Interdependent nitric oxide and hydrogen peroxide independently regulate the coix seed oil-induced triterpene acid accumulation in Ganoderma lingzhi.

Recent progress has been made in adding exogenous vegetable oils in culture media to promote bioactive metabolite production in several medicinal mushrooms, but the mechanism is still unclear. In this study, we found that the vegetable oil coix seed oil (CSO) could induce the biosynthesis of triterpene acids (TAs) and also significantly increase cytoplasmic nitric oxide (NO) and hydrogen peroxide (H2O2) concentrations in the mycelium of Ganoderma lingzhi. The change in TA biosynthesis caused by CSO could be reversed by adding NO scavenger or H2O2 scavenger, and adding NO scavenger or H2O2 scavenger resulted in the reduction of the cytoplasmic H2O2 or NO concentration under CSO treatment, respectively. Moreover, adding NO scavenger or H2O2 scavenger reversed TA biosynthesis, which could be rescued by H2O2 or NO donor, respectively. Taken together, our study indicated that both NO and H2O2 were involved in the regulation of TA biosynthesis, and CSO-activated NO and H2O2 were interdependent but independently regulated the TA biosynthesis under CSO treatment in G. lingzhi.

Determination of the Biological Efficiency and Antioxidant Potential of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), Cultivated Using Different Agro-Wastes in Malaysia.

This study investigates the cultivation of Ganoderma lucidum using different agricultural biomasses from Malaysia. Five different combinations of rubber wood sawdust, empty fruit bunch fiber, and mesocarp fiber from oil palm, alone and in combination, were used to cultivate G. lucidum. Although all the substrate combinations worked well to grow the mushroom, the highest biological efficiency was obtained from the combination of empty fruit bunch fiber with sawdust. A total yield of 27% was obtained from empty fruit bunch fiber with sawdust, followed by sawdust (26%), empty fruit bunch fiber (19%), mesocarp fiber with sawdust (19%), and mesocarp fiber (16%). The quality of mushrooms was proved by proximate analysis and detection of phenolic compounds and flavonoids. The antioxidant activity verified by DPPH, ferric-reducing ability of plasma, and ABTS analyses revealed that the empty fruit bunch fiber with sawdust had higher activity than the other substrates.

Induction of apoptosis and ganoderic acid biosynthesis by cAMP signaling in Ganoderma lucidum.

Apoptosis is an essential physiological process that controls many important biological functions. However, apoptosis signaling in relation to secondary metabolite biosynthesis in plants and fungi remains a mystery. The fungus Ganoderma lucidum is a popular herbal medicine worldwide, but the biosynthetic regulation of its active ingredients (ganoderic acids, GAs) is poorly understood. We investigated the role of 3',5'-cyclic adenosine monophosphate (cAMP) signaling in fungal apoptosis and GA biosynthesis in G. lucidum. Two phosphodiesterase inhibitors (caffeine and 3-isobutyl-1-methylxanthine, IBMX) and an adenylate cyclase activator (sodium fluoride, NaF) were used to increase intracellular cAMP levels. Fungal apoptosis was identified by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and a condensed nuclear morphology. Our results showed that GA production and fungal apoptosis were induced when the mycelium was treated with NaF, caffeine, or cAMP/IBMX. Downregulation of squalene synthase and lanosterol synthase gene expression by cAMP was detected in the presence of these chemicals, which indicates that these two genes are not critical for GA induction. Transcriptome analysis indicated that mitochondria might play an important role in cAMP-induced apoptosis and GA biosynthesis. To the best of our knowledge, this is the first report to reveal that cAMP signaling induces apoptosis and secondary metabolite production in fungi.

Effects of Exogenous Salicylic Acid on Ganoderic Acid Biosynthesis and the Expression of Key Genes in the Ganoderic Acid Biosynthesis Pathway in the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

We demonstrate herein that salicylic acid (SA) can enhance ganoderic acid (GA) accumulation in the lingzhi or reishi medicinal mushroom Ganoderma lucidum. Following treatment with different concentrations of SA, the GA content was increased 22.72% to 43.04% compared with the control group. When the fungi were treated with 200 µmol/L SA at different times, the GA content was improved 10.21% to 35.24% compared with the control group. By choosing the optimum point based on response surface methodology, the GA content could be increased up to 229.03 µg/100 mg, which was improved 66.38% compared with the control group. When the fungi were treated with 200 µmol/L SA, the transcription levels of key genes in the GA biosynthesis pathway-squalene (SQ) synthase (sqs), lanosterol (Lano; osc), and hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr)-were improved 119.6-, 3.2-, and 4.2-fold, respectively. In addition, following treatment with 100 µmol/L SA, the levels of Lano and SQ, which are intermediate metabolites of GA biosynthesis, were increased 2.8- and 1.4-fold, respectively. These results indicate that SA can regulate the expression of genes related to GA biosynthesis and increases the metabolic levels of Lano and SQ, thereby resulting in the accumulation of GA.

Efficacy of supplementation of selected medicinal mushrooms with inorganic selenium salts.

The aim of the study was to evaluate the possibility of supplementation with inorganic forms of selenium (Na2SeO4 and Na2SeO3) in concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.5 mM of three medicinal mushroom species: Agrocybe aegerita, Hericium erinaceus and Ganoderma lucidum. Tested mushroom species grew in Se additions of 0-0.6 mM (A. aegerita and H. erinaceus), while growth of G. lucidum bodies was observed for 0-0.8 mM. For the latter mushroom species, the total Se content was the highest. Content of Seorg was diverse; for control bodies it was the highest for G. lucidum (only organic forms were present), lower for A. aegerita (84% organic forms) and the lowest for H. erinaceus (56% organic forms). Accumulation of Se(IV) was generally significantly higher than Se(VI) for all tested mushroom species. There was no significant decrease of A. aegerita or G. lucidum biomass with the exception of G. lucidum bodies growing under 0.8 mM of Se species addition (15.51 ± 6.53 g). Biomass of H. erinaceus bodies was the highest under 0.2 (197.04 ± 8.73 g), control (191.80 ± 6.06 g) and 0.1 mM (185.04 ± 8.73 g) of both inorganic salts. The addition to the medium of Se salts brought about macroscopic changes in the fruiting bodies of the examined mushrooms. Concentrations exceeding 0.4 mM caused diminution of carpophores or even their total absence. In addition, colour changes of fruiting bodies were also recorded. At Se concentrations of 0.4 and 0.6 mM, A. aegerita fruiting bodies were distinctly lighter and those of H. erinaceus changed colour from purely white to white-pink.

Improved production, purification and bioactivity of a polysaccharide from submerged cultured Ganoderma lucidum.

Polysaccharides from Ganoderma lucidum showed multiple biological activities, such as immuno-modulating, antitumor, antioxidant, and hepatoprotective activity, etc. Adlay oil was added into the media to enhance polysaccharide production by submerged culture of G. lucidum in this work. The results revealed the optimal concentration of adlay oil was 1.5 % for polysaccharide production of G. lucidum. Analysis of the polysaccharide components confirmed that no novel components were biosynthesized by the addition of adlay oil. The main fraction of extracellular polysaccharide, GLEP-2, was isolated from the submerged culture broth of G. lucidum by ethanol precipitation, filtration, DEAE cellulose-52 and Sepharose CL-6B chromatography. GLEP-2, which was composed of glucose, galactose, mannose, arabinose, and rhamnose in a ratio of 332:55:32:13:3 respectively, had an average molecular weight of ~2.08 × 10(4) Da. The bioactivity tests demonstrated that GLEP-2 enhanced the T lymphocyte proliferation significantly at a concentration of 200 µg/mL and B lymphocyte proliferation at lower concentrations of 50 µg/mL. The results suggest polysaccharides from the submerged culture of G. lucidum are potential candidates for further development and possible commercial applications, especially in the pharmaceutical and functional foods industries.

A novel approach to enhancing ganoderic acid production by Ganoderma lucidum using apoptosis induction.

Ganoderma lucidum is one of most widely used herbal medicine and functional food in Asia, and ganoderic acids (GAs) are its active ingredients. Regulation of GA biosynthesis and enhancing GA production are critical to using G. lucidum as a medicine. However, regulation of GA biosynthesis by various signaling remains poorly understood. This study investigated the role of apoptosis signaling on GA biosynthesis and presented a novel approach, namely apoptosis induction, to increasing GA production. Aspirin was able to induce cell apoptosis in G. lucidum, which was identified by terminal deoxynucleotidyl transferase mediated dUPT nick end labeling assay positive staining and a condensed nuclear morphology. The maximum induction of lanosta-7,9(11), 24-trien-3α-01-26-oic acid (ganoderic acid 24, GA24) production and total GA production by aspirin were 2.7-fold and 2.8-fold, respectively, after 1 day. Significantly lower levels of GA 24 and total GAs were obtained after regular fungal culture for 1.5 months. ROS accumulation and phosphorylation of Hog-1 kinase, a putative homolog of MAPK p38 in mammals, occurred after aspirin treatment indicating that both factors may be involved in GA biosynthetic regulation. However, aspirin also reduced expression of the squalene synthase and lanosterol synthase coding genes, suggesting that these genes are not critical for GA induction. To the best of our knowledge, this is the first report showing that GA biosynthesis is linked to fungal apoptosis and provides a new approach to enhancing secondary metabolite production in fungi.

Effect of oxygen concentration in gas phase on sporulation and individual ganoderic acids accumulation in liquid static culture of Ganoderma lucidum.

Effects of oxygen concentration within 21-100% in gaseous phase on the morphology and ganoderic acids (GAs) production by Ganoderma lucidum in liquid static culture were studied. A higher oxygen concentration increased individual GAs production, and more spores and higher total GA content were obtained at an oxygen level of 80%.

Enhanced biosynthetic gene expressions and production of ganoderic acids in static liquid culture of Ganoderma lucidum under phenobarbital induction.

Static liquid culture of Ganoderma lucidum, a traditional Chinese medicinal mushroom, is a proven technology for producing ganoderic acids, which are secondary metabolites that possess antitumor properties. In this work, the addition of phenobarbital, a P450 inducer, was used to enhance the production of total and individual ganoderic acids in a two-stage cultivation involving a period of initial shake flask culture followed by static liquid culture of G. lucidum. The dosage and time of phenobarbital induction were critical for the enhanced production of ganoderic acids. The addition of 100 muM (final concentration) phenobarbital on day 5 after the shake flask culture was converted to the static liquid culture was found to be optimal, resulting in a maximal amount of total ganoderic acids of 41.4 +/- 0.6 mg/g cell dry weight and increases in the levels of ganoderic acid-Mk, -T, -S, and -Me in the treated cells by 47%, 28%, 36%, and 64%, respectively. Meanwhile, the accumulation of lanosterol, a key intermediate, was found to decrease and transcriptions of three key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase in the triterpene biosynthetic pathway were up-regulated under phenobarbital induction. This work demonstrated a useful strategy for the enhanced production of ganoderic acids by G. lucidum.

[A preliminary report on solid-state fermentation of Ganoderma lucidum with Radix Astragali containing medium].

OBJECTIVE: To test the practicability of the solid-state fermentation for medicinal fungi by fermenting Ganoderma lucidum with Radix Astragali containing medium. METHODS: Ganoderma lucidum was fermented in ordinary medium, drug-containing medium (containing Radix Astragali) and selenium-rich drug-containing medium respectively. The polysaccharide contents of fermentation products from the three kinds of culture media were tested at different time, and the changes were compared. RESULTS: The polysaccharide contents of fermentation products from the three kinds of culture media were 4.65%, 3.76% and 4.50% respectively and their relative standard deviation were 1.61%, 1.99% and 1.86% respectively. By observing the changes of the contents of polysaccharide, protein and total saponin in fermentation products from the drug-containing medium at different time, it was found that the 28th fermentation day was the time when secondary metabolism was most active, and it should be the fermented terminal point. CONCLUSION: The fermentative combination of Ganoderma lucidum and Radix Astragali is practicable.

[Effects of extracts of Chinese medicines on Ganoderma lucidum in submerged culture].

Effects of water and ethanol extracts of 10 Chinese medicines, such as Astragalus membranaceus, Coix lachryma-jobi, etc., on biomass and exopolysaccharide of Ganoderma lucidum were studied by submerged culture. The results showed: water extracts of all medicines can improve the culture of G. lucidum except of A. membranaceus, ethanol extracts of C. lachryma-jobi, Dioscorea opposita, Codonopsis pilosula, and Achyranthes bidentata( < 187.5g Medicine/L substrate) can also increase the biomass of G. lucidum, but the ethanol extracts of Angelica sinensis, Dendrobium nobile check the growth of G. lucidum. The production of exopolysa-ccharide can be improved by all the Chinese medicines and their dosage used in this experiment, Although A. sinensis, D. Nobile check the growth of G. lucidum, they could stimulate the secretion of exopolysaccharide in lower dosage. It is concluded that some Chinese medicines, such as C. lachryma-jobi, D. opposita, C. pilosula, etc. can be processed by the fermentation of G. lucidum, and bio-active compound can be produced by adding appropriate Chinese medicine in the substrate to culture G. lucidum.