360
Biochimica et Biophysica Acta, 1052(1990) 360-365
Elsevier BBAMCR 12691
Antiplatelet effects of chelerythrine chloride isolated from Zanthoxylum simulans Feng-Nien Ko 1, Ih-Sheng Chen 2, Shwu-Jen Wu 2, Lih-Gen Lee 1, Tur-Fu Haung and Che-Ming Teng 1 l Pharmacological Institute, College of Medicine, National Taiwan University, Taipei and 2 Graduate Institute of Pharmaceutical Sciences, Kaohsiung Medical College, Kaohsiung (Taiwan, China)
Key words: Chelerythrinechloride; Thromboxaneformation; Phosphoinositidebreakdown; (Z. simulans)
Chelerythrine chloride is an antiplatelet agent isolated from Zanthoxylum simulans. Aggregation and ATP release of washed rabbit platelets caused by ADP, aracbidonic acid, PAF, collagen, ionophore A23187 and thrombin were inhibited by chelerythrine chloride. Less inhibition was observed in platelet-rich plasmL The thromboxane B 2 formation of washed platelets caused by aracbidonic acid, collagen, ionophore A23187 and thrombin was decreased by chelerythrine chloride. Phosphoinositides breakdown caused by collagen and PAF was completely inhibited by chelerytiwine chloride, while that of thrombin was only partially suppressed. Chelerythrine chloride inhibited the intracellular calcium increase caused by arachidonic acid, PAF, collagen and thrombin in quin-2/AM-ioaded platelets. The cyclic AMP level of washed platelets did not elevated by chelerythrine chloride. The antiplatelet effect of chelerythrine chloride was not dependent on the incubation time and the aggregability of platelets inhibited by cholerytlwine chloride was easily recovered after sedimenfing the ldatelets by centrifugation and then the platelet pellets were resmpended. ~ n e chloride did not cause any platelet lysis, since lactate dehydrogenase activity was not found in the supernatant. These data indicate that the inlu'bitory effect of chelerythrine chloride on rabbit platelet aggregation and release reaction is due to the inhibition on tlwomboxane formation and phosphoinositides breakdown
Introduction Several factors are known to be responsible for thrombogenesis. It is now well recognized that plateletvessel wall interactions are important in the development of thrombosis and atherosclerosis [1,2]. Thus, inhibition of platelet function may be a promising approach for the prevention of thrombosis. Although. many agents have been reported to have in vitro antiplatelet effects [3-5], only few of them are clinically useful in antithrombotic therapy. So, it is very important in searching for new antiplatelet drugs for thrombotic therapy. In the screening test of the antiplatelet activity of Chinese herbs, we found aromatized quaternary benzo[c]phenanthridine alkaloids, occurring naturally in Rutaceous and P a p a v e r a c e o u s plants, especially chelerythrine chloride which isolated from Z a n t h o x y l u m
Abbreviations: PGE1, prostaglandin El; PAF, platelet-activatingfactor. Correspondence: C.M. Teng, Pharmacological Institute, College of
Medicine, National Taiwan University, No. 1, Jen-Ai Rd. Section 1, Taipei, Taiwan.
simulans possessed the most potent antiplatelet activity.
In this paper, we tried to elucidate the inhibitory mechanism of chelerythrine chloride on platelet aggregation.
Materials and Methods Materials
Chelerythrine chloride was a 2,3,7,8-oxygenated benzo[c]phenanthridine alkaloid isolated from Zanthoxylum simulans. T h e barks were refluxed with methanol and then concentrated under reduced pressure. The residue was acidified with 5% acetic acid for extraction of the base. The acidic solution was made alkaline with N H 4 O H solution, and extracted with chloroform. The chloroform solution was shaken with 5% N a O H solution, then dried with anhydrous KECO 3 and concentrated. Then, 10% HC1 was added to yield yellowish precipitate. This yellowish precipitate was recrystallized repeatedly with e t h a n o l / m e t h a n o l to obtain yellowish needles which were identified to be chelerythrine chloride (Fig. 1) [29] by IR and TLC. Thrombin (bovine) was purchased from Park Davis Co. and dissolved in 50% ( v / v ) glycerol to give a stock solution of 100 N I H units/ml. Collagen (type 1, bovine achilles tendon), obtained from Sigma Chemicals Co. was homogenized
0167-4889/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (Biomedical Division)
361 oc~ CI-
Fig. 1. Structure of chelerythrine chloride.
in 25 mM acetic acid and stored (1 mg/ml) at -70°C. Ionophore A23187 was obtained from Calbiochem-Behring Co. and dissolved in dimethyl sulfoxide (DMSO). Platelet-activating factor (PAF) was purchased from Sigma Chemicals Co. and dissolved in chloroform. Arachidonic acid, ADP, bovine serum albumin (BSA), indomethacin, EDTA (disodium salt), sodium citrate, luciferase-luciferin, Dowex-1 (100-200 mesh: ×8, chloride) resin, myo-inositol, and quin-2/AM were purchased from Sigma Chemical Co. Myo-[2-3H]in ositol and cyclic AMP RIA kits were purchased from Amersham, U.K. Thromboxane B2 RIA kits were obtained from New England Nuclear Co.
Platelet aggregation and A TP release reaction Platelet-rich plasma (PRP) was obtained from blood collected from the rabbit marginal vein, anticoagulated with sodium citrate (3.8%, 1 : 14) and centrifuged for 10 min at 90 × g and room temperature. Platelet suspension was obtained from EDTA-anticoagulated PRP according to the washing procedures described previously [6]. Platelet numbers were counted by Coulter Counter (Model ZM) and adjusted to 4.5.108 platelets/ml. The platelet pellets were finally suspended in Tyrode's solution of the following composition (mM): NaC1 (136.8), KC1 (2.8), NaHCO 3 (11.9), MgC12 (2.1), NaH2PO 4 (0.33), CaC12 (1.0) and glucose (11.2) containing bovine serum albumin (0.35%). Aggregation was measured by the turbidimetry method as described by O'Brien [7]. ATP released from platelets was detected by the bioluminescence method as described by DeLuca and McElory [8]. Both the aggregation and release of ATP were simultaneously measured by Lumi-aggregometer (Chrono-Log Co. U.S.A.) connected to two dual channel recorders. The platelet preparations were stirred at 1200 rpm. In order to eliminate the effect of the solvent on the aggregation, the final concentration of DMSO was fixed at 0.5%.
Thromboxane B 2 assay Thromboxane B2 was assayed using RIA kits according to the procedure described by the manufacturer. EDTA (2 mM) and indomethacin (50 #M) were added to stop the further formation of thromboxanes at 6 min after the addition of the inducers.
Labelling of membrane phospholipids and measurement of the production of [3H]inositol phosphate The method was modified from those of Huang and Detwiler's [9] and Neylon and Summers' [10]. EDTAPRP was centrifuged at 500 × g for 10 min, the platelet pellets were suspended in 700 /~1 of CaZ+-free and BSA-free Tyrode's solution containing 75 # C i / m l of myo-[2-3H]inositol and 1 mM EDTA. After incubation for 2 h at 37°C, the platelets were collected by centrifugation (500 × g for 4 min) and suspended in Ca z +-free Tyrode's solution. Phosphoinositides breakdown was carried out by adding aggregation inducers to 1 ml of platelet suspension in a 3.5 ml cuvette with a stirring bar driven at 900 rpm, 37°C for 6 min. An equal volume of 10% (w/v) trichloroacetic acid was added to stop the reaction. After centrifuge at 1000 × g for 10 min, 1 ml of supernatant was pooled and trichloroacetic acid was removed by washing with 5 × 2 vol. of diethyl ether. The aqueous phase containing the inositol phosphates was adjusted to pH 7-8 and diluted to 4 ml with distilled water before application to a Dowex-1 ion-exchange column for separation of the inositol phosphates as described previously by Neylon and Summers [10]. All the experiments were carried out in the presence of 5 mM LiC1 to inhibit inositol-monophosphate phosphatase. Because the levels of inositol bisphosphate and inositol trisphosphate were very low, we measured the inositol monophosphate as an index of the total inositolphosphates formation.
Estimation of platelet cyclic A M P The method of Karniguian et al. [11] was followed. Platelet suspension was warmed at 37°C for 1 min, then PGE 1 or chelerythrine chloride was added and incubated for 45 s. The incubation was stopped by adding 10 mM EDTA and immediate boiling for 5 rain. After cooling to 4°C, the precipitated protein was sedimented by centrifugation in an Eppendorf microcentrifuge (Model 5414). The supernatant (400/~1) was freeze-dried and the residue was dissolved in 100 #1 distilled water. 50 #1 of supernatant was used to determine the cyclic AMP content by radioimmunoassay kits as described by the manufacturer.
Measurement of intracellular calcium in platelets According to the method described by Rink et al. [12], EDTA-anticoagulated PRP was incubated with quin-2/AM (20/~M) for 40 min and then washed and suspended in the above Tyrode's solution. Fluorescence was measured with a Hitachi Fluorescence Spectrophotometer (Ex. 339 nm, Em. 492 nm).
Determination of cytolysis Cell lysis was judged by the presence of lactate dehydrogenase (LDH) activity in the supernatant of
362 i00
platelet suspension after centrifugation according to the method described by Wroblewski and LaDue [13].
-
90O • "~
Results
8070
-
60-
Effects of chelerythrine chloride on the aggregation, A TP release and thromboxane formation of platelets Chelerythrine chloride (10/,g/ml) inhibited the aggregation of washed rabbit platelets induced by arachidonic acid (100 /~M) and PAF (2 ng/ml) completely, and also those by ADP (20 /~M), collagen (10 /~g/ml) and ionophore A23187 (5 /~M) markedly. Although the aggregation caused by thrombin (0.1 U/ml) was more resistant to chelerythrine chloride, it was dose-dependently inhibited and was completely suppressed by chelerythrine chloride at concentration as high as 20 /,g/ml (Fig. 2). The release of ATP from platelets challenged by these inducers was also inhibited by chelerythrine chloride and this inhibition was more marked than that of platelet aggregation (Fig. 3). Incubation of chelerythrine chloride with platelets for 60 min did not cause a more pronounced inhibition than that for 3 min (data not shown). The inhibitory effect of chelerythrine chloride on platelet aggregation could be washed out easily and the aggregability of platelets was restored (Fig. 4). The inhibitory effects of chelerythrine
bJ0 gJ 5 0 -
b.,O 4-O-
Biochimica et Biophysica Acta, 1052(1990) 360-365
Elsevier BBAMCR 12691
Antiplatelet effects of chelerythrine chloride isolated from Zanthoxylum simulans Feng-Nien Ko 1, Ih-Sheng Chen 2, Shwu-Jen Wu 2, Lih-Gen Lee 1, Tur-Fu Haung and Che-Ming Teng 1 l Pharmacological Institute, College of Medicine, National Taiwan University, Taipei and 2 Graduate Institute of Pharmaceutical Sciences, Kaohsiung Medical College, Kaohsiung (Taiwan, China)
Key words: Chelerythrinechloride; Thromboxaneformation; Phosphoinositidebreakdown; (Z. simulans)
Chelerythrine chloride is an antiplatelet agent isolated from Zanthoxylum simulans. Aggregation and ATP release of washed rabbit platelets caused by ADP, aracbidonic acid, PAF, collagen, ionophore A23187 and thrombin were inhibited by chelerythrine chloride. Less inhibition was observed in platelet-rich plasmL The thromboxane B 2 formation of washed platelets caused by aracbidonic acid, collagen, ionophore A23187 and thrombin was decreased by chelerythrine chloride. Phosphoinositides breakdown caused by collagen and PAF was completely inhibited by chelerytiwine chloride, while that of thrombin was only partially suppressed. Chelerythrine chloride inhibited the intracellular calcium increase caused by arachidonic acid, PAF, collagen and thrombin in quin-2/AM-ioaded platelets. The cyclic AMP level of washed platelets did not elevated by chelerythrine chloride. The antiplatelet effect of chelerythrine chloride was not dependent on the incubation time and the aggregability of platelets inhibited by cholerytlwine chloride was easily recovered after sedimenfing the ldatelets by centrifugation and then the platelet pellets were resmpended. ~ n e chloride did not cause any platelet lysis, since lactate dehydrogenase activity was not found in the supernatant. These data indicate that the inlu'bitory effect of chelerythrine chloride on rabbit platelet aggregation and release reaction is due to the inhibition on tlwomboxane formation and phosphoinositides breakdown
Introduction Several factors are known to be responsible for thrombogenesis. It is now well recognized that plateletvessel wall interactions are important in the development of thrombosis and atherosclerosis [1,2]. Thus, inhibition of platelet function may be a promising approach for the prevention of thrombosis. Although. many agents have been reported to have in vitro antiplatelet effects [3-5], only few of them are clinically useful in antithrombotic therapy. So, it is very important in searching for new antiplatelet drugs for thrombotic therapy. In the screening test of the antiplatelet activity of Chinese herbs, we found aromatized quaternary benzo[c]phenanthridine alkaloids, occurring naturally in Rutaceous and P a p a v e r a c e o u s plants, especially chelerythrine chloride which isolated from Z a n t h o x y l u m
Abbreviations: PGE1, prostaglandin El; PAF, platelet-activatingfactor. Correspondence: C.M. Teng, Pharmacological Institute, College of
Medicine, National Taiwan University, No. 1, Jen-Ai Rd. Section 1, Taipei, Taiwan.
simulans possessed the most potent antiplatelet activity.
In this paper, we tried to elucidate the inhibitory mechanism of chelerythrine chloride on platelet aggregation.
Materials and Methods Materials
Chelerythrine chloride was a 2,3,7,8-oxygenated benzo[c]phenanthridine alkaloid isolated from Zanthoxylum simulans. T h e barks were refluxed with methanol and then concentrated under reduced pressure. The residue was acidified with 5% acetic acid for extraction of the base. The acidic solution was made alkaline with N H 4 O H solution, and extracted with chloroform. The chloroform solution was shaken with 5% N a O H solution, then dried with anhydrous KECO 3 and concentrated. Then, 10% HC1 was added to yield yellowish precipitate. This yellowish precipitate was recrystallized repeatedly with e t h a n o l / m e t h a n o l to obtain yellowish needles which were identified to be chelerythrine chloride (Fig. 1) [29] by IR and TLC. Thrombin (bovine) was purchased from Park Davis Co. and dissolved in 50% ( v / v ) glycerol to give a stock solution of 100 N I H units/ml. Collagen (type 1, bovine achilles tendon), obtained from Sigma Chemicals Co. was homogenized
0167-4889/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (Biomedical Division)
361 oc~ CI-
Fig. 1. Structure of chelerythrine chloride.
in 25 mM acetic acid and stored (1 mg/ml) at -70°C. Ionophore A23187 was obtained from Calbiochem-Behring Co. and dissolved in dimethyl sulfoxide (DMSO). Platelet-activating factor (PAF) was purchased from Sigma Chemicals Co. and dissolved in chloroform. Arachidonic acid, ADP, bovine serum albumin (BSA), indomethacin, EDTA (disodium salt), sodium citrate, luciferase-luciferin, Dowex-1 (100-200 mesh: ×8, chloride) resin, myo-inositol, and quin-2/AM were purchased from Sigma Chemical Co. Myo-[2-3H]in ositol and cyclic AMP RIA kits were purchased from Amersham, U.K. Thromboxane B2 RIA kits were obtained from New England Nuclear Co.
Platelet aggregation and A TP release reaction Platelet-rich plasma (PRP) was obtained from blood collected from the rabbit marginal vein, anticoagulated with sodium citrate (3.8%, 1 : 14) and centrifuged for 10 min at 90 × g and room temperature. Platelet suspension was obtained from EDTA-anticoagulated PRP according to the washing procedures described previously [6]. Platelet numbers were counted by Coulter Counter (Model ZM) and adjusted to 4.5.108 platelets/ml. The platelet pellets were finally suspended in Tyrode's solution of the following composition (mM): NaC1 (136.8), KC1 (2.8), NaHCO 3 (11.9), MgC12 (2.1), NaH2PO 4 (0.33), CaC12 (1.0) and glucose (11.2) containing bovine serum albumin (0.35%). Aggregation was measured by the turbidimetry method as described by O'Brien [7]. ATP released from platelets was detected by the bioluminescence method as described by DeLuca and McElory [8]. Both the aggregation and release of ATP were simultaneously measured by Lumi-aggregometer (Chrono-Log Co. U.S.A.) connected to two dual channel recorders. The platelet preparations were stirred at 1200 rpm. In order to eliminate the effect of the solvent on the aggregation, the final concentration of DMSO was fixed at 0.5%.
Thromboxane B 2 assay Thromboxane B2 was assayed using RIA kits according to the procedure described by the manufacturer. EDTA (2 mM) and indomethacin (50 #M) were added to stop the further formation of thromboxanes at 6 min after the addition of the inducers.
Labelling of membrane phospholipids and measurement of the production of [3H]inositol phosphate The method was modified from those of Huang and Detwiler's [9] and Neylon and Summers' [10]. EDTAPRP was centrifuged at 500 × g for 10 min, the platelet pellets were suspended in 700 /~1 of CaZ+-free and BSA-free Tyrode's solution containing 75 # C i / m l of myo-[2-3H]inositol and 1 mM EDTA. After incubation for 2 h at 37°C, the platelets were collected by centrifugation (500 × g for 4 min) and suspended in Ca z +-free Tyrode's solution. Phosphoinositides breakdown was carried out by adding aggregation inducers to 1 ml of platelet suspension in a 3.5 ml cuvette with a stirring bar driven at 900 rpm, 37°C for 6 min. An equal volume of 10% (w/v) trichloroacetic acid was added to stop the reaction. After centrifuge at 1000 × g for 10 min, 1 ml of supernatant was pooled and trichloroacetic acid was removed by washing with 5 × 2 vol. of diethyl ether. The aqueous phase containing the inositol phosphates was adjusted to pH 7-8 and diluted to 4 ml with distilled water before application to a Dowex-1 ion-exchange column for separation of the inositol phosphates as described previously by Neylon and Summers [10]. All the experiments were carried out in the presence of 5 mM LiC1 to inhibit inositol-monophosphate phosphatase. Because the levels of inositol bisphosphate and inositol trisphosphate were very low, we measured the inositol monophosphate as an index of the total inositolphosphates formation.
Estimation of platelet cyclic A M P The method of Karniguian et al. [11] was followed. Platelet suspension was warmed at 37°C for 1 min, then PGE 1 or chelerythrine chloride was added and incubated for 45 s. The incubation was stopped by adding 10 mM EDTA and immediate boiling for 5 rain. After cooling to 4°C, the precipitated protein was sedimented by centrifugation in an Eppendorf microcentrifuge (Model 5414). The supernatant (400/~1) was freeze-dried and the residue was dissolved in 100 #1 distilled water. 50 #1 of supernatant was used to determine the cyclic AMP content by radioimmunoassay kits as described by the manufacturer.
Measurement of intracellular calcium in platelets According to the method described by Rink et al. [12], EDTA-anticoagulated PRP was incubated with quin-2/AM (20/~M) for 40 min and then washed and suspended in the above Tyrode's solution. Fluorescence was measured with a Hitachi Fluorescence Spectrophotometer (Ex. 339 nm, Em. 492 nm).
Determination of cytolysis Cell lysis was judged by the presence of lactate dehydrogenase (LDH) activity in the supernatant of
362 i00
platelet suspension after centrifugation according to the method described by Wroblewski and LaDue [13].
-
90O • "~
Results
8070
-
60-
Effects of chelerythrine chloride on the aggregation, A TP release and thromboxane formation of platelets Chelerythrine chloride (10/,g/ml) inhibited the aggregation of washed rabbit platelets induced by arachidonic acid (100 /~M) and PAF (2 ng/ml) completely, and also those by ADP (20 /~M), collagen (10 /~g/ml) and ionophore A23187 (5 /~M) markedly. Although the aggregation caused by thrombin (0.1 U/ml) was more resistant to chelerythrine chloride, it was dose-dependently inhibited and was completely suppressed by chelerythrine chloride at concentration as high as 20 /,g/ml (Fig. 2). The release of ATP from platelets challenged by these inducers was also inhibited by chelerythrine chloride and this inhibition was more marked than that of platelet aggregation (Fig. 3). Incubation of chelerythrine chloride with platelets for 60 min did not cause a more pronounced inhibition than that for 3 min (data not shown). The inhibitory effect of chelerythrine chloride on platelet aggregation could be washed out easily and the aggregability of platelets was restored (Fig. 4). The inhibitory effects of chelerythrine
bJ0 gJ 5 0 -
b.,O 4-O-
