Japan. J. Pharmacol. 55, 51-56 (1991)

Effect of Panax Ginseng on Age-Related Changes in the Spontaneous Motor Activity and Dopaminergic Nervous System in the Rat

Hiroshi Watanabe, Hiroyuki Ohta, Lisa Imamura, Wataru Asakura, Yoshinori Matoba and Kinzo Matsumoto

Section of Pharmacology, Research Institute for Wakan-Yaku, Toyama Medical and Pharmaceutical University, Toyama 930-01, Japan

Received May 17, 1990 Accepted September 29, 1990

ABSTRACT -Effects of Panax ginseng on the spontaneous motor activity and central dopaminergic systems in old rats were investigated and compared with those in young rats. Oral intake of a 1.8% water extract of Panax ginseng for four weeks produced an increase in spontaneous motor activity during the dark period in old rats, while it caused a decrease in the activity in young rats. After the intake of ginseng extract for five weeks, it caused a significantly low dopamine utilization in the daytime in the striatum of old rats, while it produced a high dopamine utilization in the structure of young rats. Concentrations of striatal dopamine D-2 receptors in old rats were signifi- cantly lower than that in young rats, although subchronic Panax ginseng did not affect the striatal D-1 and D-2 receptors of old rats. These results suggest that subchronic in- take of ginseng extract inhibits the activity of nigro-striatal dopamine neurons in the daytime and activates spontaneous motor activity during the dark period in old rats, while it produces opposite effects in young rats.

Panax ginseng root produces various tal dopaminergic neurons, was observed in neuropharmacological effects such as changes aged rats (7). Diminution of synthesis, release in brain biogenic amines, improvement of and re-uptake of dopamine and decrease in D- learning and memory retention (1), promotion 2 dopamine receptors in the striatum were re- of recovery from fatigue (ref. 2, for review, ported in aged animals (8-11). see ref. 3), and an increase in the capacity for We investigated the effects of a water ex- intellectual performance in animals and man tract of Panax ginseng root on spontaneous (for review, see ref. 4). Little evidence, how- motor activity and nigro-striatal dopaminergic ever, has been reported on the anti-aging nervous systems in two year-old rats and com- effects of ginseng, that had been suggested by pared them with those in young rats. ancient literature (5), in experimental animals. A decrease in motor activity was shown in MATERIALS AND METHODS aged mice in association with an increase in monoamine oxidase activity in the brain (6). Young and old male Fischer 344 rats (3 and Impairment of swimming ability, which was 24 months old at the end of the experiment, similar to that seen after electrolesion of the respectively; Nippon Charles River, Tokyo) lateral hypothalamus including the nigro-stria- were used. They were kept under an alternat- ing 12 hr light and dark regimen (on, 07:00 to electrode was used to monitor the column elu- 19:00), were maintained at a constant temper- ates, and detector potential was maintained at ature (22 - 24°C) and humidity (50 - 75%), and 0.75 V vs. an Ag/AgCI reference electrode. were given a 1.8% water extract of Panax gin- seng root instead of tap water ad libitum. Receptor binding assay The dissected striata of control rats were Spontaneous motor activity homogenized in 50 volumes of ice-cold buffer Four weeks after oral intake of the Panax (50 mM Tris-HCl containing 120 mM NaCl, 5 ginseng extract, a rat housed in a cage was mM KCI, 1 mM CaCl2, 1 MM MgCl2, pH 7.4) placed on an Animex (Muromachi Kikai, Osa- using a Teflon homogenizer, centrifuged three ka) for at least 3 hr and spontaneous motor times (50,000 X g for 10 min), and finally sus- activity was recorded for the successive 24 hr pended in the same buffer. Duplicate incuba- in order to minimize the effect of arousal in a tion tubes each recieved 100 ,ul of 50 mM Tris- novel environment. The data were analyzed as HCI buffer (pH 7.4), [3H]spiperone or total activity during day-light and dark [3H]SCH 23390 (0.025 -1.0 nM) and 1 uM periods, separately. ketanserin, and 700 ,ul of striatal membranes (0.1 mg protein) (14). Incubations were car- Brain dissection ried out at 20°C for 90 min and then termi- After the intake of Panax ginseng for five nated by rapid filtration (Whatman GF/B fil- weeks (one week afer measurement of spon- ters) under vacuum with three 5-ml rinses with taneous motor activity), rats were intraperi- an ice-cold buffer (50 mM ion-free Tris-HCI, toneally treated with 250 mg/kg of a-methyl- pH 7.7). The radioactivity retained on each p-tyrosine (a-MPT), a tyrosine hydroxylase in- filter was counted by a liquid scintillation hibitor, and were killed by decapitation 4 hr counter. Specific binding was defined as the after the treatment (13:00 to 15:00). The brain excess over blanks taken in the presence of 1 was rapidly removed, placed in a cooled tem- ,uM SCH 23390 for D-1 and 1,aM plate and cut using razor blades at approx- (-)sulpiride for D-2, respectively. imately A 9.8 and 7.1 mm according to a brain atlas (12). The left striatum was excised from Drugs the slice for dopamine assay; the right, for re- One hundred grams of Panax ginseng root ceptor binding assay; and the striata were (Otane-ninjin, cultivated in Nagano Prefec- frozen in liquid nitrogen. ture, Japan) were extracted two times with 900 and 500 ml of water, respectively, at Determination of dopamine 100°C for 1 hr; and the extract was combined The striatum was homogenized in 1 ml of and freeze-dried under vacuum for two days. 0.25 N perchloric acid containing 40 ng of 3,4- The extract of Panax ginseng was prepared at dihydroxybenzylamine (DHBA) as an internal the concentration of 1.8% and given to the standard and centrifuged at 10,000 X g for 15 rat for drinking. In the present study, oral in- min. Dopamine was extracted with alumina take of 1.8% solution/rat/day corresponded and determined using HPLC with electroche- to approximately 7 g of dried Panax mical detection (13). Ten-microliter samples ginseng/kg/day, and the intake of the solution were injected into an ODS-5,um column oper- was not significantly different from that of tap ated at a flow rate of 1 ml/min. The mobile water. a -MPT methylester hydrochloride (Sig- phase consisted of 0.05 M phosphate buffer ma Chemicals) was dissolved in sterile saline. solution containing 5% (V/V) methanol, 10 The chemicals used in the HPLC were ana- ,uM EDTA and 0.2 mM sodium octylsulfate lytical grade obtained from commercially (final pH of 3.1). An LC-4B ampero- available sources. metric controller equipped with a graphite Data analysis significantly different from those in young Data were analyzed using one-way ANOVA rats. The intake of 1.8% Panax ginseng ex- with Duncan's multiple comparison test. tract for five weeks did not affect striatal dopamine levels both in old and young rats. RESULTS Four hours after treatment of old rats with 250 mg/kg of a-MPT, the striatal dopamine level Effect on spontaneous motor activity of the ginseng group was significantly higher Control old rats showed significantly lower than that of the control. On the contrary, in spontaneous motor activity than control young young rats, the Panax ginseng gourp showed rats during the dark period (19:00-7:00) (P < significantly lower striatal dopamine level than 0.05), which corresponded to the results re- the control at 4 hr after a-MPT (Fig. 2). ported previously (11). The intake of 1.8% Panax ginseng extract for four weeks in old Effect on dopamine D-1 and D-2 receptors in rats caused a significant increase in spon- the striatum taneous motor activity to the level of control In control old rats, concentrations of D-1 young rats during the dark period. In contrast, receptors (Bmax) were not significantly differ- Panax ginseng in young rats produced a signi- ent from those in control young rats, whereas ficant decrease in spontaneous motor activity the Bmax value of D-2 receptors was signifi- during the dark period. Panax ginseng did not cantly lower than that in control young rats. modify the motor activity during the light The intake of 1.8% Panax ginseng for five period (7:00-19:00) either in old or young weeks did not affect the Bmax values of D-1 rats. These results are summarized in Fig. 1. and D-2 receptors. The dissociation constant (Kd) of D-1 and D-2 receptors in old rats was Effect on striatal dopamine levels after treat- not significantly different from that in young ment with a -MPT rats and was not affected by Panax ginseng Striatal dopamine levels in old rats were not (Table 1).

Fig. 1. Effect of subchronic intake of Panax ginseng Fig. 2. Effect of subchronic intake of Panax ginseng on spontaneous motor activity in old (A, n = 10) and on dopamine levels in the striatum of old (A) and young (B, n = 5) rats. Rats were given a 1.8% water young (B) rats. Rats were given a 1.8% water extract extract of Panax ginseng root instead of tap water ad of Panax ginseng root ad libitum for 5 weeks. Striatal libitum for 4 weeks. Spontaneous motor activity was dopamine levels were determined 4 hr after intraperi- measured for 24 hr and expressed as total counts in toneal treatment with a-methyl-p-tyrosine (250 the light and dark periods separately. White column: mg/kg). White column: control gourp, striped column: control group, striped column: ginseng group. Vertical ginseng gourp. Vertical bar is the standard error of the bar is the standard error of the mean. * P < 0. 05, ** P mean (n = 3 - 5). * P < 0.05, ** P < 0.01 vs. the re- < 0.01 vs. the respective control. spective control. Table 1. Effect of subchronic intake of Panax ginseng extract on the binding of [3H]SCH 23390to D-1 receptors and [3H]spiperoneto D-2 receptors in the striatum of old rats

Rats were given a 1.8% water extract of Panax ginseng root ad libitum for 5 weeks. Concen- trations (B,,,ax, fmol/mg protein) and affinities (dissociation constant: Kd nM) were determined from individual Scatchard plots. Values are the mean of four independent experiments ± standard errors. **P < 0.01 vs. young control.

DISCUSSION tivity to the active principles of ginseng. It has been reported that neutral saponins The present study has shown that subchron- of Panax ginseng and the ginsenoside Rbl ic intake of a water extract of Panax ginseng fraction exerts an inhibitory effect on spon- restores spontaneous motor activity during the taneous movements, and that the ginsenoside dark period in old rats, while it diminishes the Rgl fraction increases alertness and spon- activity in young rats as compared with the re- taneous movements in mice (19-21). The ex- spective control. We have already shown that tract of Panax ginseng has been shown to in- the intake of 1.8% Panax ginseng extract for duce a general stimulating effect on the in- 4 months produces an increase in spontaneous tegrative nervous activity in rats (4, 22). motor activity during the dark period in mid- Panax ginseng, therefore, has several princi- life mature rats (15). These results suggest ples that produce central stimulating and/or that the effects of Panax ginseng on motor suppressing effects in animals. These active function in old rats are contrary to those in principles may have some relation to the age- young rats. The reason why Panax ginseng ex- related changes in the effects of Panax gin- tract does not affect the motor activity during seng. It is, however, unclear why the extract the light period both in old and young rats is expressed opposite effects in young adult and not clear, but it may be connected with the old rats, respectively. diurnal rhythm of spontaneous motor atctivity Striatal dopamine levels 2 to 4 hr after the in rats. treatment with a-MPT are index of dopamine Chronic treatment with haloperidol was utilization in dopaminergic neurons (23). In shown to produce behavioral supersensitivity the present study, old rats showed relatively to dopaminergic agonists and an increase in higher dopamine utilization in the striatum [3H]spiperone binding to striatal membranes than young animals in the daytime. It has in young adults but not in old rats (16). Intra- been reported that tyrosine hydroxylase activ- striatal injection of dopamine produced more ity and dopamine catabolism in the striatum rotational behavior contralateral to the side of decrease with aging (24, 25). Therefore, the the injection in young adults than in senescent present result appears to be contradictory to rats (17). On the contrary, aged rats showed those reports. This point is a problem to be much stronger sedative effects than young solved. It is well-known that the dopamine re- adults following systemic administration of ceptor agonist apomorphine inhibits and the haloperidol (18). Thus, the difference of the antagonist haloperidol increases dopamine uti- effects of Panax ginseng on spontaneous lization via pre- and postsynaptic dopamine re- motor activity between old and young rats ceptors in the nigro-striatal dopamine neurons may be due to age-related change in the sensi- of the rat (26, 27). Apomorphine produces hypermotility and stereotyped behavior such REFERENCES as sniffing and gnawing dose-dependently, and haloperidol inhibits them in rats (28). Thus, 1 Petkov, V.: Effect of ginseng on the brain bio- striatal dopamine utilization has apparently a genic monoamines and 3',5'-AMP system: Experi- ments on rats. Arzneimittelforschung28, 388-393 relation to the drug-induced behavioral (1978) changes within the limits of drugs acting on 2 Saito, H., Yoshida, Y. and Takagi, K.: Effect of dopamine receptors. The present results sug- Panax ginseng root on exhaustive exercise in mice. gest that Panax ginseng increases locomotor Japan. J. 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