Chun-Ru Cheng

Cytotoxic triterpenoids from Ganoderma lucidum.

A systematic study of the metabolites in Ganoderma lucidum led to isolation of 43 triterpenoids, six of them (1-6) are hitherto unknown. The structures of the latter were elucidated on the basis of spectroscopic studies and comparison with the known related compounds. All of the compounds were assayed for their inhibitory activities against human HeLa cervical cancer cell lines. Some compounds exhibit significant cytotoxicity, and their structure-activity relationships are discussed.

Comparison of two officinal Chinese pharmacopoeia species of Ganoderma based on chemical research with multiple technologies and chemometrics analysis.

AIM OF THE STUDY To investigate the chemical differences between Ganoderma lucidum (G. lucidum, Chizhi) and Ganoderma sinense (G. sinense, Zizhi). MATERIALS AND METHODS Thirty two batches of commercial Ganoderma samples were collected, including 20 batches of G. lucidum and 12 batches of G. sinense cultivated in different geographical regions. Chemical substances in aqueous extract and alcoholic extract, mainly polysaccharides and triterpenes respectively, were investigated. Determination of polysaccharides was carried out with a high performance liquid chromatography with an variable wavelength detector. Meanwhile, analysis of triterpenes were performed on an ultraviolet spectrophotometer, an ultra performance liquid chromatography and a rapid resolution liquid chromatograph combined with an electrospray ionization mass spectrometer. Chromatograms and spectra for all batches and reference standards of main components were obtained and used for direct comparison. Further discussion was made on the basis of the result of principal component analysis (PCA). RESULTS Significant difference of triterpenes was shown between G. lucidum and G. sinense. In 20 batches of G. lucidum, 12 main components, including eight ganoderic acids and four ganoderenic acids were identified and ten of them were quantitatively determined, with the total content from 0.249% to 0.690%. However, none of those triterpenes was found in either batch of G. sinense. As for constituents of polysaccharides, seven monosaccharides were identified and four main components among them were quantitatively determined. Difference of polysaccharides was not directly observed, but latent information was revealed by PCA and the discrimination became feasible. CONCLUSIONS G. lucidum and G. sinense were chemically different, which might result in pharmacological distinction. Preparations of traditional Chinese medicine (TCM) from Ganoderma should make accurate specification on the origin of species.

Pharmacophore-based discovery of FXR-agonists. Part II: Identification of bioactive triterpenes from Ganoderma lucidum

Graphical abstract

Macromolecular and small-molecule modulation of intracellular Aβ42 aggregation and associated toxicity

Aβ (amyloid β-peptide) has a central role in AD (Alzheimers disease) where neuronal toxicity is linked to its extracellular and intracellular accumulation as oligomeric species. Searching for molecules that attenuate Aβ aggregation could uncover novel therapies for AD, but most studies in mammalian cells have inferred aggregation indirectly by assessing levels of secreted Aβ peptide. In the present study we establish a mammalian cell system for the direct visualization of Aβ formation by expression of an Aβ(42)-EGFP (enhanced green fluorescent protein) fusion protein in the human embryonic kidney cell line T-REx293, and use this to identify both macromolecules and small molecules that reduce aggregation and associated cell toxicity. Thus a molecular shield protein AavLEA1 [Aphelenchus avenae LEA (late embryogenesis abundant) protein 1], which limits aggregation of proteins with expanded poly(Q) repeats, is also effective against Aβ(42)-EGFP when co-expressed in T-REx293 cells. A screen of polysaccharide and small organic molecules from medicinal plants and fungi reveals one candidate in each category, PS5 (polysaccharide 5) and ganoderic acid DM respectively, with activity against Aβ. Both PS5 and ganoderic acid DM probably promote Aβ aggregate clearance indirectly through the proteasome. The model is therefore of value to study the effects of intracellular Aβ on cell physiology and to identify reagents that counteract those effects.

Phenanthrenes, 9,10-dihydrophenanthrenes, bibenzyls with their derivatives, and malate or tartrate benzyl ester glucosides from tubers of Cremastra appendiculata

Eleven previously unknown compounds and 23 known compounds, including 20 phenanthrene or 9,10-dihydrophenanthrene derivatives, five bibenzyls, seven malate or tartrate benzyl ester glucosides, adenosine and gastrodin were isolated from tubers of Cremastra appendiculata. Among the obtained compounds, two are the first isolated dimers with one phenanthrene or bibenzyl unit connected to C-3 of 2,3,4,5-tetrahydro-phenanthro[2,1-b]furan moiety. In addition, 33 of these compounds were evaluated in vitro for their cytotoxic activity against two cancer cell lines. Among the compounds examined, one compound showed moderate cytotoxic activity, while five showed weak cytotoxic activity against the A549 cell line.

A reproducible analytical system based on the multi-component analysis of triterpene acids in Ganoderma lucidum

Ultra-performance liquid chromatography (UPLC) and Single Standard for Determination of Multi-Components (SSDMC) are becoming increasingly important for quality control of medicinal herbs; this approach was developed for Ganoderma lucidum. Special attention was necessary for the appropriate selection of markers, for determining the reproducibility of the relative retention times (RRT), and for the accuracy of conversion factors (F). Finally, ten components were determined, with ganoderic acid A serving as single standard. Stable system parameters were established, and with successful resolution of those issues, this analytical method could be used more broadly.

Anti-inflammatory Diterpenoids from the Root Bark of Acanthopanax gracilistylus

Five new ent-pimarane (1-3, 7, and 8) and three new ent-kaurane diterpenoids (4-6) and a new oleanane triterpene acid (9), together with 22 known compounds, were isolated from the root bark of the medicinal herb Acanthopanax gracilistylus. The structures of 1-9 were established based on the interpretation of high-resolution MS and 1D- and 2D-NMR data. The absolute configurations of 7 and 11 were determined by single-crystal X-ray diffraction and electronic circular dichroism analysis. Compounds 7 and 8 represent rare naturally occurring structures based on the devinyl ent-pimarane skeleton. Compounds 3, 10, 14, 16, and 17 exhibited potent inhibitory effects on the release of interleukin-1β (IL-1β), interleukin-8 (IL-8), and tumor necrosis factor (TNF-α) in lipopolysaccharide-stimulated peripheral blood mononuclear cells.

Identification of Metabolites of Ganoderic Acid D by Ultra-Performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry

Ganoderic acid D (GD) is the major active triterpenoid in Ganoderma lucidum, a medicinal fungus used daily. However, the metabolic fate of GD remains unknown. To know whether GD is extensively metabolized, we first investigated the metabolism of GD in vitro and in vivo. The metabolic profiles of the bile samples obtained from rats in vivo were almost the same as those obtained in vitro. Using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry, a total of 25 metabolites were identified from the bile sample. Few metabolites were found in the urine samples. These results indicated that biliary rather than renal clearance was the major route of excretion. The major metabolites were identified by comparison with the standard reference compounds. Metabolites at low concentrations were identified by interpreting the mass spectra. Both phase I and phase II metabolites were observed. The metabolic transformation included reduction, monohydroxylation, dihydroxylation, trihydroxylation, oxidation, desaturation, sulfation, and glucuronidation. The main metabolic soft spots in the chemical structure of GD were the 3-carbonyl group, angular methyl groups, the 7-hydroxy group, and the 26-carboxylic acid moiety. Overall, this study gives us an insight into the metabolism of GD, an active oxygenated tetracyclic triterpenoid.

Simultaneous Quantification of 18 Bioactive Constituents in Tripterygium wilfordii Using Liquid Chromatography-Electrospray Ionization-Mass Spectrometry

A liquid chromatography-electrospray ionization-mass spectrometry method with multiple reaction monitoring was established for simultaneous quantification of 18 bioactive constituents from the stem and root of Tripterygium wilfordii Hook. f. collected from different places in China and various commercial preparations. The chromatographic separations were achieved on an Agilent Poroshell SB-C₁₈ column (150 × 4.6 mm, 3.5 µm) with gradient elution using acetonitrile and 0.03 % formic acid aqueous solution in 45 min. Detection was performed in the positive ionization mode by monitoring the precursor-product combination. The validation of the method included tests of linearity, sensitivity, precision, repeatability, stability, and accuracy. All calibration curves showed good linearity (r > 0.9990) within the test range. The established method showed good precision and accuracy with intraday and interday variations of 2-5 % and 1-4 %, respectively, and recoveries of 95.5-104.5 %.

Pharmacokinetics of ganoderic acid D and its main metabolite by liquid chromatography-tandem mass spectrometry.

The present study aims to investigate the pharmacokinetics of ganoderic acid D (GD), a representative active triterpenoid from Ganoderma lucidum. A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of the concentrations of GD and its main metabolite (ganoderic acid B) in rat plasma. Following protein precipitation, the analytes were separated on a reversed-phase C18 column. Acetonitrile-water-acetic acid (40:60:0.01) was used at a flow-rate of 0.2ml/min. A triple quadrupole mass spectrometer equipped with an electrospray ionization source was used as the detector and was operated in the negative ion mode. Multiple reaction monitoring using the characteristic transitions was performed to quantify the analytes. The method had a lower limit of quantification of 8.19ng/ml for GD, and 8.59ng/ml for ganoderic acid B (GB). The calibration curves were demonstrated to be linear over the concentration range of 8.19-4096ng/ml and 8.59-2149ng/ml, respectively. Variations within- and between-batch were less than 6.4% and 4.6%, respectively. The extraction recovery rates ranged from 98.8 to 105.2% and 100.7 to 113.6%, respectively. The validated method was successfully applied to the quantification of GD and GB concentrations in rat plasma after oral administration (or intravenous administration) of GD preparations at a dose of 15mg/kg. The data showed that the absolute bioavailability increased from 22% to 70% after the GD suspension was changed to GD loaded solid lipid nanoparticles. In the meantime, the Cmax increased from 107.2 to 1555.6ng/ml; the tmax changed from 2.0h to 0.3h. These results are very helpful in the further studies.