Rapid Screening and Preparative Isolation of Antioxidants from Alpinia officinarum Hance Using HSCCC Coupled with DPPH-HPLC Assay and Evaluation of Their Antioxidant Activities

An efficient method using high-speed counter-current chromatography (HSCCC) coupled with DPPH-HPLC assay has been developed for rapid screening and preparative isolation of antioxidants from ethyl acetate fraction of Alpinia officinarum Hance. Target-guided by DPPH-HPLC assay, two antioxidants, galangin and kaempferide, were targeted and further separated with purities of 99.3% and 98.5% by HSCCC using petroleum ether–ethyl acetate–methanol–water (0.8 : 1 : 1 : 0.8, v/v) as the solvent system. The antioxidant activities of galangin and kaempferide were further evaluated by measuring their inhibiting effects on superoxide anion radical, hydroxyl radical, and hydrogen peroxide in different luminol chemiluminescence (CL) systems. As a result, galangin and kaempferide both showed potent antioxidant activities. Results of the present study indicated that the combinative method by offline coupling DPPH-HPLC and HSCCC could be widely applied for rapid screening and isolation of antioxidants from complex TCM extract.


Introduction
Alpinia o cinarum Hance is a medicinal plant widely distributed in Guangdong and Hainan Province, China. Galangal, the dried rhizomes of A. o cinarum, is commonly used as both a food additive and a traditional Chinese medicine (TCM) for several centuries in China, because of its health-promoting properties to treat stomach ache, colds, and swelling [1]. Phytochemical investigations indicated that avonoids, glycosides, and diarylheptanoids are the three groups of important chemical constituents of A. o cinarum [2]. Further pharmacological research showed that A. ocinarum possessed a variety of activities such as antiin ammatory, antioxidant, antibacterial, antiparasitic, and anticancer activities [3][4][5]. Especially, the avonoids exhibited signi cant antioxidant activity and have attracted a great amount of attention [6].
However, the previous isolation of antioxidant avonoids from complex plant extracts was mainly by means of repeated column chromatography over silica gel, preparative HPLC, which was a time-consuming and labor process and often led to the loss of target antioxidant avonoids due to dilution e ects or decomposition in the procedure of isolation and puri cation [7][8][9]. us, it is necessary to develop an e cient screening and target-guided separation method to screen and separate the antioxidant avonoids in this plant. Recently, a useful method using HPLC coupled with DPPH assay has been successfully developed and applied to screen the antioxidants from complex plant extracts [10,11]. High-speed counter-current chromatography (HSCCC), being as a kind of liquid-liquid partition chromatography, is an optimal choice for the preparative method, which eliminates irreversible adsorption of samples on solid support in conventional column chromatography and o ers excellent recovery of target compounds [12,13]. It has been successfully applied to the separation and puri cation of di erent kinds of natural products [14,15]. Recently, HSCCC coupled with DPPH-HPLC experiment has been successfully applied to screen and isolate antioxidants from certain biological samples [16,17].
As part of our continuous search for the antioxidants from TCM, ethyl acetate fraction of A. o cinarum was investigated and showed considerable antioxidant activity under DPPH radical assay. For rapid screening and isolating the antioxidants from the ethyl acetate fraction of A. o cinarum, an e cient method using HSCCC coupled with DPPH-HPLC assay was developed. Firstly, the ethyl acetate fraction of A. o cinarum was screened by DPPH-HPLC assay, and two antioxidant avonoids were targeted. en, the two avonoids were target-guided puri ed using HSCCC and identi ed as galangin and kaempferide ( Figure 1) by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Finally, their antioxidant activities were evaluated by di erent luminol CL systems and DPPH radical assay. is is the rst report on rapid screening and preparative isolation of two antioxidants from A. o cinarum by HSCCC coupled with DPPH-HPLC assay.

Reagents and Materials.
Analytical grade solvents used for HSCCC were purchased from Tianjin Chemical Factory (Tianjin, China). Chromatographic grade acetonitrile was used for HPLC and purchased from Siyou Special Reagent Factory (Tianjin, China). All aqueous solutions were prepared with pure water produced by Milli-Q system (Millipore, USA). e rhizomes of A. o cinarum Hance were collected in Guangxi, China, and identi ed by Dr. Jia Li (College of Pharmacy, Shandong University of Traditional Chinese Medicine).

Apparatus.
A Model GS10A-2 HSCCC apparatus (Beijing Emilion Science & Technology Co., Beijing, China) with a 230 mL multilayer coil (diameter of 1.6 mm) and with a 20 mL sample loop was employed. e β value of the preparative column ranges from 0.5 at the internal to 0.8 at the external (β � r/R, where r is the distance from the coil to the holder shaft and R is the distance between the holder axis and central axis of the centrifuge). e HSCCC system was also equipped with a Model NS-1007 pump, a Model 8823A-UV Monitor, and a Yokogawa Model 3057 portable recorder.
HPLC was carried out on a Waters Empower system (Milford, MA, USA) including a Model 600 pump, a Model 600 multisolvent delivery system, a Model 996 diode-array detector (DAD), and an Empower workstation. e ESI-MS analyses were performed on an Agilent 1100/MSG1946 (Agilent, CA, USA). e NMR data were recorded on a Varian-600 MHz NMR spectrometer (Varian, Palo Alto, USA).

DPPH Radical Assay.
e antioxidant activities of the fractions, galangin and kaempferide, were evaluated by DPPH radical assay, which was performed as described in [18]. Analytical results are presented as x ± expanded uncertainty in Table 1 [19].

DPPH-HPLC Assay.
e DPPH-HPLC assay was performed as described in [17]. In brief, the ethyl acetate fraction of A. o cinarum (2 mg/ml in methanol, 2 mL) reacted with DPPH (0.26 mg/ml in methanol, 2 mL) for 30 min at room temperature, and then the mixture was subjected to HPLC analysis. e ethyl acetate fraction of A. o cinarum (4 mg/ml in methanol) was used as a control. e peaks which are reduced or disappeared after reaction will be targeted as potent antioxidants. e HPLC chromatographic separations were accomplished with an Inertsil-ODS-SP column (150 × 4.6 mm, 5 μm) at room temperature. e mobile phase consisted of A (0.2% acetic acid in water) and B (acetonitrile) with the gradient (0-5 min, 55-68% B; 5-15 min, 68% B; 15-30 min, 68-75% B) and the HPLC chromatography was performed at a ow rate of 1.0 ml/min. e e uent was monitored at 254 nm by a photodiode-array detector.

HSCCC Separation.
We followed the methods of Lei Fang et al. [20]. e K values were determined by HPLC as follows: Approximately 2 mg of crude extract was added to the test tube, and then 2 mL of each phase of the two-phase solvent system was added. After shaken violently for several minutes, an equal volume of each phase was analyzed by HPLC to obtain the partition coe cients. e K value was calculated according to the peak area of the compound in the upper phase divided by that in the lower phase. HSCCC separation was performed as follows: Firstly, the multiplayer coiled column was lled entirely with the stationary phase (upper organic phase). en, the lower aqueous phase was pumped into the column from the head end at a suitable ow rate of 2 ml/min, while the apparatus was rotated at a speed of 850 rpm. After hydrodynamic equilibrium was reached, the sample solution was injected into the column. A UV detector was applied to continuously monitor the e uent of the column at 254 nm. Each peak fraction was collected according to the elution pro le and analyzed by HPLC. e solvents in the column were nally pushed out by pressurized nitrogen gas, and retention of the stationary phase was measured after running.

Analysis and Identi cation of the Target Compound.
e target compound from the preparative HSCCC separation was analyzed by HPLC using the chromatographic separations as described in DPPH-HPLC assay and identi ed by electrospray ionization mass spectrometry (ESI-MS) on an Agilent 1100/MSG1946 (Agilent, California, USA) and 1 H and 13 C NMR spectra on a Varian-600 NMR spectrometer (Varian, Palo Alto, USA).

Antioxidant
Capacity Assay by Luminol Chemiluminescence.
e antioxidant activity of the target compound was evaluated by measuring the scavenging percentage of superoxide anion, hydrogen peroxide, and hydroxyl radical with luminol chemiluminescence according to the previous reference [21].
). 0.5 mL sample with di erent concentrations was added to 4 mL luminol solution, and the mixture was put into detection pool. After injection of 0.2 mL pyrogallol, the change of emission intensity was recorded as a function of time.

Hydrogen Peroxide (H 2 O 2 )
. 0.5 mL sample with different concentrations was added to 4 mL luminol solution, and the mixture was put into detection pool. After injection of 0.2 mL hydrogen peroxide, the change of emission intensity was recorded as a function of time.

Hydroxyl
Radical (HO · ). 0.5 mL sample with di erent concentrations and 0.2 mL ferrisulphas were added to 4 mL luminol solution, and the mixture was put into detection pool. After injection of 0.2 mL hydrogen peroxide, the change of emission intensity was recorded as a function of time.

Data Analysis.
e scavenging percentage of the target compound was calculated according to the following formula: [(PA Blank −PA Sample )/PA Blank ] × 100%. L-ascorbic acid was used as a reference compound, and 5% ethanol was used as the blank. e value of IC 50 was calculated by Origin 8.0 Version software from the graph plotting inhibition percentage. Analytical results are presented as x ± expanded uncertainty in Table 2 [19].

Quality Assurance and Quality Control (QA/QC).
In the DPPH radical assay and antioxidant capacity assay by luminol chemiluminescence, the samples were tested and analyzed together with one analytical blank and one reference compound as the QA/QC sample. Due to the high degree of automation in the above method, three sets were prepared and analyzed at the same time as one batch. e DPPH-HPLC assay served as a procedure of qualitative analysis in the study. For QA/QC purposes, measurement of a target analyte in a set of samples was considered valid only when the RSD of the peak area in the repeatability test must not deviate more than 2.0% (n � 3).

Screening Antioxidants by DPPH-HPLC Assay.
e DPPH-HPLC assay has been used for rapid screening of antioxidants from complex mixtures, especially for TCM extract. In the assay, the peak areas of antioxidants will decrease or disappear in the HPLC chromatogram after spiking with DPPH, while the peak areas for those without antioxidant activities will not change.
It is the rst and crucial step in DPPH-HPLC assay to choose optimal HPLC conditions which allow all compounds in the complex extract to be separated completely.
e HPLC conditions were carefully optimized, including di erent mobile phases with di erent elution modes, different detection wavelengths, and di erent ow rates. e results indicated that the optimal mobile phase consisted of A (0.2% acetic acid in water) and B (acetonitrile) with the gradient (0-5 min, 55-68% B; 5-15 min, 68% B; 15-30 min, 68-75% B). e ow rate was 1.0 ml/min, and 254 nm was selected as the detection wavelength. Under optimized HPLC conditions, the ethyl acetate extract of A. o cinarum was analyzed, and the analytical results are presented in Figure 3(a). e ethyl acetate fraction of A. o cinarum spiking with DPPH was analyzed by HPLC at 254 nm and is shown in Figure 3(b) in which peaks I and II almost disappeared after spiking with the DPPH solution, while peak areas of other compounds little changed. e results indicated that only peaks I and II possessed potent antioxidant activities. us, peaks I and II were targeted and further isolated by HSCCC.

Targeted Separation of Peak I by HSCCC.
e targeted separation of peaks I and II was performed by HSCCC. e selection of a suitable two-phase solvent system is the rst and critical step for a successful separation using HSCCC. e suitable solvent system should provide an ideal range of partition coe cient (K, 0.5-2) for the target antioxidant [13]. To our best knowledge, more than 60% of avonoid derivatives were isolated by HSCCC with the solvent system of petroleum ether-ethyl acetate-methanol-water [22]. en, several kinds of solvent systems based on petroleum ether-ethyl acetate-methanol-water were assessed, and the values of K for I and II were conducted by varying volume ratios. It can be found that the K values of 0.88 and 1.45 in the solvent systems with the volume ratio of 0.8 : 1 : 1 : 0.8 (v/v) were suitable for separation of the target compounds. As shown in Figure 4, good resolution and acceptable separation time could be obtained when petroleum etherethyl acetate-methanol-water (0.8 : 1 : 1 : 0.8, v/v) was used as the two-phase solvent system. e fractions of HSCCC were collected and analyzed by HPLC ( Figure 5). As a result, 107 mg of compound I and 29 mg of compound II with the purities of 99.3% and 98.5%, respectively, were separated and puri ed in one step by the preparative HSCCC from 270 mg of the ethyl acetate extract. e retention of the  stationary phase was 74.0%, and the separation time was within 5 h in each separation run. e HPLC chromatograms of crude extract and the pure compounds are shown in Figure 4. is is the rst report on screening and targeted isolation of the major antioxidant from A. o cinarum by DPPH-HPLC combined with HSCCC, which would give an excellent example to separate active compounds from complex mixtures.

Structual Identi cation of the Target-Separated
Antioxidants. e structural identi cation of the separated antioxidants was carried out by ESI-MS, 1 H NMR, and 13 C NMR spectra as follows: Compound   13  According to the literature, compound II was identi ed as kaempferide [23].

Antioxidant Activities of Target-Isolated Compounds.
e antioxidant activities of target-isolated compounds, galangin and kaempferide, from the ethyl acetate fraction of A. o cinarum were measured by luminol chemiluminescence and DPPH radical assay in comparison with rutin as positive antioxidant. As shown in Table 2, galangin and kaempferide showed inhibitory e ects on di erent luminol CL systems including superoxide anion radical, hydroxyl radical, and hydrogen peroxide. Meanwhile, galangin and kaempferide exhibited e ective antioxidant activities against DPPH with IC 50 values of 4.2 ± 0.03 μM and 7.8 ± 0.04 μM, respectively, which were in accordance with the DPPH-HPLC experiment.

Conclusions
In this paper, an e cient method using HSCCC combinative with DPPH-HPLC assay was successfully developed for rapid screening and preparative isolation of antioxidants from ethyl acetate fraction of A. o cinarum Hance. Using this method, two antioxidants, galangin and kaempferide, were rapidly targeted and further separated with purities of 99.3% and 98.5%, respectively, which also showed signi cant antioxidant activities by luminol chemiluminescence and DPPH radical assay. e innovative potential of the method is that the antioxidants in the complex extract can be rapidly screened from the complex extract by DPPH-HPLC assay and then targetguided puri ed by HSCCC. e results of this study indicate that HSCCC coupled with DPPH-HPLC assay has a broad applicability and is a very powerful and e ective method for rapid screening and separation of the antioxidants from TCM.

Conflicts of Interest
e authors declare that there are no con icts of interest regarding the publication of this paper.