355
Atherosclerosis, 30 (1978) 355-360 @ Elsevier/North-Holland Scientific Publishers,
Ltd.
Preliminary Note EFFECT OF GARLIC ON HUMAN PLATELET AGGREGATION IN VITRO ARUN BORDIA Department
of Medicine (Cardiology),
R.N.T. Medical College, Udaipur (India)
(Received 6 March, 1978) (Revised, received 24 April, 1978) (Accepted 25 April, 1978)
In 6 healthy adults the effect of essential oil of garlic on platelet aggregation was studied in vitro with an aggreganometer. The blood was collected in a siliconized centrifuge tube containing sodium citrate. The aggregating agents used were ADP, epinephrine and collagen. In each subject aggregation was studied 3 times: (i) initial fasting control; (ii) immediately after (i) but with essential oil of garlic drawn into the syringe together with the sodium citrate; (iii) 5 days after feeding 0.5 mg of essential oil of garlic daily. Addition of essential oil of garlic inhibited in-vitro platelet aggregation induced by ADP, epinephrine or collagen; the effect was dose-related. Oral administration of garlic also decreased platelet aggregation. Thus, garlic seems to inhibit some aspects of thrombus formation. Key words:
Garlic -Herbal
pharmacology
-Platelets
- Thrombosis
Introduction We reported earlier that garlic enhances fibrinolytic activity in both man [l] and the experimental animal [ 21. In patients with acute myocardial infarction, fibrinolytic activity nearly doubled after garlic administration for 20 days during the immediate post-infarction period [3]. This led us to enquire if garlic would influence platelet aggregation, an essential step in the thrombotic process. Methods Six healthy adults between the age of 35 and 45 years were studied. They were all free from any acute or chronic disease, and had not been taking aspirin
356 TABLE 1 EFFECT OF GARLIC
OIL ON PLATELET
AGGREGATION
IN VITRO
In every case the volume of the aggregating agent was made up to 0.1 ml with isotbnic NaCl before it was added to 1 ml PRP in the aggreganometer tube. Maximum % fall in optical density (mean + SE) ADP
With garlic 10 pg 5 !Jg 2.5 pg After garlic feeding for 5 days 26 mg
Collagen
0.5 pg
1 c1g
0.5 clg
74.2 t3.5
40.2 +2.1
11.4 t6.2
48.2 r5.5
68.2 t4.9
5.2 to,5 20.0 +2.2
2.1 to.2
5.0 IO.6
3.1 to.2
12.0 21.0
24.2 Z1.6
50.0 t3.1
24.1 t2.3
41.1 ~2.3
1 .l to.1 8.0 t1.0 18.1 t1.2
54.2 ‘6.1
31.1 +4.2
57.8 r7.1
24.2 k2.6
25.2 t4.1
1 Without garlic
Epinephrine
I.47
12.0 t2.2 17.1 i2.3
or other drugs known to affect platelet aggregation. Some 18 ml of blood were drawn by free flow with a 19-gauge siliconized needle into a siliconized centrifuge tube containing 2 ml of 3.8% sodium citrate. To obtain platelet-rich plasma (PRP), the blood specimen was centrifuged slowly at room temperature at approximately 100 X g for 15 min. The supernatant platelet-rich plasma was carefully pipetted out to avoid any red cell contamination. The remaining blood was recentrifuged at approximately 1400 X g for at least 10 min to obtain platelet-poor plasma (PPP). Platelet aggregation was studied with an aggreganometer and recorder [4]. The aggregating agents used were ADP, epinephrine and collagen in different amounts as shown in Table 1. Collagen was prepared as described by Hoving [ 51. Essential oil of garlic was separated, as described earlier [l-3]. Some essential oil was mixed with the sodium citrate used for collecting the blood sample. In each subject aggregation was studied 3 times: (i) initial fasting control; (ii) immediately after (i) but with essential oil of garlic drawn into the syringe together with sodium citrate; (iii) 5 days after feeding 25 mg of essential oil of garlic daily. The fraction of separated oil was adjusted to a suitable volume with soyabean oil and prepared for administration in gelatin capsules. Rt?SUItS
As shown in Table 1 addition of the essential oil prevented platelet aggregation enhanced by ADP or epinephrine or collagen (Table 1 and Figs. 1, 2, 3, 4). Aggregation was markedly inhibited in all 6 experiments in a dose-related manner. In anotherset of ex.periments garlic oil was added directly to the separated PRP in vitro. The effect .wasfound to be essentially similar to that found when
357 0
10
ADP+tARLIC
3 MINUTES
2 D”F?ATIOk Fig. 1. Effect
i
IN
4
of essential oil of garlic on platelet
1;’
2.5 Uj
5
6
aggregation
\W”FUNE
induced
by 1.0 pg ADP.
,
: w u so 5 Y0. 100 80 L.... 0
1 DURATION
Fig. 2. Effect
2
3 IN
4
5
6
7
MINUTES
of essential oil of garlic on platelet
aggregation.produced
by 1.0 c(g epinephtie.
353 COLLAGEN + GARLIC
IOU9
0
= eo. COLLAGEN
z : u 90 L Y olOO 1
0 DURATION
2 IN
3
6
5
4
7
MINUTES
Fig. 3. Effect of essential oil of garlic on platelet aggregation produced by collagen.
, i
0
OUlAtlON
Fig.
4.
Effect of
oral
Y
I.
4
2
3
IN
MINUTES
administration
of
5
6
I 7
essential oil of garlic on platelet aggregationin vitro.
359
the oil had been taken into the sodium citrate before collecting the blood sample. Oral administration of garlic for 5 days also significantly inhibited platelet aggregation (Table 1 and Fig. 4). Discussion Garlic was advocated in the French literature of the Middle Ages for treating peripheral venous thrombosis in ‘horses. This is relevant to our recent report that essential oil of garlic enhances fibrinolytic activity in man [l-3]. In a further study on garlic, we have now examined its action on in-vitro platelet aggregation. In the present set of experiments garlic significantly inhibited platelet aggregation induced by all 3 important aggregating agents: ADP, epinephrine and collagen. Besides partially suppressing aggregation, garlic also promotes disaggregation after the peak has been reached. Platelet aggregation was reduced to a similar extent when garlic was fed orally. The mechanism by which garlic influences platelet aggregation cannot, however, be clearly defined at present. Haslam [6] suggested that ADP provides a final common pathway, through which the effect of all other clumping agents is mediated. On the other hand, it has also been suggested that catecholamines in platelets may partly be responsible for the secondary phase of ADP-induced platelet aggregation [ 71. Catecholamine release may be closely involved with the thrombotic process, particularly as it has been shown that release of catecholamines is increased after myocardial infarction [ 81. Since garlic depresses epinephrine-induced in-vitro platelet aggregation, one can expect that the thrombotic influence’of catecholamines may be inhibited by long-term garlic administration in situations of stress. It has been reported that excessive fibrinolysis is associated with the release of fibrinogen-degradation products (FDP), which inhibit platelet aggregation [ 91. As garlic significantly enhances fibrinolytic activity [ 31, it is theoretically possible that its overaction could cause platelets to aggregate through the release of FDP. We have earlier reported that administration of garlic prevents the development of experimental atherosclerosis and increases fibrinolytic activity in patients with ischaemic heart disease [2,3]. This new property of garlic, whereby it inhibits platelet aggregation, raises the possibility that a drug might ultimately be available which would prevent thrombus formation, induce thrombolysis and even influence the development of atherosclerosis. Garlic is consumed in the daily diet by a large proportion of the world’s population and, in our own studies, administration of as much as 60 g of crude garlic daily for 3 months has led neither to side-effects nor to a bleeding tendency. As such this herbal remedy seems to be clinically acceptable and safe. References 1 Bordia, A., Bansal. H.C., alimentary hyperlipemia, 2 Bordia. A., Verma, S.K.. essential oil of onion and 379.
Arora. S.K. and Singh, S.V., Effect of the essential oils of garlic and onion on Atherosclerosis, 21 (1975) 15. Vyas, A.K.. Khabya, B.L., Rathore, AS.. Bhu, N. and Bedi, H.K., Effect of garlic on experimental atherosclerosis in rabbits, Atherosclerosis, 26 (1977)
360 3 Bordia. A., Joshi, H.K.. Sanadhya, Y.K. and Bhu, N.. Effect of essential oil of garlic on serum flbrinolytic activity in patients with coronary artery disease, Atherosclerosis, 28 (19’77) 155. 4 Harms. C.S. and Triplett. D.A., Platelet aggregation. In: Laboratory Management, ASCP Press, Chicago. 1977, p. 34. 5 Hoving. T.. Aggregation of rabbit blood platelets produced in vitro by saline “extract” of tendons, Thromb. et Diath. Haemorrh., 9 (1960) 248. 6 Haslam. R.L., Role of ADP in the aggregation of human blood platelets by thrombin and by fatty acid, Nature (Land.), 202 (1964) 765. 7 Thomas, D.P.. Effect of catecholamines on platelet aggregation caused by thrombin, Nature (Lond.), 215 (1967) 298. 8 McDonald, L., Baker, C., Bray, C.. McDonald. A. and Restieaux, N.. Plasma catecholamines after cardiac infsrction. Lancet, 2 (1969) 1021. 9 Kopec, M., Budzynski. A., Stachurska. J., Wegrzynowicz, Z. and Kowalski, E., Studies on the mechanism of interference by fibrinogen degradation products (FDP) with platelet function - Role of fibrinogen in platelet atmosphere, Thromb. et Diath. Haemorrh.. 15 (1966) 476.
Atherosclerosis, 30 (1978) 355-360 @ Elsevier/North-Holland Scientific Publishers,
Ltd.
Preliminary Note EFFECT OF GARLIC ON HUMAN PLATELET AGGREGATION IN VITRO ARUN BORDIA Department
of Medicine (Cardiology),
R.N.T. Medical College, Udaipur (India)
(Received 6 March, 1978) (Revised, received 24 April, 1978) (Accepted 25 April, 1978)
In 6 healthy adults the effect of essential oil of garlic on platelet aggregation was studied in vitro with an aggreganometer. The blood was collected in a siliconized centrifuge tube containing sodium citrate. The aggregating agents used were ADP, epinephrine and collagen. In each subject aggregation was studied 3 times: (i) initial fasting control; (ii) immediately after (i) but with essential oil of garlic drawn into the syringe together with the sodium citrate; (iii) 5 days after feeding 0.5 mg of essential oil of garlic daily. Addition of essential oil of garlic inhibited in-vitro platelet aggregation induced by ADP, epinephrine or collagen; the effect was dose-related. Oral administration of garlic also decreased platelet aggregation. Thus, garlic seems to inhibit some aspects of thrombus formation. Key words:
Garlic -Herbal
pharmacology
-Platelets
- Thrombosis
Introduction We reported earlier that garlic enhances fibrinolytic activity in both man [l] and the experimental animal [ 21. In patients with acute myocardial infarction, fibrinolytic activity nearly doubled after garlic administration for 20 days during the immediate post-infarction period [3]. This led us to enquire if garlic would influence platelet aggregation, an essential step in the thrombotic process. Methods Six healthy adults between the age of 35 and 45 years were studied. They were all free from any acute or chronic disease, and had not been taking aspirin
356 TABLE 1 EFFECT OF GARLIC
OIL ON PLATELET
AGGREGATION
IN VITRO
In every case the volume of the aggregating agent was made up to 0.1 ml with isotbnic NaCl before it was added to 1 ml PRP in the aggreganometer tube. Maximum % fall in optical density (mean + SE) ADP
With garlic 10 pg 5 !Jg 2.5 pg After garlic feeding for 5 days 26 mg
Collagen
0.5 pg
1 c1g
0.5 clg
74.2 t3.5
40.2 +2.1
11.4 t6.2
48.2 r5.5
68.2 t4.9
5.2 to,5 20.0 +2.2
2.1 to.2
5.0 IO.6
3.1 to.2
12.0 21.0
24.2 Z1.6
50.0 t3.1
24.1 t2.3
41.1 ~2.3
1 .l to.1 8.0 t1.0 18.1 t1.2
54.2 ‘6.1
31.1 +4.2
57.8 r7.1
24.2 k2.6
25.2 t4.1
1 Without garlic
Epinephrine
I.47
12.0 t2.2 17.1 i2.3
or other drugs known to affect platelet aggregation. Some 18 ml of blood were drawn by free flow with a 19-gauge siliconized needle into a siliconized centrifuge tube containing 2 ml of 3.8% sodium citrate. To obtain platelet-rich plasma (PRP), the blood specimen was centrifuged slowly at room temperature at approximately 100 X g for 15 min. The supernatant platelet-rich plasma was carefully pipetted out to avoid any red cell contamination. The remaining blood was recentrifuged at approximately 1400 X g for at least 10 min to obtain platelet-poor plasma (PPP). Platelet aggregation was studied with an aggreganometer and recorder [4]. The aggregating agents used were ADP, epinephrine and collagen in different amounts as shown in Table 1. Collagen was prepared as described by Hoving [ 51. Essential oil of garlic was separated, as described earlier [l-3]. Some essential oil was mixed with the sodium citrate used for collecting the blood sample. In each subject aggregation was studied 3 times: (i) initial fasting control; (ii) immediately after (i) but with essential oil of garlic drawn into the syringe together with sodium citrate; (iii) 5 days after feeding 25 mg of essential oil of garlic daily. The fraction of separated oil was adjusted to a suitable volume with soyabean oil and prepared for administration in gelatin capsules. Rt?SUItS
As shown in Table 1 addition of the essential oil prevented platelet aggregation enhanced by ADP or epinephrine or collagen (Table 1 and Figs. 1, 2, 3, 4). Aggregation was markedly inhibited in all 6 experiments in a dose-related manner. In anotherset of ex.periments garlic oil was added directly to the separated PRP in vitro. The effect .wasfound to be essentially similar to that found when
357 0
10
ADP+tARLIC
3 MINUTES
2 D”F?ATIOk Fig. 1. Effect
i
IN
4
of essential oil of garlic on platelet
1;’
2.5 Uj
5
6
aggregation
\W”FUNE
induced
by 1.0 pg ADP.
,
: w u so 5 Y0. 100 80 L.... 0
1 DURATION
Fig. 2. Effect
2
3 IN
4
5
6
7
MINUTES
of essential oil of garlic on platelet
aggregation.produced
by 1.0 c(g epinephtie.
353 COLLAGEN + GARLIC
IOU9
0
= eo. COLLAGEN
z : u 90 L Y olOO 1
0 DURATION
2 IN
3
6
5
4
7
MINUTES
Fig. 3. Effect of essential oil of garlic on platelet aggregation produced by collagen.
, i
0
OUlAtlON
Fig.
4.
Effect of
oral
Y
I.
4
2
3
IN
MINUTES
administration
of
5
6
I 7
essential oil of garlic on platelet aggregationin vitro.
359
the oil had been taken into the sodium citrate before collecting the blood sample. Oral administration of garlic for 5 days also significantly inhibited platelet aggregation (Table 1 and Fig. 4). Discussion Garlic was advocated in the French literature of the Middle Ages for treating peripheral venous thrombosis in ‘horses. This is relevant to our recent report that essential oil of garlic enhances fibrinolytic activity in man [l-3]. In a further study on garlic, we have now examined its action on in-vitro platelet aggregation. In the present set of experiments garlic significantly inhibited platelet aggregation induced by all 3 important aggregating agents: ADP, epinephrine and collagen. Besides partially suppressing aggregation, garlic also promotes disaggregation after the peak has been reached. Platelet aggregation was reduced to a similar extent when garlic was fed orally. The mechanism by which garlic influences platelet aggregation cannot, however, be clearly defined at present. Haslam [6] suggested that ADP provides a final common pathway, through which the effect of all other clumping agents is mediated. On the other hand, it has also been suggested that catecholamines in platelets may partly be responsible for the secondary phase of ADP-induced platelet aggregation [ 71. Catecholamine release may be closely involved with the thrombotic process, particularly as it has been shown that release of catecholamines is increased after myocardial infarction [ 81. Since garlic depresses epinephrine-induced in-vitro platelet aggregation, one can expect that the thrombotic influence’of catecholamines may be inhibited by long-term garlic administration in situations of stress. It has been reported that excessive fibrinolysis is associated with the release of fibrinogen-degradation products (FDP), which inhibit platelet aggregation [ 91. As garlic significantly enhances fibrinolytic activity [ 31, it is theoretically possible that its overaction could cause platelets to aggregate through the release of FDP. We have earlier reported that administration of garlic prevents the development of experimental atherosclerosis and increases fibrinolytic activity in patients with ischaemic heart disease [2,3]. This new property of garlic, whereby it inhibits platelet aggregation, raises the possibility that a drug might ultimately be available which would prevent thrombus formation, induce thrombolysis and even influence the development of atherosclerosis. Garlic is consumed in the daily diet by a large proportion of the world’s population and, in our own studies, administration of as much as 60 g of crude garlic daily for 3 months has led neither to side-effects nor to a bleeding tendency. As such this herbal remedy seems to be clinically acceptable and safe. References 1 Bordia, A., Bansal. H.C., alimentary hyperlipemia, 2 Bordia. A., Verma, S.K.. essential oil of onion and 379.
Arora. S.K. and Singh, S.V., Effect of the essential oils of garlic and onion on Atherosclerosis, 21 (1975) 15. Vyas, A.K.. Khabya, B.L., Rathore, AS.. Bhu, N. and Bedi, H.K., Effect of garlic on experimental atherosclerosis in rabbits, Atherosclerosis, 26 (1977)
360 3 Bordia. A., Joshi, H.K.. Sanadhya, Y.K. and Bhu, N.. Effect of essential oil of garlic on serum flbrinolytic activity in patients with coronary artery disease, Atherosclerosis, 28 (19’77) 155. 4 Harms. C.S. and Triplett. D.A., Platelet aggregation. In: Laboratory Management, ASCP Press, Chicago. 1977, p. 34. 5 Hoving. T.. Aggregation of rabbit blood platelets produced in vitro by saline “extract” of tendons, Thromb. et Diath. Haemorrh., 9 (1960) 248. 6 Haslam. R.L., Role of ADP in the aggregation of human blood platelets by thrombin and by fatty acid, Nature (Land.), 202 (1964) 765. 7 Thomas, D.P.. Effect of catecholamines on platelet aggregation caused by thrombin, Nature (Lond.), 215 (1967) 298. 8 McDonald, L., Baker, C., Bray, C.. McDonald. A. and Restieaux, N.. Plasma catecholamines after cardiac infsrction. Lancet, 2 (1969) 1021. 9 Kopec, M., Budzynski. A., Stachurska. J., Wegrzynowicz, Z. and Kowalski, E., Studies on the mechanism of interference by fibrinogen degradation products (FDP) with platelet function - Role of fibrinogen in platelet atmosphere, Thromb. et Diath. Haemorrh.. 15 (1966) 476.
