing and MurA.

Pathogens embedded in biofilms are involved in many infections and are very difficult to treat with antibiotics because of higher resistance compared with planktonic cells. Therefore, new approaches for their control are urgently needed. One way to search for biofilm dispersing compounds is to look at defense strategies of organisms exposed to wet environments, which makes them prone to biofilm infections. It is reasonable to assume that mushrooms have developed mechanisms to control biofilms on their sporocarps (fruiting bodies). A preliminary screening for biofilms on sporocarps revealed several species with few or no bacteria on their sporocarps. From the edible mushroom Coprinus comatus where no bacteria on the sporocarp could be detected (3R,4S)-2-methylene-3,4-dihydroxypentanoic acid 1,4-lactone, named coprinuslactone, was isolated. Coprinuslactone interfered with quorum-sensing and dispersed biofilms of Pseudomonas aeruginosa, where it also reduced the formation of the pathogenicity factors pyocyanin and rhamnolipid B. Coprinuslactone also damaged Staphylococcus aureus cells in biofilms at subtoxic concentrations. Furthermore, it inhibited UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), essential for bacterial cell wall synthesis. These two modes of action ensure the inhibition of a broad spectrum of pathogens on the fruiting body but may also be useful for future clinical applications.

Transformation of multi-component ginkgolide into ginkgolide B by Coprinus comatus.

BACKGROUND: As the strongest antagonist of the platelet activating factor, ginkgolide B (GB) possesses anti-ischemic, anti-oxidant and anti-convulsant properties, and it is used for the treatment of thrombosis in clinical practice. Till now, GB is usually obtained from extraction of Ginkgo biloba leaves through column chromatography with an extremely low yield and high cost, which can not meet clinical requirement. Therefore, it is urgent to find a new method to prepare GB. RESULTS: In the current study, we studied the ability and mechanism to transform multi-component ginkgolide into GB by Coprinus comatus in order to enhance the GB yield. Except for ginkgolide A (GA) and GB, all the other ginkgolides in the extract were transformed by the strain. In the case of culture medium containing 20 g/L glucose, the transformation product was identified as 12% GA and 88% GB by high performance liquid chromatography-Mass spectrometry (HPLC-MS), two stage mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR). Partial GA was also transformed into GB according to the yield (76%) and the content of GA in the raw ginkgolide (28.5%). Glucose was the key factor to transform ginkgolides. When glucose concentration in medium was higher than 40 g/L, all ginkgolides were transformed into the GB. Proteomic analysis showed that C. comatus transformed ginkgolide into GB by producing 5 aldo/keto reductases and catalases, and enhancing the metabolism of glucose, including Embden-Meyerhof pathway (EMP), hexose monophophate pathway (HMP) and tricarboxylic acid (TCA). CONCLUSIONS: C. comatus could transform ginkgolides into GB when the medium contained 40 g/L glucose. When the strain transformed ginkgolides, the glucose metabolism was enhanced and the strain synthesized more aldo/keto reductases and catalases. Our current study laid the groundwork for industrial production of GB.

Assessing the immunomodulatory potential of high-molecular-weight extracts from mushrooms; an assay based on THP-1 macrophages.

BACKGROUND: Food is a potential source of immunomodulating compounds that may be used to steer immune responses towards a desired status such as reducing inflammatory disorders. However, to identify and characterize such bioactive compounds, biologically relevant and standardized assays are required. Macrophages play an important role in immunomodulation and are suited for developing cell-based assays. An assay was developed based on macrophages, in a homogeneous differentiation state, using the human monocytic cell line THP-1 previously used to assess immunomodulatory properties of low-molecular-weight allergens, hormones, dietary supplements and therapeutic drugs. RESULTS: Zymosan and mushroom polysaccharide extracts lead to a heterogeneous differentiation state of THP-1 monocytes, and these cells secrete low levels of cytokines upon stimulation. Differentiation into macrophages using a low concentration of phorbol 12-myristate 13-acetate improved responsiveness. Elevated levels of cytokines were secreted by cells in a homogenous differentiation state. In addition, it was determined that the assay performs best when using cells at a concentration of (2.5-5) × 10(5) cells mL(-1). CONCLUSION: An assay was developed suitable to distinguish the immunomodulatory properties of food compounds in a reproducible manner. It was evaluated using eight mushroom species by measuring the secretion of relevant cytokines TNF-α, IL-1ß, IL-6 and IL-10.

[Expression and characterization of Coprinus cinereus peroxidase].

OBJECTIVE: The aim of our study is to express Coprinus cinereus peroxidase (CIP) in Pichia Pastori efficiently. METHODS: We synthesized CIP gene with P. pastori codon bias by our Gene Synthesis and site-specific mutagenesis platform, using DNAWorks 3.1 program to design and optimize primers. Then, we sequenced the PCR products, inserted the correct gene into expression vector pPICZαA and transformed the linearized pPICZαA-Cip DNA into P. pastori GS115. We integrated CIP gene into the genome of P. pastori, using the α-mating factor from Sacchoramyces cerevisiae as signal peptide to direct the secretion of the recombinant protein. To obtain transformants with high CIP activity, we checked transformants by nested PCR and stained 82 positive ones on YPD agar plate with 1000 mg/L Zeocin. Then, we got 6 transforments with high resistance to Zeocin and expressed them in small scale; the one exhibiting the highest activity was chosen as engineered strain and named CIP/Gs115. RESULTS: We purified CIP from culture medium after induction with ethanol, the maximum activity reached 487.5 U/mL on the 4th day. The purified CIP exhibited maximal activity at pH 5.0 and 25 degrees C with ABTS as substrate. The enzyme had 61.5% of the maximal activity at 45 degrees C and was stable below 40 degrees C. However, the stability was drastically reduced above 45 degrees C. The recombinant CIP remained stable between pH 4.5 and 6.5. We studied the substrate specificity on different substrates with the purified enzyme, and the optimal substrates were in the order of ABTS > 2, 6-Dimethoxyphenol > guaiacol > 2, 4-Dichlorophenol > phenol. CONCLUSION: The highly secretory expression of CIP and high special activity lay the good foundation for it' s industrial applications in waste water treatment, decolouration of dyestuffs.

Illudins C2 and C3 stimulate lipolysis in 3T3-L1 adipocytes and suppress adipogenesis in 3T3-L1 preadipocytes.

The secondary metabolites illudins C2 (1) and C3 (2), obtained from the culture broth of Coprinus atramentarius, have been shown to possess antimicrobial activity. In the present study, we discovered novel biological activities of 1 and 2 in lipolysis of differentiated 3T3-L1 adipocytes and adipogenesis of 3T3-L1 preadipocytes. Compounds 1 and 2 exhibit a dose-dependent increase in glycerol release and thereby reduce intracellular lipid accumulation. The stimulatory effects of 1 and 2 on lipolysis are prevented by cAMP-dependent protein kinase (PKA) and extracellular signal-regulated kinase (ERK) inhibitors. Compounds 1 and 2 down-regulated perilipin and also affected the mRNA and protein levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). However, 1 and 2 treatment leads to a significant increase in PKA-mediated phosphorylation of HSL at S563 and S660. In addition, 1 and 2 treatment in 3T3-L1 preadipocytes induces down-regulation of the critical transcription factors, CCAAT/enhancer binding protein α and ß (C/EBPα and C/EBPß), and peroxisome proliferator activated receptor γ (PPARγ), which are required for adipogenesis, and accordingly inhibits adipogenesis. These results suggest that 1 and 2 might be useful for treating obesity due to their modulatory effects on fat by affecting adipocyte differentiation and fat mobilization.

Guanacastane-type diterpenoids with cytotoxic activity from Coprinus plicatilis.

Four new guanacastane-type diterpenoids (1-4), together with the known compound, guanacastepene E (5), were isolated from a basidiomycete of the macro-fungi, Coprinus plicatilis 82. Their structures were elucidated on the basis of extensive spectroscopic analyses, including FT-ICR-MS, UV, IR and 1D and 2D NMR experiments. The in vitro cytotoxic activities of all compounds against the human cancer cell lines HepG2, HeLa, MDA-MB-231, BGC-823, HCT 116, and U2OS were evaluated, only compound 1 exhibited significant cytotoxicities with IC(50) values ranging from 1.2 to 6.0 µM.

Isolation and biological activity of triglycerides of the fermented mushroom of Coprinus Comatus.

BACKGROUND: Although many physiological functions of Coprinus comatus have been reported, there has been no report on the antinociceptive activity of Coprinus comatus. Therefore, the objective of the present study is to demonstrate the production, isolation, and biological properties of triglycerides (TFC) of the fermented mushroom of Coprinus comatus. METHODS: The effects of TFC on cytokines levels, total antioxidant activity, antinociceptive effects in vivo, LD50 and tactile hyperalgesia were analyzed respectively. RESULTS: TFC treatment decreased the levels of cytokines and total antioxidant status (TAOS) and inhibited the acetic acid-induced abdominal constrictions in mice. In addition, TFC reduced CFA-induced tactile hyperalgesia in a dose-dependent manner and the LD50 of TFC was determined to be 400 mg/kg. However, TFC did not significantly inhibit the reaction time to thermal stimuli in the hot-plate test. CONCLUSIONS: TFC showed anti-inflammatory, antioxidant, peripheral antinociceptive and antihyperalgesic activity in various models of inflammatory pain. The data suggest that TFC may be a viable treatment option for inflammatory pain.

Inhibition of the androgen receptor activity by Coprinus comatus substances.

Prostatic adenocarcinoma is the second leading cause of death from cancer in Western men. The common prostate cancer treatments are effective in the early stages; however, advanced prostate cancer is resilient to most of these treatments. Altered androgen receptor (AR) activity caused by point mutations or signaling mechanisms that regulate AR function has been proposed as a key mechanism in the transition to the androgen-independent stage. Our previous results demonstrated that hexane extract prepared from Coprinus comatus (C. comatus) strain 734 was able to interfere with AR activity. The current study was made to further evaluate the antiandrogenic activity of the C. comatus mushroom strain 734. Activity-guided chromatography was conducted and 2 active fractions, F-32-and F-33, were found to contain substances that were able to inhibit AR-mediated reporter activity and reduce the levels of AR and prostate-specific antigen (PSA) transcripts in LNCaP cells. Fraction F-32 also inhibited the proliferation and clonigenicity of LNCaP cells. Furthermore, F-32 was able to inhibit the binding of AR to the PSA enhancer region and to inhibit Akt-mediated AR phosphorylation at Ser 213. This study illustrated the potential of substances from the C. comatus mushroom to serve as natural antiandrogenic modulators for the treatment of prostatic disorders.

The Shaggy Inc Cap medicinal mushroom, Coprinus comatus (O.F.Mull.: Fr.) Pers. (Agaricomycetideae) substances interfere with H2O2 induction of the NF-kappaB pathway through inhibition of Ikappaalpha phosphorylation in MCF7 breast cancer cells.

Breast cancer is the most commonly diagnosed cancer among women. Currently, there is no effective therapy for malignant estrogen-independent breast cancer. In our study, we used hydrogen peroxide, a well-known strong oxidative reagent capable of activating the nuclear factor kappa B (NF-kappaB) transcription factor. The IC50 value of the culinary-medicinal Shaggy Inc Cap mushroom Coprinus comatus culture liquid crude extract on MCF7 cell viability was found to be as low as 76 microg/mL, and the IC50 value of C. comatus ethyl acetate extract was only 32 microg/ mL. Our results also showed that both extracts significantly affected IkappaBalpha phosphorylation in a dose-dependent manner. The effect of ethyl acetate extract was comparable to the effect of curcumin, a known NF-kappaB pathway inhibitor, and seemed to be the most active inhibitor of H2O2-dependent IkappaBalpha phosphorylation. In addition, the data obtained showed that only ethyl acetate extract inhibited the activity of IKK complex, at close to 90% as compared to the control of the untreated sample. These results suggest that C. comatus contains potent compounds capable of inhibiting NF-kappaB function and also possibly acts as an antitumor agent.

Decolorization of the anthraquinone dye Cibacron Blue 3G-A with immobilized Coprinus cinereus in fluidized bed bioreactor.

Coprinus cinereus, which was able to decolorize the anthraquinone dye Cibacron Blue 3G-A (CB) enzymatically, was used as a biocatalyst for the decolorization of synthetic solutions containing this reactive dye. Coprinus cinereus was immobilized in both calcium alginate and polyacrylamide gels, and was used for the decolorization of CB from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when Coprinus cinereus was entrapped in calcium alginate beads, and was of about 3.84 mg g(-1) h(-1) with a 50% conversion time (t1/2) of about 2.60 h. Moreover, immobilized fungal biomass in calcium alginate continuously decolorized CB even after 7 repeated experiments without significant loss of activity, while polyacrylamide-immobilized fungal biomass retained only 67% of its original activity. The effects of some physicochemical parameters such as temperature, pH and dye concentration on decolorization performance of isolated fungal strain were also investigated.

Polarography as a technique of choice for the evaluation of total antioxidant activity: The case study of selected Coprinus Comatus extracts and quinic acid, their antidiabetic ingredient.

This study was focused on in vitro screening of the total antioxidant activity of the selected extracts of the mushroom Coprinus comatus and quinic acid, one of their antidiabetic ingredients, by an uncommon electrochemical assay. Indeed, direct current (DC) polarographic HydroxoPerhydroxo Mercury(II) Complex (HPMC) assay based on decrease of anodic limiting current originating from HPMC formation in alkaline solutions of hydrogen peroxide at potential of mercury dissolution, observed upon gradual addition of antioxidants, was applied herein for the estimation of the natural products' antioxidativity. Quinic acid was found to exhibit most promising antioxidant potential (4.0 ± 0.2%µL-1) being ≈ 2-fold more active than the screened C. comatus extract samples. Actually, such a finding puts some light on the antioxidativity of cyclic polyols, well understimated class of organic compounds, compared to aromatic (poly)phenolics. As a low cost, easy-to handle and accurate this polarographic assay may be thoroughly recommended for much broader use. [Formula: see text].

Characterization and anti-diabetic nephropathic ability of mycelium polysaccharides from Coprinus comatus.

It seems quite necessary for obtaining effective substances from natural products against the diabetic nephropathic (DN) with the presently clinical problems of accompanying side-effects and lowing life qualities. This work aimed to characterize the primary structure of Coprinus comatus mycelium polysaccharides (CMP) and investigate the abilities against DN in streptozotocin induced mice models. The results indicated that CMP could improve insulin resistance and energy metabolism, and significantly suppress dysfunction on kidney and relieve the renal oxidative stress and inflammation in DN mice. Besides, the western blot results suggested that CMP reversed renal injury by modulating the PTEN/PI3K/Akt and Wnt-1/ß-catenin pathways. The structure analysis indicated the typical characterizations with the major monosaccharide-compositions of galactose, α-pyranose configuration and proper molecular weights of 495.8 kDa possibly contributed to the anti-diabetic nephropathic effects of CMP. The results suggested that polysaccharides form C. comatus could be used as functional foods/drugs on preventing diabetic nephropathy.

Mushrooms as potential therapeutic agents in the treatment of cancer: Evaluation of anti-glioma effects of Coprinus comatus, Cantharellus cibarius, Lycoperdon perlatum and Lactarius deliciosus extracts.

Cancer incidence rates are on the increase worldwide. The most common brain cancer in adults is glioblastoma. Currently available treatment modalities are limited and natural products such as mushrooms could enhance them. Apart from nutritional value, mushrooms are an excellent source of bioactive compounds and therefore could be used to treat various disorders. The aim of the study was to assess the anti-glioma potential of selected mushrooms on U87MG, LN-18 glioblastoma and SVGp12 normal human astroglial cell lines. The materials were Cantharellus cibarius, Coprinus comatus, Lycoperdon perlatum and Lactarius delicious. Aqueous, 70 % ethanol or 95 % ethanol extracts from mushrooms were used for analysis including assessment of antioxidant activity by DPPH assay, cell viability by MTT assay, DNA biosynthesis by thymidine incorporation assay, activity of metalloproteinase by gelatin zymography and cell cycle assay by flow cytometry. Mushroom extracts influenced the viability and DNA biosynthesis of cancer cells. Activity of ethanol mushroom extracts was stronger than that of aqueous extracts. Anti-glioma mechanism consisted in inhibition of cancer cell proliferation and induction of apoptosis associated with arrest of cells in subG1 or G2/M phase of cell cycle, and inhibition of metalloproteinases activity. Among investigated mushrooms, L. deliciosus and C. comatus showed the greatest anti-glioma potential.

Antineoplastic agents 582. Part 1: Isolation and structure of a cyclobutane-type sesquiterpene cancer cell growth inhibitor from Coprinus cinereus (Coprinaceae).

Bioassay-guided (murine P388 lymphocytic leukemia and human cancer cell lines) separation of an ethyl acetate extract prepared from the inky cap fungus Coprinus cinereus led to the isolation of three new sesquiterpenes, 7,7a-diepicoprinastatin 1 (1), 14-hydroxy-5-desoxy-2S,3S,9R-illudosin (2), and 4,5-dehydro-5-deoxyarmillol (3), together with the known armillol (4). The structure and relative configuration of 1 was determined by single-crystal X-ray diffraction experiments. The structures of compounds 2, 3, and 4 were each deduced by a combination of HRMS and 1D and 2D NMR techniques. Cyclobutane 2 led to modest inhibition of the murine P388 leukemia cell line.

Pharmacodynamic action of a commercial preparation of the mushroom Coprinus comatus in rats.

The pharmacodynamic effect of a 7-day oral treatment with a suspension of Coprinus comatus at doses of 0.835 and 1.670 g/kg in rats was studied. Changes in body weight, bile secretion and hypoglycaemic action were examined together with antipyretic activity and paw oedema tests. Such treatments resulted in a significantly lower increase in the body weight of tested animals (15.73 ± 8.36 g/rat in the untreated group, 8.44 ± 8.23 g/rat (p < 0.05) and 3.18 ± 7.93 g/rat (p < 0.05), for C. comatus 0.835 and 1.67 g/kg, respectively). Hypoglycaemic action was evident only in the glucose load test (6.79 ± 0.61 to 9.70 ± 1.16 (p < 0.05) in the untreated group and 6.47 ± 0.35 to 7.27 ± 0.76 for C. comatus 1.67 g/kg). Histological examination of pancreas cross-sections suggested certain protective functions of the mushroom suspension in alloxan poisoning. In the antipyretic test, a significantly lower increase in body temperature was observed in the mushroom-pretreated rats. In the paw oedema test, no decrease in oedema induced by formalin injection was observed following treatment with C. comatus.