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Inhibiting and disaggregating effect of gel-filtered Galega officinalis L. herbal extract on platelet aggregation
Journal of Ethnopharmacology 69 (2000) 235 – 240 www.elsevier.com/locate/jethpharm
Inhibiting and disaggregating effect of gel-filtered Galega officinalis L. herbal extract on platelet aggregation Atanas Todorov Atanasov a,*, Vasil Spasov b b
a Department of Biophysics, Faculty of Medicine, Thracian Uni6ersity, 11 Armeiska Str., 6000 Stara Zagora, Bulgaria Department of Pharmacology, Faculty of Medicine, Thracian Uni6ersity, 11 Armeiska Str., 6000 Stara Zagora, Bulgaria
Received 23 September 1998; received in revised form 31 May 1999; accepted 9 August 1999
Abstract The in vitro inhibiting and disaggregating effect on platelet aggregation of a gel-fractionated herbal extract from Galega officinalis L. is examined. The obtained Sephadex G-25 filtered fraction was 35 – 36 times more active than the crude extract. The threshold concentration at which this fraction inhibits platelet aggregation (5 – 10% inhibition) by 50 mM adenosine 5%-diphosphate (ADP) is 4.5–5 mg per 1 ml platelet-rich plasma (PRP). At a concentration of 35 mg/ml PRP the fraction inhibits 50% of aggregation by ADP and at a concentration of 125 mg/ml PRP fully inhibits the aggregation of PRP by ADP. At a concentration of 40 mg/ml PRP the fraction inhibits initiation of platelet aggregation by 0.18 mg/ml collagen and at 50 mg/ml PRP inhibits the initiation of aggregation by 0.7 units/ml thrombin. The G-25 filtered fraction shows a strong disaggregating effect on aggregated PRP. At a concentration of 65–75 mg/ml PRP, the fraction is able to disaggregate the 50 – 53% of aggregated platelet-rich plasma by 50 mM ADP, and 25% of aggregated PRP by 0.18 mg/ml collagen. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Galega officinalis L.; Extract; Inhibition; Platelet aggregation
1. Introduction Galega officinalis L. is a plant used in the traditional medicinal system of Bulgaria, Italy and India (Chopra et al., 1956; Petkov, 1982) in the treatment of diabetes mellitus. A biologically active alkaloid galegine, exhibiting a hypoglycemic effect in vivo was isolated from G. officinalis (Reuter, 1963; Hoppe, 1975). In healthy normoglicemic volunteers galegine at a dose of 2 – 4 mg/kg body mass leads to reduction of blood * Corresponding author.
glucose, which begins after 3–4 h and continues for about 9 h. A hypoglycemic effect is observed in patients with diabetes mellitus (Benigni et al., 1964). The plant extract appears to have an anticoagulation effect (Erspamer, 1943; Petkov, 1982). Recent experimental results (Atanasov, 1993, 1994) show that the 2.25% crude aqueous extracts of this plant at a dose of 1.0–2.5 mg/ml platelet-rich plasma (PRP) suppress in vitro platelet aggregation induced by aggregating agents such as adenosine 5%-diphosphate (ADP), epinephrine, thrombin and collagen. The IC50 (50% inhibition effect) for aggregation by 50 mM
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 4 4 - 0
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A.T. Atanaso6, V. Spaso6 / Journal of Ethnopharmacology 69 (2000) 235–240
ADP is 1.25 mg crude extract in 1 ml PRP. The crude extracts of G. officinalis L. suppress in vivo platelet aggregation in rats for 2.5 – 3.0 h, after injection in the caudal vein of rats in a ratio of 1 part 2.5% extract per 40 parts blood (Atanasov, 1995a). The aqueous extract of G. officinalis L. has an synergetic effect on platelet aggregation in common with anti-aggregant agents such as: aspirin, dipyridamol, ticlopidine, sulfinpyrazone, indobufenum, digoxin, theophyline and other drugs with similar action (Atanasov, 1995b). Up to 1990, the biologically-active compounds with antiaggregating action have not been isolated from G. officinalis L. In the present study the effect on platelet aggregation of a Sephadex G-25 filtered fraction, obtained from crude extracts of G. officinalis L. was investigated.
water—NH3 composition of pH 7.5. The column was eluted with the same composition at 16°C. The flow-rate was 720 ml/h and the fraction from 400 to 550 ml was collected. The fraction was concentrated to (21.59 0.5) mg/ml dry matter and the effect on platelet aggregation was studied.
2.4. Isolation of human platelets Blood was taken from volunteers who had received no medication for 15 days prior to blood collection. Blood was collected in disposable syringes at a ratio of 1 part 3.8% trisodium citrate and 9 parts venous blood (Zucker, 1989). Plateletrich plasma was prepared by centrifugation (180× g for 10 min) and diluted to 300× 106 platelets per ml with autologous platelet-poor plasma (1800×g for 15 min).
2. Materials and methods
2.5. Platelet aggregation 2.1. Plant material The aerial parts of G. officinalis L. (Herba Galegae) were collected at the flowering stage between May and August 1997 in different parts of Thrace, Bulgaria. The plant was verified by Professor Ivan Assenov, and a voucher specimen (12186) has been deposited in the Herbarium of Department of Botany, Faculty of Pharmacy (University of Sofia, Bulgaria).
2.2. Preparation of extract The crude aqueous extract was obtained by maceration of 150 g dry matter in 2000 ml distilled water for 20– 24 h at 18 – 20°C. The fresh extract was adjusted to pH 8.0 with NH3 and centrifuged at 150× g after 1 h. The supernatant was filtered and concentrated at a temperature below 35°C to an extract with dry matter of about 165 – 170 mg/ml.
2.3. Gel-filtration of extract through Sephadex G-25. The concentrated extract (10 ml) was applied on a column (600 mm× 55 mm) equilibrated with
Aggregation was studied with a spectrophotometer set to operate at a wavelength of 600 nm and the results were recorded on an electronic recorder (XY-Recorder en dim. 620.02, VEB, Germany; Zucker, 1989). The extinction change that takes place during the aggregation of 400 ml PRP compared with platelet-poor plasma (whose extinction was taken as zero) after adding the aggregating agent (20 ml ADP and 40 ml collagen or thrombin) at 37°C and at a rate of stirring of 1000 rpm, was the basis of measurement for aggregating effect. The effect of the G-25 filtered fraction from G. officinalis was studied after adding 0.5– 10 ml fraction to 400 ml PRP with stirring. The aggregating agents were added after 10 min and the aggregation was monitored for a further 20 min. Aggregation in % was calculated as: (E0 − E)× 100%/(E0 − Ef), where E0 is the initial value of the extinction of PRP before the aggregating process; Ef is the final value of the extinction of PRP after finishing the aggregating process; and E is the final value of the extinction of PRP (with adding of desalted fraction or drugs) after finishing the aggregating process.
A.T. Atanaso6, V. Spaso6 / Journal of Ethnopharmacology 69 (2000) 235–240
2.6. Reagents ADP at a concentration of 1×10 − 3 M from Reanal (Hungary); human thrombin (8 units/ml) and bovine collagen (2 mg/ml) from the Research Institute of Haematology and Blood Transfusion (Sofia, Bulgaria) were used as aggregating agents.
2.7. Drugs Dipyridamole (5 mg/ml) and theophyline (10 mg/ml) from Pharmachim (Bulgaria) were used as a basis for the comparison of the effect of the column fraction of Herba Galegae.
2.8. Statistical analysis All points in the graphics are expressed as mean 9SD.
3. Results The anti-aggregating activity of the G-25 filtered fraction from G. officinalis L. was verified with ADP, collagen and thrombin. The inhibiting
237
effect of fraction on platelet aggregation initiated by 50 mM ADP is presented in Fig. 1. The threshold concentration at which the G-25 filtered fraction starts to inhibit platelet aggregation (5– 10% inhibition) by 50 mM ADP is 4.5–5.0 mg per 1 ml PRP. At a concentration of 19–20 mg/ml PRP, the fraction inhibits platelet aggregation at the same degree as dipyridamole at a dose of 25 mg/ml PRP. At a concentration of 35 mg/ml PRP, the fraction inhibits 50% of platelet aggregation (IC50). At a concentration over 125 ml/ml PRP, the 100% common (inhibition and disaggregation) effect is shown. The disaggregating effect of the G-25 filtered fraction is shown in Fig. 2. The addition of aggregating agent (50 mM ADP) to the initial PRP leads to aggregation and the decrease of free platelets in plasma from 100% (300× 106 platelets/ml) to 3–4%, and the number of associated in aggregates platelets rises to 96– 97%. A slight disaggregating effect is shown, after adding on 96–97% aggregated platelets of 25–60 mg/ml PRP G-25 filtered fraction. For example, the 25-mg/ml G-25 fraction disaggregates 96–97% aggregated platelets to 29–31% free platelets and 69–71% associated in aggregates platelets. The
Fig. 1. In vitro inhibiting effect of 35, 50, 125, 250 and 500 mg ‘G-25 filtered fraction’ of G. officinalis L. on aggregation of 1 ml platelet-rich plasma (300 ×106 platelets/ml), by 50 mM ADP (final concentration). The effect of 25 mg dipyridamol (dip) in 1 ml PRP is used for comparison. Adding of fraction or drug on platelet suspension is marked by a triangle. Values are means 9 SD for six independent experiments.
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Fig. 2. In vitro disaggregating effect of 25, 65, 125, 250 and 500 mg ‘G-25 filtered fraction’ of G. officinalis L. on aggregated platelet-rich plasma (300 × 106 platelets/ml) by 50 mM ADP (final concentration). The disaggregating effect of 500 mg theophyline (theoph) in 1 ml PRP is used for comparison. Adding of fraction or drug on platelet suspension is marked by a triangle. Values are means9SD for six independent experiments.
concentration of fraction 65 – 75 mg/ml PRP leads to 50–53% free and 50 – 47% associated in aggregates platelets (IC50). A strong disaggregating effect is shown, after adding on 96–97% aggregated platelets to 100 – 500 mg/ml PRP G-25 filtered fraction. For example, the 125-mg/ml PRP fraction leads to 68 – 71% free platelets and 32 – 29% associated in aggregates. At a concentration of over 250 mg/ml PRP, the platelets aggregates is destroyed in fullness, and the number of free platelets approaches to 298 – 300×106. The G-25 filtered fraction inhibits platelet aggregation initiated by collagen (Fig. 3) and thrombin (Fig. 4). In this case the fraction inhibits the initiation of platelet aggregation or stops further platelet aggregation, after adding to aggregated PRP. The threshold concentration, at which the fraction starts to inhibit platelet aggregation (5 – 10% inhibition) by 0.18 mg/ml collagen and 0.7 units/ ml thrombin is 10 mg/ml PRP. At a concentration of 40 mg/ml PRP, the fraction inhibits fully initiation of platelet aggregation by 0.18 mg/ml collagen, and at 50 mg/ml PRP inhibits the initiation of aggregation by 0.7 units/ml thrombin. The in-
hibiting effect of 400 mg/ml PRP theophyline is equivalent to the effect of 33 mg/ml G-25 filtered fraction on aggregation by collagen, and equivalent to the effect of 24 mg/ml G-25 filtered fraction on aggregation by thrombin. On aggregated platelets by collagen the fraction has a slight disaggregating effect—about 25%— but on aggregated platelets by thrombin the fraction has no disaggregating effect.
4. Discussion and conclusions The obtained column fraction from aqueous extract of G. officinalis L. inhibits the platelet aggregation initiated by ADP, collagen and thrombin. On the other hand our investigations show that the same fraction inhibits the platelet aggregation initiated by epinephrine, aggregating agents consists of free radicals and spontaneous platelet aggregation. Possibly the fraction inhibits aggregation through a non-specific mechanism— for example, preventing fibrinogen bridges between platelets (Karczewski and Connolly, 1997). Such mechanism of action has ticlopidine (Bruno, 1983). The IC50 for crude extract on platelet ag-
A.T. Atanaso6, V. Spaso6 / Journal of Ethnopharmacology 69 (2000) 235–240
gregation by 50 mM ADP is 1.25 mg/ml PRP (Atanasov, 1994). For the G-25 filtered fraction the IC50 is 35 mg/ml PRP. This shows that this fraction is 35–36 times more active than the crude extract. The G-25 filtered fraction has an activity
239
near to that of dipyridamole (inhibiting effect of 19–20 mg/ml PRP fraction on platelet aggregation is the same as the effect of 25 mg/ml PRP phospodiesterase inhibitor dipyridamole—Fig. 1) and two times more active than phospodiesterase
Fig. 3. In vitro inhibiting effect of 20, 30 and 40 mg ‘G-25 filtered fraction’ of G. officinalis L. on aggregation of 1 ml PRP (300 ×106 platelets/ml) by 0.18 mg collagen (final concentration). The effect of 400 mg theophyline (theoph) in 1 ml PRP is used for comparison. Adding of fraction or drug on platelet suspension is marked by a triangle. Values are means 9SD for five independent experiments.
Fig. 4. In vitro inhibiting effect of 15, 30 and 50 mg ‘G-25 filtered fraction’ of G. officinalis L. on aggregation of 1 ml PRP by 0.7 units/ml thrombin (final concentration). The effect of 400 mg theophyline (theoph) in 1 ml PRP is used for comparison. Adding of fraction or drug on platelet suspension is marked by a triangle. Values are means 9SD for five independent experiments.
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inhibitor theophyline (Fig. 2). In cases of aggregation by collagen and thrombin, G-25 filtered fraction is 12–16-fold more active than the theophyline (Figs. 3 and 4). More ever, the G-25 filtered fraction clearly shows expressed disaggregating effects on platelet aggregation by ADP and collagen. This fact remains masked in an investigation of the effect of the whole crude extract. The crude extracts and the G-25 filtered fraction do not lysis and agglutinate erythrocytes or platelets, and do not precipitate the serum proteins. This shows that the biologically-active component in extract is not a lectin as can be expected. The G-25 filtered fraction correlates with high molecular mass (over 25 kDa), as it eluates with free volume of column. The effect on aggregation is temperature and pH dependent: over 55°C, below pH 4 and over pH 11 the fraction loses its activity. This shows that the biologically-active compounds in the extract are not any of the isolated G. officinalis L. alkaloids (Hoppe, 1975), which are temperature and pH stable, and with a molecular mass below 1 kDa. It is possible the biologically-active component has a carbohydrate nature because carbohydrates (sugars and amino sugars, dextrans, heparin proteoglycans) in most cases inhibit platelet aggregation (Kinlough-Rathbone et al., 1984; Lassila et al., 1997). The ability of ‘G-25 filtered fraction’ to inhibit platelet aggregation just confirms supposed anticoagulating effects of G. officinalis L. Experimental studies in this field, however are insufficient, but imperative because ‘diabetes mellitus’ leads to disorders of blood coagulation and platelet aggregation. The isolation of the biologically-active compound constituents of the fraction is the next step towards elucidation of the mechanism of platelet inhibition.
.
References Atanasov, A., 1993. In vitro effect of water extracts of some medicinal plants from Bulgarian flora on platelet aggregation. Bulgarian Medicine 1, 17 – 20. Atanasov, A., 1994. Effect of the water extract of Galega officinalis L. on human platelet aggregation in vitro. Phytotherapy Research 8 (5), 314 – 316. Atanasov, A., 1995a. An effect of Galega officinalis L. extract on platelet aggregation in rats. Journal of Herbs, Spices and Medicinal Plants 3 (3), 71 – 76. Atanasov, A., 1995b. In vitro effect on platelet aggregation of water extracts of Galega officinalis L. in combination with some drugs. Bulgarian Medicine 3 – 4 (3), 35 – 38. Benigni, R., Capra, C., Cattorini, P.E., 1964. Galega. In: Plante Medicinali, Chimica Farmacologia e Terapia. Inverni Della Betta, Milano, pp. 626 – 632. Bruno, J.J., 1983. The mechanism of action of ticlopidine. Thrombosis Research (Suppl. 4), 59 – 67. Chopra, R.H., Nagar, S.L., Chopra, I.P., 1956. Glossary of Indian Medicinal Plants. New Delphi, India, p. 20. Erspamer, V., 1943. Droghe e principi ipoglicemizzanti vegetali. In: Quaderni di Fitoterapia, in 5. Inverni Della Beffa, Milano, pp. 23 – 26. Hoppe, H.A., 1975. Drogenkunde 1, 8th ed. Walter de Gruyter, Berlin, p. 1311. Karczewski, J., Connolly, T.M., 1997. The interaction of disagregin with the platelet fibrinogen receptor, glycoprotein IIb – IIIa. Biochemical and Biophysical Research Communications 241 (3), 744 – 748. Kinlough-Rathbone, R.L., Packham, M.A., Mustard, J.F., 1984. Effect of amino sugars on platelet aggregation and on fibrinogen binding. Thrombosis and Haemostasis (Stuttgart) 52 (1), 75 – 80. Lassila, R., Lindstedt, K., Kovanen, P.T., 1997. Native macromolecular heparin proteoglycans exocytosed from stimulated rat serosal mast cells strongly inhibit platelet-collagen interactions. Arterioscler. Thromb. Vasc. Biol. 17 (12), 3578 – 3587. Petkov, V.D., 1982. Modern Phytotherapy Medizina i Fizkultura Publ. House, Sofia, p. 307 (In Bulgarian). Reuter, G., 1963. Arginin als Vorstufe von Galegin in Galega officinalis L., Archiv der Pharmazie, 296 Bd.; N8, pp. 516 – 522. Zucker, M.B., 1989. Platelet aggregation measured by the photometric method. Methods in Enzymology 169, 118 – 133.
Inhibiting and disaggregating effect of gel-filtered Galega officinalis L. herbal extract on platelet aggregation Atanas Todorov Atanasov a,*, Vasil Spasov b b
a Department of Biophysics, Faculty of Medicine, Thracian Uni6ersity, 11 Armeiska Str., 6000 Stara Zagora, Bulgaria Department of Pharmacology, Faculty of Medicine, Thracian Uni6ersity, 11 Armeiska Str., 6000 Stara Zagora, Bulgaria
Received 23 September 1998; received in revised form 31 May 1999; accepted 9 August 1999
Abstract The in vitro inhibiting and disaggregating effect on platelet aggregation of a gel-fractionated herbal extract from Galega officinalis L. is examined. The obtained Sephadex G-25 filtered fraction was 35 – 36 times more active than the crude extract. The threshold concentration at which this fraction inhibits platelet aggregation (5 – 10% inhibition) by 50 mM adenosine 5%-diphosphate (ADP) is 4.5–5 mg per 1 ml platelet-rich plasma (PRP). At a concentration of 35 mg/ml PRP the fraction inhibits 50% of aggregation by ADP and at a concentration of 125 mg/ml PRP fully inhibits the aggregation of PRP by ADP. At a concentration of 40 mg/ml PRP the fraction inhibits initiation of platelet aggregation by 0.18 mg/ml collagen and at 50 mg/ml PRP inhibits the initiation of aggregation by 0.7 units/ml thrombin. The G-25 filtered fraction shows a strong disaggregating effect on aggregated PRP. At a concentration of 65–75 mg/ml PRP, the fraction is able to disaggregate the 50 – 53% of aggregated platelet-rich plasma by 50 mM ADP, and 25% of aggregated PRP by 0.18 mg/ml collagen. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Galega officinalis L.; Extract; Inhibition; Platelet aggregation
1. Introduction Galega officinalis L. is a plant used in the traditional medicinal system of Bulgaria, Italy and India (Chopra et al., 1956; Petkov, 1982) in the treatment of diabetes mellitus. A biologically active alkaloid galegine, exhibiting a hypoglycemic effect in vivo was isolated from G. officinalis (Reuter, 1963; Hoppe, 1975). In healthy normoglicemic volunteers galegine at a dose of 2 – 4 mg/kg body mass leads to reduction of blood * Corresponding author.
glucose, which begins after 3–4 h and continues for about 9 h. A hypoglycemic effect is observed in patients with diabetes mellitus (Benigni et al., 1964). The plant extract appears to have an anticoagulation effect (Erspamer, 1943; Petkov, 1982). Recent experimental results (Atanasov, 1993, 1994) show that the 2.25% crude aqueous extracts of this plant at a dose of 1.0–2.5 mg/ml platelet-rich plasma (PRP) suppress in vitro platelet aggregation induced by aggregating agents such as adenosine 5%-diphosphate (ADP), epinephrine, thrombin and collagen. The IC50 (50% inhibition effect) for aggregation by 50 mM
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 4 4 - 0
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A.T. Atanaso6, V. Spaso6 / Journal of Ethnopharmacology 69 (2000) 235–240
ADP is 1.25 mg crude extract in 1 ml PRP. The crude extracts of G. officinalis L. suppress in vivo platelet aggregation in rats for 2.5 – 3.0 h, after injection in the caudal vein of rats in a ratio of 1 part 2.5% extract per 40 parts blood (Atanasov, 1995a). The aqueous extract of G. officinalis L. has an synergetic effect on platelet aggregation in common with anti-aggregant agents such as: aspirin, dipyridamol, ticlopidine, sulfinpyrazone, indobufenum, digoxin, theophyline and other drugs with similar action (Atanasov, 1995b). Up to 1990, the biologically-active compounds with antiaggregating action have not been isolated from G. officinalis L. In the present study the effect on platelet aggregation of a Sephadex G-25 filtered fraction, obtained from crude extracts of G. officinalis L. was investigated.
water—NH3 composition of pH 7.5. The column was eluted with the same composition at 16°C. The flow-rate was 720 ml/h and the fraction from 400 to 550 ml was collected. The fraction was concentrated to (21.59 0.5) mg/ml dry matter and the effect on platelet aggregation was studied.
2.4. Isolation of human platelets Blood was taken from volunteers who had received no medication for 15 days prior to blood collection. Blood was collected in disposable syringes at a ratio of 1 part 3.8% trisodium citrate and 9 parts venous blood (Zucker, 1989). Plateletrich plasma was prepared by centrifugation (180× g for 10 min) and diluted to 300× 106 platelets per ml with autologous platelet-poor plasma (1800×g for 15 min).
2. Materials and methods
2.5. Platelet aggregation 2.1. Plant material The aerial parts of G. officinalis L. (Herba Galegae) were collected at the flowering stage between May and August 1997 in different parts of Thrace, Bulgaria. The plant was verified by Professor Ivan Assenov, and a voucher specimen (12186) has been deposited in the Herbarium of Department of Botany, Faculty of Pharmacy (University of Sofia, Bulgaria).
2.2. Preparation of extract The crude aqueous extract was obtained by maceration of 150 g dry matter in 2000 ml distilled water for 20– 24 h at 18 – 20°C. The fresh extract was adjusted to pH 8.0 with NH3 and centrifuged at 150× g after 1 h. The supernatant was filtered and concentrated at a temperature below 35°C to an extract with dry matter of about 165 – 170 mg/ml.
2.3. Gel-filtration of extract through Sephadex G-25. The concentrated extract (10 ml) was applied on a column (600 mm× 55 mm) equilibrated with
Aggregation was studied with a spectrophotometer set to operate at a wavelength of 600 nm and the results were recorded on an electronic recorder (XY-Recorder en dim. 620.02, VEB, Germany; Zucker, 1989). The extinction change that takes place during the aggregation of 400 ml PRP compared with platelet-poor plasma (whose extinction was taken as zero) after adding the aggregating agent (20 ml ADP and 40 ml collagen or thrombin) at 37°C and at a rate of stirring of 1000 rpm, was the basis of measurement for aggregating effect. The effect of the G-25 filtered fraction from G. officinalis was studied after adding 0.5– 10 ml fraction to 400 ml PRP with stirring. The aggregating agents were added after 10 min and the aggregation was monitored for a further 20 min. Aggregation in % was calculated as: (E0 − E)× 100%/(E0 − Ef), where E0 is the initial value of the extinction of PRP before the aggregating process; Ef is the final value of the extinction of PRP after finishing the aggregating process; and E is the final value of the extinction of PRP (with adding of desalted fraction or drugs) after finishing the aggregating process.
A.T. Atanaso6, V. Spaso6 / Journal of Ethnopharmacology 69 (2000) 235–240
2.6. Reagents ADP at a concentration of 1×10 − 3 M from Reanal (Hungary); human thrombin (8 units/ml) and bovine collagen (2 mg/ml) from the Research Institute of Haematology and Blood Transfusion (Sofia, Bulgaria) were used as aggregating agents.
2.7. Drugs Dipyridamole (5 mg/ml) and theophyline (10 mg/ml) from Pharmachim (Bulgaria) were used as a basis for the comparison of the effect of the column fraction of Herba Galegae.
2.8. Statistical analysis All points in the graphics are expressed as mean 9SD.
3. Results The anti-aggregating activity of the G-25 filtered fraction from G. officinalis L. was verified with ADP, collagen and thrombin. The inhibiting
237
effect of fraction on platelet aggregation initiated by 50 mM ADP is presented in Fig. 1. The threshold concentration at which the G-25 filtered fraction starts to inhibit platelet aggregation (5– 10% inhibition) by 50 mM ADP is 4.5–5.0 mg per 1 ml PRP. At a concentration of 19–20 mg/ml PRP, the fraction inhibits platelet aggregation at the same degree as dipyridamole at a dose of 25 mg/ml PRP. At a concentration of 35 mg/ml PRP, the fraction inhibits 50% of platelet aggregation (IC50). At a concentration over 125 ml/ml PRP, the 100% common (inhibition and disaggregation) effect is shown. The disaggregating effect of the G-25 filtered fraction is shown in Fig. 2. The addition of aggregating agent (50 mM ADP) to the initial PRP leads to aggregation and the decrease of free platelets in plasma from 100% (300× 106 platelets/ml) to 3–4%, and the number of associated in aggregates platelets rises to 96– 97%. A slight disaggregating effect is shown, after adding on 96–97% aggregated platelets of 25–60 mg/ml PRP G-25 filtered fraction. For example, the 25-mg/ml G-25 fraction disaggregates 96–97% aggregated platelets to 29–31% free platelets and 69–71% associated in aggregates platelets. The
Fig. 1. In vitro inhibiting effect of 35, 50, 125, 250 and 500 mg ‘G-25 filtered fraction’ of G. officinalis L. on aggregation of 1 ml platelet-rich plasma (300 ×106 platelets/ml), by 50 mM ADP (final concentration). The effect of 25 mg dipyridamol (dip) in 1 ml PRP is used for comparison. Adding of fraction or drug on platelet suspension is marked by a triangle. Values are means 9 SD for six independent experiments.
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Fig. 2. In vitro disaggregating effect of 25, 65, 125, 250 and 500 mg ‘G-25 filtered fraction’ of G. officinalis L. on aggregated platelet-rich plasma (300 × 106 platelets/ml) by 50 mM ADP (final concentration). The disaggregating effect of 500 mg theophyline (theoph) in 1 ml PRP is used for comparison. Adding of fraction or drug on platelet suspension is marked by a triangle. Values are means9SD for six independent experiments.
concentration of fraction 65 – 75 mg/ml PRP leads to 50–53% free and 50 – 47% associated in aggregates platelets (IC50). A strong disaggregating effect is shown, after adding on 96–97% aggregated platelets to 100 – 500 mg/ml PRP G-25 filtered fraction. For example, the 125-mg/ml PRP fraction leads to 68 – 71% free platelets and 32 – 29% associated in aggregates. At a concentration of over 250 mg/ml PRP, the platelets aggregates is destroyed in fullness, and the number of free platelets approaches to 298 – 300×106. The G-25 filtered fraction inhibits platelet aggregation initiated by collagen (Fig. 3) and thrombin (Fig. 4). In this case the fraction inhibits the initiation of platelet aggregation or stops further platelet aggregation, after adding to aggregated PRP. The threshold concentration, at which the fraction starts to inhibit platelet aggregation (5 – 10% inhibition) by 0.18 mg/ml collagen and 0.7 units/ ml thrombin is 10 mg/ml PRP. At a concentration of 40 mg/ml PRP, the fraction inhibits fully initiation of platelet aggregation by 0.18 mg/ml collagen, and at 50 mg/ml PRP inhibits the initiation of aggregation by 0.7 units/ml thrombin. The in-
hibiting effect of 400 mg/ml PRP theophyline is equivalent to the effect of 33 mg/ml G-25 filtered fraction on aggregation by collagen, and equivalent to the effect of 24 mg/ml G-25 filtered fraction on aggregation by thrombin. On aggregated platelets by collagen the fraction has a slight disaggregating effect—about 25%— but on aggregated platelets by thrombin the fraction has no disaggregating effect.
4. Discussion and conclusions The obtained column fraction from aqueous extract of G. officinalis L. inhibits the platelet aggregation initiated by ADP, collagen and thrombin. On the other hand our investigations show that the same fraction inhibits the platelet aggregation initiated by epinephrine, aggregating agents consists of free radicals and spontaneous platelet aggregation. Possibly the fraction inhibits aggregation through a non-specific mechanism— for example, preventing fibrinogen bridges between platelets (Karczewski and Connolly, 1997). Such mechanism of action has ticlopidine (Bruno, 1983). The IC50 for crude extract on platelet ag-
A.T. Atanaso6, V. Spaso6 / Journal of Ethnopharmacology 69 (2000) 235–240
gregation by 50 mM ADP is 1.25 mg/ml PRP (Atanasov, 1994). For the G-25 filtered fraction the IC50 is 35 mg/ml PRP. This shows that this fraction is 35–36 times more active than the crude extract. The G-25 filtered fraction has an activity
239
near to that of dipyridamole (inhibiting effect of 19–20 mg/ml PRP fraction on platelet aggregation is the same as the effect of 25 mg/ml PRP phospodiesterase inhibitor dipyridamole—Fig. 1) and two times more active than phospodiesterase
Fig. 3. In vitro inhibiting effect of 20, 30 and 40 mg ‘G-25 filtered fraction’ of G. officinalis L. on aggregation of 1 ml PRP (300 ×106 platelets/ml) by 0.18 mg collagen (final concentration). The effect of 400 mg theophyline (theoph) in 1 ml PRP is used for comparison. Adding of fraction or drug on platelet suspension is marked by a triangle. Values are means 9SD for five independent experiments.
Fig. 4. In vitro inhibiting effect of 15, 30 and 50 mg ‘G-25 filtered fraction’ of G. officinalis L. on aggregation of 1 ml PRP by 0.7 units/ml thrombin (final concentration). The effect of 400 mg theophyline (theoph) in 1 ml PRP is used for comparison. Adding of fraction or drug on platelet suspension is marked by a triangle. Values are means 9SD for five independent experiments.
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inhibitor theophyline (Fig. 2). In cases of aggregation by collagen and thrombin, G-25 filtered fraction is 12–16-fold more active than the theophyline (Figs. 3 and 4). More ever, the G-25 filtered fraction clearly shows expressed disaggregating effects on platelet aggregation by ADP and collagen. This fact remains masked in an investigation of the effect of the whole crude extract. The crude extracts and the G-25 filtered fraction do not lysis and agglutinate erythrocytes or platelets, and do not precipitate the serum proteins. This shows that the biologically-active component in extract is not a lectin as can be expected. The G-25 filtered fraction correlates with high molecular mass (over 25 kDa), as it eluates with free volume of column. The effect on aggregation is temperature and pH dependent: over 55°C, below pH 4 and over pH 11 the fraction loses its activity. This shows that the biologically-active compounds in the extract are not any of the isolated G. officinalis L. alkaloids (Hoppe, 1975), which are temperature and pH stable, and with a molecular mass below 1 kDa. It is possible the biologically-active component has a carbohydrate nature because carbohydrates (sugars and amino sugars, dextrans, heparin proteoglycans) in most cases inhibit platelet aggregation (Kinlough-Rathbone et al., 1984; Lassila et al., 1997). The ability of ‘G-25 filtered fraction’ to inhibit platelet aggregation just confirms supposed anticoagulating effects of G. officinalis L. Experimental studies in this field, however are insufficient, but imperative because ‘diabetes mellitus’ leads to disorders of blood coagulation and platelet aggregation. The isolation of the biologically-active compound constituents of the fraction is the next step towards elucidation of the mechanism of platelet inhibition.
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References Atanasov, A., 1993. In vitro effect of water extracts of some medicinal plants from Bulgarian flora on platelet aggregation. Bulgarian Medicine 1, 17 – 20. Atanasov, A., 1994. Effect of the water extract of Galega officinalis L. on human platelet aggregation in vitro. Phytotherapy Research 8 (5), 314 – 316. Atanasov, A., 1995a. An effect of Galega officinalis L. extract on platelet aggregation in rats. Journal of Herbs, Spices and Medicinal Plants 3 (3), 71 – 76. Atanasov, A., 1995b. In vitro effect on platelet aggregation of water extracts of Galega officinalis L. in combination with some drugs. Bulgarian Medicine 3 – 4 (3), 35 – 38. Benigni, R., Capra, C., Cattorini, P.E., 1964. Galega. In: Plante Medicinali, Chimica Farmacologia e Terapia. Inverni Della Betta, Milano, pp. 626 – 632. Bruno, J.J., 1983. The mechanism of action of ticlopidine. Thrombosis Research (Suppl. 4), 59 – 67. Chopra, R.H., Nagar, S.L., Chopra, I.P., 1956. Glossary of Indian Medicinal Plants. New Delphi, India, p. 20. Erspamer, V., 1943. Droghe e principi ipoglicemizzanti vegetali. In: Quaderni di Fitoterapia, in 5. Inverni Della Beffa, Milano, pp. 23 – 26. Hoppe, H.A., 1975. Drogenkunde 1, 8th ed. Walter de Gruyter, Berlin, p. 1311. Karczewski, J., Connolly, T.M., 1997. The interaction of disagregin with the platelet fibrinogen receptor, glycoprotein IIb – IIIa. Biochemical and Biophysical Research Communications 241 (3), 744 – 748. Kinlough-Rathbone, R.L., Packham, M.A., Mustard, J.F., 1984. Effect of amino sugars on platelet aggregation and on fibrinogen binding. Thrombosis and Haemostasis (Stuttgart) 52 (1), 75 – 80. Lassila, R., Lindstedt, K., Kovanen, P.T., 1997. Native macromolecular heparin proteoglycans exocytosed from stimulated rat serosal mast cells strongly inhibit platelet-collagen interactions. Arterioscler. Thromb. Vasc. Biol. 17 (12), 3578 – 3587. Petkov, V.D., 1982. Modern Phytotherapy Medizina i Fizkultura Publ. House, Sofia, p. 307 (In Bulgarian). Reuter, G., 1963. Arginin als Vorstufe von Galegin in Galega officinalis L., Archiv der Pharmazie, 296 Bd.; N8, pp. 516 – 522. Zucker, M.B., 1989. Platelet aggregation measured by the photometric method. Methods in Enzymology 169, 118 – 133.