A validated UHPLC-MS/MS method for pharmacokinetic and brain distribution studies of twenty constituents in rat after oral administration of Jia-Wei-Qi-Fu-Yin

Hai-Ming Ana,1, Meng-Ning Lia,1, Hua Yanga, Han-Qing Panga, Cheng Qua, Yi Xub, Run-Zhou Liua, Chao Penga, Ping Lia,∗, Wen Gaoa,∗

Abstract

A rapid ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QqQ MS/MS) approach with high sensitivity and selectivity was developed for the quantification of twenty compounds, including 9 saponins, 8 flavonoids, 2 oligosaccharide esters and 1 phenolic acid, in rat plasma and brain, which was administrated intragastrically with Jia-Wei-Qi-Fu-Yin (JWQFY), Mass spectrometric detection was operated under multiple reaction monitoring (MRM) mode. All calibration curves possessed good linearity with correlation coefficients (r2) higher than 0.9916 in
Their respective line arranges. Forintra-andinter-dayprecision, all the relative standard deviations (RSDs) at different levels were less than 14.68 %. Based on the UHPLC-QqQ MS/MS quantitative results, pharmacokinetic study and brain distribution Pharmacokinetics of multiple components in JWQFY was then successfully performed.
As a result, constituents with discrepancy structures showed diverse pharmacokinetic and distribution characteristics. For instance, ferulic acid (phenolic acid), -disinapoyl sucrose and tenuifoliside A (oligosaccharide esters) showed short Tmax (< 10 min), whereas the Tmax of ginsenosides Rb1, Rb2 and Rc (ppd-type terpenoid saponins) were much longer (> 4 h). Besides, ferulic acid, epimedin C, icariin, glycyrrhizin, ginsenoside Rb1 and ginsenoside Rg1 were considered as the potential effective ingredients of JWQFY because of their relatively high exposure to blood and brain. Our study would provide relevant information for discovery of pharmacodynamic ingredients, as well as further action mechanisms investigations of JWQFY.

Keywords:
UHPLC-MS/MS
Brain distribution

1. Introduction

Pharmacokinetic and tissue distribution studies of traditional Chinese medicines (TCMs) are of great help in predicting pharmacodynamic substances and explaining their effectiveness mechanism [1,2]. However, TCMs components generally exist low concentration and high background interference in vivo, which have put forward with higher requirements for the sensitivity and selectivity of the analytical instrument [3]. Besides, method development for simultaneous quantification of their multiple components in biological complex matrix are still facing great challenges due to the huge diversity in the category and structure, as well as the wide concentration range of compounds in TCMs [4]. Sensitive and selective analytical methods with high throughput are essential to be established when simultaneously quantifying multiple trace components in complex biological matrixes for studying on pharmacokinetics and tissue distribution of TCMs.
Presently, ultra-high performance liquid chromatography coupled with tandem mass spectrometer (UHPLC-MS/MS) has been becoming a reliable tool for pharmacokinetic research on TCMs due to its high sensitivity and wide compound coverage [5–7]. In particular, triple quadrupole tandem mass spectrometry (QqQ MS/MS) technique showed greater capability in discriminating against the co-eluting species and reducing of background interference for biological quantitative analysis with its multiple reaction monitoring (MRM) mode [8]. Therefore, UHPLC-QqQ MS/MS is widely applied in the simultaneous quantification of multiple compounds in complex matrix, especially in application to the further pharmacokinetic study as well as tissue distribution analysis of multiple components in TCMs.
Jia-Wei-Qi-Fu-Yin (JWQFY), consisting of nine original herbs of Ginseng Radix et Rhizoma (Renshen), Rehmanniae Radix Praeparata (Shudi), Angelicae Sinensis Radix (Danggui), Atractylodis Macrocephalae Rhizoma Praeparata (Chaobaizhu), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (Zhigancao), Ziziphi Spinosae Semen (Suanzaoren), Polygalae Radix Praeparata (Zhiyuanzhi), Acori Tatarinowii Rhizoma (Shichangpu) and Epimedii Folium Praeparata (Zhiyinyanghuo), is a new developing prescription for treatment on Alzheimer’s Disease. Although previous studies revealed that JWQFY showed the major effect on memory improvement and neuroprotection [9–13], its potential bioactive compounds and effective mechanism were still unclear. In present study, a rapid UHPLC-QqQ MS/MS method with high sensitivity and selectivity was established for simultaneous quantification of twenty constituents in rat plasma after oral administration of JWQFY and applied to their further studies on pharmacokinetics and brain distribution. This study could provide reliable evidence for discovery of potential pharmacodynamic ingredients, as well as valuable information for further quality evaluation and mechanisms investigations of this prescription.

2. Material and methods

2.1. Chemicals and reagents

Acetonitrile, methanol, provided by Merck (Darmstadt, Germany), and formic acid, provided ROE Scientific Inc. (Newark, DE, USA), are both in HPLC-grade. Ultra-pure water (18 M cm) was purified through a Millipore Milli-Q water purification system (Bedford, MA, USA). Other chemicals and reagents were analytical grade. Freeze-dried JWQFY powder (TQ170928) was supplied by Beijing Zhongyan Tongrentang Pharmaceutical R&D co., Ltd. (Beijing, China). JWQFY powder was dissolved using 0.5 % carboxymethyl cellulose sodium (CMC-Na) solution to prepare 0.15, 0.30, 0.45 g/mL suspension for oral administration.

2.2. Experimental animals

Male Sprague-Dawley (SD) rats (220 ± 20 g), obtained from Sino-British Sippr/BK Lab Animal Ltd. (Shanghai, China), were randomly divided into three groups. Group A (n = 5) was for the acquisition of blank plasma samples and blank brain samples, group B (n = 15) for pharmacokinetic study and group C (n = 18) for brain distribution study. All the rats were free for water and standard food and housed in a controlled environment. Only water was available for the rats 12 h before gavage. The animal experiments were approved by Department of Science and Technology Jiangsu Province (license No.: 220170272) and conducted according to the guidelines of Provision and General Recommendation of Chinese Experimental Animals Administration Legislation.

2.3. Instruments and chromatographic condition

The LC–MS/MS analysis was carried out by using a Shimadzu LC-30AD UHPLC system (Kyoto, Japan) combined with a Shimadzu 8050 QqQ MS/MS (Kyoto, Japan). Separation was performed on an Agilent Eclipse Plus C18 column (2.1 × 100 mm, 1.8 m), using 0.1 % (v/v) formic acid in water (A) and acetonitrile (B) as mobile phase. The flow rate was 0.4 mL/min, and the column temperature was set at 35 ◦C. The auto-sampler was kept at 4 ◦C and the injection volume was 5 L. An optimized gradient program was established as follows: 8% B at 0−1 min, 8–15 % B at 1−3 min, 15–20 % B at 3−5 min, 20–24 % B at 5−7 min, 24–33 % B at 7−10 min, 33–60 % B at 10−13 min, 60−100% B at 13−15 min and 100 % B at 15−17 min. The parameters of ESI source were set as follows: interface temperature, 300 ◦C; heating gas flow rate, 10 L/min; drying gas flow rate, 10 L/min; nebulizer gas flow, 3 L/min; interface voltage, 3000 V. The analyte confirmation was performed by using retention time and multi-reaction monitoring (MRM) in positive or negative ionization modes according to optimized condition of each analytes (Table 1). The dwell time of each ion pair was 40 ms. The data acquired by UHPLC-QqQ MS/MS were processed using Shimadzu Labsolutions LCMS Version 5.65 (Kyoto, Japan).

2.4. Preparation of quality control (QC) samples

Twenty analytes were accurately weighed, then dissolved and mixed in methanol to prepare stock solution. The stock solution was serially diluted with methanol to a linear concentration gradient obtain working solutions. A mixed internal standard (IS) solution was prepared in methanol with imatinib (IS1, final concentration: 25.1 ng/mL) and propafenone hydrochloride (IS2, final concentration: 3.2 ng/mL). All the solutions were stored at 4 ◦C in dark. Calibration standards were prepared by spiking the working solution and mixed IS solution into blank rat plasma or tissue homogenate. The quality control samples (in low, middle and high level) were prepared in the same way in rat plasma or brain homogenates.

2.5. Preparation of plasma and brain samples

For pharmacokinetic study, 90 L plasma sample was mixed with 10 L IS and 270 L methanol: acetonitrile 2:3 (v/v) solution. After vortexing for 5 min, the mixture was centrifuged at 13,000 rpm for 10 min. The supernatant was transferred and evaporated to dryness using a Speed Vac System (EZ-2, Gene Vac Corporation, U.K.). The residue was then dissolved with 90 L of 50 % acetonitrile, vortex-mixed for 2 min. After centrifugation at 13,000 rpm for 10 min, the supernatant was then drawn out for analysis.
For brain distribution study, each brain sample was weighed and homogenized with 2-fold volume of normal saline solution. Methanol and acetonitrile were respectively used to extracted analytes from homogenate because of the 20 analytes belong to different chemical properties. In brief, 200 L homogenized samples was added 10 L IS. Then, sample was mixed with 600 L methanol (method A) or acetonitrile (method B), then was vortexed for 5 min. After centrifugation, the supernatant was evaporated to dryness. The residue was dissolved with 100 L 50 % acetonitrile, and the subsequent steps were same with the description for the treatment of plasma samples. Liquiritin (1), tenuifoliside A (6), ginsenoside Rg1 (8), epimedin C (10), icariin (11), tenuifolin (13), ginsenoside Rc (14), ginsenoside Ro (15), jujuboside B (18), glycyrrhizin (19) and baohuoside I (20) were analyzed in methanol (method A) treated sample, while liquiritin apioside (2), ferulic acid -disinapoyl sucrose (4), isoliquiritin (5), ginsenoside Re (7), liquiritigenin (9), ginsenoside Rb1 (12), ginsenoside Rb2 (16), isoliquiritigenin (17) were analyzed in that of acetonitrile treated (method B). Optimization of sample preparations were shown in supplemental materials.

2.6. Method validation

2.6.1. Specificity

The specificity of the developed method was examined by comparing the chromatograms of blank samples, blank samples spiked with the standards of analytes and IS, and rat samples after oral administration of JWQFY.

2.6.2. Linearity and LLOQ

The calibration curves were constructed by plotting the peak area ratios of the analytes to IS versus the concentrations of the analyte using linear regression analysis (1/X as the weighing factor). The lower limit of quantification (LLOQ), of each analyte, was defined as its lowest concentration with the satisfactory accuracy error (± 20 %) and precision variation (< 20 %).

2.6.3. Accuracy and precision

Analytes in QC samples at three concentration levels (low, medium and high) were determined by the calibration curves to confirm the accuracy and precision. The intra-day and inter-day accuracy and precision were evaluated on one day and on three consecutive days respectively.

2.6.4. Matrix effect and extraction recovery

The matrix effects of 20 analytes in plasma and brain homogenate samples were conducted by calculating the peak areas ratio between post-extraction plasma spiked with analyte versus unextracted standards at three concentration levels. The extraction recoveries were assessed by comparing the ratio of peak area of analytes with IS spiked before and after extraction.

2.6.5. Stability

Short-term stability was investigated by measuring QC samples after 4 h exposure under room temperature. Autosampler stability was assessed by analyzing the QC samples after being storied in the autosampler (control as 4 ◦C) for 12 h. Freeze-thaw stability was evaluated over three freeze-thaw cycles.

2.7. Pharmacokinetic and brain distribution studies

For pharmacokinetic study, rats from group B were further randomly assigned to three groups (B1, B2, B3) and administered with JWQFY at the dose of 1.5, 3.0, 4.5 g/kg, respectively. Approximately 300 L blood was collected from each rat at 0.083 h, 0.167 h, 0.25 h, 0.5 h, 0.75 h, 1 h, 2 h, 4 h, 6 h, 9 h, 12 h and 24 h after administration, the plasma samples were obtained after centrifugation at 3000 rpm for 10 min (4 ◦C) and stored at −80 ◦C until treatment.For brain distribution study, rats from group C were administered with JWQFY at a single dose of 3.0 g/kg and they were sacrificed at 0.083 h, 0.25 h, 0.5 h, 1 h, 3 h and 6 h after administration. The brain was harvested and then store at −80 ◦C until treatment.

2.8. Data analysis

The pharmacokinetic parameters, including maximum concentration (Cmax), the time to reach the maximum concentration (Tmax), elimination half-life (t1/2), area under the plasma concentration versus time curve (AUC), clearance (CL) and apparent volume of distribution (V) were calculated by a non-compartmental pharmacokinetic analysis with the DAS 2.0 software (Drug and Statistics 2.0, Mathematical Pharmacology Professional Committee of China, Shanghai, China).

3. Results and discussion

3.1. Optimization of LC–MS/MS conditions

To achieve good separation and high efficiency, a gradient program was attentively optimized to quantify twenty constituents (the chemical structures listed in Fig. 1) in 17 min. The multiple reaction monitoring (MRM) mode was performed to obtain a better selectivity and sensitivity for analytes and IS. The MRM parameters are presented in Table 1.

3.2. Method validation

3.2.1. Selectivity

The chromatograms of representative analytes in blank biosamples spiked with analytes and IS, bio-samples after oral administration of JWQFY, and blank bio-samples are illustrated in Fig. 2 (plasma samples) and Fig. S1 (brain tissue samples). No peak with great intensity was observed in blank samples at the same retention times of all the analytes.

3.2.2. Linearity and LLOQ

The linearity results, including calibration curves, correlation coefficients, linear ranges and LLOQ of 20 analytes in plasma and brain homogenates are summarized in Table 2. The correlation coefficients (R2) of all calibration curves were not less than 0.9916, indicating that the linearity was relatively good.

3.2.3. Accuracy and precision

As showed in Table S1, the relative standard deviations (RSDs) of QC samples at three concentration levels were measured to be in the range of 0.55–14.68 % for intra-day precision and 0.95–14.21 % for inter-day precision. The accuracy of intra-day and interday were -14.47–14.83 % and -14.82–14.20 %, respectively. These results suggested the methods for the determination of analytes in rat plasma and brain samples were accurate and reproducible.

3.2.4. Matrix effect and extraction recovery

The matrix effect and extraction recovery are given in Table S2. The range of the matrix effect was 78.70–132.67 %. The extraction recovery of constituents at three concentration levels in rat plasma and brain homogenates was in the range of 82.35–123.14 %.

3.2.5. Stability

The stability of analytes in rat plasma and tissue are provided in Table S3. The results demonstrated that all analytes could remain stable after exposure at room temperature for 4 h, at room temperature in the auto-sampler for 12 h and after three freeze-thaw cycles with RSD values less than 14.76 %.

3.3. Pharmacokinetic and brain distribution studies

The validated method was further applied to investigation of pharmacokinetic parameters and brain distribution characteristics of 20 constituents in rats after oral administration of JWQFY. Mean plasma concentration-time profiles of these analytes are illustrated in Fig. 3. The pharmacokinetic parameters including Cmax, Tmax, t1/2, AUC, CL and V of the analytes were calculated by a non-compartmental pharmacokinetic analysis and the data are listed in Table 3. A common characteristic of these analytes is their Tmax were short, possibly due to the interaction of co-existing compounds in this formula. However, compounds with discrepancy structures showed different pharmacokinetic and distribution characteristics. By comparing the AUC0-∞ and Cmax of three different doses (shown in Table S4), we found the AUC0-∞ and Cmax of most constituents increased in a dose dependent manner but not proportionally. Most of the analytes could rapidly distribute into the brain and their brain distribution profiles are shown in Fig. 4.

3.3.1. Pharmacokinetic and brain distribution characteristics of flavonoids

Flavonoids, variously found in herbal medicine, have been identified as promising constituents for improving learning and memory [14]. We investigated eight flavonoids in JWQFY: Liquiritin, liquiritin apioside, isoliquiritin, liquiritigenin and isoliquiritigenin were flavonoids reported from Glycyrrhizae Radix et Rhizoma [15]. Their Tmax and t1/2 were observed at 7−17 min and 5−10 h, respectively. Liquiritigenin and isoliquiritigenin showed the phenomenon of double peaks but others appeared only one, which suggested these constituents might have multiple absorption sites, or there were compounds interactions [16]. Their AUC0-∞ values were relatively low, indicating their low exposure levels in blood. Among them, liquiritin (5.67, 10.34, 13.33 ng h/mL) and liquiritin apioside (21.34, 26.13, 43.30 ng h/mL) obtained higher AUC0-∞ than the others (< 6.81 ng h/mL), possibly because of their higher content in the formula [17]. The concentration of these ingredients in brain were not high (≤ 2.01 ng/g), demonstrating their low levels of accumulation in brain.
Flavonoids reported from Epimedii Folium have been shown to beneficially influence the brain function [18,19]. Here, epimedin C, icariin and baohuoside I were determined. The results showed these compounds were detected at the highest concentration in blood at 5−15 min and in brain at 5−15 min after administration, indicating they could be extensively absorbed in blood and quickly distributed into the brain. Compared with the other analytes, epimedin C and icariin showed relatively higher Cmax values in plasma and brain, implying their higher absorption to blood and brain. It is universally acknowledged that compounds with enough exposure to target tissue or blood are considered as the key pharmacodynamic constituents for the clinical effects [2]. Therefore, we deduced epimedin C and icariin might be the potential effective ingredients in JWQFY.

3.3.2. Pharmacokinetic and brain distribution characteristics of oligosaccharide esters

Two oligosaccharide esters with great biological interest [20,21], namely -disinapoyl sucrose and tenuifoliside A, were detected in this study. Results showed the Cmax of these constituents were relatively higher than that of the others. Their short Tmax (5−10 min) and t1/2 (3−7 h) suggesting the assimilation rate and elimination rate of these components were quick. In brain distribution study, we found although they showed obviously high Cmax in blood, their concentration in brain were not as high as the other compounds. Tenuifoliside A were not quantified in brain samples for the low content (< LLOQ).

3.3.3. Pharmacokinetic and brain distribution characteristics of phenolic acid

Ferulic acid was tested in this study. Although its Tmax (within 7 min) was short, the Cmax of ferulic acid was extremely high (259.46–310.41 ng/mL), leading to a remarkably high level of exposure in blood. These phenomena might be partially caused by the high gastric absorption efficiencies and low excretion of ferulic acid, which was consistent with the related previous literatures [22–24]. Besides, ferulic acid presented fairly good and fast distribution in brain, due to the short time (5 min) to reach the highest maximum content in brain (180.354 ng/g). These results implied the brain might be the target organ of ferulic acid, which could consequently improve cognitive skills and neurodegeneration [25]. Accordingly, ferulic acid might act as a significant component in JWQFY for treating degenerative diseases.

3.3.4. Pharmacokinetic and brain distribution characteristics of saponins

Saponins are often the principal active compounds in herbal medicines [26]. JWQFY contains abundant triterpene saponins with diverse structures and we selected some common triterpene saponins including ginsenosides probably from Ginseng Radix et Rhizoma [17], glycyrrhizin from Glycyrrhizae Radix et Rhizoma [15], tenuifolin from Polygalae Radix [17] and jujubosides from Ziziphi Spinosae Semen [27] in this research.
Ginsenosides are the most major triterpene saponins in JWQFY, they are commonly classified as 20(S)-protopanaxadiol type (PPD), 20(S)-protopanaxatriol type (PPT) or oleanolic acid type (OA) based on their aglycone structure. The PPD-type ginsenosides Rb1, Rb2 and Rc and the PPT-type ginsenosides Re, Rg1, as well as the OAtype ginsenoside Ro were evaluated. Most of saponins were poorly absorbed in the intestine, in line with the low membrane permeability caused by their physical and chemical properties, such as high molecular weight and high hydrogen-bonding capacity [28]. Another factor restricting their oral bioavailability is the rapid and extensive bile excretion. However, several saponins, including ginsenosides Rb1, Rc and Rd, are slowly excreted into the bile and, in turn, circulate for a long period [29], which is consistent with our results. The t1/2 (> 10 h) and Tmax (> 4 h) of ginsenosides Rb1, Rc and Rb2 were significantly longer than that of others, whereas the t1/2 (4−9 h) and Tmax (10−15 min) of ginsenosides Re, Rg1 and Ro were much shorter. Ginsenosides were poorly delivered into the central nervous system (CNS), mainly due to their poor membrane permeability. Ginsenosides Rc and Rb2 were not quantified in brain samples for the low content (< LLOQ). The rat brain exposure levels of ginsenosides Rb1 and Rg1 were comparatively higher than the corresponding levels for other ginsenosides. Furthermore, previous studies demonstrated ginsenosides Rb1 and Rg1 could benefit to the clinical treatment of AD through decreasing phospho-GSK3 and -amyloid formation [30,31]. Accordingly, their potentialities for treating CNS diseases should not be overlooked.
Apart from the ginsenosides, glycyrrhizin and tenuifolin, which are comprised of an OA-type pentacyclic triterpene saponin, were also analyzed. The data showed both of them could be detected immediately after oral administration in plasma and the time to peak concentration were observed at 10−40 min. Apparently, they could be quickly absorbed into blood circulatory system. The t1/2 of glycyrrhizin and tenuifolin were similar with other saponins, at about 5−9 h. In brain distribution study, glycyrrhizin showed a higher concentration level. Meanwhile, glycyrrhizin was reported with obvious effects on alleviating neuroinflammation [32], suggesting it might also be one of the important bioactive components in JWQFY.
Jujubosides A and B are generally regarded as the main constituents responsible for the effects of Ziziphi Spinosae Semen. However, in this study we found that although jujubosides A and B have analogous chemical construction and similar content in JWQFY [17], their concentration in blood and brain existed large difference. Jujuboside A was in an extremely low concentration and even could not be detected in blood and brain. Evidence implicates that jujuboside A could be absorbed into the body at only a trace amount and might not be the real active form [33]. In contrast, jujuboside B, one of the main hydrolysis products of jujuboside A, might be the real absorbed form with the specific bioactivity [33]. Our study shows that the plasma concentration of jujuboside B became highest at approximately 10 min and decreased sharply in 1 h, indicating it could be quickly absorbed and cleared from the rat plasma.

4. Conclusion

In this work, a rapid, selective, and sensitive method based on UHPLC-QqQ MS/MS technology was developed for determination of twenty components of JWQFY in rat plasma and brain, including 9 saponins, 8 flavonoids, 2 oligosaccharide esters and 1 phenolic acid. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with both positive and negative electrospray ionization. The pharmacokinetic and brain distribution characteristics of multiple ingredients in JWQFY were reported for the first time. Several obvious differences were observed in pharmacokinetic and brain distribution characteristics of the constituents owing to their different structures. For instance, ferulic acid (phenolic acid), -disinapoyl sucrose and tenuifoliside A (oligosaccharide esters) showed short Tmax (< 10 min), whereas the Tmax of ginsenosides Rb1, Rb2 and Rc (ppd-type terpenoid saponins) were much longer (> 4 h). Furthermore, ferulic acid, epimedin C, icariin, glycyrrhizin, ginsenoside Rb1 and ginsenoside Rg1 were considered as the potential pharmacodynamic compounds of JWQFY due to their relatively high exposure to blood and brain. This study could not only provide reliable evidence for discovery of the effective components, but also facilitate further research on the action mechanism of JWQFY.

References

[1] D.Y. Guo, J.L. Dang, H. Yang, Y. Fan, J.X. Cheng, Y.J. Shi, X.F. Zhang, J.B. Zou, Simultaneous determination of four flavonoids in rat plasma after oral administration of Malus hupehensis (Pamp.) Rehd. extracts by UPLC-MS/MS and its application to a pharmacokinetics study, J. Pharm. Biomed. Anal. 177 (2019), 112869.
[2] X.Y. Tang, Z.Q. Dai, Q.C. Wu, J.X. Zeng, M.X. Gao, H.H. Xiao, Z.H. Yao, Y. Dai, X.S. Yao, Simultaneous determination of multiple components in rat plasma and pharmacokinetic studies at a pharmacodynamic dose of Xian-Ling-Gu-Bao capsule by UPLC-MS/MS, J. Pharm. Biomed. Anal. 177 (2019), 112836.
[3] J.J. Zhang, P.P. Jia, X.M. Qin, Applications progress of mass spectrometry in analysis of Traditional Chinese Medicine, J. Instrum. Anal. 36 (2017) 579–587.
[4] Y.X. Zhong, X.L. Jin, S.Y. Gu, Y. Peng, K.R. Zhang, B.C. Ou-Yang, Y. Wang, W. Xiao, Z.Z. Wang, J.Y. Aa, G.J. Wang, J.G. Sun, Integrated identification, qualification and quantification strategy for pharmacokinetic profile study of Guizhi Fuling capsule in healthy volunteers, Sci. Rep. 6 (2016) 31364.
[5] K. Yang, X.M. Long, J.J. Cao, Y.J. Li, Y. Wu, X. Bai, Z.L. Sun, Z.Y. Liu, An analytical strategy to explore the multicomponent pharmacokinetics of herbal medicine independently of standards: Application in Gelsemium elegans extracts, J.Pharm. Biomed. Anal. 176 (2019), 112833.
[6] R. Liu, Q. Liu, B. Li, L. Liu, D. Cheng, X. Cai, W. Liu, W. Wang, Pharmacokinetics, bioavailability, excretion, and metabolic analysis of Schisanlactone E, a bioactive ingredient from Kadsura heteroclita (Roxb) Craib, in rats by UHPLC-MS/MS and UHPLC-Q-Orbitrap HRMS, J. Pharm. Biomed. Anal. 177 (2019), 112875.
[7] C. Liang, J. Yin, Y. Ma, X. Zhang, L. Zhang, Quantitative determination of characteristic components from compound of Lysionotus pauciflorus Maxim.By LC-MS/MS and its application to a pharmacokinetic study, J. Pharm.Biomed. Anal. 177 (2019), 112835.
[8] T.Y. Guan, Y. Liang, C.Z. Li, L. Xie, G.J. Wang, L.S. Sheng, Recent development in liquid chromatography/mass spectrometry and allied topics for Traditional Chinese Medicine research, Chin. J. Nat. Med. 9 (2011) 385–400.
[9] W.Y. Ong, Y.J. Wu, T. Farooqui, A.A. Farooqui, Qi Fu Yin-a ming dynasty prescription for the treatment of dementia, Mol. Neurobiol. 55 (2018) 7389–7400.
[10] S.Y. Wang, J.P. Liu, W.W. Ji, W.J. Chen, Q. Fu, L. Feng, S.P. Ma, Qifu-Yin attenuates AGEs-induced Alzheimer-like pathophysiological changes through the RAGE/NF-B pathway, Chin. J. Nat. Med. 12 (2014) 920–928.
[11] J.P. Liu, Q. Wang, Z. Hu, S. Wang, S. Ma, Effect the AGEs/RAGE/NF-B pathway in Alzheimer’s disease model rats of Qifu Yin, Pharmacol. Clin. Chin. Mater.Med. 31 (2015) 9–11.
[12] Z. Song, F. Yin, B. Xiang, B. Lan, S. Cheng, Systems pharmacological approach to investigate the mechanism of Acori Tatarinowii Rhizoma for alzheimer’s disease, Evid. Complement. Alternat. Med. 2018 (2018), 5194016.
[13] J.H. Cho, J.Y. Jung, B.J. Lee, K. Lee, J.W. Park, Y. Bu, Epimedii Herba: A Promising Herbal Medicine for Neuroplasticity, Phytother. Res. 31 (2017) 838–848.
[14] R.J. Williams, J.P. Spencer, Flavonoids, cognition, and dementia: actions, mechanisms, and potential therapeutic utility for Alzheimer disease, Free Radical Biol. Med. 52 (2012) 35–45.
[15] Z.B. Guan, M. Wang, Y. Cai, H.M. Yang, M. Zhao, C.J. Zhao, Rapid characterization of the chemical constituents of Sijunzi decoction by UHPLC coupled with Fourier transform ion cyclotron resonance mass spectrometry, J.Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1086 (2018) 11–22.
[16] Y.P. Wu, X.S. Meng, Y.R. Bao, S. Wang, Pharmacokinetic study of four flavones of Glycyrrhiza in rat plasma using HPLC-MS, J. Ethnopharmacol. 148 (2013) 266–270.
[17] M.N. Li, X. Dong, W. Gao, X.G. Liu, R. Wang, P. Li, H. Yang, Global identification and quantitative analysis of chemical constituents in traditional Chinese medicinal formula Qi-Fu-Yin by ultra-high performance liquid chromatography coupled with mass spectrometry, J. Pharm. Biomed. Anal.114 (2015) 376–389.
[18] Y.J. Chen, H.Y. Zheng, X.X. Huang, S.X. Han, D.S. Zhang, J.Z. Ni, X.Y. He, Neuroprotective effects of Icariin on brain metabolism, mitochondrial functions, and cognition in triple-transgenic alzheimer’s disease mice, CNS Neurosci. Ther. 22 (2016) 63–73.
[19] L.Z. He, Y.Y. Deng, J.M. Gao, L.G. Zeng, Q.H. Gong, Icariside II ameliorates ibotenic acid-induced cognitive impairment and apoptotic response via modulation of MAPK pathway in rats, Phytomedicine. 41 (2014) 74–81. [20] Y. Hu, H.B. Liao, G. Dai-Hong, P. Liu, Y.Y. Wang, K. Rahman, Antidepressant-like effects of 3,6’-disinapoyl sucrose on hippocampal neuronal plasticity and neurotrophic signal pathway in chronically mild stressed rats, Neurochem. Int. 56 (2010) 461–465.
[21] X. Wang, Y. Zhang, H. Niu, Y. Geng, B. Wang, X. Yang, P. Yan, Q. Li, K. Bi, Ultra-fast liquid chromatography with tandem mass spectrometry determination of eight bioactive components of Kai-Xin-San in rat plasma and its application to a comparative pharmacokinetic study in normal and Alzheimer’s disease rats, J. Sep. Sci. 40 (2017) 2131–2140.
[22] Z.H. Zhao, M.H. Moghadasian, Chemistry, natural sources, dietary intake and pharmacokinetic properties of ferulic acid: a review, Food Chem. 109 (2008) 691–702.
[23] Z.H. Zhao, Y. Egashira, H. Sanada, Ferulic acid is quickly absorbed from rat stomach as the free form and then conjugated mainly in liver, J. Nutr. 134 (2004) 3083–3088.
[24] A. Adam, V. Crespy, M.A. Levrat-Verny, F. Leenhardt, M. Leuillet, C. Demigne, C. Remesy, The bioavailability of ferulic acid is governed primarily by the food matrix rather than its metabolism in intestine and liver in rats, J. Nutr. 132 (2002) 1962–1968.
[25] E. Mhillaj, S. Catino, F.M. Miceli, R. Santangelo, L. Trabace, V. Cuomo, C. Mancuso, Ferulic acid improves cognitive skills through the activation of the heme oxygenase system in the rat, Mol. Neurobiol. 55 (2018) 905–916.
[26] L.P. Christensen, Ginsenosides chemistry, biosynthesis, analysis, and potential health effects, Adv. Food Nutr. Res. 55 (2009) 1–99.
[27] B. Yang, W. Dong, A.H. Zhang, H. Sun, F.F. Wu, P. Wang, X.J. Wang, Ultra-performance liquid chromatography coupled with electrospray ionization/quadrupole-time-of-flight mass spectrometry for rapid analysis of constituents of suanzaoren decoction, J. Chromatogr. B Analyt. Technol.Biomed. Life Sci. 34 (2011) 3208–3215.
[28] K. Yu, F. Chen, C. Li, Absorption, Disposition, and Pharmacokinetics of Saponins from Chinese Medicinal Herbs: What Do We Know and What Do We Need to Know More? Curr. Drug Metab. 13 (2012) 557–598.
[29] H. Liu, J. Yang, F. Du, X. Gao, X. Ma, Y. Huang, F. Xu, W. Niu, F. Wang, Y. Mao, Y. Sun, T. Lu, C. Liu, B. Zhang, C. Li, Absorption and disposition of ginsenosides after oral administration of Panax notoginseng extract to rats, Drug Metab.
Dispos. 37 (2009) 2290–2298.
[30] X.Y. Song, J.F. Hu, S.F. Chu, Z. Zhang, S. Xu, Y.H. Yuan, N. Han, Y. Liu, F. Niu, X. He, N.H. Chen, Ginsenoside Rg1 attenuates okadaic acid induced spatial memory impairment by the GSK3beta/tau signaling pathway and the Abeta formation prevention in rats, Eur. J. Pharmacol. 710 (2013) 29–38.
[31] Y. Wang, J. Liu, Z. Zhang, P. Bi, Z. Qi, C. Zhang, Anti-neuroinflammation effect of ginsenoside Rbl in a rat model of Alzheimer disease, Neurosci. Lett. 487 (2011) 70–72.
[32] J.H. Song, J.W. Lee, B. Shim, C.Y. Lee, S. Choi, C. Kang, N.W. Sohn, J.W. Shin, Glycyrrhizin alleviates neuroinflammation and memory deficit induced by systemic lipopolysaccharide treatment in mice, Molecules 18 (2013) 15788–15803.
[33] P. Song, Y. Zhang, G. Ma, Y. Zhang, A. Zhou, J. Xie, Gastrointestinal absorption and metabolic dynamics of jujuboside a, a saponin derived from the seed of Ziziphus jujuba, J. Agric. Food Chem. 65 (2017) 8331–8339.