Tohoku J. exp. Med., 1979, 128 , 175-187

Pharmacological Actions of Aconitine Alkaloids

HIROSHI SATO, CHIZUKO YAMADA, CHOHACHI KONNO, Y ASUSHI OHIZUMI, KATSUYA ENDO and HIROSHI HIKING

Pharmaceutical Institute , Tohoku University, Aoba-yama, Sendai 980

SATO, H., YAMADA, C., KONNO, C., OHIZUMI, Y., ENDO , K. and HIKING, H. Ph armacological Actions of Aconitine Alkaloids . Tohoku J. exp. Med., 1979, 128 (2), 175-187 -- Aconitine (AC) , mesaconitine (MA), hypaconitine (HA) (50 ug/kg i.v.), benzoylaconine (BA), benzoylmesaconine (BM) and benzoyl hypaconine (BH) (5 mg/kg i.v.) produced a weak temporary hypotension in rats which was blocked to some extent by atropine. In the isolated guinea pig right atria, MA and HA (3 •~ 10-8 g/ml) mediated a positive inotropic action and a

positive chronotropic action. At the higher concentration of 10-7 g/ml, AC, MA and HA exhibited the above actions followed by inhibition of contractions and disorder of the beating rate. In the isolated guinea pig ileum , AC, MA and HA (>10-6 g/ml) caused a contraction which was completely blocked by atropine. In the isolated guinea pig vas deferens, AC, MA and HA (>10-6 g/ml) elicited a contraction which was completely obliterated by phentolamine. In the isolated

guinea pig hypogastric nerve-vas deferens, the isolated rabbit mesenteric nerve- jejunum and the isolated rat phrenic nerve-diaphragm, responses induced by the electrical stimulation of the corresponding nerves were inhibited by AC, MA and HA (10-8-10-5 g/ml). Some of the mechanisms of the above actions induced by these alkaloids are discussed. These alkaloids more or less potentiated the hexobarbital anesthesia, inhibited the revolution of the wheel cage, and reduced the rectal temperature in mice, indicating that they have a depressant activity on the central nervous system. aconitines; autonomic nervous system; benozylaconines; central nervous system; pharmacological actions

The crude drug "bushi" (aconite roots), prepared from certain species of Aconitum (Ranunculaceae) of Chinese and Japanese origin, is an indispensable material in Oriental medicine which is said to have the activity to recover reduced metabolism of feeble patients and has been employed for the symptoms such as ?am, paralysis, atonia and coldness of extremities. Since the raw roots of Aconitum plants are known to be highly toxic, they are usually processed before ise by a variety of procedures to reduce the toxicity and to avoid clinical intoxica tion. In order to evaluate the therapeutic effect of the crude drug, we have first examined the quality (the alkaloid content and composition as well as the toxicity) if some raw materials and their processed preparations, thereby clarifying the

Received for publication, July 20, 1978. Pharmaceutical studies on Aconitum roots - Part 4. This paper also forms Part 9 in the series on the validity of the Oriental medicines. Abbreviations: AC, aconitine; MA, mesaconitine; HA, hypaconitine; BA, ben zoylaconine; BM, benzoylmesaconine; BH, benzoylhypaconine 175 176 H. Sato et al. changes of these parameters during processing; processing of raw roots has been shown to cause hydrolysis of the highly poisonous constituents, the aconitines, to their deacetyl derivatives, the benzoylaconines, resulting in reduction of the acute toxicity of the roots (Hikino et al. 1977). We have further investigated the pharmacological actions of some Aconitum roots (raw and processed) which are more directly related to their clinical efficacy and, in connection with them, have also examined in parallel those of the most famous principle, aconitine, and its analog benzoylaconine. As a result, it has been found that Aconitum roots possess a number of pharmacological activities some of which may be rationalized by the constituent aconitine (Hikino et al. 1979). Meanwhile, we have recognized the necessity to establish more systematically the pharmacological properties of the alkaloids in the aconitine series and benzoylaconine series, which are the main principles of the raw and processed roots, respectively, in relation to those of the crude drug. Although the pharmacological activities of Aconitum alkaloids have previously been subjected to frequent examination, the main interest has been focus sed on aconitine. However, the purities of the specimens of "aconitine" used in the earlier studies are considered to be doubtful except for a few cases, because certain commercially available preparations of "aconitine" was found to be quite impure. In addition, the main congeners of the aconitine alkaloids in the Aconitum roots which are utilized in Japan are now known to be not aconitine but mesaconitine and hypaconitine (Hikino et al. 1977). Thus, no systematic survey on the phar macological actions of these aconitine alkaloids with a sufficient purity has been performed. As part of our basic study to estimate the therapeutic effectiveness of Aconitum roots, this work describes the pharmacological actions of the six Aconitum alkaloids, the aconitines and the benzoylaconines, on the autonomic and central nervous systems.

Chart 1. Structures of Aconitum alkaloids

MATERIALS AND METHODS

The animals used were male rabbits of the white strain, male guinea pigs of the Hartley strain, male rats of the Wistar strain, and male mice of the dd strain, except for the measurement of antiulcerous activity where male mice of the ddy strain were employed. For isolation of the aconitines, the crude drug aconite roots, the dried roots of Aconitum japonicum grown in Sado, Niigata, was extracted with cold methanol to yield Aconitine Alkaloids 177 the extract which was fractionated by chromatography over polyamide , cellulose and alumina to give mesaconitine, aconitine and hypaconitine . Each of these alkaloids in dioxane-water was heated at 120•K for 40 min and purified by chromatography over silica gel to afford the corresponding deacetyl derivative (a benzoylaconine analog) . That the alkaloids were pure was ascertained by means of thin layer chromatography and gas-liquid chromatography. The Aconitum alkaloids were utilized as hydrobromides , and doses calculated as free bases.

In general, experimental procedures follow those of the previous work (Hikino et al . 1979).

Measurement of blood pressure and heart rate in rats . A rat (280-350 g) was anesthetized with urethane (1.4 g/kg s.c.) and the trachea cannulated . Arterial blood pressure and heart rate from the right common artery were recorded on a polygraph through a pressure transducer. A small polyethylene tube was inserted into the left external jugular vein in order to inject the alkaloids i.v.

Measurement of contractions and beating rate in the isolated guinea pig right atria. The right atria from a guinea pig (300-500 g) was suspended in a 50 ml bath containing Krebs-Henseleit solution bubbled with 95% 02-5% CO2 at 35•K. Contractions and beating rate of the right atria were recorded on a polygraph through a transducer. Measurement of contraction in the isolated guinea pig ileum. The ileum from a guinea pig (300-500 g) was suspended in a 10 ml bath containing Tyrode solution aerated at 28°. Contraction of the ileum was recorded on a polygraph through a transducer.

Measurement of contraction in the isolated guinea pig vas deferens. A guinea pig

(300-500 g) was stunned and the abdomen opened. The vas deferens was excised and the serous membrane carefully stripped away. The preparation was suspended in a 10 ml bath containing Tyrode solution aerated at 32•K. Contraction of the vas deferens was recorded on a polygraph through a transducer. Measurement of contraction in the isolated guinea pig hypogastric nerve-vas deferens. After the vas deferens was dissected together with the hypogastric nerve from a guinea

pig (300-500 g), the preparation was suspended in a 10 ml bath containing Tyrode solu tion bubbled with 95% 02 -5% CO, at 32°. The electrical stimulation to the hypogastric nerve was applied at 4 min intervals, at a frequency of 20 Hz with 0.1 msec duration and at supramaximal voltage for 4 see. The postganglionic stimulation of the hypogastric nerve was carried out according to the method of Birmingham (1966). Contraction of the vas deferens was recorded on a polygraph through a transducer. Measurement of motility in the isolated rabbit mesenteric nerve jejunum. According to the procedure of Finkleman (1930), a rabbit of either sex (2-3 kg) was sacrificed by cervical dislocation, and the jejunum with the mesenteric nerve quickly removed and placed in a dish containing Tyrode solution bubbled with 95% 02-5% CO2 at 37•K. The nerve was stimulated through platinum electrodes from an electric stimulator at 3 min intervals, at a frequency of 30 Hz with 0.5 msec duration and at supramaximal voltage for 4 sec. Motility of the jejunum was recorded on a kymograph.

Measurement of contraction in the isolated rat phrenic nerve-diaphragm. According to the method of Bulbring (1946), the diaphragm together with the phrenic nerve was dissected from a rat (280-350 g), and the preparation suspended in a 50 ml bath containing Tyrode solution and bubbled with 95% 0,-5% CO, at 37•K. Electric stimulation of the

phrenic nerve was applied at a frequency of 0.1 Hz with 1-4 msec duration and at supramaximal voltage. That to the skeletal muscle was applied at a frequency of 0.1 Hz with 5-10 msec duration and at supramaximal voltage. Contraction of the diaphragm was recorded on a kymograph.

Measurement of potentiation of hexobarbital anesthesia in mice. Mice (20-22 g) were used in groups of five and the test alkaloid administered s.c. After 30 min, mice were

given an i.p. injection of hexobarbital (80 mg/kg). The sleeping time was determined as the period between the disappearance and restoration of the righting reflex and compared with that of the control. Measurement of motor activity in mice. The wheel cage apparatus was used for the 178 H. Sato et al.

measurement of the spontaneous activity as described by Takagi et al. (1960). Mice

(20-22 g) were placed initially in the apparatus for 30 min, and used in groups of five. A test alkaloid was administered p.o., and the motor activity measured at 30 min intervals for 150 min and compared with that of the control. Measurement of anticonvulsion effect in mice. Mice (20-22 g) were used in groups of five, and the test alkaloid was administered p.o. After 30 min, strychnine (1.38 mg/kg) was administered i.p. The symptoms produced were observed, and the lethal rate after 24 hr was measured and compared with that of the control.

Measurement of hypothermic effect in mice. Mice (19-23 g) were used in groups of five, and the test alkaloid was administered s.c. and p.o. The rectal temperature of the mice was measured by a thermister at the distance of 2.5 cm inside from the anus. The measurement was performed at the room temperature (23-25°). The mice showing the normal rectal temperature 37.5-38.5•K were selected and used in the experiment. The rectal temperature was measured at 30 min intervals for a further 2-5 hr.

Measurement of antiulcerous effect in mice. The experiment was carried out according to the method of Yano and Harada (1973). Mice (20-22 g) were used in groups of four. After fasting for 8 hr the test alkaloid was administered p.o. After 30 min, the mice were fixed in restraint cages and immersed vertically in a water bath at 25•K to the height of the xyphoid of the animals and kept for 18 hr. The mice were killed after the stress and the isolated stomachs inflated with about 1.5 ml of 10% formalin solution for 10 min. After washing, the stomachs were cut open along the greater curvature and ulcer index was scored as 0.5, 1, 2 and 3. The sum of the ulcer indices was calculated as the percentage of that of the control.

Determination of acute toxicity in mice. Each test alkaloid was dosed to mice (20-22 g)

p.o., s.c., i.p. and i.v. The LDso values were calculated based on a 24 hr mortality response after administration by the up and down method or the Litchfield-Wilcoxon method (1949).

RESULTS

Effect on blood pressure and heart-rate in rats. In the anesthetized rats, AC,

MA and HA (50 jig/kg i.v.) produced a temporary fall in blood pressure. The hypotensive actions of these alkaloids were not inhibited by propranolol (3 mg/kg), but were to some extent blocked with atropine (1 mg/kg). The pressor actions of epinephrine (3 ƒÊg/kg), tyramine (1 mg/kg) and 1,1-dimethyl-4-phenylpiperazinium

(100ƒÊg/kg) and the hypotensive actions of isoproterenol (1 ƒÊg/kg) and acetylcholine

(10 µg/kg) were not affected by these alkaloids. The heart rate of the anesthetized rats was slightly suppressed by AC, MA and HA (50 ƒÊg/kg i.v.) but not affected by

BA, BM and BH (5 mg/kg i.v.) (Fig. 1).

Effect on contractions and beating rate in the isolated guinea pig right atria.

MA and HA showed a positive inotropic action at a concentration of 3 X 10-8 g/ml. At the higher concentration of 10-7 g/ml, AC, MA and HA exhibited a diphasic reaction, a positive inotropic action followed by a negative inotropic action. At the concentration where these alkaloids showed a positive inotropic action, the heart rate increased, and at the higher concentration where they exhibited a diphasic reaction on the contractions, disorder of the beating rate appeared after an increment. BA, BM and BH at the higher dose (10-4 g/ml) showed a negative inotropic action but did not affect the beating rate (Fig. 2, Table 1).

Effect on contraction in the isolated guinea pig ileum. While AC, MA and HA at a lower dose (10-7 g/ml) gave no influence on the isolated guinea pig ileum, they Aconitine Alkaloids 179

Fig. 1. Effect of aconitine on blood pressure and heart rate in rats . Drugs dosed at dots. BP, blood pressure; HR , heart rate; E, epinephrine 3ƒÊg/kg; Tyr, tyramine 1 mg/kg; AC, aconitine 50 ƒÊg/kg . a) Control of E, Tyr and AC. b) After propranolol (Prop) 3 mg/kg. c) After atropine (Atr) 1 mg/kg.

Fig. 2. Effect of Aconitum alkaloids on contractions and beating rate in the isolated guinea pig right atria. Drugs dosed at dots. BR, beating rate; MA, mesaconitine; BM, benzoylmesaconine. a) MA 3 x 10-8g/ml. b) MA 10-7g/ml. c) BM 10-5g/ml. d) BM 10-4g/ml.

TABLE 1. Effect of Aconitum alkaloids on the contraction and beating rate of the isolated guinea pig right atria

- , no change H. Sato et al. caused a prolonged contraction at higher doses (10-6 g/ml). The contraction of the ileum induced by these alkaloids (10-6 g/ml) was completely blocked by atropine (10-8 g/ml) (Fig. 3). BA, BM and BH (10-6 g/ml) caused no contraction.

The contraction of the ileum induced by acetylcholine (10-7 g/ml), histamine (3•~

10-7 g/ml) or BaCl2 (3 •~ 10-4 g/ml) was not affected by these test alkaloids.

Fig. 3. Effect of mesaconitine on the contraction of the isolated guinea pig ileum. Drugs dosed at dots. ACh, acetylcholine 10-9 g/ml; MA, mesaconitine 10-6g/ml. a) Control of ACh and MA. b) After atropine (Atr) 10-8 g/ml.

Effect on contraction in the isolated guinea pig vas deferens. MA (>10-5 g/ml), and AC and HA (>5•~10-5 g/ml) produced a prolonged contraction. The contraction of the vas deferens induced by these alkaloids (10-5-5 x 10-5 g/ml) was completely blocked by phentolamine (5 •~ 10-6 g/ml) (Fig. 4). BA, BM and BH

(10-4 g/ml) induced no contraction. The contraction of the vas deferens induced by norepinephrine (10-6 g/ml) and acetylcholine (10-6 g/ml) was not

affected by the test alkaloids (10-6 g/ml), but that induced by tyramine (3 •~ 10-5

g/ml) was slightly potentiated by AC, MA and HA (10-6 g/ml).

Fig. 4. Effect of mesaconitine on the contraction of the isolated guinea pig vas deferens. Drugs dosed at dots. NE, norepinephrine 10-6g/ml; MA, mesaconitine 10-5g/ml. a) Control of NE and MA. b) After phentolamine (Phen) 5 •~ 10-6g/ml.

Effect on contraction in the isolated guinea pig hypogastric nerve-vas deferens. The effect on the contraction of the vas deferens induced by the electrical stimula tion of the preganglionic fiber is shown in Fig. 5 and Table 2. AC, MA and HA (10-7 g/ml) began to suppress the contraction. AC, MA and HA (10-5 g/ml) in itially caused potentiation of the contraction followed by a prolonged inhibition which was not recovered for a long time even after washing out. The contraction began to be suppressed by BA, BM and BH at a concentration of 10-5 g/ml. The effect on the contraction induced by the transmural electrical stimulation of the postganglionic fiber was the same as the above. Effect on motility in the isolated rabbit mesenteric nervejejunum. AC, MA and HA (10-6 g/ml) markedly potentiated the pendular movement of the isolated Aconitine Alkaloids 181

Fig. 5. Effect of Aconitum alkaloids on the contraction of the isolated guinea pig hypogastric nerve-vas deferens. Drugs dosed at dots. Electrical stimulation to the preganglionic fiber applied at 20 Hz, 0.1 msec, supramaximal voltage. a) MA, mesaconitine 10-6g/ml. b) MA, mesaconitine 10-5g/ml. c) BM, benzoylmesaconine 10-5g/ml.

TABLE 2. Effect of Aconitum alkaloids on the contraction of the isolated guinea pig hypogastric nerve-vas deferens

Supramaximal electrical stimulation was applied to the preganglionic fiber at 20 Hz for 0.1 msec.

•ª, 10-50% potentiation; -, no effect; •«, 10-50% inhibition;•«•« , 50-100% inhibition.

Fig. 6. Effect of Aconitum alkaloids on the motility of the isolated rabbit mesenteric nerve-jejunum. Drugs dosed at dots. Electrical stimulation to the mesenteric nerve applied at 50 Hz, 0.5 msec, supramaximal voltage. a) AC, aconitine 10-6g/ml. b) BA, benzoylaconine 10-5g/ml.

rabbit jejunum, but BA, BM and BH (10-5 g/ml) gave no reaction (Fig. 6). The inhibitory effect on the pendular movement of the rabbit jejunum induced by the stimulation of the mesenteric nerve was almost abolished by AC, MA and HA (10-6 g/ml), but was not influenced by BA, BM and BH (10-5 g/ml). Effect on contraction in the isolated rat phrenic nerve-diaphragm. The effect of test alkaloids on the contraction of the diaphragm induced by the electrical 182 H. Sato et al.

Fig. 7. Effect of Aconitum alkaloids on the contraction of the isolated rat phrenic nerve diaphragm Drugs dosed at dots. a), b) Electrical stimulation to the phrenic nerve applied at 0.1 Hz, 1-4 msec, supramaximal voltage. c), d) Electrical stimulation to the diaphragm muscle applied at 0.1 Hz, 5-10 msec, supramaximal voltage. AC, aconitine 10-6g/ml; BA, benzoylaconine 10-5g/ml. stimulation of the phrenic nerve or diaphragm muscle is shown in Fig. 7. The contraction induced by the electrical stimulation of the phrenic nerve was markedly inhibited by AC, MA and HA (10-6 g/ml) which was not recovered by physostigmine (10-8 g/ml), while the same contraction was not affected by BA, BM and BH (10-5 g/ml). The contraction induced by electrical stimulation of the diaphragm muscle was not affected by any of the test alkaloids (10-5 g/ml). Effect on hexobarbital anesthesia in mice. HA had the most intense potentiating activity among the aconitines at doses of 1 /4 of the LD55 values. BH showed the

TABLE 3. Effect of Aconitum alkaloids on the spleeping time induced by hexobarbital in mice Aconitine Alkaloids 183 strongest activity among the benzoylaconines at doses of 1/4 of the LD50 values, marking about 3.3 times prolongation of the sleeping time. BA and BM also significantly prolonged the spleeping time induced by hexobarbital (Table 3; for LDb0i see Table 6). Effect on motor activity in mice. As tabulated in Table 4, AC, MA and HA showed a marked inhibitory activity on the revolution of the wheel cage among the test alkaloids. The inhibitory activity of MA (1 mg/kg) was the most potent and the inhibition rate of MA was about 80% of that of the control.

TABLE 4. Effect of Aconitum alkaloids on revolution activity in mice

* Inhibition was calculated as the percentage of the decrease of the mean value in a treated group divided by that of the control group.

Anticonvulsion effect in mice. None of the six test alkaloids inhibited the development of the convulsion induced by strychnine or had an influence on the i.p. LD50 of strychnine in mice.

Hypothermic effect in mice. MA (60 ƒÊg/kg s.c.) and HA (300 ƒÊg/kg s.c.) reduced rectal temperature by ca. 1•K and ca. 2•K, respectively. BA, BM and BH

(30 mg/kg s.c.) also showed hypothermic activity, the maximal falls of rectal temperature by the latter three alkaloids being 1.5-3•K. The maximal falls by these alkaloids were observed 0.5-1 hr after administration and followed by rapid recovery. The results are shown in Fig. 8.

By p.o. administration, higher doses could be applied and the maximal falls were found at later stages in these cases (Table 5).

Antiulcerous effect in mice. None of the six test alkaloids, AC, MA and HA

(1-2 mg/kg), and BA, BM and BH (30 mg/kg), prevented the ulcer formation in mice.

Acute toxicity. Generally the aconitine alkaloids developed the well-known "aconitine syndrome" . Thus, these alkaloids provoked the signs of ataxia of the extremities and respiratory paralysis in mice. The mice died in agony. AC, MA and HA produced nausea 2-3 min after the s.c. or i.p. injection. The i.v. injection of AC, MA and HA caused particularly strong respiratory paralysis in mice, and in 184 H. Sato et al.

Fig. 8. Effect of Aconitum alkaloids on rectal temperature in mice. Drugs dosed s.c. Each point represents mean for 5 animals. ƒ•-ƒ•, aconitine 60ƒÊg/kg; •œ -•œ , mesaconitine 60 ƒÊg/kg; •¢-•¢, hypaconitine, 300 ƒÊg/kg; ƒ•- - -ƒ•, benzoylacon ine 30 mg/kg; •œ---•œ, benzolymesaconine 30 mg/kg; •£---•£, benzoylhypaconine 30 mg/kg; •£-•£, aminopyrine 50 mg/kg.

TABLE 5. Effect of Aconitum alkaloids on rectal temperature in mice

TABLE 6. Acute LD50 values of Aconitum alkaloids in mice (mg/kg)

LD50 values were determined by the up and down method except for the asterisked ones which were calculated by the Litchfield-Wilcoxon me thod, 95% confidence limits being shown in parentheses. Aconitine Alkaloids 185 the mice which died respiration was rapidly arrested 30 sec after injection . BA, BM and BH slowly produced respiratory paralysis . The LD50 values of the alkaloids are shown in Table 6.

DISCUSSION

In the present investigations , we have examined the pharmacological actions of the Aconitum alkaloids , the aconitines (AC, MA and HA) and the benzoylaconines

(BA, BM and BH), on blood pressure, on the heart, on smooth muscles , on the sekeltal muscle and on the central nervous system . Among the six alkaloids, the three aconitines showed a significant activity in all of the test systems employed except for the sleeping time potentiation in aconitine , for the anticonvulsion effect and the antiulcerous effect. In the in vitro experiments , no significant difference was observed in the activity of the three alkaloids with the exception of the right atria test. In this case and in the in vivo experiments , the potency of the actions of the respective alkaloids was found to be equal in quality but to be somewhat varied in quantity. Since the toxicity of the aconitine alkaloids is variable and since the doses of the alkaloids were set in proportion to their LD 50 values in the in vivo experiments, no direct comparison of the activity was possible in certain cases. However, the pharmacological activity appears to be in the decreasing order , MA>AC>HA, which parallels that of the toxicity. This finding demonstrates that the change of the functional groups Rl and R2 in the aconitine molecule (Chart

1) causes no essential alteration in the pharmacological activity, although intro duction of a hydroxyl group at the 3ƒ¿-position and replacement of an N-ethyl by an N-methyl bring about an increase of the effect. On the other hand, hydrolysis of the acetoxyl (R3=Ac) to a hydroxyl (R3=H) (from the aconitines to the benzoylaconines) remarkably decreases the activity including the toxicity, confirming that the presence of an 0-acetyl function at the 8ƒÀ-position is essential for the occurrence of the activity.

AC, MA and HA showed a hypotensive action which was not inhibited by an adrenergic ƒÀ-blocker, but was considerably abolished with a muscarinic blocker; the hypotensive action induced by these alkaloids would be mediated in part by a muscarinic mechanism.

In the isolated guinea pig ileum, AC, MA and HA caused a strong contraction which was completely blocked by a muscarinic blocker. It is concluded, therefore, that the contraction induced by these alkaloids is caused by a muscarinic mechanism.

In order to substantiate this tentative conclusion, we have further examined the mechanism in detail, and these results will be reported elsewhere. In the isolated guinea pig vas deferens, AC, MA and HA also caused contrac tions which were completely abolished with an adrenergic ƒ¿-blocker. This finding suggests that the contraction is produced by an ƒ¿-adrenergic mechanism. The mechanism of the contraction will also be reported elsewhere.

In the isolated guinea pig hypogastric nerve-vas deferens and the isolated rabbit jejunum, responses induced by the electrical stimulation of the corresponding 186 H. Sato et al. nerve were markedly inhibited by AC, MA and HA, while the contraction of the vas deferens induced by norepinephrine and acetylcholine was not affected by these alkaloids. These results suggest that AC, MA and HA involve a neuronal blocking action. In the isolated rat phrenic nerve-diaphragm, AC, MA and HA inhibited the contraction produced by the electrical stimulation of the phrenic nerve, this inhibition not being recovered with an acetylcholine esterase inhibitor. The con- traction induced by the electrical stimulation of the muscle itself was not affected by these alkaloids. Therefore, the inhibition of the contraction induced by these alkaloids could be due to a neuronal blocking action or a succinylcholine-like ac tion. On the basis of the above results, it is concluded that the aconitines possess no direct action on smooth muscles and skeletal muscles but rather have selective blocking actions on sympathetic or motor nerves. Since the aconitines are well known to inhibit also sensory nerves, it may be said that the aconitines non- specifically inhibit the activities of peripheral nerves. The aconitines prolonged the hexobarbital-induced sleeping time, inhibited the revolution of the wheel cage, and reduced the rectal temperature in mice, suggesting that they have an inhibitory activity on the central nervous system. In the previous work, we have shown that potentiation of the hexobarbital anesthesia by the aconitines is not induced by inhibition of the metabolism of the hypnotic which is caused by the liver lesion due to the alkaloids, but is mediated by some inhibitory effect on the central nervous system (Hikino et al., 1979). As for the effect on the motor activity, however, since it is well known that the aconitines also possess a muscle relaxing activity (Inoue 1950), part of the inhibitory action of the alkaloids on the revolution of the wheel cage may be attributed to motor paralysis due to the muscle relaxation. Further inhibitory activity of the alkaloids on the central nervous system will be reported subsequently. Although the aconitine alkaloids are proved to have a number of pharmaco logical actions in the present work, it is not yet well understood how the activity contributes to the said clinical effectiveness of the crude drug, the Aconitum roots. For a more complete understanding of the therapeutic efficacy of the alkaloids, further study is required.

Acknowledgment

Thanks are due to Mrs. T. Tamada, this laboratory, for technical assistance.

References

1) Birmingham, A.T. (1966) The potentiation by anticholinesterase drugs of the responses of the guinea pig isolated vas deferens to alternate preganglionic and postganglionic stimulation. Brit. J. Pharmacol., 27, 145-156. 2) Biilbring, E. (1946) Observation on the isolated phrenic nerve diaphragm preparation of the rat. Brit. J. Pharmacol., 1, 38-61. 3) Finkleman, B. (1930) On the nature of inhibition in the intestine. J. Physiol., 70, 145-157. 4) Hikino, H., Yamada, C., Nakamura, K., Sato, H., Ohizumi, Y. & Endo, K. (1977) Aconitine Alkaloids 187

Change of alkaloid composition and acute toxicity of Aconitum roots during pro cessing. Yakugaku Zasshi, 97, 359-366. (Japanese) 5) Hikino, H., Sato, H., Yamada, C., Konno, C., Ohizumi, Y. & Endo, K. (1979) Phar macological actions of Aconitum roots . Yakugaku Zasshi, 99, 252-263. (Japanese) 6) Inoue, T. (1950) Pharmacological studies on aconitine , Aconitum japonicum and A conitum lycoctonum. Igaku Kenkyu , 20, 48-62. (Japanese) 7) Litchfield, J.T. & Wilcoxon, F. (1949) A simplified method of evaluating dose -effect experiments. J. Pharmacol. exp. Ther ., 96, 99-113. 8) Takagi, H., Ban, T., Takashima, H. & Takashima , T. (1960) Studies on the h ypnotic and anticonvulsant action of 2-methyl-3-(o-tolyl)-quinazolone-4 (MTQ). Folia pharmacol. jap., 56, 1421-1432. (Japanese) 9) Yano, S. & Harada, H. (1973) A method for the production of stress erosion in the mouse stomach and related pharmacological studies . Jap. J. Pharmacol., 23, 57-64.