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  • 1 Shanghai Normal University, Shanghai, 201418, P. R. China
  • 2 Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, 830011, P. R. China
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By application of preparative high-speed counter-current chromatography (HSCCC) to the crude quinolone alkaloids (1.1 g) from the fruit of Tetradium ruticarpum, 1-methyl-2((6Z,9Z)-pentadecadienyl)-4(1H)-quinolone (1, 8.4 mg), dihydroevocarpine (2, 27.0 mg), and 1-methyl-2-pentadecyl-4(1H)-quinolone (3, 18.8 mg) were isolated in one step with sufficient purity using the solvent system composed of hexane–ethyl acetate–methanol–water (Hex–EtOAc–MeOH–H2O, 5:2:5:3). Further purification of the subfraction was performed by amending the solvent composition and achieved another three quinolone alkaloids, i.e., 1-methyl-2-undecylquinolin-4(1H)-one (4, 13.7 mg), (Z)-1-methyl-2-(tridec-5-en-1-yl) quinolin-4(1H)-one (5, 14.0 mg) from subfraction FR3-A3-85 using Hex–EtOAc–MeOH–H2O (5:3.5:8.75:8.25), and 1-methyl-2-nonylquinolin-4(1H)-one (6, 15.1 mg) from subfraction FR3-A3-36 using Hex–EtOAc–MeOH–H2O (5:3.8:5:4.8). The relationship between the structure of the six alkaloids and their affinities for bovine serum albumin (BSA) was investigated using fluorescence titration analysis. The length and the presence of double bond of the side chain affected their binding process with BSA. The binding behavior might influence their other biological activities.

Abstract

By application of preparative high-speed counter-current chromatography (HSCCC) to the crude quinolone alkaloids (1.1 g) from the fruit of Tetradium ruticarpum, 1-methyl-2((6Z,9Z)-pentadecadienyl)-4(1H)-quinolone (1, 8.4 mg), dihydroevocarpine (2, 27.0 mg), and 1-methyl-2-pentadecyl-4(1H)-quinolone (3, 18.8 mg) were isolated in one step with sufficient purity using the solvent system composed of hexane–ethyl acetate–methanol–water (Hex–EtOAc–MeOH–H2O, 5:2:5:3). Further purification of the subfraction was performed by amending the solvent composition and achieved another three quinolone alkaloids, i.e., 1-methyl-2-undecylquinolin-4(1H)-one (4, 13.7 mg), (Z)-1-methyl-2-(tridec-5-en-1-yl) quinolin-4(1H)-one (5, 14.0 mg) from subfraction FR3-A3-85 using Hex–EtOAc–MeOH–H2O (5:3.5:8.75:8.25), and 1-methyl-2-nonylquinolin-4(1H)-one (6, 15.1 mg) from subfraction FR3-A3-36 using Hex–EtOAc–MeOH–H2O (5:3.8:5:4.8). The relationship between the structure of the six alkaloids and their affinities for bovine serum albumin (BSA) was investigated using fluorescence titration analysis. The length and the presence of double bond of the side chain affected their binding process with BSA. The binding behavior might influence their other biological activities.

1. Introduction

The fruit of Tetradium ruticarpum T. G. Hartley (formerly known as Euodia ruticarpa [A. Juss.] Benth., belonging to the family Rutaceae) has long been employed as a folk medicine in eastern Asian countries to kill pains under the name Wu-Zhu-Yu. It is listed now in Chinese Pharmacopoeia as a traditional drug to treat a variety of ailments including headache, partum hemorrhage, gastro-intestinal disorder, weakness and edema of the legs, etc. Previous intensive phytochemical isolations have discovered dozens of natural products from the plant, mainly limonoids [1], indole (with evodiamine and rutaecarpine as the major bioactive components) [2], and quinolone alkaloids [36]. Several studies demonstrated that quinolone alkaloids exhibit remarkable biological activities such as anti-inflammatory [7, 8], anti-bacteria [9], and anti-tumor growth [10, 11]. In this regard, synthetic preparation [12] and isolation of quinolone alkaloids from natural plants [1315] are still of general interest for drug development.

High-speed counter-current chromatography (HSCCC) represents a support-free liquid–liquid partition chromatographic technique [16]. Compared with conventional solid–liquid chromatography, HSCCC demonstrates significant advantages such as reduction of adsorptive loss, larger sample loading capacity, and excellent sample recovery. [17] Recently, HSCCC has been widely applied for the purification of natural flavonoids [1820], alkaloids [2123], polysaccharides [24, 25], etc.

Employing HSCCC in 2005, Liu et al. isolated only 3 quinolone alkaloids, together with the two major indole alkaloids (evodiamine and rutaecarpine) from the crude extract of T. ruticarpum [5]. To our best knowledge, pre-enrichment quinolone alkaloids would be better for the later isolation using HSCCC. Zhong et al. applied this strategy using high-performance counter-current chromatography (HPCCC) and obtained better isolation, with six quinolone compounds isolated in one step [6]. Nevertheless, HPCCC usually required higher instrumental techniques and is not commonly used. The technical parameters applied for HPCCC cannot directly be applied to HSCCC, which is now commercially available since several companies have produced their own instruments. Herein, we summarized our own efforts on purification of six natural quinolone alkaloids from T. ruticarpum (Figure 1) and evaluated their binding affinity for bovine serum albumin (BSA).

Figure 1.
Figure 1.

Chemical structure of isolated quinolone alkaloids

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00174

2. Experimental

2.1. Apparatus

HSCCC was performed using a model TBE300C high-speed counter-current chromatography (Tauto Biotech Co. Ltd., Shanghai, China). The apparatus with the maximum rotation speed of 1000 rpm was equipped with three polytetrafluoroethylene preparative coil (inner diameter [ID], 2.6 mm; total volume, 310 mL) and a 20 mL manual sample loop. The middling force invariableness flux pump TBP-5002 (Tauto Biotech Co. Ltd., Shanghai, China) was used to pump the two-phase solvent system, and a low-temperature thermostat bath (Tauto Biotech Co. Ltd., Shanghai, China) was used to control the separation temperature. The ultraviolet (UV) detector (Sanotac Scientific instruments Co, Ltd.) measured the UV absorbance, and the Easychrom-1000 (Hanbo Tec Co. Ltd., Jiangsu, China) was employed for data collection and analysis. The analytical high-performance liquid chromatography (HPLC) used was UltiMate 3000 system (Thermo Fisher, USA). Electrospray ionization (ESI) mass analyses were performed using Bruker mass spectrometer (Bruker, Switzerland). Nuclear magnetic resonance (NMR) experiments were carried out using a Bruker AVANCE 500 MHz NMR spectrometer (Bruker, Switzerland). Fluorescence spectra were recorded on a Varian Eclipse spectrofluorometer (Varian, USA).

2.2. Reagents and materials

All organic solvents used for HSCCC were of analytical grade and purchased from Runjie Chemical Co. Ltd. (Shanghai, China). The acetonitrile used for HPLC analysis was of chromatographic grade and purchased from Aladdin industrial Co. Ltd. (Shanghai, China). BSA was purchased from Sigma Co. Ltd. (MO, USA). The fruits of T. ruticarpum were purchased from Zhongyaogang, Yulin, Guangxi province, China. A voucher specimen is kept in the College of Life and Environmental Sciences, Shanghai Normal University.

2.3. Preparation of the crude sample

The fruits (0.5 kg) of T. ruticarpum were pulverized and extracted with 95% ethanol at reflux (3 × 600 mL). The total extracts were heated again and filtered immediately. The filtrate (FL1) was concentrated in vacuo to obtain around 400 mL of mixture, which was filtered again to yield filtrate. Analysis of both of fractions FR1 and FR2 confirmed that their main components are evodiamine and rutaecarpine. The concentrated filtrate (FL2) was extracted with chloroform, and around 20.0 g blown syrup (FR3) was obtained. The syrup (FR3) was subjected to separation over a silica gel column (200–300 mesh) and eluted with a solvent system of petroleum ether–ethyl acetate with a gradient polarity (4:1 to 1:2, then pure ethyl acetate) to yield 11 fractions, denoted as F1–F11. Fraction F9 (15.0 g) was chromatographed on a silica gel column (200–300 mesh) and eluted in with a solvent system petroleum ether–ethyl acetate = 1:2 to give two subfractions (FR3-A1 and FR3-A2). Finally, FR3-A2 (10.0 g) was further subjected to passage over a silica gel column (200–300 mesh) to yield FR3-A3 (3.60 g). The fraction FR3-A3 (1.1 g), which mainly contained quinolone alkaloids, was used for subsequent HSCCC separation (Figure 2 and 3).

Figure 2.
Figure 2.

Preparation procedure of the fraction FR3-A3 for HSCCC

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00174

Figure 3.
Figure 3.

HSCCC procedure of the fraction FR3-A3 (Hex = hexane, EtOAc = ethyl acetate)

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00174

2.4. Selection and preparation of two-phase solvent systems

The two-phase solvent system used was composed of hexane, ethyl acetate, methanol, and water. The designate solvent was poured together and shook rapidly, then thoroughly equilibrated at room temperature, and the two phases were separated. The system of Hex–EtOAc–MeOH–H2O (6:1.5:6:1.8, v/v), Hex–EtOAc–MeOH–H2O (5:2:5:3, v/v) was tested to a primary HSCCC separation with fraction FR3-A3, according to the K values in Table 1; the latter was selected for the primary separation. A series of two-phase solvent systems was tested to the subfractions from FR3-A3 (Table 2 for subfraction FR3-A3-85; Table 3 for subfraction FR3-A3-36). K values of the target compounds were measured by HPLC, which were expressed as the peak area of compounds in the upper phase divided by that in the lower phase.

Table 1.

The distribution constants (K values) of the target compounds in subfraction FR3-A3 at different biphasic solvent systems

Hexane–ethyl acetate–methanol–waterK value
123
6:1.5:6:1.80.961.243.94
5:2:5:33.724.119.80
Table 2.

The distribution constants (K values) of the target compounds in subfraction FR3-A3-85 at different biphasic solvent systems

Hexane–ethyl acetate–methanol–waterK valueα Value (KComp. 5 /KComp. 4)
45
5:4:5:57.3811.041.50
5:2:5:20.630.721.14
5:3.5:8.75:8.255.077.031.38
Table 3.

The distribution constants (K values) of the target compounds in subfraction FR3-A3-36 at different biphasic solvent systems

Hexane–ethyl acetate–methanol–waterK value
6
5:3:5:52.79
5:2:5:52.30
5:3.8:5:4.82.17

2.5. Separation procedure

For each separation, the multilayer column was entirely filled with the upper phase as stationary phase. Then, the lower phase as mobile phase was pumped into the column at a flow rate of 4 mL/min while the apparatus was run at a revolution speed of 815 rpm. After the mobile phase was eluted from the tail outlet, a hydrodynamic equilibrium system was established. Each sample solution was injected through the injection valve, and the mobile phase was pumped at 4 mL/min. Every fraction was collected manually according to the profile in chromatography workstation.

2.6. Purity determination of HSCCC fractions by HPLC analysis and identification of the isolated compounds

The total subfractions and purified fragments isolated by the HSCCC were analyzed with a Thermo Fisher C18 column at room temperature. Acetonitrile and water were used as the mobile phase in radiant elution mode as follows: acetonitrile, 0–1 min, 30%; 1–10 min, 30% to 70%; 10–30 min, 70% to 90%; 30–55 min, 90%; 55–60 min, 90% to 30%. The flow rate of the mobile phase was 0.8 mL/min. The effluent was monitored at 239 nm by a diode-array detector (DAD). Identification of the compounds was carried out by ESI–mass spectrometry (MS) spectrometry, using Bruker mass spectrometer (Bruker, Switzerland). NMR experiments were carried out using a Bruker Avance III 500M spectrometer with CDCl3 as solvent.

2.7. Fluorometric spectra experiments

A working solution of the isolated alkaloids (1.0 × 10−3 mol/L) was prepared by dissolving each alkaloid in ethanol. A Tris–HCl buffer (0.10 mol/L, pH = 7.4) containing 0.10 mol/L NaCl was selected to keep the pH value constant and to maintain the ionic strength of the solution. The working solution of BSA (1.0 × 10−6 mol/L) was prepared with the Tris–HCl buffer. Fluorescence spectra were measured with a Varian Eclipse spectrofluorophotometer, using 5 nm/5 nm slit widths. The excitation wavelength was 280 nm, and the emission was read at 290–500 nm.

Fluorometric experiments: 3.0 mL solution containing appropriate concentration of BSA (1.0 × 10−6 mol/L) was prepared in a quartz colorimetric utensil, and it was titrated by successive additions of a 1.0 × 10−3 mol/L stock solution of each isolated alkaloid to give a final concentration ranging from 3 to 27 × 10−6 mol/L. Titrations were done manually by using a micro injector, and then the fluorescence spectra were measured at room temperature. Each fluorescence intensity determination was repeated 3 times. Data from the fluorescence experiments were processed and described according to the binding constant formula:

logF0F/F=logKa+nlogQ
where F and F0 are current and initial fluorescence, respectively, Q is ligand concentration, Ka is the binding constant for the protein–ligand complex, and n is the number of binding sites per protein. After the fluorescence quenching, intensities on protein at 348 nm were measured.

3. Results and discussion

3.1. Pre-purification of quinolone alkaloids

It is well-known that the major alkaloids of the fruits of T. ruticarpum are evodiamine and rutaecarpine, both isolated first by Japanese scientists nearly a century ago [26]. Their content is much higher than the other components in this plant. For example, Shoji et al. isolated 6.17 g of evodiamine from 2.5 kg of fruits of T. ruticarpum by repetitive chromatography [27]. Although Liu et al. isolated evodiamine, rutaecarpine, and 3 quinolone alkaloids in a single step from the crude extract of T. ruticarpum [5], it would be advantageous to remove evodiamine and rutaecarpine before performance of HSCCC to enable enrichment of quinolone alkaloids.

As evodiamine and rutaecarpine are poorly dissolved in common organic solvents, it is assumed that recrystallization can be applied to their isolation. In fact, cooling of the ethanolic extract of T. ruticarpum yielded fraction FR1 as evodiamine (1.6 g). The filtrate FL1 was then concentrated and filtered again to remove fraction FR2 (around 11 g), which consisted mainly evodiamine and rutaecarpine. Thus, the second filtrate FL2 was nearly free of evodiamine and rutaecarpine, which benefited subsequent chromatographic enrichment of quinolone alkaloids. Finally, 3.6 g of total quinolone alkaloids (FR3-A3) was obtained, and 1.1 g of them was used in the present HSCCC purification.

3.2. Optimization of the two-phase solvent system

Some golden rules for selection of solvent system were summarized by Ito [28]. As a general rule, a successful HSCCC separation depends on a suitable two-phase solvent system that provides an ideal K value (usually between 0.5 and 2), but higher K value (up to 16) also was used in some cases [29]. Usually, Hex–EtOAc–MeOH–H2O solvent system is suitable for low to moderate polar compounds, and it was used often since it can easily be modified by amending the volume ratios of individual solvent. In the present study, two different ratios of Hex–EtOAc–MeOH–H2O were performed to determine the K value of quinolone alkaloids, which are moderate in terms of polarity. As indicated in Table 1, When Hex–EtOAc–MeOH–H2O (6:1.5:6:1.8) was used, although relatively lower K values were obtained for compounds 1–3, our pilot HSCCC separation failed to obtain good resolution. Therefore, Hex–EtOAc–MeOH–H2O (5:2:5:3) of higher K values was applied. Higher K value resulted in longer separation time, but it was still acceptable because of better isolation and purities of target compounds.

Three sets of ratio of Hex–EtOAc–MeOH–H2O solvent system were tried for the subfraction FR3-A3-85. Solvent system at a volume ratio of 5:4:5:5 gives K values higher than 7 with an α value (separation factor, Kcompound 5/Kcompound 4) of 1.50, while solvent system at a volume ratio of 5:2:5:2 gives low K values and the worse α value (1.14). Finally, solvent system at a volume ratio of 5:3.5:8.75:8.25 was applied to guarantee best separation with a suitable α value of 1.38 in an acceptable separation time. For the subfraction FR3-A3-36, the solvent system of lowest K value was chosen for HSCCC separation since this fraction consisted of less impurities than the other fraction. In summary, the optimal solvent compositions of each subfraction were listed in Table 4 according to the comparison of K values and α values.

Table 4.

The optimal solvent composition of each fraction

FractionOptimal solvent composition (Hex–EtOAc–MeOH–H2O)
FR3-A35:2:5:3
FR3-A3-855:3.5:8.75:8.25
FR3-A3-365:3.8:5:4.8

3.3. Preparative HSCCC separation

The HSCCC separation of subfraction FR3-A3 (1.1 g) was shown in Figure 4 by using the solvent system composed of Hex–EtOAc–MeOH–H2O (5:2:5:3), and the retention of stationary was 69.0%. Consequently, 1-methyl-2((6Z,9Z)-pentadecadienyl)-4(1H)-quinolone (1, 8.4 mg), dihydroevocarpine (2, 27.0 mg), and 1-methyl-2-pentadecyl-4(1H)-quinolone (3, 18.8 mg) were obtained in one step. However, two impure subfractions were collected, labeled as FR3-A3-85 and FR3-A3-36. The next HSCCC separations of above subfractions were shown in Figure 5 and 6, and three more compounds were obtained, i.e., 1-methyl-2-undecylquinolin-4(1H)-one (4, 13.7 mg) and (Z)-1-methyl-2-(tridec-5-en-1-yl) quinolin-4(1H)-one (5, 14.0 mg) from subfraction FR3-A3-85 using Hex–EtOAc–MeOH–H2O (5:3.5:8.75:8.25), and 1-methyl-2-nonylquinolin-4(1H)-one (6, 15.1 mg) from subfraction FR3-A3-36 using Hex–EtOAc–MeOH–H2O (5:3.8:5:4.8) (Figure 7).

Figure 4.
Figure 4.

HSCCC chromatogram of the fraction FR3-A3, solvent system: hexane–ethyl acetate–methanol–water (5:2:5:3); flow rate: 4 mL/min; revolution speed: 815 rpm; detection wavelength: 239 nm; retention percentage of the stationary phase: 69.0%, separation temperature: 30 °C

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00174

Figure 5.
Figure 5.

HSCCC chromatogram of the fraction FR3-A3-85, solvent system: hexane–ethyl acetate–methanol–water (5:3.5:8.75:8.25); flow rate: 3 mL/min; revolution speed: 815 rpm; detection wavelength: 239 nm; retention percentage of the stationary phase: 72.0%, separation temperature: 28 °C

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00174

Figure 6.
Figure 6.

HSCCC chromatogram of the fraction FR3-A3-36, solvent system: hexane–ethyl acetate–methanol–water (5:3.8:5:4.8); flow rate: 3 mL/min; revolution speed: 815 rpm; detection wavelength: 239 nm; retention percentage of the stationary phase: 64.0%, separation temperature: 26 °C

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00174

Figure 7.
Figure 7.

(A) HPLC chromatography of fraction FR3-A3 and each isolated compound thereof; (B) UV spectrum of each isolated compound at 239 nm. HPLC conditions: acetonitrile and water were used as the mobile phase in radiant elution mode as follows: acetonitrile, 0–1 min, 30%; 1–10 min, 30% to 70%; 10–30 min, 70% to 90%; 30–55 min, 90%; 55–60 min, 90% to 30%. The flow rate of the mobile phase was 0.8 mL/min. The effluent was monitored at 239 nm by a DAD detector. Purity: compound 1 (94.43%); compound 2 (95.94%); compound 3 (94.26%); compound 4 (97.93%); compound 5 (96.95%); compound 6 (96.20%).

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00174

3.4. Structural identification

The structural identification of isolated compounds was carried out by MS, 1H-NMR, and 13C-NMR.

Compound 1: colorless needle crystals, ESI–MS m/z 366.2797 [M + H]+, 1H-NMR (500 MHz, CDCl3) δ 8.43 (d, J = 8.0 Hz, 1H, 5-H), 7.64 (t, J = 7.7 Hz, 1H, 7-H), 7.49 (d, J = 8.6 Hz, 1H, 8-H), 7.36 (t, J = 7.4 Hz, 1H, 6-H), 6.21 (s, 1H, 3-H), 5.42–5.26 (m, 4H, 6′, 7′, 9′ 10′-H), 3.72 (s, 3H, N-CH3), 2.76 (t, J = 5.6 Hz, 2H, 1′-H), 2.73–2.65 (2.72, J = 7.8 Hz, 2H, 8′-H), 2.11–1.97 (m, 4H, 5′ & 11′-H), 1.73–1.63 (m, 2H), 1.50–1.38 (m, 4H), 1.37–1.19 (m, 6H), 0.87 (t, J = 6.7 Hz, 3H, 15′-H). 13C-NMR (125 MHz, CDCl3) δ 177.94 (4-C), 154.68 (2-C), 142.10 (8a-C), 132.13 (7-C), 130.49 (10′-C), 129.59 (6′-C), 128.67 (9′-C), 127.85 (7′-C), 126.81 (5-C), 126.72 (4a-C), 123.40 (6-C), 115.42 (8-C), 111.29 (3-C), 34.85 (1′-C), 34.21 (N-CH3), 31.63 (13′-C), 29.44 (4′-C), 29.04 (3′-C), 28.65 (2′-C), 27.34 (11′-C), 27.13 (5′-C), 25.77 (8′-C), 22.68 (14′-C), 14.17 (15′-C). Compared with the data given in refs. [1] and [5], compound 1 was identified as 1-methyl-2((6Z,9Z)- pentadecadienyl)-4(1H)-quinolone.

Compound 2, white solid, ESI–MS m/z 364.2615 [M + Na]+, 1H-NMR (500 MHz, CDCl3) δ 8.45 (dd, J = 8.0, 1.1 Hz, 1H, 5-H), 7.69–7.63 (dt, J = 8.0, 1.1 Hz, 1H, 7-H), 7.51 (d, J = 8.6 Hz, 1H, 8-H), 7.37 (t, J = 7.4 Hz, 1H, 6-H), 6.25 (s, 1H, 3-H), 3.74 (s, 3H, N-CH3), 2.72 (t, J = 7.8 Hz, 2H, 1′-H), 1.68 (quint, J = 7.6 Hz, 2H, 2′-H), 1.48–1.39 (m, 2H, 3′-H), 1.38–1.19 (m, 18H, H-4′~12′), 0.88 (t, J = 6.9 Hz, 3H, 13′-H). 13C-NMR (125 MHz, CDCl3) δ 178.03 (4-C), 154.87 (2-C), 142.17 (8a-C), 132.16 (7-C), 126.90 (5-C), 126.78 (4a-C), 123.45 (6-C), 115.41 (8-C), 111.39 (3-C), 34.95 (1′-C), 34.25 (N-CH3), 32.06 (11′-C), 29.80 (2′-C), 29.78 (3′ & 10′-C), 29.74 (4′-C), 29.62 (5′-C), 29.49 (6′-C), 29.47 (7′-C), 29.44 (8′-C), 28.77 (9′-C), (12′-C), 14.24 (13′-C). Compared with the data given in refs. [1], [3], and [12], compound 2 corresponded to dihydroevocarpine, i.e., 1-methyl-2- tridecylquinolin-4(1H)-one.

Compound 3, white solid, ESI–MS m/z 370.3123 [M + H]+, 1H-NMR (500 MHz, CDCl3) δ 8.45 (d, J = 7.9 Hz, 1H, 5-H), 7.66 (t, J = 7.7 Hz, 1H, 7-H), 7.51 (d, J = 8.6 Hz, 1H, 8-H), 7.38 (t, J = 7.4 Hz, 1H, 6-H), 6.25 (s, 1H, 3-H), 3.75 (s, 3H, N-CH3), 2.76–2.67 (m, 2H, 1′-H), 1.74–1.62 (m, 2H, 2′-H), 1.48–1.39 (m, 2H, 3′-H), 1.38–1.18 (m, 22H, 4′~14′′-H), 0.87 (t, J = 6.7 Hz, 3H, 15′-H). 13C-NMR (125 MHz, CDCl3) δ 178.02 (4-C), 154.88 (2-C), 142.17 (8a-C), 132.16 (7-C), 126.90 (5-C), 126.78 (4a-C), 123.45 (6-C), 115.41 (8-C), 111.38 (3-C), 34.94 (1′-C), 34.24 (N-CH3), 32.07 (13′-C), 29.83, 29.82, 29.80 (2 × CH2), 29.78, 29.73, 29.62, 29.49, 29.46, 29.44 (3′-C), 28.76 (2′-C), 22.82 (14′-C), 14.24 (15′-C). Compared with the data given in refs. [1] and [12], compound 3 corresponded to 1-methyl-2-pentadecyl-4(1H)-quinolone.

Compound 4, white solid, ESI–MS m/z 314.2480 [M + H]+, 1H-NMR (500 MHz, CDCl3) δ 8.44 (d, J = 8.0 Hz, 1H, 5-H), 7.65 (t, J = 7.8 Hz, 1H, 7-H), 7.50 (d, J = 8.6 Hz, 1H, 8-H), 7.36 (t, J = 7.5 Hz, 1H, 6-H), 6.23 (s, 1H, 3-H), 3.73 (s, 3H, N-CH3), 2.74–2.66 (m, 2H, 1′-H), 1.73–1.62 (m, 2H, 2′-H), 1.47–1.38 (m, 2H, 3′-H), 1.37–1.19 (m, 14H, 4′~10′-H), 0.87 (t, J = 6.8 Hz, 3H, 11′-H). 13C-NMR (125 MHz, CDCl3) δ 177.97 (4-C), 154.80 (2-C), 142.14 (8a-C), 132.12 (7-C), 126.85 (5-C), 126.76 (4a-C), 123.39 (6-C), 115.41 (8-C), 111.36 (3-C), 34.91 (1′-C), 34.22 (N-CH3), 32.02 (9′-C), 29.92 (8′-C), 29.72, 29.60, 29.45, 29.41, 28.72 (2′-C), 22.80 (10′-C), 14.23 (11′-C). Compared with the data given in ref. [1], compound 4 corresponded to 1-methyl-2-undecylquinolin-4(1H)-one.

Compound 5, white solid, ESI–MS m/z 340.2642 [M + H]+, 1H-NMR (500 MHz, CDCl3) δ 8.45 (d, J = 7.9 Hz, 1H, 5-H), 7.66 (t, J = 7.8 Hz, 1H, 7-H), 7.51 (d, J = 8.6 Hz, 1H, 8-H), 7.37 (t, J = 7.4 Hz, 1H, 6-H), 6.24 (s, 1H, 3-H), 5.40–5.30 (m, 2H, 8′ & 9′-H), 3.74 (s, 3H, N-CH3), 2.75–2.68 (m, 2H, 1′-H), 2.06–1.96 (m, 4H, 7′ & 10′-H), 1.77–1.64 (m, 2H, 2′-H), 1.49–1.39 (m, 2H, 3′-H), 1.40–1.22 (m, 10H, 4′~6′-H, 11′ & 12′-H), 0.89 (t, J = 6.3 Hz, 3H, 13′-H). 13C-NMR (125 MHz, CDCl3) δ 178.00 (4-C), 154.77 (2-C), 142.16 (8a-C), 132.15 (7-C), 130.22 (9′-C), 129.78 (8′-C), 126.91 (5-C), 126.79 (4a-C), 123.43 (6-C), 115.40 (8-C), 111.40 (3-C), 34.92 (1′-C), 34.23 (N-CH3), 32.10 (11′-C), 29.81 (5′-C), 29.39 (3′-C), 29.36 (4′-C), 29.23 (6′-C), 28.73 (2′-C), 27.27 (7′-C), 27.07 (10′-C), 22.48 (12′-C), 14.13 (13′-C). Compared with the data given in ref. [1], compound 5 corresponded to evocarpine, i.e., (Z)-1-methyl-2-(tridec-5-en-1-yl) quinolin-4(1H)-one.

Compound 6, white solid, ESI–MS m/z 286.2170 [M + H]+, 1H-NMR (500 MHz, CDCl3) δ 8.45 (d, J = 7.9 Hz, 1H, 5-H), 7.66 (t, J = 7.8 Hz, 1H, 7-H), 7.51 (d, J = 8.7 Hz, 1H, 8-H), 7.37 (t, J = 7.4 Hz, 1H, 6-H), 6.24 (s, 1H, 3-H), 3.74 (s, 3H, N-CH3), 3.48 (s, 2H), 2.75–2.67 (m, 2H, 1′-H), 1.71–1.64 (m, 2H, 2′-H), 1.47–1.39 (m, 2H, 3′-H), 1.37–1.20 (m, 10H, 4′~8′-H), 0.88 (t, J = 6.6 Hz, 3H, 9′-H). 13C-NMR (125 MHz, CDCl3) δ 178.02 (4-C), 154.91 (2-C), 142.14 (8a-C), 132.16 (7-C), 126.87 (5-C), 126.74 (4a-C), 123.45 (6-C), 115.42 (8-C), 111.34 (3-C), 34.93 (1′-C), 34.25 (N-CH3), 31.97 (7′-C), 29.56, 29.45, 29.42, 29.37, 28.74, 22.77 (8′-C), 14.20 (9′-C). Compared with the data given in ref. [3], compound 6 corresponded to 1-methyl-2-nonylquinolin-4(1H)-one.

3.5. Affinities for plasma protein assays

3.5.1 Effect of isolated compound on BSA Fluorescence Spectra

The affinities for plasma protein of the isolated compounds were evaluated with BSA. All of the six alkaloids decreased the fluorescence intensity of BSA distinctly, but the quenching efficiency of each compound was different in the same concentration gradient. Both of compounds 2 and 5 can quench BSA proportionally, but 5 quenched BSA fluorescence more strongly than 2 at the same concentration (Figure 8). The structural difference between compounds 2 and 5 might suggest that the existence of olefin on the side chain of quinolone alkaloids affected the quenching effect on BSA fluorescence. Furthermore, there were weak red shifts of λem for all tested compounds 2 (5 nm), 1 (7 nm), 3 (6 nm) etc., which means that the molecular conformation of the BSA was affected by the alkaloids isolated herein.

Figure 8.
Figure 8.

Quenching effect of 2 (left) and 5 (right) on BSA fluorescence. λex = 280 nm; BSA (1.0 × 10–6 mol/L); a–i, (3.0, 6.0, 9.0, 12.0, 15.0, 18.0, 21.0, 24.0, 27.0) × 10–6 mol/L

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00174

3.5.2 Binding constant and binding sites

Table 5 shows the binding constants (logKa) and the binding site values (n) between the alkaloids and BSA, and the relationship between logKa and n is shown in Figure 9. The values of logKa are proportional to the number of binding sites (n). It is obvious that the compounds 2, 3, and 6 with a saturated aliphatic side chain exhibit lower affinities for plasma protein. As shown in Table 5, both binding constants (logKa) and binding sites decrease after the double bond was hydrogenated (Entry 5 vs. Entry 2); therefore, it might indicate that hydrogenation of olefin in the carbon chain can weaken the affinity between those alkaloids and BSA. For the compounds with saturated aliphatic side chain, binding constants do not increase along with the increasing length of chain; optimal affinity was observed when the side chain consists of 11 carbons (compound 4).

Table 5.

Binding parameters for isolated compounds–BSA system

CompoundlogKanR
16.51821.35360.9944
24.44131.02390.9873
33.80570.92680.9943
45.47121.17480.9949
57.88641.66800.9652
64.47491.00550.9979
Figure 9.
Figure 9.

Relationship between logKa and the number of binding sites (n) between isolated compounds and BSA

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00174

3.5.3 Relationship between the affinities and the bioactivities of quinolone alkaloids

The in vitro antimycrobacterial and cytotoxic activities of the quinolone alkaloids from T. ruticarpum have been reported already, including the compounds isolated in the present work.

Wube et al. described that chain length and unsaturation of the side chain can affect the antibacterial activities of quinolone alkaloids [12]. In their research, compound 5 displayed a thirty-fold higher inhibitory effect on Mycobacterium smegmatis compared to compound 2. This is consistent with their value differences of binding constants and binding sites (Entry 5 vs. Entry 2, Table 5). Also, compound 1 showed a better activity against M. smegmatis than compound 3, in accordance with higher binding constant of compound 1. Among the quinolone alkaloids synthesized in their work, compound 5 exhibited more significant inhibitory effects on M. smegmatis than other compounds with shorter or longer chain. This is also in agreement with the higher binding constant and binding sites discovered herein. To sum up, it may suggest that higher binding affinities for BSA are associated with a better biological activity.

4. Conclusion

T. ruticarpum as one of the traditional Chinese medicines has attracted continuous research work of natural isolation, chemical synthesis, and biological evaluation. Previous study had confirmed that its major components are indole and quinolone alkaloids. In this study, by pre-treatment of the ethanolic extracts of T. ruticarpum using recrystallization, the indole alkaloids (mostly evodiamine and rutaecarpine) were removed while the quinolone alkaloids were enriched. By application of preparative HSCCC to the crude quinolone alkaloids, three quinolone alkaloids were obtained in one step with the solvent system composed of hexane–ethyl acetate–methanol–water (5:2:5:3). By amending the solvent composition, three more quinolone alkaloids were isolated from the sub-fractions. The affinities of the isolated compounds for BSA were investigated using fluorescence titration analysis. The compounds 2, 3, and 6 with a saturated aliphatic side chain exhibit lower affinities for plasma protein, while the compounds (1 and 5) with double bond show better affinities. Their affinities might affect biological activities in a positive way.

References

  • 1.

    Sugimoto, T.; Miyase, T.; Kuroyanagi, M.; Ueno, A. Chem. Pharm. Bull. 1988, 36, 4453.

  • 2.

    Son, J. K.; Chang, H. W.; Jahng, Y. Molecules 2015, 20, 10800.

  • 3.

    Tang, Y.-Q.; Feng, X.-Z.; Huang, L. Phytochemistry 1996, 43, 719.

  • 4.

    Hamasaki, N.; Ishii, E.; Tominaga, K.; Tezuka, Y.; Nagaoka, T.; Kadota, S.; Kuroki, T.; Yano, I. Microbiol. Immunol. 2000, 44, 9.

  • 5.

    Liu, R.; Chu, X.; Sun, A.; Kong, L. J. Chromatogr. A 2005, 1074, 139.

  • 6.

    Zhong, S.; Ye, H.; Peng, A.; Shi, J.; He, S.; Li, S.; Ye, X.; Tang, M.; Chen, L. Sep. Sci. Technol. 2011, 46, 869.

  • 7.

    Wang, T.-Y.; Wu, J.-B.; Hwang, T.-L.; Kuo, Y.-H.; Chen, J.-J. Chem. Biodiversity 2010, 7, 1828.

  • 8.

    Adams, M.; Kunert, O.; Haslinger, E.; Bauer, R. Planta Med. 2004, 70, 904.

  • 9.

    Tominaga, K.; Higuchi, K.; Hamasaki, N.; Hamaguchi, M.; Takashima, T.; Tanigawa, T.; Watanabe, T.; Fujiwara, Y.; Tezuka, Y.; Nagaoka, T.; Kadota, S.; Ishii, E.; Kobayashi, K.; Arakawa, T. J. Antimicrob. Chemother. 2002, 50, 547.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Paul, M.; Gafter-Gvili, A.; Fraser, A.; Leibovici, L. Eur. J. Clin. Microbiol. Infect. Dis. 2007, 26, 825.

  • 11.

    Sissi, C.; Palumbo, M. Cur. Med. Chem. - Anti-Cancer Agents 2003, 3, 439.

  • 12.

    Wube, A. A.; Hufner, A.; Thomaschitz, C.; Blunder, M.; Kollroser, M.; Bauer, R.; Bucar, F. Bioorg. Med. Chem. 2011, 19, 567.

  • 13.

    Huang, X.; Li, W.; Yang, X.-W. Fitoterapia 2012, 83, 709.

  • 14.

    Wang, X.-X.; Zan, K.; Shi, S.-P.; Zeng, K.-W.; Jiang, Y.; Guan, Y.; Xiao, C.-L.; Gao, H.-Y.; Wu, L.-J.; Tu, P.-F. Fitoterapia 2013, 89, 1.

  • 15.

    Zhao, N.; Li, Z. L.; Li, D. H.; Sun, Y. T.; Shan, D. T.; Bai, J.; Pei, Y. H.; Jing, Y. K.; Hua, H. M. Phytochemistry 2015, 109, 133.

  • 16.

    Huang, X.-Y.; Ignatova, S.; Hewitson, P.; Di, D.-L. Trends in Ana. Chem. 2016, 77, 214.

  • 17.

    Marston, A.; Hostettmann, K. J. Chromatogr. A 2006, 1112, 181.

  • 18.

    Chen, L.-J.; Games, D. E. J. Jones. J. Chromatogr. A 2003, 988, 95.

  • 19.

    Dai, X.; Huang, Q.; Zhou, B.; Gong, Z.; Liu, Z.; Shi, S. Food Chem. 2013, 139, 563.

  • 20.

    Ye, X.; Cao, D.; Song, F.; Fan, G.; Wu, F. Sep. Sci. Technol. 2016, 51, 807.

  • 21.

    Yuan, Z.; Xiao, X.; Li, G. J. Chromatogr. A 2013, 1317, 203.

  • 22.

    Li, J.; Gu, D.; Liu, Y.; Huang, F.; Yang, Y. Ind. Crops and Prod. 2013, 49, 160.

  • 23.

    Cruz, R. A.; Almeida, H.; Fernandes, C. P.; Joseph-Nathan, P.; Rocha, L.; Leitao, G. G. J. Sep. Sci. 2016, 39, 1273.

  • 24.

    Yang, W.; Wang, Y.; Li, X.; Carbohydr, P. Yu. Polym. 2015, 117, 1021.

  • 25.

    Zhou, X.-Y.; Zhang, J.; Xu, R.-P.; Ma, X.; Zhang, Z.-Q. J. Chromatogr. A 2014, 1362, 129.

  • 26.

    Asahina, Y.; Kashiwaki, K. J. Pharm. Soc. Jpn 1915, 405, 1273.

  • 27.

    Shoji, N.; Umeyama, A.; Takemoto, T.; Kajiwara, A.; Ohizumi, Y. J. Pharm. Sci. 1986, 75, 612.

  • 28.

    Ito, Y. J. Chromatogr. A 2005, 1065, 145.

  • 29.

    Friesen, J. B.; Pauli, G. F. J. Chromatogr. A 2007, 1151, 51.

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • 1.

    Sugimoto, T.; Miyase, T.; Kuroyanagi, M.; Ueno, A. Chem. Pharm. Bull. 1988, 36, 4453.

  • 2.

    Son, J. K.; Chang, H. W.; Jahng, Y. Molecules 2015, 20, 10800.

  • 3.

    Tang, Y.-Q.; Feng, X.-Z.; Huang, L. Phytochemistry 1996, 43, 719.

  • 4.

    Hamasaki, N.; Ishii, E.; Tominaga, K.; Tezuka, Y.; Nagaoka, T.; Kadota, S.; Kuroki, T.; Yano, I. Microbiol. Immunol. 2000, 44, 9.

  • 5.

    Liu, R.; Chu, X.; Sun, A.; Kong, L. J. Chromatogr. A 2005, 1074, 139.

  • 6.

    Zhong, S.; Ye, H.; Peng, A.; Shi, J.; He, S.; Li, S.; Ye, X.; Tang, M.; Chen, L. Sep. Sci. Technol. 2011, 46, 869.

  • 7.

    Wang, T.-Y.; Wu, J.-B.; Hwang, T.-L.; Kuo, Y.-H.; Chen, J.-J. Chem. Biodiversity 2010, 7, 1828.

  • 8.

    Adams, M.; Kunert, O.; Haslinger, E.; Bauer, R. Planta Med. 2004, 70, 904.

  • 9.

    Tominaga, K.; Higuchi, K.; Hamasaki, N.; Hamaguchi, M.; Takashima, T.; Tanigawa, T.; Watanabe, T.; Fujiwara, Y.; Tezuka, Y.; Nagaoka, T.; Kadota, S.; Ishii, E.; Kobayashi, K.; Arakawa, T. J. Antimicrob. Chemother. 2002, 50, 547.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Paul, M.; Gafter-Gvili, A.; Fraser, A.; Leibovici, L. Eur. J. Clin. Microbiol. Infect. Dis. 2007, 26, 825.

  • 11.

    Sissi, C.; Palumbo, M. Cur. Med. Chem. - Anti-Cancer Agents 2003, 3, 439.

  • 12.

    Wube, A. A.; Hufner, A.; Thomaschitz, C.; Blunder, M.; Kollroser, M.; Bauer, R.; Bucar, F. Bioorg. Med. Chem. 2011, 19, 567.

  • 13.

    Huang, X.; Li, W.; Yang, X.-W. Fitoterapia 2012, 83, 709.

  • 14.

    Wang, X.-X.; Zan, K.; Shi, S.-P.; Zeng, K.-W.; Jiang, Y.; Guan, Y.; Xiao, C.-L.; Gao, H.-Y.; Wu, L.-J.; Tu, P.-F. Fitoterapia 2013, 89, 1.

  • 15.

    Zhao, N.; Li, Z. L.; Li, D. H.; Sun, Y. T.; Shan, D. T.; Bai, J.; Pei, Y. H.; Jing, Y. K.; Hua, H. M. Phytochemistry 2015, 109, 133.

  • 16.

    Huang, X.-Y.; Ignatova, S.; Hewitson, P.; Di, D.-L. Trends in Ana. Chem. 2016, 77, 214.

  • 17.

    Marston, A.; Hostettmann, K. J. Chromatogr. A 2006, 1112, 181.

  • 18.

    Chen, L.-J.; Games, D. E. J. Jones. J. Chromatogr. A 2003, 988, 95.

  • 19.

    Dai, X.; Huang, Q.; Zhou, B.; Gong, Z.; Liu, Z.; Shi, S. Food Chem. 2013, 139, 563.

  • 20.

    Ye, X.; Cao, D.; Song, F.; Fan, G.; Wu, F. Sep. Sci. Technol. 2016, 51, 807.

  • 21.

    Yuan, Z.; Xiao, X.; Li, G. J. Chromatogr. A 2013, 1317, 203.

  • 22.

    Li, J.; Gu, D.; Liu, Y.; Huang, F.; Yang, Y. Ind. Crops and Prod. 2013, 49, 160.

  • 23.

    Cruz, R. A.; Almeida, H.; Fernandes, C. P.; Joseph-Nathan, P.; Rocha, L.; Leitao, G. G. J. Sep. Sci. 2016, 39, 1273.

  • 24.

    Yang, W.; Wang, Y.; Li, X.; Carbohydr, P. Yu. Polym. 2015, 117, 1021.

  • 25.

    Zhou, X.-Y.; Zhang, J.; Xu, R.-P.; Ma, X.; Zhang, Z.-Q. J. Chromatogr. A 2014, 1362, 129.

  • 26.

    Asahina, Y.; Kashiwaki, K. J. Pharm. Soc. Jpn 1915, 405, 1273.

  • 27.

    Shoji, N.; Umeyama, A.; Takemoto, T.; Kajiwara, A.; Ohizumi, Y. J. Pharm. Sci. 1986, 75, 612.

  • 28.

    Ito, Y. J. Chromatogr. A 2005, 1065, 145.

  • 29.

    Friesen, J. B.; Pauli, G. F. J. Chromatogr. A 2007, 1151, 51.

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