Attention was paid to Panax ginseng C. A. MEYER which plays an important role in Oriental medicine. Some pharmacological experiments were carried out with pure saponins [ginsenoside (GS)-Rb₁, Rb₂, Rc, Re and Rg₁] isolated from the Panax ginseng root, a mixture of ginseng saponins [ginsenoside mixture B (GMB) obtained from the lateral root (Hakumo) and crude ginsenoside K (GSK) obtained from the main root (Hakusan)], and prosapogenins (PSG), partial hydrolyzates of Rb₁, Rb₂, Rc and Rd [20R-PSG, 20S-PSG and Δ²⁰-PSG], by using specific repeatedly cold stressed (SART stressed) mice and in restraint and water immersion-stressed (RWIS) mice. A single i.p. administration of 10 mg/kg of GS or PSG gave no influence on pentobarbital-induced sleeping in non-stressed mice. The inhibition of a natural increase in body weight in SART stressed mice was markedly counteracted by administration with a daily dose of 2.5 mg/kg of Rb₁, Rc, Re, 20S-PSG or GSK for 5 consecutive days during SART stressing. A single i.p. administration of 10 mg/kg of Rb₂, Rc, Re, 20R-PSG or 20S-PSG increased the analgesic index by the modified Randall-Selitto method, and that of 20R-PSG or 20S-PSG decreased the writhing syndrome by the method of acetic acid in non-stressed mice. When SART stressed mice were used as test animals in place of non-stressed mice, the analgesic effect was further augmented. Prolonged actions were observed in SART stressed mice administered daily with 5-10 mg/kg of Rb₂, Rc, Re, 20R-PSG or 20S-PSG. When analgesic effect was tested 60 min after the last administration by the modified method of Randall-Selitto, almost the same effect as the single administration was obtained. The inhibitory effect on acetic acid writhing of Rb₁, Rb₂, Re, Δ²⁰-PSG, and GMB, which was ineffective by a single administration, in addition to Rg₁, 20R-PSG and 20S-PSG, was observed. The decrease in ACh response of the isolated SART stressed mouse duodenum was inhibited by daily administration of Rb₁, Rb₂, Rc, Re, 20S-PSG, GMB, and GSK. The increase in ACh response of the isolated RWIS mouse duodenum was inhibited by three pretreatments with Rb₁, Re, Rg₁, 20S-PSG and GMB, but not with Rb₂, Rc, or GSK. These findings suggest that the effects of ginseng saponins may be different from those of Saikosaponins reported previously. The former compounds have a weak analgesic action, and may improve some symptoms of vegetative stigmatism caused by SART stress and RWIS. The classification of GS and PSG based on their actions was attempted.

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The effect of doxapram, a respiratory stimulant, on the pentobarbital sleeping time was investigated in mice. The sleeping time induced by the intraperitoneal injection of pentobarbital was prolonged 0-120 min after the administration of doxapram (25-100 mg/kg, i.p.). The pretreatment with doxapram 60 min before had no effect on the anesthetic time induced by ether and on the sleeping time induced by the intracerebroventricular injection of pentobarbital, while increased the lethality of pentobarbital only slightly and the levels of pentobarbital in the plasma and brain significantly. The activities of pentobarbital oxidase and aminopyrine N-demethylase in the 9000×g supernatant fraction of the liver were inhibited by the pretreatment with doxapram 60 min before the test. On the other hand, 12-24 hr after the injection of doxapram the pentobarbital sleeping time was markedly shortened. Thus, the biphasic effect of doxapram, prolongation at first and shortening later, on the pentobarbital sleeping time was observed. It is possible that doxapram inhibits the hepatic microsomal drug-metabolizing enzymes without an increase in the sensitivity of the central nervous system at first and stimulates these enzymes during the second phase.

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Three methods of fractionation of ginseng radix (Panax ginseng C. A. MEYER) components for a survey of hypoglycemic principle in alloxan diabetic mice were conducted and three groups of components tested ; fat-soluble components, ginseng saponins and a third component with hypoglycemic activity. Pharmacological sequential trials of the fractionation yielded a most active fraction which was about 100-fold more effective than the original water-soluble extract of the ginseng radix. The ED₅₀ value was 0.4 mg/kg in lowering the blood level of glucose in alloxan diabetic mice. It was demonstrated that some ginseng fractions inhibited epinephrine-induced transient hyperglycemia in mice, increased glycogen content in rat liver, decreased the blood level of acetone bodies in alloxan diabetic mice, and inhibited the release of free fatty acid from rat epididymal fat pad. The results showed that hypoglycemic components existed in a new component of ginseng radix which is different from saponin.

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Some fractions extracted from ginseng radix (HAKUSAN) caused hypoglycemic effect on alloxan diabetic mice. The effect was abolished by the i.v. injection of antisera against bovine insulin. The same doses of the ginseng fraction (10-50 mg/kg) produced an increase in the blood insulin level in alloxan diabetic mice. Normal mice loaded i.p. with glucose (2 g/kg or more) showed also such an increase. Insulin release from perfused rat pancreases was stimulated by the ginseng fraction (0.2 mg/ml), but the potency was not stronger than that of the sulfonylureas. It was demonstrated that glucose-induced insulin release was marked in the presence of the ginseng fraction. Impaired insulin responses to glucose in alloxan diabetic rats were increased by the fraction (0.5 mg/ml) to or above the control responses in normal rats. The enhanced effect was observed also in the presence of 100 μg/ml cycloheximide. These results indicate that some ginseng fractions stimulated insulin release, especially glucoseinduced insulin release from pancreatic islets and thereby lowered the blood glucose level.

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The effects of calcitonin (CT) and histamine on calcium metabolism in the hepatic bile system of rats were investigated. The subcutaneous administration of CT (20 and 80 MRC mU/100g) to rats caused a significant fall in serum calcium and a marked increase in liver calcium. The subcutaneous administration of histamine (0.1 mg/100 g) to rats produced a significant decrease in serum calcium, while it did not significantly alter liver calcium. However, the effects of CT (20 or 80 MRC mU/100 g) on the serum and liver calcium was markedly prevented by the simultaneous administration of histamine (0.1 mg/100 g). On the other hand, the excretion of calcium into the bile of thyroparathyroidectomized rats intraperitoneally injected with the calcium chloride (4.0 mg Ca/100 g) was markedly increased by the administration of CT (20 or 80 MRC mU/100 g) or histamine (0.1 mg/100 g). Reversely, a remarkable elevation of bile calcium by CT was clearly prevented by the simultaneous administration of histamine (0.1 mg/100 g). The present results further support the point of view that the hypocalcemic effect of CT is based on the stimulation of calcium excretion into the bile by the hormone.

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When an isolated mouse diaphragm was incubated with an insuline-like activitypossessing protease (ILAPP), the amount of Folin-positive materials released into medium increased as the incubation proceeded. However, the digestion by ILAPP did not affect te action of epinephrine on the glycogen content in diaphragm. Comparative studies on the effects of ILAPP and insulin on the glycogen content in diaphragm incubated in the presence or absence of ouabain at different concentrations of K⁺ increased in the incubation medium, that the glycogen-increasing effect of ILAPP or insulin was observed in each buffer containing K⁺ at a concentration of 0, 0.59, or 5.9 mM, and that the additive effect of ouabain used together with ILAPP or insulin was distinctly observed in the 5.9 mM K⁺-buffer but not in the 0.59 mM-buffer or K⁺-free buffer. Thus, the effects of ILAPP resembled those of insulin. ILAPP probably idgests the surface of diaphragm at a limited portion other than an epinepherine receptor to exhibit insulin-like activity.

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Effect of proteinase inhibitors on the carrageenin-induced inflammation was studied. The formation of granulation tissue was markedly inhibited by a single injection of ε-amino-n-caproic acid n-hexyl ester (EACA hexyl ester, 300 mg/kg) into the carrageenin-air-pouch immediately after carrageenin injection, whereas repeated injections of the inhibitor starting at 12 hr, 24 hr and 48 hr after carrageenin injection were less effective, slightly effective and ineffective, respectively. A dose-dependent inhibition of both the formation of granulation tissue and the migration of polymorphonuclear leukocytes (PMNs) into the inflammatory locus was found by a single injection of EACA hexyl ester into the carrageenin-air-pouch immediately after carrageenin injection. Similarly, a single injection of L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK, 50 mg/kg) and N-α-p-tosyl-L-lysine chloromethyl ketone (TLCK, 30 mg/kg) inhibited both the formation of granulation tissue and the migration of PMNs into the inflammatory locus. These results suggest that serine proteinase inhibitors such as EACA hexyl ester, TPCK and TLCK exert their anti-inflammatory actions by interfering with the initial inflammatory reactions including the migration of PMNs into inflammatory locus after carrageenin injection.

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Both pharmacokinetics of total and free gliclazide, a potential hypoglycemic drug, were studied in the healthy (n=12) and diabetic subjects (n=11). The blood level of gliclazide was determined by a high-performance liquid chromatography, and the free gliclazide (unbound to proteins) in the serum separated by means of an ultrafiltration. The binding ratio of gliclazide to the blood proteins was about 96% during the periods of 24hr after administration of the drug. Several pharmacokinetic parameters for the blood gliclazide were derived from the decay curves of the blood drug levels. Each pharmacokinetic parameter was not changed by differences between the healthy and diabetic subjects, the total and free drug levels, and the method of administration of the drug ; each mean parameter, the elimination rate (K_e), the time to peak level (t_max), the elimination half-life (t_1/2) and the volume of distribution (Vd_β) was 0.07 hr^-1, 2.8 hr, 12.3 hr and 16.4 1 (total level), respectively. The serum from healthy subject receiving orally administered gliclazide was gel-filtered on a Sephadex G-150 column. Each fractionated serum protein of macroglobulin (IgM), γ-globulin (IgG), albumin (A) and small molecular substances (F), contained gliclazide at average of 3.7, 0.7, 82.3 and 13.2%, respectively, during the periods of 24 hr after administration. In in vitro experiment, it was found that the ratio of gliclazide-albumin binding kept a constant level at 96.5% in the range of normal protein levels (3.3-4.8 g/100 ml). In conclusion, the present result shows that the pharmacokinetics of the total blood level of gliclazide reflect the free gliclazide level, moreover, gliclazide predominantly binds with albumin in the blood and its binding ratio is not constant, but variable according to the dose-relation between the drug and the serum protein.

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The influences of bradykinin, kallidin and dipeptidyl carboxypeptidase (kininase II) on the motility of human sperm thoroughly washed with Ficoll were investigated by an objective method with multiple exposure photography technique which recorded stroboscopically the track of sperm migration. Addition of 0.1-10 ng/ml of bradykinin or kallidin to sperm suspension caused a stimulation of motility. The effect appeared by increase of the velocity of forward motile sperm, whereas the percent of motile sperm was not altered. On the contrary, higher dose of kinins suppressed the motility by decreasing the percent of motile sperm. There was a minor contamination of dipeptidyl carboxypeptidase even in the highly washed sperm suspension. This enzyme was identical with angiotensin I converting enzyme or kininase II and the major enzyme of kinin degrading activity in seminal plasma. Stimulation of sperm motility by kinin was further enhanced in the presence of SQ 14225, a specific inhibitor of dipeptidyl carboxypeptidase, by protection of added kinins from the residual dipeptidyl carboxypeptidase in the sperm suspension. Dipeptidyl carboxypeptidase level in ejaculated human semen was found to correlate to the semen quality, such as sperm density and motility, but it gave no direct influence on sperm motility.

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In the displacement study, the effects of tolbutamide on the absorption spectra and induced CD spectra of 2-(4'-hydroxyphenylazo) benzoic acid (HABA)-bovine serum albumin (BSA) complex were investigated using the samples obtained from equilibrium dialysis at pH 7.40. The binding of tolbutamide to BSA enhanced the binding of the azo form in spite of the displacement of the hydrazone form of bound HABA at the molar ratio of HABA to BSA of 0.65, causing the increase in the negative ellipticity at 445 nm, which was termed the C band. The C band could be assinged to the azo form of bound HABA.

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