The Constituents of Roots and Stems of Illigera luzonensis and Their Anti-Platelet Aggregation Effects

Phytochemical investigation of the roots and stems of Illigera luzonensis afforded two new aporphine alkaloids (1) and (2), one new bisdehydroaporphine alkaloid (3), and one new benzenoid (4), along with 28 known structures. The structures of new compounds were elucidated by spectral and MS analysis. Among the isolated compounds, (1) and (4–13) were subjected into the examination for their inhibitory effects on the aggregation of washed rabbit platelets.


Structural Elucidation of Compounds 1-4
Compound 1 was obtained as optically active syrup. The HREIMS of 1 showed a molecular ion peak at m/z 336.1113 corresponding to the molecular formula C 19 H 16 N 2 O 4 and was also corroborated by 13 C NMR spectrum which displayed 19 carbon signals. The UV spectrum exhibited absorption maxima at 220, 233 (sh), 274 (sh), 282, 308, and 316 (sh) nm was typical of the occurrence for the basic skeleton of aporphine with 1,2,9,10-tetraoxygenation [34]. The IR spectrum of 1 showed a hydroxy absorption at 3352 cm −1 , a nitrile group at 2214 cm −1 which was also proved by 13 C-NMR (δ 110.2), and two methylenedioxy absorptions at 1055 and 948 cm −1 , respectively. In the 1 H NMR spectrum of 1 (Table 1), it displayed the typical aromatic proton singlets at δ 7.63, 6.85, and 6.53 corresponding to a 1,2,9,10-tetrasubstituted aporphine alkaloid which was assigned to the H-11, H-8, and H-3 [34]. There were also two gem-coupling doublets at δ 6.11 (1H, J = 0.8 Hz) and δ 5.96 (1H, J = 0.8 Hz) characteristic for a methylenedioxy group, a D 2 O exchangeable broad singlet at δ 5.71 (1H) for a hydroxy group, and a singlet at δ 3.99 (3H) for a methoxy group. The HMBC experiment ( Figure 1) showed long-range correlations from the methoxy group to the carbon signal at C-10; and from the methylenedioxy signals (δ 6.11 and 5.96) to the carbon signals at C-1 and C-2, respectively. In addition, three mutually coupling aliphatic proton signals at δ 4.25 (1H, dd, J = 14.0, 4.8 Hz), 3.12 (1H, dd, J = 14.0, 4.8 Hz), and 2.92 (1H, t, J = 14.0 Hz) were assigned as H-6a, H-7e, and H-7a according to their chemical shifts and coupling constants. The stereochemistry of H-6a was determined as α due to the positive specific rotation of 1 [35]. Extensive interpretation of COSY, NOESY, HMQC and HMBC experimental data of 1 established all the connectivity, including the sites of the attachment of the methoxy, hydroxy, and methylenedioxy groups, to accomplish the full assignment of all 1 H and 13 C NMR signals (Table 1). On the basis of the foregoing spectral studies, the structure of 1 was determined as (S)-N-nitrile-9-hydroxy-1,2-methylenedioxy-10-dimethoxy-5,6,6a,7-tetrahydro-4Hdibenzo[de,g]quinoline and trivially named as illigeluzine A.
Compound 2 was afforded as optically active syrup. The HREIMS of 2 showed a molecular ion peak at m/z 350.1270 corresponding to the molecular formula C 20 H 18 N 2 O 4 , which was one CH 2 unit more than that of 1. The UV absorption maxima, IR absorption bands, 1 H and 13 C NMR spectra of 2 were very similar to those of 1. The only differences were one more aliphatic methylene group at δ 4.09 (1H, d, J = 17.6 Hz) and 3.73 (1H, d, J = 17.6 Hz) in 2. The location of this methylene unit was attached at the nitrogen atom according to the NOESY spectrum interpretation, in which correlations were found between δ 3.12 (H-5) and δ 4.09, and between δ 2.90 (H-7) and δ 4.09, respectively. The stereochemistry of H-6a was also determined as α due to the positive specific rotation of 2 [35]. Conclusively, the chemical structure of 2 was determined as (S)-N-acetonitrile-9-hydroxy-1,2methylenedioxy-10-dimethoxy-5,6,6a,7-tetrahydro-4H-dibenzo[de, g]quinoline and trivially named as illigeluzine B.
Compound 3 was purified as brown needles, with mp > 280 °C. The FABMS of 3 showed one pseudomolecular ion and one molecular ion peaks at m/z 617 and 616, which implied the presence of a dimeric aporphine alkaloid. The UV spectrum exhibited absorption maxima at 204, 268, 332, and 393 nm was typical of the occurrence for the basic skeleton of dehydro-aporphine alkaloid with 1,2,9,10-tetraoxygenation [34]. The IR spectrum of 3 showed a hydroxy and amino absorption band at 3382 cm −1 , and two methylenedioxy absorption band at 1056, and 952 cm −1 . In the 1 H NMR spectrum of 3, the characteristic aromatic singlets at δ 8.42, 7.07, and 6.34 corresponding to a 1,2,9,10-tetrasubstituted dehydro-aporphine alkaloid were assigned to be the H-11, -11', -3, -3', -8, and -8'. It also displayed a methylenedioxy group at δ 6.28 (4H, s), a D 2 O exchangeable hydroxy group at δ 9.07 (2H, br s), a D 2 O exchangeable amino group δ 4.54 (2H, s), and a methoxy group at δ 3.84 (6H, s), respectively. The significant spectral characteristics of 3 were the disappearances of H-7 and H-6a. The HMBC experiment ( Figure 1) exhibited a 3 J-correlation between H-8, -8' (δ 3.99) and C-7, -7', and it suggested that C-7 and -7' were quaternary carbon atoms. According to the molecular formula and the HMBC spectral analysis, the structure of 3 could be defined as a symmetric dimer of dehydroaporphine connected through C-7 and C-7'. Comprehensive interpretation of all the COSY, NOESY, HMQC and HMBC spectra of 3 established all the connectivity, including the location of the methoxy, hydroxy, and methylenedioxy groups, to accomplish the complete assignment of all 1 H and 13 C NMR signals. Therefore, the chemical structure of 3 was concluded as bisdehroactinodaphnine as shown in Figure 1. Compound 4 was isolated as white powder. The ESIMS of 4 displayed a molecular ion peak at m/z 392. The UV spectrum exhibited absorption maxima at 228 and 281 nm was typical of the occurrence for the basic skeleton of benzenoid [36]. The IR spectrum showed hydroxy and ester groups at 3457 and 1737 cm −1 . The 1 H NMR characteristics including δ 6.80 (1H, d, J = 1.8 Hz), 6.78 (1H, d, J = 8.2 Hz), and 6.68 (1H, dd, J = 8.2, 1.8 Hz) indicated the presence of 1,2,4-trisubstituted aromatic ring system. It also showed a D 2 O exchangeable hydroxy signal at δ 5.91 (1H, br s) and a methoxy signal at δ 3.86 (3H, s) which displayed NOESY correlation with H-2. In addition, two mutually coupling aliphatic methylene groups at δ 4.23 (2H, t, J = 7.2 Hz) and 2.83 (2H, t, J = 7.2 Hz), one methylene connected with carbonyl group at δ 2.28 (2H, t, J = 7.6 Hz), one terminal methyl group at δ 0.88 (3H, t, J = 6.8 Hz), and one set of long-chain alkyl methylene groups at δ 1.26 (24H, br s) constructed the phenylethyl alkanoate basic structure. Detailed analysis of the COSY and NOESY spectral data of 3 furnished the full assignment of all 1 H-NMR signals. Consequently, the structure of 4 was determined as 4-hydroxy-3-methoxyphenethyl pentadecanoate and it was named trivially as illigeraol A.

Anti-Platelet Aggregation Evaluation Bioassay
Platelets play a pivotal role in development of cardiovascular disease [37]. Arterial thrombosis is the acute complication that develops on the chronic lesions of atherosclerosis and reasons heart attack and stroke. These chronic inflammatory processes are the central pathophysiological mechanism largely driven by lipid accumulation, and provide the substrate for occlusive thrombus formation. Most current models of thrombus development propose a key role for collagen (and possibly vessel wall-derived thrombin) in initiating platelet activation in primary adherent platelets, whereas subsequent propagation of thrombin (platelet aggregation) is primarily driven by agonists released or generated from the platelet surface, including ADP, TXA2 (Thromboxane A2) and thrombin [38].
Platelets circulate in the blood of mammals and are involved in hemostasis, leading to the formation of blood clots. Too many platelets form blood clots that may obstruct blood vessels and induce strokes, myocardial infarctions, and pulmonary embolisms. Sometimes this situation also results in the blockage of blood vessels to other parts of the body, including the extremities of the arms or legs [39]. The traditional medicinal use of Illigera luzonensis is to promote the blood circulation necessary for removing blood stasis. Therefore, the purified compounds were examined for their anti-platelet aggregation bioactivity. However, due to the limited quantity of the purified compounds, only inhibitory effects on the aggregation of washed rabbit platelets were investigated. The anti-platelet aggregation effects are summarized in Tables 2 and 3. Among the tested compounds, 1, 5, 6, 7, 8, and 11-13 displayed significant inhibitory effects on the aggregation of washed rabbit platelets stimulated by arachidonic acid (AA). Compounds 4, 9, and 10 did not inhibit the rabbit platelet aggregation significantly, and therefore the data was not included in Table 2. Compounds 3, 5, and 8 were found to be the most effective compounds among the tested, with IC 50 values in the range of 0.5 and 0.2 μg/mL. Generally the significant inhibitory effects on the aggregation of washed rabbit platelets were related to the aporphine alkaloids; however, in the present study the most potent compounds 5 and 8 were benzenoids. However, the aporphine alkaloids 1, 7, and 10-12 still exhibited moderate antiplatelet aggregation bioactivity. On the other hand, the activities of these purified compounds against thrombin (Thr) and collagen (Col) induced aggregation were not as effective, with the exceptions of 1, 5-8, 12, and 13 at 100 μg/mL. Platelet activating factor (PAF) is a potent phospholipid activator and mediator of many leukocyte functions, including platelet aggregation and degranulation, inflammation, and anaphylaxis. In the present examination, 5, 7, and 12 displayed significant inhibitory effects on the aggregation of platelets stimulated by PAF. Actinodaphnine (12), which was belonged to the aporphine alkaloid, exhibited the most effective inhibition on the aggregation of washed rabbit platelets with IC 50 value in the range of 50 and 20 μg/mL.

Plant Materials
The whole plants of Illigera luzonensis Merr. were collected from Pingtung, Taiwan in January 1995. The plant was authenticated by Professor C.S. Kuoh, Department of Life Science, National Cheng Kung University, Taiwan. The voucher specimens (DG-199) have been deposited in the Department of Chemistry, National Cheng Kung University, Tainan, Taiwan.