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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 an·ti·plate·let
adj.
Acting against or destroying blood platelets.



antiplatelet

directed against or destructive to blood platelets; inhibiting platelet function.
 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 rotunda

In Classical and Neoclassical architecture, a building or room that is circular in plan and covered with a dome. The Pantheon is a Classical Roman rotunda. The Villa Rotonda at Vicenza, designed by Andrea Palladio, is an Italian Renaissance example.
, Curcuma cur·cu·ma  
n.
Any of various tropical Asian plants of the genus Curcuma, which includes turmeric and zedoary.



[New Latin Curcuma, genus name, from Arabic kurkum, saffron
 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 herbarium, collection of dried and mounted plant specimens used in systematic botany. To preserve their form and color, plants collected in the field are spread flat in sheets of newsprint and dried, usually in a plant press, between blotters or absorbent paper.  of the Department of Biology, Universiti Putra Malaysia Universiti Putra Malaysia or UPM is a public university in Malaysia. It was formerly known as Universiti Pertanian Malaysia (Malay: universiti, university; pertanian, agriculture; Malaysia). . Twelve pure compounds were isolated from the chloroform chloroform (klôr`əfôrm) or trichloromethane (trī'klôrōmĕth`ān), CHCl3  extracts of the plants by repeated chromatography on silica gel silica gel, chemical compound. It is a colloidal form of silica, and usually resembles coarse white sand. It may be prepared by partial dehydration of metasilicic acid, H2SiO3. Because it has many tiny pores, it has great adsorptive power.  (230-400 mesh) using petroleum ether--ether gradients of increasingpolarities. They were identified by spectroscopic spec·tro·scope  
n.
An instrument for producing and observing spectra.



spectro·scop
 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 epoxide /epox·ide/ (e-pok´sid) an organic compound containing a reactive group resulting from the union of an oxygen atom with two other atoms, usually carbon, that are themselves joined together. , 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 methylation,
n a phase-II detoxification pathway in the liver; methyl groups combine with toxins to rid the body of various substances.

methylation
(meth´
 of xanthorrhizol, respectively. Collagen, ADP (1) (Automatic Data Processing) Synonymous with data processing (DP), electronic data processing (EDP) and information processing.

(2) (Automatic Data Processing, Inc., Roseland, NJ, www.adp.
 and arachidonic acid arachidonic acid /arach·i·don·ic acid/ (ah-rak?i-don´ik) a polyunsaturated 20-carbon essential fatty acid occurring in animal fats and formed by biosynthesis from linoleic acid; it is a precursor to leukotrienes, prostaglandins, and  (AA) were purchased from Sigma Chemical Co. (USA).

The use of human whole blood in this study was approved by the Ethics Committee ethics committee A multidisciplinary hospital body composed of a broad spectrum of personnel–eg, physicians, nurses, social workers, priests, and others, which addresses the moral and ethical issues within the hospital. See DNR, Institutional review board.  of the Universiti Kebangsaan Malaysia Universiti Kebangsaan Malaysia (National University of Malaysia) was established in May 1970. It is located in Bangi, Selangor which is about 35 km south of Kuala Lumpur. There is also a teaching hospital in Cheras and a branch campus in Kuala Lumpur. . 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 normal saline Physiologic saline solution, see there  in the ratio of 1:1. The dried methanol extracts and the isolated compounds were each dissolved in dimethyl sulfoxide dimethyl sulfoxide (DMSO)

Colourless, nearly odourless liquid organic compound. It mixes in all proportions with water, ethanol, and most organic solvents and dissolves a wide variety of compounds (but not aliphatic hydrocarbons).
 (DMSO DMSO dimethyl sulfoxide.

DMSO
n.
Dimethyl sulfoxide; a colorless hygroscopic liquid obtained from lignin, used as a penetrant to convey medications into the tissues.


DMSO,
n.
) 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 cuvette /cu·vette/ (ku-vet´) [Fr.] a glass container generally having well-defined characteristics (dimensions, optical properties), to contain solutions or suspensions for study.

cu·vette
n.
 containing the diluted whole blood and the mixture was allowed to incubate incubate /in·cu·bate/ (in´ku-bat)
1. to subject to or to undergo incubation.

2. material that has undergone incubation.


in·cu·bate
v.
1.
 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 selective inhibition
n.
See competitive 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 thromboxane /throm·box·ane/ (-bok´san) either of two compounds, one designated A2 and the other B2. Thromboxane A2 is synthesized by platelets and is an inducer of platelet aggregation and platelet release functions and is a  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 hydroxyl group (hīdrŏk`sĭl), in chemistry, functional group that consists of an oxygen atom joined by a single bond to a hydrogen atom. An alcohol is formed when a hydroxyl group is joined by a single bond to an alkyl group or aryl 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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Dong, H., Chen, S.-X., 1998. A new antiplatelet diarylheptanoid from Alpinia blepharocalyx. J. Nat. Prod. 61, 142-144.

Ingerman-Wojenski, CM., Silver, M.J., 1984. A quick method for screening platelet dysfunctions using the whole blood lumi-aggregometer, Thro[+ or -]mb. Haemostasis hemostasis, haemostasis
the stoppage of bleeding or cessation of the circulation of the blood; stagnation of the blood in a part of the body. Also hemostasia, haemostasia.
See also: Blood and Blood Vessels

Noun 1.
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n.pl naturally occurring organic compounds that comprise two monoterpene mol-ecules; found in some essential oils; have
 from the rhizomes of Alpinia formaosana. Phytochemistry phytochemistry,
n the scientific study and classification of the chemical constituents of plants.
 20, 435-438.

Janssen, A.A., Scheffer, J.J.C., 1985. Acetoxychavicol acetate, an antifungal component of Alpinia galanga. Planta Med. 51, 507-509.

Jantan, I., Pisar, M., Sirat, H.M., Basar, N., Jamil, S., Ali, R.M., Jalil, J., 2004. Inhibitory effects of compounds from Zingiberaceae species on platelet activating factor receptor binding. Phytother. Res. 18 (12), 1005-1007.

Ko, K.L.K., Ammit, A.J., Tran, V.H., Duke, C.C., Roufogalis, B.D., 2001. Gingerols and related analogues inhibit arachidonic acid-induced human platelet serotonin release and aggregation. Thromb. Res. 103 (5), 387-397 (J. Pharm. Pharmacol. 52, 353-359).

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Shah, B.H., Nawaz, Z., Virani, S.S., Ali, I.Q., Saeed, S.A., Gilani, A.H., 1998. The inhibitory effect of cinchonine on human platelet aggregation due to blockage of calcium influx. Biochem. Pharmacol. 56, 955-960.

Shiobara, Y., Asakawa, Y., Kodoma, M., Takemoto, T., 1986. Zedoarol, 13-hydroxygermacrone and curzeone, three sesquiterpenoids from Curcuma zedoaria. Phytochemistry 25, 1351-1353.

Sirat, H.M., Masri, D., Rahman, A.A., 1993. The distribution of labdane derivatives in the Zingiberaceae of Malaysia. Phytochemistry 36, 699-701.

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 Universiti Teknologi Malaysia is the oldest public engineering and technology university in Malaysia and is known by the abbreviation UTM. The university specialises in technical studies, with separate faculties for Education, Pure Sciences, Management, and Human Resources , Skudai, Johore Bahru, Malaysia

* Corresponding author. Tel.: +60340405331; fax: +603 26983271. E-mail address: ibj@medic medic: see alfalfa. .ukm.my (I. Jantan).zerumbet, and structure activity analysis of these compounds.

Received 3 April 2007; accepted 9 August 2007
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Author:Jantan, I.; Raweh, S.M.; Sirat, H.M.; Jamil, S.; Yasin, Y.H. Mohd; Jalil, J.; Jamal, J.A.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9MALA
Date:Apr 1, 2008
Words:2488
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