Pharmacological studies of Panax Ginseng leaves.

Pharmacological properties of a crude saponin fraction (GF-DS-I) and saponins (GF-DS-II) which were obtained from Panax Ginseng leaves, were estimated by blind screening consisting of three tests : 1) neuropharmacological observations in mice, 2) tests on the respiratory and cardiovascular system in rats, and 3) tests on the guinea-pig isolated ileum. GF-DS-I appeared to have CNS-depressive, neuroleptic, analgesic, hypertensive, cholinergic, and histamine-like activities, while GF-DS-II, had CNS-depressive, neuroleptic, analgesic, hypotensive, atropine-like and papaverine-like activities. These neuroleptic activities, were examined and confirmed by tests on motor activity, exploratory movements, muscle tone, motor coordination, hypothermia and potentiation of CNS-depressant, and pole climbing, anticonvulsant and analgesic tests. No effects were seen on hemolysis. Pharmacological studies on extracts of Ginseng root, have been reported by a number of investigators (1-9), but a report on Ginseng leaves has not heretofore been seen. Chemical research on Ginseng leaves has been reported by Shibata et al. (10, 11). We have investigated Ginseng root systematically, and, in particular, pharmacological properties of saponins in Ginseng root. A crude saponin fraction (G. No. 3) and GNS being a mixture of neutral saponins consisting mainly of Ginsenoside-Rb1, -Rb2 and -Rc have been reported, in particular the pharmacological properties (12-15). In this paper, pharmacological properties of Ginsenoside-F (GF), the main component of Ginseng leaves has been studied, and compared with those of saponin fractions from Ginseng root. MATERIALS AND METHODS Preparation of extracts from Ginseng leaves Ginseng leaves were treated as shown in Fig. 1, and GF-DS-I (crude saponin fraction) and GF-DS-II (saponins prepared from GF-DS-I) were used. On acid hydrolysis, GF-DS-II afforded panaxadiol and panaxatriol. Details of the fractionation of these components from Ginseng leaves, is described by Shibata et al. (10, 11). The solution of these two components was dissolved in physiological saline. The following methods were employed. 1) Neuropharmacological observations in mice This method modified by Takagi et al. (16), is based on the work of Irwin (17). Test extracts were administered i.p. to male mice (ddy-strain), weighing 18-20g. A useful 44 H. SAITO, M. MORITA & K. TAKAGI logarithmic series for dosing is :•c 1, 2, 5, 10, •c mg/kg (from the dose which induce symptoms similar to those of a control animal to approx. LD"). Observations were made for 2 hr and then after 24 and 48 hr. Results were recorded in tabular forms (12). 2) Acute toxicity in mice Male mice (ddy-strain), weighing 18-20 g, were used in order to determine i.v., i.p. and oral LD50. Mortality was recorded 72 hr after treatment. The LD50 was calculated by the up and down method. 3) Tests on the respiratory and the cardiovascular systems in the rat After the rat had been anaesthetized with urethane-chloralose by the i.p. route, both arterial blood pressure, heart rate, and responses of respiratory system were recorded (12). Effects of extracts on respiration and cardiovascular system to a series of stimuli: acetylcholine chloride (ACh: 2 ƒÊg/kg), epinephrine hydrochloride (Epi, 5ƒÊg/kg), histamine dihydrochloride (His: 5 ƒÊg/kg), electrical stimulation (5 V, 2 sec, 30 c.p.m.) of vagal stump, and occlusion of both carotid arteries for 20 sec, were studied. Effects of atropine sulfate (Atr : 2 mg/kg), diphenhydramine hydrochloride (Diph: 3 mg/kg), propranolol hydrochloride (Prop : 250 pg/kg), phentolamine mesylate (Phent : 5 mg/kg) and hexamethonium bromide (C6: 5 mg/kg) on the responses of respiration and cardiovascular system to extracts, were also studied. FIG. 1. Separation of Panax Ginseng leaves. PHARMACOLOGY OF PANAX GINSENG LEAVES 45 4) Tests on the guinea-pig isolated ileum 2.5-3.0 cm of intestine, excised at approx. 20 cm from pylorus was suspended in Tyrode's solution and bubbled with air in a 10 ml organ bath kept at 37•Ž. The antagonism to the contraction induced by ACh, His (cumulatively added), nicotine tartarate (Nic : 2•~10-6 g/ml) and serotonin creatine sulfate (5-HT : 3 •~ 10-6 g/ml), or the antagonism of Atr (10-8 g/m1) and Diph (10-8 g/m1), to the contraction induced by test extract were studied. 5) Hemolytic tes Effects of test extracts on hemolytic activity were observed by the method of Fujita et al. (18). Pure saponins (Merck) was used as the standard. 6) Intracerebral injection in mice The method for observing the effect of test substance introduced directly into the brain in the unanaesthetized mouse in groups of 6, weighing 24-26 g, were employed. Technical details have been described by Haley et al. (19). Test extracts were dissolved in saline and 0.02 ml was injected. 7) Motor incoordination and muscle relaxation in mice 1) Rotating rod, 2) sliding angle, and 3) spring balance tests described by Takagi et al. (20) were employed for measuring the motor incoordination and myorelaxation in groups of 8 male mice weighing 20-22g. 8) Motor activity in mice 1) An activity wheel and 2) a hole cross tests described by Takagi et al. (21) were employed for motor activity. Groups of 10 male mice in a hole cross test, or a mouse in an activity wheel test, weighing 20-22 g, was placed in the test cage 10 min after the administration. Countings were continued for 1 hr, and recorded as a percent for the value which was counted in the same animal or the same group at the same time one day before the test. 9) Climbing test in mice The effect of test extracts on exploratory movement was observed in male mice in groups of 10, weighing 18-20 g, using the climbing test described by Sandberg (22). Mice were placed in a test cage for 10 min every 30 min after the administration and the number of animals climbing the net was counted. This was repeated 4 times. 10) Potentiation of hypnotic action of hexobarbital in mice Groups of 6 male mice, weighing 22-25 g, were administered test extracts by the i.p. route, and 30 min later 70 mg/kg of hexobarbital sodium was injected by the same route. Duration of loss of the righting reflex was measured. 11) Analgesic tests in mice i) Writhing induced by 0.7% acetic acid Groups of 8 male mice, weighing 20-23 g, were given test extracts orally, and 30 min later an i.p. injection of 0.7 % acetic acid was given. The number of writhings per mouse was recorded for a period of 10 min, beginning 10 min after administration of acetic acid. ii) Tail pressure test The method has been described by Takagi et al. (23). Groups of 10 male mice, weighing 46 H. SAITO, M. MORITA & K. TAKAGI 18-20 g, were given test extracts i.p. and measured 4 times every 30 min after the treatment. 12) Anticonvulsant tests in mice i) Convulsions induced by electroshock Groups of 6 male mice, weighing 20-23 g, were given test extracts i.p., and 30 min later subjected to the maximum electroshock seizure (corneal electrodes charged with 25 mA, 0.17 sec), in order to observe tonic extension of hind legs and lethal action. ii) Convulsions induced by chemicals Groups of 6 male mice, weighing 22-26 g, were given test extracts by the i.p. route 30 min before an i.v. injection of pentylenetetrazol (60 mg/kg) or an i.p. injection of strychnine nitrate (2 mg/kg). Three endpoints of time were taken : the first appearance of clonic convulsions, tonic extension of hind limbs and lethal action. 13) Hypothermia in mice Groups of 6 male mice, weighing 22-26 g with a rectal temp. of 37-38•Ž, were given test extracts i.p. and rectal temp. was recorded every 30 min for 2 hr after the treatment. 14) Ratio of reflexes in the mouse This method was described by Witkin et al. (24). Male mice in groups of 10, weighing 18-20 g, were given test extracts by the i.p. route. Corneal and pinna reflexes were observed by using a pig hair, 15, 30, 60, 90 and 120 min after administration. 15) Pole climbing test in rats Details of apparatus and training have been described in a previous paper (13). Conditioned male rats in groups of 10 (Wistar-strain), weighing 150-180 g, were tested for a period of 120 min after an i.p. injection of physiological saline. Three days later, rats given test extracts by the same route, were tested at the same intervals. 16) Motor incoordination and muscle relaxation in rats Rotating rod and suspension tests described Takagi et al. (13), were employed for measuring the motor deficit and myorelaxation in rats. RESULTS 1) Neuropharmacological observations•¬in•¬mice From the number of survivors in each group, approx. LD50 of GF-DS-I and II (i.p.) was estimated to be between 200 and 500 mg/kg. Lethal doses produced the following common symptoms for 2 hr after administration: a decrease of alertness, grooming, traction, spontaneous movement, touch response, grip tone, body tone and body temp. an appearance of passivity, piloerection, ptosis and abnormal gait. Disappearance of pain reflex, corneal reflex and pinna reflex were observed approx. 30 min after administration. A slightly extended posture with abdomen touching floor was also observed. These behavioral changes continued for 2 hr and mice died approx. 24 hr after the treatment. Effects on mice were produced with extracts in doses of less than LD.o. These changes of behavior continued for 2 hr with mice showing renewed vitality 24 hr after. These effects were dose-dependent and results are shown in Fig. 2. PHARMACOLOGY OF PANAX GINSENG LEAVES 47 2) Acute toxicity in mice LD50 of GF-DS-I and II (i.v.) was 381 and 299 mg/kg respectively. Behavioral changes observed in lethal doses : GF-DS-I produced extended posture with abdomen touching floor and abnormal gait a few min after treatment. A sedative state was seen for several min. Approx. 10 min later, swimming convulsions appeared and mice died after 15 to 25 min. GF-DS-II produced these behavioral changes faster than GF-DS-I and mice died within several min. LD50 (i.p.) was 402 and 306 mg/kg respectively, and oral LD50 was estimated to be more than 5g/kg. Surviving mice showed no change in behavior the following day. 3) Tests on the respiratory and the cardiovascular systems in the rat GF-DS-I: The arterial blood pressure of the rat lowered transiently with i.v. injection of GF-DS-I in doses between 0.5 and 2.0 mg/kg, and rose transiently with more than 5 mg/kg as shown in Fig. 3. One mg/kg lowered the mean arterial blood pressure by 12% that of normal however 5 mg/kg raised it by 9 %, in 5 rats respectively. A slight decrease in heart rate was also detected in doses of more than 1 mg/kg, but effect was not seen on respiration after these doses. Tachyphylaxis was not detected with an injecFIG. 2. Effects of GF-DS-I and II on the neuropharmacological observations in mice. The columns shows the results obtained in the neuropharmacological screening procedure of Takagi et al. (16). Length of column : From the dose which induced similar symptoms to that of control animal, to approx. lethal doses Width of column : Degrees of action Ordinate line : Approx. LD50 48 H. SAITO, M. MORITA & K. TAKAGI