Chemicals and Materials

All solvents that were used in HSCCC separation were of analytical grade. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). AB-8 macroporous resin was purchased from the Chemical Plant of Nankai University (Tianjing, China).

The whole plant of O. falcata was collected from Qinghai Lake of Qinghai Province, China, in August 2017. The plant was identified by Professor Dejun Zhang (Qinghai University). A voucher herbarium specimen was deposited at Northwest Institute of Plateau Biology (Xining, China) with the voucher number 20180809-O.

Apparatus

The HSCCC system was equipped with 3 polytetrafluoroethylene preparative coils (total volume = 280 mL). The system was equipped with a pump, a UV detector, a constant temperature-circulating implement, a sample loop, and a model N2000 workstation.

HPLC analysis was performed on an Agilent 1200 system (Agilent Technologies Co. Ltd., USA). The Agilent 1200 system was equipped with a pump, a DAD detector, a column thermostat, and an Agilent HPLC workstation.

A Bruker Avance 600 MHz (Bruker, Karlsruhe, Germany) spectrometer was used for the NMR experiments, and tetramethylsilane was used as the internal standard.

Preparation of Crude Sample

The whole fresh plant was air-dried in the shade at room temperature and ground to a fine powder. The dried powder (1 kg) was extracted with 75% ethanol under reflux three times (2 h each time). All extracting solution were combined and concentrated. The process produced 210 g crude extract. The crude extract was then suspended in distilled water and extracted with petroleum ether and ethyl acetate, sequentially, which yielded 48.8 g of the petroleum ether fraction, 102.4 g of the ethyl acetate fraction, and 57.8 g of the water fraction.

The 57.8 g water fraction was separated by AB-8 column chromatography (8 cm × 120 cm, 4.0 kg AB-8 macroporous resin). Ethanol solutions (20%, 40%, 60%, and 80%) were used consecutively to elute the resin until eluent was nearly colorless. All elution fractions were collected for further research, and 7.2 g 80% ethanol fraction was collected. The 80% fraction was used for HSCCC separation. Figure 2 summarizes the separation process of anticancer compounds from O. falcata.

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The procedure of screening and preparative isolation of compounds from O. falcata (7-hydroxyflavonone 1, 5,7-dihydroxy-4′-methoxy flavonol 2, 5,7-dihydroxyflavanone 3, 2′,4′-dihydroxychalcone 4, 2′,4′-dihydroxydihydrochalcone 5)

Citation: Acta Chromatographica Acta Chromatographica 32, 3; 10.1556/1326.2019.00672

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Determination of Anticancer Activity

The in vitro anticancer activities of the samples on SMMC-7721 were determined by the MTT-based colorimetric method [16]. Briefly, 2 × 10⁴ cells per well were plated in 200 μL of medium in 96-well plates and incubated for 24 h. Then, the medium was removed, and fresh growth medium containing different concentrations of the test samples was added to the cells. After 24 h, the growth medium was removed, the MTT reagent (0.2 mg/mL) was added, and the cells were incubated for 4 h. The medium was then discarded, and the intracellular formazan product was dissolved in 150 μL DMSO. The absorbance of each well was measured at 570 nm using an ELISA reader. Cell viability was expressed as a percentage of the cell viability of control cells.

HSCCC Separation

The selection of the two-phase solvent system was conducted according to the partition coefficient (K) of the target compounds. In the present study, the K value was determined by HPLC as follows. The crude sample powder was dissolved into an appropriate amount of a pre-equilibrated two-phase solvent system and placed into a separation funnel. The funnel was shaken vigorously to equilibrate the sample between the two phases. Then, 2 mL of each phase was evaporated to dryness. The residue was dissolved in 1 mL of methanol and subsequently analyzed by HPLC. The K value was defined as the peak area of component in the upper phase divided by the peak area of component in the lower phase.

The two-phase solvent system used here was composed of hexane–ethyl acetate–methanol–water (10:4:10:10, v/v/v/v). Each solvent was added to a separation funnel and thoroughly equilibrated at room temperature. The upper phase and lower phase were then separated and degassed via ultrasonic bath for 30 min before use. Sample solutions for HSCCC separation were prepared as follows: 200 mg of the sample was dissolved in 10 mL of the lower phase.

For each of the separation processes, the column was firstly filled entirely with the lower phase (stationary phase). Then, the mobile phase was pumped into the column at a flow rate of 1.8 mL/min. This occurred while the apparatus was rotated at 800 rpm.

After the solvent front emerged and the system established a steady-state hydrodynamic equilibrium, the sample solution was injected into the separation column. After the sample injection occurred, data were immediately collected. Fractions were collected according to the chromatogram. They were then evaporated under reduced pressure. Residues were dissolved in methanol in preparation for purity analysis by HPLC.

HPLC Analysis and Identification of Compounds

An ODS C₁₈ column (4.6 × 250 mm, 5 μm) was used to analyze the sample and each fraction of HSCCC. The HPLC mobile phases were H₂O for solvent A and CH₃OH for solvent B. They were eluted from 60% B to 65% B in 5 min, followed by 75% B in 30 min. The flow rate was 1.0 mL/min, and the detection wavelength was 254 nm. Figure 3 shows the HPLC analysis of 80% fraction.

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HPLC analysis of 80% ethanol elution and the compounds 1, 2, 3, 4, and 5. Conditions: column, ODS C₁₈ column (250 mm × 4.6 mm i.d., 5 μm); column temperature, 25 °C; mobile phase, H₂O (solvent A) and CH₃OH (solvent B); HPLC analysis, 0–5 min, linear gradient from 60 to 65% B; 5–35 min, linear gradient from 65 to 75% B; flow rate, 1.0 mL/min; detection wavelength, 254 nm

Citation: Acta Chromatographica Acta Chromatographica 32, 3; 10.1556/1326.2019.00672

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Screening of Anticancer Fraction

In order to find anticancer fraction of O. falcata, the anticancer activity was estimated with the MTT test. The anticancer activities of petroleum ether, ethyl acetate, water extract, 20% ethanol fraction, 40% ethanol fraction, 60% ethanol fraction, and 80% ethanol fraction were evaluated in vitro. The result showed that the 80% ethanol fraction decreased the viability of SMMC-7721 cells. Based on the anticancer activity, 80% ethanol fraction was treated by HSCCC to obtain active compounds.

Selection of HSCCC Conditions

A successful HSCCC separation depends on the selection of a suitable two-phase solvent system. The suitability of the system depends on the following conditions [17, 18]: (1) the settling time of the solvent system should be less than 20 s; (2) K of the compound should fall within a suitable range (0.2 < K < 5); and (3) the separation factor between the 2 compounds should be greater than 1.2.

We optimized the solvent system for HSCCC separation considering the above-stated principles. A series of common solvent systems based on ethyl acetate–methanol–water, n-butanol–ethyl acetate–water, and hexane–ethyl acetate–methanol–water were tested by changing the volume ratio of solvents to obtain the optimum conditions that could provide suitable K values. The measured K values are shown in Table 1. The solvent systems with ethyl acetate–methanol–water and n-butanol–ethyl acetate–water were tested first; large K values were obtained. The hexane–ethyl acetate–methanol–water (10:10:10:10, 8:10:10:10, and 6:10:10:10, v/v/v/v) were further studied, it was found that the compounds were mainly distributed in one phase, and good results could not be obtained. When the ratio of hexane–ethyl acetate–methanol–water was fixed at 10:8:10:10, and the K of the compound was good, but the separation factor was not good. Therefore, the ratio of ethyl acetate was decreased, and when the ratio of hexane–ethyl acetate–methanol–water was fixed at 10:4:10:10, the K of the compounds and the separation factor were good. After consideration, the solvent system of hexane–ethyl acetate–methanol–water (10:4:10:10, v/v/v/v) was finally selected for the HSCCC separation.

Under the optimized conditions, good separation results were obtained (Figure 4). In only one round of HSCCC separation, 5 fractions (A–E) were obtained in 260 min: 32.7 mg A, 34.9 mg B, 28.5 mg C, 26.7 mg D, and 22.6 mg E. The HPLC analysis of each fractions revealed that the purities of these 5 compounds (1–5) were 97.7%, 98.1%, 98.3%, 99.0%, and 98.3%, respectively (Figure 3).

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HSCCC chromatogram of 80% ethanol elution using the n-hexane–ethyl acetate–methanol–water solvent system with a volume ratio of 10:4:10:10. Conditions: stationary phase, upper phase; flow rate, 1.8 mL/min; revolution speed, 800 rpm; detection wavelength, 254 nm; retention of the stationary phase, 62%

Citation: Acta Chromatographica Acta Chromatographica 32, 3; 10.1556/1326.2019.00672

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Structural Identification

The chemical structures of the separated compounds were identified according to their ¹H and ¹³C NMR spectra and compared with the literature data. The compounds were identified as follows.

Fraction A: 7-hydroxyflavonone 1, C₁₅H₁₀O₄, ¹H NMR (500 MHz, CD₃OD) δ 7.73 (1H, d, J = 8.7 Hz, H-4), 7.49 (2H, d, J = 7.5 Hz, H-2′,6′), 7.40 (2H, t, J = 7.5 Hz, H-3′, 5′), 7.35 (1H, d, J = 7.5 Hz, H-4′), 6.51 (1H, dd, J = 8.7,1.6 Hz, H-6), 6.39 (1H, d, J = 1.6 Hz, H-8), 5.48 (1H, dd, J = 12.9, 2.3 Hz, H-2), 3.02 (1H, dd, J = 16.8, 13.0 Hz, H-3a), 2.75 (1H, dd, J = 16.8, 2.7 Hz, H-3b); ¹³C NMR (151 MHz, CD₃OD) δ 193.0 (C-4), 166.8(C-7), 165.4 (C-8a), 140.7 (C-1′), 129.9 (C-3′,5′), 129.7 (C-4′), 129.6 (C-5), 127.3 (C-2′,6′), 115.0 (C-4a), 111.9 (C-6), 103.8 (C-8), 81.0 (C-2), 45.1 (C-3). Compared with the reported data, the ¹H NMR and ¹³C NMR data are in agreement with that of 7-hydroxyflavonone in the literature [19].

Fraction B: 5,7-dihydroxy-4′-methoxy flavonol 2, C₁₅H₁₀O₄, ¹H NMR (500 MHz, CD₃OD) δ 8.02 (2H, d, J = 8.5 Hz, H-2′,6′), 6.81 (2H, d, J = 8.5 Hz, H-3′,5′), 6.49 (1H, s, H-6), 6.20 (1H, s, H-8), 3.81 (3H, s, -OCH₃); 13C NMR (151 MHz, CD₃OD) δ 177.5 (C-4), 167.0 (C-7), 162.2 (C-5), 160.7 (C-4′), 158.2 (C-8a), 146.9 (C-2), 137.5 (C-3), 130.8 (C-2′,6′), 123.6 (C-1′), 116.3 (C-3′,5′), 105.4 (C-4a), 98.6 (C-6), 95.7 (C-8), 92.71, 56.4 (-OCH₃). Compared with the reported data, the ¹H NMR and ¹³C NMR data are in agreement with that of 5,7-dihydroxy-4′-methoxy flavonol in the literature [20].

Fraction C: 5,7-dihydroxyflavanone 3, C₁₅H₁₂O₄,¹H NMR (500 MHz, CD₃OD) δ 7.44 (2H, d, J = 7.4 Hz, H-2′,6′), 7.36 (2H, t, J = 7.4 Hz, H-3′,5′), 7.32 (2H, t, J = 7.4 Hz, H-4′), 5.89 (1H, s, H-6), 5.86 (1H, s, H-8), 5.39 (1H, dd, J = 12.6, 3.6 Hz, H-2), 3.03 (1H, dd, J = 17.0,12.6 Hz, H-3a), 2.71 (1H, dd, J = 17.0, 2.6 Hz, H-3b); ¹³C NMR (151 MHz, CD₃OD) δ 197.2 (C-4), 168.8 (C-7), 165.5 (C-5), 164.6 (C-8a), 140.4 (C-1′), 129.7 (C-2′,6′), 129.6 (C-4′), 127.3 (C-3′,5′), 103.2 (C-4a), 97.2 (C-6), 96.3 (C-8), 80.4 (C-2), 44.1 (C-3). Compared with the reported data, the ¹H NMR and ¹³C NMR data are in agreement with that of 5, 7-dihydroxyflavanone in the literature [21].

Fraction D: 2′,4′ –dihydroxychalcone 4, C₁₅H₁₁O₃, ¹H NMR (500 MHz, CD₃OD) δ 7.88 (1H, d, J = 8.9 Hz, H-9), 7.71 (2H, m, H-1,2), 7.32 (2H, d, J = 6.7 Hz, H-2′,6′), 7.33 (3H, m, H-3′,4′,5′), 6.32 (1H, dd, J = 8.7, 1.5 Hz, H-8), 6.18 (1H, d, J = 1.5 Hz, H-6); ¹³C NMR (151 MHz, CD₃OD) δ 193.1 (C-3), 167.8 (C-5), 167.7 (C-7), 144.9 (C-1), 136.4 (C-1′), 133.6 (C-9), 131.6 (C-4′), 130.0 (C-(C-2′,6′), 129.6 (C-3′,5′), 121.9 (C-2), 114.4 (C-4), 109.7(C-8), 103.9 (C-6). Compared with the reported data, the ¹H NMR and ¹³C NMR data are in agreement with that of 2′,4′-dihydroxychalcone in the literature [10].

Fraction E: 2′,4′-dihydroxydihydrochalcone 5, C₁₅H₁₃O₃, ¹H NMR (500 MHz, CD₃OD) δ 7.61 (1H, d, J = 8.8 Hz, H-9), 7.16 (4H, m, H-2′,3′,5′,6′), 7.06 (1H, m, H-4′), 6.23 (1H, dd, J = 8.8,1.9 Hz, H-8), 6.14 (1H, d, J = 1.9 Hz, H-6), 3.13 (2H, t, J = 7.6 Hz, H-1), 2.90 (2H, t, J = 7.6 Hz, H-2); ¹³C NMR (151 MHz, CD₃OD) δ 205.2 (C-3), 166.4 (C-5), 166.4 (C-7), 142.5 (C-1′), 133.6 (C-8), 129.4 (C-3′,4′,5′), 127.1 (2′,6′), 114.0 (C-4), 109.1 (C-7), 103.6 (C-6), 40.5 (C-2), 31.6 (C-1). Compared with the reported data, the ¹H NMR and ¹³C NMR data are in agreement with that of 2′, 4′-dihydroxychalcone in the literature [22].

Anticancer Activities of Compounds

All compounds exhibited dose-dependent activities. The IC₅₀ values were 213.45 μg/mL, 197.74 μg/mL, 375.16 μg/mL, 17.44 μg/mL, and 136.83 μg/mL for compounds 1–5, respectively. Compounds 4 and 5 exhibited the higher anticancer activities against the cancer cells compared to the positive control (p < 0.05). In this study, 5 compounds isolated from O. falcata showed notable anticancer activities, which strongly demonstrated that all these compounds may be partially responsible for O. falcata's anticancer activity [6–8].