Antiplatelet Aggregation Coumarins from the Leaves of Murraya omphalocarpa

Using a bioactivity-guided fractionation method, two coumarins: minumicroline acetonide (1) and epimurpaniculol senecioate (2), were isolated from the leaves of Murraya omphalocarpa Hayata (Rutaceae). Compound 1 had been previously synthesized and was now isolated from natural sources for the first time, and compound 2, possessing a negative optical rotation value, is new. The structures and their stereochemistry were fully elucidated on the basis of spectroscopic and X-ray crystallographic techniques. Both compounds 1 and 2 are active in the antiplatelet aggregation assay. Interestingly, the possible acetonide artifact 1 displayed significant antiplatelet aggregation induced not only by AA and collagen but also by platelet activating factor (PAF).


Introduction
Murraya omphalocarpa Hayata (Rutaceae) is a shrub or small tree that grows wild at low altitudes in Taiwan [1]. It has been rarely studied and no traditional medicinal use has been reported before. Nine coumarins, two flavones, and phytosterols have been isolated from the fruits and leaves in previous phytochemical studies [2][3][4]. Of these, three compounds were reported to exhibit significant antiplatelet aggregation induced by arachidonic acid (AA) and collagen [4]. In the continuing phytochemical investigation, two coumarins, minumicroline acetonide (1) [5][6] and epimurpaniculol senecioate (2), were isolated from the methanoic extracts of the leaves of this plant by the way of bioactivity-guided fractionation method. Compound 2, with a negative optical rotation value, is a new compound, which is different in comparison with the previous data [7]. Compounds 1 and 2 are active in antiplatelet aggregation activity induced by AA and collagen. Furthermore, the possible acetonide artifact 1 displayed significant antiplatelet aggregation induced by platelet activating factor (PAF).

Results and Discussion
Compound 1 was isolated as colorless prisms and analyzed as C 18 H 20 O 5 (calcd. 316.1311) from the molecular ion observed at m/z 316.1311 in its HR-EI-MS. Its 1 H-NMR displayed signals of two pairs of AB-type doublets [δ 6.24 and 7.60 (each 1H, J=9.6 Hz, H-3 and H-4), and δ 7.41 and 6.87 (each 1H, J=8.8 Hz, H-5 and H-6)] as well as a methoxyl singlet at δ 3.92. This, together with its UV and IR data, suggested the presence of a 7-methoxy-8-substituted coumarin nucleus [5][6]. The two spincoupled doublets at δ 5.56 (J=8.8 Hz) and 5.01 (J=8.8 Hz) revealed the presence of two vicinal protons attached to benzylic or allylic oxygenated carbons. The terminal vinylic and allylic methyl groups were determined by 1 H-NMR signals at δ 4.95, 4.84, and 1.53, in addition to 13 C-NMR signals at δ 113.5, 141.5, and 17.6. Two three-proton singlets at δ 1.73 and 1.69, and 13 C-NMR signals at δ 109.6, 27.3, and 27.0 indicated the presence of an acetonide moiety, which is attached to C-1′ and C-2′. Based on chemical evidence, spectral data and previous literature reports [5][6], the structure of compound 1 was thus established. The physical data is described in detail in the Experimental. According to the literature [5], compound 1 had been previously synthesized and the absolute configuration was confirmed its CD spectrum. In addition, an X-ray diffraction analysis of a single crystal was undertaken. The X-ray structure ( Figure 2) confirmed that the absolute configuration of 1 is 1′R, 2′R. Compound 1 was thus isolated from natural sources for the first time and may be an extraction artifact. 24 D = +123.3 [7]. In order to confirm the stereochemistry of 2, an X-ray diffraction analysis of a single crystal was taken. The X-ray structure ( Figure 2) indicated that the relative configuration of 2 is 1′S*. Thus, the structure of 2 was fully elucidated as a new enantiomer of murpaniculol senecioate, which we have named epimurpaniculol senecioate.

Biological activity
The antiplatelet effects of these two compounds were studied on the aggregation of washed rabbit platelets induced by thrombin (Thr) (0.1 U/mL), arachidonic acid (AA) (100 μM), collagen (10 μg/mL), and platelet activating factor (PAF) (1 ng/mL). As shown in Table 1, compound 1 showed significant inhibition of AA-and collagen-induced platelet aggregation, and strong inhibition induced by PAF, which was different from other coumarins isolated from this species [4]. Compound 2 also displayed significant inhibitory activity on collagen-and AA-induced platelet aggregation. The bioactive results indicated its chemoprotective potential in cardiovascular diseases.  Platelets were preincubated with DMSO (0.5%, control), or tested compounds at 37 °C for 3 min, then Thr (0.1 U/mL), AA (100 μM), collagen (10 μg/mL) or PAF (1 ng/mL) was added.

General
Melting points were determined using a Yanagimoto micro-melting point apparatus and are uncorrected. Optical rotations were measured with a JASCO P-1020 digital polarimeter. The UV spectra were obtained on a Hitachi 200-20 spectrophotometer, and IR spectra were measured on a Hitachi 260-30 spectrophotometer. 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra were recorded with a Varian Unity-Plus NMR spectrometer in CDCl 3 using TMS as an internal standard. EI-MS were collected on a JEOL JMS-SX/SX 102A mass spectrometer having a direct inlet system. HR-EI-MS were measured on a JEOL JMS-HX 110 mass spectrometer. Si gel 60 (Merck, 230-400 mesh) was used for column chromatography. Pre-coated Si gel plates (Merck, Kieselgel 60 F-254, 0.20 mm) were used for analytical TLC, and precoated Si gel plates (Merck, Kieselgel 60 F-254, 0.50 mm) were used for preparative TLC. Spots were detected by spraying with 30% H 2 SO 4 and then heating on a hot plate.

Plant Material
Fresh leaves of M. omphalocarpa were collected from Pingtung County, Taiwan, in September 1999, and identified by Dr. Hsin-Fu Yen (Associate Researcher of Plants Taxonomy, National Museum of Natural Science, Taichung, Taiwan). A voucher specimen (Rutaceae murraya 1) was deposited in the Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China.

X-ray Structure Determination
Crystals of 1 and 2 suitable for diffraction study were all obtained from MeOH/CHCl 3 solvent mixtures (1:1). Structures were solved via direct method (SIR92) and refined with a full-matrix leastsquares program using the TeXsan [8] software package. Anisotropic refinement was carried out for all non-hydrogen atoms. Hydrogen atoms were calculated according to their idealized positions (dC- The absolute configurations of the two compounds were not determined due to lack of strong scattering atoms, however, the absolute configuration of 1 can be confirmed by association with the optical rotation data. Full crystallographic data are deposited at the Cambridge Crystallographic Data Center, 12 Union Road, Cambridge CB2, 1EZ, UK with deposition numbers CCDC 674767 (for 1) and CCDC 674768 (for 2), respectively.

Biological Assay
The platelet aggregation assay was carried out according to references [9][10].