Abstract
Elsholtzia densa Benth. var. densa (Lamiaceae) is a famous medicinal herb which has been widely used for treatment of colds, headaches, pharyngitis, fever, diarrhea, digestion disorder, rheumatic arthritis, nephritises, and nyctalopia in China. In this study, fraction of the ethyl alcohol extract of E. densa (aerial part) by different polarity solvents indicated that the ethyl acetate soluble fraction exhibited a potent 1,1-diphenyl-2-picryhydrazyl (DPPH) radical scavenging activity with the IC50 value of 148.2 μg/mL. Under the target guidance of DPPH experiment, isoquercitrin, trachelogenin, ethyl caffeate, and arctigenin were separated with purities 95.98%, 92.98%, 96.07%, and 88.83%, respectively, by a dual-mode high-speed counter-current chromatography (HSCCC) method using n-hexane–ethyl acetate–methanol–water (4.5:5:3:4, v/v/v/v) as the solvent system. In order to evaluate the scientific basis, antioxidant activity of four isolated compounds was assessed by the radical scavenging effect on DPPH radical; isoquercitrin and ethyl caffeate showed stronger antioxidant activities with IC50 values of 9.4 μg/mL and 9.2 μg/mL, respectively, while trachelogenin and arctigenin showed weak antioxidant activities with IC50 values of >500 μg/mL and 72.8 μg/mL, respectively. Results of the present study indicated that the combinative method using DPPH antioxidant assay and dual-mode HSCCC could be widely applied for rapid screening and isolating of antioxidants from complex traditional Chinese medicine extract.
Introduction
Elsholtzia densa Benth. var. densa (E. densa) belongs to the genus of Elsholtzia Willd from the Lamiaceae and is a traditional Tibetan medicine plant, distributed mainly in the high altitude area of Tibet, Qinghai, Sichuan, and Yunnan province of China [1, 2]. Previous phytochemical and pharmacological studies have demonstrated that flavonoid glycosides and hydroxycinnamic acids are the main components of the plant [3–9]. It has many bioactivities, such as antibacterial, antifungal, antiviral, analgesic, and sedative activities [9–12]. The antioxidant activity and α-glucosidase inhibitory effects of the five sub-fractions of E. densa crude extract were recently studied, and the ethyl acetate sub-fraction and petroleum ether sub-fraction showed the strongest activity, respectively [13]. In addition, we know that antioxidants have received a great amount of attention as being primary preventive ingredients against various diseases [14]. Therefore, the ethyl acetate fraction might be a good candidate for further research and development, so we performed a detailed target-guided purification and separation on ethyl acetate extract of E. densa [15].
The preparative separation and purification of complex plant extracts by traditional methods including silica-gel column chromatography [16, 17] and gel filtration chromatography [18, 19] are time-consuming, require high amounts of organic solvents, typically require numerous chromatographic steps resulting lower recovery and higher cost, and often lead to loss of activity due to dilution effects or thermal decomposition especially for antioxidants [20]. In order to avoid the abovementioned problems, simple, rapid, and effective methods to screen and purify potential antioxidants from complex extract are imperative. As for preparative method, high-speed counter-current chromatography (HSCCC) is an optimal choice, which is a solid support-free liquid–liquid partition chromatography, eliminating the irreversible adsorption of sample onto the solid support matrix that is usually encountered in conventional column chromatography [21–23]. Dual-mode HSCCC where the phase role is reversed during the separation is investigated here; it ensures elution of all the injected species from the column while the separation is still progressing after phase reversal. Dual-mode operation provides a unique way to reduce analysis time and to optimize separation. Especially, this method can elute compounds with a wide range of polarities from the column [24, 25].
The present paper describes an efficient method by coupling of DPPH radical scavenging activity and dual-mode HSCCC experiments to screen and purify antioxidant from E. densa EtOAc-soluble fraction. Fractions and four compounds antioxidant activities were estimated. Three flavonoid glycosides (isoquercitrin [I], trachelogenin [II], and arctigenin [IV]) and ethyl caffeate (III) (Figure 1) were target-guided purified by a dual-mode elution mode HSCCC method from E. densa. This study has provided a method on screening and simultaneous separation of four antioxidant compounds from E. densa by DPPH radical scavenging activity and dual-mode HSCCC.
Chemical structures of isoquercitrin (I), trachelogenin (II), ethyl caffeate (III), and arctigenin (IV)
Citation: Acta Chromatographica Acta Chromatographica 30, 3; 10.1556/1326.2017.00192
Experimental
Materials and Reagents
All solvents used for sample preparation and HSCCC separation were of analytical grade (Baishi Chemical Reagent Factory, Tianjin, China). Methanol used for HPLC was of chromatographic grade (Yuwang Chemical Reagent Factory, Shandong, China). Ultrapure water (18.25 MΩ) used in the present work was purified on a UPT-I-10 L system (Chengdu Ultrapure Technology Co., Ltd., Chengdu, China).
Ninety-six-well plates (costal 3590, corning, USA) were used in the antioxidant activity experiments. Rutin (Must Biotech Co., Ltd., Chengdu, China) as reference standards was bought for DPPH radical scavenging activity assays at a purities ≥98%.
Apparatus
The HSCCC separation was performed on a TBE-300C HSCCC instrument (Tauto Biotech Co. Ltd., Shanghai, China). The apparatus was equipped with three preparative coils connected in series (diameter of polytetrafluoroethylene [PTFE] tube = 1.9 mm; total volume = 320 mL, including the 300 mL separation volume and a 20 mL sample loop). The revolution speed of the instrument was adjustable, ranging from 0 to 1000 rpm. The system was also equipped with two TBP5002 constant flow pumps (Tauto Biotechnique Company), a UV2000D detector model (Shanghai Sanotac Scientific Instrument Co., Ltd., Shanghai, China), and a DC0506 low constant temperature bath (Tauto Biotechnique Company). EasyChrom-1000 chromatography workstation (Shanghai Sanotac Scientific Instrument Co., Ltd.) was employed to record the chromatograms.
High-performance liquid chromatography (HPLC) analysis was performed using an Agilent 1260 HPLC system, equipped with a quaternary pump (G1311A), an auto-sampler (G1329B), a thermostated column compartment (G1316A), a diode array detector (G1315B), a Zorbax Eclipse XDB-C18 analytical column (4.6 × 250 mm,5 μm), and an HPLC workstation.
The nuclear magnetic resonance (NMR) spectrometer used in this study was a Varian INOVA 600 NMR spectrometer (Varian Inc., Palo Alto, CA, USA).
Multi-well plate readers (Enspire 2300, Perkin Elmer, USA) were used in the antioxidant activity experiments.
Sample Preparation
The plant that was dried under shade at room temperature was collected from Huangzhong Country, QingHai, China in November 2013 and was positively identified by Professor Xuefeng Lu (Northwest Institute of Plateau Biology, Chinese Academy of Sciences; Xining, China).
The aerial part (4.5 kg) of E. densa was powdered and extracted for 2 h with 70% ethanol (9 L × 3) under reflux. All filtrates were combined and concentrated by rotary evaporation at 60 °C under reduced pressure, producing 480 g of crude extract. A 300 g of crude extract was dissolved in water (500 mL) and respectively partitioned with petroleum ether (PE), ethyl acetate (EtOAc), and n-butanol separately (nBuOH) and produced the 11.32 g PE-, 15.78 g EtOAc-, and 56.66 g n-BuOH-soluble fractions. Those fractions were carefully collected and concentrated by rotary evaporator.
Evaluation of Antioxidant Activity
The DPPH radical assay was performed as described [26]. DPPH was bought from Sigma-Aldrich (Nanjing Aoduofuni Biotech Co., Ltd., Nanjing, China), and DPPH radical solutions were freshly prepared in methanol every day and kept protected from light. The free radical scavenging efficiency of the fractions and isolated compounds was determined by discoloration of the DPPH radical. In brief, 10 μL of diluted sample (4 mg/mL two-fold dilution to 0.0625 mg/mL) was mixed with 190 μL DPPH methanol solution (0.2 mmol/mL) to a final volume of 200 μL. The solution of DPPH was served as a control. The absorbance was performed once at 517 nm after the mixture was kept in dark room to protect from light and incubated at 37 °C for 30 min. The antioxidant activity is expressed as percentage of DPPH radical elimination calculated according to the following formula: [(Ablank − Asample)/Ablank] × 100%, where Ablank is the absorbance of the DPPH radical solution and Asample is the absorbance of the DPPH radical solution after the addition of the sample. Sample concentration providing 50% inhibition (IC50) was calculated from the graph plotting scavenging effect. All tests were run in triplicate, and the average value was calculated.
Selection of Two-Phase Solvent System
Based on the chemical properties of target compounds, a series of two-phase solvent systems consisting of n-hexane–ethyl acetate–methanol–water was tried and tested by changing the volume ratios of the four solvents to obtain the optimum condition. The K value was confirmed using HPLC as follows. A suitable amount of crude sample was added into a series of pre-equilibrated two-phase solvent systems, and the system was then fully shaken to reach the partition equilibrium. Subsequently, the same volumes of upper and lower phase were each evaporated to dryness. The residues were then dissolved into 2 mL methanol and analyzed by HPLC. The K value was defined as the peak area of the target compound in the stationary phase divided by the peak area of the target compound in the mobile phase.
Preparation of Two-Phase Solvent System and Sample Solution
The selected two-phase solvent system was respectively prepared by adding all the solvents into a separation funnel at selected volume ratios. Each test was thoroughly equilibrated by shaking repeatedly in a separation funnel at room temperature. After being equilibrated, the upper phase and the lower phase were separated and degassed by sonication for about 30 min and cooled to room temperature before to use. The two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (4.5:5:3:4, v/v/v/v) was used for the HSCCC separation. The sample solution for the HSCCC was prepared by dissolving 200 mg of the EtOAc-soluble fraction in 10 mL the solvent system n-hexane–ethyl acetate–methanol–water (4.5:5:3:4, v/v/v/v).
HSCCC Separation Procedure
The first HSCCC separation run was carried out with a two-phase solvent composed of n-hexane–ethyl acetate–methanol–water at the volume ratio of 4.5:5:3:4 in the tail-head elution mode (the lower phase as the stationary phase). Prior to the introduction of the stationary phase to the column, the column was flushed with methanol to wash out any remaining materials. The coil column was entirely filled with the lower phase, and then apparatus was rotated at 950 rpm. After 30 min, the upper phase was pumped into the column at a flow rate of 3 mL/min. When the hydrodynamic equilibrium was established in the column, 10 mL two-phase solvent solution containing 200 mg of the EtOAc-soluble fraction was injected through the injection ring.
After 270 min, the inlet and outlet of the column were switched. The second HSCCC separation run was employed for peak1 and peak 2. The solvent system still was used, but the elution mode was changed to the head-to-tail elution mode (the upper phase as the stationary phase) in the second HSCCC separation. After the separation was completed, the apparatus was stopped and the retention of the stationary phase was measured by collecting the column contents by forcing them out of the column with pressurized air. The separation temperature was controlled at 25 °C. The eluents from the outlet of the column were continuously monitored with an ultraviolet (UV) detector at 280 nm. Each peak fraction was manually collected according to the elution profile and evaporated under vacuum. Residues were dissolved in methanol for purity analysis by HPLC.
HPLC Analysis and Identification of HSCCC Peak Fractions
Agilent 1260 HPLC system was used to analyze the EtOAc-soluble fraction and the HSCCC peak fractions. In order to achieve a best separation baseline, different mobile phase and elution model were optimized. The mobile phase was composed of water (solvent A) and methanol (solvent B). A gradient elution program was performed as follows: 0 min, 30% B; 30 min, 70% B; and 35 min, 70% B. The flow rate was 1.0 mL/min, the column temperature was 30 °C, and the detection wavelength was 280 nm.
Identification of HSCCC fractions was carried out by 1H-NMR and 13C-NMR.
Statistical Analyses
The results are expressed as the mean ± SEM. All statistical analyses were performed using GraphPad Prism 6.0 statistical software.
Results and Discussion
Screening Antioxidants by DPPH Radical Scavenging Activity Assays
Solvent extraction is usually used for isolating antioxidants from traditional Chinese medicine extract, and antioxidant activity of extracts is strongly dependent on the solvent [27]. Solvents with different polarities were used to fractionate crude ethyl alcohol extract of E. densa. The PE, EtOAc, and n-BuOH fractions were then evaluated by DPPH radical scavenging activity. The EtOAc fraction showed potent capacity to scavenge DPPH radical compared with the IC50 value of 148.2 μg/mL (Table 1). The result implies that there are antioxidants present in the ethyl acetate fraction, and then HSCCC experiments were applied to screen and isolate them.
Antioxidant activities of fractionations of different polarities in DPPH assay
| Samples | DPPH (IC50, μg/mL)a |
|---|---|
| PE fraction | 1000 |
| EtOAc fraction | 148.2 ± 0.5 |
| n-BuOH fraction | >1000 |
| Rutinb | 12.8 ± 0.04 |
Each value in the table is the mean ± standard deviation (n = 3). The IC50 value was calculated by GraphPad Prism 5.0 software.
Used as control.
HPLC Analysis
The EtOAc-soluble fraction (Figure 2) and HSCCC peak fractions (Figure 3) were analyzed by Agilent Eclipse XDB-C18 column (5 μm, 150 × 4.6 mm).
HPLC chromatogram of the EtOAc-soluble fraction from the E. densa, where peak 1 is isoquercitrin, peak 2 is impurity peak, peak 3 is trachelogenin, peak 4 is ethyl caffeate, and peak 5 is arctigenin. Conditions: Eclipse XDB-C18 column (5mm, 4.6 × 250 mm); mobile phase: methanol and water in a gradient (0–30 min, 30%–70% methanol); flow rate: 1.0 mL/min; column temperature: 30 °C; detection wavelength: 280 nm
Citation: Acta Chromatographica Acta Chromatographica 30, 3; 10.1556/1326.2017.00192
UV–DAD spectra of four targeted compounds purified by HSCCC (I: isoquercitrin, II: trachelogenin, III: ethyl caffeate, and IV: arctigenin). Conditions: Eclipse XDB-C18 column (5 mm, 4.6 × 250 mm); mobile phase: methanol and water in a gradient (0–30 min, 30%–70% methanol; 30–35 min, 70% methanol); flow rate: 1.0 mL/min; column temperature: 30 °C; detection wavelength: 280 nm
Citation: Acta Chromatographica Acta Chromatographica 30, 3; 10.1556/1326.2017.00192
HSCCC Separation
HSCCC with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (4.5:5:3:4, v/v/v/v) is performed using a dual-mode method. The HSCCC chromatogram was shown in Figure 4. Four compounds were obtained in one step separation within 6 h, which yield 25.8 mg of isoquercitrin (I) at purify of 95.98%, 8.2 mg trachelogenin (II) at purify of 92.98%, 6.9 mg ethyl caffeate (III) at 96.07%, and 5.4 mg arctigenin (IV) at purify of 88.83% based on the HPLC analysis. However, peak 2 which is an impurity peak cannot be got pure compound to be identified.
HSCCC chromatogram of peaks 1, 2, 3, 4, and 5 from EtOAc-soluble fraction of E. densa, where peak 1 is isoquercitrin (I), peak 2 is impurity peak, peak 3 is trachelogenin (II), peak 4 is ethyl caffeate (III), and peak 5 is arctigenin (IV). Two-phase solvent system consisted of n-hexane–ethyl acetate–methanol–water 4.5:5:3:4 (v/v/v/v). The stationary phase was the upper phase; the mobile phase was the lower phase. Separation conditions: flow rate, 1.5 mL/min; revolution speed, 950 rpm; detection wavelength, 280 nm; sample size, 200 mg of crude sample dissolved in 10 mL of the upper phase; separation temperature, 25 °C
Citation: Acta Chromatographica Acta Chromatographica 30, 3; 10.1556/1326.2017.00192
Structural Identification
The identification of the HSCCC fractions was confirmed by 1H-NMR and 13C-NMR. The detailed data were as follows.
Compound I was obtained as yellowish needles from methanol and was analyzed to have the molecular formula C21H20O12. Its NMR data (Table 2) were in accordance with data listed in the literature [28], and compound I was identified as isoquercitrin.
Antioxidant activities of fractionations of different polarities and isolated compounds in DPPH assay
| Samples | DPPH (IC50, μg/mL)a |
|---|---|
| Isoquercitrin | 9.4 ± 0.03 |
| Trachelogenin | 481.9 ± 0.73 |
| Ethyl caffeate | 9.2 ± 0.02 |
| Arctigenin | 72.8 ± 0.3 |
| Rutinb | 12.8 ± 0.04 |
Each value in the table is the mean ± standard deviation (n = 3). The IC50 value was calculated by GraphPad Prism 5.0 software.
Used as control.
Compound II was obtained as a pale yellow gum and was analyzed to have the molecular formula C21H24O7. Its NMR data (Table 2) were in accordance with data listed in the literature [3], and compound II was identified as trachelogenin.
Compound III was obtained as light yellow powder and was analyzed to have the molecular formula C11H12O4. Its NMR data are as follows: 1H-NMR (600 MHz, CD3OD-d4) δ: 7.56 (1H, d, J = 15.9 Hz), 7.05 (1H, dd, J = 1.5 Hz), 6.96 (1H, d, J = 8.2 Hz, H-3), 6.27 (1H, d, J = 15.9 Hz, H-8), 4.24 (2H, q, J = 7.1 Hz), 1.33 (3H, t, J = 7.1 Hz). 13C-NMR (600 MHz, CD3OD-d4) δ: 169.48, 149.79, 147.01, 146.89, 127.84, 123.03, 116.65, 115.38, 115.22, 61.55, and 14.78. Its NMR data were in accordance with data listed in the literature [21], and compound III was identified as ethyl caffeate.
Compound IV was obtained as colorless crystal and was analyzed to have the molecular formula C21H24O6. Its NMR data (Table 2) were in accordance with data listed in the literature [5], and compound IV was identified as arctigenin.
Antioxidant Activities of Target-Isolated Compounds
Antioxidant activities of target-isolated compounds from E. densa ethyl acetate fraction were measured spectrophotometrically by DPPH radical scavenging activity assay in comparison with rutin as standard antioxidant. As shown in Table 2, compound I and compound III exhibited effective antioxidant activity against DPPH with IC50 values of 9.4 μg/mL and 9.2 μg/mL, respectively, which were stronger than the positive control, rutin with IC50 value of 12.8 μg/mL, and while compound IV exhibited weak antioxidant activity with IC50 values of 72.8 μg/mL and compound II showed a weakest DPPH scavenging activities up to 500 μg/mL.
Selection of Solvent System and Other HSCCC Conditions
HSCCC is a very useful and efficient technique for separation and purification of the crude solvent extracts from natural products. However, the selection of the two-phase solvent system is the key step in HSCCC separation. A successful separation of target compounds using HSCCC requires a careful search for a suitable two-phase solvent system that could provide an ideal range of partition coefficients (K). K values in the range of 0.2 to 5.0 are generally considered to be appropriate for HSCCC separation, and the separation factor between the two components (α = K2/K1, K2 > K1) should be greater than 1.5 [22, 23].
In the present paper, a commonly used solvent system composed of n-hexane–ethyl acetate–methanol–water was selected as a basic system. According to the K values and the separation factors, it can be seen that the two-phase solvent system used here of n-hexane–ethyl acetate–methanol–water (4.5:5:3:4, v/v/v/v) was most suitable for the separation of the target compounds. The K values of the EtOAc-soluble fraction in different two-phase solvent systems were measured and summarized in Tables 3 and 4.
1H-NMR and 13C-NMR Data (600 MHz, J in Hz, and δ in ppm) of compounds I (in CD3OD-d4) and II and IV (in DMSO-d6)
| Position | Compound I (DMSO-d6) | Compound II (CD3OD-d4) | Compound IV (DMSO-d6) | |||
|---|---|---|---|---|---|---|
| δ C | δ H | δ C | δ H | δ C | δ H | |
| 1 | 133.58 | 131.23 | ||||
| 2 | 156.34 | 114.04 | 112.33 | |||
| 3 | 133.30 | 149.30 | 148.63 | |||
| 4 | 177.41 | 150.71 | 147.27 | |||
| 5 | 161.23 | 113.37 | 111.81 | 6.73 | ||
| 6 | 98.73 | 6.19 | 122.35 | 120.34 | 6.58 | |
| 7 | 164.38 | 32.38 | 3.11, 2.81 | 36.87 | 2.48 | |
| 8 | 93.54 | 6.40 | 44.70 | 2.79 | 40.79 | |
| 9 | 156.14 | 71.95 | 70.67 | |||
| 10 | 103.90 | |||||
| 1′ | 121.60 | 128.37 | 128.85 | |||
| 2′ | 115.22 | 7.58 | 115.12 | 113.41 | 6.67 | |
| 3′ | 144.83 | 149.00 | 147.38 | |||
| 4′ | 148.50 | 146.88 | 145.08 | |||
| 5′ | 116.20 | 6.85 | 116.24 | 115.26 | 6.82 | |
| 6′ | 121.16 | 7.58 | 124.23 | 121.48 | 6.63 | |
| 7′ | 42.04 | 3.35, 3.14 | 33.70 | 2.82 | ||
| 8′ | 77.56 | 45.64 | 2.48 | |||
| 9′ | 180.71 | 178.43 | 3.88, 4.07 | |||
| 3-Glc | ||||||
| 1 | 100.88 | |||||
| 2 | 74.11 | |||||
| 3 | 76.52 | |||||
| 4 | 69.94 | |||||
| 5 | 77.58 | |||||
| 6 | 60.98 | |||||
| 3-OMe | 56.71 | 3.99 | 55.31 | 3.60 | ||
| 4-OMe | 56.59 | 3.98 | 55.35 | 3.60 | ||
| 3′-OMe | 56.71 | 3.97 | 55.47 | 3.75 | ||
| 8′-OH | 5.35 | |||||
K values of target peaks measured in different HEMWat solvent systems (the upper phase as the stationary phase)
| Volume ratio | Distribution coefficient (K) | ||||
|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | |
| 1:1:1:1 | 22.86 | 13.99 | 1.61 | 1.02 | 0.95 |
| 4:5:3:4 | 51.84 | 27.29 | 1.38 | 0.64 | 0.76 |
| 4.5:5:3:4 | 33.60 | 14.36 | 1.18 | 0.39 | 0.63 |
| 5:5:3:4 | 46.24 | 23.75 | 1.77 | 1.11 | 0.88 |
| 5:5.5:3:4 | 28.37 | 17.73 | 1.60 | 0.76 | 0.86 |
| 5:6:3:4 | 35.70 | 16.59 | 0.55 | 0.20 | 0.26 |
A two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (1:1:1:1, v/v/v/v) was tested initially, and good K values could be obtained. However, it was impossible to separate peak 4 and peak 5; the separation factor used for this system was 1.07 (α0). When the volume ratio was changed to 4:5:3:4 (v/v/v/v), the problem persisted, but the separation factor was increased from 1.07 to 1.18. Therefore, increasing the proportion of n-hexane or ethyl acetate was expected to improve the separation factor of peak 4 and peak 5. When we tested the 5:5.5:3:4 and 5:6:3:4 (v/v/v/v) ratios, unfortunately, the separation factor was approximately equal to α0. When we test the 4.5:5:3:4 and 5:5:3:4, we witnessed good results; α0 was 1.62 and 1.26. All things considered, four compounds could be separated and purified by applying the solvent system n-hexane–ethyl acetate–methanol–water (4.5:5:3:4, v/v/v/v). In addition, this method would take a long time because the K values of peak 1 and peak 2 (K1 = 33.60, K2 = 14.36) were big. Thus, dual-mode elution mode was the best method. As shown in Tables 3 and 4, the K1 > K2 > 10, the separation procedure was divided into two steps. The first step, the lower phase was the stationary phase; in the tail-head elution mode, peaks 3, 4, and 5 could be separated. The second step, the upper phase was the stationary phase; in the head-tail elution mode, peak 1 could be separated.
The result as shown in Tables 4 and 5 demonstrated that the solvent system of n-hexane–ethyl acetate–methanol–water (4.5:5:3:4, v/v/v/v), in dual-mode elution mode, could be well applied to separate and purify the compounds with a large range of K values from the EtOAc-soluble fraction.
K values of target peaks measured in different HEMWat solvent systems (the lower phase as the stationary phase)
| Volume ratio | Distribution coefficient (K) | ||||
|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | |
| 1:1:1:1 | – | – | 0.23 | 0.68 | 0.41 |
| 4:5:3:4 | 0.02 | 0.04 | 0.72 | 1.55 | 1.32 |
| 4.5:5:3:4 | 0.03 | 0.07 | 0.85 | 2.53 | 1.58 |
| 5:5:3:4 | 0.02 | 0.04 | 0.56 | 0.90 | 1.14 |
| 5:5.5:3:4 | 0.04 | 0.06 | 0.63 | 1.32 | 1.16 |
| 5:6:3:4 | 0.02 | 0.06 | 0.66 | 1.94 | 1.79 |
Advantages of Dual-Mode HSCCC
In recent years, HSCCC has been widely used for the separation of various natural products. Dual-mode HSCCC has many advantages in comparison to the conventional single-mode operation. Dual-mode HSCCC ensures elution of all the injected species from the column while, unlike backflush, the separation is still progressing after phase reversal [24]. The nature of the active compound present in natural product extracts is unknown, the dual mode HSCCC is well suited for performing assay-directed fractionations, since it does not involve solid-phase adsorbents and, thus, is inherently less destructive [29]. Last but not the least, this technique can be scaled up straightforwardly, applied toward the entire range of polarity of natural products, decrease the operating time, and reduce the use of organic reagents.
Scavenging DPPH free radical for the four compounds
In this study, the antioxidant activity of compounds was determined by DPPH. It is a stable radical in a methanol solution. The reduction of DPPH as indicated below is followed by monitoring the decrease in its absorbance at a characteristic wavelength during the reaction. In its radical form, DPPH absorb at 517 nm, but upon reduction by an antioxidant (AH) or a radical species (R), the absorption disappears [30].
DPPH˙ + AH → DPPH-H + A˙
DPPH˙ + R˙ → DPPH-R
Among the antioxidants, flavonoids are an important class. They contain a number of phenolic hydroxyl groups attached to ring structures, conferring the antioxidant activity [31]. Plant polyphenols are multifunctional and can act as reducing agents, hydrogen donating antioxidants, and singlet oxygen quenchers. The chemical properties of polyphenols in terms of the availability of the phenolic hydrogens as hydrogendonating radical scavengers predict their antioxidant activity [32]. Four compounds both contain the phenolic hydroxyl groups attached to ring structures. Therefore, they demonstrated antioxidant activity. The glycosylation of flavonoids reduces their activity when compared to the corresponding aglycones [33]. The glucose is attached to the C-3 of quercetrin in the isoquercitrin molecule, which results approximately the same antioxidant activity as ethyl caffeate. The unsaturation in the C ring of flavonoids is important and allows electron delocalization across the molecule for stabilization of the aryloxyl radical. Trachelogenin and arctigenin are open-loop structure, not allowing conjugation. This structure disadvantage confers a weakening of the IC50 value compared to the heterocyclic ring of isoquercitrin with approximately one eighth the antioxidant activity. Trachelogenin has an identical number of hydroxyl groups in the same positions as arctigenin but also contains the 8′-OH. This structural difference may have a significant influence on their antioxidant activity.
Conclusions
DPPH method followed by dual-mode HSCCC experiments was successively developed for the fast screening and purification of potent radical scavengers from E. densa EtOAc-soluble fraction. The best advantage of this method is that the active compounds can be screened by a simple and efficient DPPH radical scavenging activity assay, and then target-guided separation and purification can be operated by dual-mode HSCCC. The described method has a broad applicability and is rapid, robust, and suitable for fast screening and preparing radical scavengers from crude plant extracts.
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