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molecules Article Inhibitory Effect of Triterpenoids from Panax ginseng on Coagulation Factor X Lingxin Xiong 1,2,3 , Zeng Qi 1,2 , Bingzhen Zheng 1,2 , Zhuo Li 1,2 , Fang Wang 3 , Jinping Liu 1,2, * and Pingya Li 1,2, * 1 2 3 * School of Pharmaceutical Sciences, Jilin University, Fujin Road 1266, Changchun 130021, China; [email protected] (L.X.); [email protected] (Z.Q.); [email protected] (B.Z.); [email protected] (Z.L.) National and Local Joint Engineering Research Center for Ginseng Innovative Drugs Development, Western Chaoyang Road 45, Changchun 130021, China Department of Pathogen Biology, Basic Medical College, Jilin University, Changchun 130021, China; [email protected] Correspondence: [email protected] (J.L.); [email protected] (P.L.); Tel.: +86-431-8561-9803 (J.L. & P.L.) Academic Editors: Anusha Chaparala, Lorne Hofseth and Woo-Sik Jeong Received: 23 February 2017; Accepted: 11 April 2017; Published: 24 April 2017 Abstract: Enzymes involved in the coagulation process have received great attention as potential targets for the development of oral anti-coagulants. Among these enzymes, coagulation factor Xa (FXa) has remained the center of attention in the last decade. In this study, 16 ginsenosides and two sapogenins were isolated, identified and quantified. To determine the inhibitory potential on FXa, the chromogenic substrates method was used. The assay suggested that compounds 5, 13 and 18 were mainly responsible for the anti-coagulant effect. Furthermore, these three compounds also possessed high thrombin selectivity in the thrombin inhibition assay. Furthermore, Glide XP from Schrödinger was employed for molecular docking to clarify the interaction between the bioactive compounds and FXa. Therefore, the chemical and biological results indicate that compounds 5 (ginsenoside Rg2), 13 (ginsenoside Rg3) and 18 (protopanaxtriol, PPT) are potential natural inhibitors against FXa. Keywords: ginseng; triterpenoids; coagulation factor Xa; inhibitors; thrombin; molecular docking 1. Introduction Thrombosis is a common pathology characterized by the formation of a blood clot inside a blood vessel and the obstruction of blood flow through the circulatory system that underlies three major cardiovascular diseases including acute coronary syndrome, stroke and venous thromboembolism [1]. Venous thromboembolism affects millions of people every year and is responsible for hundreds of thousands of deaths in both the United States and Europe annually [2]. Warfarin, vitamin K antagonists and low-molecular-weight heparins are the most commonly used drugs to reduce thromboembolic occurrence. Despite the prevalence of these anti-coagulants, their employment actually increases the risk of bleeding, brings about the inconvenience of frequent monitoring, interacts with many drugs and foods, as well as has slow onset of action [3], which hinders further clinical application in the treatment of thrombosis. To address the difficulties, more selective anti-coagulants are needed to meet the requirement of efficacy and safety. Coagulation factor X (FXa) is a vitamin K-dependent serine protease and is one of various enzymes involved in the process of blood coagulation cascade as a catalyst in the conversion of prothrombin to thrombin, which enables FXa to be a potent attractive target for novel anti-coagulant. Compared with the previously prevalent targeting drug thrombin (factor IIa) inhibitors, much evidence has shown that FXa inhibitors were more effective than direct thrombin inhibitors due to FXa’s upstream position from Molecules 2017, 22, 649; doi:10.3390/molecules22040649 www.mdpi.com/journal/molecules Molecules 2017, 22, 649 2 of 17 thrombin in the coagulation cascade. Selective inhibition of FXa was unable to affect the pre-existing thrombin level, and activation and aggregation of the platelets reduced the risk of bleeding when compared with traditional anti-coagulants [2]. In addition, FXa plays a vital role in amplifying the process, and is endued with the ability to produce more than one thousand thrombin molecules [2,4]. Furthermore, currently available FXa inhibitors approved by Food and Drug Administration such as rivaroxaban, apixaban and epibaxaban, have been reported to still demonstrate flaws including bleeding risks, narrow clinical applications and drug-drug interactions [2]. Thus, there is a need to develop novel FXa inhibitors with better efficacy and less side-effects. Panax ginseng C. A. Mey, the Araliaceae plant, has been considered as a health food and traditional herbal medicine in Eastern Asia including China, Korea and Bhutan over the past centuries [5]. Nowadays P. ginseng is also accessible in small doses in commercial energy beverages or herbal teas. In a previous study, water extracts of P. ginseng were reported to possess anti-coagulation effect in vitro [6]. In addition, some Chinese herbal formulas containing ginsenosides also displayed a modulatory effect on the blood coagulation system. For example, a traditional Chinese medicine remedy, Fufang Xueshuantong (including various kinds of ginsenosides) was reported to ameliorate the disorders of the blood coagulation system in a lipopolysaccharide-induced disseminated intravascular coagulation rat model via modulating the activation of the coagulation system [7]. In our previous study, we demonstrated that another traditional Chinese herbal formula Xueshuan Xinmaining Tablets (containing a total ginsenoside of ginseng stems and leaves) enhanced protective activities and anti-oxidative effect of vascular endothelial cells in vitro and removed blood stasis syndrome in murine models, indicating a potential anti-coagulation role in clinical application [8,9]. Furthermore, ginsenosides Rg1 and Rg2 have been reported to possess anti-coagulation properties in vitro [6]. Although P. ginseng and part of its major functional components (ginsenosides) have shown anti-coagulation activity in previous studies, whether ginsenosides possess anti-FXa activity and, if so, how ginsenosides interact with surrounding residues have not been reported. Therefore, we designed and conducted a series of anti-coagulation experiments to provide theoretical support for the further development of novel oral-administrated ginsenosides-based FXa inhibitors. In this study, we aimed to structurally elucidate the components of ginseng, and to determine the content of these ginsenosides through high performance liquid chromatography (HPLC) analysis. Activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) assays were performed to determine the plasma anti-coagulation activity of ginsenosides in vitro, and among ginsenosides demonstrating significant in vitro anti-coagulation effects, the in vitro bioactivities against anti-coagulation factor Xa (FXa) were assessed. Subsequently, ginsenosides that possessed the best bioactivity were molecularly docked with the receptor protein FXa via Schrödinger software to observe the ligand-protein interactions. 2. Results 2.1. Isolation and Characterization The purified products above-mentioned were each characterized by NMR analyses. The structures of compounds 1–18 were identified as follows: ginsenoside Rg1 (1, yield 0.328%), Re (2, yield 0.088%), Rf (3, yield 0.071%), Rh1 (4, yield 0.008%), Rg2 (5, yield 0.011%), Rb1 (6, yield 0.524%), Rc (7, yield 0.121%), Ro (8, yield 0.006%), F1 (9, yield 0.114%), Rb2 (10, yield 0.114%), Rb3 (11, yield 0.013%), Rd (12, yield 0.123%), Rg3 (13, yield 0.021%), 20(R)-Rg3 (14, yield 0.001%), Rh2 (15, yield 0.001%), F2 (16, yield 0.001%), protopanaxdiol (PPD, 17, yield 0.071%), protopanaxtriol (PPT, 18, yield 0.001%) [10–18]. 2.2. Determination of Content for the Compounds from Ginseng The dried ginseng root powder (1.0 g, 60 mesh sieve) was accurately weighed and was added to two volumes of water. The mixture was heated under 100 ◦ C for 2 h. Next, the filtration was added Molecules 2017, 22, 649 3 of 17 Molecules2017, 2017,22, 22,649 649 Molecules of17 17 33of to a D101 macroporous adsorption resin column eluted with water and 80% ethanol consecutively. to80% D101 macroporous adsorption resin column column eluted with with water and 80% ethanol ethanol consecutively. to aa D101 macroporous adsorption resin eluted water 80% consecutively. The ethanol elution was concentrated to the residue, which wasand dissolved in 10 mL of methanol. The 80% ethanol elution was concentrated to the residue, which was dissolved in 10 mL of methanol. The 80% ethanol elution was concentrated to the residue, which was dissolved in 10 mL of methanol. Some standard compounds were also used in this study. Ginsenoside Rg1 (110703-200726), Some standard compounds were also used in this study. Ginsenoside Rg1 (110703-200726), Some Re standard compoundsginsenoside were also used in this study. Ginsenoside Rg1 ginsenoside (110754-200822), Rb2 (111715-201203), ginsenoside Rb3(110703-200726), (111686-201203), ginsenoside Re Re (110754-200822), (110754-200822), ginsenoside ginsenoside Rb2 (111715-201203), (111715-201203), ginsenoside ginsenoside Rb3 Rb3 (111686-201203), ginsenoside ginsenoside Rd (111818-201302), ginsenosideRb2 Rg3 (110804-201504), ginsenoside Rf(111686-201203), (111719-201505), ginsenoside Rd Rd (111818-201302), (111818-201302), ginsenoside ginsenoside Rg3 Rg3 (110804-201504), (110804-201504), ginsenoside ginsenoside Rf Rf (111719-201505), (111719-201505), ginsenoside ginsenoside Rh2 (111748-200501), ginsenoside Rg2 (111779-200801), ginsenoside Ro (111903-201604), ginsenoside Rh2 Rh2 (111748-200501), (111748-200501), ginsenoside Rg2 Rg2 (111779-200801), ginsenoside ginsenoside Ro Ro (111903-201604), (111903-201604), ginsenoside protopanaxdiol (111747-200501) andginsenoside protopanaxtriol (111779-200801), (111755-200601) were purchased from the National protopanaxdiol (111747-200501) and protopanaxtriol (111755-200601) were purchased from the the protopanaxdiol (111747-200501) and protopanaxtriol (111755-200601) were purchased from Institutes for Food and Drug Control (Beijing, China). Ginsenosides Rh1, Rb1, Rc, F1, 20(R)-Rg3 and F2 National Institutes for Food and Drug Control (Beijing, China). Ginsenosides Rh1, Rb1, Rc, F1, 20(R)-Rg3 National Institutes for Food and Drug Control (Beijing, China). Ginsenosides Rh1, Rb1, Rc, F1, 20(R)-Rg3 were provided by the New Drug Research and Development Laboratory of Jilin University. and F2 F2 were were provided provided by by the the New New Drug Drug Research Research and and Development Development Laboratory Laboratory of of Jilin Jilin University. University. and The determination of the saponins was performed using a HPLC system. The detection The determination determination of of the the saponins saponins was was performed performed using using a HPLC HPLC system. system. The The detection detection The ◦ C. The amobile wavelength was 40 40 °C. phasewas wascomprised comprisedofof wavelengthwas was203 203nm. nm. Column Column temperature temperature was was The mobile mobile phase phase wavelength was 203 nm. Column temperature 40 °C. The was comprised of acetonitrile (A) and 1% acetic acid ininwater solvent (B). The gradient mode modewas wasas asfollows: follows:initial initial20% 20% acetonitrile (A) and 1% acetic acid water solvent (B). The gradient acetonitrile (A) and 1% acetic acid in water solvent (B). The gradient mode was as follows: initial 20% A linear gradient to 22% A in 25 min; linear gradient to 28% A in 55 min; linear gradient to 35% A A linear linear gradient gradient to to 22% 22% A A in in 25 25 min; min; linear linear gradient gradient to to 28% 28% A A in in 55 55 min; min; linear linear gradient gradient to to 35% 35% A A in inin A 95 95 min; linear gradient gradient to 100% 100% A Ain in135 135min. min.The Theflow flowrate rate min; linear gradienttoto to60% 60%AA Ain in112 112min; min;and and linear linear gradient gradient to to 95 min; linear gradient 60% in 112 min; and linear 100% A in 135 min. The flow rate was 1.3 mL/min. The components were identified through comparison of the retention time from the was 1.3 1.3 mL/min. mL/min. The The components components were were identified identified through through comparison comparison of of the the retention retention time time from from the the was chromatograms with of Rg1, Rg1,Re, Re,Rf, Rf,Rh1 Rh1+++Rg2, Rg2,Rb1, Rb1, Rc, Ro, F1, Rb2, chromatograms withknown knownstandards. standards.The Thecontents contents of Re, Rf, Rh1 Rg2, Rb1, Rc, Ro, F1, Rb2, chromatograms with known standards. The contents Rc, Ro, F1, Rb2, Rb3, Rd, Rg3, 20(R)-Rg3, Rh2, F2, PPD and PPT were 0.4659%, 0.1061%, 0.0908%, 0.0230%, 0.6145%, Rb3, Rd, Rg3, 20(R)-Rg3, Rh2, F2, PPD and PPT were 0.4659%, 0.1061%, 0.0908%, 0.0230%, 0.6145%, Rb3, Rd, Rg3, 20(R)-Rg3, Rh2, F2, PPD and PPT were 0.4659%, 0.1061%, 0.0908%, 0.0230%, 0.6145%, 0.1412%, 0.0017%, 0.1581%, 0.1497%, 0.0152%, 0.0342%, 0.0001%,0.0001%, 0.0001%, 0.0001%, 0.1412%, 0.0017%, 0.1581%, 0.1497%, 0.0152%,0.1546%, 0.1546%,0.0021%, 0.0021%, 0.0342%, 0.0342%, 0.0001%, 0.0001%, 0.0001%, 0.0001%, 0.1412%, 0.0017%, 0.1581%, 0.1497%, 0.0152%, 0.1546%, 0.0021%, 0.0001%, 0.0377% and 0.0958%,respectively. respectively.The Thefingerprints fingerprints ofofthe the mixed standard compounds andand theextract extract of 0.0377% and 0.0958%, The fingerprintsof themixed mixed standard compounds the extract 0.0377% and 0.0958%, respectively. standard compounds and the of ginseng are shown in Figures 1 and 2. ofginseng ginsengare areshown shown in Figures 1 and 2. in Figures 1 and 2. RR-R-Rgg33 S -R g 3 PPPPTT S -R g 3 RRdd RRbb22 RRbb33 RRcc RRoo FF11 RRhh11/R/Rgg22 72.00 74.00 76.00 78.00 80.00 82.00 84.00 86.00 88.00 90.00 92.00 94.00 96.00 98.00 100.00 102.00 104.00 106.00 108.00 110.00 112.00 72.00 74.00 76.00 78.00 80.00 82.00 84.00 86.00 88.00 90.00 92.00 94.00 96.00 98.00 100.00 102.00 104.00 106.00 108.00 110.00 112.00 MIN 20.00 20.00 30.00 30.00 40.00 40.00 50.00 50.00 60.00 60.00 70.00 70.00 MIN 80.00 80.00 90.00 90.00 MIN (min) Time Time (min) Figure2. 2.HPLC HPLC record record of of ginseng. ginseng. Figure record of ginseng. Figure 2. 100.00 100.00 110.00 110.00 120.00 120.00 Oleanic Oleanicacid acid PPD PPD F2 F2 Rh2 Rh2 R-Rg3 R-Rg3 PPT S-Rg3 PPTS-Rg3 Rd Rd Rh1/Rg2 Rh1/Rg2 Rf Rf Rg1 Rg1 Re Re 10.00 10.00 Rb1 Rb1 Rc Rc Ro Ro F1 F1 Rb2 Rb2 Rb3 Rb3 MIN (min) Time Time (min) AU AU 0.30 0.30 0.28 0.28 0.26 0.26 0.24 0.24 0.22 0.22 0.20 0.20 0.18 0.18 0.16 0.16 0.14 0.14 0.12 0.12 0.10 0.10 0.08 0.08 0.06 0.06 0.04 0.04 0.02 0.02 0.00 0.00 -0.02 -0.02 0.00 0.00 RRbb11 Figure 1.High Highperformance performanceliquid liquidchromatography chromatography (HPLC) (HPLC) record record of mixture of standards. Figure 1.1. (HPLC) recordof ofmixture mixtureof ofstandards. standards. Figure High performance liquid chromatography 130.00 130.00 Molecules 2017, 22, 649 4 of 17 2.3. In VitroMolecules Effects of 22, Ginsenosides on Human Blood Clotting Time 2017, 649 4 of 17 At final concentrations of 0.05onmg/mL, nine outTime of 18 ginseonsides showed significant 2.3. In Vitro Effects of Ginsenosides Human Blood Clotting anti-coagulant effects in vitro compared to the normal control (p < 0.05) (Figure 3) and were selected to At final concentrations of 0.05 mg/mL, nine out of 18 ginseonsides showed significant antibe further coagulant detectedeffects for their anti-FXa in vitro. Among nine3)ginsenosides with in vitro comparedactivity to the normal control (p < 0.05)the (Figure and were selected to beexcellent anti-coagulation activityfor in their vitro, Rg2, Rg3 and the ginsenosides best bioactivity (p < 0.01) further detected anti-FXa activity in PPT vitro. possessed Among the nine with excellent anti-and other coagulation activity in Rh1, vitro, F1, Rg2,Rh2, Rg3 and PPT possessed the best bioactivity (p < 0.01) and other activity ginsenosides including Rg1, F2 and PPD showed significant anti-coagulation including Rg1, Rh1, F1, Rh2, F2 and PPD showed significant anti-coagulation activity compared ginsenosides to the normal control (p < 0.05) in APTT, PT and TT tests. In contrast, the solvent group, compared to the normal control (p < 0.05) in APTT, PT and TT tests. In contrast, the solvent group, Rf Rf and 20(R)-Rg3 displayed insignificant anti-coagulation effects when compared to the normal and 20(R)-Rg3 displayed insignificant anti-coagulation effects when compared to the normal control control in all threeincoagulation parameters (p > 0.05). all three coagulation parameters (p > 0.05). 80 ** A Clotting time(s) in APTT test 70 60 ** 50 40 * * 30 ** ** ** * * * * * * 20 10 0 Clotting time (s) in PT test Groups 20 18 16 14 12 10 8 6 ** B ** ** * * * 4 2 0 Molecules 2017, 22, 649 35 5 of 17 Groups C ** 30 Clotting time (s) in TT test ** * * ** ** * 25 * * ** * 20 15 10 5 0 Groups 3. Inanti-coagulation vitro anti-coagulation activities of ginsenosides. (A) APTT (B) PTtest; test; (B) (C) TT Figure 3. Figure In vitro activities of1111 ginsenosides. (A)test; APTT PTtest. test; (C) TT * p < 0.05, ** p < 0.01 versus the normal control. APTT: activated partial thromboplastin time; PT: test. * p < 0.05, ** p < 0.01 versus the normal control. APTT: activated partial thromboplastin time; PT: prothrombin time; TT: thrombin time. prothrombin time; TT: thrombin time. 2.4. Effects of Ginsenosides on FXa Activities In Vitro Among the nine ginsenosides showing excellent anti-coagulant activities and the two ineffective ginsenosides in vitro, ginsenosides Rg2, Rg3 and PPT exhibited the best anti-FXa activities with fifty percent of inhibitory concentration (IC50) of 135.9, 126.7 and 140.7 nM, respectively (Table 1). The positive control drug showed higher activity with IC50 of 1.9 nM compared to all test ginsenosides (Figure 4). Clotting time (s) in 00 0 20 15 Groups Groups Groups 10 5 vitro Figure 3. In In vitro anti-coagulation activities of 11 11 ginsenosides. (A)(A) APTT test;test; (B) (B) PT PT test;test; (C)(C) TT TT test.test. Figure 3.vitro In anti-coagulation activities of ginsenosides. 11 ginsenosides. APTT Figure 3. anti-coagulation activities of (A) APTT test; (B) PT test; (C) TT test. 0.05, **0pp**<<p0.01 0.01 versus thethe normal control. APTT: activated partial thromboplastin time; PT:PT: Molecules 2017, 22, 649 < 0.05, < 0.01 versus normal control. APTT: activated partial thromboplastin time; ** pp *<<p0.05, ** versus the normal control. APTT: activated partial thromboplastin time; PT: 5 of 17 prothrombin time; TT:TT: thrombin time. prothrombin time; thrombin time. prothrombin time; TT: thrombin time. Groups 2.4.2.4. Effects of Ginsenosides Ginsenosides on FXa FXa Activities In Vitro Vitro Effects of Ginsenosides on FXa Activities In Vitro 2.4. Effects of on Activities In 2.4. Effects of Ginsenosides on3. InFXa Activitiesactivities In Vitro Figure vitro anti-coagulation of 11 ginsenosides. (A) APTT test; (B) PT test; (C) TT test. Among the nine ginsenosides showing excellent anti-coagulant activities and the twotwo ineffective * p the <nine 0.05, ** p ginsenosides < 0.01 versus showing the normal control. APTT: activated partial thromboplastin time; PT: Among nine showing excellent anti-coagulant activities and the ineffective Among the ginsenosides excellent anti-coagulant activities and the two ineffective prothrombin time; TT: thrombin time.Rg3 and PPT exhibited the best anti-FXa activities with fifty ginsenosides in vitro, vitro, ginsenosides Rg2, in vitro, ginsenosides Rg2, Rg3 PPT exhibited anti-FXa activities with fifty ineffective in ginsenosides Rg2, Rg3 andand PPT exhibited thethe bestbest anti-FXa activities fifty Among ginsenosides theginsenosides nine ginsenosides showing excellent anti-coagulant activities andwith the two percent of inhibitory inhibitory concentration (IC(IC 50)) 50 of) 135.9, 135.9, 126.7 andand 140.7 nM, respectively (Table 1). 1). TheThe percent of inhibitory concentration of 135.9, 126.7 140.7 nM, respectively (Table percent of concentration (IC 50 of 126.7 and 140.7 nM, respectively (Table 1). The 2.4.ginsenosides Effects of Ginsenosides on FXa Activities In Vitro ginsenosidespositive in vitro, Rg2, Rg3 and PPT exhibited the best anti-FXa activities with fifty control drug showed higher activity with IC50 50 of 1.9 nMnM compared to all all testtest ginsenosides positive control drug showed higher activity with ICof 50 1.9 of 1.9 compared to all ginsenosides positive control drug showed higher activity with IC nM compared to test ginsenosides Among the nine ginsenosides showing excellent anti-coagulant activities and the two ineffective percent of inhibitory concentration (IC50 ) ofRg2, 135.9, 126.7 and 140.7 nM, respectively (Table 1). The positive (Figure 4).ginsenosides (Figure 4). (Figure 4). in vitro, ginsenosides Rg3 and PPT exhibited the best anti-FXa activities with fifty of inhibitory concentration (IC ) ofof 140.7 nM, respectively The control drug showedpercent higher activity with IC 1.9126.7 nMandcompared to all (Table test 1). ginsenosides (Figure 4). 50 135.9, 50 Table 1. The The structures of 11 11 ginsenosides and their anticoagulation activities against coagulation positive control drug of showed activity with ICtheir 50 anticoagulation of 1.9 nM compared to all test ginsenosides Table 1. The structures of ginsenosides 11higher ginsenosides and anticoagulation activities against coagulation Table 1. structures and their activities against coagulation (Figure 4). factor Xa (FXa). (FXa). factor Xa (FXa). factor Xa Table 1. The structures of 11 ginsenosides and their anticoagulation activities against coagulation factor Table 1. The structures of 11 ginsenosides and their R3O Oanticoagulation activities against coagulation R 3 R3O OH OH Xa (FXa). factor Xa (FXa). OH OH R3O R1O O R 1 R 1O R 1O R2 R R 2 2 R2 Compound Compound Compound Compound Compound R1 R11 RR11 R R222 R2 R R R 2 HO HO HO HO Rg1 Rg1 Rg1 Rg1 22, Molecules2017, 2017, 649 Molecules 22, 649 Rg1 HO O OO O O O OH O O OH OH OHOH OH OH OH OH OH OH OH H H H H H HO Molecules 2017, 22,22, 649 Molecules 2017, 649 Molecules Molecules 2017, 2017, 22, 22, 649 649 Rh1 H Rh122, 649 MoleculesRh1 2017, Molecules 2017, 22, 649 H H H H Rh1 Rh1 Rg2 Rg2 H H Rg2 Rg2 Rg2 Rg2 HH H H Rg2 Rg2 Rg2 H HH F1 F1 F1F1 F1 Rg3 Rg3 Rg3 Rg3 Rg3 Rg3 Rg3 Rh2 Rh2 Rh2 Rh2 Rh2 Rh2 Rh2 O O OH OH OH OH HO HO O HO O O HOOHOHO OO OH OH OHO OH O O OH OH OH OH OH OH HO HO OH OH OH OH HO HO OO OH OHOH OH HO HO OO O O O O O OH OHO O OH HO OH OH OH OH OH HO OH OHOH OH OH OH OH OH OH O OHO HH H H H H Rh2 Rh2 PPD PPD H PPT PPD PPD PPT PPD PPD PPT PPT H H H H H H H PPT PPT PPT PPT PPT 20(R)-Rg3 20(R)-Rg3 20(R)-Rg3 20(R)-Rg3 HO HO 20(R)-Rg3 20(R)-Rg3 20(R)-Rg3 20(R)-Rg3 20(R)-Rg3 Rf Rf O H H HH H H HO O HO OH O O OH O OH OH HO OH HO HO HO HO OH OO OH O O OH HOO HO O HO O OH OO OH OH O OH OHO OH O OH OH OH OH OHOH OH HO HO OH OH OH OH OH O OH HO OHO O O O HO O OH OHO OH O OH OH OH OH OH OH OH OH OH HH H H H RfRf Rf Rf Rivaroxaban Rivaroxaban Rivaroxaban Rivaroxaban Rivaroxaban HH H H O O O OH OH OH OH OH OH OH OH OH OH H 334.7 334.7 334.7 334.7 334.7 H H H HO HO 235.8 235.8 235.8 235.8 6 of 17 6 of 17 H H 135.9 135.9 HH H H 135.9 135.9 135.9 135.9 H H H 135.9 135.9 135.9 OH OH HO HO HO OH HO OH OO O O OH OH O OH OH O O OH O OH OH OH OH OH OH OH OH OH OH OH OH OH OH HO HO H H HH H HH H HH HH HH H H H H H HH HH H H HO HO HHH H HH OO H H H H HHH H H H H HO HOOH OH HO HO HO OH O O HO OH O O OH OH OHO OH OH OH O OH OH OH OH OH OH OH OH OH OH H H H OH HH OH H H OH OH OH OH OH OH H H H H HOH OH H OH OH H H H H H H H HHH 227.0 227.0 227.0 227.0 227.0 227.0 227.0 227.0 227.0 126.7 126.7 348.7 348.7 348.7 348.7 348.7 348.7 348.7 348.7 348.7 425.2 425.2 425.2 425.2 425.2 425.2 425.2 425.2 425.2 339.4 339.4 339.4 339.4 339.4 140.7 339.4 339.4 140.7 339.4 339.4 140.7 140.7 140.7 HH H H H HHH 140.7 140.7 140.7 140.7 815.3 815.3 815.3815.3 HH H H 815.3 815.3 815.3 815.3 HH H H HO HO HO HO O O OH O OH O OH OH HO OH OH HO O OH HO HO O OH O O O O HO OHHO O O HO OH OH OH O O HO OH OH OH OH OH OHO OH OH OH OH O OH 126.7 126.7 126.7 126.7 126.7 126.7 126.7 H H HH HH H HOHO OHOH OHOH O O HO OHO O O HO O OHOHO OH O OHOH OH OH OHOH OH OH OH HH H H 815.3 1034.0 1034.0 1034.0 1034.0 1034.0 1034.0 1034.0 1034.0 1.91034.0 1.9 1.9 1.9 1.9 IC50 : fifty of inhibitory concentration; PPD:protopanaxdiol; protopanaxdiol; protopanaxtriol. ICpercent 50: fifty percent of inhibitory concentration; PPD: PPT: PPT: protopanaxtriol. IC50 : fifty percent of inhibitory concentration; PPD: protopanaxdiol;PPT: PPT: protopanaxtriol. protopanaxtriol. IC50: fifty percent of inhibitory concentration; PPD: protopanaxdiol; IC50: fifty percent of inhibitory concentration; PPD: protopanaxdiol; PPT: protopanaxtriol. Rivaroxaban 1.9 Rivaroxaban 1.9 Rivaroxaban 1.9 Rivaroxaban 1.9 ICIC 50:50fifty percent PPT: : fiftypercent percentof ofinhibitory inhibitoryconcentration; concentration;PPD: PPD:protopanaxdiol; protopanaxdiol; PPT:protopanaxtriol. protopanaxtriol. B IC : fifty A of IC50 50: fifty percent of inhibitory inhibitory concentration; concentration; PPD: PPD: protopanaxdiol; protopanaxdiol; PPT: protopanaxtriol. protopanaxtriol. B PPT: AA AA A A A BB BB B B of17 17 66of of 1717 6 of 666 of of 17 17 235.8 O HOHO OH OH HO HO HO OH O O HO OH O O OH OH OH OH O O OH OH OH OH OH OHOH OH OH OH OH HOH OH H OH OH OH H F2F2 O OH OH IC50 50 (nM) IC(nM) 50 (nM) IC50 (nM)IC IC50 (nM) HO HO HO OH H H OH OH HO HO HO HO OH OH HO OH HO OH O OO O O O OH OH OHOH OHOH OH OH OH OH HO HO OH OH OHOH OH OH OH OH OO F2 PPD PPD Rf Rf Rf HOH H OH OH OH HH H HOH HO H H H F2 F2 PPD OH OH OH OH HO HO Rg3 Rg3 O OH OH O O OH OHOH OH H H F1F1 F1 F1 F2F2 F2 F2 HO O O HO HO HO HO OH OOOO OOOO OH O O OHOH OH OH OH OH HO HO OH HO OH OH OH OH HO OO OH OOOO OH OH O OH O OH OHO OH OHO OOH HO OH OH OH O OH OH OOO OO O OH OHOH OOH O OH OH O OH OH OH OH OH O OH O O R33 R3 R R3 R3 HO O Rf H HO OH OH O H O 1034.0 OH OH OH Rivaroxaban 1.9 Molecules 2017, 22, 649 IC50: fifty percent of inhibitory concentration; PPD: protopanaxdiol; PPT: protopanaxtriol. 6 of 17 B A Molecules 2017, 22, 649 7 of 17 D C F E H G J I L K Figure 4. Cont. Molecules 2017, 22, 649 7 of 17 Molecules 2017, 22, 649 8 of 17 M N P O Q R S T U V Figure 4. Cont. Molecules Molecules 2017, 2017, 22, 22, 649 649 98 of of 17 17 Molecules 2017, 22, 649 9 of 17 W W X X Figure 4. The inhibition profile figures against coagulation factor X. (A,B) Rg1; (C,D) Rh1; (E,F) Rg2; (G,H) F1; (I,J) Rg3; (K,L)profile Rh2; figures (M,N) F2; (O,P)coagulation PPD; (Q,R)factor PPT;X. (S,T) 20(R)-Rg3; (U,V) Rf; (W,X) Figure The inhibition profile figures against coagulation factor X.(A,B) (A,B) Rg1;(C,D) (C,D) Rh1; (E,F) Rg2; Figure 4. 4. The inhibition against Rg1; Rh1; (E,F) Rg2; 10, OD: optical density. Rivaroxaban. lg: log (G,H)F1;F1;(I,J) (I,J)Rg3; Rg3;(K,L) (K,L)Rh2; Rh2;(M,N) (M,N)F2; F2;(O,P) (O,P)PPD; PPD;(Q,R) (Q,R)PPT; PPT;(S,T) (S,T)20(R)-Rg3; 20(R)-Rg3;(U,V) (U,V)Rf; Rf;(W,X) (W,X) (G,H) 1010 , OD:optical opticaldensity. density. Rivaroxaban. log Rivaroxaban. lg:lg:log , OD: 2.5. Selectivity Versus Thrombin 2.5.Selectivity VersusThrombin Thrombin 2.5. Versus InSelectivity the previous anti-coagulant assay in vitro, Rg2, Rg3 and PPT demonstrated excellent FXa inhibitory activity andanti-coagulant were further selected assess their selectivity versus thrombin. excellent Rg2, Rg3 and the previous anti-coagulant assayinto invitro, vitro,Rg2, Rg2,Rg3 Rg3and andPPT PPT demonstrated excellent FXa InInthe previous assay demonstrated FXa PPT displayed a high level of selectivity against thrombin with IC 50 values of 81.3, 92.6, 82.0 μM, inhibitory activity and were further selected to assess their selectivity versus thrombin. Rg2, Rg3 and inhibitory activity and were further selected to assess their selectivity versus thrombin. Rg2, Rg3 respectively, whicha were higher than that of ximelagatran with ICwith 50 values 27.1 μM 2, Figure PPT displayed high level against with IC50of values of (Table 81.3, 92.6, 82.0 5). μM, and PPT displayed a high levelof ofselectivity selectivity against thrombin thrombin IC 50 values of 81.3, 92.6, 82.0 µM, The evidence showed that Rg3 showed the best anti-coagulant activity in vitro and also demonstrated respectively, which were higher than that of ximelagatran with IC 50 values of 27.1 μM (Table 2, Figure respectively, which were higher than that of ximelagatran with IC50 values of 27.1 µM (Table 2, Figure 5).5). the highest selectivity against thrombin. The evidence showed that Rg3 showed thebest bestanti-coagulant anti-coagulant activity vitroand andalso alsodemonstrated demonstrated The evidence showed that Rg3 showed the activity ininvitro the highest selectivity against thrombin. the highest selectivity against thrombin. Table 2. Selectivity versus thrombin. Table 2. Selectivity versus thrombin. Table 2. SelectivityThrombin versus thrombin. Compound IC50 (μM) Rg2 81.3 IC50 (μM) Compound Thrombin Compound Thrombin IC50 (µM) Rg3 92.6 Rg2 81.3 Rg2 81.3 PPT 82.0 Rg3 92.6 Rg3 92.6 Ximelagatran 27.1 PPT 82.0 PPT 82.0 Ximelagatran 27.1 Ximelagatran 27.1 B A A B C C D D Figure 5. Cont. Molecules 2017, 22, 649 10 of 17 Molecules 2017, 22, 649 Molecules 2017, 22, 649 E F E 9 of 17 10 of 17 F G H G H Figure 5. The inhibition profile figures of ginsenoside (Rg2) (A,B); ginsenoside (Rg3) (C,D); protopanaxtriol Figure 5. The inhibition profile figures of ginsenoside (Rg2) (A,B); ginsenoside (Rg3) (C,D); (PPT) (E,F) and Ximelagatran (G,H) against thrombin. Figure 5. The inhibition figures of ginsenoside (Rg2) (A,B); ginsenoside (Rg3) (C,D); protopanaxtriol protopanaxtriol (PPT)profile (E,F) and Ximelagatran (G,H) against thrombin. (PPT) (E,F) and Ximelagatran (G,H) against thrombin. 2.6. Interactions between Ginsenosides and FXa Protein 2.6. Interactions between Ginsenosides and FXa Protein 2.6. Molecular Interactions docking between Ginsenosides andwas FXa Protein toclarify clarifythe theinteraction interaction modes of the Molecular dockinginvestigation investigation was performed performed to modes of the mostmost active ginsenosides inin anan anti-coagulation on FXa FXa protein andtointeraction to measure the relative Molecular docking investigation was assay performed toprotein clarifyand the modes of binding the binding most active ginsenosides anti-coagulation assay on measure the relative energies and localize accurate binding sites in the active pocket. As shown in Figure 6, Rg3 showed energies and localize accurate binding sites in the pocket. and As shown in Figure 6, Rg3 showed active ginsenosides in an anti-coagulation assay onactive FXa protein to measure the relative binding the most hydrogen nearby residues amongpocket. thebest best anti-coagulation the most hydrogen bondswith with nearby residues the anti-coagulation ginsenosides in in energies and localizebonds accurate binding sites in the among active As shown in Figure 6,ginsenosides Rg3 showed assays. Among thebinding binding amino acid among residues between ligands and thethe protein, GLY-216 bioactivity assays. Among amino acid residuesthe between ligands and protein, GLY-216 thebioactivity most hydrogen bondsthe with nearby residues best anti-coagulation ginsenosides in was commonly shared among the three three ginsenosides. Furthermore, the same GLU-97 waswas was commonly shared among the ginsenosides. Furthermore, the same residue GLU-97 bioactivity assays. Among the binding amino acid residues between ligands andresidue the protein, GLY-216 shared ligands Rg2and andRg3. Rg3.the three ginsenosides. Furthermore, the same residue GLU-97 was was commonly shared among shared by by ligands Rg2 shared by ligands Rg2 and Rg3. (A) (A) Figure 6. Cont. Molecules 2017, 22, 649 10 of 17 Molecules 2017, 22, 649 11 of 17 (B) (C) Figure 6. Interaction modes of ginsenosides 5, 5, 1313and pocket(A–C). (A–C).(A) (A)H-bonds H-bonds Figure 6. Interaction modes of ginsenosides and18 18within withinFXa FXa binding binding pocket between Rg2Rg2 (5) and FXaFXa pocket; (B)(B) H-bonds (C)H-bonds H-bondsbetween between between (5) and pocket; H-bondsbetween betweenRg3 Rg3(13) (13)and and FXa FXa pocket; pocket; (C) PPTPPT (18)(18) andand FXa pocket; light blue, ligands (Rg2, Rg3 and PPT); pink, ligands; orange, residues FXa pocket; light blue, ligands (Rg2, Rg3 and PPT); pink, ligands; orange, residues of theof the binding protein(FXa); (FXa);green greendashed dashed line, H-bond. binding protein line, H-bond. 3. Discussion 3. Discussion TheThe anti-coagulation effects ofof 1818compounds among which whichRg1, Rg1,Rg2, Rg2,Rg3, Rg3, anti-coagulation effects compoundswere wereinvestigated, investigated, among Rh2, PPD and PPT possessedsignificant significantanti-coagulation anti-coagulation activities toto Rh1,Rh1, F1, F1, Rh2, F2, F2, PPD and PPT possessed activitiesin invitro vitrocompared compared normal control < 0.05). Next, the Rg2,Rg3 Rg3and andPPT PPT that that displayed displayed the the the normal control (p <(p0.05). Next, the Rg2, the best bestanti-FXa anti-FXaactivity activity < 0.01) were further molecularly dockedwith withhuman human FXa FXa protein. The result (p <(p0.01) were further molecularly docked resultby byGlide GlideXP XPfrom from Schrödinger revealed the interactions and accurate binding sites between the bioactive FXa and Schrödinger revealed the interactions and accurate binding sites between the bioactive FXa andthe the three ginsenosides, whichfurther furthersupported supported the assays andand also also pointed out the three ginsenosides, which thedata dataofofthe thebioactivity bioactivity assays pointed out chemical modification. All All the above-mentioned evidence indicates that the potential potentialfuture futuredirection directionofof chemical modification. the above-mentioned evidence indicates Rg2, Rg3 and PPT may be potential natural FXa inhibitors. that Rg2, Rg3 and PPT may be potential natural FXa inhibitors. In a previous study of the water extract of P. ginseng, together with water extracts of the In a previous study of the water extract of P. ginseng, together with water extracts of the same same family (Araliaceae genus Panax quinquefolius L. and Panax notoginseng (Burk.) F.H. Chen), family (Araliaceae genus Panax quinquefolius L. and Panax notoginseng (Burk.) F.H. Chen), it was it was observed to significantly extend blood clotting time of activated partial thromboplastin, observed to significantly extend blood clotting time of activated partial thromboplastin, prothrombin prothrombin and thrombin in in vitro human plasma coagulation assays [6]. Similarly, the ethyl acetate and thrombin in in vitro human plasma coagulation assays [6]. Similarly, the ethyl acetate fraction of fraction of Korean red ginseng methanol extracts showed potent anti-coagulant activity via markedly Korean red ginseng methanol showed by potent anti-coagulant viareport, markedly prolonged clotting time thatextracts was measured thrombin time [19]. activity In another 0.05prolonged mg/mL clotting time that was measured by thrombin time [19]. In another report, 0.05 mg/mL (final concentration) (final concentration) Rg1 and Rg2 exhibited significantly better anti-coagulant activities in vitro Rg1compared and Rg2 to exhibited significantly better anti-coagulant activities compared to 0.1 mg/mL 0.1 mg/mL (final concentration) heparin that was choseninasvitro the positive control drug [20]. (final concentration) heparin that was chosen as the positive control drug [20]. In the present study, In the present study, nine ginsenosides exhibited significant anti-coagulation activity (Figure 3), which ninewas ginsenosides exhibited significant anti-coagulation activity (Figure 3), whichthe was consistent with consistent with the previous findings. However, not all evidence supported anti-coagulation the previous findings. However, not all evidence supported the anti-coagulation ginsenosides role of ginsenosides in the blood coagulation cascade. In contrast to our data, Wee role et al.of [19] observed in the blood coagulation cascade. In contrast to our data, Wee et al. [19] observed that ginsenosides did not inhibit the blood coagulation process. In this investigation, a 50% MeOH subfraction demonstrated higher potent inhibition against blood coagulation compared to the 100% MeOH subfraction; nevertheless, ginsenosides Rf, Rh1 and Rg3 that were present in the EtOAc fraction were weakly detected in the 50% MeOH subfraction, indicating that saponins did not contribute to the Molecules 2017, 22, 649 11 of 17 that ginsenosides did not inhibit the blood coagulation process. In this investigation, a 50% MeOH subfraction demonstrated higher potent inhibition against blood coagulation compared to the 100% MeOH subfraction; nevertheless, ginsenosides Rf, Rh1 and Rg3 that were present in the EtOAc fraction were weakly detected in the 50% MeOH subfraction, indicating that saponins did not contribute to the anticoagulation activity of the EtOAc fraction. In the current study, nine ginsenosides that showed significant anti-coagulation activity in vitro were subsequently studied for their anti-FXa effect in vitro. FXa inhibitors have emerged as new anti-coagulant drugs and when compared to previous anti-coagulants, they possess the following advantages: more rapid onset and offset of action; reduction in need for “bridging” with a parenteral anti-coagulant; less clinical monitoring; and less interactions with other drugs and food. However, for current FXa inhibitors there are still a few drawbacks, such as a lack of antidotes, short half-life affecting efficacy, as well as high acquisition costs which limits wider clinical application. Therefore, natural occurring FXa inhibitors may be an ideal substitute. Many studies have revealed that natural polypeptides isolated from organisms such as bloodsuckers and Ancylostoma caninum could act as direct inhibitors against factor Xa [21]. However, due to the near impossibility of these polypeptides being developed for oral administration, attention may focus instead on natural small molecular compounds. In a study published in 2014, a natural small molecule (glycyrrhetinic acid) derived from the Chinese herb Glycyrrhiza glabra was described in [21]. The orally administered compound was reported to significantly inhibit FXa activity in vitro with specificity, and reduce thrombus weight in rat models despite the unequalable anticoagulation property with commercially accessible drugs rivaroxaban and apixaban, suggesting that glycyrrhetinic acid could be considered as a promising natural FXa inhibitor [21,22]. In this study, ginsenosides that mainly originate from herbs of the Panax species and possess a molecular weight less than 1000 demonstrated significant inhibitory activities against FXa in vitro. Among the nine ginsenosides with significant anti-coagulation effects in vitro, Rg2, Rg3 and PPT showed the highest affinity with FXa receptors (Table 1 and Figure 4), and also displayed excellent thrombin selectivity (Table 2). FXa is structurally similar tothrombin (coagulation factor II), which may result in the binding of FXa inhibitors to thrombin and subsequent risk of bleeding. Furthermore, selectivity is an important issue for the development of FXa inhibitors [2]. Hence, there is a need to address the selectivity issue for the development of newer FXa inhibitors by understanding structural differences between FXa and thrombin, and for the reason we designed and conducted the experiment to test whether ginsenosides showing significant anti-FXa activity were unable to affect thrombin level. The data showed that Rg2, Rg3 and PPT were potent FXa inhibitors with high thrombin selectivity, indicating that these three ginsenosides may be potent anti-coagulants with less risk of bleeding. Further molecular docking by Schrödinger software confirmed the data obtained in the anti-coagulation activity assays and pointed out the specific binding protein residues around the active pocket, which may form the basis for future chemical modification in ginsenosides. Despite our finding that micromolecule direct FXa inhibitors in ginseng was accomplished in the anti-coagulation assay in vitro, and that anti-FXa and thrombin selectivity assays may reveal potent anti-coagulation activity in the future treatment of coagulation-associated disorders, nevertheless, there are some unresolved issues. First, the phenomenon that multiple ginsenosides possess anti-coagulation properties have been reported in the current research; however, the underlying action mechanism of the anti-coagulation effect of ginsenosides still remains unclarified and requires further investigation. Additionally, the unstable chemical structure of ginsenosides such as Rg3 under acidic and high-temperature conditions, especially in the stomach, may hinder its further application in scientific research and trials [20], furthermore, ginsenosides have demonstrated incomparable bioactivity with the positive drug heparin in an anti-coagulation assay in vitro and rivaroxaban in an anti-FXa test in vitro. Therefore, to develop novel anti-coagulants with better efficacy and lower side effects, future work is required. Molecules 2017, 22, 649 12 of 17 4. Materials and Methods 4.1. General A Bruker DRX 500 spectrometer (Bruker Biospin, Rheinstetten, Germany), using tetramethylsilane (TMS) as an internal standard, was used for NMR spectra. The qualitative, quantitative analyses and HPLC separations were all performed on a Waters 1525 Binary HPLC Pump equipped with Waters 2998 Photodiode Array Detector (Waters Corporation, Milford, MA, USA). SunFire Prep C18 Column (10 mm × 150 mm, silica gel particle size: 10 µm) was used for HPLC separation and Diamonsil C18 (4.6 mm × 250 mm, 5 µm) was used for HPLC analyses. Silica gel for column chromatography was provided by the Qingdao Ocean Chemical Group Co. (Qingdao, China). Pyridine-d5 was purchased from the Sigma-Aldrich Company (St. Louis, MO, USA). For plasma preparation, the micro-centrifuge was obtained from Thermo Fisher Scientific (Heraeus Fresco 21, Harz, Germany). For blood clotting time assays, a coagulometer was purchased from Beckman Coulter Inc. (Beckman ACL TOP-700, CA, USA), and the positive control drug heparin was obtained from YM Biological Technology Company Limited. For the anti-FXa activity assay, purified FXa and FXa substrate CS-11(22) were purchased from New England Biolabs (Hitchin, UK) and American Diagonostica Inc. (Stamford, CT, USA), respectively. An automated microplate reader Epoch was obtained from Bio-Tek Instruments Inc. (Winooski, VT, USA) and the positive drug rivaroxaban was obtained from Bayer Health Care AG (Wupertal, Germany). For the thrombin selectivity assay, FIIa and chromogenic substrate CS-01(38) were purchased from Hyphen BioMed (Paris, France), and the positive drug ximelagatran was purchased from AstraZeneca LP (Molndal, Sweden). For the anti-coagulation activity assay in vitro, the solvent dimethyl sulfoxide (DMSO) and heparin sodium salt were obtained from Sigma-Aldrich Company (St. Louis, MO, USA). For data analysis, the software SPSS 19.0 was obtained from SPSS Inc. (Chicago, IL, USA). 4.2. Plant Material Ginseng quinquennium, was collected in Ji’an City in Jilin Province, China, in August 2015. The plant material was identified by Professor. Pingya Li (School of Pharmaceutical Sciences, Jilin University). A voucher specimen (No. 20150928) was deposited in the School of Pharmaceutical Sciences, Jilin University. 4.3. Preparation of the Total Saponins of Ginseng The powdered air-dried root of ginseng (300 g) was extracted with 70% ethanol under reflux for 3 h and repeated for three times. The extracted solution was then concentrated under reduced pressure. The extract (63 g) was purified in an AB-8 macroporous adsorption resin column, eluted with water, 20% ethanol and 85% ethanol, respectively. Finally, the 85% ethanol elute (25.1 g) was collected as the total saponins from ginseng. 4.4. Extraction and Isolation The total saponins from ginseng were subjected to column chromatography in a silica gel column gradiently eluted with chloroform/methanol repeatedly to give eight fractions, Fraction A (chloroform/methanol (100:4, v/v)), Fraction B (chloroform/methanol (100:8, v/v)), Fraction C (chloroform/methanol (100:9, v/v)), Fraction D (chloroform/methanol(100:12, v/v)), Fraction E (chloroform/methanol (100:15, v/v)), Fraction F (chloroform/methanol (100:18, v/v)), Fraction G (chloroform/methanol (100:20, v/v)), and Fraction H (chloroform/methanol (100:30, v/v)). Compounds 17 and 18 were recrystallized from Fraction A using ethyl acetate. Compound 15 was purified from Fraction B by using reverse phase silica gel chromatography eluted with methanol/water gradiently. Fraction C was purified with octadecyl-bonded silica gel (ODS) column eluted with methanol/water (55:45, v/v) to obtain compounds 4 and 9. Compound 5 was obtained from Fraction D using column chromatography on silica gel repeatedly using acetone and ethyl acetate. Fraction E was Molecules 2017, 22, 649 13 of 17 separated on normal phase (using a mixture of chloroform, ethyl acetate, methanol and water as elution) and reverse phase (using gradient methanol/water as elution) silica gel column chromatography to obtain compounds 1, 3, 13, 14 and 16. Compounds 7, 2 and 12 were purified from Fraction F by column chromatography on silica gel repeatedly using dichloromethane and methanol, respectively. Compounds 10 and 11 were yielded from Fraction G by column chromatography on ODS, eluted using gradient methanol and water. Fraction H was separated in reverse phase (using gradient methanol/water as elution) silica gel column chromatography to obtain compounds 6 and 8. 4.5. Animal Preparation Male Sprague-Dawley rats weighing 180–220 g were purchased from the Animal Center of Norman Bethune Medical College of Jilin University in China. The rats were maintained under controlled environment (22 ± 2 ◦ C, relative humidity 40–60%, 24 h light-dark cycles, and ad libitum access to food and water). The animal experiments were conducted based on the guide for the administration of laboratory animals (Directive 86/609/EEC in the Protection of Animals Used for Experimental and Other Scientific Purposes, 1986) and were approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin University (No. SCXK-2013-0001). 4.6. Blood Collection and Preparation of Plasma Samples Rats were anaesthetized with 10% chloral hydrate (3 mL/kg). The blood obtained from the abdominal aorta was collected directly into citrated tubes containing 3.8% sodium citrate (1:9 (v/v)) and was used immediately after collection [23]. Platelet poor plasma (PRP) was prepared by centrifuging the blood at 3000 rpm for 20 min at 20 ◦ C [24]. Plasma samples with jaundice, chylus, hemolysis and blood clot were excluded prior to the assays. Plasma mixtures were 90 µL of PRP with 10 µL of tested compounds (0.5 mg/mL, dissolved in DMSO). For PRP in the solvent group, the sample was 90 µL of PRP with 10 µL of DMSO. For PRP in the normal control group, the plasma was not-treated PRP sample. For PRP in the heparin group, the sample was 90 µL of PRP with 10 µL of heparin (0.1 mg/mL), which was used as the positive control drug. 4.7. Measurement of Blood Clotting Time Blood coagulation assays were carried out using a coagulation analyzer at the Blood Center clinical laboratory in the Second Hospital affiliated to Jilin University (Changchun, China). Measurements of APTT, PT and TT were performed according to the manufacturer’s recommended protocols. The anti-coagulant activity was expressed as clotting time ± standard deviation (S.D.). Briefly, for APTT assay, 50 µL of the plasma mixture was incubated at 37 ◦ C for 60 s and the mixture was added to the APTT reagent (50 µL) and incubated at 37 ◦ C for 15 min. Finally, APTT values were recorded. For the PT assay, 50 µL of the plasma mixture was incubated at 37 ◦ C for 60 s and then added into the PT reagent (100 µL) prior to incubation at 37 ◦ C for 15 min before the PT values were recorded. For the TT assay, 80 µL of the plasma mixture was incubated at 37 ◦ C for 60 s before the addition of 80 µL of TT reagent for 100 s. TT values were determined by the coagulation analyzer. 4.8. FXa Activity Assay of Compounds In Vitro Test compounds and the positive control drug rivaroxaban were dissolved in DMSO at a concentration of 1 mM and then serially diluted to a range of 3 nM to 10 µM, respectively. 10 µL of FXa (final concentration of 0.5 nM), 40 µL of Tris buffer (adjusted to pH 7.4 with HCl containing 0.3 M NaCl and 50 mM Tris) and 10 µL of test compounds were added to the well, respectively. The negative control was performed using the same mixed solutions except that the test compound was replaced with DMSO. The positive control was composed of the same mixed solutions except that the test compound was replaced with rivaroxaban. After 15 min of incubation at 37 ◦ C, FXa substrate (40 µL, final concentration of 0.25 nM) was added and then was incubated 37 ◦ C for 25 min [3]. The optical density (OD) values at 405 nm were evaluated by an automated microplate reader. The time-absorbance Molecules 2017, 22, 649 14 of 17 curve and the slope of curve reflecting enzymatic activity were observed in test groups (Vi ), positive control group (Vi ) and negative control group (V 0 ). Inhibition rate was calculated by the following formula: Anti-FXa activity = (V 0 − Vi )/V 0 [21]. The IC50 value was subsequently calculated by SPSS 19.0. 4.9. Thrombin Inhibition In Vitro of Compounds The inhibition of thrombin was evaluated by human FIIa and chromogenic substrate CS-01(38) in 96-well microtiter plates at room temperature. Compounds 5, 13 and 18 as well as the positive reagent Ximelagatran were dissolved in DMSO to a concentration of 1mM and then serially diluted to a range of 10 µM to 100 µM, respectively. 8 µL of FIIa (3 NIH U/mL), 80 µL of Tris buffer (adjusted to pH 7.4 with HCl) containing 0.3 M NaCl and 50 mM Tris and 8 µL of test compounds were added to the well, respectively. The negative control was performed using the same mixed solutions except that tested compounds were replaced with DMSO. The positive control was performed using the same mixed solutions except that the test compounds were replaced with ximelagatran. After incubation at 37 ◦ C for 15 min, 12 µL of FIIa substrate solution (4 mM) was added and then incubated at 37 ◦ C for 25 min [3]. The anti-FIIa activity was measured at 405 nm using a microplate reader. 4.10. Molecular Docking of Rg2, Rg3 and PPT within FXa Molecular docking study of bioactive compounds was performed using GLIDE (Grid-based Ligand Docking with Energetics) (GLIDE, version 6.7, Schrödinger, LLC, New York, NY, USA) software developed by Schrödinger. Maestro (version 2015-2, Schrödinger, LLC, New York, NY, USA) was used for all the steps involving protein and ligand preparation, receptor grid generation and docking. The X-ray crystal structure of FXa (Protein Data Bank (PDB) code: 2w26) complexed with an oral and direct FXa inhibitor Bay59-7939 was retrieved from the PDB database (http://www.rcsb.org/pdb) based on a previous study [2]. The Protein Preparation Wizard in the GLIDE software was used to prepare the receptor FXa. The structure of FXa was optimized after a series of processes including assigning bond orders and water orientations, removing water molecules, adding hydrogens, creating zero-order bonds to metals and disulfide bonds. The protein was then energy minimized using a default constraint of 0.30 Å root-mean-square deviation (RMSD) using the optimized potentials for liquid simulations 3 (OPLS3) force field. When performing receptor grid generation, a present ligand in the retrieved protein-ligand complex was identified prior to setting the center and the size of the box. The grid box was limited to the size of 20 Å at the active site. Crystal coordinates of compounds (ligands) were pre-drawn in Maestro Elements (Maestro Elements, version 2.2, Schrödinger, LLC, New York, NY, USA) prior to the molecular docking study. Three-dimensional (3D) structures of all 18 compounds were generated using LigPrep module (2015-2) from the Schrödinger Suite (LLC) by assigning the bond orders and angles. In addition, these compounds were subjected to minimization using the OPLS3 force field. For GLIDE docking, the prepared structure of FXa and ligands (compounds) were imported to the workspace using GLIDE v.6.7 from the Schrödinger Suite [25–27]. Extra precision (XP) docking was carried out and the parameters of scaling factor and partial charge cutoff were set at the default values 0.80 and 0.15, respectively. At least the top ten ranking conformations for each ligand was chosen in the output tab to set the output numbers. Figures of the docking results were subsequently prepared using PyMol (Schrödinger). 4.11. Statistical Analysis All values were expressed as means ± standard deviation (SD), and one-way analysis of variance (ANOVA) and student’s t-test were performed by SPSS19.0 (SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant. Molecules 2017, 22, 649 15 of 17 5. Conclusions In summary, triterpenoids from ginseng are potential natural coagulation factor Xa (FXa)-inhibitors with high thrombin selectivity and prolongation of coagulation time. The bioactivity studies and HPLC analysis also suggested that despite the low content in total saponins, the three triterpenoids, Rg2 (5, 0.0230%), Rg3 (13, 0.0021%) and PPT (18, 0.0958%), maybe responsible for the anti-coagulant effect. Hence, the total saponins and the effective components could be used as a potential natural anticoagulation therapy. Acknowledgments: We thank Hang Su, Shuxue Zou and Zhenzhou Wang in preparing the figures of molecular docking. This work was supported by the National Science and Technology Major Project for new drug development in China (No. 2010ZX09401-305-26) and Science and Technology Development Program of Jilin Province (Grant No. 20150519015JH). Author Contributions: Pingya Li and Jinping Liu conceived and designed the experiments; Lingxin Xiong and Zeng Qi performed the experiments; Zeng Qi, Bingzhen Zheng, Zhuo Li and Fang Wang were responsible for data analysis. Lingxin Xiong wrote the paper. Pingya Li, Jinping Liu, Zhuo Li and Fang Wang assisted paper revision. Conflicts of Interest: The authors declare no conflict of interest. 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