1271
ANTIPLATELET AGGREGATION PRINCIPLES OF DENDROBIUM LODDIGESII CHIEN-CHIH CHEN,*
National Research Institute of Chinese Medicine, 2 Lane 391, Pei-1 Rd. Sec. 2, Hsintein, Taipei Hsien, Taiwan, Republic of China
IJ-GINWU, lnstitute of Traditional Medicine, Yang-Ming Medical College, Taipei, Taiwan, Republic of China FENGNIENKO, and CHE-MINGTENG
Pha-ological
Institute, College of Medicine, National Taiwan Uniwsity, Taipei, Taiwan, Republic of China
ABsmcr.-The MeOH extract of the stem of Dendrobium ldigesii was found to inhibit the aggregation of rabbit platelets induced by arachidonic acid and collagen. Two active compounds, moscatilin 111 and moscatin 121, were isolated. Moscatilin diacetate 131 also exhibited antiplatelet aggregation activity.
The stems of Dendrobium species (Orchidaceae)are used in traditional Chinese medicine as a tonic to nourish the stomach, promote the production ofbody fluid, and reduce fever (1). Pharmacological studies have revealed that an EtOH extract of D. dennunurn inhibited the reverse transcriptase and DNA polymerase-a enzymes (2), and the alkaloids, shihunidine and shisunine, from D. loddigesii, have the ability to inhibit Na', K+-ATPase (3). In the course of our studies on the development of naturally occurring antiplatelet agents, we found that an MeOH extract of the stems of D. lodigesii Rolfe exhibited antiplatelet aggregation activity. The bioassay-guided isolation of the active principles and their potencies are reported herein. In order to isolate the biologically active principles from D. loddigesii, the fractionated extracts were tested on the aggregation of washed rabbit platelets. As shown in Table 1, fractions 3 and 4 possessed strong inhibitory effects on the platelet aggregation induced by arachidonic acid (AA) and collagen. Platelet aggregation induced by AA was completely abolished by fraction 4 (50 Fg/ ml). Fraction 4 was further chromatographed on aSi gel column and two active
MeO
RoF-R
OMe
1 R=H
3 R=Ac
2
compounds, 1 and 2 , were obtained. Through spectral analysis, chemical reaction, and comparison with authentic samples (4),compounds 1 and 2 were identified as moscatilin and moscatin, respectively. Acetylation of compound 1 with Ac,O gave compound 3. The antiplatelet effects of moscatilin { 11,moscatin 121,and moscatilin diacetate E31 were studied on the aggregation of washed rabbit platelets induced by thrombin, AA, collagen, and platelet-activating factor(PAF), and the results are shown in Table 2. Compounds 1-3 strongly inhibited both AA- and collagen-induced platelet aggregations. At 100 Fg/mI, platelet aggregations induced by PAF
Wol. 57, No. 9
Journal of Natural Products
1272
TABLE 1. Effect of Fractions from Dendwbium ldigesii on the Platelet Aggregation Induced by Collagen (Col), and Platelet-Activating Factor (PAF).' Thrombin (Thr),Arachidonic Acid (AA), I
Fraction
Control . . . . . . . 1 ............ 2 ............ 3 ............ 4 ............
5 ............ 6 ............
I
Concentration (pg/ml)
200
200 200 100 50 200 100 50 20 200 200
% Aggregation
Thr
AA
0.6(4) 86.9 2 2.0(4) 0.8(3) 89.1 2 1.6(3) 3.1 (3)b 85.8 2 2.8 (3) l.0(3) 0.0 2 0.0(3)d 30.7 215.5 (4)' 45.1 k19.8 (4) 83.2 2 4.5 (3) 0.0 2 0.0(3)d 0.0 2 0.0(3)d 0.0 2 0.0 (3)d 48.1 219.7 (3) 79.0 2 4.2 (3) 72.2 2 5.8 (3)b 84.7 ? 2.8 (3) 91.9 2 0.7 (3) 88.0 2 86.4 ? 81.8 2 86.1 2
were also significantly inhibited by these three compounds. As shown in Figure 1, the antiplatelet actions of compounds 13 inhibited AA-induced platelet aggre-
PAF
Col
89.3 89.4 85.3 83.2
k 0.9(4) 2 0.2(3)
4.8 21.3 67.3 84.1 7.6 80.3
k 3.9(3p ? 7.8(3)d
2 0.6 (3)d 2 1.1(3?
92.6 91.7 91.6 85.5
2 1.7(4) 2 2.3(3) ? 1.7 (3)
2 2.8(3)b
81.7 2 3.8(3)'
2 13.8 (3) 2 2.9 (3) 2 3.6 (3)d 2 2.3 (3)'
79.2 2 2.2 (3) 87.2 2 0.8 (3)b
gation in a concentration-dependent manner. Compound 3 showed greater antiplatelet effect than the other two substances. In AA-induced platelet ag-
TABLE 2. Effect of Purified Components from Denddim ldigeszi on the Platelet Aggregation Induced by Thrombin hr), Arachidon Acid (AA),Collagen (Col), and Platelet-Activating Factor (PAF).' Compound Control . . . . . . . 1 ............
2
............
3 ............
Concentration (pg/ml)
100 50 20 10 5 2 100 50 20 10 5 2 100 20 10 5 2
% Aggregation
ThC 89.3 2 1.9 85.3 2 3.7
AA
Col
84.0 ? 6.7 0.0 2 O.Od 0.0 2 O.Od
36.4 216.3b 52.0 216.6 78.7 ? 9.0 41.0 2 9.5d
0.0 2 O.Od 0.0 2 O.Od 0.0 2 O.Od
22.8 218.6' 87.6 2 3.0 93.4 2 2.8
0.0 2 0.0 2 0.0 2 33.9 2 83.8 2
O.Od
86.4 2 3.6 0.0 2 O.Od 0.0 2 O.Od
19.6 216.0' 42.3 217.5b 58.4 215.1 78.5 2 6.3 0.0 ? O.Od
PAF 86.3 ? 2.7d 24.7 2 2.9 72.1 2 4.3b
0.0 2 O.Od
24.7 2 2.9d 72.1 2 4.3b
19.6 216.0' 42.3 ?17.5b 58.4 215.1 78.5 2 6.3 12.8 2 6.3d
36.0 2 7.Sd
O.Od O.Od 8.1d 1.2
'Platelets were preincubated with each compound or the solvent (0.5% DMSO,control) at 37" for 3 min, then thrombin (0.1 U/ml), AA (100 ph0, collagen (10 pg/ml) or PAF (2 ngiml) was added. Percentages of aggregationare presented as means 2 S.E.(n=3) and statistical significance was evaluated by Student's I test. bP
