Inhibition of human platelet aggregation and membrane lipid peroxidation by food spice, saffron Jessie Suneetha. W and Krishnakantha. T.P. * Dept of Biochemistry and Nutrition Central Food Technological Research Institute Mysore - 570 020, India.
* Corresponding author Tel: 91-821-2514876 Fax: 91-821-2517233 E. mail:
[email protected]
Abstract: The inhibitory activity of saffron extract was studied on human platelets. Platelet aggregation and lipid peroxidation were evaluated with Platelet Rich Plasma (PRP) and platelet membranes respectively obtained from blood of healthy human volunteers. Human platelets were subjected to stimulation with a variety of agonists like ADP (61 M), epinephrine (76 M), collagen (11 g/ml), calcium ionophore A 23187 (6 M) and ristocetin (1.25 g/ml) in the presence and absence of saffron extract with IC50 being 0.66, 0.35, 0.86 and 0.59 mg respectively and no inhibition with ristocetin.
The inhibitory effect was dose
dependent with concentrations varying between 0.16 to 0.80 mg and time dependent at IC50. A significant decrease was observed in malondialdehyde (MDA) formed, one of the end products of arachidonic acid metabolism and of serotonin released from dense granules of platelets at respective IC50.
Lipid
peroxidation in platelet membranes induced by iron-ascorbic acid system was inhibited by saffron extract significantly with IC50 of 0.33 mg. Hence, it may be said that aqueous extract of saffron may have component(s), which protect platelets from aggregation and lipid peroxidation. Key words: Human platelets, saffron, platelet aggregation, malondialdehyde, serotonin, platelet membranes, lipid peroxidation.
Introduction: Spices, the well-known food adjuncts, have more to them than just being added to food to enhance the taste.
One such spice being saffron, Crocus
sativus Linn. (family Iridaceae), which is one of the oldest and most expensive spice, bears importance to many cultures apart from being a popular colouring and flavouring agent in various cuisine [1]. It usually consists of dried stigma, from a bulbous perennial plant, a native of Southern Europe [2]. As per the Indian system of medicine, Ayurveda, saffron acts as an antispasmodic, aphrodisiac, carminative and a stimulative agent [3].
It helps in overcoming
problems of reproductive system as well as the gastrointestinal and urinary tracts [4]. The present investigation was under taken to study the antiaggregatory and antioxidant effect of aqueous extract of saffron on human platelets.
Materials and Methods: Adenosine diphosphate (ADP), collagen, epinephrine, calcium ionophore A 23187 and ristocetin were purchased from Sigma Chemical Co., USA. Remaining chemicals were purchased locally, which were of extra pure analytical grade. Isolation of platelets: Venous blood was collected from healthy human volunteers (who have not taken any drugs like aspirin which affect platelet aggregation at least for the past 10 days) in 3.8% tri-sodium citrate (9:1 v/v) and was used within 3 hr of collection. This citrated blood was centrifuged at 120 g for 20 min to obtain
Platelet Rich Plasma (PRP). The residual blood was again centrifuged at 1100 g for 20 min to obtain the homologous Platelet Poor Plasma (PPP). Platelet count was adjusted to 1.6 X 107 platelets per l of PRP [5]. Preparation of Saffron extract: Saffron purchased from local market (1.0 g) was ground to a fine paste with 2.5 ml water and the slurry was left over night at 4 C. Next day the slurry was centrifuged at 10000 rpm for 15 min to obtain a clear extract, with a pH of 7.4 which was used for inhibition studies. Volumes ranging between 5-50 l were used. Amount of dried material present was 16mg/ml of extract. Platelet aggregation: The aggregation experiments were carried out turbidimetrically in a Dual Path Chronolog Aggregometer. PRP of 0.45 ml was kept stirred at 1200 rpm and temperature was maintained at 37 oC as aggregation was induced by agonists like ADP (61 M), epinephrine (76 M), collagen (11 g/ml), calcium ionophore A 23187 (6 M) and ristocetin (1.25 g/ml). The change in turbidity was recorded atleast for 5 min in each case with reference to PPP using an omniscribe recorder in the presence and absence of the extract. The slopes were calculated for the control and experimental samples and
using control samples percentage inhibitions were determined for the experimental samples. Estimation of malondialdehyde (MDA) from agonist challenged platelets: To PRP aggregation (0.45 ml), 0.02 ml of 1% BHT in ethanol and 0.1 ml of 100% TCA in 3 N HCl were added and centrifuged at 10000 rpm for 10 min. After
centrifugation, 0.45 ml of supernatant was added with 0.1 ml of TBA reagent (0.12 M TBA in 0.26 M Tris-HCl) and boiled for 30 min.
The chromophore
generated was measured at 532 nm and MDA (malondialdehyde) calculated by using – (Absorbance/156) X (Total volume/mg of protein/ml) MDA was expressed in nmoles of MDA formed/mg protein/hr [6]. Estimation of serotonin in agonist challenged platelets: To PRP after aggregation (0.45 ml), 0.1 ml of 6 M TCA was added and centrifuged at 10000 rpm for 10 min. From this 0.5 ml of supernatant was mixed with 2 ml of O-phthaldialdehyde reagent (0.5% O-phthaldialdehyde in ethanol and 8 N HCl. The mixture was placed in boiling water bath for 10 min, cooled on ice and washed twice with chloroform and read at excitation 360 nm and emission 475 nm in a spectrofluorimeter. The values expressed as nmoles of serotonin released/mg protein/hr [7]. Estimation of protein in isolated platelets: Protein concentration was determined in isolated platelet membranes by modified Folin-phenol method [8].
Bovine serum albumin was used as the
standard. Preparation of platelet membranes: PRP was mixed with double the volume of saline and centrifuged at 2500 rpm for 10 min. To the platelet pellet, 5 ml of Tyrodes buffer (NaCl 137 mM, KCl 2.7 mM, NaHCO3 12 mM, EDTA 1 mM, NaH2PO4 0.4 mM, MgCl2 1 mM and glucose 5.6 mM) was added, washed twice and final wash was done with 5 ml of
0.1 M Tris-HCl (pH 7.4). The platelet pellet formed was homogenized in 0.1 M Tris-HCl and kept frozen till use [9]. Platelet membrane lipid peroxidation: Platelet membranes (1.5 mg/ml of 0.1 M Tris-HCl Buffer) were dispersed in a medium contain 0.15 M KCl, 0.025 M Tris-HCl, 2 mM ADP and 10 m FeSO4. The contents were incubated for 5 min at 37 C. Lipid peroxidation was initiated by adding 0.1 mM Ascorbic acid. The final reaction volume was 1 ml. Appropriate blanks without platelet membranes were also included and left for 30 min at 37 C. The reaction was terminated by adding 2 ml of TBA reagent (0.375% TBA in 15% TCA and 0.2 N HCl) and was again kept for 30 min in boiling water bath. The malondialdehyde formed was measured at 535 nm. Lipid peroxidation is expressed as moles of MDA formed/mg of protein/hr. MDA formed were calculated using an extinction factor coefficient of 1.56 X 105 M-1 cm-1 [10].
Results and Discussion: Platelet aggregation was carried out in the presence and absence of saffron extract with ADP, epinephrine, collagen, calcium ionophore A 23187 and ristocetin at incubation of one minute and the IC50 were determined. The inhibitory effect with ADP and calcium ionophore A 23187 were more or less similar with IC50 of 0.66 mg and 0.59 mg respectively where as with epinephrine it was 0.35 mg and collagen 0.86 mg. Epinephrine induced aggregation showed the highest sensitivity where as collagen induced aggregation showed the least sensitivity (Figure 1).
The mode of action of these platelet agonists (ADP, epinephrine and collagen) is to help in increasing the cytosolic levels of calcium either due to release from the internal stores or through calcium influx [11].
Similarly
ionophore A 23187 acts as a carrier of calcium ions across the lipid bilayers in the cell membranes, which in turn helps to increase the cytosolic calcium levels. A rise in the cytosolic levels of calcium accompanies platelet activation through stimulation of enzymes, which were otherwise not fully functional at low levels of calcium concentration present in resting platelets [12].
It is also seen that
calcium antagonists usually may not be binding to the specific receptor sites but may cause the thickening of membranes due to the insertion of these antagonists into the membrane bilayers thus affecting the calcium mobilisation [13]. The agonists ADP, epinephrine and collagen involve the activation of surface glycoprotein receptors IIb-IIIa (GP IIb-IIIa) as well as interaction with the von Willebrand factor found in the plasma [14,15] apart from increasing the cytosolic calcium levels. Also reports indicate that activation of platelets with agonists brings about changes in the cytoskeletal structure resulting in the loss of the normal discoidal shape and pseudopodal projections are seen [16]. Also ADP and epinephrine induce platelet aggregation by reducing the intraplatelet cAMP levels and inhibiting adenylate cyclase activation whereas inhibitors of platelet aggregation increase the c-AMP levels [17,18].
On the other hand,
collagen induced platelet aggregation is associated with a burst in hydrogen peroxide, a prooxidant which contributes to platelet activation [19].
This
hydrogen peroxide stimulates the arachidonic acid metabolism by contributing to the platelet production of thromboxane A2 [20]. Epinephrine induced aggregation is mediated through 2-adrenergic receptors which also decrease the PGE1 stimulated c-AMP levels [21]. But, with ristocetin platelet aggregation is mediated through von Willebrand factor’s interaction with surface glycoprotein Ib receptor (GP-Ib) [22]. Saffron extract inhibits the platelet aggregation induced by ADP, epinephrine and collagen indicating that it is likely to not only compete with the GP IIb-IIIa receptors along with these agonists but may also reduces their interaction with these receptors to cause the inhibition. Not only that, as these agonists along with A 23187 increase the cytosolic calcium levels it is likely that saffron may be interfering with the calcium signaling influx in activated platelets thus causing the inhibition of platelet aggregation. Also it may be likely that this extract prevents ADP and epinephrine reducing the increase in intraplatelet cAMP levels and adenylate cyclase stimulation. It may inhibit collagen induced aggregation by blocking the release of hydrogen peroxide, which will otherwise stimulate arachidonic acid metabolism. But ristocetin induced aggregation was not inhibited by saffron at concentrations as high as 1.5 mg and incubation time of 30 min indicating that it may not affect the GP-I b receptor. The IC50 were used to determine the effect of duration of incubation on platelet aggregation. As the incubation time was increased from 1 to 2, 4 and 8 min, a rise in the inhibitory effect was seen with ADP, epinephrine, collagen and calcium ionophore A 23187 (Table 1) indicating that the longer the interaction of
platelets with the component(s) of saffron extract more was the inhibition. Saturation point (100% inhibition) is at four min incubation for epinephrineinduced aggregation at IC50 (Table 1). A decrease in the malondialdehyde (MDA) formed between 29 to 37% was observed in the presence of this extract at IC50 (Table 2). Lipid peroxides (MDA is one of its end products) along with TXA2 increase the platelet sensitivity to agonists causing coronary heart diseases [23]. MDA formation was being effectively suppressed indicating the protective action of saffron extract. It may also be likely that the cyclo-oxygenase pathway of arachidonic acid metabolism as MDA is one of the end products [24] was being affected, thus causing a decrease in the platelet aggregation. Similarly a decrease in serotonin levels in agonist-stimulated platelets in the presence of this extract was observed. The serotonin levels decreased between 28 to 35% at IC50 (Table 2). This serotonin otherwise enhances the retention of procoagulant proteins on cell surface of stimulated platelets, thus aiding in platelet aggregation [25]. As this extract was inhibiting the serotonin levels with various agonists at their IC50, it may be likely that it is interfering with the retention or binding of these procoagulant proteins on the cell surface of stimulated platelets. Lipid bilayers of cell membranes are highly susceptible to peroxidation due to their polyunsaturated fatty acids.
Free radical reactions in the lipid
domain also result in damage to membrane proteins thus leading to alteration and impairment of membrane functions [26] resulting in adverse effects on the
biological system, which may include atherosclerosis and cancer [27]. It may also interfere with membrane surface receptors such as GP IIb / IIIa [28]. But, the presence of GP IIb / IIIa inhibitors significantly decreased the reactive oxygen species formation in activated platelets [29]. Spices, the known antioxidants are likely to exert their effect on lipid peroxidation by scavenging the reactive oxygen species which other wise would initiate the lipid peroxidation [30, 31]. The fatty acids from marine origin have been recognised to inhibit platelet aggregation by altering the redox status of the cell due to the increase in platelet glutathione peroxidase (GPx) activity, which in turn degrades the lipid hydroperoxides formed. Other wise these lipid hydroperoxides induce platelet activation through cyclooxygenation of arachidonic acid [32]. The component(s) of saffron extract inhibited lipid peroxidation in human platelet membranes induced by iron-ascorbic acid system effectively with IC50 of 0.33 mg (Figure 2) indicating that they may have antioxidant properties, which are likely to scavenge the free radicals formed during lipid peroxidation. Also like the fatty acids of marine products, component(s) of saffron may increase the platelet GPx activity, thus inhibiting the lipid hydroperoxide formation. In conclusion, as proved with other spice extracts, the aqueous extract of saffron may have potent antioxidant component(s), which are inhibiting the aggregation and lipid peroxidation in human platelets. Further experiments are required to elucidate the exact biochemical alterations taking place in platelets as a result of their interaction with the component(s) present in saffron extract, which may be causing the inhibition of platelet aggregation.
Acknowledgements: Authors thank Dr. V. Prakash, Director, CFTRI and Dr. S.G. Bhat, Head, Biochemistry and Nutrition, CFTRI, Mysore for their encouragement and keen interest in this research work. JSW thanks University Grants Commission (UGC) for providing the fellowship to carry out this work.
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Table 1: Effect of incubation time at IC50 on platelet aggregation Agonists
Amount
Inhibition (%)
IC50*- (mg)
1 min
With ADP
0.66
50.4 2.5
63.5 2.6
With Epinephrine
0.35
50.0 1.4
68.9 2.7
With Collagen
0.86
50.0 1.2
57.2 1.8
64.2 1.5 67.3 1.4
With A 23187
0.59
50.6 2.5
56.7 2.2
62.4 1.6 65.1 1.0
2 min
4 min
8 min
68.1 2.1 70.5 1.4 90.0 1.6**
100.0
* Duration of incubation is 1 min. ** Duration of incubation is 3 min. Values mean S.D of triplicates.
Table 2: Effect of saffron extract on released products formed in agonist challenged platelets at IC50 Agonist
ADP
Epinephrine
Collagen
A 23187
MDA Formed a: Without extract
250.6 22.5
138.9 11.7
288.4 20.2
218.9 25.3
With extract
174.7 20.0
88.2 8.6
204.1 18.6
150.3 17.8
30
(30.3%) (30
(36.5%)
(30
(29.2%)
(31.3%)
Serotonin Formed b: Without extract
243.3 16.1
176.5 14.8
257.0 19.7
234.2 21.3
With extract
159.6 16.1
120.9 13.4
184.5.1 15.2
164.1 17.7
(28.2%)
(31.2%)
30
(34.4%) (30
(31.5%)
(30
Values are Mean S.D of triplicates. a Values expressed in nmoles of MDA formed / mg protein / hr. b Values expressed as nmoles of serotonin released / mg protein / hr. Values in parenthesis are percentage of inhibition of the respective end products formed.
