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Effects of tetrandrine and fangchinoline on human platelet aggregation and thromboxane B2 formation
Journal of Ethnopharmacology 66 (1999) 241 – 246
Short communication
Effects of tetrandrine and fangchinoline on human platelet aggregation and thromboxane B2 formation Hack-Seang Kim a,*, Yong-He Zhang a, Lian-Hua Fang b, Yeo-Pyo Yun a, Hyung-Kyu Lee c a
Department of Pharmacology, College of Pharmacy, Chungbuk National Uni6ersity, San 48, Kaeshin-Dong, Cheongju 361 -763, South Korea b Department of Pharmacology, College of medicine, Chungbuk National Uni6ersity, San 48, Kaeshin-Dong, Cheongju 361 -763, South Korea c Natural Product Biosynthesis Research Unit, Korea Research Institute of Bioscience and Biotechnology, Taejon 305 -600, South Korea Received 26 October 1998; received in revised form 3 December 1998; accepted 3 December 1998
Abstract Tetrandrine (TET) and fangchinoline (FAN) are two major components of the radix of Stephania tetrandra. The effects of TET and FAN on human platelet aggregation and formation of thromboxane (TX) B2, a stable metabolite of TXA2, were examined in the aspect of platelet aggregation. TET and FAN inhibited platelet-activating factor (PAF)-induced human platelet aggregation. IC50 values for TET and FAN were 28.6 9 3.24 mM and 21.7 9 2.61 mM, respectively. In the PAF-receptor binding assay, neither TET nor FAN showed any inhibitory effects on the specific bindings of PAF to its receptor. TET and FAN also inhibited PAF-, thrombin- and arachidonic acid-induced thromboxane B2 formation in human washed platelet. These results indicate that TET and FAN inhibit the platelet aggregation by interfering with the intracellular messengers system, but not by inhibiting the binding of PAF to PAF-receptor on the platelet membrane directly, and the suppression of TXA2 formation by TET and FAN may be responsible for their inhibitory activities on the platelet aggregation and further on the thrombosis. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Fangchinoline; PAF-receptor binding; Platelet aggregation; Tetrandrine; Thromboxane B2
* Corresponding author. Tel.: +82-431-261-2813; fax: + 82-431-268-2732. E-mail address: [email protected] (H.-S. Kim) 0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 8 ) 0 0 2 3 7 - 2
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H.-S. Kim et al. / Journal of Ethnopharmacology 66 (1999) 241–246
1. Introduction Tetrandrine (TET) and fangchinoline (FAN), the bisbenzylisoquinoline alkaloids (Fig. 1), are the main components of the herbal remedy ‘fenfangji’ (the Radix of Stephania tetrandra S. Moore) which has been used since antiquity by Chinese physicians for the treatment of hypertension, apoplexy, etc. (Jiangsu College of New Medicine, 1975). In our previous study, TET and FAN showed inhibitory effects on experimental pulmonary thrombosis in mice and on human platelet aggregation induced by thrombin, collagen, ADP, epinephrine, arachidonic acid (AA) and A23187 (Kim et al., 1999). However, the mechanisms underlying antiplatelet aggregations of TET and FAN have not yet been fully understood. Therefore, the present study was undertaken to investigate the activities of TET and FAN on human platelet aggregation induced by platelet-activating factor (PAF), and thromboxane B2 formation induced by PAF, thrombin and AA.
2. Materials and methods
2.1. Materials TET and FAN were isolated from the root of Stephania tetrandra S. Moor (or fenfangji) and confirmed by comparing the physical – chemical properties and the 1H NMR spectra with previous reports (Yamaguchi, 1970; Lin et al., 1993). Ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(b-aminoethylether)-N,N,N%,N%-tetraacetic acid (EGTA), tris(hydroxymethyl) aminomethane (Trizma base, Sigma, St. Louis, MO, USA), bovine serum albumin (BSA), POPOP, PPO, thrombin, AA, and unlabeled C18PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) were purchased from Sigma. C18-PAF was dissolved in pure ethanol at a concentration of 10 mg/ml and stored at − 70°C as a stock solution. When in use, it was diluted in 0.9% saline containing 0.25% bovine serum albumin. AA was dissolved in 0.1 M sodium carbonate and stored in aliquots at −20°C.
[3H]PAF (TRK 743), with a specific activity of 174 Ci/mmol, and a TXB2 enzyme immunoassay kit were purchased from Amersham (UK) and the others were grade agents.
2.2. Platelet aggregation assay in 6itro The stock solutions of TET and FAN were made up by dissolution in dimethylsulfoxide (DMSO) and were further diluted in 0.9% saline for the experiments. Blood from healthy volunteers, who had not taken any drugs for at least 15 days, was collected by venopuncture and added into a plastic flask containing 3.15% sodium citrate (1:9, v/v). Platelet-rich plasma (PRP) was obtained by centrifugation of the citrated blood at room temperature for 10 min at 120×g. The platelet count was adjusted to 3.0–3.2× 108 platelets/ml and platelet aggregation was monitored in a four-channel whole blood Lumi-ionized Calcium Aggregometer (Chrono-Log, PN, USA) according to the method described by Born (1962). Human PRP (299 ml) was incubated at 37°C for 2 min in the aggregometer while stirring at 1000 rpm, and a fixed amount (1 m l) of the test drug solutions or vehicle solution was added, and this was incubated at 37°C for 3 min. After incubation, platelet aggregation was induced by the addition of 33 ml of a normal saline solution of PAF. Changes in light transmission were recorded for 7 min after stimulation with the agent. The aggregation is expressed as percent of inhibition (X) using the following equation: X (%)=
A− B × 100 A
Fig. 1. Structures of tetrandrine and fangchinoline. Tetrandrine: R = CH3. Fangchinoline: R =H.
H.-S. Kim et al. / Journal of Ethnopharmacology 66 (1999) 241–246
where A is maximal aggregation of the control and B is maximal aggregation of drug-treated PRP. The concentration of PAF (0.2 mg/ml) which exhibited maximal aggregation in the control was chosen in preliminary experiments. IC50 values were calculated by a probit method.
binding was defined as the binding of [3H]PAF which was non-displaceable with excess of cold C18-PAF. The specific binding was defined as the difference between total binding of [3H]PAF and the non-specific binding. The percent inhibition was expressed as following:
2.3. PAF-receptor binding assay % inhibition= The PAF-receptor binding assay was performed according to the procedure described by Jung et al. (1998). Five volumes of rabbit blood were drawn from the heart puncture and added into 1 volume of ACD solution (tri-sodium citrated 2.5%, citric acid 1.37% and glucose 2% in H2O). Platelet-rich plasma (PRP) was prepared by centrifugation, at 280× g for 10 min, of rabbit blood. The PRP was centrifuged at 1200×g for 10 min and the platelet pellets were washed with Trisbuffer (10 mM Tris, 150 mM NaCl, 2 mM EGTA, 0.1% glucose, pH 7.5). The washed platelets were suspended at 2×108 cells/ml in Tris –BSA buffer (10 mM Tris, 25 mM MgCl2, 75 mM KCl, 2 mM EGTA, 0.1% glucose, 0.25% BSA, pH 7.0). The PAF-receptor binding was characterized by a filtration technique. The mixtures of sample solution (3% DMSO Tris – BSA buffer solution: 10 mM Tris, 25 mM MgCl2, 75 mM KCl, 2 mM EGTA, 0.1% glucose, 0.25% BSA, pH 7.0) and the platelet suspension (final concentration 8× 107 cells/ml) were preincubated for 6 min, and [3H]PAF (final concentration 0.9 nM) was pipetted into the binding well, and the incubation was continued for 30 min. The unbound PAF was separated from bound PAF by filtration using a Millipore multiscreen assay system (membrane filter pore size, 1.2 mM). The membrane filters were presoaked with Tris – BSA buffer and each filter was successively and rapidly washed five times with Tris–BSA buffer. The filters were then dried and placed into scintillation vials containing 3 ml of toluene cocktail solution (PPO 4 g, POPOP 0.1 g in toluene). Radioactivity (dpm) was then measured in a liquid scintillation counter (Beckman LS6000TA spectrophotometer). Total binding was defined as the binding of [3H]PAF without cold C18-PAF, and non-specific
243
=
Sc − Ss × 100 Sc (Tc − Nc)− (Ts − Ns) × 100 Tc − Nc
where Sc is specific binding of control; Ss is specific binding of sample; Tc is total binding of control; Ts is total binding of sample; Nc is nonspecific binding of control; Ns is nonspecific binding of sample.
2.4. Thromboxane B2 assay in 6itro Washed platelet suspensions (WPS) were prepared from PRP according to the procedure described by Teng and Ko (1988). PRP was centrifuged at 900× g for 10 min at room temperature. The supernatant was removed and the platelet pellet was suspended in washing buffer (mM/l: NaCl 138, KCl 2.8, KH2PO4 0.8, MgCl2 0.8, Hepes 10, D-glucose 5.6, pH 7.4) containing BSA (0.35%) and centrifuged at 900× g for 10 min at room temperature. The supernatant was aspirated and the pellet was resuspended in Tyrode’s solution. The platelet count was determined automatically (Coulter Counter model T-540, Hileah, FL, USA) and adjusted to 3× 108 platelets/ml. Calcium chloride (1 mM) was added to the final platelet suspension. Human WPS (300 ml) was incubated at 37°C for 2 min in the aggregometer while stirring at 1000 rpm, and a fixed amount (1 ml) of the test drug solutions or vehicle solution was added, and this was incubated at 37°C for 3 min. After incubation of WPS with the inducer for 6 min, EDTA (2 mM) and indomethacin (50 mM) were added to halt the formation of TXB2. The mixture was centrifuged at 11 000 rpm (Mega 17R, Hanil Science Industrial, South Korea) for 2 min, and the TXB2 in the supernatant was assayed by
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H.-S. Kim et al. / Journal of Ethnopharmacology 66 (1999) 241–246 Table 1 Effects of TET and FAN on PAF-receptor bindings in rabbit plateletsa Compound
Specific binding (inhibition, %)
DMSO (control) TET (160 mM) FAN (160 mM) Magnoline (40 mM)
5319 9355 3479 9261 4255 9426 1686 9184
(0.0) (34.6) (20.0) (68.3)
a The rabbit PRP was incubated with [3H]PAF (final concentration, 0.9 nM) for 30 min. The values are expressed as mean 9S.E.M (n =3).
3.3. Effects of TET and FAN on the formation of TXB2
Fig. 2. Effects of TET and FAN on human platelet aggregation induced by PAF. The concentration of PAF was 0.2 mg/ml. The examinations were repeated three times using different PRP samples obtained from three subjects, and the values are expressed as mean 9 S.E.M.
The amount of TXB2 in resting WPS was 1.79 0.5 ng/3 × 108 platelets. After incubation of WPS with thrombin, AA and PAF for 6 min, the amount of TXB2 was increased to 386.8 98.2,
enzyme immunoassay. The concentrations which exhibited maximal aggregation in the control were chosen in preliminary experiments. 3. Results
3.1. Effects of TET and FAN on PAF-induced platelet aggregation TET and FAN inhibited PAF-induced human platelet aggregation concentration dependently (Fig. 2). IC50 values of TET and FAN were 28.6 and 21.7 mM, respectively.
3.2. Effects of TET and FAN on PAF-receptor binding As shown in Table 1, TET and FAN showed only 34.6 and 20.0% of inhibitions on PAF receptor binding assay at the concentration of 160 mM, respectively. On the contrast, the positive reference agent, magnoline, inhibited PAF-receptor binding by 68.3% at a concentration of 40 mM.
Fig. 3. Effects of TET (left) and FAN (right) on the TXB2 formation induced by PAF, AA and thrombin in human washed platelets. DMSO (0.1%, Control), TET or FAN was preincubated with WPS at 37°C for 2 min. After incubation of WPS with PAF (A, 0.2 mg/ml), AA (B, 2.1 mM) and thrombin (C, 1 U/ml) for 6 min, EDTA (2 mM) and indomethacin (50 mM) were added to halt the formation of TXB2. The values are expressed as mean 9S.E.M. (n =4). **PB0.01 compared with control. Con., control.
H.-S. Kim et al. / Journal of Ethnopharmacology 66 (1999) 241–246
451.1936.3 and 137.3 96.0 ng/3 ×108 platelets, respectively (Fig. 3). As shown in Fig. 3, TET and FAN inhibited thrombin (1 U/ml)-induced TXB2 formation by 35.2 and 50.2% at a concentration of 1.5 mM, and by 76.6 and 81.4% at a concentration of 15 mM, respectively. TET also inhibited the AA-induced TXB2 formation by 55.4 and 85.6% at concentrations of 33.0 and 50.0 mM, respectively (Fig. 3). On the other hand, FAN did not inhibit the AA-induced TXB2 formation at a concentration of 33.0 mM but, at 50.0 and 100 mM, FAN inhibited the AA-induced TXB2 formation by 61.3 and 69.2%, respectively. At a concentration of 33.0 mM, TET and FAN inhibited PAF-induced TXB2 formation by 47.5 and 44.3%, respectively.
4. Discussion In the present study, TET and FAN inhibited PAF-induced human platelet aggregation, but did not inhibit the PAF-receptor-specific bindings. Stimulants used in this experiment, such as PAF and thrombin, bind to specific cell surface receptors which result in transmembrane signalling via the phosphatidylinositol second-messenger system (Westwick et al., 1985), whereas AA acts directly on membrane cyclo-oxygenase enzyme pathways (Westwick et al., 1985). The results of receptor binding assays suggest that TET and FAN inhibit the platelet aggregation by interfering with the intracellular messengers system, but not by inhibiting the binding of PAF to PAF-receptor on the platelet membrane directly. Platelet aggregation is known to be a result of complex signal transduction cascade reactions brought about by stimulants. One of the components in the cascade is TXA2 (Hornby, 1984). The formation of TXA2 is stimulated by some aggregation inducers, such as PAF, thrombin, AA, etc. (Yu et al., 1996). TXA2 is a potent potentiator of platelet aggregation, which affects other platelets and causes them to change shape, extend pseudopods, and adhere to the platelets on the damaged surface, and makes a major contribution to the formation of some thrombi (Fuster et al., 1993). TXB2 is a stable metabolite of TXA2 (Yu
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et al., 1996). Therefore, the production of TXA2 was examined by measuring the formation of. TXB2. In this study, TET and FAN showed inhibitions of TXB2 formation induced by PAF, thrombin and AA in human WPS. These results suggest that the suppression of TXB2 formation by TET and FAN may be responsible for their inhibitory activities on platelet aggregation and, furthermore, on thrombosis. In conclusion, TET and FAN are anti-apoplexy activity components of fenfangji and their precise mechanisms need further study.
Acknowledgements This study was supported by a research grant from the Korean Ministry of Education (1996)– 1998).
References Born, G.V.R., 1962. Aggregation of blood platelets by adenosine diphosphated and its reversal. Nature 194, 927 – 929. Fuster, V., Dyken, M.L., Vokonas, P.S., Hennekens, C., 1993. Aspirin as a therapeutic agent in cardiovascular disease. Circulation 87 (2), 659 – 675. Kim, H.S., Zhang, Y.H., Yun, Y.P., 1999. Effects of tetrandrine and fangchinoline on the experimental thrombosis in mice and human platelet aggregation. Planta Medica (in press). Hornby, E.J., 1984. Evidence that prostaglandin endoperoxides can induce platelet aggregation in the absence of thromboxane A2 production. Biochemistry and Pharmacology 31, 1158 – 1160. Jiangsu College of New Medicine, 1975. Encyclopedia of Materia Medica, vol. 1. Shanghai People’s Publishing House, Shanghai, China, 268 pp. Jung, K.Y., Kim, D.S., Oh, S.R., Lee, I.S., Lee, J.J., Park, J.D., Kim, S.I., Lee, H.K., 1998. Platelet activating factor antagonist activity of ginsenosides. Biological and Pharmaceutical Bulletin 21 (1), 79 – 80. Lin, L.Z., Shieh, H.L., Angerhofer, C.K., Pezzuto, J.M., Cordell, G.A., Xue, L., Johnson, M.E., Ruangrungsi, N., 1993. Cytotoxic and antimalarial bisbenzylisoquinoline alkaloids from cyclea barbata. Journal of Natural Products 56, 22 – 29. Teng, C.M., Ko, F.N., 1988. Comparison of the platelet aggregation induced by three thrombin-like enzymes of snake venoms and thrombin. Thrombosis and Haemostasis 59, 304 – 309.
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H.-S. Kim et al. / Journal of Ethnopharmacology 66 (1999) 241–246 pp.), vol. I. Torii & Co, Ltd, Nihonbashi, Tokyo, Japan. Yu, S.M., Tsai, S.Y., Kuo, S.C., Ou, J.T., 1996. Inhibition of platelet function by A02131-1, a novel inhibitor of cGMPspecific phosphodiesterase, in vitro and in vivo. Blood 87 (9), 3758 – 3767.
Westwick, J., Scully, M.F., MacIntyre, D.E., Kakkar, V.V., 1985. Mechanisms of stimulus-response coupling in platelets. Advances in Experimental Medicine and Biology 192, 1 – 44. Yamaguchi, K., 1970. Spectra Data of Natural Products (559
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Short communication
Effects of tetrandrine and fangchinoline on human platelet aggregation and thromboxane B2 formation Hack-Seang Kim a,*, Yong-He Zhang a, Lian-Hua Fang b, Yeo-Pyo Yun a, Hyung-Kyu Lee c a
Department of Pharmacology, College of Pharmacy, Chungbuk National Uni6ersity, San 48, Kaeshin-Dong, Cheongju 361 -763, South Korea b Department of Pharmacology, College of medicine, Chungbuk National Uni6ersity, San 48, Kaeshin-Dong, Cheongju 361 -763, South Korea c Natural Product Biosynthesis Research Unit, Korea Research Institute of Bioscience and Biotechnology, Taejon 305 -600, South Korea Received 26 October 1998; received in revised form 3 December 1998; accepted 3 December 1998
Abstract Tetrandrine (TET) and fangchinoline (FAN) are two major components of the radix of Stephania tetrandra. The effects of TET and FAN on human platelet aggregation and formation of thromboxane (TX) B2, a stable metabolite of TXA2, were examined in the aspect of platelet aggregation. TET and FAN inhibited platelet-activating factor (PAF)-induced human platelet aggregation. IC50 values for TET and FAN were 28.6 9 3.24 mM and 21.7 9 2.61 mM, respectively. In the PAF-receptor binding assay, neither TET nor FAN showed any inhibitory effects on the specific bindings of PAF to its receptor. TET and FAN also inhibited PAF-, thrombin- and arachidonic acid-induced thromboxane B2 formation in human washed platelet. These results indicate that TET and FAN inhibit the platelet aggregation by interfering with the intracellular messengers system, but not by inhibiting the binding of PAF to PAF-receptor on the platelet membrane directly, and the suppression of TXA2 formation by TET and FAN may be responsible for their inhibitory activities on the platelet aggregation and further on the thrombosis. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Fangchinoline; PAF-receptor binding; Platelet aggregation; Tetrandrine; Thromboxane B2
* Corresponding author. Tel.: +82-431-261-2813; fax: + 82-431-268-2732. E-mail address: [email protected] (H.-S. Kim) 0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 8 ) 0 0 2 3 7 - 2
242
H.-S. Kim et al. / Journal of Ethnopharmacology 66 (1999) 241–246
1. Introduction Tetrandrine (TET) and fangchinoline (FAN), the bisbenzylisoquinoline alkaloids (Fig. 1), are the main components of the herbal remedy ‘fenfangji’ (the Radix of Stephania tetrandra S. Moore) which has been used since antiquity by Chinese physicians for the treatment of hypertension, apoplexy, etc. (Jiangsu College of New Medicine, 1975). In our previous study, TET and FAN showed inhibitory effects on experimental pulmonary thrombosis in mice and on human platelet aggregation induced by thrombin, collagen, ADP, epinephrine, arachidonic acid (AA) and A23187 (Kim et al., 1999). However, the mechanisms underlying antiplatelet aggregations of TET and FAN have not yet been fully understood. Therefore, the present study was undertaken to investigate the activities of TET and FAN on human platelet aggregation induced by platelet-activating factor (PAF), and thromboxane B2 formation induced by PAF, thrombin and AA.
2. Materials and methods
2.1. Materials TET and FAN were isolated from the root of Stephania tetrandra S. Moor (or fenfangji) and confirmed by comparing the physical – chemical properties and the 1H NMR spectra with previous reports (Yamaguchi, 1970; Lin et al., 1993). Ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(b-aminoethylether)-N,N,N%,N%-tetraacetic acid (EGTA), tris(hydroxymethyl) aminomethane (Trizma base, Sigma, St. Louis, MO, USA), bovine serum albumin (BSA), POPOP, PPO, thrombin, AA, and unlabeled C18PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) were purchased from Sigma. C18-PAF was dissolved in pure ethanol at a concentration of 10 mg/ml and stored at − 70°C as a stock solution. When in use, it was diluted in 0.9% saline containing 0.25% bovine serum albumin. AA was dissolved in 0.1 M sodium carbonate and stored in aliquots at −20°C.
[3H]PAF (TRK 743), with a specific activity of 174 Ci/mmol, and a TXB2 enzyme immunoassay kit were purchased from Amersham (UK) and the others were grade agents.
2.2. Platelet aggregation assay in 6itro The stock solutions of TET and FAN were made up by dissolution in dimethylsulfoxide (DMSO) and were further diluted in 0.9% saline for the experiments. Blood from healthy volunteers, who had not taken any drugs for at least 15 days, was collected by venopuncture and added into a plastic flask containing 3.15% sodium citrate (1:9, v/v). Platelet-rich plasma (PRP) was obtained by centrifugation of the citrated blood at room temperature for 10 min at 120×g. The platelet count was adjusted to 3.0–3.2× 108 platelets/ml and platelet aggregation was monitored in a four-channel whole blood Lumi-ionized Calcium Aggregometer (Chrono-Log, PN, USA) according to the method described by Born (1962). Human PRP (299 ml) was incubated at 37°C for 2 min in the aggregometer while stirring at 1000 rpm, and a fixed amount (1 m l) of the test drug solutions or vehicle solution was added, and this was incubated at 37°C for 3 min. After incubation, platelet aggregation was induced by the addition of 33 ml of a normal saline solution of PAF. Changes in light transmission were recorded for 7 min after stimulation with the agent. The aggregation is expressed as percent of inhibition (X) using the following equation: X (%)=
A− B × 100 A
Fig. 1. Structures of tetrandrine and fangchinoline. Tetrandrine: R = CH3. Fangchinoline: R =H.
H.-S. Kim et al. / Journal of Ethnopharmacology 66 (1999) 241–246
where A is maximal aggregation of the control and B is maximal aggregation of drug-treated PRP. The concentration of PAF (0.2 mg/ml) which exhibited maximal aggregation in the control was chosen in preliminary experiments. IC50 values were calculated by a probit method.
binding was defined as the binding of [3H]PAF which was non-displaceable with excess of cold C18-PAF. The specific binding was defined as the difference between total binding of [3H]PAF and the non-specific binding. The percent inhibition was expressed as following:
2.3. PAF-receptor binding assay % inhibition= The PAF-receptor binding assay was performed according to the procedure described by Jung et al. (1998). Five volumes of rabbit blood were drawn from the heart puncture and added into 1 volume of ACD solution (tri-sodium citrated 2.5%, citric acid 1.37% and glucose 2% in H2O). Platelet-rich plasma (PRP) was prepared by centrifugation, at 280× g for 10 min, of rabbit blood. The PRP was centrifuged at 1200×g for 10 min and the platelet pellets were washed with Trisbuffer (10 mM Tris, 150 mM NaCl, 2 mM EGTA, 0.1% glucose, pH 7.5). The washed platelets were suspended at 2×108 cells/ml in Tris –BSA buffer (10 mM Tris, 25 mM MgCl2, 75 mM KCl, 2 mM EGTA, 0.1% glucose, 0.25% BSA, pH 7.0). The PAF-receptor binding was characterized by a filtration technique. The mixtures of sample solution (3% DMSO Tris – BSA buffer solution: 10 mM Tris, 25 mM MgCl2, 75 mM KCl, 2 mM EGTA, 0.1% glucose, 0.25% BSA, pH 7.0) and the platelet suspension (final concentration 8× 107 cells/ml) were preincubated for 6 min, and [3H]PAF (final concentration 0.9 nM) was pipetted into the binding well, and the incubation was continued for 30 min. The unbound PAF was separated from bound PAF by filtration using a Millipore multiscreen assay system (membrane filter pore size, 1.2 mM). The membrane filters were presoaked with Tris – BSA buffer and each filter was successively and rapidly washed five times with Tris–BSA buffer. The filters were then dried and placed into scintillation vials containing 3 ml of toluene cocktail solution (PPO 4 g, POPOP 0.1 g in toluene). Radioactivity (dpm) was then measured in a liquid scintillation counter (Beckman LS6000TA spectrophotometer). Total binding was defined as the binding of [3H]PAF without cold C18-PAF, and non-specific
243
=
Sc − Ss × 100 Sc (Tc − Nc)− (Ts − Ns) × 100 Tc − Nc
where Sc is specific binding of control; Ss is specific binding of sample; Tc is total binding of control; Ts is total binding of sample; Nc is nonspecific binding of control; Ns is nonspecific binding of sample.
2.4. Thromboxane B2 assay in 6itro Washed platelet suspensions (WPS) were prepared from PRP according to the procedure described by Teng and Ko (1988). PRP was centrifuged at 900× g for 10 min at room temperature. The supernatant was removed and the platelet pellet was suspended in washing buffer (mM/l: NaCl 138, KCl 2.8, KH2PO4 0.8, MgCl2 0.8, Hepes 10, D-glucose 5.6, pH 7.4) containing BSA (0.35%) and centrifuged at 900× g for 10 min at room temperature. The supernatant was aspirated and the pellet was resuspended in Tyrode’s solution. The platelet count was determined automatically (Coulter Counter model T-540, Hileah, FL, USA) and adjusted to 3× 108 platelets/ml. Calcium chloride (1 mM) was added to the final platelet suspension. Human WPS (300 ml) was incubated at 37°C for 2 min in the aggregometer while stirring at 1000 rpm, and a fixed amount (1 ml) of the test drug solutions or vehicle solution was added, and this was incubated at 37°C for 3 min. After incubation of WPS with the inducer for 6 min, EDTA (2 mM) and indomethacin (50 mM) were added to halt the formation of TXB2. The mixture was centrifuged at 11 000 rpm (Mega 17R, Hanil Science Industrial, South Korea) for 2 min, and the TXB2 in the supernatant was assayed by
244
H.-S. Kim et al. / Journal of Ethnopharmacology 66 (1999) 241–246 Table 1 Effects of TET and FAN on PAF-receptor bindings in rabbit plateletsa Compound
Specific binding (inhibition, %)
DMSO (control) TET (160 mM) FAN (160 mM) Magnoline (40 mM)
5319 9355 3479 9261 4255 9426 1686 9184
(0.0) (34.6) (20.0) (68.3)
a The rabbit PRP was incubated with [3H]PAF (final concentration, 0.9 nM) for 30 min. The values are expressed as mean 9S.E.M (n =3).
3.3. Effects of TET and FAN on the formation of TXB2
Fig. 2. Effects of TET and FAN on human platelet aggregation induced by PAF. The concentration of PAF was 0.2 mg/ml. The examinations were repeated three times using different PRP samples obtained from three subjects, and the values are expressed as mean 9 S.E.M.
The amount of TXB2 in resting WPS was 1.79 0.5 ng/3 × 108 platelets. After incubation of WPS with thrombin, AA and PAF for 6 min, the amount of TXB2 was increased to 386.8 98.2,
enzyme immunoassay. The concentrations which exhibited maximal aggregation in the control were chosen in preliminary experiments. 3. Results
3.1. Effects of TET and FAN on PAF-induced platelet aggregation TET and FAN inhibited PAF-induced human platelet aggregation concentration dependently (Fig. 2). IC50 values of TET and FAN were 28.6 and 21.7 mM, respectively.
3.2. Effects of TET and FAN on PAF-receptor binding As shown in Table 1, TET and FAN showed only 34.6 and 20.0% of inhibitions on PAF receptor binding assay at the concentration of 160 mM, respectively. On the contrast, the positive reference agent, magnoline, inhibited PAF-receptor binding by 68.3% at a concentration of 40 mM.
Fig. 3. Effects of TET (left) and FAN (right) on the TXB2 formation induced by PAF, AA and thrombin in human washed platelets. DMSO (0.1%, Control), TET or FAN was preincubated with WPS at 37°C for 2 min. After incubation of WPS with PAF (A, 0.2 mg/ml), AA (B, 2.1 mM) and thrombin (C, 1 U/ml) for 6 min, EDTA (2 mM) and indomethacin (50 mM) were added to halt the formation of TXB2. The values are expressed as mean 9S.E.M. (n =4). **PB0.01 compared with control. Con., control.
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451.1936.3 and 137.3 96.0 ng/3 ×108 platelets, respectively (Fig. 3). As shown in Fig. 3, TET and FAN inhibited thrombin (1 U/ml)-induced TXB2 formation by 35.2 and 50.2% at a concentration of 1.5 mM, and by 76.6 and 81.4% at a concentration of 15 mM, respectively. TET also inhibited the AA-induced TXB2 formation by 55.4 and 85.6% at concentrations of 33.0 and 50.0 mM, respectively (Fig. 3). On the other hand, FAN did not inhibit the AA-induced TXB2 formation at a concentration of 33.0 mM but, at 50.0 and 100 mM, FAN inhibited the AA-induced TXB2 formation by 61.3 and 69.2%, respectively. At a concentration of 33.0 mM, TET and FAN inhibited PAF-induced TXB2 formation by 47.5 and 44.3%, respectively.
4. Discussion In the present study, TET and FAN inhibited PAF-induced human platelet aggregation, but did not inhibit the PAF-receptor-specific bindings. Stimulants used in this experiment, such as PAF and thrombin, bind to specific cell surface receptors which result in transmembrane signalling via the phosphatidylinositol second-messenger system (Westwick et al., 1985), whereas AA acts directly on membrane cyclo-oxygenase enzyme pathways (Westwick et al., 1985). The results of receptor binding assays suggest that TET and FAN inhibit the platelet aggregation by interfering with the intracellular messengers system, but not by inhibiting the binding of PAF to PAF-receptor on the platelet membrane directly. Platelet aggregation is known to be a result of complex signal transduction cascade reactions brought about by stimulants. One of the components in the cascade is TXA2 (Hornby, 1984). The formation of TXA2 is stimulated by some aggregation inducers, such as PAF, thrombin, AA, etc. (Yu et al., 1996). TXA2 is a potent potentiator of platelet aggregation, which affects other platelets and causes them to change shape, extend pseudopods, and adhere to the platelets on the damaged surface, and makes a major contribution to the formation of some thrombi (Fuster et al., 1993). TXB2 is a stable metabolite of TXA2 (Yu
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et al., 1996). Therefore, the production of TXA2 was examined by measuring the formation of. TXB2. In this study, TET and FAN showed inhibitions of TXB2 formation induced by PAF, thrombin and AA in human WPS. These results suggest that the suppression of TXB2 formation by TET and FAN may be responsible for their inhibitory activities on platelet aggregation and, furthermore, on thrombosis. In conclusion, TET and FAN are anti-apoplexy activity components of fenfangji and their precise mechanisms need further study.
Acknowledgements This study was supported by a research grant from the Korean Ministry of Education (1996)– 1998).
References Born, G.V.R., 1962. Aggregation of blood platelets by adenosine diphosphated and its reversal. Nature 194, 927 – 929. Fuster, V., Dyken, M.L., Vokonas, P.S., Hennekens, C., 1993. Aspirin as a therapeutic agent in cardiovascular disease. Circulation 87 (2), 659 – 675. Kim, H.S., Zhang, Y.H., Yun, Y.P., 1999. Effects of tetrandrine and fangchinoline on the experimental thrombosis in mice and human platelet aggregation. Planta Medica (in press). Hornby, E.J., 1984. Evidence that prostaglandin endoperoxides can induce platelet aggregation in the absence of thromboxane A2 production. Biochemistry and Pharmacology 31, 1158 – 1160. Jiangsu College of New Medicine, 1975. Encyclopedia of Materia Medica, vol. 1. Shanghai People’s Publishing House, Shanghai, China, 268 pp. Jung, K.Y., Kim, D.S., Oh, S.R., Lee, I.S., Lee, J.J., Park, J.D., Kim, S.I., Lee, H.K., 1998. Platelet activating factor antagonist activity of ginsenosides. Biological and Pharmaceutical Bulletin 21 (1), 79 – 80. Lin, L.Z., Shieh, H.L., Angerhofer, C.K., Pezzuto, J.M., Cordell, G.A., Xue, L., Johnson, M.E., Ruangrungsi, N., 1993. Cytotoxic and antimalarial bisbenzylisoquinoline alkaloids from cyclea barbata. Journal of Natural Products 56, 22 – 29. Teng, C.M., Ko, F.N., 1988. Comparison of the platelet aggregation induced by three thrombin-like enzymes of snake venoms and thrombin. Thrombosis and Haemostasis 59, 304 – 309.
246
H.-S. Kim et al. / Journal of Ethnopharmacology 66 (1999) 241–246 pp.), vol. I. Torii & Co, Ltd, Nihonbashi, Tokyo, Japan. Yu, S.M., Tsai, S.Y., Kuo, S.C., Ou, J.T., 1996. Inhibition of platelet function by A02131-1, a novel inhibitor of cGMPspecific phosphodiesterase, in vitro and in vivo. Blood 87 (9), 3758 – 3767.
Westwick, J., Scully, M.F., MacIntyre, D.E., Kakkar, V.V., 1985. Mechanisms of stimulus-response coupling in platelets. Advances in Experimental Medicine and Biology 192, 1 – 44. Yamaguchi, K., 1970. Spectra Data of Natural Products (559
.