Platelet Anti-Aggregating Triterpenoids from the Leaves of Acanthopanax senticosus and the Fruits of A. sessiliflorus

• Abstract
• Materials and Methods
• References

Abstract

Six triterpenoids, chiisanogenin, chiisanoside, ursolic acid, oleanolic acid, β-sitosterol and daucosterol, were isolated as the platelet anti-aggregating components from the leaves of Acanthopanax senticosus and the fruits of A. sessiliflorus. Chiisanogenin showed about 50-fold higher potency than acetylsalicylic acid (ASA) on U46619-induced platelet aggregation (IC₅₀: 6.21 μM) and 10 - 20-fold higher effects than ASA on epinephrine- and arachidonic acid (AA)-induced aggregation (IC₅₀: 2.50 and 4.81 μM, respectively).

The MeOH extract of the bark of Acanthopanax. sp. (Araliaceae) was reported to show strong inhibitory effects on rat platelet aggregation and 3,4-dihydroxybenzoic acid and its artifact, ethyl 3,4-dihydroxybenzoate were isolated as the active components from this plant, however, they were only very mildly effective [1], [2]. Now, activity-guided fractionation has provided six triterpenoids from the leaves of Acanthopanax senticosus and the fruits of A. sessiliflorus. The inhibitory effects of the six triterpenoids, chiisanogenin (1), chiisanoside (2), ursolic acid (3), oleanolic acid (4), β-sitosterol (5) and daucosterol (6; Fig. [1]) on rat platelet aggregation were examined. The activities were compared with the effects of acetylsalicylic acid (ASA) (Table [1]). Since rat platelets were observed not to aggregate in response to epinephrine, AA or U46619 in a concentration-dependent manner, the aggregations were induced in the presence of a threshold concentration of collagen [3], [4]. All of the tested compounds showed dose-dependent inhibitory activities on ADP-, collagen-, epinephrine-, arachidonic acid (AA)- and U46619 (PGH₂/TXA₂ receptor agonist)-induced aggregation as shown in the representative tracings (Fig. [2]). All of them including ASA were only very mildly inhibitory to ADP (IC₅₀: >1 mM, data not shown) and collagen (IC₅₀: >100 μM). Compound 1 was a much more potent inhibitor than ASA on all the other tested agonists. It showed an about 50-fold higher potency on U46619-induced aggregation (IC₅₀: 6.21 μM) and 10 - 20-fold higher potency on epinephrine- (IC₅₀: 2.50 μM) and AA- (IC₅₀: 4.81 μM) induced aggregation than ASA, whereas the saponin 2 was rather less active than ASA. Compounds 3 and 4 were almost equally potent to ASA on epinephrine-induced platelet aggregation (IC₅₀: 82.6 and 45.3 μM, respectively), however they were weaker than ASA on AA- or U46619-induced aggregation. Both compounds 5 and 6 showed 2 - 6-fold higher potency than ASA on U46619- (IC₅₀: 170 and 56.1 μM, respectively) induced aggregation. Compound 6, a glycoside of 5, was equally potent as ASA on epinephrine- and AA-induced aggregation (IC₅₀: 53.2 and 66.5 μM respectively), whereas 5, the sapogenin, showed only less than half the activity of ASA to both epinephrine- and AA-induced platelet aggregation.

The above results are suggestive that among triterpenoids, the tetracyclic rather than the pentacyclic ring system is more favorable for inhibitory activity on platelet aggregation. Introduction of one sugar unit seems to increase the hydrophilicity to the highly lipophilic triterpenoid molecule and contribute to increase the inhibitory activity as shown in compound 6, although introduction of more than three sugar units to compound 1 drastically lowered the activity as in 2. Chiisanogenin (1), the most potent inhibitor among the present compounds, might exert its effects at least partly by acting directly on PGH₂/TXA₂ receptors, since all the IC₅₀ values of 1 on epinephrine-, AA- and U46619-induced aggregation are of the same order and less than 1/90 of the IC₅₀ value on collagen-induced aggregation. If an agent inhibits platelet aggregation by interfering with the production of PGH₂/TXA₂, the IC₅₀ values on epinephrine- or AA-induced aggregation should be much lower than those for collagen and U46619. And the production of PGH₂/TXA₂ is inhibited during the secondary aggregation process as in the case of collagen-induced aggregation by ASA, the IC₅₀ values on U46619- and collagen-induced aggregation should be within the same order of potency [5]. However, the precise mechanism of action of 1 should further be investigated. Anyhow, chiisanogenin (1) might serve as a new lead for the investigation of compounds with strong anti-platelet and anti-thrombotic potential.

Materials and Methods

The leaves of A. senticosus and the fruits of A. sessiliflorus were collected at Kong Ju, Korea and identified by Prof. Seon Haeng Cho, Kong Ju University of Education, Korea. The voucher specimens (Shin 200 010-1 and Shin 9910-1, respectively) were deposited at the Herbarium of Natural Products Research Institute, Seoul National University, Seoul, Korea. The MeOH extract of the air-dried plant sample (2 kg each) was suspended in water and extracted with n-hexane, CHCl₃, EtOAc and then n-BuOH successively to afford each solvent fraction. A portion (20 g) of each fraction was applied to a silica gel (600 g, 70 - 230 mesh) column and eluted with a gradient solvent system. The CHCl₃ fr. was eluted with n-hexane and EtOAc (10 : 1 → 10 : 3) to afford compounds 5 (14 mg) and 1 (16 mg). The EtOAc fr. eluting with n-hexane-EtOAc (10 : 5 → 10 : 8) provided 3 (16 mg) and 4 (6 mg), and then eluting with EtOAc-methanol (5 : 1 → 5 : 2) gave 6 (20 mg). The n-BuOH fr. using CHCl₃-MeOH (5 : 1 → 5 : 4) afforded 2 (8 mg). Compounds 1, 2 and 4 were isolated from A. senticosus, 3 was isolated from A. sessiliflorus, and 5 and 6 were obtained from both plants. The structures of compounds 1 - 6 were confirmed as chiisanogenin (1), chiisanoside (2), ursolic acid (3), oleanolic acid (4), β-sitosterol (5) and daucosterol (6), respectively, by the direct comparison of their physical and spectral data with the literature values [6], [7], [8], [9], [10], [11], [12]. Copies of the original spectral data of all compounds are obtainable from the author of correspondence.

Blood collected from rat heart after being anesthetized with ether 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 platelet counter. Platelet aggregation responses were monitored by the changes in light transmission using an optical platelet aggregometer [13]. 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 30 sec after the sample addition. Epinephrine (1 - 4 μM)-, AA (10 - 40 μM)- and U46619 (1 - 5 μM)-induced platelet aggregations were measured in the presence of the threshold concentration of collagen (0.8 - 1.0 μg/mL) which was added 30 sec before the addition of each agent. The concentrations of the compounds causing 50 % inhibitory effects (IC₅₀) were determined with the Regression Wizard from the SigmaPlot equation library. A minimum of three experiments was performed for each test. The values of R (correlation coefficient) were allowed up to 0.9 in all tested variables.

References

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• 11 Chang I -M, Yun (Choi)  HS, Yamasaki K. Revision of ¹³C NMR assignments of β-sitosterol and β-sitosteryl-3-O-β-D-glucopyranoside isolated from Plantago asiatica seed.  Kor J Pharmacog. 1981;  12 12-4
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  Search in Google Scholar

Prof. 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, Kim J H, Lee J R. Potential inhibitors of platelet aggregation from plant sources. III.  J Nat Prod. 1987;  50 1059-64
  CrossrefPubMedSearch in Google Scholar
• 2 Yun-Choi H S, Kim S O, Kim J H, Lee J R, Cho H I. Modified smear method for screening potential inhibitors of platelet aggregation from plant sources.  J Nat Prod. 1985;  48 363-70
  CrossrefPubMedSearch in Google Scholar
• 3 Yun-Choi H S, Park K M, Pyo M K. Epinephrine induced platelet aggregation in rat platelet-rich plasma.  Thromb Res. 2000;  100 511-8
  CrossrefPubMedSearch in Google Scholar
• 4 Pyo M K, Yun-Choi H S, Hong Y -J. Antiplatelet activities of aporphine alkaloids isolated from leaves of Magnolia obovata .  Planta Med. 2003;  69 267-9
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Rand M L, Leung R, Packham M A. Platelet function assays.  Transfusion Apheresis Sci. 2003;  28 307-17
  PubMedSearch in Google Scholar
• 6 Hahn D R, Kasai R, Kim J H, Taniyasu S, Tanaka O. A new glycosyl ester of a 3,4-seco-triterpene from Korean medicinal plant, Acanthopanax chiisanensis (Araliaceae).  Chem Pharm Bull. 1984;  32 1244-7
  PubMedSearch in Google Scholar
• 7 Shirasuna K, Miyakoshi M, Mimoto S, Isoda S, Satoh Y. et al . Lupane triterpenoid glycosyl esters from leaves of Acanthopanax divaricatus .  Phytochemistry. 1997;  45 579-84
  CrossrefPubMedSearch in Google Scholar
• 8 Lee S, Shin D -S, Oh K -B, Shin K H. Antibacterial compounds from the leaves of Acanthopanax senticosus .  Arch Pharm Res. 2003;  26 40-2
  PubMedSearch in Google Scholar
• 9 Lee S, Kim B K, Cho S H, Shin K H. Phytochemical constituents from the fruits of Acanthopanax sessiliflorus .  Arch Pharm Res. 2002;  25 280-4
  CrossrefPubMedSearch in Google Scholar
• 10 Mahato S B, Kundu A P. ¹³C NMR spectra of pentacyclic triterpenoids - A compilation and some salient features.  Phytochemistry. 1994;  37 1517-75
  CrossrefPubMedSearch in Google Scholar
• 11 Chang I -M, Yun (Choi)  HS, Yamasaki K. Revision of ¹³C NMR assignments of β-sitosterol and β-sitosteryl-3-O-β-D-glucopyranoside isolated from Plantago asiatica seed.  Kor J Pharmacog. 1981;  12 12-4
  PubMedSearch in Google Scholar
• 12 Rubinstein I, Goad L J, Clague A DH, Mulheirn L J. The 220 MHz NMR spectra of phytosterols.  Phytochemistry. 1976;  15 195-200
  CrossrefPubMedSearch in Google Scholar
• 13 McNicol A. Platelet preparation and estimation of functional responses. In: Watson SP, Authi KS, editors Platelet-A Practical approach. Oirl Press at Oxford University Press 1996: pp 1-26
  Search in Google Scholar

Prof. 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