Three new alkaloids isolated from the stem tuber of Pinellia pedatisecta.

The present study was designed to determine the chemical constituents of the stem tuber of Pinellia pedatisecta. The chemical constituents were isolated and purified by various chromatographic techniques, and their structures were elucidated on the basis of physicochemical properties and spectral data. Three new alkaloids (compounds 1, 2, and 3) were obtained and identified as 9-((5-methoxypyridin-2-yl)methyl)-9H-purin-6-amine (1), 4-(2-(2, 5-dioxopyrrolidin-1-yl)ethyl)phenyl acetate (2), and N-(9-((5-methoxypyridin-2-yl)methyl)-9H-purin-6-yl)acetamide (3). These compounds were evaluated for their cytotoxicity against human cervical cancer HeLa cells. Compounds 1 and 3 significantly inhibited the proliferation of HeLa cells with IC50 values being 3.02 ± 0.54 and 7.16 ± 0.62 µmol·L-1, respectively.

Pharmacokinetic Studies of Ganoderic Acids from the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), by LC-MS/MS.

Ganoderma lucidum is a famous medicinal mushroom that has been widely used in clinical practice and as a dietary supplementa. The triterpenoid ganoderic acids are the main constituents of G. lucidum. To determine the pharmacokinetic characteristics of ganoderic acids, we developed and validated a sensitive and selective liquid chromatography-tandem mass spectrometry method to determine simultaneously the concentration of 4 representative ganoderic acids in rat plasma after oral administration of the extract from G. lucidum. Because of the similarity of their chemical structures, the 4 components exhibited similar pharmacokinetic behaviors in some aspects. However, some of the pharmacokinetic parameters and the reabsorption peaks in the plasma concentration-time curves of ganoderic acids B and E after oral administration of the extract were different from those of ganoderic acids D and A because of the metabolic transformation among the ganoderic acids. These results increase our knowledge about the use of G. lucidum.

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-1ß (IL-1ß), interleukin-8 (IL-8), and tumor necrosis factor (TNF-α) in lipopolysaccharide-stimulated peripheral blood mononuclear cells.

Activity-guided isolation of NF-κB inhibitors and PPARγ agonists from the root bark of Lycium chinense Miller.

ETHNOPHARMACOLOGICAL RELEVANCE: The root bark of Lycium chinense Miller, Lycii radicis cortex, has been used in traditional Chinese medicine (TCM) to treat different inflammation-related symptoms, such as diabetes mellitus. The pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) is a key regulator of inflammation, while the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) is a key modulator of genes involved in diabetes development. To identify putative active compound(s) from Lycii radicis cortex inhibiting NF-κB or activating PPARγ. MATERIAL AND METHODS: Using activity-guided fractionation, six extracts with different polarity, isolated fractions, and purified compounds from Lycii radicis cortex were tested for NF-κB inhibition and PPARγ activation in vitro. The structure of the purified compounds was elucidated by NMR and MS techniques. RESULTS: The ethyl acetate extract and the methanol extract of Lycii radicis cortex suppressed tumor necrosis factor alpha (TNF-α)-induced activation of NF-κB, while the dichloromethane extract activated PPARγ. Nine phenolic amide analogues, including trans-N-(p-coumaroyl)tyramine (1), trans-N-feruloyltyramine (2), trans-N-caffeoyltyramine (3), dihydro-N-caffeoyltyramine (4), three neolignanamides (5-7), and two lignanamide (8, 9), were isolated and their inhibitory potential on NF-κB was determined (1-4 were also contained in water decoction). Two of the nine isolated phenolic amides inhibited TNF-α-induced NF-κB activation. Trans-N-caffeoyltyramine was verified as the key component responsible for the NF-κB inhibition with an IC50 of 18.4µM in our cell-based test system. Activation of PPARγ was attributed to a palmitic-acid enriched fraction which displayed concentration-dependent effect ablated upon co-treatment with the PPARγ antagonist T0070907. CONCLUSIONS: Phenolic amides were confirmed as main components from Lycii radicis cortex responsible for NF-κB inhibition. Fatty acids were identified as the major plant constituent responsible for the PPARγ activation. Structure-activity relationship analysis suggests that the NF-κB inhibitory activity of trans-N-caffeoyltyramine may be attributed to its Michael acceptor-type structure (α,ß-unsaturated carbonyl group). The data of this study contribute to a better understanding of the molecular mechanism of action of Lycii radicis cortex extracts in the context of inflammation.

Metabolite identification of crude extract from Ganoderma lucidum in rats using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

The metabolism of traditional Chinese medicine is very complicated and has been a great challenge. In the present paper, a new strategy was established to study the metabolism of crude extract from Ganoderma lucidum using the highly separative and sensitive ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. Based on the investigation of the metabolism of five representative single compounds (ganoderic acid), a total of 90 metabolites were identified from the bile sample after oral administration of the crude extract. Among them, 21 compounds were identified by comparison with the reference standards, the other unknown metabolites were tentatively characterized by interpretation of the high resolution low collision energy and high collision energy mass spectra using the fragmentation rules. The metabolic characteristics and "soft spots" of the ganoderic acids were revealed. After being absorbed, the ganoderic acids from the extract could undergo extensive phases I and II metabolism in rat before excreted into the bile. The main ganoderic acids could transform from one to another through reduction, oxidation, deacetylation and desaturation reactions. Other metabolic transformation included hydroxylation, sulfation and glucuronidation. The total tendency was that the low polar ganoderic acids were transformed into the high polar metabolites to eliminate from the organism. The metabolic "soft spots" of the ganoderic acids were 3,7,15,23-carbonyl groups (or hydroxyl groups), angular methyl groups, 20(22)-double bond, 12-acetoxyl group and 26-carboxylic acid moiety. These results are considered to be important for the further investigation of G. lucidum.

Elution-extrusion counter-current chromatography separation of two new benzyl ester glucosides and three other high-polarity compounds from the tubers of Pleione bulbocodioides.

INTRODUCTION: The tubers of Pleione bulbocodioides (Franch.) Rolfe, with gastrodin and benzyl ester glucosides as main components, have been used in traditional Chinese medicine for the treatment of various cancers and bacterial infections. Up to now, their official quality control method is still inadequate, and the difficulty of obtaining these high-polarity compounds is one of the major reasons. OBJECTIVE: To develop a rapid and efficient method for preparative separation of the high-polarity compounds gastrodin and benzyl ester glucosides. METHODS: An optimised solvent system composed of n-butanol:ethanol:water (20:1:20, v/v/v) was applied for the elution-extrusion counter-current chromatography (EECCC) separation. The upper phase was used as the stationary phase, and the lower phase was used as the mobile phase at a flow rate of 1.5 mL/min, a rotation speed of 850 rpm and a temperature of 35°C. RESULTS: Five high-polarity glucosides, including two new compounds, (E)-4-ß-D-glucopyranosyloxycinnamic acid 9-(4-ß-D-glucopyranosyloxybenzyl) ester (4 mg) and (Z)-2-(2-methylpropyl)butenedioic acid bis(4-ß-D-glucopyranosyloxybenzyl) ester (9 mg), and three main components, gastrodin (87 mg), dactylorhin A (60 mg) and militarine (15 mg), with HPLC purities of 95.4%, 96.4%, 91.1%, 97.2% and 95.5% respectively, were yielded from 400 mg of the prepared sample. CONCLUSION: Elution-extrusion counter-current chromatography could be used as a useful tool for the separation of high-polarity compounds such as gastrodin and benzyl ester glucosides and the enrichment of the minor ones.

Neolignanamides, lignanamides, and other phenolic compounds from the root bark of Lycium chinense.

Seven new neolignanamides (1-7), including two pairs of cis- and trans-isomers, and a new lignanamide (8) were isolated from the EtOAc-soluble fraction of an EtOH extract of the root bark of Lycium chinense, together with 22 known phenolic compounds (9-30), four of which were obtained from the genus Lycium for the first time. Compounds 5, 6, and 7 are unusual dimers having a rare connection mode between the two cinnamic acid amide units, while compounds 6, 7, and 8 are the first naturally occurring dimers derived from two dissimilar cinnamic acid amides. The cinnamic acid amides, neolignanamides, and lignanamides possess moderate radical-scavenging activity against the DPPH (2,2-diphenyl-1-picrylhydrazyl) and superoxide radicals.

Biotransformation of gambogenic acid by Chaetomium globosum CICC 2445.

Microbial transformation of gambogenic acid (1), a caged polyprenylated xanthone isolated from the resin of Garcinia hanburyi, was carried out with Chaetomium globosum CICC 2445, after screening forty-six strains of filamentous fungi. A new caged polyprenylated xanthone, 16,17-dihydroxygambogenic acid (2), was specifically obtained, as a result of hydroxylation at C-16, and C-17. Its structure was elucidated on the basis of spectroscopic methods. The cytotoxicity of compounds 1 and 2 against HeLa tumor cell line was evaluated, with both of them being modestly active.

Simultaneous determination of atractylenolide II and atractylenolide III by liquid chromatography-tandem mass spectrometry in rat plasma and its application in a pharmacokinetic study after oral administration of Atractylodes Macrocephala Rhizoma extract.

Atractylenolide II (AII) and atractylenolide III (AIII) are the major active components in Atractylodes Macrocephala Rhizoma (AMR). In this study, a sensitive, rapid and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of AII and AIII in rat plasma using loliolide as internal standard (IS). After protein precipitation with ethyl acetate, the analytes were injected into an LC-MS/MS system for quantification. Chromatography was performed using a C(18) column, eluting with water and acetonitrile (45:55, v/v) at 0.2 mL/min. All analytes including IS were monitored under positive ionization conditions by multiple reaction monitoring with an electrospray ionization source. The validated method was successfully applied to the pharmacokinetic study of AII and AIII in rat plasma after oral administration of AMR extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine.

Chemical constituents of Spatholobus suberectus.

AIM: To investigate chemical constituents of Spatholobus suberectus Dunn. METHODS: Isolation and purification were carried out by column chromatographic methods. Compounds were characterized based on their physical characteristics and spectra data. RESULTS: Seventeen compounds were isolated from ethanol extract of S. suberectus. The structures were elucidated as prestegane B (1), (2R, 3R)-buteaspermanol (2), (+)-medioresinol (3), (2R, 3R)-3,7-dihydroxyflavanone (4), benzeneethanol (5), 4, 7, 2'-trihydroxy-4'-methoxyisoflavanol (6), naringenin (7), blumenol A (8), protocatechuic acid ethyl ester (9), liquiritigenin (10), 7, 4'-dihydroxy-8-methoxy-isoflavone (11), 3, 5, 7, 3', 5'-pentahydroxyflavanone (12), protocatechuic acid (13), glycyroside (14), 8-methylretusin-7-O-ß-D-glucopyranoside (15), 3, 3', 4', 5, 6, 7, 8-heptahydroxyflavan (16), and dulcisflavan (17). CONCLUSION: All compounds are firstly isolated from the title plant and compounds 1, 3 were isolated from the Spatholobus genus for the first time.

Macromolecular and small-molecule modulation of intracellular Aß42 aggregation and associated toxicity.

Aß (amyloid ß-peptide) has a central role in AD (Alzheimer's disease) where neuronal toxicity is linked to its extracellular and intracellular accumulation as oligomeric species. Searching for molecules that attenuate Aß aggregation could uncover novel therapies for AD, but most studies in mammalian cells have inferred aggregation indirectly by assessing levels of secreted Aß peptide. In the present study we establish a mammalian cell system for the direct visualization of Aß formation by expression of an Aß(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 Aß(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 Aß. Both PS5 and ganoderic acid DM probably promote Aß aggregate clearance indirectly through the proteasome. The model is therefore of value to study the effects of intracellular Aß on cell physiology and to identify reagents that counteract those effects.

Proteomic studies on protective effects of salvianolic acids, notoginsengnosides and combination of salvianolic acids and notoginsengnosides against cardiac ischemic-reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza and Panax notoginseng are popularly used traditional Chinese medicine for cardiovascular disorders and they are often used in the form of combination. However, mechanisms of their cardioprotective effects were still not clear. In the present study, the protective effects of salvianolic acids (SA), notoginsengnosides (NG) and combination of SA and NG (CSN) against rat cardiac ischemia-reperfusion injury were checked and the protein expression profiles of heart tissues were examined to search their possible protein targets. MATERIALS AND METHODS: The cardioprotective effects of SA, NG and CSN were checked in a rat model of ischemia-reperfusion (IR) by temporarily occluding coronary artery for 20 min followed by reperfusion. Rats were grouped into sham-operation group, IR group, IR+SA group, IR+NG group and IR+CSN group. The plasma creatine kinase (CK) activities were measured using commercial kit and the percentages of infarcted area in total ventricle tissue were calculated after nitroblue-tetrazolium (N-BT) staining of heart tissue slices. Two-dimensional protein electrophoresis (2-DE) was used to check the protein expression profiles of heart tissues. Then, proteins differentially expressed between IR group and sham-operation group were identified using matrix assisted laser desorption ionization-time of flight-mass spectrometry/mass spectrometry (MALDI-TOF MS/MS). The regulative effects of SA, NG and CSN on these IR-related proteins were analyzed. RESULTS: Treatments including SA, NG and CSN all showed cardioprotective effects against ischemia-reperfusion injury and CSN exhibited to be the best. Eighteen proteins involved in IR injury were found. These proteins are involved in pathways including energy metabolism, lipid metabolism, muscle contraction, heat shock stress, cell survival and proliferation. The regulation of these proteins by SA, NG or CSN suggested possible protein targets in their cardioprotective effects. CONCLUSIONS: SA and NG showed both similarity and difference in their protein targets involved in cardioprotective effects. The capability of CSN to regulate both protein targets of SA and NG might be the basis of CSN to show cardioprotective effects better than that of SA or NG.

Simultaneous determination of four sesquiterpenoids in Atractylodes Macrocephala Rhizoma by GC-FID: optimisation of an ultrasound-assisted extraction by central composite design.

INTRODUCTION: Atractylodes Macrocephala Rhizoma (AMR) is a traditional Chinese medicine containing several sesquiterpenoids with a series of effects. These bioactive compounds may be used as chemical markers for the quality control of AMR. It is necessary to optimise the extraction method and conditions in order to improve extraction productivity. OBJECTIVE: To develop a simple and effective method for the extraction of sesquiterpenoids from AMR and then to simultaneously determine four sesquiterpenoids, selina-4 (14), 7(11)-dien-8-one (SA), atractylenolide II (AII), atractylenolide III (AIII) and atractylenolide VII (AVII), in AMR. METHODOLOGY: Ultrasound-assisted extraction (UAE) was optimised by central composite design (CCD) to obtain the maximum efficiency. The gas chromatography method was validated and applied for the quantification of four sesquiterpenoids. RESULTS: The optimum values of factors were: particle size (120 mesh), extraction time (26 min), extraction temperature (39°C) and 31 mL of chloroform. The selectivity, linear range, limits of detection (LOD) and quantification (LOQ), accuracy, precision and repeatability of the method developed indicated its validity. The application of the method showed that the contents of four sesquiterpenoids in AMR were rather variable. CONCLUSION: The results indicated that the described GC method could be used for the quality control of AMR and its related preparations. Meanwhile, this research revealed that UAE under optimum conditions could be considered as a powerful tool for the extraction of phytochemicals from plants.

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

The farnesoid X receptor (FXR) belonging to the metabolic subfamily of nuclear receptors is a ligand-induced transcriptional activator. Its central function is the physiological maintenance of bile acid homeostasis including the regulation of glucose and lipid metabolism. Accessible structural information about its ligand-binding domain renders FXR an attractive target for in silico approaches. Integrated to natural product research these computational tools assist to find novel bioactive compounds showing beneficial effects in prevention and treatment of, for example, the metabolic syndrome, dyslipidemia, atherosclerosis, and type 2 diabetes. Virtual screening experiments of our in-house Chinese Herbal Medicine database with structure-based pharmacophore models, previously generated and validated, revealed mainly lanostane-type triterpenes of the TCM fungus Ganoderma lucidum Karst. as putative FXR ligands. To verify the prediction of the in silico approach, two Ganoderma fruit body extracts and compounds isolated thereof were pharmacologically investigated. Pronounced FXR-inducing effects were observed for the extracts at a concentration of 100 µg/mL. Intriguingly, five lanostanes out of 25 secondary metabolites from G. lucidum, that is, ergosterol peroxide (2), lucidumol A (11), ganoderic acid TR (12), ganodermanontriol (13), and ganoderiol F (14), dose-dependently induced FXR in the low micromolar range in a reporter gene assay. To rationalize the binding interactions, additional pharmacophore profiling and molecular docking studies were performed, which allowed establishing a first structure-activity relationship of the investigated triterpenes.

Fragmentation pathways of oxygenated tetracyclic triterpenoids and their application in the qualitative analysis of Ganoderma lucidum by multistage tandem mass spectrometry.

The fragmentation pathways of oxygenated tetracyclic triterpenoids from Ganoderma lucidum were systematically studied based on interpreting the mass spectra of 44 known triterpenoids using a combination of multistage tandem mass spectrometry (MS(n)) experiments and high-resolution mass spectrometry (HRMS) analysis. In negative ion mode, the fragmentation pathways of triterpenoid acids are rather characteristic. After the prominent loss of H(2) O or CO(2), cleavages take place on the A, B, C and D rings. Interestingly, the cleavage mode is highly dependent on the positions of the carbonyl groups and hydroxyl groups in the tetracyclic skeleton. Characteristic cleavage of ring A occurs in 7-oxo-11-H or 7-oxo-11-hydroxy derivatives; characteristic cleavage of ring B occurs in the 7-oxo-11-hydroxy derivatives; characteristic cleavage of ring C occurs in the 7-hydroxy-15-oxo derivatives; while the cleavage of ring D can be observed in the majority of the compounds investigated. The odd-electron species, which disobey the 'even-electron rule', are also observed and discussed in this paper. These phenomena provide an easy way to determine the tetracyclic skeleton and distinguish the isomers of the triterpenoids from each other. What is more, the fragmentation pathways of triterpenoid alcohols were also investigated in positive ion mode. The accurate masses of the product ions were determined using quadrupole orthogonal time-of-flight (QTOF) instruments. Finally, the fragmentation rules were applied to identify the components of G. lucidum. As a result, 73 triterpenoids including 11 new ones were identified. The triterpenoids were classified into six subclasses according to their different fragmentation behaviors. The application of tandem mass spectrometry was further explored.

Differential proteomic analysis of platelets suggested possible signal cascades network in platelets treated with salvianolic acid B.

BACKGROUND: Salvianolic acid B (SB) is an active component isolated from Danshen, a traditional Chinese medicine widely used for the treatment of cardiovascular disorders. Previous study suggested that SB might inhibit adhesion as well as aggregation of platelets by a mechanism involving the integrin α2ß1. But, the signal cascades in platelets after SB binding are still not clear. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, a differential proteomic analysis (two-dimensional electrophoresis) was conducted to check the protein expression profiles of rat platelets with or without treatment of SB. Proteins altered in level after SB exposure were identified by MALDI-TOF MS/MS. Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. The regulation of SB on protein levels was confirmed by Western blotting. The signal cascades network induced by SB after its binding with integrin α2ß1 was predicted. To certify the predicted network, binding affinity of SB to integrin α2ß1 was checked in vitro and ex vivo in platelets. Furthermore, the effects of SB on protein levels of hsp70, coronin-1B and intracellular levels of Ca²+ and reactive oxygen species (ROS) were checked with or without pre-treatment of platelets using antibody against integrin α2ß1. Electron microscopy study confirmed that SB affected cytoskeleton structure of platelets. CONCLUSIONS/SIGNIFICANCE: Integrin α2ß1 might be one of the direct target proteins of SB in platelets. The signal cascades network of SB after binding with integrin α2ß1 might include regulation of intracellular Ca²+ level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets.

Cytotoxicity of 9,11-dehydroergosterol peroxide isolated from Ganoderma lucidum and its target-related proteins.

The cytotoxicty of 9,11-dehydroergosterol peroxide (DHEP) isolated from the fruiting bodies of Ganoderma lucidum on HeLa cells was studied. DHEP treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with an IC50-value of 8.58 +/- 0.98 microM. Morphological changes of DHEP-treated cells indicated that DHEP induced apoptosis in HeLa cells. To identify the cellular targets of DHEP, two-dimensional electrophoresis analysis was performed to compare the protein expression profiles of DHEP-treated cells with that of control cells. Proteins altered in expressional level after DHEP exposure were identified by MALDI-TOF MS/MS. The cytotoxic effect of DHEP was associated with regulated expression of 6 proteins. Stathmin 1 might be an important target-related protein of DHEP. The regulation of stathmin 1 by DHEP treatment was also confirmed by Western blotting.

Benzyl-beta-resorcylates from Cassia obtusifolia.

The seed of Cassia obtusifolia Linn have yielded three new compounds, 2-benzyl-4,6-dihydroxy benzoic acid (1), 2-benzyl-4,6-dihydroxy benzoic acid-6-O-beta-D-glucopyranoside (2) and 2-benzyl-4,6-dihydroxy benzoic acid-4-O-beta-D-glucopyranoside (3). Their structures were determined by spectroscopic methods, including (1)D- and (2)D NMR spectroscopy, HR-ESI-MS, as well as by comparison of their spectral data with those of related compounds.

Cytotoxic constituents of chinese propolis.

A pair of new flavanol racemates (1a and 1b) and a new flavanol racemic mixture (2) were isolated from crude propolis from Henan Province, People's Republic of China. Also obtained were nine known compounds, including two flavones, four flavonols, two flavanols, and isoferulic acid. Spectroscopic analysis was employed to assign the structures of these new compounds and the absolute configurations of 1a and 1b. Cytotoxicity of the isolated compounds against the HeLa human cervical carcinoma cancer cell line was evaluated, with only compounds 1a, 1b, 2, and rhamnetin (3) being active.

Cytotoxic polyprenylated xanthones from the resin of Garcinia hanburyi.

Twelve new xanthones (1-12), a pair of new natural products (13 and 14), and 18 known related compounds were isolated from the resin of Garcinia hanburyi. The structures of 1-14 were elucidated by detailed spectroscopic analyses. A cytotoxic assay of the isolated compounds revealed that, with the exception of 2, these compounds were active against the HeLa tumor cell line.