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Isolation and effects of some ginger components on platelet aggregation and eicosanoid biosynthesis
Prostaglandins
Leukotrienes
and Medicine
25:
187-198,
1986
AND EFFECTS OF SOME GINGER COMPONENTS ON PLATELET AGGREGATION AND EICOSANOID BIOSYNTHESIS
ISOLATION
K.C. Srivastava, Department of Environmental Medicine, Institute of Community Health, Odense University, J.B. WinsMws Vej 19, DK-5000 Odense C, Denmark. ABSTRACT Aqueous ginger extract was extracted in three organic solvents viz., nhexane, chloroform and ethyl acetate with increasing polarity. The extracted materials from these solvents reduced platelet thromboxane formation from exogenous arachidonate (AA) and also inhibited platelet aggregation induced by AA, epinephrine, ADP and collagen; in this respect they were most effective against AA-induced aggregation. The extracted material in n-hexane was further resolved by thin-layer chromatography into various fractions some of which were effective in inhibiting platelet thromboxane formation and platelet g&on. Aqueous- ginger extract reduced the formation of TxEi2 from AA-label&d platelets without showing effects on platelet phospholipase activity. Thromboxane formation in labelled platelets on activation with calcium ionophore A23187 was reduced by ginger components, isolated from two TLC bands, in a dose-dependent manner (lo100 q/500 ml). At the higher dose lipoxygenase products were also reduced. Interestingly the incorporation of AA into platelet phospholipids increased in platelets treated with aqueous ginger extract. INTRODUCTION Ginger (genus Zingiber) root is used in cooking and medicine, and for this purpose it is used in fresh as well as in dry powdered form. It is preserved in syrup or candied as sweet. In the Indian subcontinent fresh ginger is widely used in the preparation of vegetable and meat dishes besides being used in the preparation of pickles. In the Indian system of medicine, Ayurveda, ginger is described to ameliorate and cure many ailments - e.g., cold, cough, fever, pain in the joints etc. The mechanism by which ginger’s medicinal properties could be accounted for is not known. However, recently we have described the effects of aqueous extract of ginger on aggregation and metabolism of arachidonic acid (AA) in piateiets. Ginger extract inhibited platelet aggregation induced by ADP, collagen, epinephrine and Presented, in part, at the IVth International Symposium on Prostaglandins, Thromboxanes and Leukotrienes in the Cardiovascular System, Halle 1984.
187
arachidonate; it did not inhibit Ca2+ ionophore A23187-induced aggregation. It reduced the formation of thromboxane (TX) and prostaglandins (PCs) in platelets (1). In this paper results are reported on the effects of some components of ginger on platelet aggregation, and on platelet cyclooxygenase- and lipoxygenase products’ formation together with the effects of aqueous extract of ginger on platelet phospholipase activity, formation of thromboxane B2 and lipoxygenase products from labelled platelets, and on the incorporation of AA into platelet phospholipids. In short, this paper describes an experimental approach to clarify the mechanism by which ginger inhibits platelet aggregation. MATERIALS AND METHODS Arachidonic acid (1-14~) (specific activity 59.3 mCi/mmol) was purchased from the Radiochemical Centre, Amersham, England. Thromboxane B2 (TxB2) was supplied free by ON0 Pharmaceutical Co., Ltd., Osaka (Japan). Calcium ionophore A23187 was a Sigma product. Thin-layer chromatography (TLC) plates were prepared in our laboratory. All reagents were of analytical reagent grade. Fractionation of ginger extract in organic solvents (with increasing polarity) nhexane, chloroform and ethyl acetate has been described (2). Ginger extract (24 ml) was diluted with 30 ml water followed by extraction in n-hexane (60 ml) by shaking and allowing to equilibrate at room temp (RT) for 30 min. The organic phase was separated from the aqueous phase (lower), the latter was adjusted to pH 3.0 with HCl and extracted in chloroform (120 ml) overnight in cold (4OC). After separating the chloroform layer, the aqueous phase was further extracted in ethyl acetate (100 ml). Organic solvents were evaporated by blowing nitrogen at 700C. Oily residues were obtained from the three extracts; they were dissolved respectively in 1000, 800 and 600 ul ethanol and stored at -200C. Thin-layer separation of ginger components. Resolution of the material present in the hexane extract was done by TLC. One hundred and fifty microliters (from 1 ml ethanolic solution of the hexane extract) were resolved by TLC using the solvent nhexane-ether-acetic acid (80:20:1, v/v). Prior to exposure to iodine vapour, two bands - one with Rf value 0.18 (yellow) and the other, application band (Rf = 0.00) were marked and scraped off. On exposing the plate to iodine, five more clearly separated bands were shown. They were marked and scraped off. The bands were extracted in chloroform-methanol-ethyl acetate (2: 1: 1, v/v). The solvent was evaporated by blowing nitrogen at 450C. The extracted material from each band was dissolved in 150 ul ethanol. This was either used as such or was concentrated prior to use in aggregation experiments. To obtain sufficient material from other TLC bands 90 ml juice (from ca. 330 g peeled ginger) were extracted as described above. The extraction was done in the same sequence, starting with n-hexane (500 ml), chloroform (ZOO ml) and finally with ethyl acetate (200 ml). About 150 ml n-hexane extract were evaporated at 70°C under nitrogen to yield 89 mg of an oily residue. This was dissolved in 1 ml chloroform, of which 150 ul were resolved by TLC as above. Before exposing the plate to iodine, TLC bands with Rf values 0.00 and 0.18 (yellow) were scraped off. Due to a higher amount of the material some more bands were shown including the seven bands mentioned above. The bands were scraped off and extracted in chloroform-methanol-ethyl acetate (2: 1: 1, v/v). The solvent was evaporated at 50°C under nitrogen and the residue dissolved in 100 ul ethanol. The extracted material from each TLC band was examined for its effect on platelet aggregation and thromboxane formation; in both experiments only 5 ul ethanolic solution of the extracted material were used. However, in aggregation experiments with collagen, epinephrine and ADP where more material was needed for inhibition of aggregat188
ion, alcohol was evaporated from the aggregation cuvette by slowly blowing N2 and the residue dissolved in 5 ul ethanol prior to incubation with platelet-rich plasma (PRP). Experiments with labelled platelets. Labelling of platelets in PRP was done for two specific purposes. The first was to examine the effect of ginger (aqueous) on labelling (incorporation) of AA into the platelet phospholipids, and the other was to examine its effect on the liberation of AA and the formation of its metabolites from labelled platelets. Thus in the first case PRP was treated with ginger prior to incubation with labelled AA; and in the second experiment platelets were first labelled with AA, separated from the plasma, washed resuspended in a buffer and then treated with ginger prior to activation with Ca 2)+ ionophore A23187 (10 uM) for the liberation of AA. The details of the method for the extraction, separation and determination of the AA metabolites have been described recently by us (3). Statistical significance was evaluated by Student’s t-test. RESULTS Effects of fractionated materials on platelet thromboxane formation from WCQXWIous AA. The materials present in the extracts with the h s&vents, VIZ., n-hexane, chloroform and ethyl acetate reduced plate~e:fr~!$kne production by 85, 67 and 66% respectively (two determinations). A TLC resolution of the material present in the hexane extract gave two bands with Rf values 0.00 and 0.18 (the latter being yellow in colour). Extracted materials from these bands reduced platelet thromboxane production significantly from exogenous arachidonate (Table 1). A few more bands were revealed in iodine vapour when a more concentrated hexane extract was used. Thus additional three bands (Rf values 0.10, 0.31 and 0.39) were revealed; they were scraped off and extracted. The extracted materials were tested for their effects on platelet thromboxane formation and platelet aggregation. Materials present in the bands inhibited thromboxane formation in platelets with a concomitant increase in the lipoxygenase products (Table 2) (Figure 4). Table 1. Effects of materials present in two TLC bands of ginger components extracted in n-hexane on platelet thromboxane formation from added arachidonate
Rf 0.00 0.18
Control 114245 165 t 73
TxB2 (picomoles/108
platelets/l5
Experimental
n
P
5 6
0.025 0.005
352 15 49 + 30
min)*
* Mean+SD Washed platelets were adjusted to lo8 platelets/200 ul suspension and incubated with either 5 ul alcohol (control) or 5 ul alcoholic solution of the separated material (experimental) prior to challenging with labelled AA (7.13 uM). Arachidonic acid metabolites were extracted after acidification with N HCl to pH 3.0, in ethyl acetate and the solvent evaporated. The residue thus obtained was subjected to TLC using solvent chloroform-methanol-acetic acid (90:80:6, v/v); TxB2 spot (Rf value 0.41) was shown in iodine, scraped off and counted in a liquid scintillation counter.
189
AOP
Eplnephrine 110 PM1 1
IZOpMl I Z=CHCI,
extract
3=Hexone
extmct
10 pl lOpi
Collagen I
2:EA
extract
3:CHClx L=Hexone
l20pglmll
1Op1
extract extract
x) pl 10 p, 10 pl w PI 20 pl 20 PI act
2Opl
Figure 2. Tracings showing the effect of ginger components extracted in the three organic solvents on platelet aggregation induced by ADP, epinephrine and collagen. lo-20 ul of an ethanolic extract were pipetted in an aggregation cuvette, the solvent evaporated by gently’ blowing nitrogen and the residue dissolved in 5 ul ethanol. To this were added 990 ul PRP, the contents mixed by stirring for 30 s and then allowed to remain at room temp for 5 min before inducing aggregation. 1 = control; aggregation was performed in the sequence of numbers on the aggregation curves.
Arochldonate Il.0 rntll I [, b =zone
no 3
c =LO”l
no 1
d =zone
no 2
A I
.!
Figure 3. Tracings showing inhibition of platelet aggregation by some ginger components separated by TLC of the extracted material in n-hexane using the solvent n-hexane-ether-acetic acid (80:20: 1, v/v). Separation profile is shown with TLC bands (zone numbers l-7) on the left and the corresponding Rf values on the right side of the TLC plate.
190
?7irtzs2 was
of inhibition
with n-hexane extract > Chloroform extract >: ethyl acetate extract. As the three solvents were used in the above sequence for extraction, it is likely that most of the active principles were extracted in n-hexane, less in chloroform and the least in ethyl acetate. Hence the degree of inhibition by a particular extract might have more to do with the sequence of extraction than with the chemical nature of the solvent used (Figure 1). However, as expected most polar substances (components) showing at the application line in the TLC system used, were extracted in ethyl acetate and least in n-hexane. Higher doses of the extracts were needed to inhibit ADP-, epinephrine- and collagen-induced aggregations. The second phase of epinephrine-induced aggregation was inhibited by the three extracts, and as expected the hexane extract was most potent. For the abolition of collagen-induced aggregation relatively larger doses were needed (Figure 2). Arochadonate _(l.OmMl
Arochidonote .Il.OmHl
Arachidonate ll.OmMl
Figure 1. Tracings showing the effect of ginger components extracted in three organic solvents on arachidonic acid-induced aggregation. Volume (u0 of a certain extract denotes the amount of alcoholic solution of the extracted material in a particular solvent. 5 ul of E.A. extract = 9 ul from 600 ul alcoholic solution of the extracted material in ethyl acetate (see Methods). Tracing 1 is controi; aggregation was performed in the sequence of numbers on the aggregation curves. In contrast to the effects of materials extracted with n-hexane, chloroform and ethyl acetate (which inhibited aggregation induced by ADP, epi#phrine, collagen and AA) only materials from a few TLC bands showed inhlbltlon of aggregation induced by the aggregating agents. Materials present in TLC bamls with Rf 0.00 and 0.18 were the most effective inhibitors. As with tha extracts in organic solvents, the materials of these two bands were most effective in AAinduced ation (Figure 3). Al materials from other TLC bands were effective in reducing pktelet TxB2 formation without inhsbiting AA-induced aggregation, this inconsistency may 191
N
a
2885 + 937 (n=6)
0.39
81
11112 480
981 f 242
546 + 188
4492
422 + 169
Experimental
co.005
P
110312 1843 (n=4)
9219 + 1076 (n=4)
11036 + 1398 (n=4)
9892 + 1417 (n=4)
11036 + 1398 (n=4)
Control
15053 + 2295
16739 + 4056
15892 + 3089
17859 + 1900
17177 + 2698
Experimental
products*
and on lipoxygenase
Lipoxygenase
TxB2 formation
co.05
co.025
co.025
<0.005
co.005
P
products from
* Counts are mainly due to HETE, the presence of other lipoxygenase products is not excluded. Platelets were pretreated with the materials contained in various fractions prior to incubation with labelled AA (9.43 uM). The values (DPM + SD) are l/8 of the total amount produced by IO8 platelets present in the incubation medium. TxB2 was resolved from other AA metabolites as mentioned earlier (Table 1); for the separation of lipoxygenase products solvent system n-hexane-ether-acetic acid (80:20:1, v/v) was used. The counts of HETE were obtained by substracting the TxB2 counts from the total counts due to HETE and HHT (HHT = TxB2) localized in a certain area of the plate in our TLC system (4). This method for the quantitative determination of HETE was used because of the lack of reference standards, that is, of HETE and HHT.
2677 + 630 (n=5)
3302 + 397 (n=6)
0.18
0.31
2993 $651 (n=5)
3302 + 397 (n=6)
Control
0.10
0.00
Rf
TxB2
Table 2. Effects of materials present in several TLC bands on platelet exogenous arachidonate
ii
TxB2’= 392 19 17 _t II*** (n = 20)
Phospholipidsb 978 + 306 868 + 254s * (n = 20)
Phospholipidsa
807 + 260 746 + 228” (n = 20)
product&
404 + 174 519 + 181*** (n = 12)
Lipoxygenase
44 + 29 155+91*** (n = 20)
AA’=Ss
of some AA
* p < 0.05 ** p < 0.01 *** p < 0.001. The values (CPM, Mean + SD) are l/6 of the total amount produced by 0.40~10~ platelets present in the incubation medium. a Counts are due to phospholipids. Thromboxane B2, prostaglandins (PCs) and other hydroxy metabolites of AA were resolved completely from the phospholipids using the solvent system ethyl acetate-isooctane-acetic acid-water (I 10:50:20:100, v/v). b Counts are due to phospholipids, TxB2, PGs and other AA-metabolites. HHT, HETE (HPETE) and AA were resolved using the solvent system n-hexane-ether-acetic acid (80:20:1, v/v) (4). c Counts are shown after substracting the background counts. 5 Includes counts mainly due to lipoxygenase products (HETE, HPETE). 88 Excess of the liberated AA. With another ginger extract preparation it was found that the lipoxygenase products were reduced. However, other effects were similar to those reported-in this table.
Control + Ginger extract
Exp. condition
Table 3. Effects of the aqueous extract of ginger on platelets phospholipase activity and on the formation metabolites produced as a result of treatment of .prelabelled platelets with A23187
be explained on the basis of the difference in concentrations of the materials used in the two experiments - viz., aggregation and incubation experiment for TxB2 determination. The volume of PRP (aggregation expt.) was ca. 5 times the volume of platelet suspension used in the incubation experiments; the same amount of material from a certain TLC band being used in both the experiments. In addition in aggregation experiments the plasma proteins might bind some of the components thus reducing further the effective concentrations. Effects of aqueous extract of ginger on A23187-induced formation of thromboxane and lipoxygenase products in platelets. The experimental design used for this purpose required the use of AA-labelled platelets which were activated by Ca2+ ionophore A23187 after treatment with ginger (experimental platelets) or with an equal volume of water (control platelets). The liberated AA was converted into various AA metabolites of which TxB2 and lipoxygenase products were determined. In the ginger-treated platelets TxB2 was significantly reduced, and at the same time lipoxygenase products and the amount of released AA increased (Table 3). Even though less counts were present in the phospholipid fraction from the gingertreated platelets (indicating an increased phospholipase activity) a reduced formation of TxB2 indicates that ginger inhibits the enzyme(s) cyclooxygenase/Txsynthetase - most probably the former (1) (Table 2). This is further supported by a reduced thromboxane B formation (measured by RIA) in ginger-treated blood after clotting (data not shownf . Effects of materials present in two TLC bands (Rf values 0.00 and 0.18) on A23187induced formation of thromboxane and lipoxy~enase products. The experimental procedure employed was similar to that described for examining the effects of aqueous extract of ginger on the endogenous formation of AA-metabolites in platelets. The effects of the materials present in the two TLC bands were examined at two dose-levels, 10 ug and 100 ug/500 ul AA-labelled platelet suspension. At both dose-levels, TxB2 was significantly reduced, and the effect was dose-dependent. Lipoxygenase products were reduced at the higher dose-level, though significant only with the material present in the band with Rf value 0.00. At the two dose-levels, inhibition of phosphoiipase activity was observed because more counts were present in the phospholipid fraction from treated platelets. As expected this effect was more pronounced at the higher dose-level (Table 4). There was observed an increased number of counts in the fraction of released AA from the treated platelets. This is in agreement to our earlier observation (and also this paper, Table 3) with aqueous ginger extract which inhibits platelet cyclooxygenase with the result that excess AA is either diverted to platelet lipoxygenase pathway and/or recovered unutilized. Table 5. Effect of the aqueous extract of ginger on the incorporation arachidonate into platelet phospholipids Exp. condition
Phospholipids
Control + Ginger extract (n = 8)
2979 + 811 3669 + 1135*
of labelled
* p < 0.01 The values are l/6 of the total amount produced by 1x109 platelets (CPM, Mean + SD) present in the incubation medium. Phospholipids (Rf = 0.00) were separated by
194
g
f;qonentd
949 + 104 1084 + 207
Control + Ginger componente (100 ug) (n=4)
7 8**
372 5+
552 302 7 I**
15 4*
335 8 4+2**
38+ 275
TxB2c
products5
459 + 125 282 + 24
666 + 123 651 + 86
321 + 46 179 + 73””
486 + 105 462 + 89
Lipoxygenase
of thromboxane A23187
products
17 12
4 6
2 4”
medium.
232 8 77 + 27”
272 352
112 152
112 182
AAcSs
and lipoxygenase
a b c Q and §S see Table 3. vHl;e b.00. value 0.18. amount produced by 0.45~109 platelets present in the incubation
1128 + 115 11782 181
1361 + 379 1431 t 345
1053 + 167 1225 ?r 149**
1064 + 108 1003 + 144
Phospholipidsb
For the explanation of z ~x<,~~~ed*~a~eri”,;“:;,m the TLC band with Rf e Extracted material from the TLC band with Rf The values (CPM, Mean 2 SD) are l/6 of the total
1247 f 341 1317 fr 327
939 + 160 1168 + 162**
9262 81 919 + 103
Phospholipidsa
Control + Ginger componente (10 ugl (n=4)
Control + Ginger componentd (100 ug) (n=4)
Control + @fiz
Exp. condition
Table 4. Effects of the materials contained in two TLC bands on the formation produced on treatment of AA-labelled platelets with calcium ionophore
TLC using solvent mixture water (110:50:20:100, v/v).
(organic
phase) ethyl acetate-isooctane-acetic
acid-
DISCUSSION Recently we have shown that aqueous extract of ginger inhibited platelet aggregation induced by several aggregating agents and this was explained by its inhibitory effect on the platelet cyclooxygenase. In ginger-treated platelets reduced amounts of prostaglandin-endoperoxides, prostaglandins and thromboxane were produced from exogenous AA. A good correlation between the anti-aggregation activity of ginger extract and its effect on platelet prostaglandin- and thromboxane synthesis was found (1). We have speculated that the mechanism behind the well-known effects of ginger in ameliorating several ailments - e.g., cold, cough, fever, pain ,in the joints etc., is due to its effect on the cyclooxygenase enzyme with the result that less PGs are produced. An elevated level of PGs is often found in association with the symptoms described above (5). In an experimental set-up where platelets are pretreated with a ginger preparation prior to addition of AA, a reduced formation of the AA metabolites may be due to one or more of the following effects: (i) ginger may inhibit the cyclooxygenase and lipoxygenase enzymes, and (ii) it may inhibit incorporation of AA into platelets. In addition, if ginger inhibited platelet phospholipase activity, the formation of thromboxane and lipoxygenase products would be reduced as a result. Ginger was found slightly to potentiate the phospholipase activity, that is, under the experimental conditions used, a slightly increased amount of AA was released from the treated- compared to control platelets. Even in this situation a reduced formation of TxB2 may indicate that ginger inhibits cyclooxygenase/Txsynthetase enzymes. In experiments where washed platelets are treated with high doses of labelled AA, large amounts of thromboxanes are produced. Inhibition of platelet cyclooxygenase by ginger should result in an increased production of lipoxygenase products due to redirection of AA from cyclooxygenaseto lipoxygenase enzyme. However, if one takes a look at Table 2, one may find that the increase of lipoxygenase products in treated platelets is not accounted for by the respective decrease of TxB2; rather more lipoxygenase products were formed than that would be expected from the decrease of thromboxane. This could either be due to potentiation of the lipoxygenase enzyme or that some other lipoxygenase products were formed in ginger-treated platelets as reported in the case of onion and garlic (5). This argument may find support from the results reported in Table 3. Increased formation of lipoxygenase products in treated platelets cannot be accounted for by the decrease of thromboxane because the counts due to TxB2 were small (~40 cpm). But there is no denying of the fact that aqueous ginger extract inhibited the utilization of released AA as more of it were recovered unused from the gingertreated platelets. A comparison of the effects of materials present in the two TLC bands (Rf values 0.00 and 0.18) on the formation of lipoxygenase products in two different experimental conditions, viz., in the first when labelled AA (9.43 u&I) was added after treatment of platelets with these compounds (Table 2), and in the second where AA-labelled platelets were treated with the garlic components and then activated by Ca2+ ionophore A23187, shows that the effects were opposite. In the first case where AA was added in higher amounts (>9 uM), large amounts of thromboxane were produced. As thromboxane was drastically reduced by the ginger components, a redirection of the substrate (AA) from the cyclooxygenase to the lipoxygenase pathway may account largely for the enhanced formation of the lipoxygenase products. In the second situation both thromboxane and lipoxygenase 196
products were reduced. The difference in the effects of the ginger components in the above two experimental conditions might be due to the mode in which the substrate (AA) is made available and/or possibly to its concentration. Since an increased incorporation of AA took place in the platelet phospholipids in the ginger-treated platelets, in the absence of an inhibitory effect on the platelet phospholipase activity, treated platelets should furnish (release) more AA resulting in a higher production of the AA metabolites. But in view of the observation that ginger inhibits the cylcooxygenase activity it would be interesting to examine the production of TxB2 in a feeding experiment.
Figure 4. Autoradiogram of the TLC plate showing the separation of some AA metabolites formed from exogenous AA by washed platelets. For the separation of AA metabolites solvent system n-hexane-ether-acetic acid (80:20:1, v/v) (ref. 4) was used. TxB2 band (Rf=O.OO) contained counts also due to prostaglandins and phospholipids. 1 = control for 2 (treated with ginger juice); 3 = control for 4 and 5 (treated respectively with materials in fractions with Rf 0.00 and 0.18). ACKNOWLEDGEMENTS The author wishes to thank the personnel of the Blood Bank, Odense University Hospital, Odense for collection of blood samples. Mrs. Ruth B. Alexandersen provided technical assistance. Mrs. Inge Bggelund and Mrs. Yrsa Kildeberg typed the manuscript. This work was supported by the Danish Medical Research Council (Grant No. 12-4832). REFERENCES 1.
Srivastava, K.C. Effects of aqueous extracts of onion, garlic and ginger on platelet aggregation and metabolism of arachidonic acid in the blood vascular system: in vitro study. Prostaglandins Leukotrienes and Medicine 13: 227-235, 1984.
2. Srivastava, K.C. Aqueous extracts of onion, garlic and ginger inhibit platelet aggregation and alter arachidonic acid metabolism. Biomedica Biochimica Acta 43: 335-346, 1984. 3. Srivastava, K.C. Evidence for the mechanism by which garlic inhibits platelet aggregation. Prostaglandins Leukotrienes and Medicine 22: 3 13-321, 1986. 4. Srivastava, K.C., Tiwari, K.P. A simple procedure for the thin-layer chromatographic separation and determination of prostaglandins and other metabolites 197
formed from 14C-arachidonic acid in human blood platelets. schrift fflr Analytische Chemie 304: 412-416, 1980. 5. Nakano, J., Koss, MC. Pathophysiologic roles of prostaglandins of aspirin-like drugs. South. Medical Journal 66: 709-723, 1973.
Fresenius
Zeit-
and the action
6. Vanderhoek, J.Y., Makheja, A.N., Bailey, J.M. Inhibition of fatty acid oxygenases by onion and garlic oils. Evidence for the machanism by which these oils inhibit platelet aggregation. Biochemical Pharmacology 29: 3169-3173, 1980.
198
Leukotrienes
and Medicine
25:
187-198,
1986
AND EFFECTS OF SOME GINGER COMPONENTS ON PLATELET AGGREGATION AND EICOSANOID BIOSYNTHESIS
ISOLATION
K.C. Srivastava, Department of Environmental Medicine, Institute of Community Health, Odense University, J.B. WinsMws Vej 19, DK-5000 Odense C, Denmark. ABSTRACT Aqueous ginger extract was extracted in three organic solvents viz., nhexane, chloroform and ethyl acetate with increasing polarity. The extracted materials from these solvents reduced platelet thromboxane formation from exogenous arachidonate (AA) and also inhibited platelet aggregation induced by AA, epinephrine, ADP and collagen; in this respect they were most effective against AA-induced aggregation. The extracted material in n-hexane was further resolved by thin-layer chromatography into various fractions some of which were effective in inhibiting platelet thromboxane formation and platelet g&on. Aqueous- ginger extract reduced the formation of TxEi2 from AA-label&d platelets without showing effects on platelet phospholipase activity. Thromboxane formation in labelled platelets on activation with calcium ionophore A23187 was reduced by ginger components, isolated from two TLC bands, in a dose-dependent manner (lo100 q/500 ml). At the higher dose lipoxygenase products were also reduced. Interestingly the incorporation of AA into platelet phospholipids increased in platelets treated with aqueous ginger extract. INTRODUCTION Ginger (genus Zingiber) root is used in cooking and medicine, and for this purpose it is used in fresh as well as in dry powdered form. It is preserved in syrup or candied as sweet. In the Indian subcontinent fresh ginger is widely used in the preparation of vegetable and meat dishes besides being used in the preparation of pickles. In the Indian system of medicine, Ayurveda, ginger is described to ameliorate and cure many ailments - e.g., cold, cough, fever, pain in the joints etc. The mechanism by which ginger’s medicinal properties could be accounted for is not known. However, recently we have described the effects of aqueous extract of ginger on aggregation and metabolism of arachidonic acid (AA) in piateiets. Ginger extract inhibited platelet aggregation induced by ADP, collagen, epinephrine and Presented, in part, at the IVth International Symposium on Prostaglandins, Thromboxanes and Leukotrienes in the Cardiovascular System, Halle 1984.
187
arachidonate; it did not inhibit Ca2+ ionophore A23187-induced aggregation. It reduced the formation of thromboxane (TX) and prostaglandins (PCs) in platelets (1). In this paper results are reported on the effects of some components of ginger on platelet aggregation, and on platelet cyclooxygenase- and lipoxygenase products’ formation together with the effects of aqueous extract of ginger on platelet phospholipase activity, formation of thromboxane B2 and lipoxygenase products from labelled platelets, and on the incorporation of AA into platelet phospholipids. In short, this paper describes an experimental approach to clarify the mechanism by which ginger inhibits platelet aggregation. MATERIALS AND METHODS Arachidonic acid (1-14~) (specific activity 59.3 mCi/mmol) was purchased from the Radiochemical Centre, Amersham, England. Thromboxane B2 (TxB2) was supplied free by ON0 Pharmaceutical Co., Ltd., Osaka (Japan). Calcium ionophore A23187 was a Sigma product. Thin-layer chromatography (TLC) plates were prepared in our laboratory. All reagents were of analytical reagent grade. Fractionation of ginger extract in organic solvents (with increasing polarity) nhexane, chloroform and ethyl acetate has been described (2). Ginger extract (24 ml) was diluted with 30 ml water followed by extraction in n-hexane (60 ml) by shaking and allowing to equilibrate at room temp (RT) for 30 min. The organic phase was separated from the aqueous phase (lower), the latter was adjusted to pH 3.0 with HCl and extracted in chloroform (120 ml) overnight in cold (4OC). After separating the chloroform layer, the aqueous phase was further extracted in ethyl acetate (100 ml). Organic solvents were evaporated by blowing nitrogen at 700C. Oily residues were obtained from the three extracts; they were dissolved respectively in 1000, 800 and 600 ul ethanol and stored at -200C. Thin-layer separation of ginger components. Resolution of the material present in the hexane extract was done by TLC. One hundred and fifty microliters (from 1 ml ethanolic solution of the hexane extract) were resolved by TLC using the solvent nhexane-ether-acetic acid (80:20:1, v/v). Prior to exposure to iodine vapour, two bands - one with Rf value 0.18 (yellow) and the other, application band (Rf = 0.00) were marked and scraped off. On exposing the plate to iodine, five more clearly separated bands were shown. They were marked and scraped off. The bands were extracted in chloroform-methanol-ethyl acetate (2: 1: 1, v/v). The solvent was evaporated by blowing nitrogen at 450C. The extracted material from each band was dissolved in 150 ul ethanol. This was either used as such or was concentrated prior to use in aggregation experiments. To obtain sufficient material from other TLC bands 90 ml juice (from ca. 330 g peeled ginger) were extracted as described above. The extraction was done in the same sequence, starting with n-hexane (500 ml), chloroform (ZOO ml) and finally with ethyl acetate (200 ml). About 150 ml n-hexane extract were evaporated at 70°C under nitrogen to yield 89 mg of an oily residue. This was dissolved in 1 ml chloroform, of which 150 ul were resolved by TLC as above. Before exposing the plate to iodine, TLC bands with Rf values 0.00 and 0.18 (yellow) were scraped off. Due to a higher amount of the material some more bands were shown including the seven bands mentioned above. The bands were scraped off and extracted in chloroform-methanol-ethyl acetate (2: 1: 1, v/v). The solvent was evaporated at 50°C under nitrogen and the residue dissolved in 100 ul ethanol. The extracted material from each TLC band was examined for its effect on platelet aggregation and thromboxane formation; in both experiments only 5 ul ethanolic solution of the extracted material were used. However, in aggregation experiments with collagen, epinephrine and ADP where more material was needed for inhibition of aggregat188
ion, alcohol was evaporated from the aggregation cuvette by slowly blowing N2 and the residue dissolved in 5 ul ethanol prior to incubation with platelet-rich plasma (PRP). Experiments with labelled platelets. Labelling of platelets in PRP was done for two specific purposes. The first was to examine the effect of ginger (aqueous) on labelling (incorporation) of AA into the platelet phospholipids, and the other was to examine its effect on the liberation of AA and the formation of its metabolites from labelled platelets. Thus in the first case PRP was treated with ginger prior to incubation with labelled AA; and in the second experiment platelets were first labelled with AA, separated from the plasma, washed resuspended in a buffer and then treated with ginger prior to activation with Ca 2)+ ionophore A23187 (10 uM) for the liberation of AA. The details of the method for the extraction, separation and determination of the AA metabolites have been described recently by us (3). Statistical significance was evaluated by Student’s t-test. RESULTS Effects of fractionated materials on platelet thromboxane formation from WCQXWIous AA. The materials present in the extracts with the h s&vents, VIZ., n-hexane, chloroform and ethyl acetate reduced plate~e:fr~!$kne production by 85, 67 and 66% respectively (two determinations). A TLC resolution of the material present in the hexane extract gave two bands with Rf values 0.00 and 0.18 (the latter being yellow in colour). Extracted materials from these bands reduced platelet thromboxane production significantly from exogenous arachidonate (Table 1). A few more bands were revealed in iodine vapour when a more concentrated hexane extract was used. Thus additional three bands (Rf values 0.10, 0.31 and 0.39) were revealed; they were scraped off and extracted. The extracted materials were tested for their effects on platelet thromboxane formation and platelet aggregation. Materials present in the bands inhibited thromboxane formation in platelets with a concomitant increase in the lipoxygenase products (Table 2) (Figure 4). Table 1. Effects of materials present in two TLC bands of ginger components extracted in n-hexane on platelet thromboxane formation from added arachidonate
Rf 0.00 0.18
Control 114245 165 t 73
TxB2 (picomoles/108
platelets/l5
Experimental
n
P
5 6
0.025 0.005
352 15 49 + 30
min)*
* Mean+SD Washed platelets were adjusted to lo8 platelets/200 ul suspension and incubated with either 5 ul alcohol (control) or 5 ul alcoholic solution of the separated material (experimental) prior to challenging with labelled AA (7.13 uM). Arachidonic acid metabolites were extracted after acidification with N HCl to pH 3.0, in ethyl acetate and the solvent evaporated. The residue thus obtained was subjected to TLC using solvent chloroform-methanol-acetic acid (90:80:6, v/v); TxB2 spot (Rf value 0.41) was shown in iodine, scraped off and counted in a liquid scintillation counter.
189
AOP
Eplnephrine 110 PM1 1
IZOpMl I Z=CHCI,
extract
3=Hexone
extmct
10 pl lOpi
Collagen I
2:EA
extract
3:CHClx L=Hexone
l20pglmll
1Op1
extract extract
x) pl 10 p, 10 pl w PI 20 pl 20 PI act
2Opl
Figure 2. Tracings showing the effect of ginger components extracted in the three organic solvents on platelet aggregation induced by ADP, epinephrine and collagen. lo-20 ul of an ethanolic extract were pipetted in an aggregation cuvette, the solvent evaporated by gently’ blowing nitrogen and the residue dissolved in 5 ul ethanol. To this were added 990 ul PRP, the contents mixed by stirring for 30 s and then allowed to remain at room temp for 5 min before inducing aggregation. 1 = control; aggregation was performed in the sequence of numbers on the aggregation curves.
Arochldonate Il.0 rntll I [, b =zone
no 3
c =LO”l
no 1
d =zone
no 2
A I
.!
Figure 3. Tracings showing inhibition of platelet aggregation by some ginger components separated by TLC of the extracted material in n-hexane using the solvent n-hexane-ether-acetic acid (80:20: 1, v/v). Separation profile is shown with TLC bands (zone numbers l-7) on the left and the corresponding Rf values on the right side of the TLC plate.
190
?7irtzs2 was
of inhibition
with n-hexane extract > Chloroform extract >: ethyl acetate extract. As the three solvents were used in the above sequence for extraction, it is likely that most of the active principles were extracted in n-hexane, less in chloroform and the least in ethyl acetate. Hence the degree of inhibition by a particular extract might have more to do with the sequence of extraction than with the chemical nature of the solvent used (Figure 1). However, as expected most polar substances (components) showing at the application line in the TLC system used, were extracted in ethyl acetate and least in n-hexane. Higher doses of the extracts were needed to inhibit ADP-, epinephrine- and collagen-induced aggregations. The second phase of epinephrine-induced aggregation was inhibited by the three extracts, and as expected the hexane extract was most potent. For the abolition of collagen-induced aggregation relatively larger doses were needed (Figure 2). Arochadonate _(l.OmMl
Arochidonote .Il.OmHl
Arachidonate ll.OmMl
Figure 1. Tracings showing the effect of ginger components extracted in three organic solvents on arachidonic acid-induced aggregation. Volume (u0 of a certain extract denotes the amount of alcoholic solution of the extracted material in a particular solvent. 5 ul of E.A. extract = 9 ul from 600 ul alcoholic solution of the extracted material in ethyl acetate (see Methods). Tracing 1 is controi; aggregation was performed in the sequence of numbers on the aggregation curves. In contrast to the effects of materials extracted with n-hexane, chloroform and ethyl acetate (which inhibited aggregation induced by ADP, epi#phrine, collagen and AA) only materials from a few TLC bands showed inhlbltlon of aggregation induced by the aggregating agents. Materials present in TLC bamls with Rf 0.00 and 0.18 were the most effective inhibitors. As with tha extracts in organic solvents, the materials of these two bands were most effective in AAinduced ation (Figure 3). Al materials from other TLC bands were effective in reducing pktelet TxB2 formation without inhsbiting AA-induced aggregation, this inconsistency may 191
N
a
2885 + 937 (n=6)
0.39
81
11112 480
981 f 242
546 + 188
4492
422 + 169
Experimental
co.005
P
110312 1843 (n=4)
9219 + 1076 (n=4)
11036 + 1398 (n=4)
9892 + 1417 (n=4)
11036 + 1398 (n=4)
Control
15053 + 2295
16739 + 4056
15892 + 3089
17859 + 1900
17177 + 2698
Experimental
products*
and on lipoxygenase
Lipoxygenase
TxB2 formation
co.05
co.025
co.025
<0.005
co.005
P
products from
* Counts are mainly due to HETE, the presence of other lipoxygenase products is not excluded. Platelets were pretreated with the materials contained in various fractions prior to incubation with labelled AA (9.43 uM). The values (DPM + SD) are l/8 of the total amount produced by IO8 platelets present in the incubation medium. TxB2 was resolved from other AA metabolites as mentioned earlier (Table 1); for the separation of lipoxygenase products solvent system n-hexane-ether-acetic acid (80:20:1, v/v) was used. The counts of HETE were obtained by substracting the TxB2 counts from the total counts due to HETE and HHT (HHT = TxB2) localized in a certain area of the plate in our TLC system (4). This method for the quantitative determination of HETE was used because of the lack of reference standards, that is, of HETE and HHT.
2677 + 630 (n=5)
3302 + 397 (n=6)
0.18
0.31
2993 $651 (n=5)
3302 + 397 (n=6)
Control
0.10
0.00
Rf
TxB2
Table 2. Effects of materials present in several TLC bands on platelet exogenous arachidonate
ii
TxB2’= 392 19 17 _t II*** (n = 20)
Phospholipidsb 978 + 306 868 + 254s * (n = 20)
Phospholipidsa
807 + 260 746 + 228” (n = 20)
product&
404 + 174 519 + 181*** (n = 12)
Lipoxygenase
44 + 29 155+91*** (n = 20)
AA’=Ss
of some AA
* p < 0.05 ** p < 0.01 *** p < 0.001. The values (CPM, Mean + SD) are l/6 of the total amount produced by 0.40~10~ platelets present in the incubation medium. a Counts are due to phospholipids. Thromboxane B2, prostaglandins (PCs) and other hydroxy metabolites of AA were resolved completely from the phospholipids using the solvent system ethyl acetate-isooctane-acetic acid-water (I 10:50:20:100, v/v). b Counts are due to phospholipids, TxB2, PGs and other AA-metabolites. HHT, HETE (HPETE) and AA were resolved using the solvent system n-hexane-ether-acetic acid (80:20:1, v/v) (4). c Counts are shown after substracting the background counts. 5 Includes counts mainly due to lipoxygenase products (HETE, HPETE). 88 Excess of the liberated AA. With another ginger extract preparation it was found that the lipoxygenase products were reduced. However, other effects were similar to those reported-in this table.
Control + Ginger extract
Exp. condition
Table 3. Effects of the aqueous extract of ginger on platelets phospholipase activity and on the formation metabolites produced as a result of treatment of .prelabelled platelets with A23187
be explained on the basis of the difference in concentrations of the materials used in the two experiments - viz., aggregation and incubation experiment for TxB2 determination. The volume of PRP (aggregation expt.) was ca. 5 times the volume of platelet suspension used in the incubation experiments; the same amount of material from a certain TLC band being used in both the experiments. In addition in aggregation experiments the plasma proteins might bind some of the components thus reducing further the effective concentrations. Effects of aqueous extract of ginger on A23187-induced formation of thromboxane and lipoxygenase products in platelets. The experimental design used for this purpose required the use of AA-labelled platelets which were activated by Ca2+ ionophore A23187 after treatment with ginger (experimental platelets) or with an equal volume of water (control platelets). The liberated AA was converted into various AA metabolites of which TxB2 and lipoxygenase products were determined. In the ginger-treated platelets TxB2 was significantly reduced, and at the same time lipoxygenase products and the amount of released AA increased (Table 3). Even though less counts were present in the phospholipid fraction from the gingertreated platelets (indicating an increased phospholipase activity) a reduced formation of TxB2 indicates that ginger inhibits the enzyme(s) cyclooxygenase/Txsynthetase - most probably the former (1) (Table 2). This is further supported by a reduced thromboxane B formation (measured by RIA) in ginger-treated blood after clotting (data not shownf . Effects of materials present in two TLC bands (Rf values 0.00 and 0.18) on A23187induced formation of thromboxane and lipoxy~enase products. The experimental procedure employed was similar to that described for examining the effects of aqueous extract of ginger on the endogenous formation of AA-metabolites in platelets. The effects of the materials present in the two TLC bands were examined at two dose-levels, 10 ug and 100 ug/500 ul AA-labelled platelet suspension. At both dose-levels, TxB2 was significantly reduced, and the effect was dose-dependent. Lipoxygenase products were reduced at the higher dose-level, though significant only with the material present in the band with Rf value 0.00. At the two dose-levels, inhibition of phosphoiipase activity was observed because more counts were present in the phospholipid fraction from treated platelets. As expected this effect was more pronounced at the higher dose-level (Table 4). There was observed an increased number of counts in the fraction of released AA from the treated platelets. This is in agreement to our earlier observation (and also this paper, Table 3) with aqueous ginger extract which inhibits platelet cyclooxygenase with the result that excess AA is either diverted to platelet lipoxygenase pathway and/or recovered unutilized. Table 5. Effect of the aqueous extract of ginger on the incorporation arachidonate into platelet phospholipids Exp. condition
Phospholipids
Control + Ginger extract (n = 8)
2979 + 811 3669 + 1135*
of labelled
* p < 0.01 The values are l/6 of the total amount produced by 1x109 platelets (CPM, Mean + SD) present in the incubation medium. Phospholipids (Rf = 0.00) were separated by
194
g
f;qonentd
949 + 104 1084 + 207
Control + Ginger componente (100 ug) (n=4)
7 8**
372 5+
552 302 7 I**
15 4*
335 8 4+2**
38+ 275
TxB2c
products5
459 + 125 282 + 24
666 + 123 651 + 86
321 + 46 179 + 73””
486 + 105 462 + 89
Lipoxygenase
of thromboxane A23187
products
17 12
4 6
2 4”
medium.
232 8 77 + 27”
272 352
112 152
112 182
AAcSs
and lipoxygenase
a b c Q and §S see Table 3. vHl;e b.00. value 0.18. amount produced by 0.45~109 platelets present in the incubation
1128 + 115 11782 181
1361 + 379 1431 t 345
1053 + 167 1225 ?r 149**
1064 + 108 1003 + 144
Phospholipidsb
For the explanation of z ~x<,~~~ed*~a~eri”,;“:;,m the TLC band with Rf e Extracted material from the TLC band with Rf The values (CPM, Mean 2 SD) are l/6 of the total
1247 f 341 1317 fr 327
939 + 160 1168 + 162**
9262 81 919 + 103
Phospholipidsa
Control + Ginger componente (10 ugl (n=4)
Control + Ginger componentd (100 ug) (n=4)
Control + @fiz
Exp. condition
Table 4. Effects of the materials contained in two TLC bands on the formation produced on treatment of AA-labelled platelets with calcium ionophore
TLC using solvent mixture water (110:50:20:100, v/v).
(organic
phase) ethyl acetate-isooctane-acetic
acid-
DISCUSSION Recently we have shown that aqueous extract of ginger inhibited platelet aggregation induced by several aggregating agents and this was explained by its inhibitory effect on the platelet cyclooxygenase. In ginger-treated platelets reduced amounts of prostaglandin-endoperoxides, prostaglandins and thromboxane were produced from exogenous AA. A good correlation between the anti-aggregation activity of ginger extract and its effect on platelet prostaglandin- and thromboxane synthesis was found (1). We have speculated that the mechanism behind the well-known effects of ginger in ameliorating several ailments - e.g., cold, cough, fever, pain ,in the joints etc., is due to its effect on the cyclooxygenase enzyme with the result that less PGs are produced. An elevated level of PGs is often found in association with the symptoms described above (5). In an experimental set-up where platelets are pretreated with a ginger preparation prior to addition of AA, a reduced formation of the AA metabolites may be due to one or more of the following effects: (i) ginger may inhibit the cyclooxygenase and lipoxygenase enzymes, and (ii) it may inhibit incorporation of AA into platelets. In addition, if ginger inhibited platelet phospholipase activity, the formation of thromboxane and lipoxygenase products would be reduced as a result. Ginger was found slightly to potentiate the phospholipase activity, that is, under the experimental conditions used, a slightly increased amount of AA was released from the treated- compared to control platelets. Even in this situation a reduced formation of TxB2 may indicate that ginger inhibits cyclooxygenase/Txsynthetase enzymes. In experiments where washed platelets are treated with high doses of labelled AA, large amounts of thromboxanes are produced. Inhibition of platelet cyclooxygenase by ginger should result in an increased production of lipoxygenase products due to redirection of AA from cyclooxygenaseto lipoxygenase enzyme. However, if one takes a look at Table 2, one may find that the increase of lipoxygenase products in treated platelets is not accounted for by the respective decrease of TxB2; rather more lipoxygenase products were formed than that would be expected from the decrease of thromboxane. This could either be due to potentiation of the lipoxygenase enzyme or that some other lipoxygenase products were formed in ginger-treated platelets as reported in the case of onion and garlic (5). This argument may find support from the results reported in Table 3. Increased formation of lipoxygenase products in treated platelets cannot be accounted for by the decrease of thromboxane because the counts due to TxB2 were small (~40 cpm). But there is no denying of the fact that aqueous ginger extract inhibited the utilization of released AA as more of it were recovered unused from the gingertreated platelets. A comparison of the effects of materials present in the two TLC bands (Rf values 0.00 and 0.18) on the formation of lipoxygenase products in two different experimental conditions, viz., in the first when labelled AA (9.43 u&I) was added after treatment of platelets with these compounds (Table 2), and in the second where AA-labelled platelets were treated with the garlic components and then activated by Ca2+ ionophore A23187, shows that the effects were opposite. In the first case where AA was added in higher amounts (>9 uM), large amounts of thromboxane were produced. As thromboxane was drastically reduced by the ginger components, a redirection of the substrate (AA) from the cyclooxygenase to the lipoxygenase pathway may account largely for the enhanced formation of the lipoxygenase products. In the second situation both thromboxane and lipoxygenase 196
products were reduced. The difference in the effects of the ginger components in the above two experimental conditions might be due to the mode in which the substrate (AA) is made available and/or possibly to its concentration. Since an increased incorporation of AA took place in the platelet phospholipids in the ginger-treated platelets, in the absence of an inhibitory effect on the platelet phospholipase activity, treated platelets should furnish (release) more AA resulting in a higher production of the AA metabolites. But in view of the observation that ginger inhibits the cylcooxygenase activity it would be interesting to examine the production of TxB2 in a feeding experiment.
Figure 4. Autoradiogram of the TLC plate showing the separation of some AA metabolites formed from exogenous AA by washed platelets. For the separation of AA metabolites solvent system n-hexane-ether-acetic acid (80:20:1, v/v) (ref. 4) was used. TxB2 band (Rf=O.OO) contained counts also due to prostaglandins and phospholipids. 1 = control for 2 (treated with ginger juice); 3 = control for 4 and 5 (treated respectively with materials in fractions with Rf 0.00 and 0.18). ACKNOWLEDGEMENTS The author wishes to thank the personnel of the Blood Bank, Odense University Hospital, Odense for collection of blood samples. Mrs. Ruth B. Alexandersen provided technical assistance. Mrs. Inge Bggelund and Mrs. Yrsa Kildeberg typed the manuscript. This work was supported by the Danish Medical Research Council (Grant No. 12-4832). REFERENCES 1.
Srivastava, K.C. Effects of aqueous extracts of onion, garlic and ginger on platelet aggregation and metabolism of arachidonic acid in the blood vascular system: in vitro study. Prostaglandins Leukotrienes and Medicine 13: 227-235, 1984.
2. Srivastava, K.C. Aqueous extracts of onion, garlic and ginger inhibit platelet aggregation and alter arachidonic acid metabolism. Biomedica Biochimica Acta 43: 335-346, 1984. 3. Srivastava, K.C. Evidence for the mechanism by which garlic inhibits platelet aggregation. Prostaglandins Leukotrienes and Medicine 22: 3 13-321, 1986. 4. Srivastava, K.C., Tiwari, K.P. A simple procedure for the thin-layer chromatographic separation and determination of prostaglandins and other metabolites 197
formed from 14C-arachidonic acid in human blood platelets. schrift fflr Analytische Chemie 304: 412-416, 1980. 5. Nakano, J., Koss, MC. Pathophysiologic roles of prostaglandins of aspirin-like drugs. South. Medical Journal 66: 709-723, 1973.
Fresenius
Zeit-
and the action
6. Vanderhoek, J.Y., Makheja, A.N., Bailey, J.M. Inhibition of fatty acid oxygenases by onion and garlic oils. Evidence for the machanism by which these oils inhibit platelet aggregation. Biochemical Pharmacology 29: 3169-3173, 1980.
198