An integration strategy combined progressive multivariate statistics with anticoagulant activity evaluation for screening anticoagulant quality markers in Chinese patent medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: The cardiovascular and cerebrovascular diseases affect human health globally. Naoxintong capsules (NXTs), a famous Chinese Patent Medicine, has been especially applied to treat cerebral infarction and coronary heart disease in clinical practice. The anticoagulant activity of this prescription plays an important role in this course of treatment. AIM OF THE STUDY: Thrombin and factor Xa (FXa) are two key targets considering the anticoagulant activity. The purpose of this investigation is to screen the quanlity markers as key thrombin and FXa inhibitors for the anticoagulant activity oriented quality control of Chinese patent medicine. MATERIALS AND METHODS: Simple multi-polar solvent extraction processes using various proportions of solvents were conducted and their thrombin/FXa inhibitory activities were evaluated in vitro. Bivariate correlation analysis (BCA), grey correlation analysis (GCA), and orthogonal partial least squares discriminate analysis (OPLS-DA) were adopted for screening the potential active markers related to the anticoagulant activity. The chemical structures of these active compounds were identified by UHPLC-Q-TOF-MS/MS and their thrombin/FXa inhibitory activity was determined. The molecular docking technology was applied to explore the interaction between the compounds and targets. The contribution of these anticoagulant active ingredients in NXT was also investigated. Last but not the least, the contents of these markers in NXT were determined by liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (HPLC-ESI-MS/MS) method. RESULTS: The results showed that the NXT extract exhibited great activity against thrombin and FXa, especially extracted by 75% methanol (v/v). Six marker compounds with potential anticoagulant activity were screened out. Therein, four of the active compounds owing thrombin inhibitory activity (paeoniflorin, lithospermic acid, salvianolic acid B, Z-ligustilide) and five of the active compounds owing FXa inhibitory activity (3,5-dicaffeoylquinic acid, rosmarinic acid, lithospermic acid, salvianolic acid B and Z-ligustilide). In addition, these active compounds accounted for a large proportion of thrombin/FXa inhibitory activity of NXTs. The binding energy also showed the strong interaction formed by close connection of the compounds to the residues of targets. CONCLUSIONS: The proposed integrated stategy could be an efficient strategy to screen potential thrombin/FXa inhibitors for the bioactivity related quanlity control of Chinese patent medicine.

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock.

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was pretreated with NaOH and washed to a neutral pH. Cellulase was added to the pretreated bamboo powder to generate the hydrolysate, which contained 30 g/L glucose and 15 g/L xylose and was used as the carbon source to prepare a medium for chemical production. When cultured in microaerobic conditions, 12.7 g/L 2,3-butanediol was produced by wildtype K. pneumoniae. In aerobic conditions, 13.0 g/L R-acetoin was produced by the budC mutant of K. pneumoniae. A mixture of 25.5 g/L 2-ketogluconic acid and 13.6 g/L xylonic acid was produced by the budA mutant of K. pneumoniae in a two-stage, pH-controlled fermentation with high air supplementation. In the first stage of fermentation, the culture was maintained at a neutral pH; after cell growth, the fermentation proceeded to the second stage, during which the culture was allowed to become acidic.

Gluconic acid production by gad mutant of Klebsiella pneumoniae.

Klebsiella pneumoniae produces many economically important chemicals. Using glucose as a carbon source, the main metabolic product in K. pneumoniae is 2,3-butanediol. Gluconic acid is an intermediate of the glucose oxidation pathway. In the current study, a metabolic engineering strategy was used to develop a gluconic acid-producing K. pneumoniae strain. Deletion of gad, resulting in loss of gluconate dehydrogenase activity, led to the accumulation of gluconic acid in the culture broth. Gluconic acid accumulation by K. pneumoniae Δgad was an acid-dependent aerobic process, with accumulation observed at pH 5.5 or lower, and at higher levels of oxygen supplementation. Under all other conditions tested, 2,3-butanediol was the main metabolic product of the process. In fed batch fermentation, a final concentration of 422 g/L gluconic acid was produced by K. pneumoniae Δgad, and the conversion ratio of glucose to gluconic acid reached 1 g/g. The K. pneumoniae Δgad described in this study is the first genetically modified strain used for gluconic acid production, and this optimized method for gluconic acid production may have important industrial applications. Gluconic acid is an intermediate of this glucose oxidation pathway. Deletion of gad, resulting in loss of gluconate dehydrogenase activity, led to the accumulation of gluconic acid in the culture broth. In fed batch fermentation, a final concentration of 422 g/L gluconic acid was produced by the K. pneumoniae Δgad strain, and the conversion ratio of glucose to gluconic acid reached 1 g/g.

Hypotensive resuscitation combined with polydatin improve microcirculation and survival in a rabbit model of uncontrolled hemorrhagic shock in pregnancy.

BACKGROUND: Obstetric hemorrhage remains a leading cause of maternal death internationally. Polydatin is an effective drug in ameliorating microcirculatory insufficiency and increasing survival rate in non-pregnant animal model of controlled hemorrhagic shock. In the present study, we investigated the effects of hypotensive resuscitation combined with Polydatin administration on microcirculation and survival rate in a clinically relevant model of uncontrolled hemorrhagic shock in pregnancy. MATERIALS AND METHODS: Twenty anesthetized New Zealand white rabbits at mid and late gestation were anesthetized, and an ear chamber was prepared to examine microvessels by intravital microscopy. Shock was induced by transecting a small artery in mesometrium, followed by blood withdrawal via the femoral artery to a mean arterial pressure (MAP) of 40-45 mm Hg. Animals were randomly divided into two groups (n=10 per group): 30 min after hemorrhage induction, hypotensive resuscitation with Ringer's solution to MAP of 60 mm Hg, followed by a single volume infusion of 4 mL/Kg of normal saline or Polydatin at 60 min after hemorrhage induction (group NS, PD). Finally all the animals received hemorrhage control and resuscitated with half of the heparinized shed blood and Ringer's solution to MAP of 80 mm Hg. RESULTS: At the end of resuscitation, compared with group NS, group PD showed significantly improved capillary perfusion as indicated by increased arteriole diameter [0.95±0.02 of baseline (PD), 0.71±0.05 of baseline (NS); P=0.000] and higher functional capillary density[95.3% ± 2.6% (PD), 57.2% ± 4.1% (NS); P=0.000]. Median survival time was significantly longer in group PD than that in group NS [4 d (PD), 2 d (NS); P=0.000]. CONCLUSIONS: On the basis of hypotensive resuscitation, Polydatin administration improved microcirculation and prolonged survival time in pregnant rabbit model of uncontrolled hemorrhagic shock.