The chemical profiling and assessment of antioxidative, antidiabetic and antineurodegenerative potential of Kombucha fermented Camellia sinensis, Coffea arabica and Ganoderma lucidum extracts.

The scientific interest in the medicinal properties of Kombucha beverages, a carbonated drink with live microorganisms, has increased recently. Hence, the aim of this study was to determine the chemical profile and to examine the antioxidant, antidiabetic and antineurodegenerative potential of unfermented and also Kombucha fermented Camellia sinensis (green tea), Coffea arabica (coffee), and Ganoderma lucidum (Reishi) extracts. The extracts were prepared as follows: the first (unfermented) set contained 1 L of water, 50 g of sucrose and 20 g of dried and ground green tea, coffee, or Reishi basidiocarp, while the second (fermented) set contained all of the aforementioned ingredients individually inoculated with Kombucha and fermented for 21 days. The chemical analysis was conducted using liquid chromatography-mass spectrometry (LC-MS). The antioxidant activity was assessed by DPPH, total reducing power (TRP), and ß-carotene bleaching assays. The inhibition of α-amylase and α-glucosidase activity was used to estimate the antidiabetic potential, while the level of inhibition of acetylcholinesterase (AChE) and tyrosinase (TYR) was used to evaluate the antineurodegenerative activity. The results suggested that the fermented extracts of green tea, coffee, and Reishi exert significant antioxidant effects, although they were lower compared to the unfermented extracts. The unfermented green tea extract exhibited the highest DPPH-scavenging activity (87.46%) and the highest preservation of ß-carotene (92.41%), while the fermented coffee extract showed the highest TRP (120.14 mg AAE per g) at 10 mg mL-1. Although the extracts did not inhibit the activity of α-amylase, they were quite effective at inhibiting α-glucosidase, especially the unfermented Reishi extract, inhibiting 95.16% (at a concentration of 10 mg mL-1) of α-glucosidase activity, which was slightly higher than the positive control at the same concentration. The most effective AChE inhibitor was unfermented green tea extract (68.51%), while the fermented coffee extract inhibited 34.66% of TYR activity at 10 mg mL-1. Altogether, these results are in accordance with the differences found in the extracts' chemical composition. Finally, this is the first report that highlights the differences in the chemical profile between the unfermented and Kombucha fermented green tea, coffee and Reishi extracts, while it also reveals, for the first time, the antineurodegenerative potential of Kombucha fermented Reishi extract. The examined extracts represent potent functional foods, while their more detailed mechanisms of action are expected to be revealed in future research.

Immunomodulatory Effects of Extract of Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) Basidiocarps Cultivated on Alternative Substrate.

Ganoderma lucidum is a medicinal mushroom exhibiting numerous health benefits primarily based on strong immunostimulatory effects. The study aimed to investigate if there were differences in effects of extracts of commercially (GC) and alternatively (wheat straw) (GA) cultivated G. lucidum basidiocarps on properties of peritoneal macrophages (PM) and monocyte-derived dendritic cells (MoDCs). Differences in immunomodulatory effects of GC/GA extracts were studied. The viability of treated PMs, their adhesive and phagocytic capability, and their capacity to produce reactive oxygen species (ROS) and NO were tested. Immature MoDCs generated from human monocytes were treated with poly I:C (10.0 µg/ml) and loxoribine (34.0 µg/ml), a selective TLR3 and TLR7 agonists, respectively, and with/without GC/GA extract (100.0 µg/ml). The effect of each combination on phenotypic properties, cytokines production by MoDCs, and their proliferation and Th polarizing capacity was studied. GA extract stimulated the metabolic and phagocytic activity of PMs, their adhesion capability, and ability to produce ROS and NO more strongly compared to GC. Both tested extracts significantly increased allostimulatory and Th1 polarization capacity of simultaneous TLR3 and TLR7-activated MoDCs, but GA extract was more effective. The extract of alternatively cultivated G. lucidum basidiocarps increased production of ROS and NO by TLR4 stimulated PMs and upregulated production of certain cytokines as well as allostimulatory and Th1 polarization capacity of MoDCs. GA extract could be a potent immunostimulatory agent for activation of MoDCs with the simultaneous engagement of TLRs, which seems to be a promising strategy for the preparation of DC-based anti-tumor vaccines.

Pleurotus ostreatus and Laetiporus sulphureus (Agaricomycetes): Possible Agents against Alzheimer and Parkinson Diseases.

Previous studies showed that some mushrooms are highly efficient in inhibiting acetylcholinesterase and tyrosinase, the increased activity of which can trigger the development of Alzheimer and Parkinson diseases. Starting from the fact that free radicals at high concentrations could cause neurodegenerative disorders as well as great interest in new, natural antineurodegenerative drugs, the goal of this study was to determine the in vitro antioxidative and neuroprotective potentials of various Pleurotus ostreatus and Laetiporus sulphureus extracts. L. sulphureus was a better antioxidative agent; it showed higher reducing power, was a more efficient scavenger of DPPH and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radicals, and was an Fe3+ reducer. The most efficient acetylcholinesterase inhibitor was hot water extract of P. ostreatus fruiting body, which was slightly weaker than the commercial preparation, galantamine. However, in comparison with α-kojic acid, tested extracts were weaker tyrosinase inhibitors. Considering that tested extracts were rich in phenols and that their amounts were in positive correlation with the extent of radical neutralization and acetylcholinesterase and tyrosinase inhibition, it is assumed that these compounds are the potential carriers of the neuroprotective activities. Owing to the significant antioxidative and antineurodegenerative capacity of these species, they can be suggested as novel nutraceuticals and pharmaceuticals.

Antioxidative, antifungal, cytotoxic and antineurodegenerative activity of selected Trametes species from Serbia.

In a last few decades mushrooms are increasingly attracting attention as functional food and sources of biologically active compounds. Several Trametes species have been used for centuries in traditional medicine of East Asia cultures, but only T. versicolor was studied sufficiently while there are less substantial data about medicinal properties of other species. Trametes versicolor, T. hirsuta and T. gibbosa were the species tested for biological activities. Antifungal potentials of extracts were assessed for clinical strains of selected Candida and Aspergillus species. ABTS and FRAP assays were used to evaluate antioxidant capacities of studied extracts. Cytotoxic activity was determined against human cervix and lung adenocarcinoma and colon carcinoma cell lines. Antineurodegenerative activity was assessed by determining the rate of acetylcholinesterase and tyrosinase activity. The presence of metabolites in extracts of mycelia and basidiocarps of studied Trametes species was analyzed by 1H NMR spectroscopy. Studied extracts showed low antifungal potential in comparison with ketoconazole. Basidiocarp extracts were more effective ABTS+ scavengers and Fe2+ reducers than mycelium ones but less effective in comparison with L-ascorbic acid. Results showed that mycelium extracts had stronger cytotoxic effects against three cancer cell lines than basidiocarp ones, and that cervix adenocarcinoma cells were the most sensitive to the extracts and commercial cytostatics. T. versicolor mycelium extract was the most effective inhibitor of acetylcholinesterase activity but double weaker than galantamine, and T. gibbosa mycelium extract was significantly better inhibitor of tyrosinase activity than kojic acid for 40.9%. Chemical analysis indicated strong synergistic action of triterpenes, sugars and polyphenols in applied assays. The results suggest that tested Trametes species have significant medicinal potentials which could be attributed to antioxidative and cytotoxic activity. Additionally both, basidiocarps and mycelia extracts can strongly inhibit activity of acetylcholinesterase and tyrosinase.

Antifungal, Antioxidative, and Genoprotective Properties of Extracts from the Blushing Bracket Mushroom, Daedaleopsis confragosa (Agaricomycetes).

This study aimed to evaluate the antifungal, antioxidative, and genoprotective potentials of various extracts of Daedaleopsis confragosa fruiting bodies and mycelia. The extracts exhibited weak fungistatic and an absence of any fungicidal activities against 7 important animal and human pathogens. Although mycelial extracts, especially the hot water extract, were more effective antifungal agents than basidiocarp extracts, they were significantly weaker than the commercial antimycotic ketoconazole. The tested extracts reduced DPPH radicals to various extents, depending on the material used for extraction and the solvent. The mycelial hot water extract was the best DPPH radical scavenger (half-maximal effective concentration, 3.42 mg mL-1), whereas the 96` ethanol extract of basidiocarps was the weakest one (half-maximal effective concentration, 5.36 mg mL-1). Phenols were the main carriers of activity, and their proportions in the tested extract mainly depended on the type and concentration of the solvent; the effect of the material used for extraction was negligible. The largest amount of phenols was detected in the 70% ethanol extract of basidiocarps (126.40 µg gallic acid equivalents mg-1) and the lowest in the basidiocarp methanol extract (19.93 µg gallic acid equivalents mg-1). Aqueous extracts of basidiocarp and mycelial extracts were not genotoxic agents but exhibited significant protective activity against hydrogen peroxide-induced DNA damage during use in leukocytes' pre- and post-treatments.

Species of Genus Ganoderma (Agaricomycetes) Fermentation Broth: A Novel Antioxidant and Antimicrobial Agent.

The bioactivity of Ganoderma lucidum basidiocarps has been well documented, but there are no data on the medicinal properties of its submerged cultivation broth nor on the other species of the genus Ganoderma. Thus the aim of this study was to test the potential antimicrobial and antioxidant activity of fermentation broth obtained after submerged cultivation of G. applanatum, G. carnosum, and G. lucidum. DPPH· scavenging ability, total phenols, and flavonoid contents were measured to determine the antioxidative potential of Ganoderma spp. fermentation filtrates, whereas their antimicrobial potential was studied using the microdilution method. DPPH· scavenging activity of G. lucidum fermentation filtrates was significantly higher than that of G. applanatum and G. carnosum, with the maximum (39.67%) obtained from strain BEOFB 432. This filtrate also contained the highest concentrations of phenols (134.89 µg gallic acid equivalents/mL) and flavonoids (42.20 µg quercetin equivalent/mL). High correlations between the activity and phenol content in the extracts showed that these compounds were active components of the antioxidative activity. G. lucidum strain BEOFB 432 was the most effective antibacterial agent, whereas strain BEOFB 434 has proven to be the most effective antifungal agent. The study showed that Ganoderma spp. fermentation filtrates are novel potent antioxidative and antimicrobial agents that could be obtained more quickly and cheaper than basidiocarps.

Genoprotective Capacity of Alternatively Cultivated Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), Basidiocarps.

Ganoderma lucidum is traditionally used in Eastern medicine to preserve vitality, promote longevity, and treat disease. It possesses immunomodulatory, antitumor, antimicrobial, and antiaging activities, among others, but one of the most important is its antioxidant property, which is the basis for other effects, because free radicals trigger many diseases. The substrate commonly used for commercial cultivation of G. lucidum is not environmentally friendly nor economically justified, so there is a need to find new alternative substrates. The aim of this study was to analyze the effect of substrate composition on the bioactivity of G. lucidum basidiocarps. G. lucidum was cultivated on 2 different substrates: (1) a mixture of wheat straw, grapevine branches, and wheat bran, and (2) wheat straw. Commercial fruiting bodies, cultivated on oak sawdust, were used as the control. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability, total phenols, and flavonoid content were determined spectrophotometrically to define the antioxidative potential of basidiocarp extracts. The comet test was performed to detect the degree of DNA damage in the cells that were exposed to G. lucidum extracts before and after the effect of oxidants. Higher antioxidative potential was observed for the extract of G. lucidum basidiocarps cultivated on wheat straw compared with that from the mixed substrate and especially with commercial ones. The alternatively cultivated basidiocarps also showed stronger antigenotoxic potential compared with commercial ones. The study showed that fruiting bodies produced on wheat straw, one of the most accessible and cheapest crop residues, are more potent antioxidant and antigenotoxic agents than commercially cultivated ones.

Effect of Selenium Enrichment of Lenzites betulinus and Trametes hirsuta Mycelia on Antioxidant, Antifungal and Cytostatics Potential.

The aim of the study was an assessment of the antioxidant, antifungal and cytotoxic potentials of L. betulinus and T. hirsuta mycelia extracts and the effect of selenium on these activities. Extracts of L. betulinus were twice as efficient in DPPH• scavenging as those of T. hirsuta. The phenol content in Se-enriched L. betulinus extracts was higher than in non-enriched extracts, in contrast to the effect of Se-enrichment on T. hirsuta extracts, and a direct correlation between the amount and DPPH• scavenging effect was observed. Ethanol extracts exhibited fungistatic but not fungicidal activity against a range of micromycetes, and mycelium enrichment with selenium inhibited this effect. Although the extracts showed low cytotoxic activity against HeLa and LS174 cells, T. hirsuta extracts, especially those enriched with selenium, had better potential. L. betulinuss extracts showed better antioxidant and antifungal activity than T. hirsuta extracts which were more active cytotoxic agents. The presence of selenium stimulated antioxidant and cytotoxic, and inhibited antifungal activity in L. betulinus, while in T. hirsuta its effect was slight.

Effects of Selenium Presence in Mycelia of Ganoderma species (Higher Basidiomycetes) on Their Medicinal Properties.

This study aimed to research the antifungal, antioxidant, and cytotoxic potential of Ganoderma applanatum and Ganoderma lucidum mycelial extracts as well as the possible effect of Se enrichment on these activities. Both Se-enriched and nonenriched extracts of G. applanatum and G. lucidum showed fungi static activity, while a fungicidal effect was not noted. The extracts exhibited significant 1,1-diphenyl-2-picryl-hydrazil radical scavenging capacity, while the effect of Se on this potential was stimulatory in G. applanatum (1.3%-33.9% without Se and 3.1%-67.1% in Se enrichment) and inhibitory in G. lucidum (1.4%-71.6% and 1.3%-48.6% without and with Se, respectively). Only phenols in G. applanatum and phenols and flavonoids in G. lucidum were holders of antioxidant activity. Cytotoxic activity against both HeLa and LS174 cell lines was very low in comparison with cis-diamminedichloroplatinum.

Potential of Pleurotus ostreatus mycelium for selenium absorption.

The aim of this study was to evaluate the effect of high selenium (Se) concentrations on morphophysiological and ultrastructural properties of Pleurotus ostreatus. Mycelium growth was good in media enriched with 5.0, 10.0, and 20.0 mg L(-1) of Se, concentration of 500.0 mg L(-1) strongly inhibited growth, and 1000.0 mg L(-1) was the minimum inhibitory concentration. Contrary to thin-walled, hyaline, branched, and anastomized hyphae with clamp-connections in the control, at Se concentrations of 100.0 and 500.0 mg L(-1), they were noticeably short, frequently septed and branched, with a more intensive extracellular matrix, and without clamp-connections. At high Se concentrations, hyphae with intact membrane, without cellular contents, with a high level of vacuolization, and with numerous proteinaceous bodies were observed. Biomass yield ranged between 11.8 g L(-1), in the control, and 6.8 g L(-1), at an Se concentration of 100.0 mg L(-1), while no production was detected at a concentration of 500.0 mg L(-1). Se content in the mycelia reached a peak (938.9 µg g(-1)) after cultivation in the medium enriched with Se at the concentration of 20.0 mg L(-1), while the highest absorption level (53.25%) was found in the medium enriched with 5.0 mg L(-1) Se.

Biological activity of Ganoderma lucidum basidiocarps cultivated on alternative and commercial substrate.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum (W. Curt.:Fr.) P. Karst. has been used for several thousand years in traditional medicine of the Far East for the treatment of a various diseases including: cancer, immunological disorders, hepatitis, arthritis, nephritis, bronchitis, asthma etc. Ganoderma species are natural sources of various antimicrobial, antioxidant and antitumor active compounds which can be alternative to commercial antibiotics, antimycotics, antioxidants and cytostatics. As the amount of wild Ganoderma lucidum basidiocarps is not sufficient, the aim of this study was to compare the bioactivity of basidiaocarps obtained on alternative substrate (wheat straw) with commercially cultivated ones. MATERIALS AND METHODS: Antimicrobial potential of the tested Ganoderma lucidum extracts was studied by disc-diffusion and microdilution method. Cytotoxic effect of tested extracts on two cancer cell lines was determined by microculture tetrazolium test. DPPH scavenging ability, total phenols and flavonoids content were measured in order to determine the antioxidative potential of Ganoderma lucidum extracts. RESULTS: Ganoderma lucidum BEOFB 434 extract was the most effective antibacterial agent, while commercial strain was the weakest. DPPH· scavenging activity of extracts of basidiocarps from alternative substrate was also significantly higher than that of commercially obtained basidiocarps. The extracts of Ganoderma lucidum BEOFB 431 exhibited the strongest cytotoxic activity significantly higher than those in commercial strain. CONCLUSION: The study showed that fruiting bodies produced on one of the most accessible and cheapest crop residue, wheat straw, are better antimicrobial, antioxidant and cytotoxic agents than those obtained on conventional substrate, oak sawdust.

Potential enrichment of medicinal mushrooms with selenium to obtain new dietary supplements.

The purpose of this study was to resolve the question of whether various selenium concentrations affect the ability of eight species, members of five genera, to produce mycelium biomass and absorb this trace element during submerged cultivation in Na2SeO3-enriched medium. The obtained results demonstrated the ability of mycelia of all of the tested species to absorb selenium at analyzed concentrations. Thus, selenium content ranged from 1.4 µg g-1 (Pleurotus eryngii) to 20.3 µg g-1 (Pleurotus ostreatus). The potential of mycelia to absorb selenium was significant, ranging from 8.1% (Lenzites betulinus) to 62.5% (P ostreatus) of its content in the medium. It may be concluded that all tested mushroom species could be used as satisfactory selenium sources due to the fact that the absorbed concentrations ranged from 15.8% (P. eryngii) to 36.9% (P. ostreatus) of the dietary selenium amount (55 µg d-1) recommended by the European Scientific Committee on Food.

Oxidative stress and species of genus Ganoderma (higher Basidiomycetes).

Oxidative stress, which is a factor in the aging process and in a series of serious disorders, arises when the reactive oxygen or nitrogen species are produced in excess and the capacity of cellular antioxidant defense is insufficient to detoxify and remove them. An internal antioxidant system is not always active enough to protect the human body from oxidative stress and, therefore, it needs the help of either synthetic or natural antioxidants. Nowadays, there is a growing interest in the substitution of synthetic antioxidants, which could have toxic and mutagen effects, with natural antioxidants. Recent studies revealed that besides their high nutritional value, mushrooms have great potential as antioxidant agents. Species of the genus Ganoderma, especially G. lucidum, are well-known medicinal mushrooms that traditionally are used in the prevention and treatment of many diseases and possess appreciable antioxidant potential.

A comparative assessment of the potential of polysaccharide production and intracellular sugar composition within Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt.:Fr.)P. Karst. (Aphyllophoromycetideae).

Ganoderma lucidum is a well-known medicinal mushroom species in which polysaccharides are one of the major sources of biological activity. The species was considered as a species-complex due to significant variations in morphological, biochemical, and genetic features among populations with a worldwide distribution. This fact was the basis for setting the aim of this research: to study intraspecific diversity in polysaccharide production and intracellular sugar composition among selected G. lucidum strains. The presence ofintraspecific diversity among 10 G. lucidum strains, from different areas worldwide, was noted. Values of produced mycelia biomass and intracellular polysaccharides were found in wide ranges (3.1 - 28.2 g L(-1) and 20.0 - 53.3 mg g(-1), respectively), while differences in extracellular polysaccharide amounts were minor (0.2 - 1.5 mg mL(-1)). The significant quantitative and qualitative differences in intracellular sugar composition were noted. Glucose was the predominant sugar in almost all strains except one (HAI 447), where sucrose was dominant. The potential of polysaccharide production and intracellular sugar composition could be one more taxonomic criterion for strain characterization within G. lucidum. The differences in intracellular sugar composition and proportions could be reflected in features of produced polysaccharides and also in their biological activities.

Morpho-physiological diversity between Lingzhi or Reishi medicinal mushroom Ganoderma lucidium (W. Curt.:Fr.) P. Karst. and G. carnosum pat.

Two weakly differentiated taxa, Ganoderma lucidum and G. carnosum, were compared in their sufficient morphological and physiological features. The obtained results showed that dimensions of basidiospores and pileocystidia were insignificantly different, while pore shape and dimensions have shown greater diversity with average diameter of 138.46 µm in G. carnosum and 238.34 µm in G. lucidum. Mycelial growth rate was higher in G. lucidum (8.39 mm day-1) than in G. carnosum (6.02 mm day-1). G. lucidum was also a slightly better producer of biomass and extracellular polysaccharides (28.16 g L-1 and 1.42 mg mL-1, respectively) than G. carnosum (23.68 g L-1 and 0.35 mg mL-1, respectively). However, a higher amount of synthesized intracellular polysaccharides was noted in G. carnosum than in G. lucidum (40.00 mg g-1 and 30.00 mg g-1 of dry biomass, respectively). Higher activity levels of Mn-oxidizing peroxidases were obtained in G. carnosum, while G. lucidum was a better laccase producer. In G. carnosum, corn stem/NH4NO3 medium with nitrogen concentration of 20 mM was the optimum for Mn-dependent peroxidase production (88.00 U L-1), while the highest versatile peroxidase activity was detected in the medium with grapevine sawdust and 10 mM of nitrogen (80.80 U L-1). Wheat straw was the best carbon source for laccase synthesis in G. lucidum (55.75 U L-1).

Interaction of trace elements and ligninolytic enzymes in Pleurotus eryngii.

Considering the fact that some trace elements have influence on the growth and physiological processes in the fungi, the aim of this study was to resolve the question of whether Fe, Zn, and Se, added to the medium in the different forms, affect activity of selected Pleurotus eryngii ligninolytic enzymes participating in mandarine peels submerged fermentation. Compared with the control, Fe and Zn showed stimulatory effect on laccase activity. Reduction in the activity was emphasized with cultivation time, but it was slower in the presence of Zn than in the presence of Fe. Among studied Se forms, SeO2 was the most unfavorable Se source for laccase activity. All tested trace elements, independent on form, showed stimulatory effect on the activity of Mn-oxidizing peroxidases on the seventh day of cultivation. Based on the results, it could be concluded that the presence of the tested trace elements can advance various biotechnological processes among which is biodegradation of agricultural wastes, potential environmental pollutants.