Interspecific hybridization between Ganoderma lingzhi and G. applanatum through protoplast fusion.

Interspecific hybridization between Ganoderma lingzhi and G. applanatum was attempted through polyethylene glycol (PEG) induced fusion technique. The protoplast isolation procedure was simplified, and we obtained a significant number of protoplasts from both Ganoderma species. The number of protoplasts obtained was 5.27 ± 0.31 × 107/mL in G. lingzhi and 5.57 ± 0.49 × 106/mL in G. applanatum. Osmotic stabilizer NaCl (0.4 M) at pH 5.8 and enzymolysis time 3.5 h have supported high frequency of protoplast regeneration. G. lingzhi and G. applanatum regeneration frequency was 1.73 ± 0.04% and 0.23 ± 0.02%, respectively. 40% of PEG induced high number of protoplast fusion the regeneration frequency was 0.09% on a minimal medium. Two hundred fifty-two fusant colonies were isolated from the following four individual experiments. Among them, ten fusants showed the mycelial morphological difference compared to their parents and other fusant isolates. The fruiting body could be generated on oak sawdust and wheat bran substrate, and a few of them showed recombined morphology of the parental strains. The highest yield and biological efficacy (BE) were recorded in GF248, while least in GF244. The hybridity of the fusant was established based on mycelia, fruiting morphology, and PCR fingerprinting. ISSR and RAPD profile analysis of ten fusants and parents depicted that fusants contained polymorphic bands, which specified the rearrangement and deletion of DNA in the fusants. A Dendrogram was constructed based on the RAPD profile, and the clustering data exhibited two major clusters: cluster I included the G. lingzhi and Cluster II, including the G. applanatum and fusant lines. Total polysaccharide (α, ß and total glucan) content was compared with fusants and parental strains. The present study highlighted the efficient methods for protoplast isolation from Ganoderma species. PEG-induced fusants showed high polymorphic frequency index, while the phenotypic characters showed high similarity to G. applanatum. A significant difference was observed in the mushroom yield and its total polysaccharide between the fusants and parental strains.

In vitro antiviral activity of medicinal mushroom Ganoderma neo-japonicum Imazeki against enteroviruses that caused hand, foot and mouth disease.

Hand, foot and mouth disease (HFMD) is a highly contagious viral disease that predominantly affects children younger than 5 years old. HFMD is primarily caused by enterovirus A71 (EVA71) and coxsackievirus A16 (CV-A16). However, coxsackievirus A10 (CV-A10) and coxsackievirus A6 (CV-A6) are being increasingly reported as the predominant causative of HFMD outbreaks worldwide since the past decade. To date, there are still no licensed multivalent vaccines or antiviral drugs targeting enteroviruses that cause HFMD, despite HFMD outbreaks are still being frequently reported, especially in Asia-Pacific countries. The high rate of transmission, morbidity and potential neurological complications of HFMD is indeed making the development of broad-spectrum antiviral drugs/agents against these enteroviruses a compelling need. In this study, we have investigated the in vitro antiviral effect of 4 Ganoderma neo-japonicum Imazeki (GNJI) crude extracts (S1-S4) against EV-A71, CV-A16, CV-A10 and CV-A6. GNJI is a medicinal mushroom that can be found growing saprophytically on decaying bamboo clumps in Malaysian forests. The antiviral effects of this medicinal mushroom were determined using cytopathic inhibition and virus titration assays. The S2 (1.25 mg/ml) hot aqueous extract demonstrated the highest broad-spectrum antiviral activity against all tested enteroviruses in human primary oral fibroblast cells. Replication of EV-A71, CV-A16 and CVA10 were effectively inhibited at 2 hours post-infection (hpi) to 72 hpi, except for CV-A6 which was only at 2 hpi. S2 also has virucidal activity against EV-A71. Polysaccharides isolated and purified from crude hot aqueous extract demonstrated similar antiviral activity as S2, suggesting that polysaccharides could be one of the active compounds responsible for the antiviral activity shown by S2. To our knowledge, this study demonstrates for the first time the ability of GNJI to inhibit enterovirus infection and replication. Thus, GNJI is potential to be further developed as an antiviral agent against enteroviruses that caused HFMD.

Adjuvanticity of Ganoderma lucidum polysaccharide liposomes on porcine circovirus type-II in mice.

Ganoderma lucidum has been widely used as a fungal, for promoting health and longevity in China and other Asian countries. Polysaccharide (PS) extracted from Ganoderma lucidum exhibits a variety of immunomodulatory activities and has the ability to induce strong immune responses. Liposomes (Lip) have been shown to be useful carriers of vaccine antigens and can be applied as a versatile delivery system for vaccine adjuvants. Here, PS and inactivated porcine circovirus type II (PCV-II) were encapsulated into Lip as a vaccine and inoculated into mice. The magnitude and kinetics of adjuvant activity were investigated. Polysaccharide-loaded liposomes (Lip-PS) could induce more efficient PCV-II-specific immune responses than other single-component formulations. The Lip-PS group displayed robust and higher titers of PCV-II-specific immunoglobulin (Ig)G antibodies and IgG subtypes as well as higher cytokine levels, furthermore, splenocytes were activated by Lip-PS. Thus, Lip-PS formulation produced vigorous humoral and cellular immune responses, with a mixed T-helper (Th)1/Th2/Th17 immune response and slight Th1 polarized cellular immune response. Overall, these results suggested that Lip-PS could provide a universal platform for vaccine design against PCV-II.

Medium composition optimization, structural characterization, and antioxidant activity of exopolysaccharides from the medicinal mushroom Ganoderma lingzhi.

To contribute towards effective exploitation and utilization of natural antioxidants, response surface methodology (RSM) was employed to optimize the medium composition for the production of exopolysaccharides from the medicinal mushroom Ganoderma lingzhi (GLEPS). An optimal medium for GLEPS production was gave through Plackett-Burman design, path of steepest ascent, and Box-Behnken design as follows: glucose (59.62 g/L), yeast extract (10.03 g/L), CaCO3 (0.2 g/L), thiamine (45.13 mg/L), KH2PO4 (1.0 g/L), peptone (1.5 g/L), Tween 80 (10.26 mL/L), ZnSO4 (0.3 g/L), mannitol (1.5 g/L), MgSO4 (0.5 g/L), and aspartate (8.86 g/L). The GLEPS yield obtained was 3.57 ±â€¯0.21 g/L-3.16-fold higher than that produced in basal medium alone. The resulting GLEPS rich in uronic acid, d-mannose, l-rhamnose, and d-glucose, was a heteropolysaccharide with high-molecular weights (475,000 kDa and 21.6 kDa, 87.97%). It was demonstrated that the GLEPS with higher carbohydrate and uronic acid contents exhibited strong in vitro antioxidant activities via radical scavenging, reductive capacity, and chelation of transition metal catalysis. These findings indicated that RSM is an efficient tool to predict the composition of culture medium required for maximizing GLEPS yield, and GLEPS had potent antioxidant activities and could be explored as a novel natural antioxidant in functional food or medicine.

Ganoderma applanatum: a promising mushroom for antitumor and immunomodulating activity.

The antitumor effect of exo-biopolymer (EXP) produced by Ganoderma applanatum was investigated using sarcoma-180 bearing mice. EXP, when administered (10-80 mg/kg body weight: BW) intraperitoneally, significantly inhibited the growth of solid tumor and increased the natural killer (NK) cell activity. A dose of 40 mg/kg BW was found to be highly effective, as it reduced the tumor formation by 39.7%, and increased the NK cell activity of splenocytes by 51.6% compared with the control group. The complement activity of EXP was increased in accordance with an increase in concentration. The phosphatase activity of macrophages was increased by 0.7-fold (200 microg/mL) compared with the control group. This EXP contained 58.9% carbohydrate and 17.1% protein. The major sugar of EXP was composed of mannose and glucose, while the protein mainly consisted of serine, glycine and aspartic acid.

Purification and identification of an actinomycin D analogue from actinomycetes associated with Ganoderma applanatum via magnetic molecularly imprinted polymers and tandem mass spectrometry.

Actinomycetes are main producers of antibiotics and targeted screening could improve the efficiency of discovering new drugs. This study describes, for the first time, the isolation of endophytic actinomycetes from the macrofungus Ganoderma applanatum. To increase the efficiency of screening, novel actinomycin D (Act D) molecularly-imprinted polymers were adsorbed to the surface of Fe3O4@SiO2 magnetic microspheres (MMIPs) and using in the isolation. A monolithic column prepared with magnetic molecularly imprinted polymers was employed to adsorb actinomycin D and its analogues for selective analysis and identification via MS/MS spectroscopy. The MMIP-monolithic column was selective for the structural features of Act D and its analogue, and the maximum loading of the MMIPs for Act D was ∼23.5 µg/g. The recognition time of the Act D was 20-30 min and had good discriminative ability. A new analogue was identified from endophytic actinomycetes KLBMP 2541, and it was purified using MMIPs comparison with MMIPs-solid phase extraction. Structural identification analysis confirmed that the new analogue was 2-methyl-actinomycin D, which has better anti-tumor activity than Act D. The presented method combines the advantages of MMIPs and MS with popular solutions to enable high affinity and selectivity screening of specific antibiotics from endophytic actinomycetes.

Evaluation of Antianxiety Potential of Four Ganoderma (Agaricomycetes) Species from India in Mice.

The genus Ganoderma consists of widespread polypore mushrooms that have traditionally been used to reduce stress and anxiety. However, scientific evidence for this is not adequate. Hence, this study was designed to investigate the anxiolytic potential of G. applanatum, G. brownii, G. lucidum, and G. philippii collected from Uttarakhand, India. Various extracts of dried, powdered basidiocarps were prepared using different solvents-namely, petroleum ether, chloroform, methanol, and distilled water-by successive Soxhlet extraction. All the extracts were tested for antianxiety activity using the elevated plus maze (EPM) model in Swiss albino mice. The results showed that the methanol extract of G. lucidum at a dose of 200 mg/kg, administered orally, shows a significant increase in the average time spent in the open arms of the EPM when compared with the control; this was comparable to the effect of the standard drug (diazepam, 2 mg/kg by mouth). This bioactive methanol extract was subjected to bioactivity-guided fractionation. The results show that the n-butanol fraction of the methanol extract evinced significant antianxiety activity at a dose of 100 mg/kg. This fraction showed the presence of phenols and flavonoids and thus was standardized with respect to total phenol content and total flavonoid content. The antianxiety activity may be the result of the phenols/flavonoids present. This study clearly demonstrated that the n-butanol fraction from the methanol extract of G. lucidum can be developed as source of new anxiolytic agents.

Recovery of ergosterol from the medicinal mushroom, Ganoderma tsugae var. Janniae, with a molecularly imprinted polymer derived from a cleavable monomer-template composite.

A semi-covalent imprinting strategy has been developed for the synthesis of molecularly-imprinted polymers specific for the fungal sterol, ergosterol, a biological precursor of vitamin D2. This imprinting approach involved a novel post-synthesis cleavable monomer-template composite, namely ergosteryl methacrylate, and resulted in the formation of an imprinted polymer that selectively and efficiently recognized ergosterol through non-covalent interactions. The derived molecularly-imprinted polymer and the corresponding non-imprinted polymer were systematically evaluated for their selectivity towards ergosterol via static and dynamic binding studies using various ergosteryl esters (e.g. ergosteryl-cinnamate, -ferulate, -coumarate, -ferulate acetate and -acetate, respectively) as competitors. Moreover, the binding capacity of the molecularly imprinted polymer for ergosterol was enhanced when the sample loading conditions involved the use of partially aqueous solvent mixtures, such as acetonitrile/water (9:1 (v/v) or 8:2 (v/v)). These attributes were exploited in a solid-phase extraction format, whereby ergosterol was obtained with excellent recoveries from an extract of the fruiting body powder of the medicinal fungus Ganoderma tsugae var. Janniae.

Fungal pretreatment improves amenability of lignocellulosic material for its saccharification to sugars.

The sugarcane bagasse was biologically pretreated with three white-rot fungi; Pleurotus florida, Coriolopsis caperata RCK 2011 and Ganoderma sp. rckk-02, individually under solid-state fermentation. P. florida, C. caperata RCK 2011 and Ganoderma sp. rckk-02 degraded lignin up to 7.91, 5.48 and 5.58%, respectively. The lignocellulolytic enzymes produced by these fungi were also monitored during solid state fermentation of sugarcane bagasse. The fungal fermented sugarcane bagasse when hydrolyzed with crude cellulases from brown-rot fungus, Fomitopsis sp. RCK2010, released comparatively 1.5-2.4 fold higher sugars than in case of untreated sugarcane bagasse. The study demonstrated that white-rot fungal pretreatment improved the amenability of plant material for enzymatic hydrolysis.

Hypoglycemic effects of Ganoderma applanatum and Collybia confluens exo-polymers in streptozotocin-induced diabetic rats.

The hypoglycemic effects of Ganoderma applanatum exo-polymer (GAE) and Collybia confluens exo-polymer (CCE) produced by submerged mycelial cultures in streptozotocin (STZ)-induced diabetic rats were investigated. Hypoglycemic effects were achieved in both the GAE- and CCE-treated groups by administration at a level of 100 mg/kg body weight (BW) daily for 3 weeks. The administration of GAE and CCE substantially reduced the plasma glucose levels by as much as 22.0% and 25.9%, respectively, when compared with the control group. The GAE and CCE also lowered the plasma total cholesterol and triglyceride levels by 20.3% and 22.5%, and by 22.7% and 25.5%, respectively. Furthermore, the activity of alanine transaminase (ALT) and aspartate transaminase (AST) was decreased by 23.2% and 20.7% in the GAE-treated group, and it was also reduced by 28.7% and 23.6% in the CCE-treated group. The results strongly demonstrate the potential of GAE and CCE in combating diabetes in experimental animals.