Chengdu Desite Bio-Technology Co., Ltd. (Chengdu, China) had a large supply of the standard compounds of chebulic acid, gallic acid, 1,2,3,4,6-O-pentagalloylglucose, protocatechuic acid, corilagin, chebulagic acid, chebulinic acid, ellagic acid, ethyl gallate and caffeic acid (IS) (purity ≥98%). Fisher Scientific (Fair Lawn, NJ, United States) made both methanol and acetonitrile (chromatographic grade) available. Formic acid (chromatographic grade) was provided by ROE (St. Louis, MO, United States). The Millipore-Q water purification system (Tianjin Xinrui Bio-Technology Co., Ltd) was used to produce demineralized water. TC was collected from Anhui province in China.

UPLC-MS/MS system was used for analysis and monitoring, which consisted primarily of an Agilent 1290 UPLC system (Agilent Technologies, Germany) and an Agilent 6,470 series triple quad (TQ) mass spectrometer (MS) (Agilent Technologies, Singapore) equipped with an electrospray ionization source.

Separation was carried out on an ACQUITY UPLC^® HSS T3 column (2.1 × 100 mm, 1.8 μm), and temperature in the column was kept at 20°C. In the mobile phase, 0.1% formic acid in water and acetonitrile were used as solvents A and B, respectively. Gradient elution was performed as follows: 1%–5% B at 0–3 min; 5%–30% B at 3–4 min; 30%–40% B at 4–8 min; 40%–100% B at 8–9 min; 100%–100% B at 9–12 min. The flow rate was set at 0.3 ml/min and the injection volume was 10 μL.

Multiple reaction monitoring (MRM) was used for quantitative analysis in negative (−) ionization mode. The mass spectrometric parameters were set as follows: drying gas (N₂) temperature was 300°C; gas (N₂) flow rate was 7 L/min; atomizer pressure was 35 psi; sheath gas flow was 11 L/min and capillary voltage was 3800 V. The specific mass spectrometric parameters of the precursor ion, product ion, fragmentor, collision energy, and ion modes of the nine compounds and IS are shown in Table 1.

Ripe fruits of Terminalia chebula Retz are yellowish grey, spherical or ovoid and about one to two inches in size, which has five lines or five ribs on the outer skin. And TC samples of this study were purchased from Bozhou Chinese herbal medicine market in Anhui province and crushed in a grinder and passed through a 60 mesh sieve. The preparation method of TC extracts was as follows: 100.0 g TC was precisely weighed and extracted with ethanol with 30% water under hot reflux for 10 times, each time for an hour. The extracts were then filtered and mixed, which was evaporated and concentrated in the decompressed state, and the TC extracts after drying were finally sprayed into a fine powder, which was stored in desiccant before analysis. Table 2 displayed the content of chebulic acid, gallic acid, protocatechuic acid, corilagin, chebulagic acid, chebulinic acid, 1,2,3,4,6-O-pentagalloylglucose, ellagic acid and ethyl gallate in TC extracts.

The standard stock solutions of 1 mg/ml were prepared by weighing 10 mg of chebulic acid, gallic acid, protocatechuic acid, corilagin, chebulagic acid, chebulinic acid, 1,2,3,4,6-O-pentagalloylglucose, ellagic acid, ethyl gallate and caffeic acid (IS). Continuous dilution with methanol produced a working solution from the stock solution.

A mixture of 20 μl serial concentration mixed working solutions of standards and 20 μl IS solutions (caffeic acid, 1,000 ng/ml) was added to 100 μl blank plasma to obtain standard curve samples. The final plasma sample concentrations were 100, 250, 500, 1,000, 2000, 5,000, 10000, 20000, 40000 ng/mL for chebulinic acid; 25, 62.5, 125, 250, 500, 1,000, 2,500, 5,000, 10000 ng/ml for chebulic acid, gallic acid, chebulagic acid and corilagin; 1, 2.5, 5, 10, 20, 50, 100, 200, 400 ng/ml for protocatechuic acid, ethyl gallate and 1,2,3,4,6-O-pentagalloylglucose; and 10, 25, 50, 100, 200, 500, 1,000, 2000, 4,000 ng/ml for ellagic acid.

Preparation of quality control (QC) samples with three concentrations (low, medium, and high) of 16, 160, and 3,200 ng/ml for ellagic acid; 1.6, 16, 320 ng/ml for protocatechuic acid, ethyl gallate and 1,2,3,4,6-O-pentagalloylglucose; 40, 400 and 8,000 ng/ml for chebulic acid, gallic acid, corilagin and chebulinic acid and 160, 1,600, 32000 ng/ml for chebulagic acid. Both QC samples and stock solutions were stored in a refrigerator at 4°C.

The plasma sample (100 μl) was sequentially spiked with 20 μl IS solutions (caffeic acid, 1,000 ng/ml); 20 μL methanol, and 10 μl formic acid, then vortex-mixed for 1 min. The mixture was precipitated protein with 1,000 μl methanol by vortex mixing for 3 min. The supernatant was collected into a 1.5 ml clean centrifuge tube after centrifugation at for 10 min 14,000 rpm, and evaporated under a nitrogen stream until dry. After that, the residue was redissolved in 100 μl methanol, vortex-mixed for 3 min, then centrifuged for 10 min at 14,000 rpm. 10 μl analyte was injected into the UPLC-MS/MS system.

Male Sprague-Dawley rats (220 ± 10) g from the Beijing HUAFUKANG Bioscience Co., Inc. (Beijing, China) were selected. In this study, six of male rats were used, given free access to water, and fasted for 12 h before the experimental period. The TC extracts were dissolved in 0.5% CMC-Na aqueous solution as a suspension at a concentration of 1 g/ml. A dose of 10 g/kg of the suspension was administered to the rats orally, the blood samples of approximately 300 μL were drawn from the fundus venous plexus of rats pre- and post-oral administration at 0.00, 0.08, 0.17, 0.25, 0.50, 0.75, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 84, 96, 120, 144 and 168 h, respectively. For analysis, the plasma supernatants collected from the centrifuged plasma were frozen at −80°C after centrifugation for 10 min at 6,000 rpm.

Data were expressed as mean ± standard deviation (mean ± SD). MassHunter Workstation software (version B.09.00) was quantitatively used to calculate the plasma concentrations of nine analytes. To evaluate the specific pharmacokinetic parameters, DAS Software (DAS 3.0, Medical College of Wannan, China) was used to process the pharmacokinetic results.

Suitable chromatographic conditions are important for the analysis of nine compounds. The effects of ACQUITY UPLC^® BEH C 18 column (2.1 × 100 mm, 1.7 μm) and ACQUITY UPLC^® HSS T3 column (2.1 × 100 mm, 1.8 μm) on chromatographic peaks and elution time were studied experimentally. Meanwhile, water (0.1% formic acid)-acetonitrile and water (0.1% formic acid)-methanol were compared to determine the optimum mobile phase. The results displayed that water (0.1% formic acid)-acetonitrile flow has a better peak shape and lower background noise than water (0.1% formic acid)-methanol flow. Nine analytes and IS were purged in 12 min using ACQUITY UPLC^® HSS T3 column at a flow phase flow rate of 0.3 mL/min, as shown in Figure 1. No interference peaks were observed.

A comparison of electrospray ionisation sources in positive and negative ionisation modes was carried out. The results showed the target substances in negative ion mode are more responsive and stable. MS/MS ion conversion monitoring in multiple reaction monitoring (MRM) mode improves the specificity of the detection method. Therefore, the nine analytes were quantitated with electron spray ionization (ESI) in the negative ionisation mode.

Protein precipitation with methanol acetonitrile and liquid-liquid extraction with ethyl acetate were used to find a suitable method for preparing plasma samples. The results showed that the methanol precipitation protein method had good extraction recoveries for the nine analytes and the matrix effects met the assay requirements. Thus, the protein precipitation method using methanol was used in the sample preparation. Furthermore, the effects of different volumes (400 μl, 600 μl, 800 μl, 1,000 μl) of precipitation solvent on extraction recovery and matrix effect were investigated, and it was found that when the volume of methanol was 1,000 μl, the extraction recovery was between 84.9% and 108.4%, and the matrix effect was between 86.4% and 115.9%, indicating that protein precipitation with methanol could obtain satisfactory extraction recovery and matrix effect.

This study examined the pharmacokinetics of nine components of TC extract administered intragastrically to rats. Figure 2 depicted the blank plasma concentration-time curve and Table 7 summarizes the corresponding pharmacokinetic parameters.

The T_1/2 values of 1,2,3,4,6-O-pentagalloylglucose, chebulic acid, gallic acid, protocatechuic acid, ellagic acid and ethyl gallate were 6.60, 1.51, 2.06, 6.52, 8.23 and 2.08 h, separately, these analytes were shown to be eliminated shortly after oral administration. However, the T_1/2 values of corilagin, chebulagic acid and chebulinic acid were 26.39, 19.98, 43.30 h, separately, and their mean residence time (MRT) were 53.45 ± 4.12, 49.78 ± 4.87 and 58.23 ± 5.63 h, separately, which demonstrated that these three analytical substances existed inside the body for a comparatively long term and may have a continuous treatment effect and enhance the clinical efficacy (Ma, 2015; Gao et al., 2016). The AUC_(0-tn) and AUC _(0-∞) values of chebulic acid, gallic acid, corilagin, chebulagic acid and chebulinic acid were a lot higher than the others, suggesting that these analytes had higher levels of plasma exposure. And the C_max of chebulic acid, gallic acid and chebulinic acid were 1927.06 ± 572.23, 2,234.40 ± 1,201.26 and 852.55 ± 433.35 ng/ml, respectively, suggesting that the concentration of those compounds was high. In combination with AUC results, chebulic acid, gallic acid and chebulinic acid might be the potential active compounds leading to the effect of TC in vivo.

Furthermore, it was found that ellagic acid and 1,2,3,4,6-O-pentagalloylglucose had double peaks phenomenon on the blank plasma concentration-time curves in Figure 2. This phenomenon may be caused by hepatointestinal circulation. And, perhaps because of the complexity of the gastrointestinal environment, where are multiple sites of absorption at different points, and their permeability of the ingredients is different (Metsugi et al., 2007; Shin et al., 2009; Müller, and Kanfer, 2011).