Effects of ginsenosides on bovine adrenal tyrosine hydroxylase

Journal of Ethnopharmacology 66 (1999) 107 – 111

Short communication

Effects of ginsenosides on bovine adrenal tyrosine hydroxylase Hack-Seang Kim a,*, Yong-He Zhang a, Lian-Hua Fang b, Myung-Koo Lee a a

Department of Pharmacology, College of Pharmacy, Chungbuk National Uni6ersity, San 48, Kaeshin-Dong, Cheongju361 -763, South Korea b Department of Pharmacology, College of Medicine, Chungbuk National Uni6ersity, San 48, Kaeshin-Dong, Cheongju, 361 -763, South Korea Received 22 July 1998; received in revised form 1 December 1998; accepted 1 December 1998

Abstract The present study was undertaken to investigate the effects of ginsenosides on bovine adrenal tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. Ginsenoside-Rb1, Rc, Re and Rg1 inhibited the TH activity by 51.5, 25.4, 31.3, 44.3 and 43.3%, respectively, at a concentration of 80 mg/ml. Ginsenoside-Rb1, Rc, Re and Rg1 exhibited noncompetitive inhibition of TH activity with a substrate L-tyrosine. From these results, it is presumed that the effects of ginsenosides on TH activity observed in vitro might be also produced in vivo, and thereby the inhibitory effects of ginsenosides on TH activity may be partially responsible for the antidopaminergic action of ginsenosides by reducing the availability of dopamine at the presynaptic dopamine receptor in vivo. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bovine adrenal; Ginsenosides; Noncompetitive inhibition; L-Tyrosine; Tyrosine hydroxylase

1. Introduction Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae) is a well-known oriental folk medicine and has been used widely for thousands of years. Many reports have provided evidences that ginseng has a variety of effects on the ner* Corresponding author. Tel.: +82-431-261-2813; fax: + 82-431-268-2732. E-mail address: [email protected] (H.-S. Kim)

vous system. It has not only stimulative and inhibitory effects on the central nervous system (Petkov, 1959; Takagi et al., 1972; Saito et al., 1977a,b), but also sedative and tranquilizing effects (Takagi et al., 1972). Ginseng saponin, as the active component of ginseng extract, modulates dopaminergic hyperactivity induced by morphine (Kim et al., 1998a), cocaine (Kim et al., 1996a) and methamphetamine (Kim et al., 1996b), as well as the climbing behavior induced by apomorphine (Kim et al., 1998c), indicating that ginseng sa-

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ponin modulates the dopaminergic receptor at both the pre- and postsynaptic sites. The major active components of ginseng saponin, the panaxadiol ginsenoside-Rb1 and the panaxatriol ginsenoside-Rg1, also modulate morphine- and methamphetamine-induced hyperactivity, but not apomorphine-induced climbing behavior, indicating that ginsenosides modulate catecholaminergic activity preferentially at pre-synaptic sites (Kim et al., 1998a,b). Tyrosine hydroxylase (EC 1.14.26.2, TH), the rate-limiting enzyme in catecholamine biosynthesis, catalyzes the formation of L-DOPA from L-tyrosine (Nagatsu et al., 1964) using L-tyrosine as a substrate and its activity is thereby closely related to the dopamine biosynthesis. In our previous report, ginseng saponin inhibited bovine adrenal TH activity (Kim et al., 1998d). Therefore, it is hypothesized that the antidopaminergic activities of ginsenosides may be associated with dopamine biosynthesis, and some single components of ginseng saponin may also have inhibitory action on TH activity. For these reasons, the present study was undertaken to investigate the effects of ginsenoside-Rb1, Rc, Re and Rg1 on bovine adrenal TH activity.

2. Materials and methods

2.1. Materials Ginsenoside-Rb1, Rc, Re and Rg1, extracted and purified from Panax ginseng, were supplied by Korea Ginseng and Tobacco Research Institute (Taejon, South Korea). L-Tyrosine, DL-6methyl-5,6,7,8-tetrahydropterin, catalase, 3,4-dihydroxybenzylamine and alumina were purchased from the Sigma (St. Louis, MO, USA). All other reagents were of reagent grade.

2.2. Bo6ine adrenal TH Bovine adrenal was obtained from the Agriculture and Livestock Development Office (Cheongju, South Korea). Bovine adrenal TH was purified with minor modification according to the method of Joh and Ross (1983). The bovine

adrenal medulla (50 g) was homogenized in a Waring Blender at low speed with 10 mM potassium phosphate buffer (pH 7.0) and filtered through cheesecloth. The filtrate was then centrifuged at 1000 × g for 5 min. The supernatant was further centrifuged at 105 000× g for 60 min. The sediment was dissolved in 20 mM potassium phosphate buffer (pH 7.0) and the solution was subjected to ammonium sulfate precipitation at 80% saturation. The 80% ammonium sulfate precipitate was dialyzed against 10 mM potassium phosphate buffer (pH 7.0) and the protein was fractionated, taking the protein which precipitated between 30 and 60% saturation in ammonium sulfate. This fraction was dialyzed against 10 mM, and the buffer TH activity obtained from the final enzyme preparation was adjusted to 1.10 nmol/ min per mg protein for the experiments.

2.3. Assay for TH TH activity was determined using L-tyrosine as substrate according to a slightly modified procedure of Nagatsu et al. (1979) as described previously (Lee and Kim, 1996; Lee and Zhang, 1996). The reaction mixture contained 1.5 M sodium acetate (pH 5.8, 20 ml), 10 mM tyrosine (10 ml), 10 mM 6-methyltetrahydropterin (10 ml), 2 mg/ml catalase (10 ml) and enzyme preparation (50 ml). The enzyme reaction took place at 37°C for 10 min, and the reaction was stopped with 600 ml of 0.5 M perchloric acid containing 100 pmol of 3,4-dihydroxybenzylamine (internal standard). After addition of 5 ml of EDTA (2%) and 1.5 ml of KH2PO4 (0.35 M), an aliquot of N-NaOH was added to adjust the pH to 8.4–8.6, and then the reaction mixture was passed through the alumina cartridges (100 mg). L-DOPA and 3,4-dihydrobenzylamine absorbed were eluted with 300 ml of 0.5 M HCl. A total of 20 ml of eluate was injected into the HPLC equipped with a CM8010 electrochemical detector (Toso, Japan) and a TSK-gel ODS 120T (5 mm, 25× 0.45 cm, Toso). The mobile phase was a 0.1 M potassium phosphate buffer (pH 3.5)–1% methanol, with a flow rate of 1 ml/min. The detector potential was set at 0.8 V against the Ag/AgCl electrode.

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Table 1 Effects of ginsenosides on bovine adrenal TH activitya Drugs (mg/ml)

TH activity (nmol/min per mg protein) (% of control)

Control Ginsenoside-Rb1 40 80 Rc 40 80 Re 40 80 Rg1 40 80

1.10 90.04 (100.0) 0.95 9 0.09 (86.8) 0.82 9 0.08 (74.6) 0.87 9 0.09 (79.3) 0.76 9 0.06 (68.7) 0.67 9 0.09 (60.7)* 0.61 9 0.07 (55.7)* 0.72 9 0.08 (65.2) 0.62 9 0.09 (56.7)*

a

The control of TH activity, 1.10 nmol/min per mg protein, was taken as 100%. The data were expressed as means9 S.E.M. of four experiments. * PB0.05 compared with control (Student’s t-test). G, ginsenoside.

Fig. 1. Inhibition of bovine adrenal TH by ginsenoside-Rg1 added in the enzyme reaction mixture. The data were plotted by linear regression analysis. Ginsenoside concentration: (1) nil; (2) 80 mg/ml.

2.4. Data analysis Protein amount was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. The values of the Michaelis constant (Km) and the maximum velocity (Vmax) were obtained using a Lineweaver – Burk plot with various concentrations of L-tyrosine.

bovine adrenal TH activity by 51.5, 25.4, 31.3, 44.3 and 43.3%, respectively, at a concentration of 80 mg/ml (Table 1). These results suggest that the inhibitory potencies of panaxatriol ginsenoside-Re and Rg1 are slightly greater than those of panaxadiol ginsenoside-Rb1 and Rc.

3.2. Effects of ginsenosides on TH kinetics 3. Results

3.1. Effects of ginsenosides on bo6ine adrenal TH Gisenoside-Rb1, Rc, Re and Rg1 inhibited the

According to the kinetic properties of bovine adrenal TH in this experiment, the values of Km and Vmax in terms of the substrate L-tyrosine were 88.09 7.2 mM and 1.0190.8 nmol/min per mg

Table 2 Effects of ginsenosides on the kinetics of bovine adrenal TH (means 9S.E.M. of five experiments) Drugs (80 mg/ml)

Km (mM)

Vm (nmol/min per mg protein)

Control

88.09 7.2

1.01 9 10.08 (Vmax)

Ginsenoside Rb1 Rc Re Rg1

0.81 90.07 0.8390.08 0.739 0.07 0.729 0.09

Ki (mM)

Type of inhibition

0.29 9 0.02 0.36 9 0.04 0.23 90.03 0.23 90.03

Noncompetitive Noncompetitive Noncompetitive Noncompetitive

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protein (n=4), respectively (Table 2). Fig. 1 shows the effect of ginsenoside-Rg1 on TH kinetics. This plot indicates noncompetitive inhibition with respect to L-tyrosine according to the definition of Lineweaver– Burk. Ginsenoside-Rb1, Rc and Re show the same pattern of plot as ginsenoside-Rg1. The Vmax values were lowered in the presence of ginsenoside-Rb1, Rc, Re and Rg1 to 0.819 0.07, 0.8390.08, 0.739 0.07 and 0.7290.09 nmol/min per mg protein (n = 4), respectively. Accordingly, the Ki values of ginsenoside-Rb1, Rc, Re and Rg1 were 0.2990.03, 0.36 90.04, 0.2390.03 and 0.279 0.04 mM (n = 4), respectively (Table 2). On the other hand, none of ginsenosides tested in this study show inhibitory effects on bovine adrenal dopamine b-hydroxylase, which catalyzes the formation of norepinephrine from dopamine (data not shown).

4. Discussion Kim et al. (1990, 1995, 1996a,b)have shown that ginseng saponin inhibits dopaminergic hyperactivity induced by morphine, cocaine and methamphetamine at the presynaptic dopamine receptor, and also exhibits an antidopaminergic property at the postsynaptic dopamine receptor, by inhibiting apomorphine-induced climbing behavior. It is also reported that ginseng saponin modulates dopaminergic activity preferentially at the pre-synaptic site (Takahashi et al., 1993; Kim et al., 1998b). Generally, it has been postulated that the drugs that reduce the availability of catecholamines in the presynaptic neuron or that block the action of the catecholamines on the postsynaptic receptor attenuate the behavioral effects, such as hyperactivity and reinforcing effects, of stimulants in the monkey and rat (Pickens et al., 1968; Wilson and Schuster, 1972). In the present study, ginsenosideRb1, Rc, Re and Rg1 noncompetitively inhibited bovine adrenal TH activity. Therefore, it could be presumed that the inhibitions of TH activity by ginsenosides in vitro might also occur in vivo, and thus it may be partially responsible for the antidopaminergic action of ginsenosides in vivo by

reducing the availability of dopamine at the presynaptic dopamine receptor. It has been reported that panaxatriol ginsenoside-Rg1 shows more potent inhibition than panaxadiol ginsenoside-Rb1 in catecholamine secretion, the Ca2 + current in adrenal chromaffin cells (Tachikawa et al., 1995; Kim et al., 1998d), morphine-induced CPP (Kim et al., 1998b), and catecholamine secretion at pre-synaptic sites (Takahashi et al., 1993). In the present study, panaxatriol ginsenoside-Rg1 and Re also show slightly greater inhibitions of TH activity than panaxadiol ginsenoside-Rb1 and Rc. It is thus presumed that the different inhibitory activities of ginsenoside-Rg1 and Rb1 on the dopaminergic receptor may result from differences in the extent of their inhibitory action on dopamine biosynthesis and/or secretion. In addition, to discuss which is the major active component of ginseng saponin on TH activity, therefore, we need further studies using several other ginsenosides simultaneously. References Joh, T. H., Ross, M. E., 1983. Preparation of catecholaminesynthesizing enzymes as immunogens for immunohistochemistry. In: Cuello, A.C. (Ed.), ImmunohistochemistryOxford IBRO Handbook. Wiley, New York, pp. 121 – 138. Kim, H.S., Jang, C.G., Lee, M.K., 1990. Antinarcotic effects of the standardized ginseng extract G115 on morphine. Planta Medica 56, 158 – 163. Kim, H.S., Kang, J.G., Rheu, H.M., Cho, D.H., Oh, K.W., 1995. Blockade by ginseng total saponin of the development of methamphetamine reverse tolerance and dopamine receptor supersensitivity in mice. Planta Medica 61, 22 – 25. Kim, H.S., Jang, C.G., Oh, K.W., Seong, Y.H., Rheu, H.M., Cho, D.H., Kang, S.Y., 1996a. Effects of ginseng total saponin on cocaine-induced hyperactivity and conditioned place preference in mice. Pharmacology Biochemistry and Behavior 53, 185 – 190. Kim, H.S., Jang, C.G., Park, W.K., Oh, K.W., Rheu, H.M., Cho, D.H., Oh, S.K., 1996b. Blockade by ginseng total saponin of methamphetamine-induced hyperactivity and conditioned place preference in mice. General Pharmacology 27, 199 – 204. Kim, H.S., Jang, C.G., Oh, K.W., Oh, S.K., Rheu, H.M., Rhee, G.S., Seong, Y.H., Park, W.K., 1998a. Effects of ginseng total saponin on morphine-induced hyperactivity and conditioned place preference in mice. Journal of Ethnopharmacology 60, 33 – 42.

H.-S. Kim et al. / Journal of Ethnopharmacology 66 (1999) 107–111 Kim, H.S., Hong, Y.T., Jang, C.G., 1998b. Effects of ginsenoside-Rg1 and Rb1 on morphine-induced hyperactivity and reinforcement in mice. Journal of Pharmacy and Pharmacology 50, 555 – 560. Kim, H.S., Zhang, Y.H., Fang, L.H., Lee, M.K., 1998c. Effect of ginseng total saponin on bovine adrenal tyrosine hydroxylase. Arch. Pharmacol. Res. 21, 782–784. Kim, H.S., Lee, J.H., Goo, Y.S., Nah, S.Y., 1998d. Effects of ginsenosides on Ca2 + channels and membrane capacitance in rat adrenal chromaffin cells. Brain Research Bulletin 46, 245 – 251. Lee, M.K., Kim, H.S., 1996. Inhibitory effects of protoberberine alkaloids from the roots of Coptis japonica on catecholamine biosynthesis in PC12 cells. Planta Medica 62, 31– 34. Lee, M.K., Zhang, Y.H., 1996. Inhibition of tyrosine hydroxylase by berberine. Medical Science Research 24, 561– 562. Lowry, O.H., Rosebrough, N J., Farr, A.L., Randall, R. J., 1951. Protein measurement with the Folin phenol reagent. Journal of Biology and Chemistry 193, 265–275. Nagatsu, T., Levitt, M., Udenfriend, S., 1964. Tyrosine hydroxylase: The initial step in norepinephrine biosynthesis. Journal of Biology and Chemistry 239, 2910–2917. Nagatsu, T., Oka, K., Kato, K., 1979. Highly sensitive assay for tyrosine hydroxylase activity by high performance liquid chromatography. Journal of Chromatography 163, 247 – 252. Petkov, V.W., 1959. Pharmacological investigation of the drug

.

111

panax ginseng C.A.Meyer. Arzneimittel-Forschung/Drug Research 9, 305 – 311. Pickens, R., Meisch, R., Dougherty, J., 1968. Chemical interactions in methamphetamine reinforcement. Psychological Reports 23, 1267 – 1270. Saito, H., Tsuchiya, M., Naka, S., Takagi, K., 1977a. Effects of Panax ginseng root on conditioned avoidance response in rats. Japanese Journal of Pharmacology 27, 509 – 616. Saito, H., Tsuchiya, M., Naka, S., Takagi, K., 1977b. Effects of Panax ginseng root on the acquisition of sound discrimination behavior in rats. Japanese Journal of Pharmacology 29, 319 – 325. Takagi, K., Saito, H., Nabata, H., 1972. Pharmacological studies of Panax ginseng root: estimation of pharmacological actions of Panax ginseng root. Japanese Journal of Pharmacology 22, 245 – 249. Takahashi, E., Kudo, K., Akasaka, Y., Miyate, Y., Tachikawa, E., 1993. Actions of saponins of red ginseng on the sympathetic nerve and effects of combination of red ginseng with other herb medicines on cardiac functions. The Ginseng Review 16, 88 – 92. Tachikawa, E., Kudo, K., Kashimoto, T., Takahashi, E., 1995. Ginseng saponins reduced acetylcholine-evoked Na + influx and catecholamine secretion in bovine adrenal chromaffin cells. Journal of Pharmacology and Experimental Therapeutics 273, 629 – 636. Wilson, M. C., Schuster, C. R., 1972. The effects of chlorpromazine on psychomotor stimulant self-administration in the rhesus monkey. Psychopharmacology 26, 115 – 126.