Investigating the anti-atherosclerotic effects and potential mechanism of Dalbergia odorifera in ApoE-deficient mice using network pharmacology combined with metabolomics.

Dalbergia odorifera (DO) is a precious rosewood species in Southern Asia, and its heartwood is used in China as an official plant for invigorating blood circulation and eliminating stasis. This study aims to evaluate the efficacy of DO on atherosclerosis (AS), and further explore its active components and potential mechanisms. The apolipoprotein-E (ApoE)-deficient mice fed a high-fat diet were used as model animals, and the pathological changes in mice with or without DO treatment were compared to evaluate the pharmacodynamics of DO on AS. The mechanisms were preliminarily expounded by combining with metabolomics and network pharmacology. Moreover, the bioactive components and targets were assessed by cell experiments and molecular docking, respectively. Our findings suggested that DO significantly modulated blood lipid levels and alleviated intimal hyperplasia in atherosclerotic-lesioned mice, and the mechanisms may involve the regulation of 18 metabolites that changed during the progression of AS, thus affecting 3 major metabolic pathways and 3 major signaling pathways. Moreover, the interactions between 16 compounds with anti-proliferative effect and hub targets in the 3 signaling pathways were verified using molecular docking. Collectively, our findings preliminarily support the therapeutic effect of DO in atherosclerosis, meanwhile explore the active constituents and potential pharmacological mechanisms, which is conducive to its reasonable exploitation and utilization.

Metabolomics and lipidomics combined with serum pharmacochemistry uncover the potential mechanism of Huang-Lian-Jie-Du decoction alleviates atherosclerosis in ApoE-/- mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Atherosclerosis (AS) is one of the main cardiovascular diseases (CVDs) leading to an increase in global mortality, and its key pathological features are lipid accumulation and oxidative stress. Huang-Lian-Jie-Du decoction (HLJDD), a representative formula for clearing heat and detoxifying, has been shown to reduce aortic lipid plaque and improve AS. However, multiple components and multiple targets of HLJDD pose a challenge in comprehending its comprehensive mechanism in the treatment of AS. AIM OF THE STUDY: This study was designed to illustrate the anti-AS mechanisms of HLJDD in an apolipoprotein E-deficient (ApoE-/-) mouse model from a metabolic perspective. MATERIALS AND METHODS: ApoE-/- mice were kept on a high-fat diet (HFD) to induce AS. Serum total cholesterol (TC), total triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were determined to evaluate the influence of HLJDD on dyslipidemia. Oil red O was used to stain mouse aortic lipid plaques, and hematoxylin and eosin (HE) staining was used to assess the pathological changes in the aortic roots. Metabolomics and lipidomics combined with serum pharmacochemistry were performed to research the HLJDD mechanism of alleviating AS. RESULTS: In this study, HLJDD treatment improved serum biochemical levels and histopathological conditions in AS mice. A total of 6 metabolic pathways (arginine biosynthesis, glycerophospholipid, sphingolipid, arachidonic acid, linoleic acid, and glycerolipid metabolism) related to 25 metabolic biomarkers and 41 lipid biomarkers were clarified, and 22 prototype components migrating to blood were identified after oral administration of HLJDD. CONCLUSION: HLJDD improved AS induced by HFD in ApoE-/- mice. The effects of HLJDD were mainly attributed to regulating lipid metabolism by regulating the metabolic pathways of glycerophospholipids, sphingolipids, arachidonic acid, linoleic acid, and glycerolipids and reducing the levels of oxidative stress by upregulating arginine biosynthesis.

Study and exploration of the pharmacokinetics of traditional Tibetan medicine Ruyi Zhenbao tablets after single and long-term administration.

Tibetan medicine is one of the oldest traditional medicine systems in the world. Taking the Ruyi Zhenbao tablet (RYZB) as an example, which is a widely used classic oral Tibetan medicine, this article discusses the pharmacokinetics of single administration and long-term treatment and analyzed its metabolic properties and tissue distribution in vivo. After single administration, blood samples were collected before administration and at different time points after administration in different groups of rats. In the study of long-term treatment effects, blood samples were collected from the animals in each group on days 1, 15, and 30 and on day 15 after withdrawal. The results showed that after a single administration, the dose change had no significant effect on the T1/2 and Tmax of agarotetrol, isoliquiritigenin, and piperine (p > 0.05). There was a certain correlation between the increase in AUC0-t and the Cmax of agarotetrol, isoliquiritigenin, piperine, and the increase in dosage, with a dose range of 0.225-0.900 g/kg. There were no significant differences in Cmax and AUC0-t of ferulic acid at different doses (p > 0.05). Meanwhile, there was no significant sex-based difference in the pharmacokinetic parameters of these four components in rats. After long-term administration, the distribution agarotetrol in various tissues of rats was kidney > liver > heart > brain; the tissue distribution in low- and medium-dose groups of isoliquiritigenin was liver > kidney > heart > brain, and in the high-dose group, kidney > liver > heart > brain. The tissue distribution of piperine in each dose group was liver > kidney > heart > brain, and that of ferulic acid in each dose group was kidney > liver > heart > brain. Through the establishment of the previously developed methodology, the pharmacokinetic properties of RYZB were analyzed after a single administration and long-term administration. Our findings confirmed this approach for the exploration and establishment of a pharmacokinetic evaluation of Tibetan medicine, to support its guiding role in clinical application, but also to accelerate research into Tibetan medicine theory and medicine and to provide a solid foundation for the translation of Tibetan medicine throughout the world.

An integrated approach for identifying the efficacy and potential mechanisms of TCM against atherosclerosis-Wu-Zhu-Yu decoction as a case study.

ETHNOPHARMACOLOGICAL RELEVANCE: Atherosclerosis (AS) is a chronic disease that is associated with high morbidity. However, therapeutic approaches are limited. Wu-Zhu-Yu decoction (WZYD) is a well-known traditional Chinese medicine prescription that is traditionally used to treat headaches and vomiting. Modern studies have demonstrated the cardiotonic effects of WZYD. However, whether WZYD can alleviate AS and its underlying mechanisms remain unclear. AIM OF THE STUDY: This study aims to investigate the antiatherosclerotic efficacy of WZYD and illustrate its potential mechanisms using an integrated approach combining in vivo and in vitro assessments, including metabolomics, network pharmacology, cell experiments, and molecular docking analyses. MATERIALS AND METHODS: In this work, an atherosclerotic mouse model was established by administering a high-fat diet to apolipoprotein-E deficient (ApoE-/-) mice for twelve weeks. Meanwhile, the mice were intragastrically administered WZYD at different dosages. Efficacy evaluation was performed through biochemical and histopathological assessments. The potential active constituents, metabolites, and targets of WZYD in atherosclerosis were predicted by metabolomics combined with network pharmacology analysis, the constituents and targets were further assessed through cell experiments and molecular docking analysis. RESULTS: WZYD decreased the lipid levels in serum, reduced the areas of aortic lesions, and attenuated intimal thickening, which had antiatherosclerotic effects in ApoE-/- mice. Metabolomics and network pharmacology approach revealed that the ten constituents (6-shogaol, evodiamine, isorhamnetin, quercetin, beta-carotene, 8-gingerol, kaempferol, 6-paradol, 10-gingerol, and 6-gingerol) of WZYD affected 24 metabolites by acting on the candidate targets, thus resulting in changes in five metabolic pathways (sphingolipid metabolism; glycine, serine and threonine metabolism; arachidonic acid metabolism; tryptophan metabolism; and fatty acid biosynthesis pathway). Cell experiments indicated that the ten key compounds showed antiproliferative effects on the vascular smooth muscle cell. Moreover, the key compounds exhibited direct interactions with the key targets, as assessed by molecular docking analysis. CONCLUSION: This study revealed that WZYD exerted therapeutic effects on atherosclerosis, and the potential mechanisms were elucidated. Furthermore, it offered a powerful integrated strategy for studying the efficacy of traditional Chinese medicine and exploring its active components and possible mechanisms.

An enhanced analytical strategy integrating offline two-dimensional liquid chromatography with high-resolution accurate mass spectrometry and molecular networking: Comprehensive characterization of HuangLian JieDu Decoction as a case study.

Comprehensive ingredient research is of great significance for understanding the effective material basis of herbal medicines, but due to the diversity and complexity of their phytochemicals, such research is challenging. Here, a multifaceted strategy was proposed to analyze and identify the composition of HuangLian JieDu Decoction based on offline two-dimensional liquid chromatography combined with ultraviolet detection and high-resolution mass spectrometry. Multiple components were separated by two-dimensional liquid chromatography, which consisted of hydrophilic interaction chromatography and reversed-phase liquid chromatography, and then further characterized by high-resolution mass spectrometry with a full mass spectrometry/precursor ion list/data-dependent secondary scan data acquisition method. For data processing, database screening and molecular networking were used to identify the components in HuangLian JieDu Decoction. The offline two-dimensional liquid chromatography combined with ultraviolet detection and a high-resolution mass spectrometry system showed good orthogonality of 76.35% and a high peak capacity of 5175, effectively separating multiple components. Finally, 527 compounds, including 164 alkaloids, 133 terpenoids, 88 flavonoids, 60 phenylpropanoids, 38 organic acids, and 44 other compounds, were characterized. This integrated approach is suitable for the comprehensive characterization of herbal medicines and other complex chemical systems.

Tanshinone IIA-Loaded Micelles Functionalized with Rosmarinic Acid: A Novel Synergistic Anti-Inflammatory Strategy for Treatment of Atherosclerosis.

Rosmarinic acid (RA) and tanshinone IIA (TA) which are effective components in Salvia miltiorrhiza show anti-inflammatory potential against atherosclerosis. Based on polysulfated propylene-polyethylene glycol (PPS-PEG), RA was grafted onto this polymer via amide bonds to form a micelle carrier for TA encapsulation: PPS-PEG-RA@TA. A potent inhibitory effect on lipopolysaccharide (LPS) -induced proliferation of endothelial cells with significant intracellular uptake was observed with this system. This could have been the result of release of TA in a reactive oxygen species (ROS) environment and stronger antioxidant effect of RA. The synergistic effect was optimized when the combination was used in a molar ratio of 1:1. Mechanistic studies showed that, compared with PPS-PEG-RA and TA+RA, PPS-PEG-RA@TA micelles could more effectively regulate the nuclear factor-kappa B (NF-κB) pathway to reduce expression of vascular cell adhesion molecule-1 (VCAM-1), inhibit the inflammatory cascade and reduce endothelial-cell injury. One month after intravenous injection of PPS-PEG-RA@TA micelles, the plaque area in murine aortic vessels was reduced significantly, and serious toxic side-effects were not observed in vivo, along with excellent biocompatibility. In summary, PPS-PEG-RA@TA micelles could achieve synergistic treatment of atherosclerosis.

Quality markers based on phytochemical analysis and anti-inflammatory screening: An integrated strategy for the quality control of Dalbergia odorifera by UHPLC-Q-Orbitrap HRMS.

BACKGROUND: Quality control, key for the clinical application of traditional Chinese medicines (TCMs), should be connected to the authentication and efficacy of TCMs. The heartwood of Dalbergia odorifera has been widely used to treat inflammation-related diseases. However, in the Chinese pharmacopeia, only the total volatile oil, which does not sufficiently reflect the clinical efficacy, is used as a quality control indicator. PURPOSE: Establishing a "phytochemical-specificity-effectiveness-Q-marker" analytical strategy to improve the quality control of D. odorifera. METHODS: Combined with biosynthetic pathway analysis, phytochemical compositions identified by UHPLC-Q-Orbitrap HRMS were used to build substantial phytochemical groups and further discover specific Q-markers. Then, lipopolysaccharide-stimulated RAW 264.7 cells were used to screen effective anti-inflammatory ingredients. Finally, a UHPLC-HRMS method was developed and validated to quantify the selected Q-markers in D. odorifera samples. RESULTS: Along the constructed biosynthetic pathways, 93 phytochemical components were identified in D. odorifera, including 7 chalcones, 13 flavanones, 21 isoflavones, 21 isoflavanones, 3 flavonols, 19 neoflavones, etc. Among them, 31 compounds representing these 6 categories were further evaluated for their anti-inflammatory activities. It revealed that the extract of D. odorifera and nine flavonoids in the noncytotoxic range could alleviated lipopolysaccharide-stimulated inflammation in RAW 264.7 cells by decreasing the production of proinflammatory mediators such as nitric oxide and interleukin-6. Notably, neoflavones, as species-specific components, exhibited superior anti-inflammatory activities among the representative compounds. Finally, 12 Q-markers (butin, liquiritigenin, eriodictyol, melanettin, naringenin, butein, genistein, 4'-hydroxy-4-methoxydalbergione, isoliquiritigenin, 2,4-dihydroxy-5-methoxybenzophenone, medicarpin, and pinocembrin), which reflect specificity and effectiveness, were successfully quantified in 10 batches of samples from different origins. The origins and consistency of D. odorifera could be efficiently discriminated by hierarchical cluster analysis (HCA). CONCLUSION: The analysis strategy that combines phytochemical analysis with anti-inflammatory screening clarified the therapeutic material basis and discovered Q-markers, which possibly offers a more comprehensive quality assessment of D. odorifera.

Simultaneously qualitative and quantitative analysis of the representative components in Kadsura heteroclita stem by UHPLC-Q-Orbitrap HRMS.

Kadsura heteroclita (Roxb) Craib stem (KHS) is a medicinal plant used for the treatment of rheumatism arthritis diseases in Tujia ethnomedicine. Thus far, the complex chemical compositions in KHS are not clear, and the levels of the major compounds in KHS are not well understood. In this study, a novel UHPLC-Q-Orbitrap HRMS method was established for the simultaneous quali-quantitative analysis of KHS. A total of 204 compounds were identified, including triterpenoids, lignans, sesquiterpenes, fatty acids, phenolic acids, and flavonoids, more than 100 of which were first discovered in KHS. Using the same method, 12 representative bioactive components were successfully quantified. The method was fully validated by linearity, LOD, LOQ, precision, stability, recovery, and matrix effects, and it was applied to quantify the 12 representative compounds in 4 batches of KHS. As this method enables retrospective data analysis and has no upper limit to the number of analytes in a single run, it can be applied to quantify more active components of KHS in the future.

Identifying potential active components of walnut leaf that action diabetes mellitus through integration of UHPLC-Q-Orbitrap HRMS and network pharmacology analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Walnut leaf (WL) is a hypoglycemic herbal medication with blood glucose-lowering activity that can affect diabetes mellitus (DM). However, the active components of WL and the mechanisms by which these compounds affect DM are unclear. AIM OF STUDY: This study aimed to determine these effective ingredients and elucidate the potential mechanisms by which they affect DM via ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) coupled with network pharmacology analysis. MATERIALS AND METHODS: First, UHPLC-Q-Orbitrap HRMS was utilized to identify components of WL. Second, the putative targets of the components were identified and predicted based on chemical similarity and online databases. Third, the key candidate targets and potential active components were identified through topological analysis of a component-disease target interaction network. Finally, interactions between active components and therapeutic targets were confirmed by molecular docking analysis. RESULTS: One hundred and thirty components were identified in WL, among which 38 were considered potentially bioactive, as they showed hypoglycemic effects. Among these 38, 8 key active components possessed high similarities and shared 4 targets with approved drugs. These findings were confirmed by molecular docking analysis. CONCLUSION: The approach combining UHPLC-Q-Orbitrap HRMS with network pharmacology analysis is a rapid and effective tool to identify potentially bioactive constituents in medicinal plants and prescriptions.

Pharmacokinetics, bioavailability, excretion, and metabolic analysis of Schisanlactone E, a bioactive ingredient from Kadsura heteroclita (Roxb) Craib, in rats by UHPLC-MS/MS and UHPLC-Q-Orbitrap HRMS.

Schisanlactone E (SE) is a bioactive ingredient extracted from the stem of Kadsura heteroclita (Roxb) Craib. SE has various pharmacological activity such as anti-tumor and anti-leukemia effects. However, its absorption, distribution, metabolism, and excretion have rarely been examined. In this study, new quali-quantitative analytical methods were developed for metabolic and pharmacokinetic studies of SE in rats. A UHPLC-MS/MS method was developed to determine SE in rat plasma, urine, and feces. Samples were precipitated with methanol and analyzed in multiple reaction monitoring mode. The established method was validated and applied to the pharmacokinetics, bioavailability, and excretion analysis of SE after oral (6 mg/kg) or intravenous (2 mg/kg) administration. The absolute oral bioavailability of SE was approximately 79.3%. After oral administration, SE was mainly excreted via feces with a rate of 41.7% for 48 h. SE could not be detected in urine. Furthermore, a UHPLC-Q-Orbitrap HRMS method was developed for the metabolite screening of SE in rat plasma, urine, and feces. Metabolites were extracted by solid phase extraction and analyzed with full MS/dd-MS2 scan mode. As a result, 15 metabolites including 11 phase I and 4 phase II metabolites were identified by a three-step analytical strategy. The carboxyl group, the five membered ring, and the six membered α,ß-unsaturated lactone ring of SE could be predicted as the main metabolic sites. This study provides comprehensive insights into the pharmacokinetic and metabolic profiles of SE, and would be valuable for future development and utilization of SE and Kadsura heteroclita.

Identification and Quantification of Bioactive Compounds in Diaphragma juglandis Fructus by UHPLC-Q-Orbitrap HRMS and UHPLC-MS/MS.

Diaphragma juglandis fructus is the dry wooden diaphragm inside walnuts and a byproduct in food processing of walnut kernels. The purpose of our research is to enrich the information on compounds in Diaphragma juglandis fructus to further discover and exploit its potential nutritional value. In this study, new quali-quantitative analytical approaches were developed to identify and determine bioactive compounds in Diaphragma juglandis fructus. Two-hundred compounds, including hydrolyzable tannins, flavonoids, phenolic acids, and quinones, were identified by UHPLC-Q-Orbitrap HRMS, more than 150 of which were first discovered in Diaphragma juglandis fructus. Among them, 21 major dietary polyphenols with health-promoting effects were successfully quantified using UHPLC-MS/MS, with total contents of 2.88-6.18 mg/g. This successful characterization and quantification of bioactive compounds in Diaphragma juglandis fructus gives a better understanding of its potential nutritional value and supports efficiently developing and reusing it instead of discarding it as agrofood waste.

Constituents of Mediterranean Spices Counteracting Vascular Smooth Muscle Cell Proliferation: Identification and Characterization of Rosmarinic Acid Methyl Ester as a Novel Inhibitor.

SCOPE: Aberrant vascular smooth muscle cell (VSMC) proliferation is involved in atherosclerotic plaque formation and restenosis. Mediterranean spices have been reported to confer cardioprotection, but their direct influence on VSMCs has largely not been investigated. This study aims at examining rosmarinic acid (RA) and 11 related constituents for inhibition of VSMC proliferation in vitro, and at characterizing the most promising compound for their mode of action and influence on neointima formation in vivo. METHODS AND RESULTS: RA, rosmarinic acid methyl ester (RAME), and caffeic acid methyl ester inhibit VSMC proliferation in a resazurin conversion assay with IC50 s of 5.79, 3.12, and 6.78 µm, respectively. RAME significantly reduced neointima formation in vivo in a mouse femoral artery cuff model. Accordingly, RAME leads to an accumulation of VSMCs in the G0 /G1 cell-cycle phase, as indicated by blunted retinoblastoma protein phosphorylation upon mitogen stimulation and inhibition of cyclin-dependent kinase 2 in vitro. CONCLUSION: RAME represses PDGF-induced VSMC proliferation in vitro and reduces neointima formation in vivo. These results recommend RAME as an interesting compound with VSMC-inhibiting potential. Future metabolism and pharmacokinetics studies might help to further evaluate the potential relevance of RAME and other spice-derived polyphenolics for vasoprotection.

Piperine Congeners as Inhibitors of Vascular Smooth Muscle Cell Proliferation.

Successful vascular healing after percutaneous coronary interventions is related to the inhibition of abnormal vascular smooth muscle cell proliferation and efficient re-endothelialization. In the search for vascular smooth muscle cell anti-proliferative agents from natural sources we identified piperine (1), the main pungent constituent of the fruits from Piper nigrum (black pepper). Piperine inhibited vascular smooth muscle cell proliferation with an IC50 of 21.6 µM, as quantified by a resazurin conversion assay. Investigations of ten piperamides isolated from black pepper fruits and 15 synthesized piperine derivatives resulted in the identification of three potent vascular smooth muscle cell proliferation inhibitors: the natural alkaloid pipertipine (4), and the two synthetic derivatives (2E,4E)-N,N-dibutyl-5-(3,5-dimethoxyphenyl)penta-2,4-dienamide (14) and (E)-N,N-dibutyl-3-(naphtho[2,3-d][1,3]dioxol-5-yl)acrylamide (20). They showed IC50 values of 3.38, 6.00, and 7.85 µM, respectively. Furthermore, the synthetic compound (2E,4E)-5-(4-fluorophenyl)-1-(piperidin-1-yl)penta-2,4-dien-1-one (12) was found to be cell type selective, by inhibiting vascular smooth muscle cell proliferation with an IC50 of 11.8 µM without influencing the growth of human endothelial cells.

Identification and characterization of [6]-shogaol from ginger as inhibitor of vascular smooth muscle cell proliferation.

SCOPE: Vascular smooth muscle cell (VSMC) proliferation is involved in the pathogenesis of cardiovascular disease, making the identification of new counteracting agents and their mechanisms of action relevant. Ginger and its constituents have been reported to improve cardiovascular health, but no studies exist addressing a potential interference with VSMC proliferation. METHODS AND RESULTS: The dichloromethane extract of ginger inhibited VSMC proliferation when monitored by resazurin metabolic conversion (IC50 = 2.5 µg/mL). The examination of major constituents from ginger yielded [6]-shogaol as the most active compound (IC50 = 2.7 µM). In the tested concentration range [6]-shogaol did not exhibit cytotoxicity toward VSMC and did not interfere with endothelial cell proliferation. [6]-shogaol inhibited DNA synthesis and induced accumulation of the VSMC in the G0 /G1 cell-cycle phase accompanied with activation of the nuclear factor-erythroid 2-related factor 2 (Nrf2)/HO-1 pathway. Since [6]-shogaol lost its antiproliferative activity in the presence of the heme oxygenase-1 (HO-1) inhibitor tin protoporphyrin IX, HO-1 induction appears to contribute to the antiproliferative effect. CONCLUSION: This study demonstrates for the first time inhibitory potential of ginger constituents on VSMC proliferation. The presented data suggest that [6]-shogaol exerts its antiproliferative effect through accumulation of cells in the G0 /G1 cell-cycle phase associated with activation of the Nrf2/HO-1 pathway.

Porous silicon optical microcavity biosensor on silicon-on-insulator wafer for sensitive DNA detection.

Silicon-on-insulator (SOI) wafer is one of the most appealing platforms for optical integrated circuit with the potential to realize high performance Ultra Large Scale Integration (ULSI) and device miniaturization. In this work, based on simulations to obtain appropriate optical properties of a porous silicon microcavity (PSM), we successfully fabricated a highly efficient PSM on SOI wafer by electrochemical etching for DNA detection at optical wavelength 1555.0 nm. The narrow resonance peak with a full width at half maximum about 26.0 nm in the reflectance spectrum gives a high Q factor which causes high sensitivity for sensing performance. The sensitivity of this sensor is investigated through 19-base pair DNA hybridization in the PSM by surface modification using a standard cross link chemistry method. The red shift of the reflectance spectra shows a good linear relationship with complementary DNA concentration, ranging from 0.625 to 12.500 µM, and the detection limit is 43.9 nM. This optical PSM on SOI is highly sensitive, fast responsive, easy to fabricate and low-costly, that will broadly benefit to develop a new optical label-free biosensor on SOI wafer and has a great potential for biochips based on integrated optical devices.

Structural determination of seven new triterpenoids from Kadsura heteroclita by NMR techniques.

Seven new triterpenoids, namely heteroclitalactones G-M (1-7), were isolated from the ethanol extract of the stems of Kadsura heteroclita. Structures of these compounds were characterized by extensive 1D and 2D NMR spectroscopic analyses.

HPLC method for the determination and pharmacokinetic studies of four triterpenoids in rat plasma after oral administration of Ganoderma lucidum extract.

Four major triterpenoids (ganoderic acids C(2), B, K and H) in rat plasma after oral administration of G. lucidum extract were analyzed quantitatively by high-performance liquid chromatography (HPLC). Plasma samples taken from rats were acidified with hydrochloric acid and extracted with dichloromethane-ethyl acetate (90:10). The chromatographic separation was achieved on an Agilent Zorbax SB-C(18) column (250 x 4.6 mm, 5 microm) at 35 degrees C, with a linear gradient of acetonitrile and 0.03% aqueous phosphoric acid (v/v), at a flow rate of 1.0 mL/min. The four triterpenoids and internal standard (hydrocortisone) were detected at a wavelength 252 nm. All calibration curves showed good linearity (r(2) > 0.99) within test ranges. The relative deviation of this method was less than 10% for intra- and inter-day assays, and the accuracy ranged from 89 to 108%. The extract recovery for the four triterpenoids and internal standard ranged from 95 to 67%, and the QC samples were found to be stable according to the results of the stability study. This is the first report on determination of the major triterpenoids in rat plasma after oral administration of G. lucidum extract and the results provided a firm basis for clarifying the pharmacological effect of G. lucidum and evaluating the clinical applications of this medicinal fungus.

[Study on influence of processing methods on chemical constituents in Radix Paeoniae Alba].

OBJECTIVE: The influence of processing methods on chemical constituents in Radix Paeoniae Alba was observed. METHOD: A HPLC method was used for analyzing the changes of eight major constituents, namely gallic acid, paeoniflorin sulfonate, catechin, paeoniflorin sulfonate, albiflorin, paeoniflorin, benzoic acid, pentagalloylglucose and benzoylpaeoniflorin, with the three processing procedures of decorticating, boiling and fumigating by burning of sulphur. Analysis was performed using a Zorbax SB-C18 column (4.6 mm x 250 mm, 5 microm) with the mixture of acetonitrile (A) and 0.015% phosphoric acid solution as mobile phase in gradient mode. The detection wavelength was set at 230 nm and the column temperature was at 30 degrees C. RESULT: Except for gallic acid and pentagalloylglucose, the other constituents decreased during procedure of decorticating and boiling. Fumigating by burning of sulphur would produce a new compound, paeoniflorin sulfonate, which was a byproduct from the reaction of paeoniflorin with SO2. CONCLUSION: The significant changes were produced in chemical constituents of Radix Paeoniae Alba during three processing procedures. Therefore, the processing of Radix Paeoniae Alba should be strictly controlled and standardized.

New triterpenoids from Kadsura heteroclita and their cytotoxic activity.

Phytochemical investigations of the stem of Kadsura heteroclita (Roxb) Craib (Schizandraceae) resulted in the isolation and structure elucidation of six new triterpenoidal compounds named heteroclitalactones A-E (1-5) as well as heteroclic acid (6) and heteroclitalactone F (7), which was isolated for the first time from a natural source, and the six known compounds schisanlactone E (8), cycloartenone (9), schisandronic acid (10), nigranoic acid (11), changnanic acid (12) and schisanlactone B (13), respectively. The structures of these compounds were characterized by extensive 1D and 2D NMR spectral analyses. The majority of these triterpenoids showed moderate cytotoxic activity against the human tumor cell lines Bel-7402, BGC-823, MCF-7 and HL-60. Among the compounds tested, heteroclitalactone D (4) showed the strongest cytotoxic activity against the HL-60 cells with an IC50 of 6.76 microM.

Four new lignans from the stems of Kadsura heteroclita.

Two new C(18) dibenzocyclooctadiene lignans named heteroclitalignans A and B (1 and 3), one new C(19) homolignan named heteroclitalignan C(4) and one new natural product named heteroclitalignan D(2), together with the four known lignans kadsurarin (5), kadsulignan E (6), d-epigalbacin, and meso-dihydroguaiaretic acid were isolated from the stems of Kadsura heteroclita. The structures of these compounds were characterized by extensive 1D and 2D NMR spectral data analysis.