Two New Amides from Physochlainae Radix.

In this article, two undescribed amides (1-2) with an unusual (2-formyl-5-hydroxymethyl)pyrroyl-butylamine moiety were obtained from the Physochlainae Radix. Comprehensive spectroscopic studies, including NMR and HR-ESI-MS, coupling with spectroscopic data comparisons were used to determine structures. Anti-inflammatory assay results showed that new amides possessed significant inhibitory activities of the NO production of LPS-induced RAW 264.7 cells, with IC50 values of 17.52±1.68 µM and 20.37±2.42 µM, respectively.

[Sleep-improving mechanism of Chaiqin Ningshen Granules in insomnia rats: based on hippocampal metabonomics].

With the ultra high performance liquid chromatography-quadruple-electrostatic field orbitrap high resolution mass spectrometry(UHPLC-Q Exactive Orbitrap-MS)-based metabonomics technology, this study aims to analyze the effect of Chaiqin Ningshen Granules(CNG) on endogenous metabolites in insomnia rats of liver depression syndrome and explore the sleep-improving mechanism of this prescription. Parachlorophenylalanine(PCPA, ip) and chronic stimulation were combined to induce insomnia of liver depression pattern in rats, and the effect of CNG on the macroscopic signs, hemorheology, and neurotransmitters in the hippocampus of insomnia rats of liver depression syndrome was observed. After the administration, rat hippocampus was collected for liquid chromatography-mass spectrometry(LC-MS) analysis of the metabolomics. Principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA), and orthogonal partial least squares discriminant analysis(OPLS-DA) were employed for analyzing the metabolites in rat hippocampus and screening potential biomarkers. MetPA was used to yield the related metabolic pathways and metabolic networks. The results show that the drugs can significantly improve the mental state, liver depression, and blood stasis of rats, significantly increase the content of 5-hydroxytryptamine(5-HT) and gamma aminobutyric acid(GABA) in hippocampus(except low-dose CNG), and significantly reduce the content of glucose(Glu)(except low-dose CNG). Among them, estazolam and high-dose CNG had better effect than others. Metabolomics analysis yielded 27 potential biomarkers related to insomnia. MetPA analysis showed 4 metabolic pathways of estazolam in intervening insomnia and 3 metabolic pathways of high-dose CNG in intervening insomnia, involving purine metabolism, glycerophospholipid metabolism, histidine metabolism, and caffeine metabolism. CNG can alleviate insomnia by regulating endogenous differential metabolites and further related metabolic pathways. The result lays a basis for further elucidating the mechanism of CNG in improving sleep.

[Mechanism of different processed products of Codonopsis pilosula on spleen deficiency rats based on 1H-NMR metabonomics].

Based on1 H-NMR metabonomics,the effects of Codonopsis pilosula,rice-fried C. pilosula and honey-fried C. pilosula on spleen-asthenia rats were compared,and the mechanism was discussed in this study. The rat model of spleen deficiency was established by weight-bearing swimming and fasting every other day. The effects of different processed products of C. pilosula on the body weight and swimming time of rats were observed. At the end of administration,the gastrocnemius muscle of the right leg of rats was collected and detected by1 H-NMR,and the mechanism of different processed products of C. pilosula in improving spleen deficiency was preliminarily investigated by multivariate statistical analysis. The results showed that C. pilosula,honey-fried C. pilosula and rice-fried C. pilosula could significantly prolong the swimming time( P<0. 05). There was no significant difference in the body weight of rats with spleen deficiency. The results of metabonomics showed that honey-processed C. pilosula could significantly decrease levels of leucine,isoleucine,alanine,acetate,glutamate,succinate,anserine,dimethylamine,dimethylglycine,creatine,phosphorylcholine,glycerophosphorylcholine,taurine,inosine,fumate,hypoxanthine and lactate,but increase levels of glucose,glycine,compared with model group. Therefore,honey-fried C. pilosula has the best efficacy on spleen deficiency syndrome in rats by regulating glycometabolism,amino acid metabolism,lipid metabolism and nucleotide metabolism.

Preparation of Astragalus membranaceus lectin and evaluation of its biological function.

Astragalus membranaceus lectin (AML) was abstracted as a supposedly novel agglutinin of 67 kDa from the seeds of Astragalus membranaceus. The seeds of Astragalus membranaceus were treated with acetate, ammonium sulfate precipitation, and purified by HiTrap SP XL ion column and Superdex G25 gel filtration chromatography to obtain the AML. AML contained 16.4% sugar, ~70% polar amino acids and ~30% hydrophobic amino acids. The AML exhibited agglutination activity toward human and animal erythrocytes, particularly human blood type O and rabbit erythrocytes. It also exhibited acid/alkali resistance and thermal denaturation above 64°C. Compared with human normal liver HL-7702 cells, different concentrations of AML (6.25, 12.50, 25.00 and 50.00 µg/ml) exhibited superior inhibitory effects on the growth of SGC-7901, HepG2 and H22 carcinoma cell lines, and displayed marked antibacterial effects on bacteria; the half maximal inhibitory concentration for B. dysenteriae, S. aureus and E. coli were 85.4, 80.2 and 65.3 µg/ml, respectively.

Therapeutic effects of 5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanone (LM49) in an experimental rat model of acute pyelonephritis by immunomodulation and anti-inflammation.

Antibiotics are still the primary therapy for acute pyelonephritis (APN); rarely, natural polyphenols are also used. LM49 is a novel marine bromophenol derivative displaying strong anti-inflammatory effects. We investigated the therapeutic efficacy of LM49 in an experimental rat model of APN. The model was established by injecting 0.5 mL Escherichia coli (ATCC 25922, 108 CFU/mL) into the urinary bladders of Sprague Dawley rats. This model showed increased kidney viscera indices and renal bacterial growth scores, as well as pathological changes in kidneys. We also performed a broth microdilution antimicrobial susceptibility test of the E. coli strain. Both norfloxacin and LM49 treatment reduced kidney viscera indices and decreased microbial counts in urine cultures and kidney homogenates in APN rats. However, in vitro experiments showed that LM49 did not directly inhibit bacteria. Rather, LM49 treatment inhibited inflammatory cell infiltration or abscess and improved tissue lesions in the renal medullary junction, renal pelvis, and calyx, and high-dose LM49 treatment inhibited the production of inflammatory interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in serum. CD4+ T cells were higher in the LM49 groups treated with high, medium, and low doses than in the model group, whereas only high-dose LM49 treatment increased the number of CD8+ T cells, as compared with that in the model group. However, LM49 treatment did not influence hematological parameters. Our results show that LM49 therapeutic effects in an APN animal model may be achieved by regulating immune responses and inhibiting inflammatory mediators, suggesting it as a candidate APN treatment.

An Optimized and Feasible Preparation Technique for the Industrial Production of Hydrogel Patches.

For hydrogel patches, the laboratory tests could not fully reveal the existing problems of full scale of industrial production, and there are few studies about the preparation technique for the industrial manufacturing process of hydrogel patches. So, the purpose of this work was to elucidate the effects of mainly technological operation and its parameters on the performance of hydrogel patches at the industrial-scale production. The results revealed the following: (1) the aqueous phase was obtained by polyvinylpyrrolidone (PVP) along with tartaric acid dissolved in purified water, then feeding this into a vacuum mixer as a whole in one batch, thus extended the crosslinking reaction time of hydrogel paste (matrix) and allowed the operation of coating/cutting-off to be carried out easily, and there was no permeation of backing layer; (2) the gel strength of the hydrogel patches increased with the increase of working temperature, however, once the temperature exceeded 35 ± 2 °C, the hydrogel paste would lose water severely and the resultant physical crosslinking structure which has lower gel/cohesive strength would easily bring gelatinization/residues during application; (3) the relative humidity (RH) of the standing-workshop was dynamically controlled (namely at 35 ± 2 °C, keeping the RH at 55 ± 5% for 4 days, then 65 ± 5% for 2 days), which would make patches with satisfactory characteristics such as better flexibility, higher adhesive force, smooth flat matrix surface, and without gelatinization/residues and warped edge during the using process; (4) the aging of the packaged hydrogel patches was very sensitive to storage temperature, higher temperature, higher gel strength and lower adhesiveness. The storage temperature of 10 ± 2 °C could effectively prevent matrix aging and adhesion losing, which would also facilitate the expiration date of patches extended obviously. In conclusion, this work provides an optimized and feasible preparation technique for the industrial production of the hydrogel patches and establishes the hydrogel patches as a novel carrier for transdermal drug delivery.

[Modern research and application of Longgu].

Longgu is the fossil of ancient mammals which was used as a common kind of mineral medicine. Longgu is always used to treat neurological diseases. Currently, the quality standard of Longgu is incomplete. Moreover, because of the non-renewable nature of the resource and the increase of national protection of fossils, the clinical application of Longgu is facing a series of problems. As the discovery of the ingredient and the development of forging technology researchers launched to search the substitutes of Longgu. The article summarizes the usage and the study of Longgu, in order that we can discuss the modern usage and substitutability of Longgu.

Protective effects of hyperoside against H2O2-induced apoptosis in human umbilical vein endothelial cells.

The vascular endothelium is important in the physiological homeostasis of blood vessels. Increasing evidence demonstrates that oxidative stress­induced endothelial damage is involved in the pathogenesis of several cardiovascular diseases, including atherosclerosis. Hyperoside, one of major active components from Apocynum venetum L. (Luo­Bu­Ma), which is a traditional Chinese herbal medicine commonly used for the prevention of cardiovascular diseases, exhibits diverse bioactivities, including anti­inflammatory and antioxidant effects. In the present study, the protective effects of hyperoside against hydrogen peroxide (H2O2)­induced apoptosis of human umbilical vein endothelial cells (HUVECs) were investigated. The results demonstrated that hyperoside significantly prevented the loss of cell viability, the increase of endothelial Ca2+ content and apoptosis in H2O2­induced HUVECs. Additionally, reverse transcription-polymerase chain reaction and western blot analysis revealed that hyperoside significantly decreased the mRNA expression levels of B­cell lymphoma (Bcl)­2 associated X protein (Bax), cleaved caspase­3 and phosphorylated­p38, while increasing the mRNA expression of Bcl­2 in H2O2­induced HUVECs. The present findings suggested that hyperoside has protective effects against H2O2­induced apoptosis in HUVECs and serves a key role in the prevention of cardiovascular diseases.

iTRAQ-based quantitative proteomic analysis of the anti-apoptotic effect of hyperin, which is mediated by Mcl-1 and Bid, in H2O2-injured EA.hy926 cells.

Endothelial injury has been implicated in the pathogenesis of many cardiovascular diseases, including thrombotic disorders. Hyperin (quercetin-3-O-galactoside), a flavonoid compound and major bioactive component of the medicinal herb Apocynum venetum L., is commonly used to prevent endothelium dysfunction. However, its mode of action remains unclear. To the best of our knowledge, we have for the first time investigated the protective effect hyperin exerts against H2O2-induced injury in human endothelium-derived EA.hy926 cells using isobaric tags for relative and absolute quantitation (iTRAQ)­based quantitative proteomic analysis. The results showed that H2O2 exposure induced alterations in the expression of 250 proteins in the cells. We noted that the expression of 52 proteins associated with processes such as cell apoptosis, cell cycle and cytoskeleton organization, was restored by hyperin treatment. Of the proteins differentially regulated following H2O2 stress, the anti-apoptotic protein, myeloid cell leukemia-1 (Mcl-1), and the pro-apoptotic protein, BH3-interacting domain death agonist (Bid), exhibited marked changes in expression. Hyperin increased Mcl-1 expression and decreased that of Bid in a dose-dependent manner. In addition, flow cytometric analysis and western blot analysis of the apoptosis-related proteins, truncated BID (tBid), cleaved caspase-3, cleaved caspase-9, Fas, FasL and caspase-8, demonstrated that the rate of apoptosis and the pro-apoptotic protein levels were decreased by hyperin pre­treatment. In the present study we demonstrate that hyperin effectively prevents H2O2­induced cell injury by regulating the Mcl­1­ and Bid-mediated anti­apoptotic mechanism, suggesting that hyperin is a potential candidate for use in the treatment of thrombotic diseases.

Propylene glycol-embodying deformable liposomes as a novel drug delivery carrier for vaginal fibrauretine delivery applications.

The purpose of this work was to develop and characterize the fibrauretine (FN) loaded propylene glycol-embodying deformable liposomes (FDL), and evaluate the pharmacokinetic behavior and safety of FDL for vaginal drug delivery applications. FDL was characterized for structure, particle size, zeta potential, deformability and encapsulation efficiency; the ability of FDL to deliver FN across vagina tissue in vitro and the distribution behavior of FN in rat by vaginal drug delivery were investigated, the safety of FDL to the vagina of rabbits and rats as well as human vaginal epithelial cells (VK2/E6E7) were also evaluated. Results revealed that: (i) the FDL have a closed spherical shape and lamellar structure with a homogeneous size of 185±19nm, and exhibited a negative charge of -53±2.7mV, FDL also have a good flexibility with a deformability of 92±5.6 (%phospholipids/min); (ii) the dissolving capacity of inner water phase and hydrophilicity of phospholipid bilayers of deformable liposomes were increased by the presence of propylene glycol, this may be elucidated by the fluorescent probes both lipophilic Nile red and hydrophilic calcein that were filled up the entire volume of the FDL uniformly, so the FDL with a high entrapment capacity (were calculated as percentages of total drug) for FN was 78±2.14%; (iii) the permeability of FN through vaginal mucosa was obviously improved by propylene glycol-embodying deformable liposomes, no matter whether the FN loaded in liposomes or not, although FN loaded in liposomes caused the highest permeability and drug reservoir in vagina; (iv) the FN mainly aggregated in the vagina and uterus, then the blood, spleen, liver, kidney, heart and lungs for vaginal drug delivery, this indicating vaginal delivery of FDL have a better 'vaginal local targeting effect'; and (v) the results of safety evaluation illustrate that the FDL is non-irritant and well tolerated in vivo, thereby establishing its vaginal drug delivery potential. These results indicate that the propylene glycol-embodying deformable liposomes may be a promising drug delivery carrier for vaginal delivery of fibrauretine.

[Anti-inflammatory active fraction screening and mechanism of unripe Forsythiae Fructus based on ¹H-NMR metabolomics].

To screen the anti-inflammatory active fraction of unripe Forsythiae Fructus, and elucidate the action mechanism, water decoction, ethyl acetate portion, n-butanol portion and residue water extracts of unripe Forsythiae Fructus were administered into rats for continuously 15 days. The acute lung injury inflammatory model was established to observe the section structure of lung tissues. Levels of IL-6, TNF-α, IL-1ß and IL-10 in bronchoalveolar lavage fluid were determined by ELISA kits, and changes in endogenous metabolites in serum were analyzed based on 1H-NMR metabolomics. The results showed that ethyl acetate portion of unripe Forsythiae Fructus had a better anti-inflammatory activity against acute lung injury, and could suppress the release of inflammatory factors of IL-6, TNF-α, IL-1ß, significantly reduce contents of creatine, ß-OH-butyrate, succinate, lysine, valine, isoleucine and glutamine, and elevate the content of GPC in serum. Ethyl acetate portion was proved to be the main fraction of anti-inflammatory activity from the perspective of endogenous metabolites in serum, and played an anti-inflammatory role by regulating creatine metabolism, choline metabolism, branched-chain amino acid metabolism and TCA cycles. This study could lay a foundation for studying pharmacodynamic material basis of unripe Forsythiae Fructus.

Anti-tumoral effects of a trypsin inhibitor derived from buckwheat in vitro and in vivo.

Native buckwheat, a common component of food products and medicine, has been observed to inhibit cancer cell proliferation in vitro. The aim of the present study was to evaluate the in vitro and in vivo anti-tumoral effects of recombinant buckwheat trypsin inhibitor (rBTI) on hepatic cancer cells and the mechanism of apoptosis involved. Apoptosis in the H22 cell line induced by rBTI was identified using MTT assays, DNA electrophoresis, flow cytometry, morphological observation of the nuclei, measurement of cytochrome C and assessment of caspase activation. It was identified that rBTI decreases cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies and DNA fragmentation. rBTI-induced apoptosis occurred in association with mitochondrial dysfunction, leading to the release of cytochrome C from the mitochondria to the cytosol, as well as the activation of caspase-3, -8 and -9. In conclusion, the results of the present study suggested that rBTI specifically inhibited the growth of the H22 hepatic carcinoma cell line in vitro and in vivo in a concentration-dependent and time-dependent manner, while there were minimal effects on the 7702 normal liver cell line. In addition, rBTI­induced apoptosis in H22 cells was, at least in part, mediated by a mitochondrial pathway via caspase-9.

[Chemical constituents from Crepis crocea].

Thirteen compounds were isolated from the ethyl acetate fraction of Crepis crocea by column chromatographies on silica gel, Sephadex LH-20 and semi-preparative HPLC. The structures were elucidated on the basis of spectral analysis as tectorone I (1), 8ß- (2-methyl- 2-hydroxy-3-oxobutanoyloxy) -glucozaluzanin C (2), tectoroside (3), luteolin-7-O-glucoside (4), cosmosiin (5), esculetin (6), 3,4-dihydroxybenzaldehyde (7), trans-4-hydroxycinnamic acid (8), Caffeic acid (9), methyl p-hydroxyphenyllactate (10), ethylp- hydroxyphenyllactate (11), cis-3,4-dihydroxy-ß-ionion (12). All the compounds, except for compounds 4 and 9, were isolated from this plant for the first time, and tectorone I (1) is a new natural product.

[HPLC fingerprint of total flavonoids of Folium Apocyni Veneti].

OBJECTIVE: To establish a sensitive and specific HPLC fingerprint for the quality controlling of total flavonoids of Folium Apocyni Veneti. METHOD: HPlC analysis was performed on a Kromasil C18 column (4.6 mm x 250 mm, 5 microm) with the mixture of solvent A [acetonitrile-phosphoric acid (95:5)] and solvent B (0.05% phosphoric acid) in gradient mode at a flow rate of 1.0 mL x min(-1). The detection wavelength was set at 360 nm. The column temperature was set at 25 degrees C and the injection volume was 20 microL. RESULT: The chromatographic fingerprint of total flavonoids was established which showed 17 characteristic peaks from 7 patches of total flavonoids products. The similarity from different patches was 0.95-1.00 analyzed by the software of 'Computer-aided Similarity Evaluation' and showed high similitude in peak numbers and the retention time. Moreover, comparison of the HPLC profiles of the total flavonoids with the corresponding Folium Apocyni Veneti leaves indicated that they were closely related to each other. CONCLUSION: The chromatographic fingerprint of the total flavonoids with high specificity and can be used to control its quality and assure the homogenicity for each patch of the total flavonoids.