Communication between nerves and mast cells is a prototypic demonstration of neuro-immune interaction. Recently, we used an in vitro co-culture approach comprising cultured murine superior cervical ganglia (SCG) and rat basophilic leukemia (RBL) cells to study this interaction. Previously, we concentrated mainly on the activation signal from neurites to mast cells (RBL). However, it is proposed that mast cell-nerve communication is not a one-sided relationship but a bi-directional one. In the present work, we studied the communication from mast cells to neurites. We observed that binding of anti-IgE receptor antibodies to mast cells increases calcium ion concentration [Ca2+]i in SCG neurites. This indicates that mast cell-nerve communication is bi-directional. Confocal fluorescence microscopic images indicated that [Ca2+]i in neurites increased after an increase of [Ca2+]i in mast cells. The lag-time of neurite activation was several times longer than that of mast cell activation. The correlation coefficient between the lag-times for mast cell and nerve activation was calculated to be 0.81. In addition, the fluorescence images showed that calcium signals in SCG neurites were able to extend to a long distance (100—200 μm) from the site where mast cells (RBL) attached to neurites.
The role of the sugar chain on the fibrin affinity property of tissue plasminogen activator (t-PA) was investigated using two variants of wild type t-PA (WT t-PA I and WT t-PA II) and mutant type t-PA (mt-PA; Gln117 t-PA I and Gln117 t-PA II), whose sugar chains have different structures. In terms of fibrin affinity, Gln117 t-PA was higher than WT t-PA; moreover, Type II was higher than Type I. Bindings mediated via finger domain (F mode) and kringle 2 domain (K2 mode) were distinguished using ε-amino caproic acid (EACA). Consequently, F mode and K2 mode bindings were inhibited by the sugar chains at Asn117 and 184, respectively. These results were assumed to be due to the steric hindrance of the sugar chains.
We have undertaken four basic in vitro studies and an animal experiment to obtain information about the antioxidant activities of buckwheat hull extract (BWHE). In the in vitro studies, BWHE scavenged super oxide anion produced in the xanthine/xanthine oxidase system (IC50=11.4 μg phenolic compound/ml), and strongly inhibited autoxidation of linoleic acid (IC50=6.2 μg phenolic compound/ml). Low-density lipoprotein (LDL) oxidation induced by Cu2+ ion was also protected by BWHE. In the animal experiment, ddY mice were fed a standard diet supplemented with 0.75% BWHE for 14 d. In blood, liver and brain of the mice TBARS and fluorescent substance concentration were significantly decreased compared with those of non-treated mice. SOD like activity in serum also significantly rose by BWHE treatment. BWHE was shown to be effective for protecting biological systems against various oxidative stresses in vitro, and to have antioxidant activity in vivo.
Inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP), which has a sequence similarity to the heavy chains of the inter-alpha-trypsin inhibitor (ITI) family, is a novel glycoprotein found in human plasma. We prepared two clones (1A4 and 6E11) of anti-IHRP mouse monoclonal antibody. Both of them recognized the C-terminal 35-kDa fragment which was produced by plasma kallikrein-digestion of IHRP. We developed a sandwich ELISA for measurement of the plasma IHRP concentration with the monoclonal antibody coated microtiter plate and the anti-N-terminal 57-kDa fragment of IHRP rabbit polyclonal antibody (anti-GP57). We found that the average concentration of IHRP in the plasma of healthy donors was 101.3±31.8 μg/ml (average±S.D.). The IHRP concentration in the plasma of patients with inflammatory disorders was slightly increased (137.5±40.2 μg/ml: average±S.D.). Together with the previous data indicating the induction of the porcine mRNA, which is thought to be the species counterpart of human IHRP mRNA, in the liver after resuscitation from cardiogenic shock, we propose that IHRP is a member of the acute-phase protein family.
We studied the effects of bile acid sulfonate analogs, namely, 3α,7α,12α-trihydroxy-5β-cholane-24-sulfonate (C-sul), 3α,7α-dihydroxy-5β-cholane-24-sulfonate (CDC-sul), and 3α,7β-dihydroxy-5β-cholane-24-sulfonate (UDC-sul), on serum and liver cholesterol levels, cholesterol 7α-hydroxylase activity, and biliary bile acid composition in hamsters fed cholesterol. Of the three analogs studied, UDC-sul slightly but significantly decreased free, esterified, and total cholesterol concentrations in the serum. UDC-sul and CDC-sul reduced liver total cholesterol levels by 25% and 18%, respectively, particularly in the esterified cholesterol fraction. Analysis of biliary bile acids showed the presence of the administered analogs, indicating that sulfonate analogs efficiently participate in enterohepatic cycling. The proportion of cholic acid was increased in all groups fed sulfonate analogs, but the ratio of glycine to taurine conjugated bile acids (G/T) was elevated only in UDC-sul feeding hamsters. There was no significant change in cholesterol 7α-hydroxylase activity in hamsters fed C-sul or CDC-sul, while UDC-sul slightly stimulated the enzyme activity compared to the control. The UDC-sul induced decrease in serum and liver cholesterol concentrations may be secondary to enhanced bile acid synthesis. This is supported by the increased cholesterol 7α-hydroxylase activity and elevated G/T ratio in biliary bile acids observed following UDC-sul administration.
The inhibitor for the serine protease activity of plasma hyaluronan binding protein (PHBP) was purified from human plasma by polyethylene glycol (PEG) fractionation, diethylaminoethyl (DEAE)-Sephacel ion-exchange chromatography, Phenyl Toyopearl 650M hydrophobic chromatography, Bio Gel A-0.5 m gel-filtration and hydroxyapatite chromatography. The serine protease activity of PHBP was measured with Boc-Phe-Ser-Arg-methylcoumarine amide (MCA) as the synthetic substrate of PHBP. The results of the amino acid sequence analyses of the purified PHBP inhibitor indicated that it was C1 inhibitor of the serpin family. C1 inhibitor formed a complex with PHBP, suggesting that it is the actual inhibitor of PHBP in human plasma. On the other hand, dextran sulfate and phosphatidylethanolamine enhanced the auto-fragmentation and the serine protease activity of pro-PHBP, but kaolin did not. These results suggested that the serine protease activity of PHBP was regulated in a similar manner to that of factor XII of the coagulation system.
Zinc is an essential heavy metal and is more abundant in human prostate and kidney than in other tissues. The effects of zinc on the invasion activity of human prostate and renal cancer cell lines, PC-3, LNCaP and SKRC-1, were investigated in vitro using a Transwell cell-culture chamber and were compared with specific protease inhibitors for MMPs, uPA and AP-N, respectively. The invasion activity of PC-3 cells was effectively suppressed by zinc and by all protease inhibitors in a dose-dependent manner. The invasion activity of LNCaP cells was almost unaffected by these inhibitors. In SKRC-1 cells, the invasion activity was strongly suppressed by MP03, although a moderate inhibition by zinc and bestatin was observed. The purified AP-N activity was strongly inhibited by zinc at a concentration similar to that suppressing the invasion activity of PC-3 cells and this inhibition by zinc was apparently competitive. Although the purified uPA activity was also inhibited by zinc, this inhibition was uncompetitive. AP-N was expressed abundantly on the membrane fraction of PC-3 cells among these cells tested, while its expression on the membrane fraction of SKRC-1 cells was weaker than that of PC-3 cells. The expression of uPA was also highest on the membrane fraction of PC-3 cells. These results suggest that AP-N and uPA may be involved in the invasion of human prostate cancer cells and that zinc probably participates in the invasion and metastasis of cancer cells through the regulation of the enzymatic activity of AP-N and uPA in human cancerous prostate.
We have previously reported that pur α, known to be a regulator of DNA replication and transcription, links neural BC1 RNA to microtubules via dendrite-targeting RNA motifs. Here we demonstrate the subcellular localization of pur proteins within the brain. Pur proteins were detected in neurons but not in glia. Immunohistochemical staining was prominent in perikarya and proximal dendrites and also extended into primary dendritic processes, but no significant signals were detected in the distal regions of dendrite. When homogenates of mouse brain were fractionated, pur α was most concentrated in the microsomal pellet. Consistently, pur α co-fractionated with free polysomes as well as with membrane-bound polysomes and the association with polysomes was mediated by binding ribosomal subunits. Levels of ribosomes with pur α progressively increased during postnatal development of the brain.
γ-Thujaplicin and β-dolabrin, the constituents of the wood of Thujopsis dolabrata SIEB. Et ZUCC. Var. hondai showed strong in vitro cytotoxic effects against the human stomach cancer cell lines KATO-III and Ehrlich’s ascites carcinoma. The cytotoxic effects of the two compounds against both tumor cell lines were clear when cell growth was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. γ-Thujaplicin and β-dolabrin at 0.32 μg/ml inhibited cell growth of human stomach cancer KATO-III by 85 and 67%, and Ehrlich’s ascites carcinoma by 91 and 75%, respectively. There is no large difference in cytotoxicity between these compounds, but the activity of γ-thujaplicin was slightly more potent than that of β-dolabrin. On the other hand, hinokitiol acetate did not show a cytotoxic effect, suggesting that at least a part of the mechanism of the cytotoxic effect of hinokitiol-related compounds is due to metal chelation between the carbonyl group at C-1 and the hydroxyl group at C-2 in the tropolone skeleton of these molecules. The acute toxicities [50% lethal dose (LD50) value: intraperitoneal injection, Van der Waedem] of γ-thujaplicin and β-dolabrin in mice were 277 mg/kg and 232 mg/kg, respectively.
Costunolide is an active compound isolated from the root of Saussurea lappa Clarks, a Chinese medicinal herb, and is considered a therapeutic candidate for various types of cancers. Nevertheless, the pharmacological pathways of costunolide are still unknown. In this study, we investigate the effects of costunolide on the induction of apoptosis in HL-60 human leukemia cells and its putative pathways of action. Using apoptosis analysis, measurement of reactive oxygen species (ROS), and assessment of mitochondrial membrane potentials, we show that costunolide is a potent inducer of apoptosis, and facilitates its activity via ROS generation, thereby inducing mitochondrial permeability transition (MPT) and cytochrome c release to the cytosol. ROS production, mitochondrial alteration, and subsequent apoptotic cell death in costunolide-treated cells were blocked by the antioxidant N-acetylcystein (NAC). Cyclosporin A, a permeability transition inhibitor, also inhibited mitochondrial permeability transition and apoptosis. Our data indicate that costunolide induces the ROS-mediated mitochondrial permeability transition and resultant cytochrome c release. This is the first report on the mechanism of the anti-cancer effect of costunolide.
The proteases encoded by herpesviruses including herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) are attractive targets for antiviral drug development because of their important roles in viral replication. We randomly screened a chemical compound library for inhibitory activity against HSV-1 protease. 1,4-Dihydroxynaphthalene and three naphthoquinones were found to be potent inhibitors of HSV-1 protease with IC50 values of 6.4 to 16.9 μM. Inhibitory mode analysis of the compounds against HSV-1 protease suggested that, in spite of structural similarities, only 1,4-dihydroxynaphthalene was a competitive inhibitor, whereas the three naphthoquinones were noncompetitive inhibitors. Among all assayed dihydroxynaphthalene derivatives in the chemical compound library, 1,4-dihydroxynaphthalene proved to be the most potent inhibitor of HSV-1 protease. Therefore, the two hydroxyl groups located at positions 1 and 4 on the naphthalene structure seemed essential for exertion of a potent inhibitory activity against HSV-1 protease. In addition, we have found that these compounds are also potent inhibitors of HCMV protease with extremely low micromolar IC50 values. This differed from the results of inhibitory mode analysis of HSV-1 protease, 1,4-dihydroxynaphthalene was a noncompetitive inhibitor of HCMV protease, and three naphthoquinones were competitive inhibitors. These compounds showed no effective inhibitory activity against several mammalian serine proteases (trypsin, chymotrypsin, kallikrein, plasmin, thrombin and Factor Xa) at 100 μM. These results suggest that 1,4-dihydroxynaphthalene and three naphthoquinones may be useful in the development of nonpeptidic antiherpesvirus agents.
We compared the effects of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] and its analog, 1α,25-dihydroxy-16-ene-vitamin D3 [1α,25(OH)2-16-ene-D3], as well as their interactions with 17-β estradiol (E2) on osteoblastic function in our human normal (HOB) and osteosarcoma SaOS-2 cell models representing two different stages of differentiation, the more differentiated HOB+DEX cells and SaOS+DEX cells, and the corresponding less differentiated HOB-DEX and SaOS-DEX cells. The differential effects of 1α,25(OH)2D3 and 1α,25(OH)2-16-ene-D3 and the modulation by E2 on ALP activity in HOB-DEX and HOB+DEX cells were small but significant. The most significant effects were seen in SaOS+DEX cells, in which 1α,25(OH)2-16-ene-D3 was 100-fold more potent than 1α,25(OH)2D3, the maximal enhancement being exerted at 0.1 nM and 10 nM, respectively. E2 enhanced the stimulatory effects of both compounds, with ALP being increased 2-fold at 0.1 nM (p<0.001). Osteocalcin (OC) production in HOB-DEX cells was stimulated 1.3 to 1.4-fold by 1α,25(OH)2D3 and 1α,25(OH)2-16-ene-D3 at a concentration of 0.01 nM, with E2 inhibiting the effect of 1α,25(OH)2-16-ene-D3. In SaOS-DEX and SaOS+DEX cells, 1α,25(OH)2D3 and 1α,25(OH)2-16-ene-D3 stimulated OC production 1.6-fold at 0.1 nM with E2 slightly enhancing the effect of 1α,25(OH)2D3. Western blot analysis of 1α,25(OH)2D3 receptor (VDR) levels showed that in SaOS+DEX cells, the effect of 1α,25(OH)2D3 was larger than that of 1α,25(OH)2-16-ene-D3. These results show that 1α,25(OH)2-16-ene-D3 is biologically active in human osteoblasts.
Gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol, showed selective cytotoxicity against tumor cells with higher sensitivity than normal cells such as hepatocytes and keratinocytes. To elucidate the difference in sensitivity between normal and tumor cells to gallic acid, we studied whether the inhibitor of gallic acid-induced apoptosis existed or not. A serum-free conditioned medium, prepared from high density rat primary cultured hepatocytes and cytoplasm of hepatocytes, prevented gallic acid-induced apoptosis. In contrast, hepatomas and hepatic cell lines such as dRLh-84, PLC/PRF/5, HLE, and HUH and two other kinds of tumor cell, HeLa and KB, scarcely generated such an inhibitor in either their conditioned medium or their cells. Biochemical characterization of the inhibitors revealed that the inhibitor in the hepatocyte conditioned medium was completely inactivated by heating at 65°C for 10 min. Its molecular weight was estimated at 150—250 kDa by gel filtration column chromatography, indicating that the inhibitor may be a protein-like substance. These results suggest that the generation of a large amount of the inhibitor may endow hepatocytes with insensitivity to gallic acid. In conclusion, the difference in the amount of the inhibitors generated by hepatocytes and tumor cells should contribute to the underlying mechanism in the difference in sensitivity of cells to gallic acid.
Acute toxicity (24 h) and general behavior in mice of a lignan from Justicia hyssopifolia, a β-D-glucoside (elenoside), was studied, and the cytotoxic activity was performed. Elenoside (arylnaphthalene lignan) in mice showed a moderate toxicity order (305 mg/kg) and central depressive properties at doses of 25, 50, and 100 mg/kg. It also displayed cytotoxic activity in a range of concentration of 10-5−10-4M when studied in the human tumor cell line panel of the US National Cancer Institute (NCI). The results indicated that elenoside has central depressant effects, and the cytotoxic activity of elenoside suggests that this compound and its genin derivatives merit further investigation as antitumoral drugs.
The aqueous ethanol extract of Myricae Cortex (bark of Myrica rubra SIEB. et ZUCC., Myricaceae) showed in vitro testosterone 5α-reductase inhibitory activity and in vivo anti-androgenic activity using growth of flank organ in castrated Syrian hamsters and/or hair regrowth after shaving in testosterone-treated C57Black/6CrSlc mice. Three constituents, myricanone, myricanol, and myricetin were identified as the main active principles.
Methanol extract (RM-ext) obtained from the dried rhizome of Rheum undulatum was screened for activity in experimental models of type I allergy. RM-ext exhibited the inhibition on 48-h homologous passive cutaneous anaphylaxis (PCA) in rats and an antigen-induced histamine release from rat peritoneal mast cells. Among nine stillbenes isolated from RM-ext, seven inhibited the histamine release. Rhapontigenin (compound 1), piceatannol (2) and piceatannol 3'-β-D-glycoside (6) with oral administration showed the inhibition on PCA. Compounds 1 and 2 exhibited the inhibitory effect on sheep red blood cell-induced delayed-type hyper sensitivity (SRBC-DTH) of type IV allergic model. These results indicated that the rhizome of Rheum undulatum inhibits the allergic reactions and that these inhibitory effects may be partially attributable to the stillbenes mentioned above.
The methanol soluble portion of black cumin oil, which is prepared by compression of seeds of Nigella sativa L., showed inhibitory effects on arachidonic acid (AA)-induced platelet aggregation and blood coagulation. By bioactive assay of AA-induced platelet aggregation, the methanol soluble part was purified to isolate a new compound 2-(2-methoxypropyl)-5-methyl-1,4-benzenediol (1) and two known compounds, thymol (2), carvacrol (3), having very strong inhibitory activity. Further, we then examined the isolated compounds (1—3) and eight related compounds by the screening test for AA-induced platelet aggregation. Compounds possessing aromatic hydroxyl and acetoxyl group had more potent activity than aspirin, which is well known as a remedy for thrombosis.
Amentoflavone and three other flavonoids were isolated from the ethanol extract of Selaginella sinensis. Amentoflavone showed potent antiviral activity against respiratory syncytial virus (RSV), with an IC50 of 5.5 μg/ml. The contents of amentoflavone in nine species of Selaginella were determined by reversed-phase HPLC. S. sinensis showed a higher content of 1.13%.
Intracellular disposition and cytotoxicity of macromolecular conjugate of mitomycin C (MMC) with transferrin (TF) were examined in the human hepatoma cell line HepG2 cell and normal cultured rat hepatocyte. The conjugate (TF-MMC) was specifically bound to the HepG2 cell as well as TF. The number of the binding site and the association constant of TF-MMC in the HepG2 cell were 396000±31000 molecules/cell and 3.24×107±0.58×107M-1, respectively. No difference in the binding parameters of TF-MMC and TF can be detected in the HepG2 cell. The association constant for the TF receptor was almost identical between HepG2 cell and hepatocyte, however, the numbers of the binding site of TF-MMC and TF in the HepG2 cell were from 40-times to 50-times greater than those in the hepatocyte. Furthermore, TF-MMC was internalized into the HepG2 cell and the hepatocyte as well as TF. The rates of internalization of TF-MMC and TF into the HepG2 cell were nearly identical to those into the hepatocyte. However, the levels of the internalization into the HepG2 cell were remarkably higher than those into the hepatocyte because the number of receptors in the HepG2 cell was larger than that in the hepatocyte, and the rate of release from the HepG2 cell was slower than that from the hepatocyte. TF-MMC inhibited the growth of the HepG2 cells. The 50% growth inhibition (GI50) of TF-MMC against the HepG2 cell was 0.9 μg MMC/ml, which was a little higher than that of MMC (GI50=0.5 μg/ml). These results indicated that the TF-MMC might be useful for delivery of MMC to the HepG2 cell.
The purpose of this study was to evaluate the pharmacokinetics of lansoprazole enantiomers and contribution of cytochrome P450 enzymes to enantioselective metabolism in dogs. The mean Cmax and area under the curve (AUC) values of (+)-lansoprazole were 4—5 times greater than those of (−)-lansoprazole following oral administration of 30-mg racemic lansoprazole to dogs. The CLtot/F values of (+)-lansoprazole were significantly smaller than those of (−)-lansoprazole (p<0.05). The mean unbound fraction of (−)-lansoprazole was significantly greater than that of the (+)-lansoprazole. The amount of (+)-lansoprazole remaining was significantly greater than that of the (−)-lansoprazole after incubation of racemic lansoprazole in dog liver microsomes. When the effects of ticlopidine or ketoconazole on the metabolism of lansoprazole were studied using dog liver microsomes, ticlopidine significantly inhibited the formation of 5-hydroxylansoprazole, but not another metabolite, lansoprazole sulfone; however ketoconazole significantly inhibited formation of both metabolites. When the amount of (+)- and (−)-enantiomers remaining was measured in the presence and absence of ticlopidine, the amount of (+)-lansoprazole was significantly greater than that of the (−)-lansoprazole. On the other hand, there was no significant difference between the amount of (+)- and (−)-enantiomers remaining in combination with ketoconazole. These results suggest that the enantioselective pharmacokinetics of lansoprazole enantiomers are probably ascribable to their enantioselective protein binding and/or metabolism, and among the cytochrome P450 enzymes, CYP3A contributed to the enantioselective metabolism of lansoprazole.
The effect of drug lipophilicity on in vivo iontophoretic transdermal absorption was evaluated. Non-steroidal anti-inflammatory drugs (NSAIDs) were selected as model drugs with a wide range of lipophilicity: salicylic acid (SA), ketoprofen (KP), naproxen (NP) and indomethacin (IM). Cathodal iontophoresis of NSAIDs was conducted in rats (0.625 mA/cm2; 90 min), and drug concentrations in skin, cutaneous vein and systemic vein were determined. Skin concentrations of NSAIDS were higher in the case of lipophilic drugs (SA=KP=NP<IM), whereas cutaneous plasma concentrations decreased with an increase in lipophilicity (SA>KP=NP>IM). Additionally, the dependence of drug lipophilicity on systemic plasma concentration was similar to cutaneous plasma concentration. The transfer rate from skin to cutaneous vein (RSC) was calculated from the arterio-venous plasma concentration difference of drug in the skin. Normalized RSC by skin concentration (RSC/XS) yielded a negative correlation with the logarithm of n-octanol/buffer partition coefficient (Log P at pH 7.4), suggesting that transfer of NSAIDs from skin to cutaneous vein decreased with increasing lipophilicity (SA>KP=NP>IM). This correlation means that drug partitioning between stratum corneum and viable epidermis might be a dominant step.
The gastric acidity of young to elderly Japanese subjects from 1989 to 1999 was assessed and compared with that obtained in 1984, using GA-Test capsules containing acid-dissolving granules of riboflavin. The percentage of achlorhydric subjects increased with age as observed before, however, an over all decrease in all age categories year by year was noted. The percentage of achlorhydric subjects aged 50 years in 1995—1999 was about 40%, which was lower than that (60%) in 1984. However, such a chronological change was not observed when the percentage of achlorhydric subjects was determined according to birth year, indicating that it is related to the birth year of subjects. The percentage of achlorhydric subjects correlated with infection by Helicobacter pylori. Considering the high percentage of achlorhydric elderly, bioavailability and bioequivalence studies should be performed taking into consideration the effects of gastric acidity on the in vivo performance of drug products.
We investigated the inhibitory effect of the sialyl Lewis-X (sLeX) analog, GSC-150, on hepatic metastasis of the human colon carcinoma derived cell line, KM12-HX, which highly expresses sLeX antigen on the cell surface. The number of cancer nodules found in BALB/c nude mouse liver 6 weeks after intrasplenic injection of KM12-HX cells was significantly reduced by co-administration of GSC-150. The amount of [3H]thymidine-labeled KM12-HX cells distributed in liver was also significantly reduced by GSC-150 co-administration in lipopolysaccharide (LPS)-treated mice at 48 h after administration of the tumor cells, while GSC-150 did not reduce the amount of HX cells distributed at 30 min. Considering our previous report that the initial phase of the distribution of KM12-HX cells in liver is governed by their being trapped in the hepatic microvessels because of their large size (Mizuno et al., J. Hepatol., 28, 865-877, 1998), these results suggest that GSC-150 does not inhibit this first-pass trapping by microvessels, but inhibits the subsequent process which is more directly related to final metastasis. GSC-150 inhibited the adhesion of KM12-HX cells to tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVECs). These findings imply that the anti-metastatic effect of GSC-150 in vivo could be explained by its inhibition of cell-cell interactions between cancer and host cells.
We have demonstrated that oral administration of a Kampo formulation, Byakko-ka-ninjin-to (Bai-Hu-Jia-Ren-Sheng-Tang), inhibited IgE-mediated triphasic skin reaction, including immediate phase response (IPR), late phase response (LPR) and very late phase response (vLPR), in passively sensitized mice with anti-DNP IgE antibody. Variant formulations of Byakko-ka-ninjin-to without Gypsum Fibrosum (Sekko), Glycyrrhizae Radix (Kanzo) or Oryzae Semen (Kobei) attenuated the inhibitory effect as compared with that of Byakko-ka-ninjin-to. The decreased effect of Byakko-ka-ninjin-to without Kanzo was restored by the addition of Kanzo to the variant formulations before oral administration, while the decreased effect of Byakko-ka-ninjin-to without Sekko could not be recovered by the addition of Sekko. Comparison of HPLC profiles of variant formulations without one crude drug with that of original Byakko-ka-ninjin-to revealed that some peaks could be detected only when five constituent crude drugs were simultaneously present during the preparation of Byakko-ka-ninjin-to formulation. Since elimination of Sekko from the Byakko-ka-ninjin-to constituents attenuated the efficacy although it did not show any activity per se, mutual interaction of Sekko with other constituents during the preparation may result in the production of new components. These findings suggest that the effect of Byakko-ka-ninjin-to formulation on cutaneous inflammatory disease can differ from the sum of the effect of the individual constituents.