We investigated the induction of apoptosis in cultured human fetal membrane cells infected with influenza virus type A. We found that influenza virus yield in supernatants of primary cultured chorion and amnion cells prepared from human fetal membranes increased 6 h after infection. Chromosomal DNA was fragmented into oligonucleosomes at 48 h after influenza virus infection in chorion cells but not in mock-infected chorion cells, mock-infected amnion cells or influenza virus-infected amnion cells. The DNA fragmentation in influenza virus-infected chorion cells was evident at 24 h after infection and depended on the multiplicity of infection at 48 h. Incubating influenza virus-infected chorion cells with ribavirin, an inhibitor of viral RNA synthesis, reduced the increase in virus yield and simultaneously blocked DNA fragmentation. These results suggest that both chorion and amnion cells become infected with influenza virus, but that influenza virus infection induces apoptosis in chorion, but not typical apoptosis characterized by DNA ladder formation in amnion cells. We further observed that influenza virus replication is associated with the induction of apoptosis.
Asp-hemolysin is a hemolytic toxin from Aspergillus fumigatus and is a specific binding protein with high affinity for oxidized low density lipoprotein (Ox-LDL). As a first step in clarifying the structure–function relationship of Asp-hemolysin, we expressed Asp-hemolysin in Escherichia coli (E. coli) as a fusion protein with a maltose-binding protein (MBP) and purified it by affinity chromatography on an amylose resin. The apparent molecular size of the protein produced by E. coli was approximately 57 kDa, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This is consistent with the predicted molecular size of 56.9 kDa for a fusion protein which includes 14.2 kDa of Asp-hemolysin and 42.7 kDa from MBP. The purified recombinant Asp-hemolysin showed an immunoreactivity with the anti-Asp-hemolysin antibody as revealed by western blot analysis. Furthermore, in dot blot analysis, MBP-Asp-hemolysin fusion protein exhibited binding activity to Ox-LDL as did native Asp-hemolysin.
Lactoferricin (LFcin) hydrolyzed from lactoferrin (LF), a major 80 kDa iron-binding protein in milk and other exocrine secretions, was characterized as a potent activator of protein kinase CK2 (CK2) in vitro. Human LFcin (hLFcin) at 0.5 µg stimulated approx. 5-fold CK2 activity [phosphorylation of 60S acidic ribosomal proteins (P0, P1, P2) and Hsp90 (p98)] in a manner similar to other functional proteins with oligo-Arg clusters, such as salmine A1, sperm histone H2B and HIV-1 Rev. Interestingly, this stimulatory effect of hLFcin was significantly reduced when it was phosphorylated by A-kinase in vitro. These results suggest that (i) hLFcin acts as a potent CK2 activator in vitro; and (ii) the stimulatory effect of hLFcin on CK2 activity is regulated by its phosphorylation by A-kinase in vitro.
Four sesquiterpenes isolated from Jasonia glutinosa D.C. (Asteraceae), namely lucinone, glutinone, 5-epi-kutdtriol and kutdtriol, have been evaluated for their in vitro anti-inflammatory activity in cellular systems generating cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) metabolites. None of the compounds assayed had a significant effect on leukotriene C4 (LTC4)-release from calcium ionophore-stimulated mouse peritoneal cells. However, the release of prostaglandin E2 (PGE2) by mouse peritoneal cells stimulated with calcium ionophore was inhibited by these compounds, although with less potency than the reference drug indomethacin (IC50=0.24 µM). The IC50 values of the active compounds were: lucinone 42.69 µM, glutinone 3.61 µM, 5-epi-kutdtriol 1.28 µM and kutdtriol 39 µM. Of the tested compounds, only glutinone (IC50=24 µM) showed a significant effect on thromboxane B2 (TXB2)-release induced by calcium ionophore in human platelets, although with less potency than the reference drug ibuprofen (IC50=1.27 µM).
We studied the inhibitory effect of the green sap of Ixeris dentata (IXD) on compound 48/80-induced anaphylaxis-like response in a murine model. IXD dose-dependently inhibited the anaphylaxis-like response induced by compound 48/80 in mice. IXD inhibited the anaphylaxis-like fatal response at the dose of 0.1 g/kg by 75%. IXD had a significant inhibitory effect on compound 48/80-induced ear swelling response at the doses of 0.05 and 0.1 g/kg. IXD (0.1 g/kg) also inhibited passive cutaneous anaphylaxis mediated by anti-dinitrophenyl IgE by 81.45%. When IXD was given as a pretreatment at concentrations ranging from 0.005 to 0.1 g/l, the histamine release from rat peritoneal mast cells induced by compound 48/80 was reduced in a dose-dependent manner. These results indicate that IXD may possess antianaphylactic activity.
In this study, the antiinflammatory effects of 50, 100 and 200 mg/kg doses of extract obtained from Cladonia rangiformis, so-called C-1, were investigated. The effects of C-1 on the acute phase of inflammation were studied in formaldehyde-induced edema. A cotton-pellet granuloma test was used to investigate the effects of C-1 on chronic inflammation. The antiedema potency of C-1 was compared with indomethacin. C-1 at the doses mentioned above showed 33.8% (p<0.005), 36.1% (p<0.005), 43.1% (p<0.001) inhibition, respectively. The corresponding antiinflammatory effect for indomethacin was determined as 72% (p<0.001). 200 mg/kg C-1 and 10 mg/kg indomethacin decreased the formation of granuloma tissue induced by cotton-pellet method at a rate of 57.3% (p<0.005) and 52.1% (p<0.005), respectively. It was seen that C-1 was more effective on chronic inflammation than on acute inflammation.
The effects of carbachol, cholecystokinin octapeptide (CCK-8), secretin, prostaglandin E2 (PGE2), and second mediator-like substances (A23187, phorbol 12-myristate 13-acetate, and dibutyryl cAMP) on mucus secretion from cultured gastric epithelial cells were investigated. Gastric mucus was measured by an enzyme-linked lectin assay with soybean agglutinin and wheat germ agglutinin. Intracellular cAMP and Ca2+ were measured with a cAMP assay kit and an image analysis system using fura-2-loaded cells, respectively. Secreted mucus induced by any combination of receptor agonists was almost equal to the summation of each stimulated mucus secretion. On the other hand, combined stimulation with second mediator-like substances secreted mucus synergistically. These results suggest the existence of interactions among receptors for mucus secretion. Based on these results, the secretagogue induced intracellular cAMP and free calcium ([Ca2+]i) levels were measured in cultured gastric epithelial cells incubated with secretagogues. Secretin and PGE2 induced cAMP accumulation, and carbachol and CCK-8 induced a [Ca2+]i increase. To confirm these results, the effects of protein kinase A and C inhibitors and intracellular calcium chelator on mucus secretion were investigated. An intracellular calcium chelator inhibited the mucus secretion induced not only by carbachol and CCK-8 but also by secretin and PGE2. These results suggest that the [Ca2+]i plays an important role in mucus secretion through cAMP accumulation.
Sixteen flavonoids including flavonols, flavones, flavanonol and catechins, and five aromatic compounds were examined for their ability to scavenge superoxide radical (O2−) generated enzymatically in a xanthin–xanthinoxidase system and non-enzymatically in a phenazine methosulfate–NADH system. Pyrogallol, gallic acid and its ester, were much more efficient in scavenging O2− than catechol. The superiority of pyrogallol over catechol in the flavonoidal nucleus is apparent from the much higher O2− scavenging activity of myricetin and epigallocatechin, which contain 3′, 4′, 5′-trihydroxyl substitution in the B-ring, compared to quercetin and epicatechin, which contain 3′, 4′-dihydroxyl substitution, respectively. The strong O2− scavenging ability of pyrogallol appears to function even in the A-ring, as in baicalein, and also in the form of a pyrogalloyl ester at the C-3 position in the C-ring, as in epicatechin gallate and epigallocatechin gallate. It can be concluded that the pyrogallol moiety is an active component of flavonoids for displaying high O2− scavenging activity. Flavonoids and aromatics were also examined to correlate their O2− scavenging activity with their oxidizability, which was measured on the basis of electrochemical redox potential and the reducing ability of the Cu2+ ion. Aromatics such as pyrogallol, gallic acid and its ester, and flavonoids such as baicalein, epicatechin gallate and epigallocatechin gallate, in which the O2− scavenging activity is enhanced by the presence of a pyrogallol moiety which does not belong to the B-ring, reduced the correlation between the higher O2− scavenging activity and the lower redox potential. The O2− scavenging activity was well correlated with the Cu2+ reducing ability of flavonoids and aromatics.
The present study was undertaken to measure 1, 4-dihydropyridine (DHP) receptor binding sites in vivo in the mesenteric artery and other tissues of spontaneously hypertensive rats (SHR) and to examine the effect of nifedipine and cilnidipine. Specific in vivo binding of (+)-[3H]PN 200-110 in the SHR mesenteric artery was dose dependently reduced by oral administration of nifedipine at relatively low doses. Oral administration of cilnidipine (6.09 µmol/kg) significantly reduced the specific in vivo binding of (+)-[3H]PN 200-110 in the mesenteric artery, aorta, and myocardium. A significant reduction in (+)-[3H]PN 200-110 binding was seen at 1—12 h in the mesenteric artery and at 1—7 h in the aorta and myocardium. In contrast, oral administration of nifedipine (28.9 µmol/kg) markedly reduced in vivo (+)-[3H]PN 200-110 binding in all tissues of SHR at 1—6 h, and the degree and time course of the reduction did not differ much among the tissues. The area under the curve (AUC) for receptor occupancy vs. time was calculated from the reduction rate (%) of specific in vivo (+)-[3H]PN 200-110 binding. The ratio (1.4 or 1.7) of the AUCmesenteric artery to AUCaorta or AUCmyocardium after oral administration of cilnidipine was greater than the corresponding value (1.1) for nifedipine. In conclusion, the present study demonstrates that cilnidipine, but not nifedipine, may occupy 1, 4-DHP receptors in the small artery in a more selective and sustained manner than in other tissues of SHR, and thus such receptor binding specificity may be responsible for the long-lasting hypotensive effect of this drug.
The anti-proliferative activity of the somatostatin analog RC-160 is limited by its short serum half life. To circumvent this limitation, fatty acids of chain lengths ranging from 4 to18 were individually conjugated to the N-terminal residue of RC-160. The lipophilized derivatives of RC-160 were synthesized, purified and characterized. The anti-proliferative activity of lipophilized-RC-160 on the human breast carcinoma cell line MCF-7, was evaluated in vitro. The long chain lipopeptides like pamitoyl-RC-160 exhibited significantly higher anti-proliferative activity on MCF-7 cells (p<0.001), relative to RC-160. The affinity of RC-160 towards somatostatin receptors remained unaltered by pamitoylation. However, the observed increase in bioactivity was manifested within an optimum range of chain length of thelipoppetide. Increasing the peptide hydrophobicity beyond this range reduced the bioactivity of lipophilized-RC-160. Accordingly, stearoyl-RC-160, manifested lower anti-neoplastic activity and receptor affinity relative to pamitoyl-RC-160 and RC-160 itself. The signaling pathways underlying the antineoplastic activity of these lipopeptides were found to be similar to RC-160. Pamitoyl-RC-160 displayed enhanced inhibition of protein tyrosine kinase activity and intracellular cAMP levels in MCF-7 cells, relative to butanoyl-RC-160 or RC-160 itself. Pamitoyl-RC-160 also displayed greater resistance towards trypsin and serum degradation than RC-160. Lipophilization of RC-160 with long chain fatty acids like pamitic acid improves its stability and anti-proliferative activity, thereby improving the scope of enhancing its therapeutic index. However, the optimization of peptide hydrophobicity seems to be a crucial factor governing the efficacy of bioactive lipopeptides.
Shosaiko-to is a Kampo medicine used for the treatment of chronic hepatitis in Japan. Lately, over 200 cases of interstitial pneumonia have been reported resulting from Shosaiko-to therapy, and the number of cases increased when patients were administrated interferon (IFN)-α at the same time. However, the mechanisms of this Shosaiko-to implicated interstitial pneumonia are not fully understood. In this study, we examined by flow cytometry analysis the in vitro effects of 7 phenolic compounds (lignans and flavonoids), which were detected from human urine after administration of Shosaiko-to, and IFN-α on inducing apoptosis in human lung fibroblasts and peripheral blood mononuclear cells (PBMCs). Among the 7 compounds, baicalein and medicarpin (10 µg/ml) showed significant apoptosis-inducing effects on human PBMCs. In human lung fibroblasts, medicarpin exhibited a significantly higher activity to induce apoptosis compared to the control, and the percentage of cells undergoing apoptosis showed time- and dose-dependent increases. Baicalein (0.1 and 1 µg/ml), liquiritigenin (10 µg/ml) and davidigenin (10 µg/ml) also showed significant effects after 96 h treatment. Whereas, baicalin, oroxylin A and wogonin did not show any effect on inducing apoptosis in PBMCs and fibroblasts. Baicalein and medicarpin significantly inhibited the growth and reduced the viability of lung fibroblasts. IFN-α had no apoptosis-inducing effect, and it did not show synergistic interaction with any of the compounds derived from Shosaiko-to on inducing apoptosis in both human lung fibroblasts and PBMCs. These results suggested that phenolic compounds found in human post-administrative urine of Shosaiko-to, especially baicalein and medicarpin, exhibited a direct effect on human lung fibroblasts and immune cells to induce apoptosis.
We have previously reported that restraint stress inhibits small intestinal motility in rats, and that the adrenergic β3-antagonist SR59230A administration recovered the inhibition. In the present study, we compared the effects of restraint stress and norepinephrine on small intestinal motility using α- and β-adrenergic antagonists. SR59230A did not recover the norepinephrine-induced inhibition of small intestinal motility. The norepinephrine-induced inhibition of small intestinal motility was recovered after administration of the α2-antagonist yohimbine, but not by α1-, β1-, and β2-antagonists. Considering these results, it is reasonable to assume that the mechanisms of inhibition of small intestinal motility due to restraint stress and norepinephrine treatment are different.
The formation of 7-oxo-Δ8-tetrahydrocannabinol (7-Oxo-Δ8-THC) from 7α- or 7β-hydroxy-Δ8-THC (7α- or 7β-OH-Δ8-THC) was found in hepatic microsomes of monkeys. The activity in 7β-OH-Δ8-THC was stereoselectively 2.5- to 4.6-fold higher than that from 7α-OH-Δ8-THC. The oxidative activities of 7α- and 7β-OH-Δ8-THC to 7-Oxo-Δ8-THC were inhibited to 35% and 10%, respectively, of the control value by the antibody against P450GPF-B (CYP3A), a major enzyme responsible for the formation of 7-Oxo-Δ8-THC in guinea pigs. In the Lineweaver-Burk double-reciprocal plot analysis, testosterone 6β-hydroxylase activity was competitively inhibited by 7β-OH-Δ8-THC. Two cytochrome P450 enzymes, called P450JM-D and P450JM-E, were purified from hepatic microsomes of Japanese monkeys. P450JM-E, assumed to be CYP3A8, immunologically reacted with the antibody against P450GPF-B and showed high forming activity of 7-Oxo-Δ8-THC from 7-OH-Δ8-THC. On the other hand, 7-Oxo-Δ8-THC forming activity of P450JM-D, assumed to be CYP2C, was less than 10% of that of P450JM-E (CYP3A8). Oxygen-18 (18O) derived from atmospheric oxygen was incorporated into about 40% of the corresponding ketone formed from 7α-OH-Δ8-THC or 8β-OH-Δ9-THC by P450JM-E (CYP3A8), although the incorporation of the stable isotope into the oxidized metabolite from 7β-OH-Δ8-THC or 8α-OH-Δ9-THC was negligible. These results indicate that P450JM-E (CYP3A8) is a major enzyme of the oxidation of 7-OH-Δ8-THC in monkey hepatic microsomes. The oxidation mechanism may proceed as follows: the α- and β-epimers of 7-OH-Δ8-THC or 8-OH-Δ9-THC may be converted to ketone through dehydration of an enzyme-bound gem-diol by P450JM-E (CYP3A8), although this stereoselective dehydration differentiates between two epimers.
We investigated the estrogenic activities of isoflavone derivatives in competition binding assays with human estrogen receptor (hER) α or hER β protein, and in a gene expression assay using a yeast system. Coumestrol binds as strongly as 17β-estradiol to both hERs. Biochanin A, 5-OMe-genistein, formononetin, and tectorigenin bind well to hER β, but significant binding to hER α is only observed with 5-OMe-genistein, formononetin and tectorigenin. The binding of 7-OMe-genistein and irisolidone is poor to both receptors. Among the glucosides, sissotorin binds both receptors and the binding is stronger than genistin. Coumestrol induces transcription as strongly as genistein. Tectorigenin also induces transcription with both hERs. Though biochanin A, 5-OMe-genistein, 7-OMe-genistein, irisolidone and formononetin slightly induce transcription with hER β, they act as antagonists in the induction of transcription by 17β-estradiol. The results show that methylation or glucosidation of isoflavones generally inhibits their phytoestrogenic activities.
To determine the effect of climbazole on hepatic microsomal cytochrome P450 (P450) and drug-metabolizing enzymes, four different P450 isoforms (CYP2B1, 3A2, 2E1, and 2C12) were examined in female Long–Evans rats. Treatment of rats with climbazole resulted in the induction of P450 content. Climbazole both induced and inhibited aminopyrine N-demethylase activity, but not erythromycin N-demethylase activity. Uridine 5′-phosphate (UDP)-glucuronosyl transferase and glutathione S-transferase activities were also increased with climbazole treatment. Immunoblot analyses revealed that climbazole induces CYP2B1 and CYP3A2 at the lower dose examined, but it failed to increase CYP2B1 at the higher dose. Northern blot analysis revealed that climbazole markedly increases P450 2B1 mRNA. These results indicate that climbazole induces and inhibits P450-dependent drug-metabolizing enzymes in vivo and may have the dose-differential effect on CYP2B1 in rat liver.
When ginsenoside Rg3 was anaerobically incubated with human fecal microflora, all specimens metabolized ginsenoside Rg3 to ginsenoside Rh2 and protopanaxadiol. The main metabolite was ginsenoside Rh2. 20(S)-ginsenoside Rg3 was quickly transformed to 20(S)-ginsenoside Rh2 or 20(S)-protopanaxadiol in an amount 19-fold that compared with the transformation of 20(R)-ginsenoside Rg3 to 20(R)-ginsenoside Rh2 or 20(R)-protopanaxadiol. Among the bacteria isolated from human fecal microflora, Bacteroides sp., Eubacterium sp., and Bifidobacterium sp. metabolized ginsenoside Rg3 to protopanaxadiol via ginsenoside Rh2. However, Fusobacterium sp. metabolized ginsenoside Rg3 to ginsenoside Rh2 alone. Among ginsenoside Rg3 and its metabolites, 20(S)-protopanaxadiol and 20(S)-ginsenoside Rh2 exhibited the most potent cytotoxicity against tumor cell lines, 20(S)- and 20(R)-protopanaxadiols potently inhibited the growth of Helicobacter pylori, and 20(S)-ginsenoside Rh2 inhibited H+/K+ ATPase of rat stomach.
We compared the pharmacological actions of the high and low molecular mass fractions of Sho-saiko-to using a murine immunologically induced liver injury model to estimate the roles of these fractions in the expression of the pharmacological action. In a Bacillus Calmette–Guérin (BCG)/lipopolysaccharide (LPS)-induced liver injury model, Sho-saiko-to and both of its fractions significantly reduced the increases in the aminotranseferase levels in serum. They also reduced the increase in the nitric oxide (NOx) level in serum. On the other hand, Sho-saiko-to and its high molecular mass fraction suppressed the increase in plasma NOx level in an LPS-induced endotoxin shock model but its low molecular mass fraction did not. These results suggest the possibility that both fractions act hepatoprotectively in a different manner. We believe that these results can help to elucidate the mechanism of action of ingredients in Sho-saiko-to.
When kalopanaxsaponin K (KPK) from Kalopanax pictus was incubated for 24 h at 37 °C with human intestinal microflora, KPK was mainly metabolized to kalopanaxsaponin I (KPI) via kalopanaxsaponin H (KPH) rather than via kalopanaxsaponin J (KPJ), and then transformed to kalopanaxsaponin A (KPA) and hederagenin. Bacteroides sp., and Bifidobacterium sp. and Fusobacterium sp. transformed KPK to KPI and KPA and hederagenin via KPH or KPJ. However, Lactobacillus sp. and Streptococcus sp. transformed KPK to KPI, KPA, and hederagenin only via KPJ. The metabolite KPA of KPK showed potent antirheumatoid arthritis activity.
The hepatoprotective effects of the hot water (SRHW) and methanolic (SRM) extracts from the roots and stems of Salacia reticulata were examined using an oxidative stress-induced liver injury model. Both SRHW and SRM extracts (400 mg/kg, p.o.) significantly suppressed the increase in glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities in carbon tetrachloride (CCl4)-treated mice. These extracts also inhibited CCl4-induced thiobarbituric acid-reactive substance (TBA-RS) formation, which indicates increased lipid peroxidation in the liver. A good correlation (r=0.945, p<0.01) was observed between the amount of phenolic compounds in the extracts and their inhibitions of TBA-RS formation. The IC50 values of the extracts on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging were less than 10 µg/ml and the antioxidative activities of six phenolic compounds from the roots of S. reticulata were examined. Mangiferin, (−)-4′-O-methylepigallocatechin, and (−)-epicatechin-(4β→8)-(−)-4′-O-methylepigallocatechin, which a principal phenolic compounds, showed potent scavenging activity on DPPH radicals and their concentrations required for 50% reduction of 40 µM DPPH radicals were 5.9, 10, and 3.2 µM, respectively. On the other hand, against the CCl4-induced serum GOT and GPT elevations and TBA-RS formation in mice, mangiferin and (−)-4′-O-methylepigallocatechin showed potent activity at a dose of 100 mg/kg, but (−)-epicatechin-(4β→8)-(−)-4′-O-methylepigallocatechin did not. These results suggest that the antioxidative activity of the principal phenolic compounds is involved in the hepatoprotective activity of S. reticulata.
The effects of Astragali radix extract on interleukin (IL)-6 and tumor necrosis factor (TNF)-α productions, prostaglandin E2 (PGE2) biosynthesis, and leukotriene C4 (LTC4) production from lipopolysaccharide (LPS)-stimulated human amnion cells were investigated. Amnion cells produced detectable amounts of both IL-6 and TNF-α under LPS-stimulated conditions. Astragalus extract inhibited the production of IL-6. However, TNF-α production was not inhibited by the extract on L929 cytotoxicity assay. Treatment of amnion cells with LPS for up to 24 h resulted in an increase in PGE2 release in a concentration- and time-dependent manner. The extract (150 mg/ml) significantly inhibited the output of PGE2 by amnion cells (p<0.01). The arachidonate lipoxygenase metabolite (LTC4) was increased by LPS treatment of amnion cells. Astragalus extract (30 mg/ml) inhibited LTC4 production by approximately 65% throughout the culture period. These results suggest that Astragali radix extract may have a role in inhibiting bacterial infection-associated preterm labor by suppressing the productions of IL-6, PGE2, and LTC4 by human amnion cells.
In the course of screening natural products for anti-acetylcholinesterase (AChE) activity, we found that a total methanolic extract of the underground parts of Caragana chamlague (Leguminosae) had significant inhibition towards AChE. Bioactivity-guided fractionation of the total methanolic extract resulted in the isolation and identification of two active stilbene oligomers, (+)-α-viniferin (1) and kobophenol A (2). Both 1 and 2 inhibited AChE activity in a dose-dependent manner, and the IC50 values of 1 and 2 were 2.0 and 115.8 µM, respectively. The AChE inhibitory activity of 1 was specific, reversible and noncompetitive.
Four sesquiterpenes, β-selinene, isocurcumenol, nootkatone and aristolone and one triterpene, oleanolic acid were isolated from the ethylacetate fraction of the rhizomes of Cyperus rotundus and tested for their ability to modulate γ-aminobutyric acid (GABAA)-benzodiazepine receptor function by radioligand binding assays using rat cerebrocortical membranes. Among these compounds, only isocurcumenol, one of the newly identified constituents of this plant, was found to inhibit [3H]Ro15-1788 binding and enhance [3H]flunitrazepam binding in the presence of GABA. These results suggest that isocurcumenol may serve as a benzodiazepine receptor agonist and allosterically modulate GABAergic neurotransmission via enhancement of endogenous receptor ligand binding.
We recently discovered that the triterpene acid compound dehydrotrametenolic acid promotes adipocyte differentiation in vitro and acts as an insulin sensitizer in vivo. This natural product has been isolated from dried sclerotia of Poria cocos Wolf (Polyporaceae), a well-known traditional Chinese medicinal plant. We examined the effects of dehydrotrametenolic acid on plasma glucose concentration in obese hyperglycemic db/db mice. Dehydrotrametenolic acid can reduce hyperglycemia in mouse models of noninsulin-dependent diabetes mellitus (NIDDM) and act as an insulin sensitizer as indicated by the results of the glucose tolerance test. These terpenoids and thiazolidine type of antidiabetic agents such as Ciglitazone, although structurally unrelated, share many biological activities: both induce adipose conversion, activate peroxisome proliferator-activated receptor γ (PPAR γ) in vitro, and reduce hyperglycemia in animal models of NIDDM. Dehydrotrametenolic acid is a promising candidate for a new type of insulin-sensitizing drug. This finding is very important for the development of insulin sensitizers that are not of the thiazolidine type.
To investigate the effects of honey on the pharmacokinetics of glycyrrhizin and glycyrrhetic acid, administration of glycyrrhizin or glycyrrhetic acid with and without honey was carried out in rabbits in a randomized crossover design. An in vitro study using rabbit fecal flora was employed to elucidate the mechanism of the interaction. HPLC methods were used for the determination of glycyrrhizin, glycyrrhetic acid and 3-dehydroglycyrrhetic acid concentrations in serum and feces. Paired and unpaired Student's t-tests were used for statistical comparisons for in vivo and in vitro studies, respectively. Our study indicated that the area under the curve (AUC0—t) of glycyrrhetic acid was significantly enhanced by 53% when honey was concomitantly given with glycyrrhizin, whereas that of glycyrrhizin was not significantly altered. Nevertheless, lack of effect was observed when honey was concurrently given with glycyrrhetic acid. Fecal study indicated that both the hydrolysis of glycyrrhizin to glycyrrhetic acid and subsequent oxidation of glycyrrhetic acid to 3-dehydroglycyrrhetic acid were significantly affected in the presence of honey to result in more glycyrrhetic acid available for absorption. It could be concluded that honey significantly affected the gastrointestinal metabolism of glycyrrhizin and resulted in the increased glycyrrhetic acid exposure. Therefore, honey might enhance the efficacy and adverse effects of glycyrrhizin.
Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in Japan by imitating skin lightening creams commercially available in the U.S.A. and European Union. In our hospital, HQ ointments consisting of 5 or 10% HQ, 1.6% l(+)-ascorbic acid (AsA), 0.5% (w/w) Na2SO3, 10% (v/w) glycerin and hydrophilic ointment have been prepared. However, various problems have been observed including chromatic aberration of HQ ointments, relatively large variability of efficacy, and undesirable side effects although they were mild. Herein, the pharmaceutical and clinical properties of the HQ ointments were evaluated. HQ ointments were highly effective for treatment of various types of skin pigmentation. Chromatic aberration occurred during 3 months of storage, but this could be suppressed by storage at 4 °C. Chromatic aberration was independent of prescribed HQ content, and was not explained by alterations of HQ or p-benzoquinone (p-BQ) contents. Unexpectedly, removal of both antioxidants resulted in suppression of chromatic aberration, but an increase in p-BQ content. Acidification by removal of Na2SO3 only was further effective for the suppression of chromatic aberration, but with a decrease of p-BQ content except in the initial period. Chromatic aberration was due to water soluble material and insoluble material both formed by co-existence of HQ and p-BQ at a molecular ratio of 5 : 3 to 1 : 1. 1H-NMR analysis elucidated that the water soluble material was not HQ or p-BQ, and the insoluble material was a complex of HQ and p-BQ with non-covalent binding.
The liposomal phase transition temperature was monitored in unstirred suspensions using a differential scanning calorimeter. The main and pre-transition temperatures under conditions of stirring were measured by the change in 90° light scattering using a fluorescence spectrophotometer. Both methods show the same main transition temperature either with or without stirring. Temperature sensitive liposomes were made of DPPC (dipalmitoylphosphatidylcholine), DMPC (dimylisitoylphosphatidylcholine) or DSPC (distearoylphosphatidylcholine). The calcein release profile from the liposomes depends on the stirring time of the liposome suspension at the main transition temperature. For 1 h incubation, the leakage profile with and without stirring is similar. It had been hypothesized that temperature sensitive liposomes released drug at the main-transition temperature. However, calcein leakage from liposomes is observed also at the pre-transition temperature. Thus, a liposomal encapsulated drug will likely leak from DPPC liposomes at body temperature (37 °C), even if the liposomes were designed to have a higher main transition temperature.
The effects of composition of applied solutions, containing water, ethanol (EtOH) and l-menthol (LM) as penetration enhancers, on the in vitro permeation of morphine hydrochloride (MPH) through excised hairless rat skin were examined in finite application experiments. Three of the five different applied solutions contained almost saturated LM and two contained levels of LM below the limit of solubility. Despite similar pseudo steady-state fluxes (maximum fluxes observed) of MPH from the solutions, lag time for the permeation of MPH from the saturated systems was shorter than that from the unsaturated systems. Lag times for the permeation of EtOH and LM from the saturated systems were also shorter than those from the unsaturated systems. Thermodynamic activity of LM is important for the enhancing effect against MPH permeation. At the beginning for the permeation experiment, the activity of LM in the unsaturated systems was lower than that in the saturated solutions. As the skin permeability of EtOH was higher than that of other components, the content of EtOH in the applied solution gradually decreased with time, while the activity of LM increased eventually showing a sufficient enhancing effect. Solvent drag effect was not important for the permeation of MPH, since penetration rate of MPH was independent of the time course of that of EtOH. The amount of LM migrating into skin appeared to be the most important parameter for the penetration-enhancing effect of the mixed system in the in vitro permeation of MPH through excised hairless rat skin.
EtOH-soluble fraction from leaves of Laurus nobilis (bay leaves) possessed the highest alkyl peroxy radical (ROO⋅) scavenging activity among 120 kinds of herbs and edible plants, using the bioassay system which could determine the viability of Staphylococcus aureus 209p by ROO⋅ cytotoxicity. After EtOH-soluble fraction was partitioned with chloroform, ethylacetate, n-butanol and water, the ethylacetate-soluble fraction (L-EA) possessing the highest scavenging activity was further fractionated by Silica gel, Sephadex LH-20 and semi-preparative HPLC analysis on μ-Bondapak C18 reverse phase, and a major flavonol (L-EA-IIa-3-H2) in leaves of L. nobilis was isolated. According to the ultraviolet-visible absorption spectra, L-EA-IIa-3-H2 was thought to be 3, 5, 7, 3′-OH or 3(5), 7, 3′, 4′-OH flavonol. After acid hydrolysis of the fraction, L-EA-IIa-3-H2 was found to consist of quercetin and glucose, and was confirmed by one- or two-dimensional (1D or 2D)-NMR to be isoquercitrin. In addition, the ROO⋅ scavenging activity of L-EA-IIa-3-H2 was supported by ESR and its activity was found to be comparable to that of other well-known antioxidants such as epigallocatechin and resveratrol, and higher than that of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and ascorbic acid.
The antipruritic effects of orally administered 1, 4-naphthoquinone derivatives and related compounds on compound 48/80-induced scratching behavior in mice were studied. 2-Hydroxy-3-(2-hydroxyethyl)-1, 4-naphthoquinone, ferulic acid, 2, 2′-methylenebis(3-hydroxy-1, 4-naphthoquinone), and 2, 2′-ethylidenebis(3-hydroxy-1, 4-naphthoquinone) (impatienol) all exhibited significant antipruritic activity. However, 2-methoxy-3-(2-hydroxyethyl)-1, 4-naphthoquinone (balsaquinone), which was isolated from a natural source for the first time, did not show any activity. The present results indicate that these compounds are promising for treating allergic diseases with chronic and severe pruritus.
The effects of human platelets on interleukin (IL)-8 production from human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) stimulated with the fungal (1→3)-β-d-glucan schizophyllan (SPG) were examined using ELISA. PBMCs/PMNs in the presence of platelets and SPG enhanced IL-8 production in comparison with those in the presence of either platelets or SPG. IL-8 production was dependent on the concentration of platelets and incubation time, and the activity reached the maximal level at 18 h of incubation. These activities were also observed with the addition of platelets prestimulated with SPG to PBMCs. Addition of SPG directly enhanced expression of P-selectin on platelet membrane surfaces. These results suggest that platelets play a key role in the cytokine production of leukocytes induced by fungal (1→3)-β-d-glucans and might be mediated, at least in part, by P-selectin.
In a previous study, we focused on estrogenic activity of the hexane extract of diesel exhaust particles (DEP). The extract of hexane was first fractionated to acidic, phenolic and neutral portions according to their chemical properties, of which the neutral fraction was fractionated by column chromatography on silica gel. The chemical structures of compounds in these fractions were then analyzed. It was found that the neutral fraction of the hexane extract of DEP contains dibenzothiophene derivatives, one of which, 4, 6-dimethyldibenzothiophene, possesses estrogenic activity.
A novel resveratrol tetramer, vaticanol C, isolated from the stem bark of Vatica rassak markedly suppressed cell growth through induction of apoptosis, which was characterized by nuclear changes and DNA ladder formation, in three different human colon cancer cell lines.