Antiplatelet Activities of Aporphine Alkaloids Isolated from Leaves of Magnolia obovata

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Five aporphine alkaloids, N-acetylanonaine (1), N-acetylxylopine (2), N-formylanonaine (3), liriodenine (4), and lanuginosine (5) as the antiplatelet constituents, were isolated from the methanol extract of leaves of Magnolia obovata. This is the first reported occurrence of 2 and 3 from genus Magnolia and 5 was isolated from this plant for the first time. Compounds 1, 2 and 3 showed 60 ∼ 264-fold stronger inhibitory effects than acetylsalicylic acid (ASA) to rat platelet aggregation induced by collagen, epinephrine, arachidonic acid (AA), or U46619.

The inhibitory effects of five aporphine alkaloids (Fig. [1]), N-acetylanonaine (1), N-acetylxylopine (2), N-formylanonaine (3), liriodenine (4), and lanuginosine (5) isolated from the leaves of M. obovata Thunberg (Magnoliaceae), on rat platelet aggregation were examined and compared with the effects of acetylsalicylic acid (ASA) (Table [1]). All of the tested compounds showed dose-dependent inhibitory activities to ADP, collagen, epinephrine, arachidonic acid (AA) and U46619 (PGH₂/TXA₂ receptor agonist) induced platelet aggregation. Since rat platelets were observed not to aggregate in response to epinephrine, AA or U46619 in a concentration dependent manner [1], [2], the aggregations were induced in the presence of a threshold concentration of collagen. The inhibitory effects of 1, 2, and 3 were approximately 130-, 190-, and 220-fold, respectively, stronger on rat platelet aggregation induced by epinephrine, 264-, 178-, and 244-fold, respectively, more potent on AA-induced aggregation, and 94-, 92-, and 89-fold, respectively, higher on U46619-induced aggregation than those of ASA. The effects of 1, 2, and 3 on ADP- or collagen-induced aggregation were milder than those on epinephrine-, AA- or U46619-induced aggregation, although they were stronger than ASA. However, oxoaporphines, 4 and 5 showed only very mild effects to all the stimulators tested.

Human platelet-rich plasma (PRP) was pretreated with ASA (1 mM) to block the endogenous production of prostaglandins including TXA₂ [3] and then stimulated with U46619. The three aporphine alkaloids 1, 2 and 3 (IC₅₀; 64, 47. and > 400 μM, respectively) were much less inhibitory to U46619 on human platelets than rat platelets, while α₂-antagonists, yohimbine and pentolamine (IC₅₀ >100 μM) were not inhibitory to U46619-induced aggregation. These results suggest that compounds 1 and 2 exert their effects by inhibiting both the production and receptor binding of TXA₂ while compound 3 mostly inhibits the production of TXA₂.

Materials and Methods

The leaves of M. obovata were collected and identified by Prof. Hyung Joon Chi, Natural Products Research Institute, Seoul National University and the specimens (NAPRI 970 725 - 237) were deposited at the Herbarium of the Institute. The MeOH extract of the leaves (5 kg) of M. obovata (M. hypoleuca) was partitioned between CHCl₃ and H₂O and then the CHCl₃ layer was further partitioned between n-hexane and 90 % MeOH. 60 g of the MeOH fraction was subjected to silica gel (1.2 kg) column chromatography eluting with CHCl₃ containing increasing proportions of MeOH [CHCl₃ : MeOH (95 : 9) → (2 : 8)] and were divided into six subfractions M-I to M-VI according to the eluting order. Compounds 1 (53 mg) and 2 (20 mg) from M-III, 3 (6 mg) from M-II, 4 (15 mg) and 5 (8 mg) from M-V were obtained through silica gel column chromatography eluting with n-hexane : EtOAc (5 : 5), CHCl₃ : MeOH (98 : 2) and/or EtOAc : MeOH (9 : 1), repeatedly. TLC were performed with n-hexane : EtOAc (5 : 5) for compounds 1 (R_f : 0.44), 2 (R_f:0.42), and 3 (R_f : 0.46), and with EtOAc:MeOH (100 : 1) for compounds 4 (R_f : 0.75), and 5 (R_f : 0.72) and detected with Dragendorff reagent. These alkaloids were identified by the direct comparison of their physical and spectral data with the literature values [4], [5], [6], [7], [8], [9]. Copies of the original spectral data from all compounds are obtainable from the author of correspondence.

Blood collected from rat hearts after surgery using a syringe containing 0.1 volume of 2.2 % sodium citrate, was centrifuged at 200 g for 10 min to obtain PRP. The supernatant PRP was diluted with saline to adjust the number of platelets (400 - 450 × 10⁶ platelets/mL) with the aid of a platelet counter. Human blood was collected by vein puncture into 3.8 % trisodium citrate (9 : 1, v/v) from volunteers who had not taken any medicine for at least 2 weeks, PRP was obtained as described above for rat PRP and platelet-poor plasma (PPP) was prepared from the residue by centrifugation at 900 g for 30 more min. The platelet number was adjusted to ∼300 × 10⁶ platelets/mL by mixing PRP and PPP. After 3 min pre-incubation of the adjusted PRP, sample or vehicle was added and an aggregation inducing agent [ADP (2 - 5 μM) or collagen (2 - 5 μg/mL)] was added at 1 min after the sample addition on a platelet aggregometer. Epinephrine (1 - 4 μM), AA (10 - 40 μM) and U46619 (1 - 5 μM) induced platelet aggregations were also measured in the presence of the threshold concentration of collagen (0.8 - 1.0 μg/mL).

The concentrations of the compounds causing 50 % inhibitory effects (IC₅₀) were determined from the Regression Wizard from the SigmaPlot equation library. A minimum of three experiments was performed for each tests. Values of R (correlation coefficient) up to 0.9 were allowed in all tested variables.

Acknowledgements

The authors are grateful for the financial supports of the Korea Health 21 R&D Project, Ministry of Health and Welfare (01-PJ2-PG6-01NA01-0002) and for the fellowship awarded to M.K. Pyo from the BK 21 project supported from Ministry of Education, Republic of Korea.

References

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Hye Sook Yun-Choi

Natural Products Research Institute

Seoul National University

Seoul 110-460

Korea

Phone: +82-2-740-8923

Fax: +82-2-766-7818

Email: hsyun@snu.ac.kr

References

• 1 Yun-Choi H S, Park K M, Pyo M K. Epinephrine-induced platelet aggregation in rat platelet-rich plasma.  Thrombosis Research. 2000;  100 511-8
  CrossrefPubMedSearch in Google Scholar
• 2 Hanasaki K, Nakano T, Arita H. Two phasic generation of thromboxane A₂ by the action of collagen on rat platelets.  Thrombosis Research. 1987;  46 425-36
  CrossrefPubMedSearch in Google Scholar
• 3 Halushka P V. Thromboxane A₂ receptors: Where have you gone?.  Prostaglandins and Other Lipid Mediators. 2000;  60 175-89
  PubMedSearch in Google Scholar
• 4 Johns S R, Lamberton JA and Sioumis A A. Alkaloids of Xylopia papuana (Annonaceae).  Australian Journal of Chemistry. 1968;  21 1383
  PubMedSearch in Google Scholar
• 5 Ito K, Asai S I. Studies on the alkaloids of Magnoliaceous plants. XXXX. Alkaloids of Magnolia obovata Thunb. on the bases of leaves and roots.  Yakugaku Zasshi. 1974;  94 729-34
  PubMedSearch in Google Scholar
• 6 Sashida Y, Suugiyama R, Iwasaki S, Shimomura H, Itokawa H, Fujita M. Studies on the components of M. obovata Thunb. V. Neutral and acidic components of the heartwood.  Yakugaku Zasshi. 1976;  96 659-62
  PubMedSearch in Google Scholar
• 7 Bhaumik P K, Mukhrjee B, Juneau J P, Bhacca N S, Mukherjee R. Alkaloids from leaves of Annona squamosa .  Phytochemistry. 1979;  18 1584-6
  CrossrefPubMedSearch in Google Scholar
• 8 Nonato M G, Garson M J, Truscott R J, Carver J A. ¹H-NMR assignments of annonaine and xylopine derivatives from Talauma gitingensis .  Journal of Natural Products. 1990;  53 1623-7
  CrossrefPubMedSearch in Google Scholar
• 9 Pachaly P, Adnan A Z, Will G. NMR-assignment of N-acylaporphine alkaloids from Tinospora crispa .  Planta Medica. 1992;  58 184-7
  PubMedSearch in Google Scholar

Hye Sook Yun-Choi

Natural Products Research Institute

Seoul National University

Seoul 110-460

Korea

Phone: +82-2-740-8923

Fax: +82-2-766-7818

Email: hsyun@snu.ac.kr