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ELISA for the determination of saikosaponin a, an active component of Bupleuri Radix.

In order to quantify saikosaponin a (SSA), one of the major active components of Bupleuri Radix, a competitive and indirect ELISA method was developed. High titer rabbit polyclonal antibodies (pAbs) were raised against a conjugate of SSA and bovine serum albumin, coupled with a periodate oxidation method. SSA competitively inhibited the binding of rabbit anti-SSA pAbs to SSA-ovalbumin on the solid phase, a coated antigen on the well. The quantity of pAbs bound to the well was monitored using a peroxidase-conjugated anti-rabbit IgG as a secondary antibody, and tetramethylbenzidine solution as a substrate. The measuring range extended from 50 pg/ml to 20 ng/ml of SSA, with a detection limit of 40 pg/ml (5.13 pM). Antibodies showed some cross-reactivity with saikosaponin c (12.74%). However, the antibodies showed only slight cross-reactivities with saikosaponin d (0.3%), which differs from SSA only in the stereochemistry of the 16-hydroxyl group, and the artificial saikosaponins, saikosaponin b1 (2.1%) and saikosaponin g (0.53%). The specific and sensitive ELISA is especially suited for determination of SSA in samples when only small quantities of materials can be extracted for analysis.

Animals

[Inactivation and toxoiding of biologically-active components of Bordetella pertussis by tea catechins].

An ability of tea catechins known as agents for the disinfection to bacteria and viruses were tested on application for toxoiding biologically active components of Bordetella pertussis. The effects on the activities and antigenicity of filamentous hemagglutinin (FHA) and pertussis toxin (PT) were investigated. The activities of FHA and PT were inactivated by catechins at approximately 10(3) times lower dose (0.2 mM) compared with that of formalin. The activity of inactivated FHA was recovered by dialysis against Tris-HCl buffer, pH 8.0, containing glutathione or Tris-HCl buffer, pH 6.0. But the activity of inactivated PT was not recovered. Antigenicity of catechin-treated antigens were investigated by immunization to mice. The sera from mice immunized by catechin-treated FHA or PT were contained antibody against not only catechin-treated but also non-treated FHA or PT. These results suggest that antigenicity of FHA or PT was not destroyed by the treatment with catechin. We prepared pertussis-component vaccines by treatment of several catechins on the condition that FHA or PT activity was not recovered. Higher efficacy were found on the vaccines made by treatment of epicatechin, epicatechin gallate, or epigallocatechin than those by formalin. The vaccine prepared by using epigallocatechin gallate had significant efficacy as well as that by formalin treated one. From these results, it is suggested that tea leaf catechins were effective agents for toxoiding of vaccine components.

Animals

Mangafodipir trisodium injection, a new contrast medium for magnetic resonance imaging: in vitro metabolism and protein binding studies of the active component MnDPDP in human blood.

The binding to human serum proteins of MnDPDP (manganese(II) dipyridoxyl diphosphate), the active component of the magnetic resonance imaging contrast medium mangafodipir trisodium injection (Teslascan) was studied in ultrafiltration experiments. Sera from three males and three females were incubated with 86 microM [14C]MnDPDP for 60 min at room temperature (20-23 degrees C), followed by centrifugation through filters with a cut-off of 30 kDa. Analysis of the filtrates and the initial incubation mixtures for manganese, by ICP-AES, and for DPDP and its dephosphorylated metabolites DPMP (dipyridoxyl monophosphate) and PLED (dipyridoxyl ethylenediamine diacetate) by liquid scintillation counting, showed a clear difference in protein binding of manganese and the ligands under these conditions. Only 2.2 +/- 1.8% (mean +/- S.E.; n = 6) of DPDP, DPMP and PLED were bound to protein, whereas 26.9 +/- 2.9% (mean +/- S.E.; n = 6) of manganese was bound to protein. No binding of DPDP, DPMP or PLED to blood cells was observed when whole blood, containing either heparin or EDTA as anticoagulant, was spiked with [14C]MnDPDP and the cell-free fraction and the lysed cell fraction analysed by liquid scintillation counting. The extent of protein binding of manganese corresponded well with results from an in vitro metabolism study, in which MnDPDP was added to heparinized human whole blood, showing that approximately 25% of DPDP, DPMP or PLED were not bound to manganese. The in vitro metabolism study revealed that transmetallation with zinc was nearly complete within 1 min, and that dephosphorylation is a sequential process going from DPDP to the monophosphate DPMP, and then to the fully dephosphorylated compound PLED.

Blood Proteins

The response of human platelets to activated components of the complement system.

Many in vivo platelet responses are considered to be mediated, directly or indirectly, by activated components of the complement system. These include the secretion of proteolytic enzymes and the assembly of key enzymes of the coagulation and fibrinolytic pathways. In this review, Peter J. Sims and Therese Wiedmer summarize the known interactions of human platelets with the complement system and discuss the implications of these interactions for platelet hemostatic function within the vasculature.

Blood Platelets

[Studies on Kochiae Fructus. I. Antipruritogenic effect of 70% ethanol extract from kochiae fructus and its active component].

The antipruritogenic effect of the 70% ethanol extract obtained from Kochiae Fructus (fruits of Kochia scoparia) and its active components were investigated on a compound 48/80-induced pruritogenic model in male ddY strain mice. The extract (200, 500 mg/kg, p.o.) inhibited the scratching behavior as a pruritogenic indicator. Oleanolic acid oligoglycoside, momordin Ic, isolated from the extract also exhibited the inhibition. These results suggest that Kochiae Fructus could be used as an antipruritogenic agent and its inhibitory effect may be partially attributed to momordin Ic.

Animals

Relationship between the antidiarrhoeal effects of Hange-Shashin-To and its active components.

This study was designed to examine the relationship between the antidiarrhoeal effects of Hange-Shashin-To (TJ-14) and its active components. Oral treatment with TJ-14 at 1000 mg/kg significantly inhibited castor oil-induced diarrhoea. Both the 50% methanol eluate fraction (fraction III) and the methanol eluate fraction (fraction IV) showed antidiarrhoeal effects at oral doses of 68 mg/kg and 63 mg/kg, respectively, corresponding to 1000 mg/kg of TJ-14. TJ-14 (1000 mg/kg, p.o.) showed a significant increase in blood corticosterone levels. Increased blood corticosterone was noted after the oral administration of 63 mg/kg of fraction IV. The inhibitory activity of TJ-14 on cyclooxygenase-2 (COX-2) was also observed in fractions III and IV. The main component of fraction III was Scutellariae Radix-derived baicalin. Fraction IV contained Glycyrrhizae Radix-derived glycyrrhizin and isoliquiritin, Coptidis Rhizoma-derived berberine, coptisine and palmitine. Ginseng Radix-derived saponins were also present in fraction IV. These compounds inhibited castor-oil induced diarrhoea at oral doses of 10 or 30 mg/kg. Thus, the present results indicate that Scutellariae Radix, Glycyrrhizae Radix, Ginseng radix and Coptidis Rhizoma-derived components are involved in the antidiarrhoeal action of TJ-14.

Animals

Relative effectiveness coefficient: a quality characteristic of toothpastes containing active components.

It has been proposed that the parameter of relative effectiveness coefficient (REC) be used for the qualitative assessment of toothpastes containing active ingredients. REC is the ratio between the concentration of the active component in water eluates obtained after three minutes and adequately prolonged (up to the reaching of equilibrium state) dispersion of the toothpaste in distilled water at a 1:4 ratio (condition simulating the use of toothpaste in the oral cavity). The change in REC after storage following its production, as well as testing the toothpaste stability at high and low temperatures, provides an evidence for deviations in its quality. REC was applied for the assessment of toothpastes containing 0.5% zinc citrate as an active ingredient.

Aluminum Oxide

[Protective effect of active components extracted from radix Astragali on human erythrocyte membrane damages caused by reactive oxygen species].

A study has been made on the protective activities of active components extracted from Radix Astragali by using reactive oxygen species initiated lipid peroxidation of purified human erythrocyte membrane. The results show that the total flavonoids of Astragalus and total saponins of Astragalus can significantly inhibit the membrane lipid peroxidation generated by O2.-, H2O2 and UV rays, while the total polysaccharide of Astragalus possesses weaker protective activity.

Drugs, Chinese Herbal

Presence of a serine protease in the complement-activating component of the complement-dependent bactericidal factor, RaRF, in mouse serum.

A 100-kDa protein was isolated from the complement-activating component of mouse Ra-reactive factor. It generated a 29-kDa chain upon reduction. Partial amino acid sequences of the 29-kDa chain were determined after fragmentation with CNBr. A 386-bp-long probe was synthesized by the polymerase chain reaction with oligonucleotide primers designed by reference to the amino acid sequence. Using this probe, we cloned a 3.7-kb DNA from a mouse liver cDNA library. The amino acid sequence of the 29-kDa chain deduced from the nucleotide sequence of this cDNA was consistent with the amino acid sequences determined. The full sequence was found to have characteristics of a serine protease and exhibited 29% and 30% homologies with those of light chains of the C1r and C1s subcomponents of complement, respectively.

Amino Acid Sequence

Improved retention of the EMS activation component (EMSAC) in adult CPR education.

This study was undertaken to determine whether using a model-telephone to simulate the emergency medical services activation component (EMSAC) during adult cardiopulmonary resuscitation (CPR) training practice would lead to better retention of this component during end-of-class assessment. In a prospective randomized manner, 233 medical professionals and lay-persons taking American Heart Association (AHA) CPR classes were evaluated for EMSAC retention during CPR skills performance at the end of class. During the assessment correct versus incorrect activation of EMS was noted. Subject response by age, exposure to previous CPR training, and medical professional or lay-person status was examined. Differences in results among instructors also were examined. Overall, those in the group using the model-telephone remembered to activate EMS correctly more frequently than those in the group not using the phone (69 vs 52%, P = 0.009). The < 30 age group was unaffected by the use of the phone (P = 0.85). The group between 30 and 50 years of age did significantly better with the use of the phone (P = 0.007), as did those 50 years of age and older (P = 0.03). Previous CPR training did not affect the response (P = 0.18). We conclude that use of the model-telephone improved EMSAC retention significantly overall except in the < 30 year-old age group. We recommend using the model-telephone in future adult CPR classes.

Adolescent

[Analysis of the informational and activating components of reinforcement in a conditioned reflex experiment].

The reinforcement in adaptive behaviour performs at least two functions: satisfaction of specific need (activational component) and estimation of signal significance of the conditioned stimulus while comparing the nervous model of the stimulus with current afferentation (informational component). In usual conditioning experiments with permanent reinforcement these components concide and therefore do not differ. However, they may be separated in the case of probabilistic reinforcement by changing stimuli contingency and shifting motivational level. With this aim the diagrams of probabilistic stimuli and reactions space, semantic and pragmatic matrices are worked out, allowing to estimate correlations of these parameters of conditioning experiments. Comparative psychophysiological investigations showed the possibility of determination of species peculiarities in behavioural choice strategies among various animals under above mentioned conditions.

Adaptation, Physiological

Glycyrrhizin, an active component of licorice roots, reduces morbidity and mortality of mice infected with lethal doses of influenza virus.

The antiviral effect of glycyrrhizin (GR), an active component of licorice roots, was investigated in mice infected with influenza virus A2 (H2N2). When mice that had been exposed to 10 50% lethal doses of the virus were treated intraperitoneally with 10 mg of GR per kg of body weight 1 day before infection and 1 and 4 days postinfection, all of the mice survived over the 21-day experimental period. At the end of this period, the mean survival time (in days) for control mice treated with saline was 10.5 days, and there were no survivors. The grade of pulmonary consolidations and the virus titers in the lung tissues of infected mice treated with GR were significantly lower than those in the lung tissues of infected mice treated with saline. GR did not show any effects on the viability or replication of influenza virus A2 in vitro. When splenic T cells from GR-treated mice were adoptively transferred to mice exposed to influenza virus, 100% of the recipients survived, compared to 0% survival for recipient mice inoculated with naive T cells or splenic B cells and macrophages from GR-treated mice. In addition, the antiviral activities of GR on influenza virus infection in mice were not demonstrated when it was administered to infected mice in combination with anti-gamma interferon (anti-IFN-gamma) monoclonal antibody. These results suggest that GR may protect mice exposed to a lethal amount of influenza virus through the stimulation of IFN-gamma production by T cells, because T cells have been shown to be producer cells of IFN-gamma stimulated with the compound.

Adoptive Transfer

Approximative molecular weight of the active component in toxoplasmin.

The approximative molecular weight of toxoplasmin -- a skin reactive extract from Toxoplasma gondii -- was estimated by ultrafiltration through different Amicon membranes. The activity of the filtered and unfiltered product was compared with the aid of intradermal test on humans. The results indicate that the molecular weight of an active component in toxoplasmin is in the range from 10 000 to 50 000.

Antigens

Localization of the genes for the 100-kDa complement-activating components of Ra-reactive factor (CRARF and Crarf) to human 3q27-q28 and mouse 16B2-B3.

Human and mouse genes for the complement-activating component (P100) of Ra-reactive factor, a novel bactericidal factor (CRARF and Crarf), were mapped to R-banded metaphase chromosomes by fluorescence in situ hybridization with human and mouse P100 cDNA 2.7 and 2.0 kb long, respectively. The localization of fluorescent signals showed that CRARF and Crarf mapped to human 3q27-q28 and mouse 16B2-B3, respectively. This evidence is consistent with the previous assumption that the distal portion of the long arm of human chromosome 3 is homologous to the proximal portion of mouse chromosome 16.

Animals

Migration behavior and separation of active components in Glycyrrhiza uralensis Fisch and its commercial extract by micellar electrokinetic capillary chromatography.

A micellar electrokinetic capillary chromatography (MECC) method for the separation and determination of five components, glycyrrhizic acid (GA), glycyrrhetinic acid (GTA) and 3,4'-dimethoxy-5-hydroxychalone, fermononetin and isoliquiritigenin, in extracts of Glycyrrhiza uralensis Fisch root was developed. Migration behavior of these analytes was studied by the systematic examination of the borate and sodium dodecyl sulfate (SDS) concentrations in the run buffer. The optimum separation for these analytes was achieved using 10 mmol l(-1) of tetraborate and 25 mmol l(-1) of SDS as the running buffer, with 17 kV of applied voltage. All experiments were performed using a 50.0 cm (42.4 cm effective length)x75 microm I.D. of fused-silica capillary. The apparent pKa values of GA and GTA and the binding constants for the association between the above five analytes and SDS were calculated in this study. A comparison of the extraction efficiency for GA and GTA from Glycyrrhiza uralensis Fisch root was made using ethanol, distilled water and chloroform as extraction reagents, respectively. These results provided very useful information to select the proper solvent to extract the desired components in Glycyrrhiza uralensis Fisch. The MECC method established in this paper was employed to analysis the above five active components.

Acids

Comparison of fluorescence and photodynamic activities of whole hematoporphyrin derivative and its enriched active components.

The in vivo biologic activities of the hematoporphyrin derivative (Photofrin) and the enriched, so-called "active fraction" (Photofrin II) were determined by measuring the necrosis produced in implanted tumors in DBA/2Ha mice exposed to various total doses of light (20-100 J/cm2) after ip administration of 10 mg/kg standard doses of either Photofrin or Photofrin II. Total relative percentage increase in fluorescence in tumor tissue, as compared to fluorescence in control tissue, also was measured for both Photofrin and Photofrin II. In response to total light doses (630 nm) of 40-100 J/cm2, mice that received Photofrin had comparable amounts of tumor necrosis to those mice that received Photofrin II. At doses of 40-60 J/cm2, 80% tumor destruction resulted, and at 80-100 J/cm2, tumor destruction was 100%. However, at a total light dose of 20 J/cm2, the tumors that received Photofrin II exhibited 60-80% tumor necrosis, whereas those animals that received Photofrin had only small areas of patchy necrosis associated with signs of vascular thrombosis and hemorrhage into the surrounding perivascular stroma. A 25.2% total increase in maximal tissue fluorescence over that in controls was observed for animals that received Photofrin II, as compared to 13.9% for those animals that received Photofrin. It is concluded that the greater demonstrable efficacy of treatment with Photofrin II, as compared to treatment with Photofrin, is due to enrichment of those nonpolar hydrophobic components of the hematoporphyrin derivative mixture that are thought to be primarily responsible for the in vivo biologic activities.

Animals

Two isoforms of the mouse ether-a-go-go-related gene coassemble to form channels with properties similar to the rapidly activating component of the cardiac delayed rectifier K+ current.

HERG, the human ether-a-go-go-related gene, encodes a K(+)-selective channel with properties similar to the rapidly activating component of the delayed rectifier K+ current (IKr). Mutations of HERG cause the autosomal-dominant long-QT syndrome (LQTS), presumably by disrupting the normal function of IKr. The current produced by HERG is not identical to IKr, however, and the mechanism by which HERG mutations cause LQTS remains uncertain. To better define the role of Erg in the heart, we cloned Merg1 from mouse genomic and cardiac cDNA libraries. Merg1 has 16 exons and maps to mouse chromosome 5 in an area syntenic to human chromosome 7q, the map locus of HERG. We isolated three cardiac isoforms of Merg1: Merg1a is homologous to HERG and is expressed in heart, brain, and testes, Merg1a' lacks the first 59 amino acids of Merg1a and is not expressed abundantly, and Merg1b has a markedly shorter divergent N-terminal cytoplasmic domain and is expressed specifically in the heart. The Merg1 isoforms, like HERG, produce inwardly rectifying E-4031-sensitive currents when heterologously expressed in Xenopus oocytes. Merg1a and HERG produce currents with slow deactivation kinetics, whereas Merg1a' and Merg1b currents deactivate more rapidly. Merg1b coassembles with Merg1a to form channels with deactivation kinetics that are more rapid than those of Merg1a or HERG and nearly identical to IKr. In addition, a homologue of Merg1b is present in human cardiac and smooth muscle. Thus, we have identified a novel N-terminal Erg isoform that is expressed specifically in the heart, has rapid deactivation kinetics, and coassembles with the longer isoform in Xenopus oocytes. This N-terminal Erg isoform may determine the properties of IKr and contribute to the pathogenesis of LQTS.

Amino Acid Sequence