Antibacterial lanostane triterpenoids from Ganoderma tsugae.

A phytochemical investigation on the 80% EtOH extract of the fruiting bodies of Ganoderma tsugae resulted into the isolation of two previously undescribed lanostane triterpenoids, 7,11-dioxo-3ß-acetyloxy-26,27-dihydroxy-lanosta-8,24-diene (1) and 7,20-dioxo-3ß-acetyloxy-11ß,15α-dihydroxy-22,23,24,25,26,27-hexanorlanosta-8-ene (2), togeher with one known lanostane triterpenoid ganodermanontriol (3). Structural elucidation of all the compounds were performed by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy. All the triterpenoids were in vitro evaluated for their antibacterial activities against six pathogenic microorganisms. Compound 3 exhibited some activities against three Gram positive bacteria with MIC values less than 30 µg/ml.

Hepatoprotective Activity of Ethanol Extract of Rice Solid-State Fermentation of Ganoderma tsugae against CCl4-Induced Acute Liver Injury in Mice.

Ganoderma tsugae is well known as a medicinal mushroom in China and many Asian countries, while its fermentation technique and corresponding pharmacological activity are rarely reported. In this study, a wild G. tsugae strain (G42) with high triterpenoid content was screened from nine strains by rice solid-state fermentation, and 53.86 mg/g triterpenoids could be produced under optimized conditions; that is, inoculation amount 20%, fermentation temperature 27 °C, and culture time 45 days. The hepatoprotective activity of G42 ethanol extract was evaluated by CCl4-induced liver injury in mice, in which changes in the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), oxidation-related factors, and inflammatory cytokines in serum or liver samples demonstrated the therapeutic effect. In addition, the ethanol extract of G42 reduced the incidence of necrosis and inflammatory infiltration, and decreased protein expression levels of phosphor-nuclear factor-κB (NF-κB), interleukin-Iß (IL-1ß), and nuclear factor erythroid-2-related factor 2 (NRF2). The chemical composition of the ethanol extract was analyzed by high-resolution mass spectrometry and molecular networking. Three main triterpenoids, namely platycodigenin, cucurbitacin IIb, and ganolecidic acid B were identified. This work provided an optimized fermentation method for G. tsugae, and demonstrated that its fermentation extract might be developed as a functional food with a hepatoprotective effect.

Ganoderma tsugae suppresses the proliferation of endometrial carcinoma cells via Akt signaling pathway.

Ganoderma is one of the common medicinal mushrooms in traditional Chinese medicine. Previous researches have unveiled the multifaceted biological activity of Ganoderma extract. Ganoderma tsugae has been investigated the potential on curing prostate, colon, lung, epidermoid, breast and ovarian cancers, but not including endometrial cancer. Endometrial cancer is a gynecological malignant tumor with serious drug resistance problem in clinical cancer treatment. This study aimed to demonstrate the first study of Ganoderma on treating endometrial cancer. The Ganoderma tsugae ethanol extract (GTEE) could suppress the proliferation of endometrial cancer cells HEC-1-A, KLE, and AN3 CA. GTEE also induced G1/S phase arrest and mitochondria-mediated apoptosis in endometrial cancer cells. Furthermore, the Akt signaling pathway could be suppressed by GTEE. Therefore, our results suggest for the first time that GTEE has the potential to be an adjuvant therapeutic agent in the treatment of endometrial cancer.

Chinese Herbal Medicine Ganoderma tsugae Displays Potential Anti-Cancer Efficacy on Metastatic Prostate Cancer Cells.

Resistance to the current therapies is the main clinical challenge in the treatment of lethal metastatic prostate cancer (mPCa). Developing novel therapeutic approaches with effective regimes and minimal side effects for this fatal disease remain a priority in prostate cancer study. In the present study, we demonstrated that a traditional Chinese medicine, quality-assured Ganoderma tsugae ethanol extract (GTEE), significantly suppressed cell growth and metastatic capability and caused cell cycle arrest through decreasing expression of cyclins in mPCa cells, PC-3 and DU145 cells. GTEE also induced caspase-dependent apoptosis in mPCa cells. We further showed the potent therapeutic efficacy of GTEE by inhibiting subcutaneous PC-3 tumor growth in a xenograft model. The in vitro and in vivo efficacies on mPCa cells were due to blockade of the PI3K/Akt and MAPK/ERK signaling pathways associated with cancer cell growth, survival and apoptosis. These preclinical data provide the molecular basis for a new potential therapeutic approach toward the treatment of lethal prostate cancer progression.

Importance of a Laccase Gene (Lcc1) in the Development of Ganoderma tsugae.

In this study, a novel laccase gene (Lcc1) from Ganoderma tsugae was isolated and its functions were characterized in detail. The results showed that Lcc1 has the highest expression activity during mycelium development and fruit body maturation based on the analysis of Lcc1 RNA transcripts at different developmental stages of G. tsugae. To investigate the exact contribution of Lcc1 to mycelium and fruit body development in G. tsugae, Lcc1 transgenic strains were constructed by targeted gene replacement and over-expression approaches. The results showed that the lignin degradation rate in Lcc1 deletion mutant was much lower than the degradation efficiency of the wild-type (WT), over-expression and rescue strains. The lignin degradation activity of G. tsugae is dependent on Lcc1 and the deletion of Lcc1 exerted detrimental influences on the development of mycelium branch. Furthermore, the study uncovered that Lcc1 deletion mutants generated much shorter pale grey fruit bodies, suggesting that Lcc1 contributes directly to pigmentation and stipe elongation during fruit body development in G. tsugae. The information obtained in this study provides a novel and mechanistic insight into the specific role of Lcc1 during growth and development of G. tsugae.

Ganoderma tsugae Inhibits the SREBP-1/AR Axis Leading to Suppression of Cell Growth and Activation of Apoptosis in Prostate Cancer Cells.

Recent research suggests that the activation of lipid biosynthesis (lipogenesis) is linked with prostate cancer (PCa) malignancy. Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcriptional regulator controlling lipogenesis. Moreover, androgen receptor (AR) has been well defined to play an important role in lethal PCa aggressiveness from androgen-responsive to castration-resistant status. In this study, we showed that the quality-assured Ganoderma tsugae ethanol extract (GTEE), a Chinese natural and herbal product, significantly inhibited expression of SREBP-1 and its downstream genes associated with lipogenesis in PCa cells. Through inhibiting SREBP-1, GTEE reduced the levels of intracellular fatty acids and lipids in PCa cells. Importantly, GTEE also downregulated the expression of AR and prostate-specific antigen (PSA) in both androgen-responsive and castration-resistant PCa cells. By blocking the SREBP-1/AR axis, GTEE suppressed cell growth and progressive behaviors, as well as activating the caspase-dependent apoptotic pathway in PCa cells. These data provide a new molecular basis of GTEE for the development of a potential therapeutic approach to treat PCa malignancy.

UPLC-ESI-MS/MS-based widely targeted metabolomics reveals differences in metabolite composition among four Ganoderma species.

The Chinese name "Lingzhi" refers to Ganoderma genus, which are increasingly used in the food and medical industries. Ganoderma species are often used interchangeably since the differences in their composition are not known. To find compositional metabolite differences among Ganoderma species, we conducted a widely targeted metabolomics analysis of four commonly used edible and medicinal Ganoderma species based on ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Through pairwise comparisons, we identified 575-764 significant differential metabolites among the species, most of which exhibited large fold differences. We screened and analyzed the composition and functionality of the advantageous metabolites in each species. Ganoderma lingzhi advantageous metabolites were mostly related to amino acids and derivatives, as well as terpenes, G. sinense to terpenes, and G. leucocontextum and G. tsugae to nucleotides and derivatives, alkaloids, and lipids. Network pharmacological analysis showed that SRC, GAPDH, TNF, and AKT1 were the key targets of high-degree advantage metabolites among the four Ganoderma species. Analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes demonstrated that the advantage metabolites in the four Ganoderma species may regulate and participate in signaling pathways associated with diverse cancers, Alzheimer's disease, and diabetes. Our findings contribute to more targeted development of Ganoderma products in the food and medical industries.

Activation of Nrf2/HO-1 signaling: An important molecular mechanism of herbal medicine in the treatment of atherosclerosis via the protection of vascular endothelial cells from oxidative stress.

Introduction: Recently, Nrf2/HO-1 has received extensive attention as the main regulatory pathway of intracellular defense against oxidative stress and is considered an ideal target for alleviating endothelial cell (EC) injury. Objectives: This paper aimed to summarized the natural monomers/extracts that potentially exert protective effects against oxidative stress in ECs. Methods: A literature search was carried out regarding our topic with the keywords of "atherosclerosis" or "Nrf2/HO-1" or "vascular endothelial cells" or "oxidative stress" or "Herbal medicine" or "natural products" or "natural extracts" or "natural compounds" or "traditional Chinese medicines" based on classic books of herbal medicine and scientific databases including Pubmed, SciFinder, Scopus, the Web of Science, GoogleScholar, BaiduScholar, and others. Then, we analyzed the possible molecular mechanisms for different types of natural compounds in the treatment of atherosclerosis via the protection of vascular endothelial cells from oxidative stress. In addition, perspectives for possible future studies are discussed. Results: These agents with protective effects against oxidative stress in ECs mainly include phenylpropanoids, flavonoids, terpenoids, and alkaloids. Most of these agents alleviate cell apoptosis in ECs due to oxidative stress, and the mechanisms are related to Nrf2/HO-1 signaling activation. However, despite continued progress in research on various aspects of natural agents exerting protective effects against EC injury by activating Nrf2/HO-1 signaling, the development of new drugs for the treatment of atherosclerosis (AS) and other CVDs based on these agents will require more detailed preclinical and clinical studies. Conclusion: Our present paper provides updated information of natural agents with protective activities on ECs against oxidative stress by activating Nrf2/HO-1. We hope this review will provide some directions for the further development of novel candidate drugs from natural agents for the treatment of AS and other CVDs.

Ganoderma tsugae induced ROS-independent apoptosis and cytoprotective autophagy in human chronic myeloid leukemia cells.

The medicinal fungus Ganoderma, known in Chinese as Lingzhi or Reishi, traditionally has various medicinal uses and has been employed in cancer treatment in Asia for centuries. This study used ethanol-extracted Ganoderma tsugae (GT) and examined its antitumor activities on human chronic myeloid leukemia cells as well as its molecular mechanism of action. Treatment with GT (200-400 µg/mL) significantly reduced cell viability and caused G2/M arrest in K562 cells. In addition, GT induced mitochondrial and death receptor mediated apoptosis, correlated with DNA fragmentation, followed by cytochrome c release, caspase-3/8/9 activation, PARP cleavage, Fas activation, Bid cleavage, and Bax/Bcl-2 dysregulation. Cytoprotective autophagy was found to be induced by GT, as was revealed by increased LC3-II accumulation, Beclin-1/Bcl-2 dysregulation, acidic vesicular organelle formation, and p62/SQSTM1 activation. Notably, pretreatment of cells with the autophagy inhibitors 3-MA and CQ enhanced GT-induced apoptosis. Interestingly, reactive oxygen species production in cells was not triggered by GT administration; equally, the antioxidant N-acetylcysteine was found to be incapable of preventing apoptosis and autophagy induced by GT treatment. Finally, this study discovered that cytoprotective autophagy induced by GT was associated with EGFR and PI3K/AKT/mTOR signaling cascade suppression. In summary, GT demonstrated antitumor activity against human chronic myeloid leukemia.

Proteomic profiling of Ganoderma tsugae ethanol extract-induced adipogenesis displaying browning features.

Ganoderma is classified as a top grade traditional Chinese medicine for promoting human health by regulating 'vital energy'. Its potency towards metabolism and energy homeostasis, particularly, metabolic adaptations of adipocytes, needs to be re-evaluated through an evidence-based study. Here, the triterpenoid-rich Ganoderma tsugae ethanol extract (GTEE) was found to contribute towards adipogenesis accompanied with elevated intracellular lipid metabolic flux. Additionally, proteomic profiling revealed GTEE-upregulated mitochondrial remodeling and chemical energy redox modifications, which display UCP1-positive browning fat-selective features and a NADH-mediated adaptive mechanism. GTEE-treated mice with diet-induced obesity also resulted in the amelioration of white adipocyte hypertrophy and the appearance of UCP1-positive browning adipocytes. Our novel findings unravel that GTEE could promote intracellular metabolic flexibility and plasticity followed by the induction of adipocyte browning.

Triterpenoids and an alkamide from Ganoderma tsugae.

Ganoderma tsugae is a medicinal mushroom. In a continual study on the bioactive constituents of this fungus, a new lanostanoid, 3ß-acetoxy-16α-hydroxy-24ξ-methyl-5α-lanosta-8,25-dien-21-oic acid, named tsugaric acid F (1) and a novel palmitamide, N-(3'α,4'ß-dihydroxy-2'ß-(hydroxymethyl)-1'ß-(cyclobutyl)palmitamide (2) were isolated and characterized from the fruit bodies of G. tsugae, and three novel seco-lanostanoids, 3,4-seco-8α,9α-epoxy-5α-lanosta-21-oic acid 3,4 lactone (5), 3,4-seco-5ß-lanosta-7,9(11),4(29)-trien-3,21-dioic acid-3-methyl ester (6), 3,4-seco-5ß-lanosta-7,9(11),4(29)-trien-3,21-dioic acid (7), and a known compound, 3-oxo-5α-lanosta-8-en-21-oic acid (4) were prepared from 3. The structures of new compounds, 1, 2, 5-7 were determined by spectroscopic methods. Compounds 1 and 4 showed inhibitory effects on xanthine oxidase (XO) with an IC50 values of 313.3 ± 80.0 and 43.9 ± 29.9 µM, respectively when 7 exhibited potent inhibitory effect on superoxide anion generation in rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB) with an IC50 values of 1.3 ± 0.2 µM. Compounds 4-7 showed weak cytotoxic activities against PC3 cells. These results indicated that 4 and 7 may be used as cancer chemopreventive agents.

Potent In Vitro Protection Against PM2.5-Caused ROS Generation and Vascular Permeability by Long-Term Pretreatment with Ganoderma tsugae.

Epidemiological studies show increased particulate matter (PM[Formula: see text]) particles in ambient air are correlated with increased myocardial infarctions. Given the close association of capillaries and alveoli, the dysfunction is caused when inhaled PM[Formula: see text] particles come in close proximity to capillary endothelial cells. We previously suggested that the inhalation of PM[Formula: see text] diesel exhaust particles (DEP) induces oxidative stress and upregulates the Nrf2/HO-1 pathway, inducing vascular permeability factor VEGFA secretion, which results in cell-cell adherens junction disruption and PM[Formula: see text] transmigratation into circulation. Here, we minimized the level that PM[Formula: see text] traveled in the bloodstream by pre-supplementing with a traditional Chinese medicine (TCM) Ganoderma tsugae DMSO extract (GTDE) prior to PM[Formula: see text] exposure. Our results show that PM[Formula: see text] caused alterations in enzyme activities and cellular anti-oxidant balance. We found decreased glutathione levels, a reduced cellular redox ratio, increased ROS generation and cytotoxicity in the cellular fractions. The oxidative stress caused DNA damage and apoptosis, likely causing downstream molecular events that trigger vasculature permeabilization and, eventually, cardiovascular disorders. Our results show long-term GTDE treatment increased endogenous glutathione level, while PM[Formula: see text]-reduced glutathione levels and the cellular redox ratio. GTDE was protective against the genotoxic and apoptotic effects initiated by PM[Formula: see text] oxidative stress. Vascular permeability revealed that PM[Formula: see text] only accumulated on the surface of cells after GTDE treatment; no penetration was detected. After two weeks of GTDE treatment, VEGFA secretion was significantly reduced in human umbilical vein endothelial cells (HUVEC) and endothelial cell migration was blocked. Our results suggest GTDE prevents PM[Formula: see text] transmigration into the bloodstream, and the resultant dysfunction, by inhibiting oxidative stress production and endothelial permeability.

Chemical characteristics and anti-proliferation activities of Ganoderma tsugae polysaccharides.

Polysaccharides were extracted by hot-water and hot-alkali from four forms of Ganoderma tsugae including mature and baby Ling chih, mycelium and filtrate. Different profiles of proximate composition and monosaccharide constituents, and element contents were found in the extracted polysaccharides from different extractions and different forms. The molecular weight distributions of polysaccharides were 2.8×10(4)-6.5×10(5)Da and their infrared spectra were comparable. The hot-alkali extracted polysaccharides exhibited better anti-proliferation on IMR32 cells than the hot-water extracted polysaccharides, which were in turn more effective than the hot-water extracts. Besides, most hot-water extracts and both extracted polysaccharides exhibited an anti-proliferation effect on Hep G2 cells. However, the hot-water extracts showed less effective in anti-proliferation of IMR32 and Hep G2 cells. Based on the anti-tumor effects, both polysaccharides could be prepared for use in the formulation of nutraceuticals and functional foods.

Preparation of nano/submicrometer Ganoderma tsugae and its mutagenic potencies and cytotoxicity.

This study explored the feasibility of preparing nano/submicrometer particles from Ganoderma tsugae to enhance the contents of bioactive compounds and to assess its mutagenic potencies and cytotoxicity. Hot-water extract, a common product, was employed as a reference. After 3 h of media milling, almost all of the particles were smaller than 1 µm with a number-mean diameter of 0.11 µm. There were about 62% particles smaller than 0.1 µm in terms of number of particles. Scanning electron microscopy (SEM) confirmed the presence of particles at nano/submicrometer scale. The content of 1→3-ß-D-glucan in nano/submicrometer G. tsugae was 3.5 times of that in hot-water extract. Both nano/submicrometer and hot-water extract G. tsugae exhibited no mutagenic potential to Salmonella Typhimurium tester strains. Cell toxicity test also confirmed the safety of both nano/submicrometer and hot-water extract G. tsugae. The effect of media milling on the structural change of hyphae was also discussed.

Xanthine oxidase inhibitory lanostanoids from Ganoderma tsugae.

Two new lanostanoids, 3α-acetoxy-22-oxo-5α-lanosta-8,24-dien-21-oic acid, named tsugaric acid D (1) and 16α-hydroxy-3-oxo-5α-lanosta-6,8,24(24(1))-trien-21-oic acid, named tsugaric acid E (2) were isolated from the fruit bodies of Ganoderma tsugae. The structures 1 and 2 were determined by spectroscopic methods. Compound 1 and known compounds 3 and 6 exhibited significant inhibitory effects on xanthine oxidase (XO) activity with an IC50 values of 90.2±24.2, 116.1±3.0, and 181.9±5.8 µM, respectively. Known compound 5 was able to protect human keratinocytes against damage induced by UVB light, which showed 5 could protect keratinocytes from photodamage. The 1 and 5 µM 1 combined with 5 µM cisplatin, respectively, enhanced the cytotoxicity induced by cisplatin. It suggested that 1 and 5 µM 1 combined with low dose of cisplatin may enhance the therapeutic efficacy of cisplatin and reduce side effect and cisplatin resistant.

The triterpenoids of Ganoderma tsugae prevent stress-induced myocardial injury in mice.

Ganoderma mushrooms (Lingzhi in Chinese) have well-documented health benefits. Ganoderma tsugae (G. tsugae), one of the ganoderma species, has been commercially cultivated as a dietary supplement. Because G. tsugae has high antioxidant activity and because oxidative stress is often associated with cardiac injury, we hypothesized that G. tsugae protects against cardiac injury by alleviating oxidative stress. We tested the hypothesis using a work-overload-induced myocardial injury model created by challenging mice with isoproterenol (ISO). Remarkably, oral G. tsugae protected the mice from ISO-induced myocardial injury. Moreover, the triterpenoid fraction of G. tsugae, composed of a mixture of nine structurally related ganoderic acids (GAs), provided cardioprotection by inhibiting the ISO-induced expression of Fas/Fas ligand, oxidative stress, and apoptosis. The antioxidant activity of GAs was tested in cultured cardio-myoblast H9c2 cells against the insult of H2O2. GAs dissipated the cellular reactive oxygen species imposed by H2O2 and prevented cell death. Our findings uncovered the cardioprotective activity of G. tsugae and identified GAs as the bioactive components against cardiac insults.

Chemical constituents from fruiting bodies of Ganoderma tsugae (Ⅱ) / 中草药

Objective To study the chemical constituents from the fruiting bodies of Ganoderma tsugae. Methods To isolate the compounds by silica gel and Sephadex LH-20 column chromatography and to elucidate their structures by means of spectral analyses. Results Eight triterpenoids were obtained from EtOAc fraction of EtOH extract and identified as ganoderiol A (Ⅰ), ganodermanontriol (Ⅱ), ganodermatriol (Ⅲ), ganoderic acid C (Ⅳ), ganoderic acid A (Ⅴ), lucidone A (Ⅵ), lucidenic acid C (Ⅶ), and lucidenic acid LM1 (Ⅷ). Conclusion Compounds Ⅰ-Ⅷ are all isolated from G. tsugae for the first time.