The pharmacological studies of central effects of herb paeony root, chiefly of its component paeoniflorin (Pae), were carried out. Herb paeony root is frequently used in chinese medicine. As licorice is one of the crude drugs which are frequently used in combination with paeony, combined effects of Pae with licorice component FM 100 were examined in order to investigate the combined effects of both crude drugs. Mice were used in these tests. The pharmacological effects of Pae are given as follows. The acute toxicity was very low. Sedative symptom was found. Loss of righting reflex was obtained in the rat by intraventricular administration. Sleeping duration induced by hexobarbital was prolonged. Writhing symptom induced by intraperitoneal administration of AcOH was inhibited. Weak hypothermia and weak anticonvulsive effect to pentylenetetrazol were found. The pharmacological effects of FM 100 are given as follows. Analgesia was found in both writhing test and tail-pressure test. This action was also obtained in the rat by intraventricular administration in the method which consists in pressing base of the fingers of the hind feet. Marked hypothermic and antipyretic effects and weak anticonvulsive effect to pentylenetetrazol were found by intraperitoneal administration. The combined effects of Pae with FM100 were shown as synergistic in almost all experiments.
Pharmacological studies of peony root, chiefly of its component paeoniflorin (Pae), were carried out : anti-inflammatory effect, inhibitory effect on gastric juice secretion, preventive effect on stress ulcer, antidiuretic effect and combined effects with licorice component FM100 had been studied. Pae showed a weak anti-inflammatory effect significantly inhibiting the rat paw edema induced by carrageenin and showing a tendency to inhibit dextran- or α-chymotrypsininduced edema and exudation of dye into the abdominal cavity of the mouse. Pae had also a preventive effect on stress ulcer in the rat. The combined effects of Pae with FM100 were obtained as synergistic in the tests to evaluate inhibitory effect on gastric and anti-inflammatory effect by intraperitoneal administration. In so far as our tests were concerned, the extract of peony root showed little effects by oral administration ; marked effects could not be obtained even by intraperitonael administration.
Pharmacological studies of peony component paeoniflorin (Pae) were carried out as for its effects on circulatory and respiratory systems, on isolated smooth muscle and skeletal muscle and on smooth muscle organs in situ. The effects of licorice component FM100 on isolated organs were examined as well. Pae showed no antitussive effect on the guinea pig and scarcely affected the respiration of the guinea pig. Pae showed hypotensive effect on the guinea pig which can be thought to be based on its peripheral vasodilation. Direct action on heart muscle and antifibrillatory effect on ouabain-induced arrhythmia in the guinea pig were scarcely found. Pae showed vasodilation of coronary vessel and hind limb of the dog. Relaxation and inhibition of movement and tonus of smooth muscle organs such as rat stomach or rat uterus were also found. The experiment in isolated organs revealed anti-oxytocic action in rat uterus only, suggesting some organ specificity. The relaxing effect of Pae on smooth muscle is assumed to be mainly musculotropic. FM100 showed anti spasmodic action in isolated guinea pig intestine and rat uterus, being synergistic with Pae. Pae is decomposed into desbenzoylpaeoniflorin and benzoic acid, but both compounds scarcely showed such pharmacological effects as Pae did.
A semi-quantitative trial was carried out in order to clarify the therapeutic effects of herb peony root and licorice root in chinese medicine as objectively as possible. As shown in Table II and IV, abdominal pain and "Fukukin Koren" (syndrome like stiffness of abdominal muscle, specific observation in chinese medicine) were analysed first of all as therapeutic effects of peony. Accordingly, antispasmodic, analgesic, sedative, and antiinflammatory effects were expected as pharmacological ones. The therapeutic effects of licorice were analysed as soothing pain first of all, expecting the similar pharmacological effects to peony. On the other hand, the pharmacological effects of paeoniflorin (Pae) and FM100 reported in foregoing 3 papers were tried to be expressed by means of scoring procedure based on the comparison with the value of acute toxicity (LD30) as shown in Table VII. As to Pae, central depressive effects such as sedation etc., antispasmodic effect and anti-inflammatory effect, and as to FM100, analgesic effect, antispasmodic effect and inhibitory effect on gastric juice secretion were obtained as main effects respectively. The therapeutic effects of paeony coincided well with the effects of Pae in the experimental pharmacological tests. Pae was thought to be the effective component of paeony. From the pharmacological effects of FM100, important pharmacological supports were obtained for the therapeutic effects of licorice and both of the crude drugs in combination were characterized as pharmacologically synergistic.
During studies on the stability of vitamin D2 (D2) with various excipients, it was found that D2 is readily isomerized in powders prepared with CaHPO4 or talc, yielding isocalciferol and isotachysterol, and that this isomerization is catalyzed by surface acid on the excipients. In this paper, the isomerization reaction was examined in n-hexane solutions of D2 in which natural aluminum silicate (pKa -8.2 to -5.6), CaHPO4 (pKa 0.8 to 1.5) or talc (pKa⪈1.5) was suspended. The end products were isotachysterol and a small amount of isocalciferol, and the initial isomerization rates of D2 were found to be dependent on the acid strength (pKa) and acidity of the solid surfaces. The rates were independent of the initial concentration of D2, if excess D2 remained in the liquid phase. The apparent activation energy in this isomerization reaction was about 25 kcal mole-1. When n-butylamine was chemisorbed on solid surfaces, the rates decreased proportionally with increasing amine content.
Stability of vitamin D2 (D2) in powder preparations mixed with various excipients was examined in the presence or absence of air. In a preparation with CaHPO4 (acid strength of surface pKa 0.8 to 1.5), containing 5% D2 and 1% butylated hydroxyanisole (BHA), the D2 loss was 72% in vacuum (1 mmHg) and 78% in air under low humidity after a 5 days storage at 37°. With lactose (pKa 4.0 to 4.8), the D2 loss was very small under the same conditions. These results show that the degradation of D2 mixed with CaHPO4 proceeds through isomerization rather than by oxidation. Such phenomenon is generally observed with excipients such as synthetic aluminum silicate, magnesium trisilicate, talc or CaSO4, having a pKa of 3.3. When the preparations were stored under high humidity, the stability of D2 increased markedly because the surface acidity of the excipients decreased through adsorption of moisture during storage. As isomerization products of D2, a new isomer was found together with isocalciferol and isotachystreol.
In a previous study, it was found that vitamin D2 (I) in powders prepared with excipients such as talc or CaHPO4, which have a high surface acidity, is readily isomerized yielding a new isomer (III) together with isocalciferol (II) and isotachysterol (IV). In this paper, the physicochemical properties of III were determined. Compound III, C28H44O, [α]22D+40.8° in CHCl3, was isolated from the other isomers through alumina column chromatography, and was crystallized as a p-phenylazobenzoate, C41H52O2N2, mp 152-153°, [α]23D+359.4° in CHCl3. III absorbs at 286.5 mμ (ε, 38000) with shoulders both at 276 mμ and 298 mμ in isopropanol. Infrared and nuclear magnetic spectral resonance data, as well as the results of optical rotatory dispersion and circular dichroism suggest that the structure of III is 9, 10-secoergostatetraene-(1 : 10, 5 : 6, 7 : 8, 22 : 23)-3α-ol, which is a cis isomer of II. III is redily isomerized to II by iodine and to IV by boron trifluoride, both in n- hexane solutions.
This paper deals quantitatively with the relationship between the surface acidity of excipients and isomerization of vitamin D2 (I) and its isomers, involving isocalciferol (II), 5, 6-cis-isocalciferol (III), isotachysterol (IV), precalciferol (V), 5, 6-trans-calciferol (VI), and tachysterol (VII). Isomerization rates and products were examined in hexane solution of each isomer in which CaHPO4 (pKa 0.8 to 1.5) was suspended. The results showed that the isomerization products are I→II and IV, III→II and IV, VI→II and IV, VII→II and IV, V→I, II, and IV (I is thermal transformation product of V), II→IV. Similar results were obtained when natural aluminum silicate or talc was used. The isomerization rates of vitamin D2 and its isomers were intimately related with "Bronsted acidity, " rather than "Lewis acidity, " on the solid surfaces. Natural aluminum silicate was base-exchanged with sodium chloride solution, and was re-activated in air at 300°. This sample exhibited little diminution in Lewis acidity although its activity in D2 isomerization decreased markedly with decreasing Bronsted acidity. On the other hand, catalys tsample which had been saturated with perylene, that gives colored complexes with typical Lewis acids, was found to be still effective for this isomerization. In a powder preparation, 5, 6-cis-isocalciferol (III) was obtained together with II and IV as the isomerization products of vitamin D2 (I). This differs from the results obtained suspension samples.
This paper presents the analytical technique utilized in this series of experiments. Experiments were carried out on n-hexane solution containing vitamin D2 (I) and/or its isomers involving isocalciferol (II), 5, 6-cis-isocalciferol (III), isotachysterol (IV), precalciferol (V), 5, 6-trans-calciferol (VI) and tachysterol (VII). A mixture containing 0.1 mg of each component was chromatographed on a column (1×10 cm) of weakened alumina containing 5% water. The chromatogram was developed with 16.5% ether in n-hexane. The eluate fractions of each component were : II and VI (25-45 ml), IV and V (50-70 ml), I, III and VII (75-95 ml). Components, separated quantitatively from the mixtrue, were determined by ultraviolet spectrophotometry in isopropanol. Fractions containing nonseparable components were analyzed by means of SbCl3 coloration after the mixtures were treated with maleic anhydride (MA). It was found that I, V, VI, and VII formed an adduct with MA in benzene within two hours at 80°, while II, III and IV give a negative reaction with MA, and that the adducts are uncolored with SbCl3 reagent. The use of the difference in reaction rates with MA, therefore, made it possible to determine each component in the above fractions within a 10% error.
Examination was made on the relationship between acid strength (pKa) of solid surfaces and isomerization of vitamin D2 (D2). A mixtures of D2 and other powdered vitamins was stored at 37° in vacuum (1 mmHg). It was found that D2 was readily isomerized by vitamins such as ascorbic acid, folic acid, thiamine hydrochloride, or pyridoxine hydrochloride, as well as by talc or CaHPO4, because each vitamin has a pKa of 2.0 to 4.0 in the dry state. Nicotinamide and calcium pantothenate, both having a pKa of 4.8 to 6.8, did not isomerize D2. D2 was isomerized also by citric acid or phosphoric acid in dry preparations. It is undesirable, threfore, to use any of the above acids as a synergist for an antioxidant to prevent the oxidation of D2. The main isomerization products of D2 were isocalciferol, 5, 6-cis-isocalciferol, and isotachysterol.
The effects of certain compounds which have the ability to reduce the surface acidity of excipients were examined in the isomerization of vitamin D2 (D2). Ethanolamines and polyoxyethylene (POE) compounds, such as polyethylene glycol 4000 (PEG), POE (20) stearate, or POE (23) lauryl ether, were used as stabilizers. The relationship between the acid strength of solid surfaces and the isomerization of D2 was quantitatively examined in n-hexane solutions of D2 in which natural aluminum silicate, CaHPO4, or talc deactivated with PEG were suspended. The isomerization rates of D2 decreased with increasing amount of PEG coated on the solids, and isomerization ceased when their acid strength had a pKa 3.3. In powder preparations, D2 isomerization was not observed when the excipient surfaces were reduced by the amines to a pKa 4.8. For example, in a preparation with talc (pKa⪈1.5) containing 0.5% D2 and 0.1% BHA, the D2 loss was 90% after a storage of 5 days at 37°, but only 8% after even 30 days with the addition of 1% triethanolamine. Coating agents, such as shellac, ethocel or celullose acetate phthalate, were unable to prevent D2 isomerization.
Effect of an anti-tumor compound, 1-(2-methylbutyl)-3-methyl-6, 7-methylenedioxyisoquinoline·HCl (B-15) on nucleic acids and protein synthesis of Sarcina lutea and synchronized HeLa cells was investigated ; the following results were obtained. A selective inhibitory activity against the incorporation of 3H-thymidine into DNA of Sarcina lutea was observed with B-15 at concentrations which were not, or only slightly, inhibitory in the bacterial viability test. On the other hand, the rapidly labeled DNA in the nucleus of Sarcina lutea was not degraded. In synchronized HeLa cells, the incorporation of 3H-thymidine into DNA was inhibited immediately after addition of B-15 and the extent of inhibition was greater than in the case of the incorporation of 3H-uridine into RNA or 14C-leucine into proteins. Moreover, if added at the stage of DNA synthesis, B-15 inhibited both the incorporation of 3H-thymidine into DNA and division of the cells. However, the inhibition of DNA synthesis was rapidly reversed when B-15 was removed from the culture medium. From these results, it seems that the primary action of B-15 on the intact cells is a selective inhibition of DNA synthesis.
Three new triterpenes, named 25-O-acetylcimigenol (I), dehydroxy-15-O-methyl cimigenol (VI) and 15-O-methylcimigenol (VIII), were obtained from the hypogenous part of Cimicifuga acerina and their structures were determined. I, mp 193-194°, [α]15D+39.3°, C32H50O6, leads to cimigenol (II) and cimigenyl diacetate (V), of which structures were determined, by hydrolysis and acetylation, respectively. I forms a diketo derivative (III), mp 211-212°, C32H46O6 and a monoketo derivative (IV), mp 175-175.5°, C32H48O6 by·oxidation. VI, mp 222-223°, [α]7D+38.5°, C31H48O4 forms an acetate (VII), mp 186-187°, [α]7D+42.6°, C38H50O5, which is obtained from 15-O-methylcimigenol (VIII), on acetylation and dehydration. VIII, mp 199.5-200.5°, [α]5D+38.9°, C31H50O5, forms an acetate (IX), mp 124-125°, [α]5D+45.7°, C33H52O6 and a keto derivative (X), mp 156-157°, C31H48O5, by acetylation and oxidation, respectively. VIII, also, is synthesized from cimigenyl diacetate II (V) by reaction with dry methanol and conc. H2SO4 following hydrolysis.
The ring carbon at 1-position in 1-(methylsulfonyl) phthalazine (I) is active to nucleophilic reagents, same as that in 1-(methylsulfonyl)-4-phenylphthalazine, and reacts with sodium methoxide, butylamine, aniline, hydrazine, and hydroxylamine to form 1-methoxy-(VIa), 1-butylamino-(VIb), 1-anilino-(VIc), 1-hydrazino-(VId), and 1-hydroxylaminophthalazine (VIe). VIa to VIe were identified with samples synthesizes from 1-chlorophthalazine (II) and the above reagents (Table I). I also reacts with active methylene compounds such as ethyl cyanoacetate, ethyl acetoacetate, phenylacetonitrile, diethyl malonate, malononitrile, and acetonitrile to form ethyl α-cyano-1-phthalazineacetate (VIIIa), ethyl α-acetyl-1-phthalazineacetate (VIIIb), α-phenyl-1-phthalazineacetonitrile (VIIIc), diethyl 1-phthalazinemalonate (VIIId), and 1-phthalazinemalononitrile (VIIIe). The reaction of I and phenylacetonitrile afforded, besides VIIIc, a structurally unknown substance of mp 244-246°, while the reaction of I and acetonitrile produced a minute amount of a substance assumed to be bis (1-phthalazinyl) acetonitrile (VIIIg') and not the anticipated 1-phthalazineacetonitrile (VIIIg) (Tables II and III). I differs from 1-(methylsulfonyl)-4-phenylphthalazine in its reaction with ketone carbanion and the phenyl group in 4-position was found to affect the direction of this reaction. Reaction of I in dehyd. benzene, in the presence of sodium amide, with ketones afforded 1-(1-phthalazinyl)-2-propanone (Xc) and 2-(1-phthalazinyl)-3-pentanone (Xd) in respective yield of 8% and 14% from the reaction with acetone and diethyl ketone, same as in the reaction of II with these ketones. Reaction of I with acetophenone, propiophenone, and cyclohexanone failed to produce the expected compounds and only XIVa-x, XIVb, and XIVe were obtained (Table VI). The same reaction of I with ketones in the presence of sodium hydroxide also produced XIVa-e and not the anticipated Xa-e. Prolonged reaction of I and acetophenone also gave XIVa-x, besides XIVa. The structure of XIVa-x is now being examined (Table VII). The structure of XVa to XVe was presumed from the analytical values and from IR, UV, NMR, and mass spectral data of XIVa to XIVe and the compounds (XVa to XVe) obtained by conversion of their -SO2CH3 group with -CN. For example, XIVd was assumed to be 2-[1, 2-dihydro-2-(1-phthalazinyl)-4-(methylsulfonyl)-1-phthalazinyl]-3-pentanone and XVd to be 3, 4-dihydro-4-(1-methyl-2-oxobutyl)-3-(1-phthalazinyl)-1-phthalazinecarbonitrile (Chart 3).
The plant considered to be the source of the crude drug, Wujiapi, was examined morphologically. Many plants are known to be the source plant of this drug and they are in both Asclepiadacea and Araliacea families. Morphological examinations were made with special stress on the characteristics of these two families and the plant, which was being examined for constituent principles, was found to have the characteristics of Periploca sepium BUNGE of Asclepiadacea family, which is found in Chinese literature (as the source of this crude drug).
In order to clarify the source plant of the crude drug, Wujiapi, whose constituents were being examined, the components of the root bark of the seven kinds of this plant was comparatively examined by thin-layer and gas-liquid chromatography. It was thereby clarified that 4-methoxysalicylaldehyde, α- and β-amyrin, α- and β-amyrin acetates, periplogenin, and cymarose are contained in the Periploca genus plants in the Asclepiadacea family, while these are totally absent in the plants of the Araliacea family. The crude drug whose components had been examined is the so-called "Bei-wujiapi" and its original plant is assumed to be Periploca sepium BGE. from the known literature but further examinations are being made. Examination of components in P. calophylla FALCON was undertaken for the first time in the present series of work.
Body distribution of iron [59Fe] chondroitinsulfate was examined by intravenous injection of its colloid in mice, either by radioactivity counting after autopsy or by a whole-body autoradiography. 1) The labelled iron chondroitinsulfate after intravenous injection is phagocytized by the reticuloendothelial system cells, mainly the liver, and iron is transported to hemopoietic organs such as the spleen and bone marrow, and then further transits to erythrocytes. 2) Changes in the dose administered results in the speed of phagocytosis by the liver and uptake into erythrocytes, the greater the speed, the smaller the dose. Naturally, the abosolute amount of iron taken up is greater, the larger the dose. 3) A part of excess iron is excreted through the small intestine into feces but there is hardly any excretion into urine. 4) It was found through whole-body autoradiography that iron transits to villi in the small intestine and also to the skin. 5) Distribution of iron in various organs found by radioactivity counting after autopsy corresponded well with the result of whole-body autoradiography.
Studies were made on the interaction of 4-nitroquinoline 1-oxide with phenol and its methyl substituted derivatives in solution, and simultaneous participation of charge transfer and hydrogen bonding was deduced. New absorption bands were observed in the visible difference spectra of mixed systems of phenols and the N-oxide compound, which were ascribed to π-π* transitions. From the analysis of the difference spectra it was revealed that 1 : 1 complexes were formed, and the values of λmax, ε, K, and ΔH were determined. The participation of π-π type charge transfer was indicated by the following results. (1) A linear relationship was found between ET values for the phenol-4-nitroquinoline 1-oxide complex and Z values of solvents. (2) Corresponding relationships were found between ID values of phenols and the hv values of complexes formed between phenols and 4-nitroquinoline 1-oxide in carbon tetrachloride. (3) Examination of K values revealed that the complex formation is hindered by multiple substitution of methyl groups into the benzene ring of phenol. Participation of hydrogen bonding in this interaction was demonstrated by the smallness of K value in the mixed system of anisole as compared with that of the mixed system of phenol and also by the lower frequency shift of the O-H stretching band of phenol caused by the addition of the N-oxide compound. Possible conformation of the phenol-4-nitroquinoline 1-oxide complex was proposed.
Gastrin (I) and gastrone-like substance (II) were extracted from the mucosa of the third stomach of the finback whale (Balaenoptera physalus L.). I is dialyzable and negative to both Ninhydrin and biuret reactions, but positive to the chlorimino reaction. II is non-dialyzable, negative to Ninhydrin reaction, and positive to both biuret and chlorimino reaction. II contained 6-7% of hexose and 8-9% of amino sugar, and was assumed to be a glycoprotein or some glycoproteins. The gastric secretion was elicited by the intravenous administration of 0.5-10 μg/kg of I in the Schild rats, and the same response was elicited by the subcutaneous administration of 10-100 μg/kg of I in the Heidenhain pouch dogs. The inhibition of gastric secretion was observed by the intravenous doses of more than 60 μg/kg of II in the Shay method using pylorus-ligated rats.
Gastric secretory effect and other pharmacological effect of tetragastrin, a synthetic gastrin-like substance, were examined. Tetragastrin did not show any severe acute toxicity in mice and rabbits, but showed respiratory stimulation, retching, and vomiting in dogs. The gastric secretion in Heidenhain pouch dogs was increased by the subcutaneous administration of tetragastrin and also increased in Schild rats by its subcutanous or intravenous administration. In chloralose anesthetized dogs, tetragastrin produced an increase in volume flow of bile and pancreatic juice, and stimulated the motility of stomach or pyloric sphincter.
The bulkiness of fine powders in centrifugal fields was investigated, using CaCO3, Al2O3, and other pharmaceuticals having different particle size. The cylindrical tube filled with a known weight of powder was set to the rotor driven by a variable-speed electric motor, and whirled until the apparent volume of powder no longer changed. The apparent volume of the powder varied with the magnitude of centrifugal force. The relation between ε, the porosity, and F, the average centrifugal force acting on a single particle, was given by the following equation for each material [numerical formula] where k and n are constants. Tapping experiments were also carried out on the same powders. The final porosity was varied with the magnitude of tapping impact which is the function of falling height, H. The data for each material were correlated by [numerical formula] where m is the mass of a single particle, g is the gravitational acceleration, and h and n' are constants. These results support the assumption that the bulkiness of a fine powder is affected by a balance between the adhesion force of particle and the external force acting on an individual particle.
Synthesis of indolizine derivatives was carried out using derivatives other than α-picoline, i.e., 2-pyridineacetonitrile, and halogenoketones. Reaction of 2-pyridineacetonitrile (I) with bromoacetone or phenacyl bromide resulted in the progress of intramolecular cyclization in the first step and 2-methyl-3-indolizinecarbonitrile (II), mp 101-102°, and 2-phenyl-3-indolizinecarbonitrile (IV), mp 102-103°, were obtained. Reaction of II with acetic anhydride afforded 1-acetyl-2-methyl-3-indolizinecarbonitrile (VI), mp 135-136.5°. The great activity of I is assumed to be the effect of electron-attracting CN group in its molecule.
The powders (80-100 mesh) of Fossilia Ossis Mastodi (Japanese name "Ryukotsu") were extracted with ethylether and, from the soluble portion a crystalline component (I) and some oils (II) were isolated. I showed mp 205.5°, [α]20D+24.8° and was examined by gas chromatography and infrared spectrum. It was identified with d-borneol. II are now under investigation. Using aqueous infusion of the powders above mentioned, amino acid and fatty acid components were examined by gas chromatography and paper partition chromatography. Acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid were identified from the aqueous solution. Amino acids are at present under study.