1. Introduction
As traditional Chinese medicine (TCM) is gradually adopted by the world, their effects have attracted more and more attention of researchers from various fields. Among those effects, antioxidant activity is important for many diseases. For example, some studies have shown that the anticancer activities of TCM components are directly or indirectly related to their antioxidant activity [
1]. The antioxidant activity of TCM components is also closely related to many pharmacological effects, such as neuroprotective [
2,
3], antidepressant [
4,
5], anti-inflammatory [
6], anticancer [
7], hypoglycemic, and hypolipidemic [
8]. Therefore, the research on antioxidant activity has become more and more important in TCM research.
As the strongest reducing agent in metal ions, ferrous ions are closely related to human aging and can produce strong oxidizing hydroxyl radicals and equivalent oxidizing power by reducing H
2O
2 [
9]. The active components in TCM can chelate ferrous ions to form a stable complex and prevent ferrous ions from being oxidized or mediating other oxidation reactions. The formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) was mediated by ferrous ions chelated by some components of TCM [
10,
11,
12]. It was found that tannic acid could block Fenton reaction by chelating ferrous ions to provide antioxidant activity, and the complex of tannic acid with Fe(II) would not react with H
2O
2 [
13]. Therefore, the ferrous ions chelating ability of TCM is inextricably linked to its antioxidant activity. The antioxidant activity of TCM can be evaluated from the aspect of chelating ferrous ions [
14].
Ultraviolet and visible (UV/VIS) spectrophotometry does not have enough specificity for determining the ability of components in the mixtures to chelate ferrous ions [
15,
16]. Moreover, the method possesses some disadvantages, including the consumption of more reagents and the complicated operation of metal-chelating assays. Recently, high-performance liquid chromatography coupled with post-column derivation (HPLC-PCD) was developed to determine the iron ions reduction ability of samples [
17]. However, the post-column derivation made the operation more complicated. The capillary electrophoresis (CE) has the advantages of low solvent consumption and high efficiency, so it was widely applied in screening enzyme inhibitors, complex samples analysis, and derivatization [
18,
19,
20] in recent years. At present, the in-capillary ABTS
+-CE-DAD method [
21] and DPPH-CE-DAD method [
22,
23] were established to evaluate the antioxidant activity of TCM. Based on these methods, an in-capillary [Fe(ferrozine)
3]
2+-CE-DAD was proposed to evaluate the ferrous ions chelating ability of TCM.
Sophora japonica is a medium-sized deciduous tree commonly found in China and many other countries. Flos Sophorae (FS) and Flos Sophorae Immaturus (FSI) are dry flowers and flower buds of Sophora japonica, respectively [
24]. Many studies have shown that the antitumor, anti-inflammatory [
25], antioxidant and radical scavenging [
26], antibacterial [
27], and other important activities of FSI and FS are related to their large amount of flavonoids. At present, the determination methods for the ability of FSI(FS) to chelate ferrous ions are mainly limited to UV spectrophotometry. Therefore, an in-capillary [Fe(ferrozine)
3]
2+-CE-DAD method was established for screening active components and determining the total ferrous ions chelating ability of FSI(FS).
The extraction methods of TCM make a great effect on its efficacy and activities. There are many extraction methods for TCM, including heating reflux [
28], ultrasound [
29], far-infrared [
30], microwave [
31], and ionic liquid-based pressurized liquid extraction (IL-PLE) [
32], in which Microwave-assisted extraction (MAE) has the advantages of high extraction efficiency and is environment friendly. Furthermore, MAE is a reliable extraction method with high stability and reproducibility, which makes it superior to other traditional extraction methods [
33]. Therefore, the MAE was combined with in-capillary analysis methods to evaluate the quality of FSI(FS).
Taking activity as an important aspect of quality control of traditional Chinese medicine is the trend of the development of TCM. To our knowledge, there is no literature on the application of in-capillary “[Fe(ferrozine)3]2+”-CE-DAD method for determining the ability of TCM to chelate ferrous ions. Therefore, we used the microwaves to assist in the extraction of FSI(FS) and in-capillary -CE-DAD method to evaluate the ability of extracts to chelate ferrous ions via screening the antioxidants and determining IC50 of the extracts. Factors affecting extraction and separation were optimized. The results of method validation proved that the microwave-assisted extraction (MAE) combined with in-capillary [Fe(ferrozine)3]2+-CE-DAD method was sensitive, reliable, highly efficient, and easy to operate and can be used for quality control of TCM.
3. Materials and Methods
3.1. Chemicals and Reagents
The reference components, including rutin, kaempferol-3-rutinoside, narcissoside, and quercetin (more than 99% pure), were purchased from Chengdu Must Bio. Sci. and Tec. Co. Ltd. (Chengdu, China). The purity of the biological reagent ferrozine was 99%. Ferrous sulfate (FeSO4·7H2O) and citric acid purchased from Commio Chemical Testing Co. Ltd. (Tianjin, China). were of analytical grade. Deionized water was achieved from a Milli-Q system (Millipore, Milford, MA, USA). Acetonitrile (ACN) and methanol (MeOH) were bought from Merck (Chromatographic, Germany).
3.2. Preparation of Analytical Samples
All the standards and stock solution were prepared in methanol, and the mixed standard solution of each concentration was diluted by methanol. Ferrozine was dissolved in deionized water to 4 mmol·L−1 (mM) and stored at 4 °C. Ferrous sulfate was dissolved in deionized water to 4 mM before each use. The citric acid was dissolved in water to 4 mg·mL−1 and then diluted with water.
The extraction of samples was performed by Multiwave PRO SOLV (Anton Paar Co. Ltd., Graz, Austria). The operating condition was the mode of keeping power (no setting climb time) in which the temperature was optimized by adjusting the power. After 1 g of medicinal powder (through 80 mesh sieve) was extracted in 10 mL 50% (v/v) MeOH for 7.5 min at 750 w power, the weightlessness was compensated by adding the corresponding solvent. Subsequently, the mixture was cooled at room temperature and then centrifuged at 1000× g for 10 min. Finally, the supernatant was directly diluted with the corresponding extraction solvent to a crude drug concentration of 5 mg·mL−1 and kept at room temperature before analysis.
3.3. Instruments and CE Condition
All samples were analyzed by an Agilent 7100 capillary electrophoresis (CE) system (Waldbronn, Germany) with a Diode Array Detector. All analytes were separated on a fused silica capillary with a diameter of 50 µm and a total length of 50.2 cm (effective length of 42.2 cm) (Ruifeng, Handan, Hebei, China). Before the first use, the new capillary was activated by sequentially flushing with 1 M NaOH, 0.1 M NaOH, and deionized water. Before the samples were injected, 0.1 M NaOH for 1 min, deionized water for 1 min, and the background electrolyte (BGE) for 2 min were used to rinse the capillary in sequence. All samples were injected under 50 mbar. After the last analysis of one day, the analytical system was washed with 0.1 M NaOH and deionized water for 10 min. BGE needs to be updated every three runs to ensure that the method can be reproduced.
3.4. The Conditions and Principle of Method
The conditions, including pH of running buffer; the concentration of disodium phosphate, sodium dodecyl sulfate (SDS), β-cyclodextrin (β-CD), and ACN; the separation voltage, and cartridge temperature, were optimized. According to the principle of the traditional ferrozine method [
35,
37], after 4 mM ferrous sulfate was injected into CE system for 3 s, the samples extracted by microwave were injected for 5 s, incubated for 300 s, then 4 mM ferrozine was injected for 2 s, and the analytes were separated. In this process, ferrozine had a strong ability to chelate ferrous ions and was commonly used as an indicator for determining the ability of TCM to chelate ferrous ions. The active components of FSI(FS) would compete with ferrozine for chelating ferrous ions in the capillary. By comparing with the corresponding electropherogram of blank, the peak area of the active components or the chelate [Fe(ferrozine)
3]
2+ would be reduced. All of the six compounds had strong absorption at 260 nm, and [Fe(ferrozine)
3]
2+ also had absorption at 562 nm, so the detection wavelength was 260 nm.
3.5. Method Validation
Based on optimized extraction and separation conditions, active components were screened by comparing the electrophoretograms of in-capillary mixed ferrous sulfate, sample extracts, ferrozine with the electrophoretograms of in-capillary mixed sample extracts, and deionized water.
To verify the feasibility of the method, the calibration curves were fitted by injecting six concentrations of the mixed standard solution. The limit of detection (LOD) and the limit of quantification (LOQ) were obtained by electropherograms of the standard solution. A known concentration of the mixed standard solution was added into the extraction tank and then extracted together with the crude drug to obtain recoveries. On this basis, low, medium, and high concentration of mixed standard solution were used for evaluating the precision and stability for 24 h. Stability for 24 h was determined at six timepoints in one day. The accuracy of precision in one day or three days and remains of stability was the ratio of the measured concentration to the actual concentration.
4. Conclusions
The microwave-assisted extraction combined with in-capillary [Fe(ferrozine)3]2+-CE-DAD method was successfully established to screen active components and evaluate the ability of traditional Chinese medicine to chelate ferrous ions. The microwave extraction adopted in this study can be considered as an eco-friendly, green, and fast technique for the extract preparation because of less consumption of organic reagents, shorter extraction time, and higher extraction efficiency. Besides, it contributed to enhance the ability of the FSI and FS extracts to chelate ferrous ions. Rutin and Quercetin were screened as the target compounds to chelate ferrous ions in Flos Sophorae Immaturus and Flos Sophorae. Compared with the traditional methods, the online method of integrating reaction and detection not only extremely simplified the whole analysis procedure but also increased the sensitivity and reduced the consumption of reagent. Moreover, in-capillary screening of active components from TCM provides a comprehensive evaluation for the chelation of ferrous ions. Consequently, the proposed MAE extraction combined with in-capillary [Fe(ferrozine)3]2+-CE-DAD method can be a practical and effective technology for the evaluation of the activity of TCM. Furthermore, it can be used for quality control of TCM and can provide certain method strategies for industrial applications.