Neurite Outgrowth-Inducing Drimane-Type Sesquiterpenoids Isolated from Cultures of the Polypore Abundisporus violaceus MUCL 56355.

Abundisporin A (1), together with seven previously undescribed drimane sesquiterpenes named abundisporins B-H (2-8), were isolated from a polypore, Abundisporus violaceus MUCL 56355 (Polyporaceae), collected in Kenya. Chemical structures of the isolated compounds were elucidated based on exhaustive 1D and 2D NMR spectroscopic measurements and supported by HRESIMS data. The absolute configurations of the isolated compounds were determined by using Mosher's method for 1-4 and TDDFT-ECD calculations for 4 and 5-8. None of the isolated compounds exhibited significant activities in either antimicrobial or cytotoxicity assays. Notably, all of the tested compounds demonstrated neurotrophic effects, with 1 and 6 significantly increasing outgrowth of neurites when treated with 5 ng/mL NGF.

The study of laccase immobilization optimization and stability improvement on CTAB-KOH modified biochar.

BACKGROUND: Although laccase has a good catalytic oxidation ability, free laccase shows a poor stability. Enzyme immobilization is a common method to improve enzyme stability and endow the enzyme with reusability. Adsorption is the simplest and common method. Modified biochar has attracted great attention due to its excellent performance. RESULTS: In this paper, cetyltrimethylammonium bromide (CTAB)-KOH modified biochar (CKMB) was used to immobilize laccase by adsorption method (laccase@CKMB). Based on the results of the single-factor experiments, the optimal loading conditions of laccase@CKMB were studied with the assistance of Design-Expert 12 and response surface methods. The predicted optimal experimental conditions were laccase dosage 1.78 mg/mL, pH 3.1 and 312 K. Under these conditions, the activity recovery of laccase@CKMB was the highest, reaching 61.78%. Then, the CKMB and laccase@CKMB were characterized by TGA, FT-IR, XRD, BET and SEM, and the results showed that laccase could be well immobilized on CKMB, the maximum enzyme loading could reach 57.5 mg/g. Compared to free laccase, the storage and pH stability of laccase@CKMB was improved greatly. The laccase@CKMB retained about 40% of relative activity (4 °C, 30 days) and more than 50% of relative activity at pH 2.0-6.0. In addition, the laccase@CKMB indicated the reusability up to 6 reaction cycles while retaining 45.1% of relative activity. Moreover, the thermal deactivation kinetic studies of laccase@CKMB showed a lower k value (0.00275 min- 1) and higher t1/2 values (252.0 min) than the k value (0.00573 min- 1) and t1/2 values (121.0 min) of free laccase. CONCLUSIONS: We explored scientific and reasonable immobilization conditions of laccase@CKMB, and the laccase@CKMB possessed relatively better stabilities, which gave the immobilization of laccase on this cheap and easily available carrier material the possibility of industrial applications.

Species Prioritization Based on Spectral Dissimilarity: A Case Study of Polyporoid Fungal Species.

Biological species collections are critical for natural product drug discovery programs. However, prioritization of target species in massive collections remains difficult. Here, we introduce an untargeted metabolomics-based prioritization workflow that uses MS/MS molecular networking to estimate scaffold-level distribution. As a demonstration, we applied the workflow to 40 polyporoid fungal species. Nine species were prioritized as candidates based on the chemical structural and compositional similarity (CSCS) metric. Most of the selected species showed relatively higher richness and uniqueness of metabolites than those of the others. Cryptoporus volvatus, one of the prioritized species, was investigated further. The chemical profiles of the extracts of C. volvatus culture and fruiting bodies were compared, and it was shown that derivative-level diversity was higher in the fruiting bodies; meanwhile, scaffold-level diversity was similar. This showed that the compounds found from a cultured fungus can also be isolated in wild mushrooms. Targeted isolation of the fruiting body extract yielded three unknown (1-3) and six known (4-9) cryptoporic acid derivatives, which are drimane-type sesquiterpenes with isocitric acid moieties that have been reported in this species. Cryptoporic acid T (1) is a trimeric cryptoporic acid reported for the first time. Compounds 2 and 5 exhibited cytotoxicity against HCT-116 cell lines with IC50 values of 4.3 and 3.6 µM, respectively.

Pigmentation Levels Affect Melanoma Responses to Coriolus versicolor Extract and Play a Crucial Role in Melanoma-Mononuclear Cell Crosstalk.

Melanoma, the malignancy originating from pigment-producing melanocytes, is the most aggressive form of skin cancer and has a poor prognosis once the disease starts to metastasize. The process of melanin synthesis generates an immunosuppressive and mutagenic environment, and can increase melanoma cell resistance to different treatment modalities, including chemo-, radio- or photodynamic therapy. Recently, we have shown that the presence of melanin pigment inhibits the melanoma cell response to bioactive components of Coriolus versicolor (CV) Chinese fungus. Herein, using the same human melanoma cell line in which the level of pigmentation can be controlled by the L-tyrosine concentration in culture medium, we tested the effect of suppression of melanogenesis on the melanoma cell response to CV extract and investigated the cell death pathway induced by fungus extract in sensitized melanoma cells. Our data showed that susceptibility to CV-induced melanoma cell death is significantly increased after cell depigmentation. To the best of our knowledge, we are the first to demonstrate that CV extract can induce RIPK1/RIPK3/MLKL-mediated necroptosis in depigmented melanoma cells. Moreover, using the co-culture system, we showed that inhibition of the tyrosinase activity in melanoma cells modulates cytokine expression in co-cultured mononuclear cells, indicating that depigmentation of melanoma cells may activate immune cells and thereby influence a host anticancer response.

Constituents from the Fruiting Bodies of Trametes cubensis and Trametes suaveolens in Vietnam and Their Anti-Inflammatory Bioactivity.

It is reported that various fungi have been used for medicine and edible foods. The tropical Trametes genus is popular and well-known in Vietnam for its health effects and bioactivities. In this study, the fruiting bodies of the edible fungi T. cubensis and T. suaveolens were collected in Vietnam. The preliminary bioactivity screening data indicated that the methanol extracts of the fruiting bodies of T. cubensis and T. suaveolens displayed significant inhibition of superoxide anion generation and elastase release in human neutrophils. Therefore, the isolation and characterization were performed on these two species by a combination of chromatographic methods and spectrometric analysis. In total, twenty-four compounds were identified, and among these (1-3) were characterized by 1D-, 2D-NMR, and HRMS analytical data. In addition, the anti-inflammatory potentials of some purified compounds were examined by the cellular model for the inhibition of superoxide anion generation and elastase release in human neutrophils. Among the isolated compounds, (5,14), and (19) displayed significant anti-inflammatory potential. As the results suggest, the extracts and isolated compounds from T. cubensis and T. suaveolens are potential candidates for the further development of new anti-inflammatory lead drugs or natural healthy foods.

Protein-Bound Polysaccharides from Coriolus Versicolor Fungus Disrupt the Crosstalk Between Breast Cancer Cells and Macrophages through Inhibition of Angiogenic Cytokines Production and Shifting Tumour-Associated Macrophages from the M2 to M1 Subtype.

BACKGROUND/AIMS: The tumour microenvironment is rich in multiple cells that influence cancer development. Among them, macrophages are the most abundant immune cells, which secrete factors involved in carcinogenesis. Since protein-bound polysaccharides (PBP) from the Coriolus versicolor fungus are believed to inhibit the growth of cancers, in the present study, we investigated whether these PBP influence crosstalk between triple-negative 4T1 breast cancer cells and RAW 264.7 macrophages. METHODS: 4T1 cells were cultured in conditioned media (CM) collected after: stimulation of the macrophages with PBP (CM-PBP) or incubation of non-treated macrophages (CM-NT). A co-cultured model of both cell lines was also employed to investigate the crosstalk between the cells. Cell viability was measured using the MTT assay. The levels of cytokines and chemokines were determined by ELISA methods. Commercial assay kits were used to assess the activity of both arginase 1 and inducible nitric oxide synthase (iNOS) and the level of cell migration. RESULTS: The results revealed that CM-NT promotes proliferation and migration of 4T1 cells, and increases the secretion of pro-angiogenic factors (VEGF, MCP-1) by cancer cells. In contrast, CM-PBP inhibits 4T1 cell growth and migration, decreases the secretion of pro-angiogenic factors (VEGF, MCP-1) and upregulates the production of pro-inflammatory mediators (IL-6, TNF-α) with certain anti-tumoral properties Moreover, PBP-treated CM significantly decreases the level of M2 macrophage markers (arginase 1 activity, IL-10 and TGF-ß concentrations), but upregulates iNOS activity and IL-6 and TNF-α production, which are M1 cell markers. CONCLUSION: The results suggest that PBP suppress the favourable tumour microenvironment by inhibiting the crosstalk between 4T1 cells and macrophages through the regulation of production of angiogenic and inflammatory mediators, and modulating the M1/M2 macrophage subtype.

Highly Modified Lanostane Triterpenes from the Wood-Rot Basidiomycete Ganoderma colossus: Comparative Chemical Investigations of Natural and Artificially Cultivated Fruiting Bodies and Mycelial Cultures.

The wood-rot basidiomycete Ganoderma colossus has been chemically investigated. Comparative analyses of the natural fruiting body, artificially cultivated fruiting bodies, and mycelial cultures resulted in the isolation, in total, of 13 new highly modified lanostanes, ganocolossusins A-H (1-8) and ganodermalactones T-X (9-13), together with 23 known compounds (14-36). There were significant overlaps of the same compounds among the three different states of the fungal materials. Ganocolossusin D (4) displayed the most potent antimalarial activity against Plasmodium falciparum K1 (multi-drug-resistant strain) with an IC50 value of 2.4 µM, while it was noncytotoxic to Vero cells at 50 µg/mL.

Bioactive Phenolic Compounds of Two Medicinal Mushroom Species Trametes versicolor and Stereum subtomentosum as Antioxidant and Antiproliferative Agents.

Medicinal mushrooms have tremendous potential in production of bioactive compounds with diverse bioactivities while the biochemical potential of some specific mushroom strains (autochthonous for the region) in production of specific bioactive agents may be of the main importance in a continuous search for novel strains with supreme activities all over the world. In this study, the ethanolic (EtOH) and water (H2 O) extracts of wild-growing polypore mushroom species were investigated: Trametes versicolor (L.) Lloyd and Stereum subtomentosum Pouzar. This study was designed to determine total phenol (TP), flavonoid (TF) and protein content (TPR) as well as LC/MS/MS phenolic profile related to in vitro antioxidant, antiproliferative (MTT assay) (AP) and DNA fragmentation properties. The H2 O extracts expressed better antioxidant scavenging potential than EtOH showing the highest activity for the T. versicolor (IC50 =5.6 µg/mL, IC50 =0.6 µg/mL for DPPH. and OH. , respectively) while O2 .- activity achieved the best activity for S. subtomentosum (IC50 =4.1 µg/mL). In contrary, the highest AP activity was obtained for the EtOH extracts of S. subtomentosum (IC50 =141.1 µg/mL). The EtOH extracts of both species showed the highest TP, TF and TPR content. Obtained results of DNA degradation indicate genotoxicity potential of the extracts at high concentration. The LC/MS/MS detection showed that the majority of analyzed extracts contained phenolic acids, p-hydroxybenzoic and protocatechuic acid. The obtained results suggest that analyzed medicinal mushroom species, T. versicolor and S. subtomentosum, could be of potential interest as new sources of strong natural antioxidants as well as antiproliferative agents in the future.

Extract from the Coriolus versicolor Fungus as an Anti-Inflammatory Agent with Cytotoxic Properties against Endothelial Cells and Breast Cancer Cells.

Chronic inflammation is a well-recognised tumour-enabling component, which includes bioactive molecules from cells infiltrating the tumour microenvironment and increases the risk of cancer progression. Since long-term use of the currently available anti-inflammatory drugs used in cancer therapy causes numerous side effects, the aim of this study was to investigate the effect of an extract isolated from the Coriolus versicolor fungus (CV extract) on HUVEC endothelial cells and MCF-7 breast cancer cells in a pro-inflammatory microenvironment mimicked by lipopolysaccharide (LPS). The cells were simultaneously stimulated with the LPS and CV extract. After co-treatment, the cell viability, generation of reactive oxygen species (ROS), wound-healing assay, production of the pro-inflammatory and pro-angiogenic factors (interleukin (IL) 6, IL-8, and metalloproteinase (MMP) 9)), as well as expression of Toll-like receptor (TLR) 4 and phosphorylated IκB (p-IκB) were evaluated. The results showed that the CV extract inhibited IL-6, IL-8, and MMP-9 production by the LPS-stimulated cells. This effect was accompanied by a decrease in TLR4 and p-IκB expression. The CV extract also had anti-migratory properties and induced a cytotoxic effect on the cells that was enhanced in the presence of LPS. The observed cytotoxicity was associated with an increase in ROS generation. We conclude that the CV extract possesses cytotoxic activity against cancer cells and endothelial cells and has the ability to inhibit the expression of the pro-tumorigenic factors associated with inflammation.

Cloning, overexpression, purification, and modeling of a lectin (Rhinocelectin) with antiproliferative activity from Tiger Milk Mushroom, Lignosus rhinocerus.

A lectin gene from the Tiger Milk Mushroom Lignosus rhinocerus TM02® was successfully cloned and expressed via vector pET28a in Escherichia coli BL21(DE3). The recombinant lectin, Rhinocelectin, with a predicted molecular mass of 22.8 kDa, was overexpressed in water-soluble form without signal peptide and purified via native affinity chromatography Ni-NTA agarose. Blast protein analysis indicated the lectin to be homologous to jacalin-related plant lectin. In its native form, Rhinocelectin exists as a homo-tetramer predicted with four chains of identical proteins consisting of 11 beta-sheet structures with only one alpha-helix structure. The antiproliferative activity of the Rhinocelectin against human cancer cell lines was concentration dependent and selective. The IC50 values against triple negative breast cancer cell lines MDA-MB-231 and breast cancer MCF-7 are 36.52 ± 13.55 µg mL-1 and 53.11 ± 22.30 µg mL-1 , respectively. Rhinocelectin is only mildly cytotoxic against the corresponding human nontumorigenic breast cell line 184B5 with IC50 value at 142.19 ± 36.34 µg mL-1 . The IC50 against human lung cancer cell line A549 cells is 46.14 ± 7.42 µg mL-1 while against nontumorigenic lung cell line NL20 is 41.33 ± 7.43 µg mL-1 . The standard anticancer drug, Doxorubicin exhibited IC50 values mostly below 1 µg mL-1 for the cell lines tested. Flow cytometry analysis showed the treated breast cancer cells were arrested at G0/G1 phase and apoptosis induced. Rhinocelectin agglutinated rat and rabbit erythrocytes at a minimal concentration of 3.125 µg mL-1 and 6.250 µg mL-1 , respectively.

Optimization of the Cellulase-Ultrasonic Synergistic Extraction Conditions of Polysaccharides from Lenzites betulina.

Lenzites betulina has been recognized as a rich source of chemical components, including polysaccharides, sterides and sugar alcohols. In this study, cellulase-ultrasonic synergistic extraction method was applied to extract polysaccharides from L. betulina, and the response surface methodology was used to optimize the extraction conditions. The eight basic factors affecting extraction yield were evaluated by Plackett-Burman design (PBD). Then, the four important factors significantly affecting the yield of polysaccharides from L. betulina, including enzymolysis temperature, enzymolysis time, ultrasound time and ultrasound temperature, were optimized by Box-Behnken design (BBD). Maximum extraction yield of L. betulina polysaccharides was 13.64±0.09 % at a cellulase dosage of 0.8 %, enzymolysis temperature of 60 °C, enzymolysis time of 180 min, pH of 4.5, liquid-solid ratio of 45 ml/g, ultrasound power of 300 W, ultrasound time of 20 min and ultrasound temperature of 45 °C. Subsequently, the characteristic structure of crude polysaccharides was determined by FT-IR. Results indicated that cellulase-ultrasonic synergistic treatment is suitable for L. betulina polysaccharides extraction, and it has good prospect for development and utilization.

Changes of Phytochemical Components (Urushiols, Polyphenols, Gallotannins) and Antioxidant Capacity during Fomitella fraxinea⁻Mediated Fermentation of Toxicodendron vernicifluum Bark.

The stem bark of Toxicodendron vernicifluum (TVSB) has been widely used as a traditional herbal medicine and food ingredients in Korea. However, its application has been restricted due to its potential to cause allergies. Moreover, there is limited data available on the qualitative and quantitative changes in the composition of its phytochemicals during fermentation. Although the Formitella fraxinea-mediated fermentation method has been reported as an effective detoxification tool, changes to its bioactive components and the antioxidant activity that takes place during its fermentation process have not yet been fully elucidated. This study aimed to investigate the dynamic changes of urushiols, bioactive compounds, and antioxidant properties during the fermentation of TVSB by mushroom F. fraxinea. The contents of urushiols, total polyphenols, and individual flavonoids (fisetin, fustin, sulfuretin, and butein) and 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG) significantly decreased during the first 10 days of fermentation, with only a slight decrease thereafter until 22 days. Free radical scavenging activities using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulfonic acid) (ABTS), and ferric reducing/antioxidant power (FRAP) as an antioxidant function also decreased significantly during the first six to nine days of fermentation followed by a gentle decrease up until 22 days. These findings can be helpful in optimizing the F. fraxinea⁻mediated fermentation process of TVSB and developing functional foods with reduced allergy using fermented TVSB.

Cytotoxic Drimane Sesquiterpenoids Isolated from Perenniporia maackiae.

A chemical investigation of a basidiomycetes fungus, Perenniporia maackiae, led to the discovery of 12 drimane sesquiterpenoids, including seven new constituents (1-7). The elucidation of the structures was performed via interpreting extensive spectroscopic methods, including ECD calculations. Among all isolated compounds, 1, 2, and 6 exhibited cytotoxicity toward six carcinoma cells, including ACHN, HCT-15, MDA-MB-231, NCI-H23, NUGC-3, and PC-3 cells, with half-maximal inhibition of cell proliferation values of 1.2-6.0 µM.

Selective in vitro inhibition of Leishmania donovani by a semi-purified fraction of wild mushroom Grifola frondosa.

The current study was designed to assess the anti-leishmanial effect of a semi-purified fraction of wild mushroom Grifola frondosa against Leishmania donovani, in vitro. A total of five extracts from three wild mushrooms [Grifola frondosa (family, Meripilaceae) Laetiporus sulphurous (family, Polyporaceae) and Meripilus giganteus (family, Meripilaceae) were explored for novel anti-leishmanial leads against promastigotes. The ethanol extract of G. frondosa was selected as the most efficient against L. donovani promastigotes (IC50: 93.9 µg/mL). A semi-purified fraction was obtained from an active ethanol extract of G. frondosa and found to inhibit the survival of promastigotes of L. donovani (MHOM/IN/83/AG83) significantly (IC50: 20.37 µg/mL) and it also had some effect against L. major LV39 (MRHO/Sv/59/P strain) and L. tropica WR683 (MHOM/SU/58/OD) strains at higher concentrations (IC50: 46.08 µg/mL and 53.79 µg/mL respectively). The semi-purified fraction also interfered in lipid biosynthesis, altered parasite morphology and induced apoptosis in L. donovani promastigotes. The semi-purified fraction was also effective against intracellular amastigotes in infected macrophages and enhanced the release of nitric oxide and pro-inflammatory cytokines, in vitro. Interestingly, the 50% inhibitory concentration of the semi-purified fraction against the intracellular amastigotes (IC50: 2.48 µg/mL) was much lower in comparison to promastigotes (IC50: 20.37 µg/mL). The semi-purified fraction was found to inhibit the intracellular amastigotes slightly more efficiently in comparison to conventional anti-leishmanial drugs; sodium antimony gluconate, amphotericin B, miltefosine and paromomycin and noticeably non-toxic towards host splenocytes. The findings of the present study established that G. frondosa might be a natural resource for development of a new anti-leishmanial lead.

Delamination of plastic-coated waste paper by enzymes of the white rot fungus Dichomitus squalens.

Many paper products are coated with plastic to improve their quality and stability. However, this limits recycling and recovery options and the plastic-coated waste paper is mostly disposed in landfills. Such practices are uneconomical and contrary to sustainable waste management. In this work enzymes of the white rot fungus Dichomitus squalens were investigated for possible delamination of plastic-coated waste paper. Enzymes were found capable to release the polyethylene foil from plastic-coated paper which resulted in 88.6-91.5% mass loss. The delamination rate, however, was depended on the ratio between plastic-coated paper and volume of enzyme filtrate. Results of a consequent experiment showed that enzymes are also efficient when plastic-coated paper is treated in a sequencing batch reactor resulting in 88.2-90.6% mass loss. The system was fully functional up to the 5th cycle; afterwards, the delamination rate reduced due to high thickness of the waste paper sludge. The enzyme activity, however, was still very high; with the laccase activity at the end of the experiment above 900 U/L and manganese peroxidase above 250 U/L. Our results demonstrated, that plastic-coated waste paper has the potential to be efficiently recovered instead of being disposed in landfills.

Comparative nutritional and mycochemical contents, biological activities and LC/MS screening of tuber from new recipe cultivation technique with wild type tuber of tiger's milk mushroom of species Lignosus rhinocerus.

Tiger's milk mushroom is known for its valuable medicinal properties, especially the tuber part. However, wild tuber is very hard to obtain as it grows underground. This study first aimed to cultivate tiger's milk mushroom tuber through a cultivation technique, and second to compare nutritional and mycochemical contents, antioxidant and cytotoxic activities and compound screening of the cultivated tuber with the wild tuber. Results showed an increase in carbohydrate content by 45.81% and protein content by 123.68% in the cultivated tuber while fat content reduced by 13.04%. Cultivated tuber also showed an increase of up to 64.21% for total flavonoid-like compounds and 62.51% of total ß-D-glucan compared to the wild tuber. The antioxidant activity of cultivated tuber and wild tuber was 760 and 840 µg mL-1, respectively. The cytotoxic activity of boiled water extract of cultivated tuber against a human lung cancer cell line (A549) was 65.50 ± 2.12 µg mL-1 and against a human breast cancer cell line (MCF7) was 19.35 ± 0.11 µg mL-1. ß-D-glucan extract from the purification of boiled water extract of cultivated tuber showed cytotoxic activity at 57.78 ± 2.29 µg mL-1 against A549 and 33.50 ± 1.41 µg mL-1 against MCF7. However, the ß-glucan extract from wild tuber did not show a cytotoxic effect against either the A549 or MCF7 cell lines. Also, neither of the extracts from cultivated tuber and wild tuber showed an effect against a normal cell line (MRC5). Compound profiling through by liquid chromatography mass spectrometry (LC/MS) showed the appearance of new compounds in the cultivated tuber. In conclusion, our cultivated tuber of tiger's milk mushroom using a new recipe cultivation technique showed improved nutrient and bioactive compound contents, and antioxidant and cytotoxic activities compared to the wild tuber. Further investigations are required to obtain a better quality of cultivated tuber.

Fruit Extract from Pyropolyporus fomentarius (L. ex Fr.) Teng Induces Mitochondria-Dependent Apoptosis in Leukemia Cells but Enhances Immunomodulatory Activities of Splenic Lymphocytes.

Pyropolyporus fomentarius (L. ex Fr.) Teng is a unique woody mushroom due to its medicinal value with numerous pharmacological activities. This study presented the potential antitumor and immunomodulatory properties of ethanol extract of P. fomentarius. The results showed that P. fomentarius extract inhibited the viability of murine leukemia L1210 cells in a dose-dependent manner with IC50 value of 69.35 µg/ml. Flow cytometry analysis demonstrated that the extract induced apoptosis in L1210 cells. Additionally, the decline of mitochondrial membrane potential was observed as well as the changes of caspase-3, caspase-9, Bcl-2, and Bax, suggesting that proapoptosis effect of the extract involved mitochondria-related pathway. Simultaneously, the P. fomentarius extract significantly enhanced the proliferation and activation of splenic lymphocytes in a dose-dependent manner. This P. fomentarius extract has potential applications as a natural antitumor agent with immunomodulatory activity.

Phytochemical profiles and inhibitory effects of Tiger Milk mushroom (Lignosus rhinocerus) extract on ovalbumin-induced airway inflammation in a rodent model of asthma.

BACKGROUND: Lignosus rhinocerus (L. rhinocerus), which is known locally as Tiger Milk mushroom, is traditionally used in the treatment of asthma by indigenous communities in Malaysia. However, to date, its efficacy on asthma has not been confirmed by scientific studies and there is also sparse information available on its active constituents. In this study, the volatile constituent of L. rhinocerus hot water extract was investigated using gas chromatography mass spectrometry (GC-MS). The potential effects of L. rhinocerus extract for anti-asthmatic activity was further investigated on ovalbumin (OVA)-sensitized asthmatic Sprague Dawley rats. METHODS: Sequential extraction using five solvents (petroleum ether, diethyl ether, hexane, ethyl acetate and methanol) was conducted prior to GC-MS analysis. Male Sprague Dawley rats were divided into the following four groups of five animals each: 1) normal rats, 2) sensitization plus OVA-challenged rats 3) sensitization plus OVA-challenged with L. rhinocerus treatment and 4) sensitization plus OVA-challenged with dexamethasone treatment. The levels of immunoglobulin E (IgE) in the serum and T-helper 2 cytokines, including interleukin (IL)-4, IL-5 and IL-13, in bronchoalveolar lavage fluid (BALF), as well as eosinophil infiltration in the lungs, were investigated. RESULTS: GC-MS analysis revealed the presence of five main groups (alkane, fatty acids, benzene, phenol and dicarboxylic acid) with a total of 18 constituents. Linoleic acid (21.35 %), octadecane (11.82 %) and 2,3-dihydroxypropyl elaidate (10.47 %) were present in high amounts. The extract significantly ameliorated the increase in total IgE in serum and IL-4, IL-5 and IL-13 levels in BALF and also effectively suppressed eosinophils numbers in BALF while attenuating eosinophil infiltrations in the lungs. CONCLUSION: L. rhinocerus hot water extract has the potential to be used as an alternative for the treatment of acute asthma.

Laccase Gene Family in Cerrena sp. HYB07: Sequences, Heterologous Expression and Transcriptional Analysis.

Laccases are a class of multi-copper oxidases with industrial potential. In this study, eight laccases (Lac1-8) from Cerrena sp. strain HYB07, a white-rot fungus with high laccase yields, were analyzed. The laccases showed moderate identities to each other as well as with other fungal laccases and were predicted to have high redox potentials except for Lac6. Selected laccase isozymes were heterologously expressed in the yeast Pichia pastoris, and different enzymatic properties were observed. Transcription of the eight laccase genes was differentially regulated during submerged and solid state fermentation, as shown by quantitative real-time polymerase chain reaction and validated reference genes. During 6-day submerged fermentation, Lac7 and 2 were successively the predominantly expressed laccase gene, accounting for over 95% of all laccase transcripts. Interestingly, accompanying Lac7 downregulation, Lac2 transcription was drastically upregulated on days 3 and 5 to 9958-fold of the level on day 1. Consistent with high mRNA abundance, Lac2 and 7, but not other laccases, were identified in the fermentation broth by LC-MS/MS. In solid state fermentation, less dramatic differences in transcript abundance were observed, and Lac3, 7 and 8 were more highly expressed than other laccase genes. Elucidating the properties and expression profiles of the laccase gene family will facilitate understanding, production and commercialization of the fungal strain and its laccases.

Enzymatic degradation of lignin-carbohydrate complexes (LCCs): model studies using a fungal glucuronoyl esterase from Cerrena unicolor.

Lignin-carbohydrate complexes (LCCs) are believed to influence the recalcitrance of lignocellulosic plant material preventing optimal utilization of biomass in e.g. forestry, feed and biofuel applications. The recently emerged carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) has been proposed to degrade ester LCC bonds between glucuronic acids in xylans and lignin alcohols thereby potentially improving delignification of lignocellulosic biomass when applied in conjunction with other cellulases, hemicellulases and oxidoreductases. Herein, we report the synthesis of four new GE model substrates comprising α- and É£-arylalkyl esters representative of the lignin part of naturally occurring ester LCCs as well as the cloning and purification of a novel GE from Cerrena unicolor (CuGE). Together with a known GE from Schizophyllum commune (ScGE), CuGE was biochemically characterized by means of Michaelis-Menten kinetics with respect to substrate specificity using the synthesized compounds. For both enzymes, a strong preference for 4-O-methyl glucuronoyl esters rather than unsubstituted glucuronoyl esters was observed. Moreover, we found that α-arylalkyl esters of methyl α-D-glucuronic acid are more easily cleaved by GEs than their corresponding É£-arylalkyl esters. Furthermore, our results suggest a preference of CuGE for glucuronoyl esters of bulky alcohols supporting the suggested biological action of GEs on LCCs. The synthesis of relevant GE model substrates presented here may provide a valuable tool for the screening, selection and development of industrially relevant GEs for delignification of biomass.