Arch. Pharm. Res. (2014) 37:1394–1402 DOI 10.1007/s12272-014-0422-5
RESEARCH ARTICLE
Antithrombotic and antidiabetic flavonoid glycosides from the grains of Sorghum bicolor (L.) Moench var. hwanggeumchal Phi-Hung Nguyen • Vu Viet Dung • Bing Tian Zhao Young Ho Kim • Byung Sun Min • Mi Hee Woo
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Received: 16 January 2014 / Accepted: 25 May 2014 / Published online: 24 June 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract A phytochemical study of the grains of S. bicolor, resulting in the isolation of twelve flavonoid glycosides 1–12. Their chemical structures were elucidated on the basis of spectroscopic (1D and 2D NMR) and MS data analyses. All compounds were tested on thrombin time (TT) assay and a-glucosidase assay in order to assess their inhibitory effects on blood coagulation and a-glucosidase enzyme. At the concentration of 500 lg/mL, compounds 3, 4, 7 and 10 possessed the potential effects on blood coagulation with inhibitory percentage of 197, 152, 120 and 158 %, respectively, whereas aspirin, which used as a positive control, indicated 181 and 138 % inhibition at 500 and 375 lg/mL, respectively. Furthermore, compounds 3, 4, 7, 9 and 10 also displayed strong inhibitory effects on aglucosidase enzyme, with 85.2, 55.7, 43.9, 52.7 and 65.2 % inhibition at 100 lg/mL, respectively, whereas acarbose, as a positive control, possessed only 38.7 % at the same concentration. Taken together, our data suggest that S. bicolor and its flavonoid-enrich extracts could be considered as supplemental and or functional foods having beneficial effects against blood coagulation-induced ischemia, possibly thromboembolism disease, as well as diabetes.
Electronic supplementary material The online version of this article (doi:10.1007/s12272-014-0422-5) contains supplementary material, which is available to authorized users. P.-H. Nguyen V. V. Dung B. T. Zhao B. S. Min M. H. Woo (&) College of Pharmacy, Catholic University of Daegu, Hayang 712-702, Gyeongbuk, Korea e-mail: [email protected] Y. H. Kim Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Korea
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Keywords Sorghum bicolor Anticoagulants Thrombin inhibitors a-Glucosidase inhibitors Flavonoid glycosides
Introduction Thromboembolic disorders (TD) are the major causes of morbidity and mortality. Arterial thrombosis is the most common cause of myocardial infarction and ischemic stroke, whereas deep vein thrombosis can lead to pulmonary embolism (Mackman 2008). In the U.S., pulmonary embolism causes almost 300,000 deaths per annum (Heit 2005). Moreover, it is estimated that 12 % of annual deaths occurring in France, Germany, Italy, Spain, Sweden and the UK are due to venous thromboembolism (VTE) (Cohen et al. 2007). Great advances have been made in understanding the molecular and cellular basis of thrombus formation in the past few decades, with anticoagulants remaining as the cornerstone for the prevention and treatment of TD (Weitz 2004). Mechanism studies revealed that Factor Xa, a key component of the prothrombinase complex, first stimulates the conversion of the inactive zymogen, prothrombin, to the active serine protease, thrombin. Thrombin, in turn, catalyzes the conversion of soluble fibrinogen to insoluble strands of fibrin, which leads to platelet activation and the release of adenosine diphosphate, serotonin and thromboxane A2 through the cleavage of protease-activated receptor 1 (PAR1) on the platelet surface. This in conjunction with simultaneous platelet activation by exposed subendothelial collagen leads to platelet aggregation, fibrin and blood cell agglutination and thus thrombus formation (Coughlin 2000, Hamilton 2009). The inhibition of thrombin generation, activation or both is therefore a logical target in the treatment of TD. Unfractionated heparin (UFH), low molecular weight heparin
Antithrombotic and antidiabetic flavonoid glycosides
Re la tive Throm bi n Ti m e (%)
Fig. 1 Inhibitory effect of the n-Hexane, EtOAc, BuOH fractions, the H2O residue and the total 95 %-EtOH extract of the grains of S. bicolor on TT activity assay. TT of individual samples was measured at a final concentration of 500 lg/mL. The experiment was performed in triplicates
1395 Thrombin Time assay
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Fig. 2 Chemical structure of isolated compounds (1–12) from the grains of S. bicolor
(LMWH) and fondaparinux (an AT-dependent factor Xa [FXa] inhibitor) bind to antithrombin (AT), enhance its protease inhibition activity, and exert anticoagulant effects (Bauer and Rosenberg 1991; Rao et al. 1993). However, its use is accompanied by some side effects, frequently requiring the monitoring of partially activated thromboplastin time and ultimately resulting in other hemorrhagic complications (Nader et al. 2001). Although it is well established that aspirin still provides an effective secondary prevention of ischemic cardiovascular disorders, this drug can produce major drawbacks, such as hemorrhagic events and upper gastrointestinal bleeding (Lee and Kim 2005). Thus, the search for alternative anticoagulants with reduced side effects is still needed and urgent. As part of an investigation aimed as anticoagulant agents from plants, we found that an EtOAc-soluble extract of the grains of S. bicolor exhibited significant activities on both thrombin time (TT) and a-glucosidase assays (Fig. 1). Thus, a directly bioassay-guided isolation of the EtOAcsoluble extract of the grains of this plant has led to the isolation of a series of flavonoid glucosides (1–12) as
active principles (Fig. 2). In addition, the inhibitory effects of the isolated compounds (1–12) on a-glucosidase were also investigated.
Materials and methods General procedures The optical rotations were determined on a Rudolph Autopol AP 589 polarimeter using a 100 mm glass microcell. The IR spectra were recorded on a Nicolet 6700 FT-IR (Thermo electron Corp.). UV spectra were recorded in MeOH using a Shimadzu spectrometer. The NMR spectra were recorded in methanol-d4 (CD3OD), pyridine-d5 (C5D5N) on Varian OXFORD-AS 400 MHz instrument (PaloAlto, CA, USA) with TMS as the internal standard at the Department of Pharmacy, Catholic University of Daegu, Korea. All mass experiments were performed on a Micromass QTOF2 (Micromass, Wythenshawe, UK) mass spectrometer. Silica Gel (Merck, 63–200 lm particle size)
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and RP-18 (Merck, 150 lm particle size) were used for column chromatography. For thin-layer chromatography (TLC), pre-coated TLC was carried out on Silica Gel 60 F254 and RP-18 F254 plates from Merck. HPLC runs were carried out using a Gilson system with a UV detector and an Optima Pak C18 column (10 9 250 mm, 10 lm particle size, RS Tech Corp., Korea). Reagents p-Nitrophenyl-a-D-glucopyranoside (pNPG), a-glucosidase (0.1 Unit), Acarbose, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent were purchased from Sigma Chemical Co. Mouse fibroblast (3T3-L1) cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), Trypsin–EDTA, penicillin/streptomycin/amphotericin (10,000 U/mL, 10,000 lg/mL, and 2,500 lg/mL, respectively), and fetal bovine serum (FBS) were obtained from Gibco BRL, Life Technologies (USA). All other chemicals and solvents were of analytical grade commercially available. Plant material The grains of Sorghum bicolor (L.) Moench var. hwanggeumchal (S. bicolor) were provided by National Institute of Crop Science of Miryang, Korea, in February 2011. A voucher specimen (20100523) has been deposited at the College of Pharmacy, Catholic University of Daegu, Republic of Korea. Extraction and isolation The grains of S. bicolor (10 kg) were extracted with 95 % ethanol (EtOH) at room temperature (18 L 9 3 times) for 1 week. The solvent extract was concentrated under reduced pressure to give the EtOH extract (210 g). The concentrated EtOH extract was suspended in H2O (1 L) and partitioned successively with n-hexane (1.5 L 9 4 times, 96 g), EtOAc (1.5 L 9 4 times, 48.6 g), n-BuOH (1.5 L 9 4 times, 38.5 g) and H2O-soluble fractions (30.5 g), respectively. The n-hexane, EtOAc, n-BuOH and H2O-soluble fractions were tested on TT assay and a-glucosidase assay. Among those, the EtOAc fraction showed the highest activities. Thus, the EtOAc fraction (108.6 g) was subjected to silica gel column chromatography (15 9 60 cm; 63–200 lm particle size), using gradient solvents of CH2Cl2:MeOH (100:1 ? 0:1), yielded six combined fractions (F.1 to F.6), according to their TLC profiles. These fractions were assayed for blood coagulants and a-glucosidase enzyme inhibition. Strong active fractions 2, 4 and 5 were continuously chromatographed for activity-guided isolation. Fraction 5 (F.5, 7.3 g) was further
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chromatographed on a RP-18 column with gradient solvent of MeOH:H2O (50:50 ? 95:5) to afford ten sub-fractions (F.5.1 to F.5.10). The sub-fraction F.5.5 (300 mg) was re-chromatographed on a RP-18 column with an isocratic solvent of MeOH:H2O (40:60) to yield compound 1 (81.6 mg). The subfraction F.5.4 (75.2 mg) was further purified by an open RP18 column with a gradient solvent of MeOH:H2O (20:80 ? 30:70), and afforded compound 2 (10.6 mg). The sub-fraction F.5.3 (644.9 mg) was also re-chromatographed on an open RP-18 column using a gradient solvent of MeOH:H2O (20:80 ? 70:30) to yield compounds 3 (10.1 mg), 7 (12.7 mg), 9 (9.6 mg), and 10 (9.0 mg), respectively. Further purification of subfraction F.5.6 (355 mg) by semi-preparative Gilson HPLC, using an isocratic solvent system of 40 % MeOH in H2O for over 40 min [RS Tech Optima Pak C18 column (10 9 250 mm, 10 lm particle size); mobile phase MeOH/H2O containing 0.1 % formic acid (0–40 min: 40 % MeOH, 40–42 min: 40–100 % MeOH, 42–50 min: 100 % MeOH); UV detections at 205 and 254 nm], yielded compound 5 (17.1 mg, tR = 31.5 min) and compound 8 (2.5 mg, tR = 38.7 min). Compound 6 (151.6 mg) was purified from fraction 2 by using an open RP18 column (3.5 9 15 cm) and eluting with a gradient solvent system of 50 % MeOH in H2O. Fraction 4 (F.4, 2.0 g) was also chromatographed on a RP-18 column (4.0 9 20 cm), with a stepwise gradient solvent of MeOH in H2O (50:50 ? 50:0), afforded compound 4 (15 mg), and three sub-fractions (F.4.1–F.4.3). The purification of sub-fraction F.4.2 (105.5 g), which was prepared by preparative Gilson HPLC using an isocratic solvent system of 35 % MeOH in H2O for over 40 min [RS Tech Optima Pak C18 column (10 9 250 mm, 10 lm particle size); mobile phase MeOH/ H2O containing 0.1 % formic acid (0–40 min: 40 % MeOH, 40–42 min: 40–100 % MeOH, 42–50 min: 100 % MeOH); UV detections at 205 and 254 nm], resulted in the isolation of compound 11 (16.1 mg, tR = 33.1 min) and compound 12 (41.5 mg, tR = 35.4 min), respectively. Quercetin-7-O-b-D-glucoside (1) Yellow powder; positive FAB-MS m/z 465.10 [M ? H]? (calcd. for C21H20O12, 464.10); 1H-NMR (400 MHz, CD3OD) dH: 7.75 (1H, d, J = 2.0 Hz, H-20 ), 7.65 (1H, dd, J = 8.4, 2.0 Hz, H-60 ), 6.87 (1H, d, J = 8.4 Hz, H-50 ), 6.74 (1H, d, J = 2.0 Hz, H-8), 6.45 (1H, d, J = 2.0 Hz, H-6), 5.04 (1H, d, J = 7.2 Hz, H-100 ), 3.92–3.30 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b); 13C-NMR (100 MHz, CD3OD) dC: 146.4 (C-2), 137.8 (C-3), 177.6 (C-4), 162.3 (C-5), 100.3 (C-6), 164.6 (C-7), 95.7 (C-8), 157.9 (C-9), 106.4 (C-10), 124.1 (C-10 ), 116.3 (C-20 ), 149.1 (C-30 ), 148.9 (C-40 ), 116.4 (C-50 ), 122.0 (C-60 ), 101.8 (C100 ), 74.9 (C-200 ), 78.0 (C-300 ), 71.5(C-400 ), 78.5 (C-500 ), 62.6 (C-600 ).
Antithrombotic and antidiabetic flavonoid glycosides
Luteolin-7-O-b-D-glucoside (2) Yellow powder; positive FAB-MS m/z 449.10 [M ? H]? (calcd. for C21H20O11, 448.10); 1H-NMR (400 MHz, CD3OD) dH: 7.33 (1H, dd, J = 8.0, 2.0 Hz, H-60 ), 7.31 (1H, d, J = 2.0 Hz, H-20 ), 6.80 (1H, d, J = 8.0 Hz, H-50 ), 6.69 (1H, d, J = 2.0 Hz, H-8), 6.50 (1H, s, H-3), 6.40 (1H, d, J = 2.0 Hz, H-6), 5.06 (1H, d, J = 7.6 Hz, H-100 ), 3.83–3.40 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b); 13 C-NMR (100 MHz, CD3OD) dC: 161.1 (C-2), 122.2 (C3), 182.8 (C-4), 163.6 (C-5), 99.9 (C-6), 165.6 (C-7), 94.8 (C-8), 157.7 (C-9), 105.9 (C-10), 119.3 (C-10 ), 115.6 (C20 ), 149.5 (C-30 ), 150.6 (C-40 ), 113.1 (C-50 ), 102.9 (C-60 ), 100.4 (C-100 ), 73.5 (C-200 ), 76.7 (C-300 ), 70.0 (C-400 ), 77.2 (C-500 ), 61.2 (C-600 ). Luteolin-30 -O-b-D-glucoside (3) Yellow powder; positive FAB-MS m/z 471.10 [M ? Na]? (calcd for C21H20O11, 448.10); 1H-NMR (400 MHz, CD3OD) dH: 7.41 (1H, d, J = 2.0 Hz, H-20 ), 7.15 (1H, dd, J = 8.4, 2.0 Hz, H-60 ), 6.76 (1H, d, J = 2.0 Hz, H-8), 6.74 (H, d, J = 8.4 Hz, H-50 ), 6.50 (1H, s, H-3), 6.29 (1H, d, J = 2.0 Hz, H-6), 4.94 (1H, d, J = 7.6 Hz, H-100 ), 3.92–3.17 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b); 13 C-NMR (100 MHz, CD3OD) dC: 162.0 (C-2), 105.2 (C3), 182.4 (C-4), 158.6 (C-5), 99.2 (C-6), 170.0 (C-7), 93.9 (C-8), 158.0 (C-9), 105.5 (C-10), 124.2 (C-10 ), 114.2 (C20 ), 149.4 (C-30 ), 147.9 (C-40 ), 116.8 (C-50 ), 118.9 (C-60 ), 101.4 (C-100 ), 73.5 (C-200 ), 78.7 (C-300 ), 70.0(C-400 ), 77.2 (C500 ), 62.2 (C-600 ). Quercetin-3-O-b-D-glucoside (4) Yellow amorphous powder; positive ESI–MS m/z 487.10 [M ? Na]? (calcd for C21H20O12, 464.10); 1H-NMR (400 MHz, CD3OD) dH: 7.82 (1H, d, J = 2.0 Hz, H-20 ), 7.57 (1H, dd, J = 8.0, 2.0 Hz, H-60 ), 6.85 (1H, d, J = 8.0 Hz, H-50 ), 6.30 (1H, d, J = 2.0 Hz, H-8), 6.12 (1H, d, J = 2.0 Hz, H-6), 5.04 (1H, d, J = 7.6 Hz, H-100 ), 3.85–3.45 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b); 13 C-NMR (100 MHz, CD3OD) dC: 158.1 (C-2), 130.2 (C3), 180.2 (C-4), 162.8 (C-5), 100.3 (C-6), 164.6 (C-7), 97.7 (C-8), 160.4 (C-9), 105.4 (C-10), 122.2 (C-10 ), 115.4 (C20 ), 146.6 (C-30 ), 149.5 (C-40 ), 121.8 (C-50 ), 120.9 (C-60 ), 104.6 (C-100 ), 75.6 (C-200 ), 76.9 (C-300 ), 71.3 (C-400 ), 76.7 (C-500 ), 62.5 (C-600 ); Taxifolin-7-O-b-D-glucoside (5) Yellow powder; positive ESI–MS m/z 467.11 [M ? H]? (calcd for C21H22O12 466.11); 1H-NMR (400 MHz, pyridine-d5) dH: 7.37 (1H, d, J = 1.6 Hz, H-20 ), 7.08 (1H, dd,
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J = 8.0, 1.6 Hz, H-60 ), 6.98 (1H, d, J = 8.0 Hz, H-50 ), 6.15 (1H, d, J = 2.0 Hz, H-6), 6.08 (1H, d, J = 2.0 Hz, H-8), 5.26 (1H, d, J = 7.6 Hz, H-100 ), 4.90 (1H, d, J = 11.2 Hz, H-2), 4.55 (1H, d, J = 11.2 Hz, H-3), 4.07–3.54 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b); 13 C-NMR (100 MHz, pyridine-d5) dC: 85.4 (C-2), 73.8 (C3), 199.9 (C-4), 167.2 (C-5), 98.3 (C-6), 164.8 (C-7), 96.8 (C-8), 163.9 (C-9), 103.6 (C-10), 129.8 (C-10 ), 116.9 (C20 ), 147.8 (C-30 ), 148.6 (C-40 ), 117.0 (C-50 ), 120.8 (C-60 ), 101.9 (C-100 ), 73.8 (C-200 ), 78.8 (C-300 ), 71.4 (C-400 ), 79.5 (C-500 ), 62.6 (C-600 ). Taxifolin (6) Yellow amorphous powder; positive ESI–MS m/z 305.25 [M ? H]? (calcd for C15H12O7, 304.25); 1H-NMR (400 MHz, CD3OD) dH: 6.89 (1H, d, J = 2.0 Hz, H-20 ), 6.79 (1H, dd, J = 8.0, 2.0 Hz, H-60 ), 6.76 (1H, d, J = 8.0 Hz, H-50 ), 6.15 (1H, d, J = 2.0 Hz, H-6), 6.13 (1H, d, J = 2.0 Hz, H-8), 4.92 (1H, d, J = 11.6 Hz, H-2), 4.50 (1H, d, J = 11.6, H-3), 13C NMR (100 MHz, CD3OD) dC: 85.0 (C-2), 73.6 (C-3), 199.9 (C-4), 164.8 (C-5), 98.3 (C-6), 167.2(C-7), 96.8 (C-8), 163.97 (C-9), 103.6 (C10), 129.8 (C-10 ), 116.9 (C-20 ), 147.8 (C-30 ), 148.6 (C-40 ), 117.0 (C-50 ), 120.9 (C-60 ). Taxifolin-30 -O-b-D-glucoside (7) Yellow powder; positive FAB-MS m/z 489.10 [M ? Na]? (calcd. for C21H22O12, 466.10); 1H-NMR (400 MHz, CD3OD) dH: 6.89 (1H, d, J = 2.0 Hz, H-20 ), 6.79 (1H, dd, J = 8.0, 2.0 Hz, H-60 ), 6.76 (1H, d, J = 8.0 Hz, H-50 ), 6.15 (1H, d, J = 2.0, H-6), 6.13 (1H, d, J = 2.0, H-8), 4.92 (1H, d, J = 11.2 Hz, H-2), 4.50 (1H, d, J = 11.2, H-3), 4.07–3.54 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b); 13 C-NMR (100 MHz, CD3OD) dC: 85.0 (C-2), 73.6 (C-3), 199.9 (C-4), 164.8 (C-5), 98.3 (C-6), 167.2 (C-7), 96.8 (C8), 164.0 (C-9), 103.6 (C-10), 129.8 (C-10 ), 116.9 (C-20 ), 147.8 (C-30 ), 148.6 (C-40 ), 117.0 (C-50 ), 120.9 (C-60 ), 101.9 (C-100 ), 73.8 (C-200 ), 78.8 (C-300 ), 71.4 (C-400 ), 79.5 (C-500 ), 62.6 (C-600 ). Aromadendrin-3-O-b-D-glucoside (8) Yellow powder; UV (MeOH) kmax nm: 223, 293; IR (KBr) mmax cm-1: 3253 (OH), 1635 (C = O), 1614, 1598, 1517 (C = C), 1253, 1161, 1071 (C–O); positive FAB-MS m/ z 473.11 [M ? Na]? (calcd. for C21H22O11, 450.11); 1HNMR (400 MHz, CD3OD) dH: 7.36 (2H, d, J = 8.4 Hz, H-20 , 60 ), 6.81 (2H, d, J = 8.4 Hz, H-30 , 50 ), 5.91 (1H, d, J = 2.0 Hz, H-6), 5.89 (1H, d, J = 2.0 Hz, H-8), 5.23 (1H, d, J = 10.4 Hz, H-2), 4.97 (1H, d, J = 10.4 Hz, H-3), 4.38 (1H, d, J = 7.6 Hz, H-100 ), 3.87–3.24 (6H, m, H-200 , H-300 ,
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H-400 , H-500 , H-600 a, H-600 b); 13C-NMR (100 MHz, CD3OD) dC: 82.6 (C-2), 76.2 (C-3), 195.2 (C-4), 164.5 (C-5), 93.4 (C-6), 168.1 (C-7), 95.4 (C-8), 163.3 (C-9), 101.6 (C-10), 127.5 (C-10 ), 129.5 (C-20 , 60 ), 115.3 (C-30 , 50 ), 158.3 (C-40 ), 101.6 (C-100 ), 73.5 (C-200 ), 76.5 (C-300 ), 70.2 (C-400 ), 77.2 (C-500 ), 61.6 (C-600 ). 5,7,30 ,50 -tetrahydroxy-flavanone-7-O-b-D-glucoside (9) Yellow powder; UV (MeOH) kmax nm: 215, 282 and 334; IR (KBr) mmax cm-1: 3385 (OH), 1696, 1635 (C = O), 1580, 1528 (C = C), 1446, 1344, 1242, 1073 (C–O), and 857; positive FAB-MS m/z 473.11 [M ? Na]? (calcd for C21H22O11, 450.11); 1H-NMR (400 MHz, CD3OD) dH: 6.83 (1H, br, s, H-40 ), 6.70 (2H, d, J = 0.8 Hz, H-20 , 60 ), 6.33 (1H, d, J = 2.0 Hz, H-8), 6.04 (1H, d, J = 2.0 Hz, H-6), 5.22 (1H, dd, J = 12.0, 2.8 Hz, H-2), 4.68 (1H, d, J = 7.2 Hz, H-100 ), 3.87–3.39 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b), 3.17 (1H, dd, J = 12.0, 17.2 Hz, H-3a), 2.74 (1H, dd, J = 2.8, 17.2 Hz, H-3b); 13C-NMR (100 MHz, CD3OD) dC: 80.1 (C-2), 42.5 (C-3), 197.6 (C4), 162.3 (C-5), 100.4 (C-6), 164.6 (C-7), 95.7 (C-8), 157.3 (C-9), 106.4 (C-10), 124.1 (C-10 ), 116.3 (C-20 ), 149.1 (C30 ), 148.9 (C-40 ), 161.4 (C-50 ), 116.4 (C-60 ), 101.84 (C-100 ), 74.9 (C-200 ), 78.0 (C-300 ), 71.5 (C-400 ), 78.5 (C-500 ), 62.6 (C600 ).
P.-H. Nguyen et al.
3.96–3.30 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b), 3.05 (1H, dd, J = 11.2, 15.6 Hz, H-3a), 2.68 (1H, br, d, J = 15.6 Hz, H-3b); 13C-NMR (100 MHz, CD3OD) dC: 80.3 (C-2), 46.3 (C-3), 193.2 (C-4), 161.6 (C-5), 100.2 (C6), 166.6 (C-7), 99.5 (C-8), 166.0 (C-9), 106.8 (C-10), 131.8 (C-10 ), 114.7 (C-20 ), 146.8 (C-30 ), 146.4 (C-40 ), 116.3 (C-50 ), 119.3(C-60 ), 103.9 (C-100 ), 74.6 (C-200 ), 78.4 (C-300 ), 71.2 (C-400 ), 77.4 (C-500 ), 62.5 (C-600 ). (2S)-eriodictyol-5-O-b-D-glucoside (12) Yellow amorphous powder; UV kmax (MeOH) nm: 232 and 284; CD (c 0.5 MeOH): [h]250 –2.68, [h]270 –1.75; [h]300 ?8.98, [h]310 ?1.15; negative ESI–MS m/z 449.25, [M H]- (calcd for C22H24O10, 450.25); 1H-NMR (400 MHz, CD3OD) dH: 6.92 (1H, br, s, H-20 ), 6.81 (1H, d, J = 8.0 Hz, H-50 ), 6.79 (1H, br, d, J = 8.0 Hz, H-60 ), 6.47 (1H, br, s, H-6), 6.13 (1H, br, s, H-8), 5.24 (1H, br, d, J = 12.4 Hz, H-2), 4.78 (1H, br, d, J = 7.2 Hz, H-100 ), 3.96–3.30 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b), 2.93 (1H, dd, J = 12.4, 16.8 Hz, H-3a), 2.70 (1H, br, d, J = 16.8 Hz, H-3b); 13C-NMR (100 MHz, CD3OD) dC: 80.2 (C-2), 46.2 (C-3), 192.1 (C-4), 162.2 (C-5), 100.4 (C6), 166.9 (C-7), 99.5 (C-8), 166.4 (C-9), 107.0 (C-10), 131.6 (C-10 ), 114.7 (C-20 ), 146.7 (C-30 ), 146.4 (C-40 ), 116.3 (C-50 ), 119.3 (C-60 ), 104.9 (C-100 ), 74.6 (C-200 ), 78.5 (C-300 ), 71.2 (C-400 ), 77.0 (C-500 ), 62.5 (C-600 ).
Eriodictyol-7-O-b-D-glucoside (10) Thrombin time (TT) assay Yellow powder; positive ESI–MS m/z 451.11 [M ? H]? (calcd for C21H22O11, 450.11); 1H-NMR (400 MHz, CD3OD) dH: 6.88 (1H, br, s, H-20 ), 6.75 (2H, br, s, H-50 , 60 ), 6.45 (1H, br, s, H-6), 6.11 (1H, br, s, H-8), 5.29 (1H, dd, J = 3.2, 12.4 Hz, H-2), 4.75 (1H, d, J = 7.6 Hz, H-100 ), 3.90 (1H, br, d, J = 12.4 Hz, H-600 a), 3.71 (1H, m, H-600 b), 3.52-3.38 (4H, m, H-200 , H-300 , H-400 , H-500 ), 3.05 (1H, dd, J = 12.4, 17.6 Hz, H-3a), 2.70 (2H, dd, J = 2.8, 17.6 Hz, H-3b); 13C-NMR (100 MHz, CD3OD) dC: 80.7 (C-2), 44.1 (C-3), 193.6 (C-4), 164.9 (C-5), 97.0 (C-6), 167.0 (C-7), 98.0 (C-8), 164.6 (C-9), 105.0 (C-10), 131.5 (C-10 ), 114.8 (C-20 ), 147.0 (C-30 ), 146.6 (C-40 ), 116.3 (C-50 ), 119.4 (C60 ), 101.3 (C-100 ), 74.7 (C-200 ), 78.3 (C-300 ), 71.2 (C-400 ), 77.8 (C-500 ), 62.4 (C-600 ). (2R)-eriodictyol-5-O-b-D-glucoside (11) Yellow amorphous powder; UV kmax (MeOH) nm: 232 and 284; CD (c 0.5 MeOH): [h]250 ?7.20, [h]270 ?9.10; [h]310 –2.05; negative ESI–MS m/z 449.40, [M - H]- (calcd for C22H24O10, 450.40); 1H-NMR (400 MHz, CD3OD) dH: 6.92 (1H, br, s, H-20 ), 6.79 (2H, br, s, H-50 , 60 ), 6.42 (1H, br, s, H-6), 6.13 (1H, br, s, H-8), 5.29 (1H, br, d, J = 11.2 Hz, H-2), 4.85 (1H, d, J = 7.6 Hz, H-100 ),
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To assess anticoagulant activity of sample, the effect of sample on TT was determined using an Auto Blood Coagulation Analyzer (Sysmex CA-540, Japan), according to the manufacturer’s instructions. Briefly, 50 lL of human thrombin (Sigma, St. Louis, MO, USA) was preincubated for 10 min at 37 °C with 10 lL of individual samples dissolved in DMSO before mixing with 50 lL of 20 mM CaCl2 and 100 lL of standard human plasma (Siemens, Marburg, Germany). DMSO and aspirin dissolved in DMSO were used as negative and positive controls, respectively. The time period required for clot formation was measured by the Auto Analyzer. The TT–inhibitory activity (%) = (coagulation time of sample/coagulation time of negative control) 9 100. The experiment was performed in triplicates. In vitro a-glucosidase inhibition assay The a-glucosidase inhibition assay was performed as described in Table 1 (Supplemental material). Briefly, 20 lL of a-glucosidase (0.25 unit/mL, Sigma) was mixed with 65 lL of phosphate buffer (50 mM, pH 6.8) and 15 lL of individual inhibitors dissolved in DMSO, prior to
Antithrombotic and antidiabetic flavonoid glycosides
preincubation at 37 °C for 10 min. Sequentially, 100 lL of p-nitrophenyl a-D-glucopyranoside (3 mM) as a substrate was added to the mixture. The reaction was performed at 37 °C for 30 min. a-glucosidase activity was determined by measuring release of p-nitrophenol from p-nitrophenyl a-D-glucopyranoside at 405 nm. Acarbose was used as a positive control, and all assays were conducted in triplicates. The a-glucosidase inhibitory activity of each sample was calculated by the following equation: Inhibition ratio ð%Þ ¼½1 ðSample OD Blank 2 ODÞ= ðControl OD Blank 1 ODÞ 100; where OD is absorbance at 405 nm.
Results and discussion A phytochemical study on the EtOAc-soluble extract of the grains of S. bicolor using an in vitro TT assay and repeated column chromatographic separation yielded twelve flavonoid glycosides 1–12 (Fig. 2). Compounds 1 and 4 were isolated as yellow powder. Their 1H and 13C NMR spectra revealed an identical flavonol skeleton bearing a sugar moiety. The ESI–MS spectrum of compound 4 presented the ion [M ? H]? at 465, and the molecular formula of C21H20O12 was inferred from 1H and 13C NMR revealing a quercetin aglycone and one glucose sugar unit in the molecule. The b-configuration of the glucose was drawn from a coupling constant of the anomeric proton (5.04, 1H, d, J = 7.6 Hz). Thus, compound 4 was identified as quercetin-3-O-b-D-glucopyranoside, which was identical to the published data (Wu et al. 2008). All 1H, 13C, and MS data of compound 1 were identical with those of 4. Further analysis of the HMBC experiment revealed the correlations between the anomeric proton at dH 5.04 (1H, d, J = 7.6 Hz, H-100 ) and C–7 (164.6). Therefore, compound 1 was identified as quercetin-7-O-b-D-glucoside (Markham, 1982). The 1H and 13C NMR spectra of compounds 2 and 3 were identical with those of 1 and 4, except only for an additional aromatic singlet-proton at dH 6.50 (1H, s) that is assignable for H-3 of the flavones (Lu and Yeap Foo 2000; Orhan et al. 2012). A detailed comparison of their 1H and 13 C NMR data with published literatures led us to identify the structures of 2 and 3 as luteolin-7-O-b-D-glucoside and luteolin-30 -O-b-D-glucoside, respectively (Orhan et al. 2012). Compounds 5–8 were also isolated as yellow powder, their 1H NMR spectra indicated that they were derivatives of 3-O-flavanones (Agrawal et al. 1980) (flavanonol type) with proton signals at dH at 4.88–4.92 (1H, d, J = 11.2 Hz, H-2) and 4.50–4.55 (1H, d, J = 11.2 Hz, H-3), and two corresponding carbons at dC 85.0–85.4 (C-2), 73.6-73.8
1399
(C-3). The 1H and 13C-NMR data of compound 6 are in good agreement with that reported for dihydro-quercetin (taxifolin) with an ABX system at dH 6.89 (1H, d, J = 1.6 Hz, H-20 ), 6.79 (1H, dd, J = 2, 8, H-60 ) and 6.76 (1H, d, J = 8.2 Hz, H-50 ); this is due to a 30 ,40 -disubstitution of ring B and a typical meta-coupled pattern for H-6 and H-8 protons (d 6.15 and 6.13, d, J = 2.0 Hz). The negative ESI–MS of compound 6 gave a quasi-molecular ion peak [M-H]- at m/z 301.10, which is compatible with the molecular formula C15H12O7. The 13C-NMR spectra of compounds 5, 7, and 9 gave 21 carbons, of which 15 carbons were consistent to aglycone and the remaining 6 carbon signals were of b-D-glucoside units [dC 101.9 (C100 ), 73.8 (C-200 ), 78.8 (C-300 ), 71.4 (C-400 ), 79.5 (C-500 ), 62.6 (C-600 )]. A detailed analysis of their HMBC experiments along with the comparison of their 1H, 13C NMR, and MS data with those of published literatures (Fossen et al. 1998; Shen and Theander 1985, Slimestad et al. 1994), led to identify the structures of compounds 5, 7, and 8 to be taxifolin-7-O-b-D-glucoside, taxifolin-30 -O-b-D-glucoside, and aromadendrin-3-O-b-D-glucoside, respectively. The 1H and 13C NMR spectra of compound 9 showed to have a flavanone aglycone with dH 5.22 (1H, dd, J = 12.0, 2.8 Hz, H-2), 3.17 (1H, dd, J = 12.0, 16.0 Hz, H-3eq), and 2.74 (1H, dd, J = 2.8, 16.0 Hz, H-3ax), with corresponding carbons at dC 80.1 (C-2), 42.5 (C-3), and a conjugated carbonyl carbon at dC 197.6 (C-4) (Hu et al. 2012). In addition, a b-D-glucopyranoside moiety was observed [dH 4.68 (d, J = 7.2 Hz, H-100 ), 3.39-3.87 (6H, m, H-200 to H-600 ), and dC 101.8 (C-100 ), 74.9 (C-200 ), 78.0 (C-300 ), 71.5 (C-400 ), 78.5 (C-500 ), 62.6 (C-600 )]. In addition, the 1H-NMR spectrum of compound 9 presented three singlet protons at dH 6.04 (1H, d, J = 2.0 Hz, H-6) and 6.33 (1H, d, J = 2.0 Hz, H-8),6.83 (1H, br, s, H-40 ), a doublet proton at dH 6.70 (2H, d, J = 0.8 Hz, H-20 and H-60 ), which disclosed a 5,7,30 ,50 -tetrahydroxy-dihydropyrone core of a flavanone aglycone. A detailed comparison with literature data led to the identification of compound 9 to be 5,7,30 ,50 tetrahydroxy-flavanone-7-O-b-D-glucoside (Moretti et al. 1998). Compound 10 was also isolated as a flavanone glucoside. The 1H and 13C NMR data of 10 were identical with those of 9; detailed comparison with the reported data (Pan et al. 2008), led to the identification of compound 10 to be eriodictyol-7-O-b-D-glucoside. It was known that different retention behavior of flavanone diastereomers in HPLC with the RP-C18 column was primarily affected by a different stereochemistry at C-2 (Li et al. 2007). Compounds 11 and 12 were isolated as stereoisomers with differences at (2R) and (2S)-configurations. The 1H-1H coupling constants between H-2/H-3a (12.4 Hz) and H-3a/H-3b (16.8 Hz) of compound 12 were different with those of compound 11 (11.2 Hz and 15.7 Hz, respectively). Compounds 11 and 12 contained one chiral
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Fig. 3 Inhibitory effect of isolated compounds (1–12) from S. bicolor on TT activity assay
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% inhibition
Fig. 4 Inhibitory effect of isolated compounds (1–12) from S. bicolor on aglucosidase activity assay
250% 200% 150% 100% 50% 0%
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
center at C-2 position, which possessed either R or S configurations according to the stereochemical environment around the C-2. The absolute configuration of a flavanone compound can be presumed from the sign of its circular dichroism (CD) (Slade et al. 2005). The absolute configuration at C-2 of compound 11 was inferred to be R by the CD spectrum, which had positive Cotton effects near 250 and 270 nm, and negative Cotton effects near 310 nm. Whereas compound 12 possessed positive Cotton effects near 300 and 310 nm, and negative Cotton effects near 250 and 270 nm, in its CD spectrum, indicating S configuration for compound 12. Thus, compounds 11 and 12 were identified as (2R)-eriodictyol-5-O-b-D-glucoside and (2S)eriodictyol-5-O-b-D-glucoside, respectively (Hu et al. 2012). The interaction between platelets and blood vessels is important in the development of thrombosis and cardiovascular diseases (Gorden 1981). Uncontrolled platelet aggregation is critical in arterial thrombosis, leading to ischemia and may cause life-threatening disorders, such as
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Thrombin Time assay
α-glucosidase activity
heart attacks and stroke. Hence, in the treatment and prevention of these cardiovascular diseases the inhibition of thrombus formation is of fundamental importance. To assess the antithrombotic effects, the anticoagulant action of isolated compounds 1–12 was determined using an in vitro TT assay, and the results are presented in Fig. 3. Among them, luteolin-30 -O-b-D-glucoside (3) possessed the most potency with 197 % inhibition stronger than aspirin (positive control, 181 % inhibition) at the same concentration of 500 lg/mL. The following eriodictyol-7O-b-D-glucoside (10), quercetin-3-O-b-D-glucoside (4) and taxifolin-30 -O-b-D-glucoside (7) displayed 158, 152 and 120 % inhibition, respectively, whereas aspirin possessed only 138 % inhibition at 375 lg/mL. It is well known that a-glucosidase is an exo-type carbohydrase, which catalyzes the release of a-D-glucose from the non-reducing end of oligosaccharide and disaccharides (Frandsen and Svensson 1998). Because small intestinal aglucosidase is crucial for dietary carbohydrate digestion, it has been proposed that a-glucosidase inhibitors can retard
Antithrombotic and antidiabetic flavonoid glycosides
carbohydrate digestion and glucose absorption (Bertozzi and Kiessling 2001); therefore, it may be useful in the treatment of carbohydrate-mediated diseases, such as diabetes and obesity. Interestingly, at the concentration of 100 lg/mL, all compounds showed inhibitory effects on aglucosidase activity with an inhibition percentage at around 40 % comparable with the positive control (acarbose) having 38.7 % inhibition at the same concentration (Fig. 4). Compound 3, which had the most potency possessed 85.2 % inhibition, was followed by compounds 10 (65.2 %), 4 (55.7 %) and 7 (44.9 %). S. bicolor is an important food crop in semiarid parts of Africa and Asia. It has been increasingly used as an ‘‘ancient grain’’ and gluten free food ingredient in the United States. This growth in popularity is mainly due to agronomic advantages, such as high drought tolerance, high yields, low cost and potential health benefits including slow starch digestibility, cardiovascular disease reduction, antioxidant activity, anti-inflammatory and anti-carcinogenic properties (Barros et al. 2012). Our finding clearly demonstrates the effects of S. bicolor on cardiovascular disease reduction and its components which are responsible for the activity. Thus, we may suggest that S. bicolor and its flavonoid-enrich extracts could be considered as supplemental and or functional foods having beneficial effects against blood coagulation-induced ischemia, possibly thromboembolism disease, as well as obesity. Acknowledgments This work was supported by Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (Grant #110133-3), and the BK 21 Plus Program of the Ministry of Education, Science and Technology, Korea. The authors are grateful to S. H. Kim and collaborators at the Korea Basic Science Institute (Daegu) for measuring the mass spectra. Conflict of interest The authors declare no any actual and potential conflict of interest including any financial, personal or other relationships.
References Agrawal, P.K., S.K. Agarwal, and R.P. Rastogi. 1980. Dihydroflavonols from Cedrus deodara. Phytochemistry 19: 893–896. Barros, F., J.M. Awika, and L.W. Rooney. 2012. Interaction of tannins and other sorghum phenolic compounds with starch and effects on in vitro starch digestibility. Journal of Agricultural and Food Chemistry 60: 11609–11617. Bauer, K.A., and R.D. Rosenberg. 1991. Role of antithrombin III as a regulator of in vivo coagulation. Seminars in Hematology 28: 10–18. Bertozzi, C.R., and L.L. Kiessling. 2001. Chemical glycobiology. Science 291: 2357–2364. Cohen, A.T., G. Agnelli, F.A. Anderson, J.I. Arcelus, D. Bergqvist, J.G. Brecht, I.A. Greer, J.A. Heit, J.L. Hutchinson, A.K. Kakkar, D. Mottier, E. Oger, M.M. Samama, and M. Spannagl. 2007.
1401 Venous thromboembolism (VTE) in Europe. The number of VTE events and associated morbidity and mortality. Thrombosis and Haemostasis 98: 756–764. Coughlin, S.R. 2000. Thrombin signalling and protease-activated receptors. Nature 407: 258–264. Fossen, T., A.T. Pedersen, and M. Andersen. 1998. Flavonoids from red onion (Allium cepa). Phytochemistry 47: 281–285. Frandsen, T., and B. Svensson. 1998. Plant a-glucosidases of the glycoside hydrolase family 31. Molecular properties, substrate specificity, reaction mechanism, and comparison with family members of different origin. Plant Molecular Biology 37: 1–13. Gorden, J.L. 1981. Platelets in perspective: In Platelets in Biology and Pathology. Amsterdam: Elsevier. Hamilton, J.R. 2009. Protease-activated receptors as targets for antiplatelet therapy. Blood Reviews 23: 61–65. Heit, J. A., Cohen, A.T., and Anderson, F.A. On behalf of the VTE Impact Assessment Group. Estimated annual number of incident and recurrent, non-fatal and fatal venous thromboembolism (VTE) events in the US. Blood, (2005). Hu, Q., D.D. Zhang, L. Wang, H. Lou, and D. Ren. 2012. Eriodictyol7-O-glucoside, a novel Nrf2 activator, confers protection against cisplatin-induced toxicity. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 50: 1927–1932. Lee, K.A., and M.S. Kim. 2005. Antiplatelet and antithrombotic activities of methanol extract of Usnea longissima. Phytotherapy research : PTR 19: 1061–1064. Li, W., X. Xu, H. Zhang, C. Ma, H. Fong, R. Van Breemen, and J. Fitzloff. 2007. Secondary metabolites from Andrographis paniculata. Chemical & Pharmaceutical Bulletin 55: 455–458. Lu, Y., and L. Yeap Foo. 2000. Flavonoid and phenolic glycosides from Salvia officinalis. Phytochemistry 55: 263–267. Mackman, N. 2008. Triggers, targets and treatments for thrombosis. Nature 451: 914–918. Markham, K.R. 1982. Techniques of flavonoid identification. London: Academic Press. Moretti, C., M. Sauvain, C. Lavaud, G. Massiot, J. Bravo, and V. Munoz. 1998. A novel antiprotozoal aminosteroid from Saracha punctata. Journal of Natural Products 61: 1390–1393. Nader, H. B., Pinhal, M. A., Bau, E. C., Castro, R. A., Medeiros, G. F., Chavante, S. F., Leite, E. L., Trindade, E. S., Shinjo, S. K., Rocha, H. A., Tersariol, I. L., Mendes, A., and Dietrich, C. P. Development of new heparin-like compounds and other antithrombotic drugs and their interaction with vascular endothelial cells. Brazilian Journal of Medical and Biological Research = Revista Brasileira de Pesquisas Medicas e Biologicas/Sociedade Brasileira de Biofisica [et al.], 34:699–709 (2001). Orhan, F., O. Baris, D. Yanmis, T. Bal, Z. Guvenalp, and M. Gulluce. 2012. Isolation of some luteolin derivatives from Mentha longifolia (L.) Hudson subsp. longifolia and determination of their genotoxic potencies. Food Chemistry 135: 764–769. Pan, J., S. Zhang, L. Yan, J. Tai, Q. Xiao, K. Zou, Y. Zhou, and J. Wu. 2008. Separation of flavanone enantiomers and flavanone glucoside diastereomers from Balanophora involucrata Hook. f. by capillary electrophoresis and reversed-phase high-performance liquid chromatography on a C18 column. Journal of Chromatography A 1185: 117–129. Rao, L.V., S.I. Rapaport, and A.D. Hoang. 1993. Binding of factor VIIa to tissue factor permits rapid antithrombin III/heparin inhibition of factor VIIa. Blood 81: 2600–2607. Shen, Z., and O. Theander. 1985. Flavonoid glycosides from needles of Pinus massoniana. Phytochemistry 24: 155–158. Slade, D., D. Ferreira, and J.P. Marais. 2005. Circular dichroism, a powerful tool for the assessment of absolute configuration of flavonoids. Phytochemistry 66: 2177–2215.
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RESEARCH ARTICLE
Antithrombotic and antidiabetic flavonoid glycosides from the grains of Sorghum bicolor (L.) Moench var. hwanggeumchal Phi-Hung Nguyen • Vu Viet Dung • Bing Tian Zhao Young Ho Kim • Byung Sun Min • Mi Hee Woo
•
Received: 16 January 2014 / Accepted: 25 May 2014 / Published online: 24 June 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract A phytochemical study of the grains of S. bicolor, resulting in the isolation of twelve flavonoid glycosides 1–12. Their chemical structures were elucidated on the basis of spectroscopic (1D and 2D NMR) and MS data analyses. All compounds were tested on thrombin time (TT) assay and a-glucosidase assay in order to assess their inhibitory effects on blood coagulation and a-glucosidase enzyme. At the concentration of 500 lg/mL, compounds 3, 4, 7 and 10 possessed the potential effects on blood coagulation with inhibitory percentage of 197, 152, 120 and 158 %, respectively, whereas aspirin, which used as a positive control, indicated 181 and 138 % inhibition at 500 and 375 lg/mL, respectively. Furthermore, compounds 3, 4, 7, 9 and 10 also displayed strong inhibitory effects on aglucosidase enzyme, with 85.2, 55.7, 43.9, 52.7 and 65.2 % inhibition at 100 lg/mL, respectively, whereas acarbose, as a positive control, possessed only 38.7 % at the same concentration. Taken together, our data suggest that S. bicolor and its flavonoid-enrich extracts could be considered as supplemental and or functional foods having beneficial effects against blood coagulation-induced ischemia, possibly thromboembolism disease, as well as diabetes.
Electronic supplementary material The online version of this article (doi:10.1007/s12272-014-0422-5) contains supplementary material, which is available to authorized users. P.-H. Nguyen V. V. Dung B. T. Zhao B. S. Min M. H. Woo (&) College of Pharmacy, Catholic University of Daegu, Hayang 712-702, Gyeongbuk, Korea e-mail: [email protected] Y. H. Kim Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Korea
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Keywords Sorghum bicolor Anticoagulants Thrombin inhibitors a-Glucosidase inhibitors Flavonoid glycosides
Introduction Thromboembolic disorders (TD) are the major causes of morbidity and mortality. Arterial thrombosis is the most common cause of myocardial infarction and ischemic stroke, whereas deep vein thrombosis can lead to pulmonary embolism (Mackman 2008). In the U.S., pulmonary embolism causes almost 300,000 deaths per annum (Heit 2005). Moreover, it is estimated that 12 % of annual deaths occurring in France, Germany, Italy, Spain, Sweden and the UK are due to venous thromboembolism (VTE) (Cohen et al. 2007). Great advances have been made in understanding the molecular and cellular basis of thrombus formation in the past few decades, with anticoagulants remaining as the cornerstone for the prevention and treatment of TD (Weitz 2004). Mechanism studies revealed that Factor Xa, a key component of the prothrombinase complex, first stimulates the conversion of the inactive zymogen, prothrombin, to the active serine protease, thrombin. Thrombin, in turn, catalyzes the conversion of soluble fibrinogen to insoluble strands of fibrin, which leads to platelet activation and the release of adenosine diphosphate, serotonin and thromboxane A2 through the cleavage of protease-activated receptor 1 (PAR1) on the platelet surface. This in conjunction with simultaneous platelet activation by exposed subendothelial collagen leads to platelet aggregation, fibrin and blood cell agglutination and thus thrombus formation (Coughlin 2000, Hamilton 2009). The inhibition of thrombin generation, activation or both is therefore a logical target in the treatment of TD. Unfractionated heparin (UFH), low molecular weight heparin
Antithrombotic and antidiabetic flavonoid glycosides
Re la tive Throm bi n Ti m e (%)
Fig. 1 Inhibitory effect of the n-Hexane, EtOAc, BuOH fractions, the H2O residue and the total 95 %-EtOH extract of the grains of S. bicolor on TT activity assay. TT of individual samples was measured at a final concentration of 500 lg/mL. The experiment was performed in triplicates
1395 Thrombin Time assay
200% 180% 160% 140% 120% 100% 80% 60% 40% 20% 0%
R1
R1 OH
R3
O
OH R2
O
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R3
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1
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OH
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OH OH
O
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5
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10 11 12
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H
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OH
H
OH
O-β-D-gluc
OH
H
OH
O-β-D-gluc
R4 OH OH
Fig. 2 Chemical structure of isolated compounds (1–12) from the grains of S. bicolor
(LMWH) and fondaparinux (an AT-dependent factor Xa [FXa] inhibitor) bind to antithrombin (AT), enhance its protease inhibition activity, and exert anticoagulant effects (Bauer and Rosenberg 1991; Rao et al. 1993). However, its use is accompanied by some side effects, frequently requiring the monitoring of partially activated thromboplastin time and ultimately resulting in other hemorrhagic complications (Nader et al. 2001). Although it is well established that aspirin still provides an effective secondary prevention of ischemic cardiovascular disorders, this drug can produce major drawbacks, such as hemorrhagic events and upper gastrointestinal bleeding (Lee and Kim 2005). Thus, the search for alternative anticoagulants with reduced side effects is still needed and urgent. As part of an investigation aimed as anticoagulant agents from plants, we found that an EtOAc-soluble extract of the grains of S. bicolor exhibited significant activities on both thrombin time (TT) and a-glucosidase assays (Fig. 1). Thus, a directly bioassay-guided isolation of the EtOAcsoluble extract of the grains of this plant has led to the isolation of a series of flavonoid glucosides (1–12) as
active principles (Fig. 2). In addition, the inhibitory effects of the isolated compounds (1–12) on a-glucosidase were also investigated.
Materials and methods General procedures The optical rotations were determined on a Rudolph Autopol AP 589 polarimeter using a 100 mm glass microcell. The IR spectra were recorded on a Nicolet 6700 FT-IR (Thermo electron Corp.). UV spectra were recorded in MeOH using a Shimadzu spectrometer. The NMR spectra were recorded in methanol-d4 (CD3OD), pyridine-d5 (C5D5N) on Varian OXFORD-AS 400 MHz instrument (PaloAlto, CA, USA) with TMS as the internal standard at the Department of Pharmacy, Catholic University of Daegu, Korea. All mass experiments were performed on a Micromass QTOF2 (Micromass, Wythenshawe, UK) mass spectrometer. Silica Gel (Merck, 63–200 lm particle size)
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and RP-18 (Merck, 150 lm particle size) were used for column chromatography. For thin-layer chromatography (TLC), pre-coated TLC was carried out on Silica Gel 60 F254 and RP-18 F254 plates from Merck. HPLC runs were carried out using a Gilson system with a UV detector and an Optima Pak C18 column (10 9 250 mm, 10 lm particle size, RS Tech Corp., Korea). Reagents p-Nitrophenyl-a-D-glucopyranoside (pNPG), a-glucosidase (0.1 Unit), Acarbose, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent were purchased from Sigma Chemical Co. Mouse fibroblast (3T3-L1) cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), Trypsin–EDTA, penicillin/streptomycin/amphotericin (10,000 U/mL, 10,000 lg/mL, and 2,500 lg/mL, respectively), and fetal bovine serum (FBS) were obtained from Gibco BRL, Life Technologies (USA). All other chemicals and solvents were of analytical grade commercially available. Plant material The grains of Sorghum bicolor (L.) Moench var. hwanggeumchal (S. bicolor) were provided by National Institute of Crop Science of Miryang, Korea, in February 2011. A voucher specimen (20100523) has been deposited at the College of Pharmacy, Catholic University of Daegu, Republic of Korea. Extraction and isolation The grains of S. bicolor (10 kg) were extracted with 95 % ethanol (EtOH) at room temperature (18 L 9 3 times) for 1 week. The solvent extract was concentrated under reduced pressure to give the EtOH extract (210 g). The concentrated EtOH extract was suspended in H2O (1 L) and partitioned successively with n-hexane (1.5 L 9 4 times, 96 g), EtOAc (1.5 L 9 4 times, 48.6 g), n-BuOH (1.5 L 9 4 times, 38.5 g) and H2O-soluble fractions (30.5 g), respectively. The n-hexane, EtOAc, n-BuOH and H2O-soluble fractions were tested on TT assay and a-glucosidase assay. Among those, the EtOAc fraction showed the highest activities. Thus, the EtOAc fraction (108.6 g) was subjected to silica gel column chromatography (15 9 60 cm; 63–200 lm particle size), using gradient solvents of CH2Cl2:MeOH (100:1 ? 0:1), yielded six combined fractions (F.1 to F.6), according to their TLC profiles. These fractions were assayed for blood coagulants and a-glucosidase enzyme inhibition. Strong active fractions 2, 4 and 5 were continuously chromatographed for activity-guided isolation. Fraction 5 (F.5, 7.3 g) was further
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chromatographed on a RP-18 column with gradient solvent of MeOH:H2O (50:50 ? 95:5) to afford ten sub-fractions (F.5.1 to F.5.10). The sub-fraction F.5.5 (300 mg) was re-chromatographed on a RP-18 column with an isocratic solvent of MeOH:H2O (40:60) to yield compound 1 (81.6 mg). The subfraction F.5.4 (75.2 mg) was further purified by an open RP18 column with a gradient solvent of MeOH:H2O (20:80 ? 30:70), and afforded compound 2 (10.6 mg). The sub-fraction F.5.3 (644.9 mg) was also re-chromatographed on an open RP-18 column using a gradient solvent of MeOH:H2O (20:80 ? 70:30) to yield compounds 3 (10.1 mg), 7 (12.7 mg), 9 (9.6 mg), and 10 (9.0 mg), respectively. Further purification of subfraction F.5.6 (355 mg) by semi-preparative Gilson HPLC, using an isocratic solvent system of 40 % MeOH in H2O for over 40 min [RS Tech Optima Pak C18 column (10 9 250 mm, 10 lm particle size); mobile phase MeOH/H2O containing 0.1 % formic acid (0–40 min: 40 % MeOH, 40–42 min: 40–100 % MeOH, 42–50 min: 100 % MeOH); UV detections at 205 and 254 nm], yielded compound 5 (17.1 mg, tR = 31.5 min) and compound 8 (2.5 mg, tR = 38.7 min). Compound 6 (151.6 mg) was purified from fraction 2 by using an open RP18 column (3.5 9 15 cm) and eluting with a gradient solvent system of 50 % MeOH in H2O. Fraction 4 (F.4, 2.0 g) was also chromatographed on a RP-18 column (4.0 9 20 cm), with a stepwise gradient solvent of MeOH in H2O (50:50 ? 50:0), afforded compound 4 (15 mg), and three sub-fractions (F.4.1–F.4.3). The purification of sub-fraction F.4.2 (105.5 g), which was prepared by preparative Gilson HPLC using an isocratic solvent system of 35 % MeOH in H2O for over 40 min [RS Tech Optima Pak C18 column (10 9 250 mm, 10 lm particle size); mobile phase MeOH/ H2O containing 0.1 % formic acid (0–40 min: 40 % MeOH, 40–42 min: 40–100 % MeOH, 42–50 min: 100 % MeOH); UV detections at 205 and 254 nm], resulted in the isolation of compound 11 (16.1 mg, tR = 33.1 min) and compound 12 (41.5 mg, tR = 35.4 min), respectively. Quercetin-7-O-b-D-glucoside (1) Yellow powder; positive FAB-MS m/z 465.10 [M ? H]? (calcd. for C21H20O12, 464.10); 1H-NMR (400 MHz, CD3OD) dH: 7.75 (1H, d, J = 2.0 Hz, H-20 ), 7.65 (1H, dd, J = 8.4, 2.0 Hz, H-60 ), 6.87 (1H, d, J = 8.4 Hz, H-50 ), 6.74 (1H, d, J = 2.0 Hz, H-8), 6.45 (1H, d, J = 2.0 Hz, H-6), 5.04 (1H, d, J = 7.2 Hz, H-100 ), 3.92–3.30 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b); 13C-NMR (100 MHz, CD3OD) dC: 146.4 (C-2), 137.8 (C-3), 177.6 (C-4), 162.3 (C-5), 100.3 (C-6), 164.6 (C-7), 95.7 (C-8), 157.9 (C-9), 106.4 (C-10), 124.1 (C-10 ), 116.3 (C-20 ), 149.1 (C-30 ), 148.9 (C-40 ), 116.4 (C-50 ), 122.0 (C-60 ), 101.8 (C100 ), 74.9 (C-200 ), 78.0 (C-300 ), 71.5(C-400 ), 78.5 (C-500 ), 62.6 (C-600 ).
Antithrombotic and antidiabetic flavonoid glycosides
Luteolin-7-O-b-D-glucoside (2) Yellow powder; positive FAB-MS m/z 449.10 [M ? H]? (calcd. for C21H20O11, 448.10); 1H-NMR (400 MHz, CD3OD) dH: 7.33 (1H, dd, J = 8.0, 2.0 Hz, H-60 ), 7.31 (1H, d, J = 2.0 Hz, H-20 ), 6.80 (1H, d, J = 8.0 Hz, H-50 ), 6.69 (1H, d, J = 2.0 Hz, H-8), 6.50 (1H, s, H-3), 6.40 (1H, d, J = 2.0 Hz, H-6), 5.06 (1H, d, J = 7.6 Hz, H-100 ), 3.83–3.40 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b); 13 C-NMR (100 MHz, CD3OD) dC: 161.1 (C-2), 122.2 (C3), 182.8 (C-4), 163.6 (C-5), 99.9 (C-6), 165.6 (C-7), 94.8 (C-8), 157.7 (C-9), 105.9 (C-10), 119.3 (C-10 ), 115.6 (C20 ), 149.5 (C-30 ), 150.6 (C-40 ), 113.1 (C-50 ), 102.9 (C-60 ), 100.4 (C-100 ), 73.5 (C-200 ), 76.7 (C-300 ), 70.0 (C-400 ), 77.2 (C-500 ), 61.2 (C-600 ). Luteolin-30 -O-b-D-glucoside (3) Yellow powder; positive FAB-MS m/z 471.10 [M ? Na]? (calcd for C21H20O11, 448.10); 1H-NMR (400 MHz, CD3OD) dH: 7.41 (1H, d, J = 2.0 Hz, H-20 ), 7.15 (1H, dd, J = 8.4, 2.0 Hz, H-60 ), 6.76 (1H, d, J = 2.0 Hz, H-8), 6.74 (H, d, J = 8.4 Hz, H-50 ), 6.50 (1H, s, H-3), 6.29 (1H, d, J = 2.0 Hz, H-6), 4.94 (1H, d, J = 7.6 Hz, H-100 ), 3.92–3.17 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b); 13 C-NMR (100 MHz, CD3OD) dC: 162.0 (C-2), 105.2 (C3), 182.4 (C-4), 158.6 (C-5), 99.2 (C-6), 170.0 (C-7), 93.9 (C-8), 158.0 (C-9), 105.5 (C-10), 124.2 (C-10 ), 114.2 (C20 ), 149.4 (C-30 ), 147.9 (C-40 ), 116.8 (C-50 ), 118.9 (C-60 ), 101.4 (C-100 ), 73.5 (C-200 ), 78.7 (C-300 ), 70.0(C-400 ), 77.2 (C500 ), 62.2 (C-600 ). Quercetin-3-O-b-D-glucoside (4) Yellow amorphous powder; positive ESI–MS m/z 487.10 [M ? Na]? (calcd for C21H20O12, 464.10); 1H-NMR (400 MHz, CD3OD) dH: 7.82 (1H, d, J = 2.0 Hz, H-20 ), 7.57 (1H, dd, J = 8.0, 2.0 Hz, H-60 ), 6.85 (1H, d, J = 8.0 Hz, H-50 ), 6.30 (1H, d, J = 2.0 Hz, H-8), 6.12 (1H, d, J = 2.0 Hz, H-6), 5.04 (1H, d, J = 7.6 Hz, H-100 ), 3.85–3.45 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b); 13 C-NMR (100 MHz, CD3OD) dC: 158.1 (C-2), 130.2 (C3), 180.2 (C-4), 162.8 (C-5), 100.3 (C-6), 164.6 (C-7), 97.7 (C-8), 160.4 (C-9), 105.4 (C-10), 122.2 (C-10 ), 115.4 (C20 ), 146.6 (C-30 ), 149.5 (C-40 ), 121.8 (C-50 ), 120.9 (C-60 ), 104.6 (C-100 ), 75.6 (C-200 ), 76.9 (C-300 ), 71.3 (C-400 ), 76.7 (C-500 ), 62.5 (C-600 ); Taxifolin-7-O-b-D-glucoside (5) Yellow powder; positive ESI–MS m/z 467.11 [M ? H]? (calcd for C21H22O12 466.11); 1H-NMR (400 MHz, pyridine-d5) dH: 7.37 (1H, d, J = 1.6 Hz, H-20 ), 7.08 (1H, dd,
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J = 8.0, 1.6 Hz, H-60 ), 6.98 (1H, d, J = 8.0 Hz, H-50 ), 6.15 (1H, d, J = 2.0 Hz, H-6), 6.08 (1H, d, J = 2.0 Hz, H-8), 5.26 (1H, d, J = 7.6 Hz, H-100 ), 4.90 (1H, d, J = 11.2 Hz, H-2), 4.55 (1H, d, J = 11.2 Hz, H-3), 4.07–3.54 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b); 13 C-NMR (100 MHz, pyridine-d5) dC: 85.4 (C-2), 73.8 (C3), 199.9 (C-4), 167.2 (C-5), 98.3 (C-6), 164.8 (C-7), 96.8 (C-8), 163.9 (C-9), 103.6 (C-10), 129.8 (C-10 ), 116.9 (C20 ), 147.8 (C-30 ), 148.6 (C-40 ), 117.0 (C-50 ), 120.8 (C-60 ), 101.9 (C-100 ), 73.8 (C-200 ), 78.8 (C-300 ), 71.4 (C-400 ), 79.5 (C-500 ), 62.6 (C-600 ). Taxifolin (6) Yellow amorphous powder; positive ESI–MS m/z 305.25 [M ? H]? (calcd for C15H12O7, 304.25); 1H-NMR (400 MHz, CD3OD) dH: 6.89 (1H, d, J = 2.0 Hz, H-20 ), 6.79 (1H, dd, J = 8.0, 2.0 Hz, H-60 ), 6.76 (1H, d, J = 8.0 Hz, H-50 ), 6.15 (1H, d, J = 2.0 Hz, H-6), 6.13 (1H, d, J = 2.0 Hz, H-8), 4.92 (1H, d, J = 11.6 Hz, H-2), 4.50 (1H, d, J = 11.6, H-3), 13C NMR (100 MHz, CD3OD) dC: 85.0 (C-2), 73.6 (C-3), 199.9 (C-4), 164.8 (C-5), 98.3 (C-6), 167.2(C-7), 96.8 (C-8), 163.97 (C-9), 103.6 (C10), 129.8 (C-10 ), 116.9 (C-20 ), 147.8 (C-30 ), 148.6 (C-40 ), 117.0 (C-50 ), 120.9 (C-60 ). Taxifolin-30 -O-b-D-glucoside (7) Yellow powder; positive FAB-MS m/z 489.10 [M ? Na]? (calcd. for C21H22O12, 466.10); 1H-NMR (400 MHz, CD3OD) dH: 6.89 (1H, d, J = 2.0 Hz, H-20 ), 6.79 (1H, dd, J = 8.0, 2.0 Hz, H-60 ), 6.76 (1H, d, J = 8.0 Hz, H-50 ), 6.15 (1H, d, J = 2.0, H-6), 6.13 (1H, d, J = 2.0, H-8), 4.92 (1H, d, J = 11.2 Hz, H-2), 4.50 (1H, d, J = 11.2, H-3), 4.07–3.54 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b); 13 C-NMR (100 MHz, CD3OD) dC: 85.0 (C-2), 73.6 (C-3), 199.9 (C-4), 164.8 (C-5), 98.3 (C-6), 167.2 (C-7), 96.8 (C8), 164.0 (C-9), 103.6 (C-10), 129.8 (C-10 ), 116.9 (C-20 ), 147.8 (C-30 ), 148.6 (C-40 ), 117.0 (C-50 ), 120.9 (C-60 ), 101.9 (C-100 ), 73.8 (C-200 ), 78.8 (C-300 ), 71.4 (C-400 ), 79.5 (C-500 ), 62.6 (C-600 ). Aromadendrin-3-O-b-D-glucoside (8) Yellow powder; UV (MeOH) kmax nm: 223, 293; IR (KBr) mmax cm-1: 3253 (OH), 1635 (C = O), 1614, 1598, 1517 (C = C), 1253, 1161, 1071 (C–O); positive FAB-MS m/ z 473.11 [M ? Na]? (calcd. for C21H22O11, 450.11); 1HNMR (400 MHz, CD3OD) dH: 7.36 (2H, d, J = 8.4 Hz, H-20 , 60 ), 6.81 (2H, d, J = 8.4 Hz, H-30 , 50 ), 5.91 (1H, d, J = 2.0 Hz, H-6), 5.89 (1H, d, J = 2.0 Hz, H-8), 5.23 (1H, d, J = 10.4 Hz, H-2), 4.97 (1H, d, J = 10.4 Hz, H-3), 4.38 (1H, d, J = 7.6 Hz, H-100 ), 3.87–3.24 (6H, m, H-200 , H-300 ,
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H-400 , H-500 , H-600 a, H-600 b); 13C-NMR (100 MHz, CD3OD) dC: 82.6 (C-2), 76.2 (C-3), 195.2 (C-4), 164.5 (C-5), 93.4 (C-6), 168.1 (C-7), 95.4 (C-8), 163.3 (C-9), 101.6 (C-10), 127.5 (C-10 ), 129.5 (C-20 , 60 ), 115.3 (C-30 , 50 ), 158.3 (C-40 ), 101.6 (C-100 ), 73.5 (C-200 ), 76.5 (C-300 ), 70.2 (C-400 ), 77.2 (C-500 ), 61.6 (C-600 ). 5,7,30 ,50 -tetrahydroxy-flavanone-7-O-b-D-glucoside (9) Yellow powder; UV (MeOH) kmax nm: 215, 282 and 334; IR (KBr) mmax cm-1: 3385 (OH), 1696, 1635 (C = O), 1580, 1528 (C = C), 1446, 1344, 1242, 1073 (C–O), and 857; positive FAB-MS m/z 473.11 [M ? Na]? (calcd for C21H22O11, 450.11); 1H-NMR (400 MHz, CD3OD) dH: 6.83 (1H, br, s, H-40 ), 6.70 (2H, d, J = 0.8 Hz, H-20 , 60 ), 6.33 (1H, d, J = 2.0 Hz, H-8), 6.04 (1H, d, J = 2.0 Hz, H-6), 5.22 (1H, dd, J = 12.0, 2.8 Hz, H-2), 4.68 (1H, d, J = 7.2 Hz, H-100 ), 3.87–3.39 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b), 3.17 (1H, dd, J = 12.0, 17.2 Hz, H-3a), 2.74 (1H, dd, J = 2.8, 17.2 Hz, H-3b); 13C-NMR (100 MHz, CD3OD) dC: 80.1 (C-2), 42.5 (C-3), 197.6 (C4), 162.3 (C-5), 100.4 (C-6), 164.6 (C-7), 95.7 (C-8), 157.3 (C-9), 106.4 (C-10), 124.1 (C-10 ), 116.3 (C-20 ), 149.1 (C30 ), 148.9 (C-40 ), 161.4 (C-50 ), 116.4 (C-60 ), 101.84 (C-100 ), 74.9 (C-200 ), 78.0 (C-300 ), 71.5 (C-400 ), 78.5 (C-500 ), 62.6 (C600 ).
P.-H. Nguyen et al.
3.96–3.30 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b), 3.05 (1H, dd, J = 11.2, 15.6 Hz, H-3a), 2.68 (1H, br, d, J = 15.6 Hz, H-3b); 13C-NMR (100 MHz, CD3OD) dC: 80.3 (C-2), 46.3 (C-3), 193.2 (C-4), 161.6 (C-5), 100.2 (C6), 166.6 (C-7), 99.5 (C-8), 166.0 (C-9), 106.8 (C-10), 131.8 (C-10 ), 114.7 (C-20 ), 146.8 (C-30 ), 146.4 (C-40 ), 116.3 (C-50 ), 119.3(C-60 ), 103.9 (C-100 ), 74.6 (C-200 ), 78.4 (C-300 ), 71.2 (C-400 ), 77.4 (C-500 ), 62.5 (C-600 ). (2S)-eriodictyol-5-O-b-D-glucoside (12) Yellow amorphous powder; UV kmax (MeOH) nm: 232 and 284; CD (c 0.5 MeOH): [h]250 –2.68, [h]270 –1.75; [h]300 ?8.98, [h]310 ?1.15; negative ESI–MS m/z 449.25, [M H]- (calcd for C22H24O10, 450.25); 1H-NMR (400 MHz, CD3OD) dH: 6.92 (1H, br, s, H-20 ), 6.81 (1H, d, J = 8.0 Hz, H-50 ), 6.79 (1H, br, d, J = 8.0 Hz, H-60 ), 6.47 (1H, br, s, H-6), 6.13 (1H, br, s, H-8), 5.24 (1H, br, d, J = 12.4 Hz, H-2), 4.78 (1H, br, d, J = 7.2 Hz, H-100 ), 3.96–3.30 (6H, m, H-200 , H-300 , H-400 , H-500 , H-600 a, H-600 b), 2.93 (1H, dd, J = 12.4, 16.8 Hz, H-3a), 2.70 (1H, br, d, J = 16.8 Hz, H-3b); 13C-NMR (100 MHz, CD3OD) dC: 80.2 (C-2), 46.2 (C-3), 192.1 (C-4), 162.2 (C-5), 100.4 (C6), 166.9 (C-7), 99.5 (C-8), 166.4 (C-9), 107.0 (C-10), 131.6 (C-10 ), 114.7 (C-20 ), 146.7 (C-30 ), 146.4 (C-40 ), 116.3 (C-50 ), 119.3 (C-60 ), 104.9 (C-100 ), 74.6 (C-200 ), 78.5 (C-300 ), 71.2 (C-400 ), 77.0 (C-500 ), 62.5 (C-600 ).
Eriodictyol-7-O-b-D-glucoside (10) Thrombin time (TT) assay Yellow powder; positive ESI–MS m/z 451.11 [M ? H]? (calcd for C21H22O11, 450.11); 1H-NMR (400 MHz, CD3OD) dH: 6.88 (1H, br, s, H-20 ), 6.75 (2H, br, s, H-50 , 60 ), 6.45 (1H, br, s, H-6), 6.11 (1H, br, s, H-8), 5.29 (1H, dd, J = 3.2, 12.4 Hz, H-2), 4.75 (1H, d, J = 7.6 Hz, H-100 ), 3.90 (1H, br, d, J = 12.4 Hz, H-600 a), 3.71 (1H, m, H-600 b), 3.52-3.38 (4H, m, H-200 , H-300 , H-400 , H-500 ), 3.05 (1H, dd, J = 12.4, 17.6 Hz, H-3a), 2.70 (2H, dd, J = 2.8, 17.6 Hz, H-3b); 13C-NMR (100 MHz, CD3OD) dC: 80.7 (C-2), 44.1 (C-3), 193.6 (C-4), 164.9 (C-5), 97.0 (C-6), 167.0 (C-7), 98.0 (C-8), 164.6 (C-9), 105.0 (C-10), 131.5 (C-10 ), 114.8 (C-20 ), 147.0 (C-30 ), 146.6 (C-40 ), 116.3 (C-50 ), 119.4 (C60 ), 101.3 (C-100 ), 74.7 (C-200 ), 78.3 (C-300 ), 71.2 (C-400 ), 77.8 (C-500 ), 62.4 (C-600 ). (2R)-eriodictyol-5-O-b-D-glucoside (11) Yellow amorphous powder; UV kmax (MeOH) nm: 232 and 284; CD (c 0.5 MeOH): [h]250 ?7.20, [h]270 ?9.10; [h]310 –2.05; negative ESI–MS m/z 449.40, [M - H]- (calcd for C22H24O10, 450.40); 1H-NMR (400 MHz, CD3OD) dH: 6.92 (1H, br, s, H-20 ), 6.79 (2H, br, s, H-50 , 60 ), 6.42 (1H, br, s, H-6), 6.13 (1H, br, s, H-8), 5.29 (1H, br, d, J = 11.2 Hz, H-2), 4.85 (1H, d, J = 7.6 Hz, H-100 ),
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To assess anticoagulant activity of sample, the effect of sample on TT was determined using an Auto Blood Coagulation Analyzer (Sysmex CA-540, Japan), according to the manufacturer’s instructions. Briefly, 50 lL of human thrombin (Sigma, St. Louis, MO, USA) was preincubated for 10 min at 37 °C with 10 lL of individual samples dissolved in DMSO before mixing with 50 lL of 20 mM CaCl2 and 100 lL of standard human plasma (Siemens, Marburg, Germany). DMSO and aspirin dissolved in DMSO were used as negative and positive controls, respectively. The time period required for clot formation was measured by the Auto Analyzer. The TT–inhibitory activity (%) = (coagulation time of sample/coagulation time of negative control) 9 100. The experiment was performed in triplicates. In vitro a-glucosidase inhibition assay The a-glucosidase inhibition assay was performed as described in Table 1 (Supplemental material). Briefly, 20 lL of a-glucosidase (0.25 unit/mL, Sigma) was mixed with 65 lL of phosphate buffer (50 mM, pH 6.8) and 15 lL of individual inhibitors dissolved in DMSO, prior to
Antithrombotic and antidiabetic flavonoid glycosides
preincubation at 37 °C for 10 min. Sequentially, 100 lL of p-nitrophenyl a-D-glucopyranoside (3 mM) as a substrate was added to the mixture. The reaction was performed at 37 °C for 30 min. a-glucosidase activity was determined by measuring release of p-nitrophenol from p-nitrophenyl a-D-glucopyranoside at 405 nm. Acarbose was used as a positive control, and all assays were conducted in triplicates. The a-glucosidase inhibitory activity of each sample was calculated by the following equation: Inhibition ratio ð%Þ ¼½1 ðSample OD Blank 2 ODÞ= ðControl OD Blank 1 ODÞ 100; where OD is absorbance at 405 nm.
Results and discussion A phytochemical study on the EtOAc-soluble extract of the grains of S. bicolor using an in vitro TT assay and repeated column chromatographic separation yielded twelve flavonoid glycosides 1–12 (Fig. 2). Compounds 1 and 4 were isolated as yellow powder. Their 1H and 13C NMR spectra revealed an identical flavonol skeleton bearing a sugar moiety. The ESI–MS spectrum of compound 4 presented the ion [M ? H]? at 465, and the molecular formula of C21H20O12 was inferred from 1H and 13C NMR revealing a quercetin aglycone and one glucose sugar unit in the molecule. The b-configuration of the glucose was drawn from a coupling constant of the anomeric proton (5.04, 1H, d, J = 7.6 Hz). Thus, compound 4 was identified as quercetin-3-O-b-D-glucopyranoside, which was identical to the published data (Wu et al. 2008). All 1H, 13C, and MS data of compound 1 were identical with those of 4. Further analysis of the HMBC experiment revealed the correlations between the anomeric proton at dH 5.04 (1H, d, J = 7.6 Hz, H-100 ) and C–7 (164.6). Therefore, compound 1 was identified as quercetin-7-O-b-D-glucoside (Markham, 1982). The 1H and 13C NMR spectra of compounds 2 and 3 were identical with those of 1 and 4, except only for an additional aromatic singlet-proton at dH 6.50 (1H, s) that is assignable for H-3 of the flavones (Lu and Yeap Foo 2000; Orhan et al. 2012). A detailed comparison of their 1H and 13 C NMR data with published literatures led us to identify the structures of 2 and 3 as luteolin-7-O-b-D-glucoside and luteolin-30 -O-b-D-glucoside, respectively (Orhan et al. 2012). Compounds 5–8 were also isolated as yellow powder, their 1H NMR spectra indicated that they were derivatives of 3-O-flavanones (Agrawal et al. 1980) (flavanonol type) with proton signals at dH at 4.88–4.92 (1H, d, J = 11.2 Hz, H-2) and 4.50–4.55 (1H, d, J = 11.2 Hz, H-3), and two corresponding carbons at dC 85.0–85.4 (C-2), 73.6-73.8
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(C-3). The 1H and 13C-NMR data of compound 6 are in good agreement with that reported for dihydro-quercetin (taxifolin) with an ABX system at dH 6.89 (1H, d, J = 1.6 Hz, H-20 ), 6.79 (1H, dd, J = 2, 8, H-60 ) and 6.76 (1H, d, J = 8.2 Hz, H-50 ); this is due to a 30 ,40 -disubstitution of ring B and a typical meta-coupled pattern for H-6 and H-8 protons (d 6.15 and 6.13, d, J = 2.0 Hz). The negative ESI–MS of compound 6 gave a quasi-molecular ion peak [M-H]- at m/z 301.10, which is compatible with the molecular formula C15H12O7. The 13C-NMR spectra of compounds 5, 7, and 9 gave 21 carbons, of which 15 carbons were consistent to aglycone and the remaining 6 carbon signals were of b-D-glucoside units [dC 101.9 (C100 ), 73.8 (C-200 ), 78.8 (C-300 ), 71.4 (C-400 ), 79.5 (C-500 ), 62.6 (C-600 )]. A detailed analysis of their HMBC experiments along with the comparison of their 1H, 13C NMR, and MS data with those of published literatures (Fossen et al. 1998; Shen and Theander 1985, Slimestad et al. 1994), led to identify the structures of compounds 5, 7, and 8 to be taxifolin-7-O-b-D-glucoside, taxifolin-30 -O-b-D-glucoside, and aromadendrin-3-O-b-D-glucoside, respectively. The 1H and 13C NMR spectra of compound 9 showed to have a flavanone aglycone with dH 5.22 (1H, dd, J = 12.0, 2.8 Hz, H-2), 3.17 (1H, dd, J = 12.0, 16.0 Hz, H-3eq), and 2.74 (1H, dd, J = 2.8, 16.0 Hz, H-3ax), with corresponding carbons at dC 80.1 (C-2), 42.5 (C-3), and a conjugated carbonyl carbon at dC 197.6 (C-4) (Hu et al. 2012). In addition, a b-D-glucopyranoside moiety was observed [dH 4.68 (d, J = 7.2 Hz, H-100 ), 3.39-3.87 (6H, m, H-200 to H-600 ), and dC 101.8 (C-100 ), 74.9 (C-200 ), 78.0 (C-300 ), 71.5 (C-400 ), 78.5 (C-500 ), 62.6 (C-600 )]. In addition, the 1H-NMR spectrum of compound 9 presented three singlet protons at dH 6.04 (1H, d, J = 2.0 Hz, H-6) and 6.33 (1H, d, J = 2.0 Hz, H-8),6.83 (1H, br, s, H-40 ), a doublet proton at dH 6.70 (2H, d, J = 0.8 Hz, H-20 and H-60 ), which disclosed a 5,7,30 ,50 -tetrahydroxy-dihydropyrone core of a flavanone aglycone. A detailed comparison with literature data led to the identification of compound 9 to be 5,7,30 ,50 tetrahydroxy-flavanone-7-O-b-D-glucoside (Moretti et al. 1998). Compound 10 was also isolated as a flavanone glucoside. The 1H and 13C NMR data of 10 were identical with those of 9; detailed comparison with the reported data (Pan et al. 2008), led to the identification of compound 10 to be eriodictyol-7-O-b-D-glucoside. It was known that different retention behavior of flavanone diastereomers in HPLC with the RP-C18 column was primarily affected by a different stereochemistry at C-2 (Li et al. 2007). Compounds 11 and 12 were isolated as stereoisomers with differences at (2R) and (2S)-configurations. The 1H-1H coupling constants between H-2/H-3a (12.4 Hz) and H-3a/H-3b (16.8 Hz) of compound 12 were different with those of compound 11 (11.2 Hz and 15.7 Hz, respectively). Compounds 11 and 12 contained one chiral
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Fig. 3 Inhibitory effect of isolated compounds (1–12) from S. bicolor on TT activity assay
P.-H. Nguyen et al.
Re la tive Throm bin Tim e (%)
1400
% inhibition
Fig. 4 Inhibitory effect of isolated compounds (1–12) from S. bicolor on aglucosidase activity assay
250% 200% 150% 100% 50% 0%
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
center at C-2 position, which possessed either R or S configurations according to the stereochemical environment around the C-2. The absolute configuration of a flavanone compound can be presumed from the sign of its circular dichroism (CD) (Slade et al. 2005). The absolute configuration at C-2 of compound 11 was inferred to be R by the CD spectrum, which had positive Cotton effects near 250 and 270 nm, and negative Cotton effects near 310 nm. Whereas compound 12 possessed positive Cotton effects near 300 and 310 nm, and negative Cotton effects near 250 and 270 nm, in its CD spectrum, indicating S configuration for compound 12. Thus, compounds 11 and 12 were identified as (2R)-eriodictyol-5-O-b-D-glucoside and (2S)eriodictyol-5-O-b-D-glucoside, respectively (Hu et al. 2012). The interaction between platelets and blood vessels is important in the development of thrombosis and cardiovascular diseases (Gorden 1981). Uncontrolled platelet aggregation is critical in arterial thrombosis, leading to ischemia and may cause life-threatening disorders, such as
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Thrombin Time assay
α-glucosidase activity
heart attacks and stroke. Hence, in the treatment and prevention of these cardiovascular diseases the inhibition of thrombus formation is of fundamental importance. To assess the antithrombotic effects, the anticoagulant action of isolated compounds 1–12 was determined using an in vitro TT assay, and the results are presented in Fig. 3. Among them, luteolin-30 -O-b-D-glucoside (3) possessed the most potency with 197 % inhibition stronger than aspirin (positive control, 181 % inhibition) at the same concentration of 500 lg/mL. The following eriodictyol-7O-b-D-glucoside (10), quercetin-3-O-b-D-glucoside (4) and taxifolin-30 -O-b-D-glucoside (7) displayed 158, 152 and 120 % inhibition, respectively, whereas aspirin possessed only 138 % inhibition at 375 lg/mL. It is well known that a-glucosidase is an exo-type carbohydrase, which catalyzes the release of a-D-glucose from the non-reducing end of oligosaccharide and disaccharides (Frandsen and Svensson 1998). Because small intestinal aglucosidase is crucial for dietary carbohydrate digestion, it has been proposed that a-glucosidase inhibitors can retard
Antithrombotic and antidiabetic flavonoid glycosides
carbohydrate digestion and glucose absorption (Bertozzi and Kiessling 2001); therefore, it may be useful in the treatment of carbohydrate-mediated diseases, such as diabetes and obesity. Interestingly, at the concentration of 100 lg/mL, all compounds showed inhibitory effects on aglucosidase activity with an inhibition percentage at around 40 % comparable with the positive control (acarbose) having 38.7 % inhibition at the same concentration (Fig. 4). Compound 3, which had the most potency possessed 85.2 % inhibition, was followed by compounds 10 (65.2 %), 4 (55.7 %) and 7 (44.9 %). S. bicolor is an important food crop in semiarid parts of Africa and Asia. It has been increasingly used as an ‘‘ancient grain’’ and gluten free food ingredient in the United States. This growth in popularity is mainly due to agronomic advantages, such as high drought tolerance, high yields, low cost and potential health benefits including slow starch digestibility, cardiovascular disease reduction, antioxidant activity, anti-inflammatory and anti-carcinogenic properties (Barros et al. 2012). Our finding clearly demonstrates the effects of S. bicolor on cardiovascular disease reduction and its components which are responsible for the activity. Thus, we may suggest that S. bicolor and its flavonoid-enrich extracts could be considered as supplemental and or functional foods having beneficial effects against blood coagulation-induced ischemia, possibly thromboembolism disease, as well as obesity. Acknowledgments This work was supported by Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (Grant #110133-3), and the BK 21 Plus Program of the Ministry of Education, Science and Technology, Korea. The authors are grateful to S. H. Kim and collaborators at the Korea Basic Science Institute (Daegu) for measuring the mass spectra. Conflict of interest The authors declare no any actual and potential conflict of interest including any financial, personal or other relationships.
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