Cell death mechanisms induced by synergistic effects of halofuginone and artemisinin in colorectal cancer cells.

Our previous study found that the combination of halofuginone (HF) and artemisinin (ATS) synergistically arrest colorectal cancer (CRC) cells at the G1/G0 phase of the cell cycle; however, it remains unclear whether HF-ATS induces cell death. Here we report that HF-ATS synergistically induced caspase-dependent apoptosis in CRC cells. Specifically, both in vitro and in vivo experiments showed that HF or HF-ATS induces apoptosis via activation of caspase-9 and caspase-8 while only caspase-9 is involved in ATS-induced apoptosis. Furthermore, we found HF or HF-ATS induces autophagy; ATS can't induce autophagy until caspase-9 is blocked. Further analyzing the crosstalk between autophagic and caspase activation in CRC cells, we found autophagy is essential for activation of caspase-8, and ATS switches to activate capase-8 via induction of autophagy when caspase-9 is inhibited. When apoptosis is totally blocked, HF-ATS switches to induce autophagic cell death. This scenario was then confirmed in studies of chemoresistance CRC cells with defective apoptosis. Our results indicate that HF-ATS induces cell death via interaction between apoptosis and autophagy in CRC cells. These results highlight the value of continued investigation into the potential use of this combination in cancer therapy.

[Evaluation of genetic diversity and population structure of genus Tripterygium based on SSR markers].

The genus Tripterygium is an immune suppressor in the Chinese traditional medicines. Due to the habitat destruction and anthropogenic over-exploitation, the wild genus Tripterygium plants have decreased dramatically in recent years or even been endangered. It is critical to evaluate and protect genus Tripterygium wild resource. In this research, simple sequence repeat (SSR) molecular markers were applied to the investigation of the genetic diversity and genetic structure of 28 populations for genus Tripterygium (396 samples from 9 provinces in China). We found a high level of genetic diversity (percentage of polymorphic loci PPL = 77.29%, Shannon's information index I = 0.639 4; Nei's expected heterozygosity H = 0.359 9) and high genetic differentiation among the populations (gene flow N_m = 0.228 7). Based on Nei's genetic distance, the phylogenic tree of populations was constructed and 28 populations were divided into 6 clusters according to STRUCTURE clustering analysis. T. hypoglaucumwas was mainly divided into 3 clusters, including Sichuan, Yunnan and Guizhou- Chongqing. T. regelii was separated to cluster 4, while T. wilfordii was divided into two clusters: the transition type LQ and NY were divided into cluster 5, and the others were in cluster 6. These results provide a theory basis for the conservation of wild resource, research of genetic polymorphism and molecular marker for assisted breeding of genus Tripterygium.

[Studies on difference of chemical compositions in plant species of Tripterygium genus].

We studied the content of chemical compositions and correlation among species of Tripterygium genus by principal component analysis(PCA) and variance analysis(ANOVA), and we also studied the difference among the 3 species.Using [BMIm]PF6 ionic liquid-based ultrasonic-assisted extraction, we determined the contents of 11 compounds including wilforgine, wilforzine, triptophenolide, wilforine, triptoquinone A, triptolide, tripterin, egallocatechin, epigallocatechin, catechin, and epicatechin in 28 batches of the Tripterygium species by HPLC and PCA. Partial least squares analysis (PLS) and ANOVA were also performed to verify the results.The analysis results of PCA and PLS showed that three species of Tripterygium genus were clustered into three regions respectively, and triptoquinone A was the important factor which affected the aggregation of these three species.There was a significant difference among the contents of 11 chemical components in the three species(P<0.000 1).These results indicated that there was a certain correlation between the chemical compositions and the classification of the species, and the difference of the chemical compositions among the three species was obvious. In this work, the content determination method is rapid and accurate, and the analysis method is simple and convenient, which provides a reference for the classification, the efficacy and the toxicity of the species.

Cantharimide and Its Derivatives from the Blister Beetle Mylabris phalerata Palla.

Eleven new monoterpenoids including three 1-methyl cantharimide-type derivatives (1-3), five 1,2-dimethyl cantharimide-type derivatives (4, 5, 7-9), and three 1-hydroxymethyl-2-methyl cantharimide-type derivatives (10-12), together with seven known cantharimides (6, 13-18), were isolated from Mylabis phalerata Palla. The planar structures and absolute configurations of compounds 1-14 were fully elucidated on the basis of spectroscopic analysis, ECD spectra, single-crystal X-ray diffraction analysis, and chemical methods. Compounds 6, 15, 16, and 18 were found to be potent inhibitors of HBV virus, with IC50 values of 62, 42, 58, and 19 µM.

[Species differences in caffeine metabolism in liver microsomes of rats and mice].

Caffeine and its metabolic products play an important role in clinical applications. An ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS/MS) method was applied to systemically study the caffeine metabolism in liver microsomes of rats and mice, and comprehensively evaluate caffeine metabolites in vitro and metabolism differences between species. The caffeine metabolites and metabolism differences between species in liver microsomes of rats and mice were analyzed by UPLC-Q-TOF-MS/MS high resolution mass spectrometry system and metabolitepolite software. The results showed that in addition to the demethylated and oxidized products in previous analysis, methylated, double oxidized, dehydrated and decarbonylated metabolites were also found in caffeine metabolism in liver microsomes of rats and mice, with significant difference in metabolism in vitro between rats and mice. The demethylated metabolite M2(C7H8N4O2) and decarbonylated metabolite M6(C7H10N4) in metabolism in vitro of mice were not found in rats, and the in vitro metabolite M7(C8H12N4O5) in rats were not found in mice. There was significant species difference in caffeine metabolism in vitro between rats and mice, providing important reference value for the further metabolism study and safety evaluation of caffeine.

[Progress on molecular biology of Isaria farinosa, pathogen of host of Ophiocordyceps sinensis during the artificial culture].

Isaria farinosa is the pathogen of the host of Ophiocordyceps sinensis. The present research has analyzed the progress on the molecular biology according to the bibliometrics, the sequences (including the gene sequences) of I. farinosa in the NCBI. The results indicated that different country had published different number of the papers, and had landed different kinds and different number of the sequences (including the gene sequences). China had published the most number of the papers, and had landed the most number of the sequences (including the gene sequences). America had landed the most numbers of the function genes. The main content about the pathogen study was focus on the biological controlling. The main content about the molecular study concentrated on the phylogenies classification. In recent years some protease genes and chitinase genes had been researched. With the increase of the effect on the healthy of O. sinensis, and the whole sequence and more and more pharmacological activities of I. farinosa being made known to the public, the study on the molecular biology of the I. farinosa would be deeper and wider.

[Chemical constituents from Mylabris phalerata and their cytotoxic activity in vitro].

Ten compounds were isolated from Mylabris phalerata by using preparative HPLC and column chromatography over MCI gel. On the basis of physical-chemical properties, NMR and MS data analysis, the compounds were identified as 5'-[(1 R,2 R,3 S,6R)-1-hydroxymethyl-2-methyl-3,6-epoxycyclohexane-1,2-dicarboximide]- ethyl-2'-methyl-2'-butenoate (1),cantharidin (2), cyclo-(L-Pro-L-Ala) (3), cyclo-(R-Pro-R-Leu) (4), cyclo-(S-Pro-R-Leu) (5), cyclo-(D-Pro-L-Tyr) (6), indole-3-aldehyde (7), 3-indoleacetic acid (8), valerolactam (9), and 4-hydroxyphthalid (10).Compound 1 was a new compound, and compounds 2-10 were obtained from this genus for the first time. Compounds 1-9 were subjected to cytotoxic activity on HCT-116, HepG2, BGC-823, NCI-H1650, A2780 cell lines, and only compound 2 showed inhibitory effect on all cancer cell lines.

Wilfordonols A-D: four new norsesquiterpenes from the leaves of Tripterygium wilfordii.

Four new norsesquiterpenes wilfordonols A-D (1-4), along with three known compounds, sarmentol B (5), boscialin (6), and (+)-loliolide (7), were isolated from the leaves of Tripterygium wilfordii Hook.f.. The structures of the new compounds were elucidated on the basis of their spectroscopic analysis, and the absolute configuration of the compounds was confirmed by CD and modified Mosher's method. At a concentration of 10 µM, compounds 4, 6, and 7 inhibited signal transducer and activator of transcription 1 translocation by 34.27 ± 1.02%, 48.93 ± 1.76%, and 70.31 ± 2.20%, respectively.

Prenylated benzoylphloroglucinols and xanthones from the leaves of Garcinia oblongifolia with antienteroviral activity.

An acetone extract of the leaves of Garcinia oblongifolia showed antiviral activity against enterovirus 71 (EV71) using a cytopathic effect inhibition assay. Bioassay-guided fractionation yielded 12 new prenylated benzoylphloroglucinols, oblongifolins J-U (1-12), and five known compounds. The structures of 1-12 were elucidated by spectroscopic analysis including 1D- and 2D-NMR and mass spectrometry methods. The absolute configurations were determined by a combination of a Mosher ester procedure carried out in NMR tubes and ECD calculations. Compared to ribavirin (IC50 253.1 µM), compounds 1, 4, and 13 exhibited significant anti-EV71 activity in vitro, with IC50 values of 31.1, 16.1, and 12.2 µM, respectively. In addition, the selectivity indices of these compounds were 1.5, 2.4, and 3.0 in African green monkey kidney (Vero) cells, respectively.

Simultaneous determination of nine nucleosides and nucleobases in different Fritillaria species by HPLC-diode array detector.

A sensitive and reliable HPLC-diode-array detector method was developed for the first time to simultaneously determine nine nucleosides and nucleobases including uracil, cytidine, guanine, uridine, thymine, inosine, guanosine, thymidine and adenosine in 13 different Fritillaria species. The analysis was performed on a BaseLine C18 column with a gradient of acetonitrile in water at a flow rate of 0.8 mL/min. The diode-array detector wavelength was set at 260 nm for the UV detection of nucleosides and nucleobases. Satisfactory separation of these compounds was obtained in less than 40 min. The optimized method provided good linear relation (r(2)>0.9995 for all the investigated analytes), satisfactory precision (RSD <1.51%) and good recovery (from 97.64 to 101.16%). The established method was successfully applied to simultaneous determination of nine nucleosides and nucleobases in 61 batches of samples from 13 Fritillaria species collected from different habitats in China, which could be helpful to control the quality of Fritillaria bulbs.

Process parameters and morphology in puerarin, phospholipids and their complex microparticles generation by supercritical antisolvent precipitation.

The aim of the study was to develop and evaluate a new method for the production of puerarin phospholipids complex (PPC) microparticles. The advanced particle formation method, solution enhanced dispersion by supercritical fluids (SEDS), was used for the preparation of puerarin (Pur), phospholipids (PC) and their complex particles for the first time. Evaluation of the processing variables on PPC particle characteristics was also conducted. The processing variables included temperature, pressure, solution concentration, the flow rate of supercritical carbon dioxide (SC-CO2) and the relative flow rate of drug solution to CO2. The morphology, particle size and size distribution of the particles were determined. Meanwhile Pur and phospholipids were separately prepared by gas antisolvent precipitation (GAS) method and solid characterization of particles by the two supercritical methods was also compared. Pur formed by GAS was more orderly, purer crystal, whereas amorphous Pur particles between 0.5 and 1microm were formed by SEDS. The complex was successfully obtained by SEDS exhibiting amorphous, partially agglomerated spheres comprised of particles sized only about 1microm. SEDS method may be useful for the processing of other pharmaceutical preparations besides phospholipids complex particles. Furthermore adopting a GAS process to recrystallize pharmaceuticals will provide a highly versatile methodology to generate new polymorphs of drugs in addition to conventional techniques.

[Osmotically controlled oral drug delivery systems: an update review].

Osmotically controlled oral drug delivery systems (OCODDSs) utilize osmotic pressure for controlled delivery of active agents. The release of drugs from osmotic systems is governed by various formulations and processing factors such as solubility and pressure of the core components, properties of the semi-permeable membrane. In the present review, the references on OCODDSs have systematically been summarized in the following aspects: prescription design, industrial processing and equipments, methods for quality evaluation, and general situation of application. Prospect of applying the osmotic-pump technology into Chinese patent drugs is also discussed.

[Determination of anthraquinone in Semen Cassiae from different regions by HPLC].

OBJECTIVE: To determine the content of 7 anthraquinones in Semen Cassiae. METHOD: A HPLC method was developed, with Inertsil ODS-3 column, acetonitrile and 0.1% H3PO4 solution as mobile phases in gradient elution. The detection wavelength wasset at 278 nm, and the flow rate was 0.8 mL x min(-1). RESULT: Recoveries of all 7 anthraquinones were between 95%-105%. The content of the anthraquinones in crude drug produced in different habitation were different. CONCLUSION: The method is convenient and accurate, which provides the foundation for the research of Semen Cassiae.

[Neuroprotective effects of water extracts of American Ginseng on SH-SY5Y cells apoptosis induced by Abeta25-35].

OBJECTIVE: To study the neuroprotective effect of water extracts of American Ginseng (WEAG) on Abeta25-35-induced SH-SY5Y cells apoptosis in Alzheimer's Disease cellular model. METHODS: The optimal concentration and treating time of Abeta25-35 for Alzheimer's Disease cellular model as well as those of WEAG were measured by flow cytometer. In addition, the cell viability was measured by MTT test and the morphology of SH-SY5Y cells was observed by Hoechst 33258 staining. RESULTS: Treated by Abeta25-35 50 micromol/L 72 h later, SH-SY5Y cells turned rounder, aggregated and were positively stained with fluorochrome Hoechst 33258. Cells displayed a typical sub-diploid peak in flow cytometry, and the percentage of apoptosis reaches (37.30 +/- 0.69)% (P < 0.05 as compared with the control group) (1.56 +/- 0.80)%. When incubated with Abeta 50 micromol/L and different doses (0.5, 1, 5 mg/ml) of WEAG for 72h, the characteristics of apoptosis as measured by FCM dose-dependently declined to (16.71 +/- 1.08)%, (10.52 +/- 2.11)% and (3.39 +/- 1.65)%, respectively (P < 0.05 as compared with the model group). CONCLUSION: Water extracts of American Ginseng have markedly neuroprotective effects on SH-SY5Y cells apoptosis induced by Abeta25-35.

Paeoniflorin exerts neuroprotective effects by modulating the M1/M2 subset polarization of microglia/macrophages in the hippocampal CA1 region of vascular dementia rats via cannabinoid receptor 2.

BACKGROUND: Cerebral hypoperfusion is a pivotal risk factor for vascular dementia (VD), for which effective therapy remains inadequate. Persistent inflammatory responses and excessive chemotaxis of microglia/macrophages in the brain may accelerate the progression of VD. Endocannabinoids are involved in neuronal protection against inflammation-induced neuronal injury. Cannabinoids acting at cannabinoid receptor 2 (CB2R) can decrease inflammation. Based on the identification of paeoniflorin (PF) as a CB2R agonist, we investigated the neuroprotective and microglia/macrophages M1 to M2 polarization promoting effects of PF in a permanent four-vessel occlusion rat model. METHODS: One week after surgery, PF was intraperitoneally administered at a dose of 40 mg/kg once a day for 28 successive days. The effects of PF on memory deficit were investigated by a Morris water maze test, and the effects of PF on hippocampal neuronal damage were evaluated by light microscope and electron microscope. The mRNA and protein expression levels of key molecules related to the M1/M2 polarization of microglia/macrophages were assessed by RT-qPCR and Western blotting, respectively. RESULTS: Administration of PF could significantly attenuate cerebral hypoperfusion-induced impairment of learning and memory and reduce the morphological and ultrastructural changes in the hippocampal CA1 region of rats. Moreover, PF promoted an M1 to M2 phenotype transition in microglia/macrophages in the hippocampus of rats. In addition to its inhibitory property against proinflammatory M1 mediator expression, such as IL-1ß, IL-6, TNF-α and NO, PF dramatically up-regulated expression of anti-inflammatory cytokines IL-10 and TGF-ß1. Importantly, CB2R antagonist AM630 abolished these beneficial effects produced by PF on learning, memory and hippocampus structure in rats, as well as the polarization of microglia/macrophages to the M2 phenotype. Additionally, PF treatment significantly inhibited cerebral hypoperfusion-induced mTOR/NF-κB proinflammatory pathway and enhanced PI3K/Akt anti-inflammatory pathway. Effects of PF on these signaling pathways were effectively attenuated when rats were co-treated with PF and AM630, indicating that the mTOR/NF-κB and PI3K/Akt signaling pathways were involved in the PF effects through CB2R activation. CONCLUSION: These findings demonstrated PF exerts its neuroprotective effect and shifts the inflammatory milieu toward resolution by modulation of microglia/macrophage polarization via CB2R activation.

Cytotoxic prenylated phenolic compounds from the twig bark of Garcinia xanthochymus.

Three new hydroxylated xanthones with prenyl or geranyl substituents, compounds 1-3, were isolated from the twig bark of Garcinia xanthochymus, along with the four known compounds 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone (4), 1,3,5,6-tetrahydroxy-4,7,8-triprenylxanthone (5), garciniaxanthone E (6), and 6-prenylapigenin (7). Their structures were elucidated by extensive spectroscopic analysis, including 1D- and 2D-NMR as well as HR-MS experiments. All compounds showed moderate cytotoxicities against breast cancer (MDA-MB-435S) and lung adenocarcinoma (A549) cell lines, but lacked antifungal activity against Candida albicans.

[Effect of combination glycyrrhiizin and triptolide on TNF-alpha and IL-10 in serum of collagen induced arthritis rats].

OBJECTIVE: To observe the effects of combination therapy with glycyrrhizin (GL) and triptolide (TP) on collagen-induced arthritis (CIA) rats. METHOD: Sixty male SD rats were randomly divided into 6 groups: the model group, the TP group, the GL group, and combination 1, 2, 3 groups. The models were induced by collagen type II. The arthritis index (AI) and the edema rate were detected as curative effect, and the level of antibodies to collagen, TNF-alpha and IL-10 were measured by ELISA. RESULT: The combination therapy with GL and TP significantly reduced the paw edema and arthritis index of CIA rats (P <0. 01 ), and the combination therapy can increase the level of IL-10, while decrease the level of TNF-alpha, and the level of antibodies to collagen decreased too (P <0.05, P <0.01). CONCLUSION: Combine 26.78 mg x kg(-1) GL with 13.40 microg x kg(-1) TP can significantly inhibited the CIA, and the effect equal to the dosage of 17. 86 microg x kg(-1) TP. It supports the possible of GL in combination with TP to reduce the dose and side effects related to TP.

Halofuginone dually regulates autophagic flux through nutrient-sensing pathways in colorectal cancer.

Autophagy has a key role in metabolism and impacts on tumorigenesis. Our previous study found that halofuginone (HF) exerts anticancer activity in colorectal cancer (CRC) by downregulating Akt/mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway. But whether and how HF regulates autophagy and metabolism to inhibit cancer growth remains an open question. Here, we unveil that HF activates ULK1 by downregulation of its phosphorylation site at Ser757 through Akt/mTORC1 signaling pathway, resulting in induction of autophagic flux under nutrient-rich condition. On the other hand, HF inactivates ULK1 by downregulation of its phosphorylation sites at Ser317 and Ser777 through LKB1/AMPK signaling pathway, resulting in autophagic inhibition under nutrient-poor condition. Furthermore, Atg7-dependent autophagosome formation is also induced under nutrient-rich condition or blocked in nutrient-poor environment, respectively, upon HF treatment. More interestingly, we also found that HF inhibits glycolysis under nutrient-rich condition, whereas inhibits gluconeogenesis under nutrient-poor condition in an Atg7-dependent manner, suggesting that autophagy has a pivotal role of glucose metabolism upon HF treatment. Subsequent studies showed that HF treatment retarded tumor growth in xenograft mice fed with either standard chow diet or caloric restriction through dual regulation of autophagy in vivo. Together, HF has a dual role in autophagic modulation depending on nutritional conditions for anti-CRC.

High-performance thin-layer chromatographic fingerprints of isoflavonoids for distinguishing between Radix Puerariae Lobate and Radix Puerariae Thomsonii.

The roots of Pueraria lobata (Wild.) Ohwi and Pueraria thomsonii Benth have been officially recorded in all editions of Chinese Pharmacopoeia under the same monograph 'Gegen' (Radix Puerariae, RP). However, in its 2005 edition, the two species were separated into both individual monographs, namely 'Gegen' (Radix Puerariae Lobatae, RPL) and 'Fenge' (Radix Puerariae Thomsonii, RPT), respectively, due to their obvious content discrepancy of puerarin, the major active constituent. In present paper, the fingerprint of high-performance thin-layer chromatography (HPTLC) combining digital scanning profiling was developed to identify and distinguish the both species in detail. The unique properties of the HPTLC fingerprints were validated by analyzing ten batches of Pueraria lobata and P. thomsonii samples, respectively. The common pattern of the HPTLC images of the roots of Pueraria spp. and the respective different ratios of the chemical distribution can directly discern the two species. The corresponding digital scanning profiles provided an easy way for quantifiable comparison among the samples. Obvious difference in ingredient content and HPTLC patterns of the two species questioned their bio-equivalence and explained that recording both species separately in the current edition of Chinese Pharmacopoeia (2005 edition) is reasonable due to not only the content of major constituent, puerarin, but also the peak-to-peak distribution in the fingerprint and integration value of the total components. Furthermore, the HPTLC fingerprint is also suitable for rapid and simple authentication and comparison of the subtle difference among samples with identical plant resource but different geographic locations.