Ganoderic acid A ameliorates renalfibrosis by suppressing the expression of NPC1L1

Objective: The study aimed to explore the protective mechanism of Ganoderic acid A (GAA) in renal fibrosisand to verify that GAA can ameliorate renal fibrosis by regulating the Niemann-pick C1-like 1 (NPC1L1) gene. Methods:Transforming growth factor beta1 (TGF-β1) was used to treat Human Kidney-2 (HK-2) cells to establish a renal fibrosismodel. The differentially expressed genes in the control (CTRL) group, TGF-β1 group, and TGF-β1 + GAA group werescreened via transcriptome sequencing technology and verified by qPCR and Western blot experiments. The NPC1L1gene overexpression plasmid was constructed. The expression levels of N-cad, E-cad, and Slug-related proteins inCTRL, TGF-β1, TGF-β1+GAA (25 μg/mL), and TGF-β1+GAA (25 μg/mL) + NPC1L1 Overexpression (OE) groupswere detected by qPCR and Western blot analysis. Western blot analysis was used to identify the extracellular matrixassociated proteins Tenascin-C, α-SMA, and fibrosis-related protein Collagen I. Fibrosis marker protein Fibronectinwas detected and quantified by immunofluorescence. Results: Transcriptomic sequencing revealed that TGF-β1stimulation led to 267 differentially regulated genes, with 118 up-regulated and 149 down-regulated, while furthermodulation of 213 genes, comprising 112 up-regulated and 101 down-regulated genes, was observed in the GAAintervention group. The target gene in these processes was found to be NPC1L1 by investigations using GeneOntology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). qPCR and Western blot resultsconfirmed that TGF-β1 increased NPC1L1 expression, which was attenuated by GAA. Additionally, TGF-β1upregulated N-cad and Slug. However, GAA reversed this effect and NPC1L1 overexpression partially rescued theGAA effect. TGF-β1 also decreased E-cad expression, reversed by GAA, and NPC1L1 overexpression antagonized thisreversal. Furthermore, TGF-β1 promoted Collagen I, α-SMA, and Tenascin-C expression, and GAA reduced theselevels, effects that were reversed by NPC1L1 overexpression. Immunofluorescence results showed that TGF-β1increased fibronectin expression, which was decreased by GAA, and increased by NPC1L1 overexpression.Conclusion: GAA ameliorates renal fibrosis by antagonizing NPC1L1 gene expression inhibiting epithelialmesenchymal transition and reducing extracellular matrix formation.

Rapid Simultaneous Determination of Four Ganoderic Acids in Ganoderma(Chinese Name:Lingzhi)by Direct Infusion-Multiple Reaction Monitoring Cubed

Attributing to the rapid demand expansion for the edible medicinal materials in the market,the limited throughput of highperformance liquid chromatography-multiple reaction monitoring(HPLC-MRM)cannot fully address the measurement workload for a huge number of testing samples.Hence,it is urgent to pursue more efficient approaches for the quality evaluation.Because of the greater selectivity of MRM cubed(MRM^(3))over MRM,there might be a chance to omit the time-intensive LC separation.In current study,we attempted to develop a direct infusion(DI)-MRM^(3) program,and the applicability was thereafter assessed through simultaneous determination of four ganoderic acids(GAs)in one of the most famous tonic herbal medicines namely Ganoderma(Chinese name:Lingzhi).Primary parameters such as Q1>Q3>QLIT ion transitions,collision energy(CE),and excitation energy were optimized by programming online energy-resolved mass spectrometry with authentic compounds.A single DI-MRM measurement merely costed four minutes,and in spite of the wide occurrences of isomers,satisfactory selectivity was achieved.Method validation assays demonstrated the method to be sensitive,precise,accurate,and reproducible.The quantitative results from DI-MRM^(3) were also justified by conducting LC-MRM measurements in parallel.Significant differences occurred for the content patterns between the two original sources namely Ganoderma lucidum and G.sinense,and,moreover,either cultivar or harvest time showed dramatical influence on the quantitative features of the four targeted GAs.More importantly,DI-MRM3 is a meaningful analytical option for rapid quantitative analysis of herbal medicines,because of the comparable reliability,nonetheless,less consumptions of both measurement time and solvent,compared with LC-MRM.

Determination of ganoderic acids in rat plasma after oral administration of total triterpenoids from Ganoderma lucidum

To investigate the absorption of total triterpenoids from Ganoderma lucidum in rats. HPLC-DAD and LC-MS methods were used to identify ganoderic acids in rat plasma after oral administration of total triterpenoids from G. lucidum by comparing their HPLC retention behaviors, UV absorption spectra, and mass spectra with authentic samples. Five ganoderic acids, ganoderic acid C2, C6, G, B and A were simultaneously detected in rat plasma. Ganoderic acids can be directly absorbed into circulation after oral administration of total triterpenoids from G. lucidum in rats.

Hydrodynamic Shear Stress Affects Cell Growth and Metabolite Production by Medicinal Mushroom Ganoderma lucidum

By investigating the shear effect on submerged cultivation of a traditional Chinese medicinal herb Ganoderma lucidum, a relatively high cell concentration of 13.8 g·L-1 by dry mass was obtained in bioreactor at an impeller tip speed (ITS) of 0.51m·s-1. At an ITS of 0.51,1.02 and 1.53m·s-1, a maximal production titer of intracellular polysaccharide was 2.64, 2.20 and 2.28g·L-1 and that of ganoderic acid was 306, 299 and 273g·L-1, respectively. Under these ITSs, the maximal mean projected area of dispersed hyphae was 3.70, 2.54 and 2.13×104μm2, and that of pellets was 0.91, 0.67 and 0.55mm2, respectively. The information obtained is useful for efficient submerged cultivation of mushrooms on a large scale.

Quantitative determination of ganoderic acid T in rat plasma by a sensitive RP-HPLC method and its application in pharmacokinetic studies

A selective and sensitive HPLC method has been developed and validated for the quantification of ganoderic acid T in rat plasma. A reverse-phase column was used with UV detection at 245 nm. The mobile phase consisted of methanol-water-acetic acid （95.5：4.5：0.5, v/v/v） run at a flow rate of 1 mL/min. Testosterone propionate was used as the internal standard. The standard curve was linear over the range of 0.05-50 ~tg/mL in rat plasma with a linear correlation coefficient greater than 0.999. The lower limit of quantification of this assay was 20 ng/mL. The recoveries of samples at 0.05, 2 and 40 μg/mL were 97.6%, 98.4% and 103.2%, respectively. The relative standard deviations of intra- and inter-day assay variations were less than 8.2%. The usefulness of the assay was confirmed by the successful analysis of plasma samples from a pharmacokinetics study in rats. Following a single dose of ganoderic acid T （5 mg/kg for i.v. or 14 mg/kg for p.o.）, the main pharmacokinetic parameters by intravenous injection were： t1/2α （5.46±1.25） min; t1/2β：（227.18±11.40） min; CL： （1.09±0.16） mL/（kg·min）; A UC0-12 h： （3939.13±311.14） μg.min/mL; AUC0-12h （4681.96±710.70）μg.min/mL and the absolute bioavailability was 41.98%±2.40%. This method is simple, sensitive, and accurate. It is suitable for pharmacokinetic studies of ganoderic acid in rats.

Studies on the Triterpenoid Constituents of the Spores from Ganoderma lucidum Karst

Five compounds were isolated from the ether-soluble fraction of the spores of Gano- derma lucidum.On the basis of their chemical properties and spectral data(MS,UV,IR,~1H and ^(13)CNMR),they were identified as 3,7,11,12,15,23-hexaoxo-5α-lanosta-8-en-26-oic acid(Ⅰ),gano- deric acid B(Ⅱ),C(Ⅲ),D(Ⅳ)and ganodermanontriol(Ⅴ).Compound Ⅰ is a new natural product, named ganosporeric acid A.Compounds Ⅱ,Ⅲ,Ⅳ and Ⅴ are known compounds and were obtained for the first time from the spores of Ganoderma lucidum.Pharmacological experiments showed that ganosporeric acid A has an activity for lowering the levels GPT in mice with liver injury by CCl_4 and GaNI and exhibits heptoprotective effects.

Ganoderic acid A protects lens epithelial cells from UVB irradiation and delays lens opacity

A contributory role of oxidative stress and protection by antioxidant nutrients have been suspected in cataract formation.Ganoderic acid A(GAA),an effective lanostane triterpene,is widely reported as an antioxidant.The aim of this study is to investigate the potential effects of GAA on cataract formation.After lens epithelial cells(LECs)were exposed to UVB radiation for different periods,cell viability,apoptosis-related protein levels,malondialdehyde(MDA)and superoxide dismutase(SOD)activities were monitored.We found that cell viability,the Bcl-2/Bax ratio and SOD activity were increased,while Cleaved caspase-3 levels and MDA activity were decreased compared with those in UVB-impaired LECs after GAA treated.Furthermore,GAA activated PI3 K/AKT in UVB-impaired LECs and effectively delayed the occurrence of lens opacity in vitro.In conclusion,these findings demonstrated that GAA exhibited protective functions in SRA01/04 cells and rat lenses against UVB-evoked impairment through elevating cell viability and antioxidant activity,inhibiting cell apoptosis,activating the PI3 K/AKT pathway and delaying lens opacity.

Germplasm resources and secondary metabolism regulation in Reishi mushroom(Ganoderma lucidum)

Ganoderma lucidum is a valuable medical macrofungus with a myriad of diverse secondary metabolites,in which triterpenoids are the major constituents.This paper introduced the germplasm resources of genus Ganoderma from textual research,its distribution and identification at the molecular level.Also we overviewed G.lucidum in the components,the biological activities and biosynthetic pathways of ganoderic acid,aiming to provide scientific evidence for the development and utilization of G.lucidum germplasm resources and the biosynthesis of ganoderic acid.

Submerged fermentation production and characterization of intracellular triterpenoids from Ganoderma lucidum using HPLC-ESI-MS

As the main bioactive metabolites of Ganoderma lucidum, triterpenoids have various pharmacological effects. In this paper, the nutritional requirements and culture conditions of a submerged culture of G. lucidum were optimized using the response surface methodology; maximum mycelia biomass and intracellular triterpenoid production reached 1.87 g/100 ml and 93.21 mg/100 ml, respectively, for a culture consisting of wort 4.10% （0.041 g/ml） and yeast extract 1.89% （0.0189 g/ml）, pH 5.40. For the first time, we established that wort, which is cheap and abundant, can replace the more commonly used glucose as the sole source of carbohydrate. Using high-performance liquid chromatography-electrospray ionization-mass spectrometry （HPLC-ESI-MS）, 10 major ganoderic acids were tentatively identified based on the predominant fragmentation pathways with the elimination of H20 and CO2, as well as cleavage of the D-ring.