The dried root of Polygonum multiflorum Thunb (He-Shou-Wu in Chinese) is one of the commonly used traditional Chinese medicines (TCMs) officially recorded in Chinese Pharmacopoeia. Clinically, He-Shou-Wu was used as a tonic and anti-aging agent in many remedies [1]. The major bioactive compounds in He-Shou-Wu have been reported to be stilibene and polyphenols. These compounds have multiple effects, such as antioxidation [2, 3], radical scavenging activity [4], lipid regulation [5, 6], hair growing effect of resting hair follicles [7], inhibition of advanced glycation end product formation [8] and neuroprotection [9–13]. Therefore, analysis of these compounds will be helpful to control the quality of Polygonum multiflorum. However, many analytical methods including HPLC [14–16], UPLC [17], GC [18] and CE [19, 20] only focused on the analysis of anthraquinones with hepatoxic activity and stilibenes in organic solvent extract. There has been few report for determination of bioactive compounds in water extract of He-Shou-Wu [21, 22], but LC analysis of hydrophilic compounds is still a challenge. Actually, water decoction, usually contains a lot of hydrophilic components, is the major administration form of TCMs. Therefore, analysis of hydrophilic compounds is beneficial to well understand active components in water extracts of TCMs.
CE analysis is usually performed in aqueous buffer system, which is easily used for analysis of hydrophilic components. In addition, CE also has the advantages of low consumption of reagent and sample, short analysis time and high efficiency [23, 24]. Furthermore, a variety of separation modes such as CZE, MEKC, MEEKC and NACE could analyze compounds with different characteristics. To the best of our knowledge, no CE method was reported for analysis of hydrophilic bioactive compounds in He-Shou-Wu. This study firstly developed a pressurized liquid extraction and short-end injection MEKC method for simultaneous determination of seven hydrophilic bioactive compounds, including hypaphorine (1), 2,3,5,4’-tetrahydroxystilbene 2-O-β-D-glucoside (2), epicatechin (3), proanthocyanidin B2 (4), proanthocyanidin B1 (5), catechin (6) and gallic acid (7) in water extract of He-Shou-Wu.
Experiment
Chemicals, reagents, and materials
Catechin (>98%), epicatechin (>98%) and gallic acid (>98%) were purchased from Shanghai Winherb Medical S&T Development Co. Ltd (Shanghai, China). Proanthocyanidin B1 (>95%) and proanthocyanidin B2 (>95%) were purchased from Chengdu Biopurify Phytochemicals Co. Ltd (Chengdu, China). Adenosine was purchased from Sigma (St. Louis, MO, USA). Hypaphorine and 2,3,5,4’-tetrahydroxystilbene 2-O-β-D-glucoside (THSG) were separated and purified in our laboratory (98%, determined by HPLC). The chemical structures of the analytes and internal standard (IS) with were shown in Figure 1.
Sodium dodecyl sulfate (SDS) was purchased from USB (Cleveland, OH, USA). Sodium phosphate monobasic was purchased from Riedel-de Haën (Seelze, Germany). Hydroxypropyl-β-cyclodextrin (HP-β-CD) was purchased from DeLi Biochemical (Xian, China), poly (ethylene glycol) (PEG, Mw=1,450) was purchased from Sigma (St. Louis, MO, USA). Hydroxypropyl methylcellulose–E5 (HPMC-E5) was purchased from Colorcon (Shanghai, China). Sodium hydroxide of analytical grade was purchased from Labscan (Bangkok, Thailand). Deionized water was prepared using a Millipore Milli-Q Plus system (Millipore, Bedford, MA, USA).
The materials of He-Shou-Wu were collected and identified by Prof. Li Shaoping, one of the correspondence authors. The voucher specimens of these samples were deposited at the Institute of Chinese Medical Sciences, University of Macau, Macao, China.
Sample preparation
The extraction was performed by pressurized liquid extraction (PLE) on a Dionex ASE 200 system (Dionex, Sunnyvale, CA, USA) under the optimized conditions reported before [21]. In brief, powder (0.5 g) was mixed with diatomaceous earth in a proportion of 1:2 and placed into an 11 mL stainless steel extraction cell. The extraction cell was extracted under the optimized condition: Solvent, water; particle size, 80–96 μm; pressure, 1500 psi; temperature, 40°C; Static time, 10 min; number of cycle, 1. After PLE extraction, the extract was diluted to a certain volume in 25 mL volumetric flask with water. Before injection, the extract was filtered through a 0.45 μm filter (Millipore, Ireland) and mixed with IS in a proportion of 4:1.
Each Standard was dissolved in water as stock solution at the concentration of 1 mg/mL (10 mg/mL for THSG), and diluted to appropriate concentration, then mix with IS in a proportion of 4:1 before use.
MEKC analysis
All analysis was performed on an Agilent HP 3D CE instructment (Agilent Technologies, Palo Alto, CA, USA) using “Short-end injection” mode. A fused-silica capillary (64.5 cm × 75 μm id, 8.5 cm effective length; Agilent Technologies) was used throughout this study. The running buffer containing 50 mM phosphate, 90 mM SDS and 2.0% HP-β-CD was adjusted to pH 2.5 using phosphate acid. The buffer was filtered through 0.45 μm filter before it was transferred to the inlet/outlet vials. A 15 kV voltage was applied and pressure injection was 25 mbar for 3 s. The detection wavelength was 210 nm and the temperature was maintained at 20°C. The new capillary was first flushed with 1 M NaOH, 0.1 M NaOH and water for 20 min. For each run, the capillary was conditioned by rinsing with 0.1 M NaOH, water and running buffer for 3 min, respectively. Adenosine (80 μg/mL of final concentration) was used as IS.
Calibration curves, limit of detection and quantification
Stock solutions of reference compounds were prepared and diluted to appropriate concentrations with water, then mixed with 400 μg/mL of adenosine solution in a proportion of 4:1. At least seven concentrations of the solution were analyzed in two replicates, and the calibration curves were constructed by plotting the peak area ratio of individual standard to IS versus the concentration of each analyte. LOD and LOQ for each analyte were determined at an S/N of about 3 and 10, respectively.
Precision, repeatability and recovery
Intra- and inter-day variations were chosen to determine the precision of the developed method. For intra-day variation test, three levels of the mixed standards solution was analyzed for six replicates (n=6) within one day, while for inter-day variations test, the three levels was examined in duplicates for consecutive 3 days (n=6). Variations were expressed as RSD.
The repeatability of the method was determined by analyzing three levels (0.4 g, 0.5 g and 0.6 g) of sample HN for three replicates and represented as RSD. The recovery was performed by adding known amount of individual standards into a certain amount of sample HN. The mixture was extracted and analyzed for three replicates.