As it is important to determine the optimal serum concentration of schizophyllan (SPG) when it is used as an anti-cancer drug, we devised a solid-phase ELISA. We also developed a sandwich ELISA using murine anti-SPG monoclonal antibody as the first antibody and rabbit anti-SPG serum as the second antibody in order to improve the detection sensitivity. This assay was able to determine SPG concentrations over 1.0ng/ml and the absorbance at 490 nm was directly proportional to the SPG concentration. SPG in rabbit serum, obtained after intravenous and intramuscular injection (SPG ; 10 mg/kg), was determined by this sandwich ELISA. Furthermore, the sensitivity of this ELISA method was compared with that of the Limulus test. The Limulus test is able to detect SPG in physiological saline (pH 6.3) at concentrations greater than 1.0 μg/ml, but the sensitivity increased when SPG was dissolved in alkaline solution (pH 12.0), enabling SPG to be measured almost down to 1.0 ng/ml. These data suggest that our sandwich ELISA may be used for the measurement of SPG in blood or tissue.
4-Isopropenyltoluene (8) was administered orally to rabbits and the following four optically active metabolites, 2-(p-tolyl) propanoic acid (10), p-(1-carboxyethyl) benzoic acid (11), 2-hydroxy-2-(p-tolyl) propanoic acid (12), and 2-(p-tolyl)-1, 2-propanediol (13) were isolated from urine in addition to an optically inactive metabolite, 4-isopropenylbenzoic acid (9). The enantiomeric ratios of the metabolites 10 and 11 were respectively R/S=33 : 67 and 39 : 61 (S predominance), whereas those of the metabolites 12 and 13 possessing a tertiary hydroxyl group were R/S=77 : 23 and 84 : 16 respectively (R predominance). The presumed metabolic pathways of 8 in rabbits leading to these metabolites are discussed.
Isofatty acid-containing phosphatidylglycerol (Fr. 7-C), isolated from Bacillus stearothermophilus UBT8038, enhances the induction of concanavalin A (Con A)-activated suppressor T (Ts) cells in a dose dependent manner (0.01-1μg/ml). Its further biological properties on mouse mixed lymphocyte reaction (MLR) has been demonstrated. Fr. 7-C (0.01-1 μg/ml) suppressed the MLR at 4 d in a dose-dependent manner when added at the start of splenocyte cultivation. Moreover, Fr. 7-C was effective in preventing the generation of cytotoxic T lymphocytes after the MLR. On the other hand, this fraction significantly enhanced the induction of Ts cells in the MLR carried out in any of the antigen-specific, antigen-nonspecific and major histocompatibility complex antigen-nonrestricted fashions. Fr. 7-C increased prostaglandin E2 (PGE2) release approximately 2-fold in the culture supernatant of Con A-activated splenocytes, and PGE2 release decreased dose-dependently when cultured with indomethacin. The inhibitory effect by Fr. 7-C on the MLR was abrogated by the addition of indomethacin. The enhancement by Fr. 7-C on Ts cell induction was blocked by indomethacin in a dose dependent manner. These results strongly suggest that Fr. 7-C suppresses the MLR via the enhancement of antigen-nonspecific Ts cell induction mediated at least partly by PGE2.
Cytotoxic actions of alloxan are thought to be caused by the hydroxyl radical (HO·) generated in a cyclic reaction involving alloxan and its reduction product, alloxan radical (HA·). The generation of HA· and the oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) were observed in the reaction system of alloxan with NADPH in the presence of purified NADPH : cytochrome P-450 reductase [EC 220.127.116.11] (fp2) from rat liver microsomes. Both the generation of HA· and the oxidation of NADPH were increased with increasing the concentrations of alloxan or fp2. These results indicate that NADPH-linked HA·generation was catalyzed by fp2. The HA·was generated in the reaction of alloxan with a homogenate of rat islet cells. The generation of HA· in the reaction of alloxan with the homogenate of rat islet cells was markedly diminished by an immunogloblin fraction (Ig) of anti-fp2 serum, but not by control-Ig. The anti-fp2-Ig also inhibited the generation of HA· in the reaction system of alloxan with NADPH in the presence of fp2. When the homogenate of islet cells from phenobarbital-treated rats was used, the generation of HA· was signiflcantly increased in comparison with that from the control rats. However, the homogenate of islet cells from 3-methylcholanthrene-treated rats did not have such a stimulative effect. These results suggest that the reduction of alloxan to HA· was catalyzed by fp2 presented in islet cells.
Two acidic polysaccharides, called rehmannan FS-I and rehmannan FS-II, were isolated from the raw root of Rehmannia glutinosa LIBOSCHITZ. They were homogeneous on electrophoresis and gel chromatography, and their molecular masses were estimated to be 5.8×104 and 6.6×104, respectively. Rehmannan FS-I is composed of L-arabinose : D-galactose : L-rhamnose : D-galacturonic acid in the molar ratio of 68 : 40 : 4 : 84 : 3 ; rehmannan FS-II is composed of L-arabinose : D-galactose : L-rhamnose : D-galacturonic acid in the molar ratio of 18 : 15 : 4 : 33, in addition to small amounts of O-acetyl groups. About two-thirds (rehmannan FS-I) and about one half (rehmannan FS-II) of the hexuronic acid residues exist as methyl esters. Methylation analysis of the carboxyl-reduced derivatives and nuclear magnetic resonance studies indicated that their structural features include mainly both arabino-3, 6-galactan and rhamno-galacturonan type structural units. Both polysaccharides showed remarkable reticuloendothelial system-potentiating activity in a carbon clearance test.
A water-insoluble, alkali-soluble, glucan (AM-APP), [α]D + 160° in 0.4м NaOH, was isolated from the alkaline extract of the fruiting bodies of Amanita muscaria. The results of chemical and spectroscopic investigations indicate that AM-APP is a linear (1→3)-α-D-glucan with a molecular weight estimated by gel chromatography of about 42000. Its carboxymethylated product (AM-APP-CM) showed potent antitumor activity against sarcoma 180 in mice, although the native polysaccharide (AM-APP) had little effect. The distribution of carboxymethyl groups in the molecule was analyzed by gas chromatography and mass spectrometry. The degree of substitution of carboxymethyl groups was 0.95 and the substituents were located at O-2, at O-4, at O-6, at O-2 and O-6, and at O-4 and O-6 on glucose.
Human chorion, but not amnion, tissue explants produced substantial quantities of neutrophil chemoattractant in response to interleukin 1 (IL-1) and tumor necrosis factor α (TNFα). This suggested that chorion is one of the chemoattractant producing tissues. Therefore, the biochemical properties and the regulation of a chemoattractant in human chorionic cells were examined. IL-1α and TNFα stimulated human chorionic cells to produce neutrophil chemotactic factor in both a dose- and time-dependent manner. This chemotactic factor was a heat-stable and trypsin-sensitive protein with an apparent molecular weight of 10000, and it was also immunologically identified as a chemotactic cytokine of the human IL-8 family. Immunohistochemical observations with IL-1α-and TNFα-treated chorion explants indicated that trophoblasts and stromal cells, including fibroblast-like and macrophage-like cells, but not endothelial cells, were characterized as IL-8-producing cells. From these observations, it is very likely that both IL-1 and TNFα may participate in the production of chemotactic factor/IL-8 in pre-term parturition, accompanied by an intraamniotic infection, along with their known promotive effect on the production of matrix metalloproteinases, which is connected with the destruction of matrix components of fetal membranes.
Benidipine and amlodipine, 1, 4-dihydropyridine calcium channel blocking drugs, are long-acting antihypertensive and antianginal drugs. In the present study, the vascular-selectivity and duration of action of benidipine were determined in vitro and in vivo, and compared with those of amlodipine. The relaxing effect of benidipine on the canine coronary artery precontracted by KCl (55 mм) was about 40 times that of amlodipine. The negative inotropic effect of benidipine in the electrically-stimulated canine right ventricular papillary muscle was about twice that of amlodipine. The potency ratios of the vasorelaxing effect in the coronary artery and the negative inotropic effect in papillary muscle were 1300 for benidipine and 67 for amlodipine, respectively. In anesthetized dogs, the maximum hypotensive effect and the duration of action of 3μg/kg (i.v.) benidipine was almost the same as those of 500 μg/kg (i.v.) amlodipine. The duration of the hypotensive action of benidipine at 10 μg/kg (i.v.) was almost the same as that of amlodipine at 1500 μg/kg (i.v.). Amlodipine at 1500 μg/kg (i.v.) reduced mean blood pressure and left ventricular dp/dt max immediately after its administration, whereas such transient falls were not observed after the administration of benidipine at 10μg/kg (i.v.). These results suggest that benidipine possesses a stronger vasodilating effect and a higher vascular-selectivity, compared with amlodipine.
The acute effects of DX-9386, a traditional Chinese medicinal preparation (ginseng, polygala, acorus and hoelen), were studied on learning and memory performances in passive and active avoidance tests in normal, as well as in learning-impaired, mice. A single oral administration of the prescription had no effect on memory registration, consolidation or retrieval or on motor activity in normal mice. DX-9386 reduced the ethanol-induced impairment of memory registration in the step-down test and also tended to ameliorate the scopolamine-induced memory registration deficit in the same test. The preparation reduced the number of spontaneous responses in the active avoidance test. The preparation also prolonged the disappearance of righting reflex induced by pentobarbital. These results suggest that DX-9386 ameliorates the impairment effect of ethanol on learning and memory processes and also exhibits a sedative effect.
We studied the effects of neurotrophic factors on neurite extension and branching of embryonic chick sensory neurons in culture after damage to their filopodia by an irradiating laser beam. The factors employed were nerve growth factor (NGF), basic fibroblast growth factor (bFGF), ganglioside GM1 (GM1), laminin (LAM), and combinations of bFGF plus NGF, and LAM plus NGF. Neither NGF, bFGF, nor NGF plus bFGF affected either the neurite extension or the amount of branching of damaged and intact neurites. Tenμg/ml of GM1 tended to enhance the extension of damaged neurites. One hundred μg/ml of LAM significantly enhanced the elongation of damaged neurites but not intact ones. One hundred μg/ml of LAM with 10 ng/ml of NGF significantly elongated the neurite lengths of both damaged and intact neurites. These results suggest that LAM plays an important role in neurite extension after damage to peripheral sensory neurons and that NGF modulates the effect of LAM.
DX-9386 is a traditional Chinese prescription consisting of ginseng, polygala, acorus and hoelen. The effect of chronic oral treatment with this preparation on learning behaviors and lipid peroxide concentration was studied in senescence accelerated mouse (SAM). SAM P8, a senescence prone of SAM, and SAM R1, a senescence resistant substrain of SAM, were started on a diet containing 1% of DX-9386 from the age of 2 months. All the experiments were performed at the age of 10 months. The prescription ameliorated the memory disorders of SAM P8, as evaluated in a step down test as well as a spatial memory test. The preparation, however, did not affect the learning behaviors in SAM R1. DX-9386 reduced the elevated levels of lipid peroxide in the serum and liver of SAM P8, while it did not alter that in SAM R1. These results suggested that DX-9386 slowed the aging process of SAM P8 in terms of learning behaviors and lipid peroxidation.
Ginseng extract and its active component, saponin, were administered orally to nephrectomized rats for 90 d, and changes in blood and urine parameters and renal tissue lesions were assessed. Rats given saponin showed a significant decrease in the concentrations of blood urea nitrogen, creatinine and methylguanidine and a significant increase in total protein and albumin in the blood, with reduced urinary excretion of protein. There was also slight amelioration of the degree of mesangial proliferation, the severity of extratubular lesions and glomerular sclerotic lesions, and the extent of tubular interstitial lesions. However, these histological changes were inconspicuous in nephrectomized rats given ginseng extract.
The potential usefulness of oil in water (O/W) lipid emulsions as parenteral drug delivery system for lipophilic drugs was examined in tumor-bearing rats. A model lipophilic drug, sudan II (PCoct=226000), was formulated in five lipid emulsions consisting of soybean oil and various surfactants. Compared with HCO-60 micellar and plasma solutions of sudan II, the blood concentration of sudan II was markedly elevated by administration as a lipid emulsion. However, the distribution of sudan II to the liver, lungs, spleen, and adipose tissue was not altered, and that to the brain, heart, kidneys, muscle, and tumor was slightly decreased. To understand these results, pharmacokinetic analysis was performed using a newly derived compartmental model, and moreover, the organ distribution clearance was analyzed. It was suggested that the oil particles deliver the incorporated drug selectively to the liver, lungs, and spleen, and the speed of delivery could be surpressed by using HCO-60. However, in the case of sudan II, its rapid release from the oil particles after i.v. injection prevented a drastic alteration in the distribution of sudan II. The simulation studies suggested that a considerable decrease in the release rate or an increase in partition coefficient (experimentally more than 108) would be required for delivery.
The absorption of an antibiotic, latamoxef sodium (LMOX), following the rectal administration of a suppository containing adjuvants was investigated. A lipophilic base (Witepsol H15) was used. The rectal absorption of LMOX following the administration of a suppository without adjuvants was very low. Diclofenac sodium (DF) was used as an absorption promoter ; it enhances rectal membrane permeability. The blood level of LMOX following the addition of DF (10 mg) to the base was increased only about 1.3-fold compared with that achieved with LMOX alone (difference not significant) ; even with a higher dose of DF, the absorption of LMOX was not sufficient. The release rate of LMOX from the base was slow. When Tween 80, a non-ionic surfactant, was added to improve the release rate of LMOX, the rate was sufficiently increased. The rectal absorption of LMOX on the addition of both Tween 80 and DF was markedly increased compared to that achieved with LMOX alone or with DF. These results indicate that the rectal absorption of LMOX after administration by a suppository was sufficiently improved by enhancing both the release rate from the base and the membrane permeability of the rectum. Lymphatic uptake and blood levels of LMOX were also investigated after the rectal administration of the LMOX preparation containing both Tween 80 and DF ; the lymphatic uptake of LMOX was significantly enhanced compared with the LMOX preparation in which only DF was used as an adjuvant. The mechanism whereby adjuvants lead to the absorption of a non-absorbable drug, and the subsequent drug transportation routes through the membrane are discussed.
Unconjugated steroids were extracted with dichloromethane from the serum of pre-term neonates. Conjugated steroid fraction in the residue was separated and purified by high performance liquid chromatography. The purified steroid fraction was subjected to enzyme (arylsulfatase from Helix pomatia) or acid hydrolysis. Liberated steroid was analyzed by gas chromatography-mass spectrometry (GC/MS) and gas chromatography-Fourier transformation infrared spectroscopy (GC/FT-IR). All of the results obtained from GC/MS and GC/FT-IR analysis were identical with those of authentic 16-dehydropregnenolone (3β-hydroxy-5, 16-preguadien-20-one, 16-DHP). This is the first report to demonstrate the presence of 16-DHP in human blood.
The binding properties of Sudlow's site-specific drugs to glycosylated bovine serum albumin (G-BSA) (1.25 mol glucose per mol of albumin) have been investigated using the circular dichroism (CD) method. Site I-specific drugs, phenylbutazone and warfarin, and site II-specific drugs, flufenamic acid and ibuprofen, were used. The induced ellipticities of phenylbutazone, flufenamic acid and ibuprofen-G-BSA complexes diminished and those of warfarin complex were enhanced in comparison with those for the intact bovine serum albumin (BSA) complexes. These CD change suggests that the glycosylation of BSA at the primary modification site influences the binding properties of the site-specific drugs to serum albumin.
Antibody for (1→6)-branched (1→3)-β-D-glucan was prepared using rodents. An antitumor (1→6)-β-monoglucosyl branched (1→3)-β-D-glucan (GRN : grifolan) was conjugated with bovine serum albumin and used as an immunogen. The antibody titer in serum was determined by ELISA using biotin-conjugated GRN. Administration of the antigen raised the antibody titer only in the rabbit, with mouse and rat showing no significant antibody titer for the glucan. The antigen specificity of the anti-GRN antibody was determined by competitive ELISA. The rabbit anti-GRN antibody bound to structurally related antitumor (1→6)-branched (1→3)-β-D-glucans such as lentinan, schizophyllan and SSG, whereas it did not react with linear (1→3)-β-D-glucan, curdlan or GRN-derivatives obtained by periodate-oxidation and Smith degradation. These facts strongly suggest that the hapten site of the antibody was the monoglucosyl branched moiety of (1→3)-β-D-glucan. These results indicate that this antibody would be a useful probe for the detection of (1→6)-branched antitumor glucans administered to the host.
Semotiadil fumarate, a novel benzothiazine calcium antagonist, was given alone or in combination with either enalapril or trichlormethiazide to conscious, spontaneously hypertensive, rats daily for 2 weeks. Systolic blood pressure and heart rate were recorded 24h before the start of the regimen and then every 2 and 24h after the 1st, 3rd, 7th, 10th and 14th doses. When given alone, the antihypertensive effects of semotiadil (10mg/kg, p.o.) and enalapril (5mg/kg, p.o.) first became apparent after the 3rd dose and thereafter the effects appared to develop daily although this effect had waned by the time of the next dose. When given in combination, however, these drugs appeared to potentiate each other and after the 7th dose, the antihypertensive effect persisted. Trichlormethiazide (30mg/kg, p.o.) alone failed to exert any significant antihypertensive effect and in combination was not always additive to that of semotiadil. In contrast to the effect on blood pressure, the heart rate remained resistant to all these drugs. These results indicate that combined daily dosing of semotiadil, especially with enalapril, each at relatively low doses may be able to control hypertension in a continuous manner.
To compare proteinuric potentials among angiotensin II (ANG II) and its fragments, [des-Asp1]-angiotensin II (ANG III) and [des-Asp1, des-Arg2]-angiotensin II (ANG IV), the peptide was intravenously infused for 30 min at doses of 0.015, 0.05, 0.15, 0.45 and 1.45 nmol/kg body weight/min. The infusion of ANG II and ANG III increased the fractional clearance of albumin in a dose-dependent manner : most extensively for ANG II, and moderately for ANG III. In contrast, the infusion of ANG IV hardly showed any proteinuric action, even at the maximal dose of 1.45 nmol/kg body weight/min. These results denoted that the cleaving of the N-terminal aspartic acid1 from ANG II weakened the proteinuric action in the glomeruli, and the further cleaving of the N-terminal arginine2 from ANG III led to a complete loss of proteinuric action in the glomeruli.
Two flavonoids, 3, 5, 6, 7, 8, 3', 4'-heptamethoxyflavone (HEPTA) and natsudaidain isolated from Citrus plants (Rutaceae), produced a positive inotropic effect (PIE) on guinea-pig papillary muscle. Natsudaidain (pD2 4.98±0.07) was more potent than HEPTA (pD2 4.33±0.08), but the maximum PIE of HEPTA was greater than that of natsudaidain. The PIE of HEPTA was completely inhibited by reserpinization of the guinea pig, and partially inhibited by metoprolol and carbachol. The carbachol inhibition was omitted by atropine. The mechanism of PIE of HEPTA is accounted for catecholamine release from cardiac tissue.
Sennoside A content in hot-water extracts from 17 varieties of rhubarb obtained from the market was measured, and their respective cathartic effects were also examined in mice. A positive correlation was confirmed between the sennoside A content and cathartic effects in our experiments. Furthermore, the effects of Shisendaio ( ?? ?? ?? ?? ), with a higher sennoside A content, and Kinmondaio ( ?? ?? ?? ?? ), with a lower sennoside A content, exhibited endotoxin-induced disseminated intravascular coagulation (DIC) which related closely to Oketsu symptoms, and these effects were examined in rats. Kinmondaio exhibited weak inhibition on both reducing erythrocyte deformability and prolonging euglobulin lysis time (ELT) in DIC rats. From our results, it is possible to evaluate rhubarb's cathartic effect, one of its main drug effects, by examining the rhubarb's sennoside A content. However, it is difficult to estimate the cathartic effect according to the general market name of Rhubarb. No dramatic effect was found on the experimental models used for Oketsu symptoms such as endotoxin-induced DIC.
A cyclosporine derivative, dihydrocyclosporine D, was used for the evaluation of milk fat globule membrane (MFGM) as an emulsifier of lipophilic cyclopeptides. As compared with olive oil formulation, MFGM emulsion significantly enhanced the blood and lymphatic fluid concentrations of the cyclosporine derivative after intraduodenal dosing in rats. Thus, it was suggested that MFGM can be used as an intestinal absorption enhancer of cyclosporines.
The genetic similarities of four species of Glycyrrhiza plants were determined by DNA analyses of random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). The phylogenic trees were constructed from the genetic similarities estimated from RAPD and RFLP profiles. These results indicated that G. glabra and G. uralensis, rich in glycyrrhizin, are more closely related to each other than to G. echinata or to G. pallidiflora.
The effect of phenyl- and benzylphthalimide analogs on tumor necrosis factor (TNF)-α production by a human leukemia cell line, HL-60, stimulated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was investigated. Though non-substituted phenyl- and benzyl-phthalimide had no effect on TNF-α production after TPA-induction, introduction of methyl group (s) onto the phenyl group resulted in the appearance of potent enhancing activity on TNF-α production.