Giant hornet (Vespa mandarinia) venomous phospholipases. The purification, characterization and inhibitory properties by biscoclaurine alkaloids.

Two species of giant hornet phospholipase B (PLB), alpha and beta, were purified from the venom of Vespa mandarinia. The purification procedure was simplified by two steps of column chromatographies, Sephadex G-100 and SP-Sepharose. The molecular sizes of PLB alpha and beta were 29.5 and 26.0 kDa, respectively. The isoelectric point of alpha and beta enzymes were pH 10.6 and 10.7, respectively. The temperature optimum for egg yolk lecithin was a broad peak at 40-60 degrees C for both enzymes. Amino acid compositions of both enzymes were high contents of aspartic acid, glycine, leucine, lysine and other aliphatic amino acids. Cystine was similar amounts to other species of phospholipases (PLs). The K(m) values of alpha and beta enzymes were 8.29 and 7.53 mg/ml for egg yolk lecithin, respectively. In the catalytic specificity for L-alpha-phosphatidylcholine-beta-oleoil-gamma-palmitoil, the K(m) values of alpha enzyme for gamma-palmitoil and beta-oleoil residues were 0.528 and 1.392 mM, respectively. While the K(m) values of beta enzyme for gamma-palmitoil and beta-oleoil residues were 7.91 and 2. 68 mM, respectively. Both alpha and beta enzymes were inhibited strongly by cepharanthine. The lecithin hydrolysis of alpha enzyme was competitively inhibited, but beta enzyme was uncompetitive. Cepharanthine also inhibited noncompetitively PLA(2)s of bovine pancreas, bee venom and Naja mossambica mossambica.

Potent antiperoxidation activity of the bisbenzylisoquinoline alkaloid cepharanthine: the amine moiety is responsible for its pH-dependent radical scavenge activity.

The bisbenzylisoquinoline alkaloid cepharanthine, which has been considered to exhibit antiperoxidation activity due to its membrane stabilizing effect, was found to scavenge radicals such as .OH and DPPH (1,1-diphenyl-2-picrylhydrazyl) in solution, and to inhibit lipid peroxidation in mitochondria and liposomes by Fe2+/ADP. The antiperoxidation activity of cepharanthine in rat liver mitochondria initiated by Fe2+/ADP at pH 7.4 was much greater than that of alpha-tocopherol, its half-inhibitory concentration being about 23 microM. However, cepharanthine was effective only at neutral pH values such as pH 7.4, not in a moderately acidic pH region below pH 6.5. Accordingly, the neutral form of the deprotonated amine moiety in the tetrahydroisoquinoline ring is concluded to be responsible for the radical scavenging activity of cepharanthine. There are two amine moieties in the cepharanthine molecule, but we specified the effective amine moiety from the antiperoxidation activities of the imine analogs of cepharanthine.

Differences in tissue angiotensin II-forming pathways by species and organs in vitro.

Angiotensin (Ang) II plays an important role in cardiovascular homeostasis, not only in the systemic circulation but also at the tissue level, and is involved in the remodeling of the heart and vasculature under pathological conditions. Although alternative Ang II-forming pathways are known to exist in various tissues, the details of such pathways remain unclear. The aim of this study was to examine tissue Ang II-forming activities and to identify the responsible enzyme in several organs (lung, heart, and aorta) in various species (human, hamster, rat, rabbit, dog, pig, and marmoset). Among the organs examined, the lung contained the highest Ang II-forming activity. The responsible enzyme for pulmonary Ang II formation was angiotensin I-converting enzyme (ACE) in all of the species except the human lung, in which a chymaselike enzyme was dominant. In the heart, the highest total Ang II-forming activity was observed in humans, and a chymaselike enzyme was dominant in all of the species except rabbit and pig. Aorta exhibited a relatively high total Ang II-forming activity, with a predominance of chymaselike activity in all of the species except rabbit and pig, in which ACE was dominant. Our results indicate that there were remarkable differences in Ang II-forming pathways among the species and organs we examined. To study the pathophysiological roles of ACE-independent Ang II formation, one should choose species and/or organs that have Ang II-forming pathways similar to those in humans.

Bisbenzylisoquinoline alkaloids inhibit tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin.

Bisbenzylisoquinoline alkaloids, cepharanthine, berbamine and isotetrandrine, were isolated from Stephania cepharantha Hayata. These compounds inhibit arachidonic acid-induced inflammation in mice. We have found that cepharanthine inhibits tumor promotion after topical application and oral administration in two-stage carcinogenesis in mouse skin. Furthermore, topical application of berbamine (2 mumol/mouse) and isotetrandrine (2 mumol/mouse) markedly suppressed the tumor-promoting effect of 12-O-tetradecanoylphorbol-13-acetate (1 microgram) in mouse skin initiated with 7,12-dimethylbenz[a]anthracene (50 micrograms), at a grade corresponding to that of cepharanthine.

[Effect of cepharanthin on the lethality and cardiovascular disorder by Mamushi, Agkistrodon halys blomhoffi, snake venom].

Effects of Cephranthin on the lethality to mice and the cardiovascular disorder in rat induced by Mamushi (Agkistrodon halys blomhoffi) snake venom were studied. The LD50 of Mamushi snake venom was 1.22 mg/kg in mice by i.p. administration. Cepharanthin activated slightly the lethal activity when it was administered just after injection of the venom. However, this drug was effective against the venom at 4 to 5 times the lethal dose, if it was administered at the optimal interval. Mamushi snake venom and Cepharanthin showed a lethality and dose relationship that were clearly prolonged by Cepharanthin. In rat, Mamushi snake venom administered intraperitoneally produced an irreversible decrease of blood pressure accompanied by capillary hemorrhage. Moreover, it suppressed the heart rate. On the electrocardiogram, prolongation of ventricular contraction, extension of the relative refractory period and reduction of diastole were induced by the venom. These observations show that the venom suppresses cardiac functions inotropically and chronotropically. Cepharanthin shortened the refractory period and prolonged the diastole, indicating that it activated cardiac function. It was able to produce recovery from several cardiac disorders induced by Mamushi snake venom, for example, increase of blood pressure with inhibition of capillary hemorrage, release of heart rate suppression, partial recovery of the amplitudes of P and R waves, and shortening of extensive relative refractory period. These cardiac recoveries by Cepharanthin will be reflected in the suppression of lethality.

Cepharanthine inhibits two-stage tumor promotion by 12-O-tetradecanoylphorbol 13-acetate and mezerein on skin tumor formation in mice initiated with 7,12-dimethylbenz[a]anthracene.

Cepharanthine, isolated from Stephania cepharantha, is one of the bisbenzylisoquinoline-type alkaloids. We have found that it inhibits tumor promotion after topical application in two-stage carcinogenesis in mouse skin. Epidermal ornithine decarboxylase activities inhibited by topical application of cepharanthine, with an 5 micrograms/mouse) and mezerein (5 micrograms/mouse) were found to be inhibited by topical application of cepharanthine, with a ED50 of 1.2 mumol and 1.4 mumol respectively. These inhibitory effects of cepharanthine are considered to be related to its antitumor activity in two-stage carcinogenesis in mouse skin. Cell-mediated immunosuppression by TPA was unaffected by topical application of cepharanthine. A diet containing 0.005% cepharanthine (about 0.5 mg mouse-1 day-1) slightly suppressed the two-stage promotion of skin tumors by twice-weekly applications of 2.5 micrograms TPA for 2 weeks (first stage) followed by twice-weekly applications of 2.5 micrograms mezerein for 23 weeks (second stage) in ICR mice following initiation by 50 micrograms 7,12-dimethylbenz[a]anthracene. Oral administration of cepharanthine inhibits the tumor promotion in two-stage carcinogenesis in mouse skin.

Structure-activity relationships of 4'-O-substituted 1-benzylisoquinolines with respect to their actions on the cell membrane of blood platelets and erythrocytes.

4'-O-Substituted 1-benzyl-2-methyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolines inhibited the collagen-induced activation (aggregation and ATP secretion) of rabbit platelets and transformed the shape of human erythrocytes in a dose-dependent manner. Both effects increased with increasing number of carbon atoms of the 4'-substituted hydrocarbon moiety. The incorporation of these compounds into the erythrocyte membrane was also dependent on the number of carbon atoms of the 4'-substituted radical. The most potent phenoxy derivative suppressed arachidonic acid release from membrane phospholipids but had no effect on arachidonic acid metabolism in platelets. This indicated that the effects are comparable to those of a natural bisbenzylisoquinoline, cepharanthine. These effects appear to be due to a perturbing action on the membrane lipid bilayer.

[Mode of the anti-allergic action of cepharanthine on an experimental model of allergic rhinitis].

Cepharanthine, a biscouclaurine alkaloid of Stephania cepharantha, has been used for various clinical purposes. Cepharanthine was known to inhibit histamine release from mast cells obtained from sensitized animals. In vitro studies suggested that the mechanism of action of cepharanthine may be ascribed to the membrane stabilizing action. The membrane stabilization may be attained by reducing the elasticity of the membrane. However, in vivo mechanisms of the anti-allergic action of cepharanthine have not been examined. In the present in vivo study, the anti-allergic action of cepharanthine was examined using experimental allergic rhinitis in rats. The locally administered cepharanthine solution (0.1 mg/ml), by perfusing the nasal cavity, inhibited the dye leakage and an increase in lysosomal enzyme activity due to antigen stimulation. This is the reason for the membrane stabilization by cepharanthine. In the metyrapone (20 mg/kg, s.c., 5 days) pretreated rats of the rhinitis models, the anti-allergic action of cepharanthine was weaker. On the other hand, the effect of ketotifen was not altered by such an effect. The experimental results suggest that the anti-allergic mechanism of cepharanthine might be exerted by its membrane stabilizing action and by stimulation of the pituitary-adrenotropic function.

[Effects of cepharanthine on experimental nasal allergy].

The anti-allergic actions of cepharanthine were examined using experimental nasal allergy rats (rhinitis models). In the actively sensitized rhinitis models, leaks of pontamine sky blue (PSB) dye in the perfusate of the nasal cavity were suppressed by cepharanthine treatment. Leaks of PSB dye in the perfusate were also suppressed by ketotifen treatment. beta-Glucuronidase activity in the perfusate was lower in the cepharanthine group and in the ketotifen group. In the passively sensitized rhinitis models, leaks of PSB dye in the perfusate were suppressed by cepharanthine treatment. Leaks of PSB dye in the perfusate in the ketotifen group were also suppressed. However, beta-glucuronidase activity was not different among the three groups. Cepharanthine (0.025-25 mg/kg body weight) and ketotifen (0.1-10 mg/kg body weight) inhibited leaks of PSB into the perfusate in a dose-dependent manner. These results suggest that cepharanthine may be clinically effective for treating patients with nasal allergy, and its anti-allergic mechanism may be the same as that of ketotifen.

[Stimulation of pituitary-adrenocortical system by cepharanthine].

We observed that cepharanthine might exert its anti-allergic action by stimulating the secretion of corticosterone. The present experiments were carried out to investigate stimulation of the pituitary-adrenocortical system by cepharanthine. Administration of cepharanthine to rats produced increases in plasma and adrenal corticosterone levels. Administration of cepharanthine to propranolol pretreated rats also produced increases in plasma and adrenal corticosterone levels and plasma ACTH level. The elevation of corticosterone level induced by cepharanthine was considered to be the specific effect of cepharanthine. Cepharanthine did not increase plasma corticosterone level in rats in the state of dexamethasone suppression of the pituitary-adrenocortical system, in which the level was lowered. Administration of cepharanthine to Bordetella pertussis vaccine induced beta-adrenergic blocked rats also produced increases in plasma and adrenal corticosterone levels. The production and release of corticosterone from an adrenal cell suspension were not influenced by cepharanthine in vitro. These results suggest that cepharanthine stimulates the pituitary-adrenotropic function.