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Abstract

A new isohopane triterpenoid (1) and two known triterpenoids (2-3) were isolated from the roots of Rubia akane (Rubiaceae). The molecular formula C₃₀H₄₆O₄ of 1 was determined by HRESIMS. Detailed NMR spectroscopic data analysis suggested that compound 1 is a new isohopane triterpenoid with a ketone moiety at C-16. Based on the key NOE correlations of H-3/H-5 and H-21/H₃-28, compound 1 was determined as 3β-hydroxy-16-oxo-21β-isohop-22(29)-en-24-oic acid. The anticoagulant activities of new isohopane 1 were evaluated by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of thrombin (Factor IIa, FIIa) and activated factor X (FXa). The effects of 1 on expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated in tumor necrosis factor-α activated human umbilical vein endothelial cells. Treatment with 1 (200 μM) resulted in the prolongation of aPTT and PT and the inhibition of relative thrombin (28%) and FXa (29%) activities. In addition, 1 inhibited thrombin-catalyzed fibrin polymerization (18% inhibition at 100 μM) and platelet aggregation (21.8% inhibition at 100 μM). Compound 1 also elicited anticoagulant effects in mice in a dose-dependent manner ranging from 18.8 to 94.0 μg/mouse. In addition, treatment with 1 resulted in significant reduction of the PAI-1 to t-PA ratio (25% decreased). Collectively, the new isohopane triterpenoid 1 possesses antithrombotic activities and offers a basis for the development of a new anticoagulant agent.

Keywords

isohopane triterpenoid Rubiaceae coagulation cascade fibrinolysis

The plant genus Rubia, a member of the Rubiaceae family, contains about 70 species. The roots of Rubia spp. are widely used as a traditional medicine for the treatment of cancer, tuberculosis, rheumatism, contusion, menstrual disorder, liver fluke, and dysentery.^1,2 For decades, phytochemicals of Rubia spp. have attracted attention due to their potent bioactivities. Anthraquinones, naphtoquinones, cyclic hexapeptides, triterpenoids, and lignans have been reported as constituents of this genus. Among them, cyclic hexapeptides, including RA-700, showed anticancer activity¹ and triterpenoids, including 2-deoxyrubianol F, inhibited PDGF-induced proliferation.³ Rubia akane (Nakai) is widely distributed in East Asia.⁴ The roots of R. akane exhibit pharmacological effects, including antiplatelet,⁵ antitumor,⁶ and antimicrobial⁷ activities. Although many bioactive constituents of the genus Rubia have been reported, phytochemical study on R. akane is limited. The objective of this study was to explore small molecule bioactive compounds from R. akane. In this study, a new isohopane tripterpene (1) and two known oleanane triterpenes (2-3) have been isolated, and their structures have been elucidated using spectroscopic methods including 1D- and 2D-NMR experiments. The relative configuration of 1 was determined by NOE data interpretation. So far, there are no studies on the anticoagulant activity of 1. Activated factor X (FXa) forms prothrombinase complex on phosphatidylserine containing surface, which is responsible for the conversion of prothrombin to thrombin.⁸ Thrombin generation is a central event in hemostasis, which regulates blood coagulation potential.⁹ Therefore, in the current study, we have examined the anticoagulant activity of 1 in the production of FXa and thrombin, and their effects on fibrinolytic activity.

Compound 1 was isolated as white amorphous solid and exhibited a positive HRESIMS ion peak at m/z 493.3291 [M+Na]⁺ (calcd 493.3294), consistent with the molecular formula C₃₀H₄₆O₄Na. The ¹H NMR spectrum of 1 showed signals corresponding to one exomethylene at δ _H 4.82 (2H, d, J = 16.93 Hz, H₂-29); one oxygenated methine at δ _H 4.67 (1H, t, H-3); two methines at δ _H 3.00 (1H, m, H-21) and 2.64 (1H, d, J = 11.51 Hz, H-17); eight methylenes at δ _H 2.50 to 1.15; and six methyl groups at δ _H 1.75 (3H, s, H₃-30), 1.66 (3H, s, H₃-23), 1.06 (3H, s, H₃-26), 0.97 (3H, s, H₃-27), 0.94 (3H, s, H₃-25), and 0.72 (3H, s, H₃-28). The ¹³C NMR spectroscopic data showed 30 carbon signals, including one ketone (δ _C 210.7), one carboxylic acid (δ _C 181.3), and one exomethylene (δ _C 148.2, 109.5). These NMR spectroscopic data were typical of hopane- or isohopane-type triterpenoid. Except for a ketone moiety at δ _C 210.7, the 1D NMR spectroscopic data of 1 were similar to those of 3α-hydroxyisohop-22(29)-en-24-oic acid.¹⁰ The attachment of the ketone at C-16 was confirmed via key HMBC cross-peaks from H₂-15 (δ _H 2.50, 1.86) and H-17 (δ _H 2.64) to C-16 (Figure 1). The HMBC cross-peaks from H₃-23 to C-24 and from H-17/H-21 to C-22 confirmed the connection of the carboxylic acid at C-24 and the location of the exomethylene moiety at C-29. The key HMBC cross-peaks were observed from six methyl groups (H₃-23, H₃-25, H₃-26, H₃-27, H₃-28, and H₃-30) to quaternary carbons C-4, C-10, C-8, C-14, C-18, and C-22, respectively. The relative configuration of 1 was assigned on the basis of NOESY correlations. The key NOE correlations between H₃-28 and H-21 proved the isohopane skeleton of 1. The NOE correlations between H-3 and H-5 indicated that the H-3 proton should be α-oriented. The NOE cross-peaks between H₃-4 and H₃-10 confirmed that H₃-4 was β-oriented. Therefore, the structure of new compound 1 was determined to be 3β-hydroxy-16-oxo-21β-isohop-22(29)-en-24-oic acid.

Figure 1

Chemical structure (a) and key COSY, HMBC (b), and NOESY correlations (c) of 1.

Two known oleanane triterpenoids, 30-norhederagenin (2) and 2α,3β,24-trihydroxyolean-12-en-28-oic acid (3), were identified by comparison of their NMR spectroscopic data and specific rotation value with those in the literature.^11,12

Effects of Compound 1 on Clotting Times and Bleeding Times

The anticoagulant activities of 1 were tested using activated partial thromboplastin time (aPTT) and prothrombin time (PT) assays in human plasma (Table S1). Although the anticoagulant activities of 1 were weaker than those of heparin or warfarin, aPTT (4.2 s increased) and PT (7.8 s increased) were significantly prolonged by 1 at 200 µM. The results indicate that 1 could also inhibit the common coagulation pathway. To confirm these in vitro results, in vivo tail bleeding times were determined (Table S1). Because an average body weight is 20 g and an average blood volume is 2 mL, 1 administered (18.8, 47.0, or 94.0 µg/mouse) produced a concentration of approximately 10, 30, or 50 µM in peripheral blood, respectively. As shown in Table S1, tail bleeding times were significantly prolonged (about 1.5 times) by 1 in comparison to the controls. The anticoagulation in response to 1 was also observed ex vivo in mice with dose-dependent prolongation of the aPTT (16.8 s increased at 94.0 µg/mouse) and PT (8.7 s increased at 94.0 µg/mouse) (Table S1).

Effects of Compound 1 on Thrombin-Catalyzed Platelet Aggregation and Fibrin Polymerization

Incubation of human plasma with 1 resulted in a significant decrease (18% inhibition at 100 µM) in the maximal rate of fibrin polymerization (Figure 2a). To confirm the additional mode of antithrombotic activity of compound 1, thrombin-catalyzed platelet aggregation assay was performed. As shown in Figure 2b, treatment with 1 resulted in significantly inhibited mouse platelet aggregation (21.8% inhibition at 100 µM) induced by thrombin (final concentration: 3 U/mL) in a concentration-dependent manner.

Figure 2

Effects of 1 on fibrin polymerization in human plasma. (a) Thrombin-catalyzed fibrin polymerization at the indicated concentrations of 1. The results are V _max values expressed as percentages vs controls. (b) Effect of 1 on mouse platelet aggregation induced by 3 U/mL thrombin. D = 0.2% DMSO is the vehicle control. Data represent the mean ± SEM of 3 independent experiments performed in triplicates. *P < 0.05 vs Th alone.

Effects of Compound 1 on the Activities of Thrombin and FXa

In order to elucidate the mechanism responsible for the inhibition of coagulation by 1, the inhibitory effects of 1 on the activities of thrombin and FXa were measured using chromogenic substrates. Results are shown in Figure 3a, treatment with 1 resulted in dose-dependent inhibition of the amidolytic activity of thrombin, indicating direct inhibition of thrombin activity by the anticoagulant (maximum 28%). Direct thrombin inhibitor, argatroban, was used as a positive control. In addition, we also investigated the effects of 1 on the activity of FXa. Compound 1 inhibited the activity of FXa (maximum 29%) (Figure 3b). Direct FXa inhibitor, rivaroxaban, was used as a positive control. These results are consistent with those of our antithrombin assay. The antithrombotic mechanisms of 1 are due to the inhibition of fibrin polymerization and/or the intrinsic/extrinsic pathway.

Figure 3

Effects of 1 on inactivation and production of thrombin and factor Xa. (a) Inhibition of thrombin by 1 was measured using a chromogenic assay. (b) Inhibition of factor Xa by 1 was monitored using a chromogenic assay. Argatroban (a) or rivatoxaban (b) was used as positive control. D = 0.2% DMSO is the vehicle control. *P < 0.05 vs 0.0001 (a, b).

Effects of Compound 1 on Secretion of PAI-1 or t-PA Protein

In order to determine the direct effects of 1 on tumor necrosis factor (TNF)-α-stimulated secretion of plasminogen activator inhibitor type 1 (PAI-1), human umbilical vein endothelial cells (HUVECs) were cultured in media with or without 1 in the absence or presence of TNF-α for 18 hours. As shown in Figure 4a, treatment with 1 reduced about 18.7% of TNF-α-induced secretion of PAI-1 from HUVECs, and these decreases became significant at 1 100 µM. TNF-α does not have a significant effect on tissue-type plasminogen activator (t-PA) production,¹³ and the balance between plasminogen activators and their inhibitors reflects net plasminogen-activating capacity^14
-16; therefore, we investigated the effect of TNF-α with 1 on the secretion of t-PA from HUVECs. The results obtained were consistent with those of a previous study reporting a modest decrease in the production of t-PA by TNF-α in HUVECs.¹⁷ This decrease was not significantly altered by treatment with 1 (Figure 4b). Collectively, these results indicated that the PAI-1/t-PA ratio was increased by TNF-α and that 1 prevented this increase (25% decreased) (Figure 4c).

Figure 4

Effects of 1 on secretion of plasminogen activator inhibitor type 1 and tissue-type plasminogen activator. (a) Human umbilical vein endothelial cells were cultured with 1 in the absence or presence of tumor necrosis factor-α (10 ng/mL) for 18 h and plasminogen activator inhibitor type 1 concentrations in culture media were determined. (b) Human umbilical vein endothelial cells were cultured with 1 in the absence or presence of tumor necrosis factor-α (10 ng/mL) for 18 h and tissue-type plasminogen activator concentrations in culture media were determined. (c) Plasminogen activator inhibitor type 1/tissue-type plasminogen activator ratio in tumor necrosis factor-α activated human umbilical vein endothelial cells from (a) and (b). D = 0.2% DMSO is the vehicle control. *P < 0.05 vs tumor necrosis factor-α alone or D; n.s., not significant.

The genus Rubia has been studied extensively, and there are several reports on the chemical constituents of R. akane and their bioactivities. Anthraquinone, lignan, and cyclopeptide have been isolated from R. akane. The arborinane- and oleanane-type triterpenoids, such as rubiarbonol D, rubiprasin A, and rubiprasin B, were discovered from the species.² However, isohopane triterpenoid is the first report on the chemical constituent in R. akane. In this study, we demonstrated the antithrombotic, anticoagulant, and antiplatelet effects of new isohopane triterpenoid 1 in human endothelial cells and mice. Therefore, the antithrombotic and antiplatelet activities by 1 provide a basis for the development of an anticoagulant agent.

According to the previous studies, antiplatelet activity by thrombin, ADP, and PAF was confirmed with anthraquinones isolated from R. akane.⁵ In the current study, a new isohopane triterpenoid 1 was isolated from R. akane. To date, isohopane triterpenoids have been rarely found in Rubia spp. and the biological study on isohopanes is limited. Isohopane triterpene isolated from the genus Carissa was demonstrated to have cytotoxic activity against human cancer cells.¹⁸ However, there is no report on the anticoagulant activity of isohopane triterpenoid. In our experiment, the new isohopane triterpene 1 prolonged PT and aPTT values and 1 resulted in a significant decrease in the maximal rate of fibrin polymerization and inhibited platelet aggregation without cytotoxicity on human endothelial cells. Compound 1 inhibited the extrinsic and intrinsic blood coagulation pathways through the inhibition of FXa and thrombin production in HUVECs. Compound 1 suppressed 25% PAI-1/t-PA ratio induced by TNF-α. These results add to the previous work on the topic and should be of interest to develop a natural product-derived anticoagulant agent.

Experimental

General

IR spectrum was recorded on a Bruker ALPHA FT-IR spectrometer. NMR experiments were conducted using a Bruker DMX 300 (¹H-300 MHz, ¹³C-75 MHz) and Bruker DMX 600 (¹H-600 MHz, ¹³C-150 MHz) spectrometers (Karlsruhe, Germany). Optical rotations were recorded using a JASCO DIP-1000 (Tokyo, Japan) and mass spectrometric data were obtained utilizing a SYNAPT G2 Waters mass spectrometer (Manchester, United Kingdom). Vacuum-liquid chromatography (VLC) was performed using silica gel (Merck, 70-230 mesh). MPLC was carried out employing Biotage Isolera reversed phase C18 SNAP Cartridge KP-C18-HS and normal phase SNAP Cartridge KP-Sil (Biotage AB, Uppsala, Sweden). HPLC separation was performed using a Gilson system with a UV detector and Phenomenex C18 column (250 × 21.2 mm, 5 µm). TLC was performed on glass plates precoated with silica gel 60 F254 and RP-18 F254 (Merck).

Plant Material

The roots of Rubia akane were purchased from Korea traditional medicine market (Daejeon, Korea) in 2017. A voucher specimen (CNU 20170726) was deposited at the Pharmacognosy Laboratory of the College of Pharmacy, Chungnam National University (Daejeon, Korea).

Extraction and Isolation

The roots of R. akane (4.9 kg) were extracted with EtOH (8 L × 3) at room temperature for 6 days. The EtOH extract (158.2 g) was suspended in H₂O and partitioned with CH₂Cl₂. The CH₂Cl₂ fraction (78.9 g) was subjected to silica gel VLC and eluted with n-hexane/EtOAc (10:0, 5:1) and CHCl₃/MeOH (40:1, 20:1, 10:1, 5:1, and 0:10) to obtain 6 fractions (MC1-MC6). Fr. MC4 (9.8 g) was further divided into 9 fractions (MC4-1-MC4-9) using MPLC (C₁₈ SNAP Cartridge KP-C₁₈-HS, 400 g) with MeCN/H₂O (37:63, 37:63 → 100:0). Fr. MC4-7 (404.8 mg) was applied to C₁₈ HPLC with isocratic MeOH/H₂O (78:22) to afford 3 (13.2 mg) and 4 other subfractions (MC4-7-1-MC4-7-4). Compound 2 (5.0 mg) was isolated from MC4-7-4 (15.2 mg) by biphenyl HPLC using a mobile phases of MeOH/H₂O (70:30 → 90:10). Fr. MC4-8 (850.0 mg) was separated by preparative HPLC using a MeCN/H₂O (80:20) solvent system to afford 5 subfractions (MC4-8-1-MC4-8-5). Compound 1 (5.2 mg) was acquired from Fr. MC4-8-5 (17.0 mg) using C₁₈ column eluting with MeCN/H₂O (60:40 → 90:10).

3β-Hydroxy-16-Oxo-21β-Isohop-22(29)-En-24-Oic Acid (1)

White amorphous solid.

${[α]}_{D}^{21}$ : −10 (c 0.12, MeOH).

FT-IR (ATR) V_max : 3319, 2943, 2831, 1450, 1415, 1020 cm⁻¹.

UV/Vis λ_max (MeOH) nm (log ε): 200 (3.89).

¹H NMR (600 MHz, pyridine-d ₅): 4.82 (2H, d, J = 16.93 Hz, H₂-29), 4.67 (1H, t, H-3), 3.00 (1H, m, H-21), 2.64 (1H, d, J = 11.51 Hz, H-17), 2.50 (1H, d, J = 12.42 Hz, H₂-15), 2.20 (1H, m, H-13), 1.92 (1H, d, J = 12.29 Hz, H-5), 1.86 (1H, d, J = 12.42 Hz, H₂-15), 1.75 (3H, s, H₃-30), 1.72 (1H, m, H₂-1), 1.66 (3H, s, H₃-23), 1.55 (1H, m, H₂-19), 1.40 (1H, m, H₂-19), 1.36 (1H, m, H-9), 1.15 (1H, m, H₂-1), 1.06 (3H, s, H₃-26), 0.97 (3H, s, H₃-27), 0.94 (3H, s, H₃-25), 0.72 (3H, s, H₃-28)

¹³C NMR (150 MHz, pyridine-d ₅): 210.7 (C-16), 181.3 (C-24), 148.2 (C-22), 109.5 (C-29), 75.8 (C-3), 65.6 (C-17), 54.9 (C-4), 52.6 (C-18), 51.9 (C-5), 50.7 (C-8, 9, 15), 49.3 (C-13), 43.2 (C-14), 42.3 (C-21), 41.1 (C-19), 39.6 (C-1), 37.1 (C-10), 33.7 (C-7), 28.3 (C-11, 20), 24.1 (C-2), 22.0 (C-6), 21.9 (C-30), 21.4 (C-12), 17.9 (C-27), 17.5 (C-26), 16.7 (C-25, 28), 12.4 (C-23)

HRESIMS: m/z 493.3291 [M+Na]⁺ calcd for C₃₀H₄₆O₄Na: 493.3294

Biological Section

Detailed methods for the biological evaluation in this study are provided in Supplemental data.

Supplemental Material

Supplemental data - Supplemental material for Antithrombotic and Antiplatelet Activities of New Isohopane Triterpene From the Roots of Rubia akane

Supplemental material, Supplemental data, for Antithrombotic and Antiplatelet Activities of New Isohopane Triterpene From the Roots of Rubia akane by InWha Park, Wonhwa Lee, Hyelim Kim, Khong Trong Quan, DaYoung Kim, Jong-Sup Bae, and MinKyun Na in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by research fund from Chungnam National University.

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Supplementary Material

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