Using a validated high-performance liquid chromatographic (HPLC) method, the pharmacokinetics of multi-constituents in Huangqin-Tang decoction were simultaneously studied both in the compound prescription and in each single herb decoction. At different intervals (0, 1, 2, 4, 8, 12, 24, 36, 48 h) after oral administration of the Huangqin-Tang decoction or a single herb decoction at a dose of 10 g·kg−1, the concentrations of the constituents and their metabolites: baicalin (BG), wogonoside (WG), oroxylin-A-glucuronide (OG), baicalein (B), wogonin (W), oroxylin-A (O), paeoniflorin (PF), paeonimetabolin-I (PM-I), liquiritin (LG), liquiritigenin (L), glycyrrhizic acid (GL) and glycyrrhetinic acid (GA), were detected in the rat plasma. A new metabolite—3,5,7,2′,6′-penta hydroxy flavone (visidulin I, VD-I) was found in rat plasma after oral administration of Huangqin-Tang or a single herb Huangqin decoction, and the quality was identified by HPLC and LC/MS. The pharmacokinetic parameters of the constituents and metabolites in the compound prescription and single herb decoctions were compared. All concentration–time curves corresponded to the one-compartment model. The constituents of BG, WG, OG, VD-I and LG had higher Cmax and AUC0—lim in the compound prescription than in the single herb decoction, and WG had significant difference. The constituents of PF, W and O only had a higher AUC0—lim in the compound prescription, and O had a significant difference. It was concluded, in brief, that there were obvious differences in the pharmacokinetic parameters of most constituents (especially constituent WG) between the compound prescription and single herb decoction. The constituents in the compound prescription had delayed absorption and elimination, a longer residence time in the body, and higher Cmax and AUC0—lim, than those in the single herb decoction. Therefore, they were more efficient and durable, making them promising to exerting pharmacological effects in vivo.
Synthetic decarboxylated S-adenosyl-L-methionine (dcAdoMet), a mixture of the absolute configuration of S and R at the sulfonium center, was evaluated as a substrate for the measurement of spermidine synthase activity. The diastereomers were separated by HPLC with an isocratic elution, and the constant for racemization at the sulfur was determined to be 2.4×10−6 s−1 at 37 °C and pH 1.5 for the first-eluted biologically active isomer (S-dcAdoMet) and 2.0×10−6 s−1 for the second-eluted biologically inactive isomer (R-dcAdoMet). The peak area ratio of S-dcAdoMet to R-dcAdoMet of 48 to 52 in HPLC supported the different racemization constants. Similar substrate activity of dcAdoMet to that of S-dcAdoMet was demonstrated by enzymatic spermidine synthesis. It was shown from the result that the racemized [methyl-14C]dcAdoMet prepared in this report was useful for measuring spermidine synthase activity.
Bacillus cereus sphingomyelinase belongs to the Mg2+-dependent neutral sphingomyelinase, which hydrolyses sphingomyelin to phosphocholine and ceramide, and acts as an extracellular hemolysin. The triplet residues, His151–Asp195–His296, of the enzyme are highly conserved among bacterial and mammalian Mg2+-dependent neutral sphingomyelinases. The triplet residues converge on the active-site pocket of the 3D model of the enzyme. To investigate the function of these residues in the acid-base catalysis, we introduced several mutations for each residue by site-directed mutagenesis. Hemolytic and hydrolytic activities of the enzyme, abolished by the mutations at Asp195 and His296, revealed that these residues are critical for the catalytic function. The effect of the divalent metal cations on the pH dependency of the hydrolytic activities indicates that His296 corresponds to the most acidic ionizable group as a general base. The mutagenesis at His151 was also deleterious; however, the H151A and H151Q mutant enzymes partially retained their activities. The H151A mutation affected the most basic ionizable group, suggesting that His151 may act as a general acid in catalysis. By the structural basis of the 3D model, Asp195 must maintain not only the appropriate spatial arrangement but also pKas of His151 and His296. Taking into consideration all of these, we proposed the acid-base catalytic mechanism of B. cereus sphingomyelinase.
Mucus is an important factor in gastric mucosal protection against acid, pepsin and various factors such as alcohol and nonsteroidal anti-inflammatory drugs. MUC5AC is a gel-forming mucin secreted from gastric surface mucous cells. However, little is known about expression of the MUC5AC gene. We examined developmental changes in rat MUC5AC mRNA expression and the effect of glucocorticoid on MUC5AC mRNA expression in infant rat gastric mucosa. Expression levels of MUC5AC mRNA in the stomach of 0 to 30-d-old and 8-week-old (adult) rats were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and by in situ hybridization. We also examined pepsinogen C (PgC) and F (PgF) mRNA expression by RT-PCR. The expression of MUC5AC mRNA increased from 10 d of age, which was about one week earlier than that of PgC mRNA. The expression of PgF mRNA decreased as that of PgC mRNA increased. The injection of hydrocortisone induced PgC mRNA expression in the infant rat stomach, whereas MUC5AC and PgF mRNA expression decreased. These results suggest that developmental changes of MUC5AC mRNA expression differ from those of Pgs, and are not induced by glucocorticoid.
Conditions that perturb the function of the endoplasmic reticulum (ER) lead to an accumulation of proteins and subsequent induction of several responses, such as an increased expression of ER-resident chaperones involved in protein folding and activation of c-jun N-terminal kinase (JNK). These responses are mediated by a transmembrane kinase/ribonuclease, IRE1, which transduces the signal from the ER lumen to the cytosol. Although nuclear transcription factor-κB (NF-κB) is also activated by ER stress, whether this response depends on IRE1 is unknown. In this study, we show that IRE1 is involved in the activation of NF-κB induced by ER stress. NF-κB was activated by ER stress-inducing agents, thapsigargin and tunicamycin. The activation was inhibited by a dominant-negative IRE1. In addition, a dominant-negative TRAF2 also suppressed the activation of NF-κB by ER stress. These results suggest that ER stress-induced NF-κB activation is also mediated by the IRE1-TRAF2 pathway, as well as JNK activation.
Phorbol myristate acetate (PMA)-induced superoxide radical (O2−)-production in guinea pig peritoneal polymorphonuclear leukocytes (PMNs) was significantly lower than that in peripheral cells. To determine the role of phosphotyrosine proteins in the lower O2− production, the effect of ST638 and genistein, tyrosine kinase inhibitors, on PMA-induced O2− production in peritoneal PMNs was examined. PMA-induced O2−-production of the cells was increased by the pretreatment with ST638 or genistein, the increment depending on the inhibitor concentration. The p47phox level in the plasma membrane of PMA-stimulated PMNs was increased by the pretreatment with ST638, although the phosphorylated p47phox level in the cells was not altered by ST638. On the other hand, PMA-induced O2−-production of peripheral PMNs was not affected by the pretreatment with ST638, but that of cytochalasin B (CB)-primed peripheral PMNs significantly increased by further treatment with ST638. The phosphotyrosine protein level of peritoneal PMNs was higher than that of the peripheral cells, especially in cytosolic proteins including 50—60 and 70—85 kDa proteins, and that of the CB-primed peripheral cells was also higher than that of the intact cells in similar cytosolic proteins to those above. Further treatment of CB-primed peripheral cells with ST638 resulted in a lower level of phosphotyrosine proteins. These findings suggest that phosphorylation of some protein(s) at specific tyrosine residues inhibits the translocation of p47phox to the plasma membrane from the cytosol, resulting in lower O2−-generation in casein-induced peritoneal exudate PMNs.
To clarify the mechanism of extracellular matrix (ECM) remodeling in bovine endometrium, we investigated the regulation of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases-3 (TIMP-3) in bovine endometrial stromal cells (BESCs) on type-I collagen gel. When BESCs were seeded onto the center of collagen gel placed in culture dishes, the cells proliferated and formed multiple cell layers after 2 weeks. Under this culture condition, the production of progelatinase B/promatrix metalloproteinase (proMMP)-9 was augmented, which was not occurred with monolayered BESCs on plastic dishes. The mRNA expression of progelatinase A/proMMP-2 was not changed, but proMMP-2 activation was augmented. Furthermore, the level of prostromelysin-1/proMMP-3 mRNA was decreased, whereas the gene expression of TIMP-3 tended to increase in BESCs cultured on collagen gel. When BESCs cultured on collagen gel were treated with transforming growth factor-β1 (TGF-β1), the levels of proMMP-9 in the medium and TIMP-3 mRNA were augmented, but the mRNA expression of proMMP-3 was further suppressed. However, the expression and activation of proMMP-2 were not changed by TGF-β1 in BESCs cultured on either plastic or collagen-gel dishes. These results suggest that the expression of MMPs-2, 3 and 9 and TIMP-3 is likely to be discoordinately regulated due to interaction with collagen and/or TGF-β1 in bovine endometrium, and thereby different sets of MMPs may be associated with ECM remodeling during implantation and placentation in vivo.
Penicillin binding proteins (PBPs) are penicillin-sensitive DD-peptidases catalyzing the terminal stages of bacterial cell wall assembly. We identified a Dictyostelium discoideum gene that encodes a protein of 522 amino acids showing similarity to Escherichia coli PBP4. The D. discoideum protein conserves three consensus sequences (SXXK, SXN and KTG) that are responsible for the catalytic activities of PBPs. The gene product prepared in the cell-free translation system showed carboxypeptidase activity but the activity was not detected in the presence of penicillin G. These results demonstrate that the D. discoideum gene encodes a eukaryotic form of penicillin-sensitive carboxypeptidase.
Although it is well established that estrogen inhibits bone resorption, its effects on bone formation remain controversial. We studied the effects of intermittent and continuous treatment with estrogen on bone formation in vitro using long term cultures of SaOS-2 cells under conditions that permit mineralization. SaOS-2 cells cultured in dexamethasone, ascorbic acid and β-glycerophosphate for up to 17 d formed mineralized bone nodules as visualized by von Kossa staining. Electron microscopic analysis of ultrathin sections of representative mineralized nodules showed the presence of mineral deposits, collagen fibrils and osteocytes. Both the mineralized nodule numbers and areas increased exponentially with time of culture after addition of β-glycerophophate at day 8. Intermittent addition of 17β-estradiol (E2) for 6 h or 24 h of every 48 h starting at day 3 or day 8 to the end of culture period resulted in a specific time- and dose-dependent stimulation of mineralized bone nodule number and area, and alkaline phosphatase activity which were accompanied with increase in cell numbers. On the other hand, continuous treatment with E2 added every 48 h had no effect. The estrogen receptor alpha (ERα) mRNA expression was stimulated after 6 or 24-h (intermittent), but not after 48-h (continuous) treatment with E2. The stimulatory effect of E2, when added intermittently, but not continuously, on differentiation and bone formation in human osteoblasts in culture may be relevant to previous reports of stimulatory effects of E2 on bone formation in vivo.
This work studied the antinociceptive effects of the hydroalcoholic extracts (HAEs) from Erythrina velutina (Ev) and Erythrina mulungu (Em) in three experimental models of nociception in mice. The extract was administered intraperitoneally to female mice at the doses of 200 and 400 mg/kg. Inhibitions of abdominal contractions were observed with the doses of 200 (88.6%; 86.8%) and 400 (95.5%; 83.5%) mg/kg of E. velutina and E. mulungu, respectively, as compared to controls. E. velutina and E. mulungu, at both doses, reduced the nociception produced by formalin in the 1st and 2nd phases and this effect was not reversed by the pretreatment with naloxone. In the hot plate test an increase of the reaction time was observed only at 60 (Ev=18.0±2.2; Em=20.8±2.52) and 90 min (Ev=20.4±1.71; Em=23.7±2.32) after the treatment with E. velutina and E. mulungu at the dose of 400 mg/kg as compared to controls (T60=11.1±0.74; T90=11.9±0.86). This effect was not reversed by naloxone. We conclude that E. velutina and E. mulungu presents antinociceptive effects, which are independent of the opioid system.
We previously reported stimulatory effects of endogenous and exogenous nitric oxide (NO) on gastric acid secretion. In the present study, we investigated effects of NO donors on release of histamine, which is related to acid secretion, in isolated rat gastric mucosal cells. NO donors such as (±)-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexanamide (NOR 1) and sodium nitroprusside significantly augmented the histamine release. It was inhibited by 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-amide (carboxy-PTIO), a NO scavenger, and 6-(phenylamino)-5,8-quinolinedione (LY83583), a soluble guanylate cyclase inhibitor. Dibutyryl cyclic GMP also stimulated histamine release. These results suggest that NO donors act on cyclic GMP pathway in isolated gastric mucosal cells, resulting in facilitation of histamine release. NO may stimulate gastric acid secretion through histamine release from the histamine-containing cells, possibly enterochromaffin-like cells.
We previously identified pyruvate dehydrogenase kinase 4 (PDK4) mRNA as a most rapidly induced mRNA by fibrates and suggested the possibility that the coupled induction of PDK4 and reduction of serum triglyceride and fatty acid levels can cause protein degradation in muscles. To investigate whether the drugs that are known to have a risk of rhabdomyolysis induce PDK4 mRNA in skeletal muscle, the effects of statins and new quinolon anti-bacterial drugs on the expression levels of the mRNA were examined using mice and cultured cells. Several statins and new quinolon anti-bacterial drugs solely induced PDK4 mRNA in the muscle as efficiently as fibrates and at least some combinations were synergistic. The present results suggest that induction of PDK4 mRNA is involved in the drug induced acute rhabdomyolysis when the muscle is restricted to use fatty acids as a major energy source.
Anti-asthmatic property of curcumin (diferuloylmethane), a natural product from the rhizomes of Curcuma longa, has been tested in a guinea pig model of airway hyperresponsiveness. We sensitized guinea pigs with ovalbumin (OVA) to develop certain characteristic features of asthma: allergen induced airway constriction and airway hyperreactivity to histamine. Guinea pigs were treated with curcumin during sensitization (to examine its preventive effect) or after developing impaired airways features (to examine its therapeutic effect). Status of airway constriction and airway hyperreactivity were determined by measuring specific airway conductance (SGaw) using a non-invasive technique, constant-volume body plethysmography. Curcumin (20 mg/kg body weight) treatment significantly inhibits OVA-induced airway constriction (p<0.0399) and airway hyperreactivity (p<0.0043). The results demonstrate that curcumin is effective in improving the impaired airways features in the OVA-sensitized guinea pigs.
Duchenne muscular dystrophy is known to be caused by a defective gene of dystrophin, a 427-kDa cytoskeletal protein, but the effective therapeutic drug is presently unavailable. We previously reported that a trypsin-like protease designated as dystrypsin is markedly activated in the muscle microsomal fraction immediately before onset of the clinical signs in mdx mice, a dystrophin-deficient hereditary animal model for human Duchenne muscular dystrophy. In order to examine the possible participation of dystrypsin in the occurrence of the disease, we investigated the therapeutic effects of dystrypsin inhibitors on the occurrence and progress of muscular dystrophy. Here, we show that camostat mesilate, a low-molecular-weight inhibitor of trypsin-like proteases, including dystrypsin, is a candidate drug for Duchenne muscular dystrophy.
A large number of studies have demonstrated that the presence of eosinophils in the lungs of patients with pulmonary fibrosis correlates with poor prognosis or resistance to therapy. However, direct evidence of the relationship between the influx of eosinophil and pulmonary fibrosis has not yet been described experimentally. In this article, pulmonary fibrosis was induced by different doses of bleomycin (BLM) and using different aged rats. On selected days afterwards, the lungs were lavaged and harvested for analyzing fibrosis, eosinophil influx and cytokine expression. There was a significant relationship between eosinophilia and the pulmonary fibrosis (r=0.98, p<0.01). In spite of the fact that there was no significant increase in hydroxyproline of the lung, eosinophil influxes of bronchoalveolar lavage fluid (BALF) was maximal 7 d after BLM administration. Moreover, there were similar patterns among transforming growth factor beta (TGF)-β1, hepatocyte growth factor (HGF) and eosinophil influx of BALF in that they were dependent on dose of BLM and age. These findings, taken together, have suggested the causal correlation of eosinophilia during the early stage with subsequent pulmonary fibrosis. The possible role of eosinophils in the pathogenesis of pulmonary fibrosis might contribute to not only TGF-β1 but also HGF production.
Diethyldithiocarbamate (DDTC) has been shown to induce cytotoxicity in several different systems. We examined whether the DDTC-induced cytotoxicity was via apoptosis, or in relation to intracellular glutathione (GSH) in various murine and human leukemia cell lines. The cells most sensitive to DDTC-induced cytotoxicity were P388 lymphoid neoplasma cells and NALM-6, a B cell line of acute lymphocytic leukemia (ALL). The next level of susceptible cells included J774.1, having a macrophage function, HL-60 premyelocytic leukemia cells, MOLT-4, an acute lymphoblastic leukemia cell, and Jurkat, a T-cell leukemia. U937 (expressing many monocyte-like characteristics), K562 erythroleukemia and K562/DXR (a multidrug-resistant clone derived from K562) were almost unaffected by DDTC. P388 was also highly susceptible to H2O2, a most useful exogenous reactive oxygen species generator, and was lower in intracellular total GSH content than other leukemia cells. DDTC-induced cytotoxicity was closely related to intracellular GSH, but the level of cellular GSH did not always correlate with H2O2-induced cytotoxicity in this experiment. K562 had a higher intracellular total GSH content and showed lower susceptibility to DDTC and H2O2, but with the combination of DDTC and DL-buthionine-(S,R)-sulfoximine (BSO), cytotoxicity increased significantly. The ratio of GSH/GSSG in P388 was reduced by DDTC or H2O2. H2O2-induced cytotoxicity was completely blocked by catalase (CAT), while it was enhanced by superoxide dismutase (SOD). CAT or SOD did not affect DDTC-induced cytotoxicity. N-Acetylcysteine (NAC: 1 mM), a vanguard substance of GSH, and aurintricarboxylic acid (ATA: 100 μM), an endonuclease inhibitor, ameliorated DDTC-induced cytotoxicity and apoptosis. In conclusion, we suggest that DDTC-induced cytotoxicity was via an oxidative shift in the intracellular redox state, and accompanied the activation of endonuclease through apoptosis in leukemia cell lines.
Information on the comparative digestibility of food allergens and non-allergenic proteins is crucial when stability to digestion is to be used as a criterion to assess the allergenic potential of novel proteins. Preheating effect on in vitro digestibility has not been fully examined. In this study we investigated the preheating effect of in vitro digestibility of several proteins and their proteolytic fragments in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Five major food allergens, ovalbumin (OVA), ovomucoid (OVM), β-lactoglobulin (BLG), bovine serum albumin (BSA), soybean trypsin inhibitor (STI), four proteins of unproven allergenicity, horseradish peroxidase (HRP), ribulose-1,5-bisphosphate carboxylase/oxidase (RBC), phosphinothricin acetyltransferase (PAT) and zein from corn, and plant lectin, concanavalin A (Con A) were preheated (at 100 °C for 5 min) or not preheated, and then digested in SGF or SIF. Food allergens were relatively stable in both SGF and SIF. Among the allergens, digestibility of OVA in both SGF and SIF was markedly decreased, and BLG and STI were relatively stable after preheating. Digestibility of ConA in SGF and SIF was markedly decreased by preheating. Digestibility of non-allergenic proteins in SGF was higher than the allergenic proteins. From these results, because of the marked increase of the digestibility in several proteins by preheating, systematic information concerning the effect of food treatment on protein digestion is necessary to assess the relationship between allergenic potential and the digestibility of food protein.
To investigate whether Bak Foong Pills (BFP), a well-known gynaecological tonic, has a direct effect on the central nervous system, we employed the in vivo electrochemical detection technique, fast cyclic voltammetry (FCV), to measure the dopamine release from the mesolimbic structure-amygdala of both male and female rats. The results showed that intracerebroventricular BFP (0.75, 1.5 μg) treatment promoted dopamine release from the amygdala in both female and ovariectomized female rats. The BFP-induced response appeared within 5 min after addition of BFP and lasted for at least 40 min. However, no effect of BFP was observed in male rats for an observed period of up to 60 min. The results suggest that BFP may have gender-specific beneficial effect on dopaminergic functions of the amygdala.
This study was conducted to clarify the effect of the n-butanol fraction from the anomalous fruits of Gleditsia sinensis LAM. (NBGS) on experimental allergic rhinitis. NBGS (100, 200, 400 mg/kg, p.o.) dose-dependently inhibited nasal symptoms (sneezing and nasal rubbing) and dye leakage induced by antigen challenge into the nasal cavity of actively sensitized rats. Significant effects were observed at doses of 200 and 400 mg/kg. NBGS (200, 400 mg/kg) also showed a clear inhibition of sneezing and an inhibitory tendency on nasal rubbing induced by histamine in normal rats. At 400 mg/kg, it significantly reduced dye leakage induced by histamine into the nasal cavity of rats. Terfenadine (10 mg/kg, p.o.), an antihistaminic drug, clearly inhibited the nasal symptoms and the amount of dye leakage induced by antigen or histamine. Furthermore, NBGS significantly reduced in vitro histamine release from rat peritoneal mast cells triggered by compound 48/80 at concentrations of 30 and 100 μg/ml. These results suggest that NBGS may be clinically effective in alleviating the nasal symptoms of allergic rhinitis, probably by inhibiting both histamine release from mast cells and nasal vascular permeability.
The present study was designed to evaluate central inhibitory effects of the essential oil from Acori graminei Rhizoma (AGR), the dry rhizomes of Acorus gramineus SOLANDER (Araceae) upon fragrance inhalation (aroma therapy). Preinhalation of the oil markedly delayed the appearance of pentylenetetrazole-induced convulsion. Furthermore, inhalation impressively inhibited the activity of γ-aminobutyric acid (GABA) transaminase, a degrading enzyme for GABA as the inhalation period was lengthened. The GABA level was significantly increased and glutamate content was significantly decreased in mouse brain by preinhalation of the essential oil. The above results suggest that the anticonvulsive effect of this AGR oil is originated by the enhancement of GABA level in the mouse brain, because convulsion depends partially on GABA concentration which can be properly preserved by inhibiting GABA transaminase. Moreover, fragrance inhalation progressively prolonged the pentobarbital-induced sleeping time as inhalation time was lengthened. Ten hour inhalation corresponded almost to the effect (145% increase) of oral administration (60 mg/kg). This sedative effect after inhalation or oral administration of AGR essential oil suggests that this oil may act on the CNS via the GABAergic system. The inhibitory activity of preinhalation of the essential oil on lipid peroxidation, to which the anticonvulsive action is attributed, also supported the above results, confirming and amplifying our previous reports on the CNS inhibitory effects of AGR.
A 35% EtOH extract of the fruits of Chaenomeles sinensis, long utilized as a folk medicine for cough, significantly inhibited the pruritogenic agent compound 48/80 (COM)-induced scratching behavior in mice. Antipruritic activity-guided fractionation and purification yielded active quercetin, apigenin, and catechin derivatives, which exhibited significant inhibitory effects on COM-induced scratching behavior. To the best of our knowledge, apigenin (5), apigenin 7-glucronide (6), and apigenin 4′-methoxy-7-glucronide (acacetin 7-glucronide) (7) were isolated from the fruits of C. sinensis for the first time. The active fraction and these compounds also inhibited serotonin-, platelet activating factor-, and prostaglandin E2-induced scratching behavior, but did not inhibit histamine-induced scratching behavior or locomotive behavior. This study also showed that the fruits of C. sinensis could be used to treat allergic itching sensation.
Effects of major intestinal metabolites of ginsenosides, including compound K (IH-901, 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol), compound Y (IH-902, 20-O-[α-L-arabinopyranosyl (1→6)-β-D-glucopyranosyl]-20(S)-protopanaxadiol), and ginsenoside Mc (IH-903, 20-O-[α-L-arabinofuranosyl (1→6)-β-D-glucopyranosyl]-20(S)-protopanaxadiol), on acute stress-induced plasma corticosterone levels were studied in mice. Intracerebroventricularly (i.c.v.) administered compound K (1 μg) attenuated the i.c.v. injection stress-induced increase in plasma corticosterone level, and this inhibitory effect was not affected by co-administered NG-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor. Compound K administered intraperitoneally affected neither the i.c.v. injection stress- nor the immobilization stress-induced increase in plasma corticosterone levels. Compound K and ginsenoside Mc did not affect plasma corticosterone levels induced by the two stress modalities used in this study.
The bioassay-guided fractionation of the methylene chloride soluble portion of a methanol extract of Machilus thunbergii bark led to the isolation of four known lignans, machilin A (1), meso-monomethyl dihydroguaiaretic acid (2), nectandrin A (3) and nectandrin B (4), which exhibited potent inhibitory activity on melanin biosynthesis in cultured B-16 mouse melanoma cells (IC50: 39.9, 15.1, 19.4 and 37.8 μM, respectively).
Four known flavonoids and two galloyl glucoses isolated from the stem-bark of Juglans mandshurica (Juglandaceae), namely taxifolin (1), afzelin (2), quercitrin (3), myricitrin (4), 1,2,6-trigalloylglucose (5), and 1,2,3,6-tetragalloylglucose (6), were evaluated for their anti-complement activity against complement system. Afzelin (2) and quercitrin (3) showed inhibitory activity against complement system with 50% inhibitory concentrations (IC50) values of 258 and 440 μM. 1,2,6-Trigalloylglucose (5) and 1,2,3,6-tetragalloylglucose (6) exhibited anti-complement activity with IC50 values of 136 and 34 μM. In terms of the evaluation of the structure–activity relationship of 3,5,7-trihydroxyflavone, compounds 2, 3, and 4 were hydrolyzed with naringinase to give kaempferol (2a), quercetin (3a), and myricetin (4a) as their aglycones, and these were also tested for their anti-complement activity. Of the three aglycones, kaempferol (2a) exhibited weak anti-complement activity with an IC50 value of 730 μM, while quercetin (3a) and myricetin (4a) were inactive in this assay system. Among the compounds tested, 1,2,3,6-tetragalloylglucose (6) showed the most potent anticomplement activity (IC50, 34 μM).
Mongolian plants were screened for their influence on α-amylase activity in mouse plasma. Methanolic extracts of Geranium pratense, Rhodiola rosea, Ribes pullchelum and Vaccinium uliginosum inhibited the enzyme activity in isolated mouse plasma by greater than 40% and the effect was concentration dependent. Vaccinium uliginosum also showed a depressive effect on elevation of postprandial blood glucose to some extent.
Blood glucose levels are routinely obtained by invasive and painful methods using glucose meters and test strips. The development of less invasive or non invasive techniques would be beneficial for diabetes patients. In this study, a noninvasive method was evaluated using the back diffusion of glucose across skin with or without permeation enhancement methods. An in vitro model was utilized. The stratum corneum (SC) was the predominant barrier for both back and forward diffusion of glucose across skin. Surfactants with various charges and essential oils (cyclic monoterpenes) were used as chemical enhancers to promote the back diffusion of glucose. A cationic surfactant (benzalkonium chloride) showed the highest enhancement, followed by anionic and nonionic surfactants. d-Limonene and 1,8-cineole dispersed in appropriate proportions of ethanol could enhance the glucose diffusion after pretreatment of the skin surface. Electroporation, defined as a physical method, significantly increased the amount of glucose that diffused back. The percentages of diffused glucose by 300 V (volts) and 500 V high voltage pulses on skin for 10 min were found to be 45 and 75 times greater than the control group, respectively.
We have examined the influence of liver disease on drug absorption from the liver surface membrane, regarded as the first barrier for drug targeting to the liver. The main purpose of this study is to examine the possibility of direct liver surface application as a drug targeting method. We employed rats intoxicated with carbon tetrachloride (CCl4) or D-galactosamine (GAL) as the liver disease model, and examined drug absorption characteristics after application to the liver surface, by utilizing a cylindrical diffusion cell. In the liver-intoxicated rats, about 90% of a low molecular weight drug, phenolsulfonphthalein (PSP), as a model was absorbed from the liver surface in 6 h, similar to the normal rats (no treatment). Although the absorption rate was increased in the CCl4 group, whereas slightly retarded absorption was observed in GAL group, there should be no serious problem for the clinical use of liver surface application. The PSP absorption from the liver surface in the CCl4 group was indicated to obey first-order kinetics by elimination profile from the diffusion cell. The first-order absorption rate constant Ka values of PSP from the liver surface, obtained by a compartment model and elimination profile, were increased 1.3-fold in the CCl4 group compared to the control. Moreover, we performed drug application to the liver surface in the peritoneal cavity to assume clinical use. The Ka of PSP in the CCl4 group was about 4-fold larger than in the normal group, implying the importance of estimating changes in peritoneal drug absorption as a result of liver disease. Consequently, it is expected that there will be no marked decline in the absorption rate from the liver surface in a liver disease state, leading us to apply this administration method for liver targeting.
KW-3902 (a newly synthesized adenosine A1-receptor antagonist) has potent diuretic and renal protective activities and was formulated in lipid dispersion systems, i.e., lipid emulsions and liposomes. The objective of the present study was to evaluate the carrier potential of these lipid dispersion systems, which is explained here as the ability of the formulation to retain the drug in its dispersed phase. The relative affinity of the drug to the formulation, Kf/b, was defined as a parameter in order to assess the performance of the formulations and was obtained from the in vitro blood component binding study. The results indicated that KW-3902 showed higher relative affinity to the liposome formulation than to the lipid emulsion. Moreover, the total amount of drug retained in the dispersion system depended on both Kf/b and the dosing volume. The usefulness of the parameter, Kf/b, was discussed as an indicator for a carrier potential to understand the properties of the formulations.
The present study was undertaken to examine the effects of the absorption enhancer saponin on the intrarenal distribution of 5-fluorouracil (5-FU) following the kidney surface application of 5-FU in rats. We selected an experimental system utilizing a cylindrical diffusion cell attached to the kidney surface. The intrarenal concentration of 5-FU 120 min after right kidney surface application of 5-FU with saponin at concentrations of 0.25 and 1 mg/ml was modified. Among four sites in the right kidney, the concentration of 5-FU under the diffusion cell was selectively increased by saponin. These results suggest it may be possible to control the intrarenal distribution of the drug following its application with an absorption enhancer on the kidney surface.
We investigated the gene expression of GAPDH, cytochrome P450 isoforms (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7), UGT-dependent glucuronosyl transferase isoforms (UGT1A6 and UGT1A9), and ABC transporter (ABCB1) after exposure to 0.1, 0.5, and 2.5% dimethyl sulfoxide (DMSO). GAPDH mRNA levels after exposure were less than 2 times higher or lower than control levels at all DMSO concentrations. CYP1A1, CYP1B1, CYP2A6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, and UGT1A9 mRNA levels were less than 2 times higher or lower than control levels at 0.1% and 0.5% DMSO. CYP1A2, UGT1A6, and ABCB1 mRNA levels were less than 2 times higher or lower than control levels at all DMSO concentrations. CYP2E1 and CYP3A4 mRNA levels were 2.5—3.2 times and 2.0—3.3 times higher than control levels at 2.5% DMSO, respectively. UGT1A9 mRNA levels were 0.2—0.5 times lower than control levels at 2.5% DMSO. Exposure to β-naphthoflavone reduced CYP1A1 mRNA levels in a concentration-dependent manner for donors 1 and 2. Exposure to β-naphthoflavone reduced CYP1A2 mRNA levels in a concentration-dependent manner for all donors. However, exposure to β-naphthoflavone increased CYP1B1 mRNA levels for donors 2 and 3 at 2.5% DMSO. Exposure to rifampicin reduced CYP3A4 mRNA levels for all donors at 0.5% and 2.5% DMSO. Exposure to rifampicin also reduced CYP3A5 and CYP3A7 mRNA levels for donors 1 and 3 at 0.5% and 2.5% DMSO. In conclusion, a DMSO concentration of 0.1% or less may be appropriate for studying induction after drug exposure.
The protective effect of Shengmai San (SMS) on oxidative damage in cultured PC12 cells was studied as a model of an antioxidant-based composite formula usable for the treatment of oxidative stress-related complex disorders. SMS, a traditional Chinese herbal medicine, has previously been shown to prevent cerebral oxidative injury in rats. Neuronal model PC12 cells were incubated with SMS for defined periods, chased with H2O2 for 30 min at 37 °C, and subjected to an ELISA-based assay for determining the protein carbonyl content, and a Comet assay for DNA single strand breaks (SSBs). The results showed that both protein carbonyl content and DNA SSBs increased in PC12 cells after the H2O2 chase in a concentration-dependent manner. Both H2O2-dependent carbonyl formation and DNA damage were markedly prevented in the cells pretreated with SMS, and the SMS effects were dependent on both the SMS concentration and the period of pre-incubation with SMS before the H2O2 abuse. At the same time, cell viability was enhanced in the SMS-pretreated cells after the H2O2 abuse compared to the control cells as determined by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It is concluded that SMS functions not only as a simple antioxidant but also as a modulator of cellular antioxidant defense.
A cell growth inhibitory effect of drupanin and baccharin, ingredients of propolis, was found in human cancer cell lines. These compounds induced apoptosis in the cells characterized by morphological and nucleosomal DNA fragmentation analysis. Their effects were less potent compared with that of artepillin C, which is a known anticancer compound from propolis. Importantly, HL60 cells were more sensitive to drupanin than were Con A-stimulated peripheral blood lymphocytes, whereas the potency of artepillin C was the opposite of that of drupanin.
Retinoic acid (RA), a potent inducer of cell differentiation, and N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide), a potent inducer of apoptosis, are well known as anticancer agents that are administered orally to patients for leukemia, breast and prostate cancer, respectively. However, it has not been studied whether both retinoids are effective on metastatic cancer. In mice implanted with M5076 cells, murine reticulum cell sarcoma survival times were prolonged by i.v. treatment of RA and 4-HPR entrapped in liposomes containing soybean-derived sterylglucoside mixture (SG), which accumulates in liver. In contrast, free RA and 4-HPR were inactive. These results indicate that RA and 4-HPR in SG-liposomes exhibit anticancer efficacy on metastatic cancers, and may have great potential for clinical use in the treatment of various cancers.