Inhibitory effect of compounds from Zingiberaceae species on human platelet aggregation.

Introduction

In a previous paper, we reported the ability of compounds from several Zingiberaceae species to displace [sup.3.H]PAF-specific binding from washed rabbit platelets (Jantan et al., 2004). The methanol extracts of these species showed strong antiplatelet aggregation activity in human whole blood in vitro. This paper reports the antiplatelet aggregation activity of compounds previously isolated from five Zingiberaceae species, namely, Alpinia mutica, Kaempferia rotunda, Curcuma xanthorrhiza. Curcuma aromatica and Zingiber

Materials and methods

The rhizomes of C. xanthorrhiza (AR 415), C. aromatica (AR 424), K. rotunda (AR 436) and Z. zerumbet (AR 478), and the fruit of Alpinia mutica (AR 457) were collected from Johore Bahru, Malaysia, and their voucher specimens were deposited at the Herbarium of the Department of Biology, Universiti Putra Malaysia. Twelve pure compounds were isolated from the chloroform extracts of the plants by repeated chromatography on silica gel (230-400 mesh) using petroleum ether--ether gradients of increasingpolarities. They were identified by spectroscopic techniques and by comparison with published data (Sirat et al., 1993; Itokawa et al., 1988; Shiobara et al., 1986; Janssen and Scheffer, 1985). Xanthorrhizol epoxide, l-acetyl-2-methyl-5-(l',5'-dimethylhex-4'-enyl)benzene and 1-methoxy-2-methyl-5-(l',5'-dimethylhex-4'-enyl)benzene were prepared by epoxidation, acetylation and methylation of xanthorrhizol, respectively. Collagen, ADP and arachidonic acid (AA) were purchased from Sigma Chemical Co. (USA).

The use of human whole blood in this study was approved by the Ethics Committee of the Universiti Kebangsaan Malaysia. Blood was collected from volunteers who were selected based on the criteria that they were healthy, non-smokers, had not taken any medications, including aspirin, within the last 2 weeks and had not taken any food within the last 8h. Whole blood (20 ml) was withdrawn from the right arm of a subject into a vacutainer containing 3.8% sodium citrate. The blood and the anticoagulant were thoroughly mixed by inverting the vacutainer several times. The blood sample was diluted with normal saline in the ratio of 1:1. The dried methanol extracts and the isolated compounds were each dissolved in dimethyl sulfoxide (DMSO) to obtain stock solutions of 20, 10, 5 and 2.5 [micro]g/[micro]L. Five microliters of the stock solutions was added to a cuvette containing the diluted whole blood and the mixture was allowed to incubate at 37 [degress]C for 4 min prior to the addition of AA (0.5 mM), ADP (10 [micro]M) or collagen (2[micro]ml). The total volume of the mixture was 1 ml. The final concentrations of the sample in the mixture were 100, 50, 25 and 12.5[micro].g/ml.

Platelet aggregation was measured by a Whole Blood Lumi-Aggregometer (Chrono-Log Corp., Havertown, PA) using an electrical impedance method (Ingerman-Wojenski and Silver, 1984). The mean platelet aggregation in whole blood was measured as a change in impedance over 6 min after the addition of inducers by comparison to that of a control group impedance (Challen et al., 1982). A mixture containing 0.5% DMSO in the diluted whole blood was used as control. Aspirin was used as the positive control. The final concentration of DMSO in the whole blood was 0.5% to eliminate the effect of the solvent on the aggregation (Dong and Chen, 1998).

Each sample was measured in triplicate and the data are presented as means[+ or -]SE. A one-way analysis of variance was used for multiple comparisons and if significant variation occurred between treatment groups, the mean values for inhibitors were compared with those for controls by Student's t test. p<0.05 was considered to be statistically significant. The [IC.sub.50]values of the compounds were obtained from at least three determinations.

Results and discussion

The methanol extracts of the fruit of A. mutica and the rhizomes of K. rotunda, C. xanthorrhiza, C. domestica and Z. zerumbet showed strong antiplatelet aggregation activity at 100 [micro]g/ml in human whole blood in vitro, with all extracts exhibiting 100% inhibition. Twelve compounds isolated from these species were investigated for their effects on platelet aggregation of human whole blood (Fig. 1). Aspirin, a potent cyclooxygenase inhibitor, was used as a positive control in the bioassay (Lloyd and Bochner, 1996). Table 1 shows the % inhibitory effects of the isolated compounds and synthesized derivatives of xanthorrhizol at various concentrations. The compounds showed dose-dependent responses. Among all tested compounds, four compounds, i.e. zerumbone (10), xanthorrhizol (11), curcumin (12) and xanthorrhizol epoxide (13) showed strong inhibition on platelet aggregation caused by all three inducers (AA, collagen and ADP), with inhibitory effects ranging from 64.7% to 100% at 100[micro]gml.

[FIGURE 1 OMITTED]

The [IC.sub.50] values of the active compounds with the mean values of three measurements are shown in Table 2. Curcumin (12) was the most effective antiplatelet compound; it inhibited AA-, collagen- and ADP-induced platelet aggregation with IC[sub.50]50 values of 37.5, 60.9 and 45.7 [micro]M, respectively. The result for AA-induced aggregation by compound 12 was in accordance with the result from a previous study by Shah et al. (1998). However, the latter reported that compound 12 preferentially inhibited AA- and PAF-induced aggregation, whereas much higher concentrations of the compound were required to inhibit aggregation induced by other agonists. 5,6-Dehydroka-wain (5) and 3-deacetylcrotepoxide (9) showed selective inhibitory activity of platelet aggregation induced by AA with IC[sub.50]50 values of 83.8 and 78.1 [micro.M], respectively, while flavokawain A (7), 2',3',4',6'-tetrahydroxychal-cone (4) and crotepoxide (8) fairly inhibited ADP-induced aggregation (IC[sub.50]50; 146.9-216.2 [micro,M] but not that of other inducers. Cardamonin (3) and flavokawain B (6) on the other hand, strongly inhibited platelet aggregation induced by AA and ADP but not by collagen. The strong inhibition of compounds (3) and (5) against AA-induced aggregation was in accordance with the results from a previous study by Dong and Chen (1998). The IC[sub.50]50 values of all the compounds evaluated were higher than that of aspirin (25.3 [micro.M].

Table 1. Percentage inhibition of compounds of Zingiberaceae species
on platelet aggregation in human whole blood induced by arachidonic
acid (AA) (0.5 mM), collagen (2[micro.g/ml]) collagen (2[micro.g.ml)
and ADP(10[micro.M]) (conectration of sample in reaction mixture=
10[micro.g.ml]

Compound                          Plant         AA
                                  species

Pinocembrine (1)                  Alpinia        100.0 [+ or -] 0.0 (b)
                                  mutica

Alpinetin (2)                     Alpinia          0.0 [+ or -] 0.0
                                  mutica

Cardamonin (3)                    Alpinia        100.0 [+ or -] 0.0 (a)
                                  mutica

2',3',4/,6'-TetrahydroxychaIcone  Alpinia         25.0 [+ or -] 0.7
(4)                               mutica

5,6-Dehydrokawain (5)             Alpinia        100.0 [+ or -] 0.0 (a)
                                  mutica

Flavokawain B (6)                 Alpinia        100.0 [+ or -] 0.0 (c)
                                  mutica

Flavokawain A (7)                 Kaempferia       0.0 [+ or -] 0.0
                                  rotunda

Crotepoxide (8)                   Kaempferia      21.4 [+ or -] 0.7
                                  rotunda

3-Deacetylcrotepoxide (9)         Kaempferia             100.0 +0.0 (b)
                                  rotunda

Zerumbone (10)                    Zingiber       100.0 [+ or -] 0.0 (a)
                                  zerumbet

Xanthorrhizol (11)                Curcuma                100.0+ 0.0 (c)
                                  xanthorrhiza

Curcumin (12)                     Curcuma       100.0  [+ or -] 0.0 (c)
                                  aromatica

Xanthorrhizol epoxide (13)        -              100.0 [+ or -] 0.0 (a)

l-Acetyl-2-methyl-5-              -              100.0 [+ or -] 0.0 (b)
(r,5'-dimethylhex-4'enyl)
benzene (14)

l-Methoxy-2-methyl-5-             -                0.0 [+ or -] 0.0
(1,5'-dimethylhex-4'enyl)
benzene (15)

#Aspirin                          -              100.0 [+ or -] 0.0

Compound                          Collagen

Pinocembrine (1)                           11.1+0.6

Alpinetin (2)                     10.6 [+ or -] 0.9

Cardamonin (3)                     0.0 [+ or -] 0.0

2',3',4/,6'-TetrahydroxychaIcone          11.01+0.3
(4)

5,6-Dehydrokawain (5)             27.8 [+ or -] 0.9

Flavokawain B (6)                 33.3 [+ or -] 0.2

Flavokawain A (7)                          14.6+2.1

Crotepoxide (8)                   26.8 [+ or -] 1.4

3-Deacetylcrotepoxide (9)          6.3 [+ or -] 1.4

Zerumbone (10)                    64.7 [+ or -] 0.7

Xanthorrhizol (11)                81.3 [+ or -] 0.7

Curcumin (12)                              83.8+0.7 (b)

Xanthorrhizol epoxide (13)        81.3 [+ or -] 0.7 (c)

l-Acetyl-2-methyl-5-              62.5 [+ or -] 0.7
(r,5'-dimethylhex-4'enyl)
benzene (14)

l-Methoxy-2-methyl-5-             32.5 [+ or -] 2.1
(1,5'-dimethylhex-4'enyl)
benzene (15)

#Aspirin                          31.3 [+ or -] 0.7

Compound                          ADP

Pinocembrine (1)                  78.6 [+ or -] 0.7 (a)

Alpinetin (2)                             42.9+1.4

Cardamonin (3)                    57.1 [+ or -] 2.1

2',3',4/,6'-TetrahydroxychaIcone  71.4 [+ or -] 0.6 (c)
(4)

5,6-Dehydrokawain (5)             40.0 [+ or -] 2.1

Flavokawain B (6)                         100.0+l.l (b)

Flavokawain A (7)                 57.1 [+ or -] 0.3

Crotepoxide (8)                   57.1 [+ or -] 0.3

3-Deacetylcrotepoxide (9)          0.0 [+ or -] 0.0

Zerumbone (10)                    100.0 [+ or -] 0.0 (c)

Xanthorrhizol (11)                 78.6 [+ or -] 0.7 (b)

Curcumin (12)                               85.1+0.7 (b)

Xanthorrhizol epoxide (13)        100.0 [+ or -] 0.0 (a)

l-Acetyl-2-methyl-5-               50.4 [+ or -] 0.7
(r,5'-dimethylhex-4'enyl)
benzene (14)

l-Methoxy-2-methyl-5-              49.4 [+ or -] 3.2
(1,5'-dimethylhex-4'enyl)
benzene (15)

# Aspirin                          42.9 [+ or -] 1.0

# Aspirin was used as a positive control. Concentration of aspirin in
reaction mixture: 25 [micro.g/ml. Values are presented as means
[+ or -]SE in=3); [.sup.a] p<0.05. [.sup.b]p<0.01 and [.sup.c]p<0.001 as
compared with the respective control.

Table 2. [IC.sub.50] values [micro.M] of compounds on platelet
aggregation induced by arachidonic acid (AA) (0.5mM), collagen
(2 [micro.g/ml]) and ADP (10[micro.M])

Compound                                   AA

Pinocembrine (1)                          82.8 [+ or -] 4.1

Cardamomin (3)                            72.6 [+ or -] 2.8

2',3',4',6'-Tetrahydroxychalcone (4)      -

5,6-Dehydrokawain (5)                     78.1 [+ or -] 2.8

Flavokawain B (6)                        106.3 [+ or -] 3.5

Flavokawain A (7)                        -

Crotepoxide (8)                          -

3-Deacetylcrotepoxide (9)                 83.8 [+ or -] 3.5

Zerumbone (10)                           102.6 [+ or -] 4.0

Xanthorrhizol (11)                       104.5 [+ or -] 5.3

Curcumin (12)                             37.5 [+ or -] 3.9

Xanthorrhizol epoxide (13)               117.5 [+ or -] 5.8

l-Acetyl-2-methyl-5-                     103.9 [+ or -] 4.8
(l',5'-dimethylhex-4'enyl) benzene (14)

Aspirin                                   26.6 [+ or -] 2.5

Compound                                 Collagen

Pinocembrine (1)                         -

Cardamomin (3)                           -

2',3',4',6'-Tetrahydroxychalcone (4)     -

5,6-Dehydrokawain (5)                    -

Flavokawain B (6)                        -

Flavokawain A (7)                        -

Crotepoxide (8)                          -

3-Deacetylcrotepoxide (9)                -

Zerumbone (10)                           177.5 [+ or -] 5.7

Xanthorrhizol (11)                       212.9 [+ or -] 5.2

Curcumin (12)                             60.9 [+ or -] 3.4

Xanthorrhizol epoxide (13)               173.1 [+ or -] 3.7

l-Acetyl-2-methyl-5-                     194.3 [+ or -] 5.5
(l',5'-dimethylhex-4'enyl) benzene (14)

Aspirin                                  -

Compound                                 ADP

Pinocembrine (1)                         128.5 [+ or -] 6.2

Cardamomin (3)                            220.1 [+ or -]3.6

2',3',4',6'-Tetrahydroxychalcone (4)     175.0 [+ or -] 5.6

5,6-Dehydrokawain (5)                    -

Flavokawain B (6)                        94.0 [+ or -] 3.1

Flavokawain A (7)                        216.2 [+ or -] 2.9

Crotepoxide (8)                          146.9 [+ or -] 5.3

3-Deacetylcrotepoxide (9)                -

Zerumbone (10)                           102.6 [+ or -] 6.0

Xanthorrhizol (11)                       249.8 [+ or -] 7.1

Curcumin (12)                             45.7 [+ or -] 4.7

Xanthorrhizol epoxide (13)               119.5 [+ or -] 5.0

l-Acetyl-2-methyl-5-                     334.1 [+ or -] 4.7
(l',5'-dimethylhex-4'enyl) benzene (14)

Aspirin                                  -

Data represent mean [+ or -] SE of three independent experiments
performed in triplicate.

The compounds showed significant inhibition of platelet aggregation induced by many inducers. The selective inhibition of 5,6-dehydrokawain (5) and 3-deacetylcrotep-oxide (9) against AA-induced platelet aggregation indicatesthat the antiplatelet mechanism is probably is probably similar to that of aspirin, i.e. due to the inhibition of thromboxane A2 formation. However, the mechanism of action of these compounds requires further investigation, as the effect of other inducers has not been determined.

Structure--activity analysis of the compounds revealed that the 5-methoxyflavanone derivative (compound 2) showed no significant inhibition on platelet aggregation by all three inducers (Table 1). This was in accordance with the results from a previous study in which 4', 7-dihydroxy-5-methoxyflavanone was not active against AA-, collagen-, ADP- and ristocetin-induced aggregation (Dong and Chen, 1998). The substitution of methoxy group at C-5 of compound 2 with a hydroxyl group (i.e. compound 1) resulted in a significant increase of activity against AA-and ADP-induced aggregation, indicating a possible strong hydrogen bonding. The significant antiplatelet effect of chalcones (compound 3, 4, 6 and 7) against AA- and ADP-induced aggregation was in accordance with the results from a previous study in which chalcone derivatives, in general, were significantly active against aggregation induced by many agonists (Ko et al., 2001). Deacetylation of crotepoxide at C-3 (compound 9) resulted in a decrease in loss of activity against ADP-induced aggregation but a strong increase in AA-induced aggregation. Epoxidation and acetylation of xanthorrhizol did not produce derivatives with significant changes in antiplatelet activity; however, methylation resulted in a compound with weak activity against aggregation induced by all agonists.

The results indicate that curcumin, cardamonin, pinocembrine, 5,6-dehydrokawain and 3-deacetylcrotepoxide isolated from Zingiberacea species were relatively strong inhibitors of platelet aggregation. Further studies need to be carried out to investigate further the structure-activity relationship of the active compounds and to find the lead structures with maximum inhibitory activity.

Acknowledgements

This work was supported by a grant (IRPA 09202065-EA165) from the Ministry of Science, Technology and Innovation, Malaysia.

References

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I. Jantan, S.M. Raweh, H.M Sirat, S. Jamil, Y.H. Mohd Yasin, J. Jalila, J.A. Jamal

(a) Department of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia

(b) Department of Chemistry, Universiti Teknologi Malaysia, Skudai, Johore Bahru, Malaysia

* Corresponding author. Tel.: [phone]; fax: [phone]. E-mail address: [e-mail] (I. Jantan).zerumbet, and structure activity analysis of these compounds.

Received 3 April 2007; accepted 9 August 2007