Article pubs.acs.org/JAFC
Plasma Pharmacokinetics, Bioavailability, and Tissue Distribution of Four C‑Glycosyl Flavones from Mung Bean (Vigna radiata L.) Seed Extracts in Rat by Ultrahigh-Performance Liquid Chromatography− Tandem Mass Spectrometry Yan Bai, Qili Zhang, Baoyu Wang, Meiyan Zhang, Yan Xu, Shasha Li, Yunli Zhao,* and Zhiguo Yu* Department of Pharmaceutical Analysis, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, People’s Republic of China ABSTRACT: The main polyphenols in mung bean (Vigna radiata L.) seed (MBS), an edible legume with various biological activities, are C-glycosyl flavones (vitexin, isovitexin, isovitexin-6″-O-α-L-glucoside, and dulcinoside). In our study, a validated ultrahigh-performance liquid chromatography−tandem mass spectrometry (UHPLC−MS/MS) method was developed to quantitate the concentrations of four C-glycosyl flavones from MBS extracts in the plasma and various tissues of rats and successfully applied to study their pharmacological profile and tissue distribution in vivo. Four C-glycosyl flavones were rapidly absorbed after oral administration, achieving a Cmax at around 1.5 h, and they could be distributed widely and rapidly in tested tissues. The concentrations of four C-glycosyl flavones in all of the tested tissues decreased obviously in 4 h, which indicated that there was not a trend of long-term accumulation of them. This is the first time to report on pharmacokinetic and tissue distribution studies of four C-glycosyl flavones in rat. The results provided a significative basis for the application of MBS. KEYWORDS: mung bean seeds, C-glycosyl flavones, pharmacokinetics, bioavailability, tissue distribution, UPLC−MS/MS
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INTRODUCTION Mung bean (Vigna radiata L.) seed (MBS) is a common edible legume in many countries.1 The sprouts and seeds of mung beans are popular as a fresh salad vegetable in Southeast Asia and Western countries. MBS contains abundant nutrients with biological activities, such as proteins, fatty acid, minerals, phenolic acids, and flavonoids.2 In the recent years, many studies have reported that MBS exhibits various bioactivities, including anti-inflammatory,3 hypolipidemic,4 antioxidant,5,6 myocardial preservation,7,8 detoxication,9 hepatoprotective,10 and antidiabetic11 effects. These effects could be related to the constituents in MBS. Previous phytochemical investigations on MBS indicated that it contained a series of C-glycosyl flavones, such as vitexin (V), isovitexin (IV), isovitexin-6″-O-α-L-glucoside (IVG), and dulcinoside (D) (structures in Figure 1).7 In addition, vitexin and isovitexin were found to possess antiinflammatory,3 myocardial preservation,7 and inhibitory effects on the formation of advanced glycation endproducts.12 In recent years, the studies of pharmacokinetics and tissue distribution of the main constituents in food have become essential to understand before studying in vivo effects.13,14 Although four C-glycosyl flavones have numerous beneficial pharmacological activities, as mentioned above, it should be noted that most of the results were obtained in vitro. The studies of pharmacokinetics and tissue distribution play a critical role to evaluate if those in vitro effects can be translated in vivo. Until now, in stark contrast to the pharmacological studies of MBS, very few reports are related to the pharmacokinetic and tissue distribution studies of the multiple components of MBS in vivo. There are just a few studies about the quantitative analysis of vitexin or isovitexin in biological samples.15,16 Furthermore, therapeutic effects of herbal © 2017 American Chemical Society
medicines or functional foods are usually based on the interactions of multiple ingredients, and quantifying one constituent was insufficient.17 To date, there was no method reported for the simultaneous quantification of V, IV, IVG, and D in rat biological matrices, such as plasma, intestine, heart, spleen, liver, lung, stomach, kidney, and brain. This work was aimed to develop and validate a liquid chromatography−tandem mass spectrometry (LC−MS/MS) method for the simultaneous quantification of four C-glycosyl flavones in rat biological matrices. The method was substantiated to be of immense use for preclinical studies, owing to the sensitivity (0.12 ng/mL) and relatively short run time (6 min).
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MATERIALS AND METHODS
Plant Material and Chemicals. MBS (V. radiata L.) was purchased from Carrefour supermarket in China, 2016. Reference standards of V, IV, IVG, and D were isolated and purified from our laboratory in our previous studies.7 Their structures were identified by nuclear magnetic resonance (NMR) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR−MS) spectroscopic analyses. The internal standard (IS) puerarin (structure in Figure 1) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity was above 98.0%, which was tested by high-performance liquid chromatography−diode array detection (HPLC−DAD). Ethanol for the extraction was purchased from Qingdao Haiyang Chemical Co., Ltd. The water used during ultra performance liquid chromatography Received: Revised: Accepted: Published: 5570
May 3, 2017 June 17, 2017 June 19, 2017 June 19, 2017 DOI: 10.1021/acs.jafc.7b02053 J. Agric. Food Chem. 2017, 65, 5570−5580
Article
Journal of Agricultural and Food Chemistry
Figure 1. Product ion scan spectra, chemical structures, monitored transitions, cone voltage (CV), and collision energy (CE) of vitexin, isovitexin, isovitexin-6″-O-α-L-glucoside, dulcinoside, and puerarin (IS). (UPLC) analysis was purchased from Wahaha Group Co., Ltd. (Hangzhou, China). LC−MS-grade formic acid and methanol were purchased from Fisher Scientific, Pittsburgh, PAU.S.A. Instrument. An Acquity UPLC system (Waters Corp., Milford, MA, U.S.A.), coupled with a cooling autosampler and a column oven was used for UPLC analysis. The chromatographic separation was performed on a Thermo Hypersil GOLD C18 column (2.1 × 50 mm, 1.9 μm) protected by a Van Guard BEH C18 column (2.1 × 5 mm, 1.7 μm) at 35 °C. Water with 0.1% formic acid (A) and methanol (B) was used as the mobile phase with gradient elution (0−3.0 min, 25% B; 3.0−5.0 min, 25−40% B; 5.0−5.5 min, 40−25% B; and 5.5−6.0 min, 25% B) with a 0.3 mL/min flow rate and a 2 μL injection volume. Mass spectrometry (MS) was carried out on a Waters Xevo TQ-S mass spectrometer (Waters Corp., Milford, MA, U.S.A.) with an electrospray ionization (ESI) interface. The positive-ion multiple reaction monitoring (MRM) mode was used for mass spectrometric analysis. The following setups of the analyzers were used: capillary voltage, 3.0 kV; desolvation temperature, 350 °C; ion source temperature, 150 °C; cone gas flow rate, 150 L/h; and desolvation gas flow rate, 700 L/h. The precursor-to-product ion pairs, collision energy, and cone voltage for four C-glycosyl flavones and puerarin are listed in Figure 1. All data were acquired by the use of Masslynx NT 4.1 software (Waters Corp., Milford, MA, U.S.A.). Animals. The study was approved by the Animal Ethics Committee of Shenyang Pharmaceutical University, and the protocol was approved by the Animal Ethics Committee of the institution. Male Sprague
Dawley (SD) rats (200−240 g) were brought from the Experimental Animal Center of Shenyang Pharmaceutical University. They are housed under suitable conditions (relative humidity of 55 ± 10% and temperature of about 25 °C). All of the rats fasted for 12 h before the experiment. Preparation of Oral (p.o.) and Intravenous (i.v.) Administration Solution of MBS Extracts. The extraction method used was as described by Bai et al.7 To calculate the administration dose, the contents of the four C-glycosyl flavones in MBS extracts were quantitatively analyzed by the standard curve method. The contents of V, IV, IVG, and D in the MBS extracts were 2.29, 5.32, 1.70, and 0.13 mg/g, respectively. The p.o. administration solution (2 g/kg of MBS extracts, equivalent to 4.58 mg/kg of V, 10.64 mg/kg of IV, 3.40 mg/kg of IVG, and 0.26 mg/kg of D) was prepared with normal saline. The i.v. administration solution (25 mg/kg of MBS extracts, equivalent to 57.25 μg/kg of V, 133.00 μg/kg of IV, 42.50 μg/kg of IVG, and 3.25 μg/kg of D) was suspended in 0.5% sodium carboxymethyl cellulose prepared. Preparation of Standard Solutions, Calibration, and Quality Control (QC) Samples. Individual stock solutions of four analytes (V, IV, IVG, and D) at a concentration of 10.0 μg/mL V, 25.0 μg/mL IV, 8.0 μg/mL IVG, and 0.6 μg/mL D were prepared by dissolving the four C-glycosyl flavones in methanol. The stock solution was serially diluted with methanol to obtain a series of standard mixture working solutions to prepare the calibration and QC samples. A 200 ng/mL solution for pueararin was prepared in methanol. All of the solutions were kept at 4 °C and brought to room temperature before use. 5571
DOI: 10.1021/acs.jafc.7b02053 J. Agric. Food Chem. 2017, 65, 5570−5580
Article
Journal of Agricultural and Food Chemistry
Figure 2. Typical MRM chromatograms of the four C-glycosyl flavones and pueararin in plasma: (A) blank plasma, (B) blank plasma spiked with the four analytes at LLOQ and IS, and (C) plasma sample collected at 4 h after a single p.o. dose of 2 g/kg of MBS extracts.
Table 1. Regression Equations, Linear Ranges, Correlation Coefficients, and LLOQs of the Four C-Glycosyl Flavones in Rat Plasma and Tissues matrix plasma
heart
liver
spleen
lung
kidney
stomach
intestine
analyte V IV IVG D V IV IVG D V IV IVG D V IV IVG D V IV IVG D V IV IVG D V IV IVG D V IV IVG
calibration curve y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y
= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =
0.0276x 0.1035x 0.0118x 0.1115x 0.0252x 0.1131x 0.0089x 0.1259x 0.0225x 0.1053x 0.0079x 0.1090x 0.0221x 0.1130x 0.0081x 0.1232x 0.0249x 0.1209x 0.0083x 0.1210x 0.0203x 0.1336x 0.0061x 0.1021x 0.0344x 0.2192x 0.0086x 0.1095x 0.0174x 0.1156x 0.0086x
+ 0.0041 + 0.0067 + 0.0059 + 0.0072 + 0.0194 − 0.0307 + 0.0780 + 0.0081 + 0.0013 + 0.0090 + 0.0022 + 0.0406 + 0.0257 + 0.0796 − 0.0037 − 0.0032 + 0.0007 + 0.0106 + 0.0012 + 0.0053 + 0.0042 − 0.0027 + 0.0002 + 0.0268 + 0.0176 + 0.0098 + 0.0005 + 0.0227 + 0.0093 + 0.0610 + 0.0022
range (ng/mL)
correlation coefficient (r)
LLOQ (ng/mL)
2−250 5−625 1.6−200 0.12−15 2−250 5−625 1.6−200 0.12−15 2−250 5−625 1.6−200 0.12−15 2−250 5−625 1.6−200 0.12−15 2−250 5−625 1.6−200 0.12−15 2−250 5−625 1.6−200 0.12−15 2−250 5−625 1.6−200 0.12−15 2−250 5−625 1.6−200
0.9965 0.9987 0.9979 0.9969 0.9994 0.9976 0.9969 0.9989 0.9997 0.9999 0.9997 0.9987 0.9996 0.9996 0.9997 0.9997 0.9987 0.9963 0.9986 0.9994 0.9995 0.9999 0.9994 0.9963 0.9925 0.9964 0.9992 0.9994 0.9917 0.9947 0.9992
2 5 1.6 0.12 2 5 1.6 0.12 2 5 1.6 0.12 2 5 1.6 0.12 2 5 1.6 0.12 2 5 1.6 0.12 2 5 1.6 0.12 2 5 1.6
5572
DOI: 10.1021/acs.jafc.7b02053 J. Agric. Food Chem. 2017, 65, 5570−5580
Article
Journal of Agricultural and Food Chemistry Table 1. continued matrix
analyte D V IV IVG D
brain
calibration curve y y y y y
= = = = =
0.1108x 0.0317x 0.1526x 0.0101x 0.1391x
+ + + + +
0.0069 0.0039 0.0355 0.0073 0.0008
range (ng/mL)
correlation coefficient (r)
LLOQ (ng/mL)
0.12−15 2−250 5−625 1.6−200 0.12−15
0.9998 0.9963 0.9984 0.9997 0.9997
0.12 2 5 1.6 0.12
Table 2. Intra- and Interday Precision, Accuracy, Recovery, and Matrix Effect of the Four C-Glycosyl Flavones from QC Samples Prepared in Rat Plasma and Tissues (n = 6) matrix plasma
analyte V
IV
IVG
D
heart
V
IV
IVG
D
liver
V
IV
IVG
D
spleen
V
IV
IVG
D
lung
V
concentration (ng/mL)
intraday RSD (%)
interday RSD (%)
accuracy RE (%)
4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25
8.87 7.71 0.93 11.73 6.16 5.02 11.94 8.42 4.88 13.27 10.79 7.20 9.64 6.43 1.22 10.60 7.02 3.69 11.11 8.62 2.01 12.41 8.73 2.10 9.81 8.38 2.48 11.31 8.67 4.03 10.98 6.39 2.75 13.21 7.41 1.93 10.67 6.93 1.70 9.84 5.37 1.39 10.77 6.82 2.96 12.86 7.77 4.20 10.97 7.84
8.31 9.70 3.39 12.59 5.95 2.84 4.18 9.57 3.22 10.42 4.47 5.53 8.33 5.90 0.91 9.61 5.44 2.13 10.09 6.42 3.68 9.88 7.27 3.31 10.00 7.42 3.29 10.89 7.56 2.22 9.66 4.66 1.01 12.38 8.63 2.00 9.55 4.37 0.88 10.31 8.66 3.24 11.72 7.42 3.95 11.08 8.71 5.09 13.67 8.58
4.58 −1.94 0.25 6.67 1.44 3.97 −5.73 5.67 0.36 −1.39 1.25 1.39 5.34 3.86 −1.70 4.03 3.92 2.77 3.74 −2.64 2.54 −4.70 1.77 3.82 3.55 2.87 2.80 4.72 3.79 −2.71 4.66 2.90 3.01 5.81 2.73 3.33 −3.58 2.99 −1.07 7.05 1.72 −6.83 3.96 2.20 3.17 −4.88 2.69 2.69 5.04 4.00
5573
recovery (%, mean ± SD)
matrix effect (%, mean ± SD)
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
91.33 ± 3.91 85.45 ± 3.58 88.91 ± 9.12 91.02 ± 4.96 85.39 ± 5.17 96.20 ± 6.34 97.32 ± 2.77 87.20 ± 3.75 91.98 ± 8.62 95.89 ± 8.92 88.48 ± 5.71 96.03 ± 8.20 98.63 ± 2.70 107.75 ± 4.82 101.46 ± 1.88 89.62 ± 4.04 102.29 ± 8.39 97.66 ± 2.55 90.20 ± 1.04 85.49 ± 0.42 92.67 ± 2.51 94.60 ± 3.35 85.45 ± 5.49 99.89 ± 2.73 98.63 ± 2.26 90.21 ± 3.49 94.82 ± 2.17 89.63 ± 1.94 90.38 ± 2.82 94.04 ± 1.99 89.33 ± 2.09 88.08 ± 5.50 85.43 ± 1.57 92.98 ± 2.77 94.18 ± 4.92 90.30 ± 1.43 102.32 ± 2.64 98.78 ± 3.72 89.64 ± 4.43 100.10 ± 2.00 96.36 ± 3.77 87.37 ± 7.60 104. 55 ± 3.27 87.85 ± 4.02 99.96 ± 3.31 89.52 ± 4.64 103.37 ± 3.75 88.11 ± 1.05 98.73 ± 2.61 109.49 ± 2.21
79.93 77.42 83.08 87.16 93.17 79.41 91.87 85.76 93.50 75.13 92.83 86.03 89.89 90.80 91.01 99.02 94.40 97.61 82.14 95.24 88.78 92.23 93.59 91.66 96.89 87.13 83.24 87.42 89.60 84.88 92.66 83.60 86.03 92.07 80.34 79.98 96.72 87.66 90.46 82.09 85.49 91.33 88.18 86.02 90.09 86.47 79.96 82.05 90.52 84.67
7.67 3.08 6.68 2.64 3.38 2.56 4.64 4.86 3.87 10.23 5.25 2.64 3.42 0.71 2.44 0.82 3.23 2.85 2.53 3.83 3.08 7.85 9.34 6.03 2.55 0.22 4.13 3.58 4.03 3.40 5.67 2.70 4.48 2.78 6.70 2.68 3.42 1.38 2.87 3.03 1.28 3.71 4.68 7.46 2.49 4.02 3.72 2.84 1.91 4.08
DOI: 10.1021/acs.jafc.7b02053 J. Agric. Food Chem. 2017, 65, 5570−5580
Article
Journal of Agricultural and Food Chemistry Table 2. continued matrix
analyte IV
IVG
D
kidney
V
IV
IVG
D
stomach
V
IV
IVG
D
intestine
V
IV
IVG
D
brain
V
IV
IVG
D
concentration (ng/mL)
intraday RSD (%)
interday RSD (%)
accuracy RE (%)
200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12
4.02 9.86 8.88 2.01 10.44 7.02 5.43 11.23 7.73 5.01 9.61 6.19 3.03 11.05 6.86 1.39 9.28 5.51 3.18 10.66 6.96 4.84 9.20 7.24 2.98 8.78 6.55 2.21 10.97 7.92 4.31 13.69 8.81 2.97 9.99 4.62 3.33 8.60 4.72 1.84 10.01 7.93 3.17 13.82 8.02 3.00 9.68 6.49 3.35 10.11 7.37 1.85 10.89 7.49 4.33 13.03 8.63 4.84
3.66 10.79 6.63 2.58 9.37 8.68 4.34 10.00 7.68 4.98 10.30 7.34 4.44 10.62 7.71 2.07 10.99 7.28 3.32 9.87 7.86 2.94 10.71 6.68 4.60 8.73 4.42 3.38 11.44 8.68 2.81 12.47 7.70 3.62 10.82 7.29 2.61 11.25 7.71 0.86 10.74 8.69 2.94 10.67 7.45 2.62 10.59 7.82 2.77 11.84 6.62 2.07 9.67 5.88 2.96 10.78 7.88 4.69
−3.79 2.87 2.41 2.69 5.72 3.67 −4.05 2.81 2.94 3.00 2.96 5.23 4.74 −3.96 2.85 −4.05 7.66 6.29 −5.83 5.29 3.87 2.94 −4.92 3.60 2.74 4.88 3.06 −2.86 3.96 4.44 2.86 4.75 −3.22 2.74 −5.81 3.72 1.02 7.75 −2.62 2.74 2.91 −3.55 0.72 3.87 0.99 1.69 −4.82 3.71 5.86 4.44 3.09 −0.76 −6.67 2.53 3.56 2.80 −1.73 3.61
recovery (%, mean ± SD) 94.90 92.24 90.30 96.17 96.75 89.99 93.73 98.99 79.82 85.63 98.69 81.23 84.16 91.78 86.06 88.47 91.73 89.86 86.33 85.59 81.80 84.39 93.29 84.21 89.73 96.81 77.92 88.07 83.61 78.34 86.11 90.82 84.82 73.08 92.08 85.76 88.97 94.84 80.60 86.77 84.54 80.69 88.73 92.27 81.11 86.72 96.79 87.91 80.63 95.64 83.31 84.65 89.83 86.04 83.36 90.53 79.28 72.06
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.12 4.92 8.46 3.04 4.32 4.97 3.95 4.66 0.01 3.90 1.17 2.92 6.61 3.77 2.98 5.51 3.91 2.40 3.27 4.37 2.23 1.98 3.00 2.64 3.81 3.24 3.62 6.47 7.12 4.06 7.78 3.04 4.58 3.50 4.32 3.41 2.01 3.25 2.05 4.27 3.21 2.22 4.38 3.91 1.05 2.76 2.34 5.44 7.87 3.61 4.05 2.40 8.08 8.35 8.87 3.49 2.69 1.78
matrix effect (%, mean ± SD) 92.15 93.67 105.43 86.97 87.46 91.52 91.87 94.44 95.15 87.80 99.09 92.03 85.67 88.03 85.14 86.15 89.58 92.68 94.07 95.05 93.12 85.73 101.11 89.97 98.89 103.47 96.56 100.75 100.81 98.03 88.43 94.76 89.92 98.74 90.35 104.82 86.24 96.73 96.83 98.79 102.08 96.63 87.05 97.39 99.30 90.95 96.36 103.01 87.60 95.63 103.61 99.37 101.17 86.53 89.44 98.72 92.67 88.61
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.85 3.69 6.75 4.23 2.86 3.29 3.99 3.07 1.39 3.58 2.31 4.59 3.36 5.02 6.39 2.71 2.03 3.87 3.82 1.61 4.59 0.58 3.30 4.42 6.63 1.93 2.41 0.24 8.03 4.37 2.64 3.44 4.24 2.16 0.93 7.70 5.55 4.46 3.05 3.03 3.82 4.74 4.27 3.82 0.64 6.06 2.22 3.39 2.53 3.41 4.49 2.82 3.42 2.60 4.36 4.48 8.06 2.87
prepare final concentrations of 2, 10, 25, 50, 100, and 250 ng/mL for V, 5, 25, 62.5, 125, 250, and 625 ng/mL for IV, 1.6, 8, 20, 40, 80, and
A six point calibration curve was prepared by freshly spiking the appropriate working solution into blank plasma and various tissues to 5574
DOI: 10.1021/acs.jafc.7b02053 J. Agric. Food Chem. 2017, 65, 5570−5580
Article
Journal of Agricultural and Food Chemistry
Table 3. Stability of the Four C-Glycosyl Flavones in Rat Plasma and Tissue Homogenates under Different Storage Conditions (n = 6) storage at −20 °C for 15 days
3 freeze−thaw cycles matrix plasma
analyte V
IV
IVG
D
heart
V
IV
IVG
D
liver
V
IV
IVG
D
spleen
V
IV
IVG
D
lung
V
IV
IVG
D
6 h at room temperature
autosampler for 12 h
concentration (ng/mL)
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24
0.29 3.01 8.38 2.00 4.34 1.63 0.26 3.49 5.72 0.06 0.38 6.12 1.38 3.69 7.82 4.47 3.96 8.12 0.76 4.85 4.62 1.03 3.88 6.51 1.84 4.27 9.82 2.31 5.15 8.65 0.28 3.82 9.74 0.03 1.77 4.39 1.26 3.88 6.73 0.84 3.48 8.77 1.32 3.83 4.69 0.22 1.68 4.77 1.11 3.79 8.72 1.74 5.88 4.72 2.66 3.81 8.90 0.07
3.75 1.11 −1.17 8.83 −0.67 1.98 −3.13 1.67 −1.15 4.86 4.58 −1.04 4.40 −1.08 3.28 5.83 −2.63 1.17 8.82 5.49 −4.55 8.10 6.63 3.05 −8.67 4.73 2.88 −9.01 4.38 4.08 12.42 −8.76 −4.46 9.87 −5.77 2.66 8.57 −3.02 1.11 4.85 −5.74 6.69 −10.63 7.45 4.62 −6.74 9.82 −3.51 9.64 6.44 −1.39 6.72 4.38 −5.94 10.48 −6.61 4.34 9.86
0.31 5.30 11.83 1.17 3.27 8.41 0.25 4.69 7.41 0.04 0.29 5.61 0.93 2.66 4.82 2.68 6.62 4.83 1.38 3.82 5.59 0.02 0.89 4.79 1.35 5.37 8.75 2.58 6.48 9.84 0.05 2.64 4.73 1.02 2.94 5.88 1.82 2.88 9.67 3.76 6.49 6.53 2.30 3.34 5.68 1.86 4.88 3.07 2.86 3.49 5.50 2.37 3.77 5.71 1.89 0.27 7.83 0.74
5.04 −0.38 0.42 8.28 1.11 0.14 −2.92 1.77 1.52 3.77 0.83 −1.39 9.08 −3.21 1.01 10.05 −5.51 5.15 8.37 3.52 2.94 −7.09 3.86 4.72 8.71 −6.86 3.90 9.62 −5.81 7.44 10.91 4.13 2.18 −8.31 5.77 −1.93 6.49 3.81 −2.04 5.93 4.70 3.95 −5.99 2.81 1.79 4.83 3.72 −2.02 4.59 −3.91 1.85 10.77 4.31 −3.86 8.95 3.75 −1.03 7.64
0.31 6.82 9.38 1.89 3.32 3.97 0.30 3.07 7.28 0.03 0.26 6.31 1.20 4.38 3.29 1.92 5.69 8.77 0.99 3.21 4.63 0.10 4.72 4.29 1.93 3.85 5.93 3.79 6.77 10.04 0.06 2.88 6.77 0.97 3.68 7.83 1.33 3.49 9.85 2.55 3.89 8.91 0.16 4.80 11.67 3.76 3.88 4.70 1.24 4.38 5.77 2.66 5.86 9.60 0.48 3.87 8.54 3.48
1.75 1.21 0.58 4.17 0.78 0.84 −3.18 3.03 1.17 −10.42 5.42 0.69 −9.88 5.42 4.43 8.70 3.23 −1.80 3.72 0.29 1.72 −5.02 5.69 −4.70 10.82 −7.43 6.76 9.56 5.73 2.75 8.77 5.73 −2.06 7.73 4.92 5.04 8.05 6.62 −3.63 7.58 6.04 −4.48 6.69 10.54 3.82 9.73 −2.69 −3.60 8.81 2.70 −3.92 4.79 5.92 6.61 −7.07 3.71 0.83 6.89
0.53 5.27 3.82 1.26 5.68 8.44 0.37 2.99 4.61 0.05 0.30 4.67 1.91 6.62 4.92 0.86 3.48 5.65 2.37 2.90 4.08 1.04 3.66 6.08 2.66 4.85 7.53 1.49 3.62 5.93 0.89 6.41 3.80 0.06 2.73 3.92 1.04 4.86 3.79 2.05 5.66 8.93 1.94 4.48 6.68 1.25 4.65 6.90 0.05 3.42 6.88 1.29 4.86 8.77 3.52 9.01 6.06 2.39
7.25 1.39 1.29 8.67 1.78 0.65 2.19 3.67 0.42 −6.25 0.75 −0.69 6.94 5.42 3.84 −8.79 6.04 1.83 −7.59 3.18 2.86 −5.94 −0.83 2.81 −9.77 4.81 −2.18 8.71 3.92 −1.66 7.02 3.07 1.94 −6.96 3.21 0.91 10.82 8.73 −4.03 9.89 5.01 0.75 7.84 8.59 −5.66 −9.99 4.73 −5.04 7.69 10.00 −4.71 11.21 8.62 1.47 −9.86 6.88 2.81 8.70
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Journal of Agricultural and Food Chemistry Table 3. continued storage at −20 °C for 15 days
3 freeze−thaw cycles matrix
kidney
analyte
V
IV
IVG
D
stomach
V
IV
IVG
D
intestine
V
IV
IVG
D
brain
V
IV
IVG
D
6 h at room temperature
autosampler for 12 h
concentration (ng/mL)
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12 4 25 200 10 62.5 500 3.2 20 160 0.24 1.5 12
0.92 3.77 3.48 7.69 9.33 2.86 4.93 9.80 3.66 4.71 3.92 0.20 3.76 6.55 2.44 8.60 11.04 8.38 2.94 5.79 0.28 3.87 5.69 1.28 3.44 8.75 3.77 2.95 5.80 2.21 4.86 6.63 1.81 8.83 5.62 0.08 1.93 4.79 2.60 3.87 8.77 0.65 5.66 6.66 2.32 7.94 11.48 0.09 3.21 4.58
6.73 −5.52 10.01 −8.42 4.41 −7.64 4.82 −2.91 8.73 5.31 −1.50 5.41 −3.77 2.02 10.93 6.67 −2.94 9.71 5.83 2.81 7.62 −4.40 3.01 −9.92 7.21 3.80 −8.71 7.53 3.78 −7.64 5.82 8.60 −11.84 7.51 8.44 −9.72 5.73 −6.82 11.05 −5.82 4.79 8.08 4.71 −2.85 9.84 8.66 −5.73 10.06 6.49 −8.77
5.62 5.43 2.48 4.72 7.99 2.33 5.90 9.88 1.77 3.96 8.84 4.60 6.58 6.77 2.85 5.57 5.96 2.99 3.05 1.73 1.25 3.58 6.77 1.39 4.86 7.99 2.07 7.72 9.49 1.84 5.79 8.03 2.87 3.07 4.94 0.87 3.96 4.40 1.77 7.83 8.33 4.36 4.97 4.02 0.92 2.99 10.92 0.39 3.74 4.77
2.74 0.96 −6.66 3.75 −1.84 5.93 2.70 −1.07 11.76 4.82 2.82 −9.09 4.72 3.86 10.74 2.89 −6.74 8.72 7.05 5.91 −9.84 8.32 −1.25 −7.73 5.02 2.98 −6.72 4.21 0.21 −8.88 4.62 0.92 9.62 −3.77 5.81 11.94 −5.92 1.01 4.69 −8.27 2.96 7.62 5.38 −2.97 3.88 −1.29 0.46 8.74 −5.62 2.70
4.90 9.09 2.65 6.68 8.88 3.70 2.86 8.56 2.33 4.64 6.80 2.40 3.86 5.99 1.35 3.59 9.69 2.49 9.40 12.38 2.99 0.93 5.87 0.94 4.58 9.63 2.74 3.88 6.97 2.84 4.86 9.68 2.84 5.97 9.81 1.12 2.42 6.87 3.80 2.95 5.74 1.73 5.97 7.96 3.96 6.06 10.08 3.44 5.85 6.86
8.92 −2.73 7.77 −3.88 1.41 6.02 −5.83 2.00 11.91 8.92 −3.80 8.73 4.87 2.91 9.66 −4.56 6.92 2.91 −3.99 0.74 8.33 5.82 −4.61 7.92 −2.94 1.05 9.03 −5.21 4.92 8.04 4.82 −1.73 7.42 3.85 2.36 9.03 −5.82 1.82 8.73 −4.48 −2.81 9.50 2.84 −5.55 8.20 2.91 −1.04 10.30 4.81 −5.87
3.86 5.00 1.34 5.83 8.01 0.44 4.33 7.52 1.03 2.87 6.43 2.66 4.89 10.48 1.74 3.86 2.58 0.64 4.83 5.91 1.92 4.06 8.94 2.86 4.59 9.48 0.83 3.86 2.44 0.18 3.97 2.77 0.52 4.84 9.69 1.42 5.67 5.98 2.21 3.46 3.10 1.77 4.87 2.85 0.76 7.84 4.82 3.75 5.83 5.91
4.81 −2.01 6.82 5.73 4.44 8.05 6.91 −5.65 −10.78 3.48 −2.06 4.86 6.07 −1.88 7.63 3.33 2.07 −6.77 3.02 −0.55 9.03 −7.88 2.96 10.04 −3.95 2.11 5.82 3.85 4.61 −6.76 2.09 4.82 5.58 5.42 −2.99 6.03 7.44 2.06 5.82 4.79 3.86 −12.03 4.02 −5.20 8.76 7.73 −5.05 8.08 −6.60 5.21
to a 100 μL portion of plasma or tissue homogenate. After vortexing for 1 min, 200 μL of methanol was added, followed by vortexing for 5 min and centrifuging at 10000g for 5 min. The supernatant was evaporated to dryness at 35 °C using a slight stream of nitrogen. The residue was redissolved in 100 μL incipient mobile phase, vortexed for 3 min, and centrifuged at 10000g for 10 min. A 2 μL aliquot of the final solution was injected into the UPLC−MS/MS system for analysis.
200 ng/mL for IVG, and 0.12, 0.6, 1.5, 3, 6, and 15 ng/mL for D. QC samples at low, middle, and high concentrations (4, 25, and 200 ng/mL for V, 10, 62.5, and 500 ng/mL for IV, 3.2, 20, and 160 ng/mL for IVG, and 0.24, 1.5, and 12 ng/mL for D) were also prepared separately in the same way. Sample Pretreatment. Biological samples stored at −20 °C were taken out and thawed at room temperature before use. A total of 20 μL of pueararin solution and 10 μL of methanol were added 5576
DOI: 10.1021/acs.jafc.7b02053 J. Agric. Food Chem. 2017, 65, 5570−5580
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Figure 3. Mean plasma concentration−time curves of the four C-glycosyl flavones in rats after (A) oral administration of MBS extracts and (B) intravenous administration of MBS extracts. Method Validation. The developed method was validated according to the United States Food and Drug Administration (FDA) Bioanalytical Method Validation Guide.18 The selectivity of the method was assessed by analyzing six different sources of blank plasma and tissues, blank plasma and tissues spiked with four C-glycosyl flavones and puerarin, and plasma and tissues obtained after oral or intravenous administration of MBS extracts. Six calibration standards in duplicate were analyzed for three independent runs. The calibration curves, described as y = ax + b, were assessed by plotting the peak area ratios of four C-glycosyl flavones to puerarin against the nominal concentrations by the use of a 1/x2 weighted least squares linear regression model. The method sensitivity was determined by the lower limit of quantification (LLOQ). The criterion for precision [relative standard deviation (RSD)] and accuracy [relative error (RE)] were required to be within ±15%. Precision (RSD) and accuracy (RE) were assessed by analyzing QC samples at low, medium, and high concentrations by the use of six replicates on 3 consecutive days.
The extraction recovery and matrix effects of the four C-glycosyl flavones was calculated according to the following formulas: extraction recovery peak areas of extracted samples = × 100% mean peak areas of spike after extraction samples
matrix effect =
peak areas of spike after extraction samples mean peak areas obtained in pure reference standard solution × 100%
The stability of four C-glycosyl flavones and puerarin was assessed by analyzing six replicates of QC samples at three concentrations under different conditions: 3 freeze−thaw cycles, stored at −20 °C for 15 days, 6 h at room temperature, and stored in the autosampler (4 °C) for 12 h. 5577
DOI: 10.1021/acs.jafc.7b02053 J. Agric. Food Chem. 2017, 65, 5570−5580
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± ± ± ± ± 63.40 0.01 0.47 42.45 48.76 ± ± ± ± ±
IVG
222.92 0.04 0.88 120.97 127.14 ± ± ± ± ±
140.22 0.01 0.25 47.12 47.25 IV
± ± ± ± ± ± ± ± ± ±
F (%) =
19.50 1.58 3.63 103.42 126.21
D
4.95 0.20 2.00 45.50 65.70
178.73 0.04 0.38 34.10 34.18
61.94 0.01 0.14 11.00 11.04
744.08 0.04 0.89 220.87 221.46 V
intravenous (mean ± SD)
± ± ± ± ± 70.80 1.04 1.18 120.36 139.34
IVG
17.90 0.34 0.44 37.78 51.11
RESULTS AND DISCUSSION Optimization of Chromatographic and Mass Conditions. Methanol−water, methanol−water with formic acid, acetonitrile−water, and acetonitrile−water with formic acid were investigated. Methanol was used as the organic phase because it improved the UPLC resolution compared to acetonitrile. For the MS conditions, parameters including collision energy (CE), declustering potential (DP), cell exit potential (CXP), and entrance potential (EP) were optimized and the other parameters were adopted from the recommended value of the instrument. Both positive- and negative-ion detection modes were conducted to find a more sensitive ionization mode. They represented lower background noise and a stronger signal intensity in the positive-ion mode than the negative-ion mode. Thus, intense and stable protonated molecular ion peaks of four C-glycosyl flavones and pueararin were observed in the positive ionization mode ([M + H]+ for V, IV, IVG, D, and IS at m/z 433.17, 433.16, 594.98, 579.31, and 417.17, respectively). Figure 1 shows the main fragmentation pathways and product ions for four C-glycosyl flavones and pueararin, from which we could find that four C-glycosyl flavones and pueararin have similar MS/MS fragmentation behaviors, such as m/z 433.17 [M + H]+ → 282.97 [M + H − C5H10O5]+ for V, m/z 433.16 [M + H]+ → 282.89 [M + H − C5H10O5]+ for IV, m/z 594.98 [M + H]+ → 433.26 [M + H − C6H10O5]+ for IVG, m/z 579.31 [M + H]+ → 432.67 [M + H − C6H11O4]+ for D, and m/z 417.17 [M + H]+ → 296.84 [M + H − C4H8O4]+ for IS. Method Validation. Selectivity. Representative ion chromatograms of blank rat plasma, blank rat plasma spiked with the four C-glycosyl flavones (at LLOQs) and pueararin, and rat
IV
± ± ± ± ± ± ± ± ± ± 36.05 2.01 4.08 158.71 160.66
6.63 0.09 0.27 32.41 31.19
148.49 1.56 5.02 927.65 980.48 V parameter
Cmax (μg/L) Tmax (h) t1/2Z (h) AUC0−t (μg L−1 h−1) AUC0−∞ (μg L−1 h−1)
AUC0i.g. −∞ × dose i.v. × 100% i.v. AUC0 −∞ × dose i.g.
■
60.02 0.62 1.58 160.60 46.00
oral (mean ± SD)
Table 4. Pharmacokintic Parameters of the Four C-Glycosyl Flavones in Rats Following p.o. (2 g/kg) or i.v. (25 mg/kg) Administration of MBS Extracts
16.75 0.04 0.81 9.00 9.24
D
3.05 0.01 0.20 2.26 2.33
Dilution integrity was tested to evaluate the dilution effect on the experiment by a 5-fold dilution of the upper limit of quantification (ULOQ) concentration with the blank biological matrix (plasma and tissue homogenate) for six replicates with acceptable precision and accuracy (RSD < 20%, and RE < ±20%). Pharmacokinetics. For pharmacokinetic studies, male SD rats were randomly divided into two groups, each consisting of five subgroups (n = 8). The first group was administrated with MBS extract solution by tail intravenous injection, and the second group received an oral administration of MBS extract solution. Blood samples (about 250 μL) were obtained approaching at 0 (predose), 5, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480, 720, and 1440 min for p.o. administration and 0 (predose), 2, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, and 360 min for i.v. administration from the fosse orbital vein in heparinized tubes. The blood samples were immediately centrifuged at 10000g for 10 min to obtain the plasma and then stored at −80 °C for analysis. Tissue Distribution. Male SD rats were randomly divided into four groups (6 animals each) for tissue sample collection at 0, 0.5, 1.5, and 4 h after p.o. administration of a dose (2 g/kg) of MBS extracts. Rats were euthanized by excess ether, and tissues were harvested, including the heart, spleen, liver, lung, intestine, stomach, kidney, and brain, which were thoroughly rinsed with ice-cold physiological saline solution to remove blood and wiped dry with filter paper. The accurately weighed tissues were homogenized with normal saline (1:4, w/v) and stored at −80 °C until analysis by UPLC−ESI−MS/MS. Calculation. The calculation of the pharmacokinetic parameters in rats were carried out by the use of statistics software DAS 2.0 software package (Chinese Pharmacological Society). All data were expressed as the mean ± standard deviation (SD). Absolute bioavailability (F) of the four analytes was calculated according to the following formula:
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DOI: 10.1021/acs.jafc.7b02053 J. Agric. Food Chem. 2017, 65, 5570−5580
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Figure 4. Mean concentrations of the four C-glycosyl flavones in various tissues at indicated time points after oral administration of MBS extracts (n = 6).
Stability. The data of stability are shown in Table 3, indicating that the four C-glycosyl flavones and pueararin were stable in the biological matrix after 3 freeze−thaw cycles, at room temperature for 6 h, at −20 °C for 15 days, and at 4 °C for 12 h in processed samples. Pharmacokinetic Analysis and Bioavailability. The developed and validated UPLC−MS/MS method was successfully applied for simultaneous determination of four C-glycosyl flavones after i.v. or p.o. administration of MBS extracts. The mean plasma concentration−time profiles of four C-glycosyl flavones are presented in Figure 3, and the estimated pharmacokinetic parameters were summarized in Table 4. As seen in Figure 3A and Table 4, V, IV, and D exhibited similar pharmacokinetic behaviors as a result of their similar chemical structures. However, t1/2 values of V, IV, IVG, and D were 4.08, 5.02, 1.18, and 3.63 h, respectively, which indicates that IVG is eliminated significantly faster than the other C-glycosyl flavones in vivo after p.o. administration of the MBS extracts, owing to different types of glycosidic bonds (α or β). As seen in Figure 3B and Table 4, the pharmacokinetic behaviors of IV, IVG, and D in rats after i.v. administration of MBS extracts were similar. However, the pharmacokinetic behaviors of V was markedly different from the other C-glycosyl flavones (the t1/2 values of V, IV, IVG, and D were 0.38, 0.89, 0.88, and 0.81 h, respectively) possibly as a result of different types of glycosylation sites (C-8 or C-6) of the analytes, which indicates that V is eliminated significantly faster than the other C-glycosyl flavones in vivo after i.v. administration of the MBS extracts. The absolute bioavailabilities (F, %) of V, IV, IVG, and D for p.o. were different (5.82, 5.53, 1.37, and 17.07%, respectively), and the pharmacokinetic behaviors of V and IV in the MBS extracts were different from that obtained for their pure forms
plasma sample 4 h after a p.o. dose of 2 g/kg of MBS extracts are illustrated in Figure 2. The retention times of V, IV, IVG, D, and IS were 3.29, 4.22, 2.20, 5.03, and 1.22 min, respectively. Cross interference and endogenous interference were not found at the retention times of the four C-glycosyl flavones and puerarin. Linearity and LLOQ. The regression equations, correlation coefficients, LLOQs, and linear ranges of the four analytes are shown in Table 1. In the literature, there is no LC−MS/MS method reported for simultaneous determination of V, IV, IVG, and D. The calibration curves for the four C-glycosyl flavones showed good linearity (r > 0.9917) over a certain range. The LLOQs for V, IV, IVG, and D were 2, 5, 1.6, and 0.12 ng/mL, respectively, which indicate that the method is sensitive for quantitative analysis of the four C-glycosyl flavones. Precision and Accuracy. The intra- and interday precision and accuracy of four C-glycosyl flavones are displayed in Table 2. At each concentration level, the RSD was not more than 13.82% and the accuracy did not exceed ±7.75% for any of the analytes, indicating that this method was accurate, reproducible, and reliable. Extraction Recovery and Matrix Effect. The mean extraction recoveries and matrix effect for the four C-glycosyl flavones in rat plasma/tissue homogenate are displayed in Table 2. The mean extraction recoveries of the four C-glycosyl flavones and pueararin ranged from 72.06 to 99.02%, indicating that the sample preparation method was acceptable. The matrix effects for the four C-glycosyl flavones and pueararin ranged from 85.14 to 109.49%. The precision values of the four C-glycosyl flavones and pueararin were lower than 9.12%, indicating that the matrix effect of our C-glycosyl flavones and pueararin is negligible. 5579
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Journal of Agricultural and Food Chemistry or in other extracts.15,16 These differences were likely due to the complexity of herbal medicines or functional foods. Tissue Distribution. The results showed that the distribution of four C-glycosyl flavones occurred widely and rapidly in various tissues within the time course examined (Figure 4). All of the analytes were detected in tested tissues at 0.5 h after the administration of MBS extracts. Then, the highest concentrations of V, IV, IVG, and D were detected at 1.5 h after administration in all of the tested tissues. Moreover, the concentrations of four C-glycosyl flavones in all of the tested tissues decreased obviously in 4 h, which indicated that there was not a trend of long-term accumulation of these four compounds. The results showed that the analytes exhibited the maximum concentration in stomach and intestine, which may be related to the oral administration. Four C-glycosyl flavones exhibited a higher concentration in the kidney, heart, spleen, liver, and lung, indicating that the perfusion rate and blood flow of the organ played a critical role in the distribution of four C-glycosyl flavones. In addition, four C-glycosyl flavones could be detected in the brain, which indicated that these four compounds could transfer across the blood−brain barrier. In conclusion, a specific and sensitive UPLC−MS/MS method for the simultaneous analysis of V, IV, IVG, and D after p.o. or i.v. administration of MBS extracts to rats was developed and validated. The advantages of the method presented are having a relatively simple sample preparation procedure and a short analysis time of 6.0 min per sample. This was the first time reporting a UPLC−MS/MS method on the simultaneous quantification of four C-glycosyl flavones in rat biological matrices, such as plasma, heart, spleen, liver, kidney, intestine, lung, stomach, and brain. The pharmacokinetic data indicated that the bioavailabilities of V, IV, IVG, and D were 5.82, 5.53, 1.37, and 17.07%, respectively. The pharmacokinetic characteristics and bioavailabilities of the four C-glycosyl flavones showed significant difference as a result of their different types of glycosidic bonds (α or β) and glycosylation sites (C-8 or C-6). In the tissue distribution study, the four C-glycosyl flavones could be distributed widely and rapidly in tested tissues and transfer across the blood−brain barrier. The research provided a significative basis for the application of MBS.
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AUTHOR INFORMATION
Corresponding Authors
*Telephone/Fax: +86-24-43520580. E-mail:
[email protected]. *Telephone/Fax: +86-24-43520580. E-mail:
[email protected]. ORCID
Zhiguo Yu: 0000-0003-1493-8932 Notes
The authors declare no competing financial interest.
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