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Online coupling of the Ussing chamber, solid-phase extraction and high-performance liquid chromatography for screening and analysis of active constituents of traditional Chinese medicines
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Online coupling of the Ussing chamber, solid-phase extraction and high-performance liquid chromatography for screening and analysis of active constituents of traditional Chinese medicines Dandan Wang a,1, Jinxiang Zeng b,1, Wei Xiang a, Manni Yin a, Guoyue Zhong b,∗, Zhining Xia a,∗ a
School of Pharmaceutical Sciences, Chongqing University, Chongqing, 401331, China Research Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang, 330004, China
b
a r t i c l e
i n f o
Article history: Received 17 June 2019 Revised 18 August 2019 Accepted 22 August 2019 Available online xxx Keywords: Traditional Chinese medicines Active constituents Flavonoid Ussing chamber-SPE–HPLC Online screening Intestinal absorption
a b s t r a c t A semi-automated online platform was established successfully for preliminary screening of potential active flavonoids of traditional Chinese medicines (TCMs) in multicomponent system. Online coupling of the in vitro intestinal absorption model, solid phase extraction (SPE) and high-performance liquid chromatography (HPLC) was actualized at the first time. The Ussing chamber model was selected to absorb the constituents of TCMs. A mini chromatographic column filled with C18 was used as a SPE column for online enrichment of flavonoids. HPLC was applied to analyze the constituents screened by platform. With the use of rutin as a model flavonoid, the specifications of SPE column, eluting solvent, elution time and flow rate of eluent were systematically investigated to optimize online system. Under the optimal conditions, the linear range of rutin was 0.125–368 μg/mL with the correlation coefficient (R2 ) greater than 0.9947. The limit of detection (LOD) was as low as 0.0500 μg/mL and the limit of quantification (LOQ) was 0.125 μg/mL. The intra-day relative standard deviation (RSD) and inter-day RSD was 2.5% and 3.8%, respectively. The recoveries of rutin in the intestinal absorption samples ranged from 93.2% to 94.0%. Finally, the online system was applied to screen the potential active flavonoids of Scutellaria baicalensis Georgi (Huangqin, HQ) and Polygoni Cuspidati Rhizoma et Radix (Huzhang, HZ). A total of 14 flavonoids of these two TCMs were identified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS), and 12 flavonoids of them were screened as the potential active components by online Ussing chamber-SPE–HPLC. In comparison with offline method and gavage in rats, the online system can screen the active constituents from TCMs more accurately and completely. The results demonstrated that the online system was reliable and sufficiently accurate for screening and determination of the potential active flavonoids of TCMs in multicomponent system. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Traditional Chinese medicines (TCMs) possess a long history and remarkable curative effect. TCMs have been widely applied in prevention and treatment of diseases, relief of anxiety, weight loss and etc. [1]. In virtue of their powerful medicinal properties, TCMs have been spread beyond China and Asia, with increasing worldwide acceptance [2]. However, the unclear therapeutic basis of most TCMs has become a bottleneck to restrict the development
∗
1
Corresponding authors. E-mail addresses: [email protected] (G. Zhong), [email protected] (Z. Xia). These authors contributed equally to this work.
of TCMs. Bioactive components are the basis of TCMs pharmacodynamics and the important source of new drug development [3–5]. Screening of active constituents is an indispensable part in clarifying the therapeutic material basis of TCMs and searching for active ingredients producing pharmacodynamic effects in TCMs [6]. Hence, it is very important for the modernization research of TCMs to screen the active constituents from TCMs rapidly and effectively. The classical study method of active ingredients of TCMs is to extract total components, extract a single component, and then determine its active ingredients through pharmacological experiments [7–9]. Although this method based on phytochemistry can screen the active constituents from TCMs, it has complicated steps, high labor intensity and low probability of success. Moreover, the chemical constituents of TCMs are complex and diverse. The
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pharmacodynamic material basis of TCMs does not simply consider the additive effects of all the components but treats the TCMs as a potentially synergistic combination of its various active components [10]. In the complex matrixes of TCMs, some components have pharmacological activity in vitro, but they may be transformed and lose their function in vivo, and some components even cannot be absorbed by organisms [11]. Therefore, it is urgent to develop a universal, effective, quick and practicable method or platform for preliminary screening of the active components of TCMs in multicomponent system. Oral administration is an important route of administration for TCMs, and the small intestine is the main site for absorption of oral drugs. [12]. Only the components absorbed into the body have the opportunity to play a pharmacological role and can be defined as the real active ingredients. The ingredients that can be absorbed will be quickly and effectively screened by intestinal absorption models. In recent years, intestinal absorption models such as in vivo animal model [11,13], everted rat gut sac model [14], in situ single-pass intestinal perfusion [15], Ussing chamber model [16,17] and cell model [18,19] have been widely used to study the absorption kinetics, absorption mechanism, effective absorption sites, factors affecting absorption and bioavailability of active ingredients. In particularly, the Ussing chamber model has been used to study the potential active components in complex matrix of TCMs, and these components were detected by highperformance liquid chromatography-mass spectrometry (HPLC–MS) [17]. Regrettably, the processing of intestinal absorbent sample is complicated, samples need to be removed protein, centrifuged, enriched and re-dissolved before monitoring and analysis. Despite the high sensitivity of HPLC–MS, the analysis of highly complex samples like the intestinal absorbent samples of TCMs is often error prone due to the species diversity and content differentia of components in TCMs [20]. To avoid this problem, the components in intestinal absorption samples of TCMs will be purified and enriched before detection [18]. Solid phase extraction (SPE) is the predominant technique to enrich the components, which has been widely combined with HPLC for enrichment, clean-up and detection of samples. However, most of the SPE methods reported are used in offline mode [21,22]. The major problems of offline SPE are risks of sample loss and contamination [23]. Thus, online coupling of the intestinal absorption model, SPE and HPLC detection will bring advantages for screening active constituents from TCMs. On the other hand, flavonoids are a large group of polyphenols that occur ubiquitously in TCMs. Flavonoids have attracted substantial attention for their various biological activities. Baicalein, a well-known naturally occurring flavonoid, is the main constituent of Scutellaria baicalensis Georgi (Huangqin, HQ). Baicalein possesses many pharmacological and biological properties, such as anti-inflammatory, anti-cancer, anti-neurotoxic and anti-angiogenic [24]. Polydatin is a main flavonoid of Polygoni Cuspidati Rhizoma et Radix (Huzhang, HZ) with the pharmacological and biological properties of anti-inflammatory, anti-oxidative and anti-cancer [25]. However, the biopotency of flavonoids is not only determined by its activity, but also by absorption, distribution, metabolism, and excretion [24]. For oral drugs, only the components absorbed by organisms can give full play to therapeutic effect. Therefore, it is significant to screen the potential active flavonoids from TCMs by intestinal absorption model. In present work, a semi-automated online platform was established successfully for preliminary screening of potential active flavonoids of TCMs in multicomponent system. The Ussing chamber model as the intestinal absorption model was applied to absorb and screened the potential active components of TCMs. A mini-hollow chromatographic column filled with C18 was used as a SPE column. Furthermore, the experimental conditions of online enrichment and elution of flavonoids were systematically investi-
gated. Finally, the online system was applied in preliminary screening of potential active flavonoids of Scutellaria baicalensis Georgi (Huangqin, HQ) and Polygoni Cuspidati Rhizoma et Radix (Huzhang, HZ). Potential active components screened by online system of two TCMs were identified by UPLC-MS/MS analysis. Moreover, the accuracy and reliability of the online screening system was compared with those of offline method and gavage in rats. 2. Materials and methods 2.1. Reagents and materials Scutellaria baicalensis Georgi (Huangqin, HQ) and Polygoni Cuspidati Rhizoma et Radix (Huzhang, HZ) were obtained from Lotus pond traditional Chinese medicine market in Chengdu (Sichuan, China), authenticated by professor Zhining Xia (Chongqing University). Rutin (purity >96%) was obtained from Adamas Reagent Co., Ltd. (Shanghai, China). Sodium chloride (NaCl), potassium chloride (KCl), calcium chloride dihydrate (CaCl2 ·2H2 O), sodium dihydrogen phosphate dihydrate (NaH2 PO4 ·2H2 O), magnesium sulfate anhydrous (MgSO4 ), sodium hydrogen carbonate (NaHCO3 ) and dglucose were obtained from Aladdin Reagents (Shanghai, China). Methanol and formic acid were of chromatographic grade and obtained from Kermel Reagent (Tianjin, China). All solutions used for HPLC were filtered through a 0.22 μm membrane filter to use. 2.2. Animals The male Sprague-Dawley rats (200–250 g) were purchased from the Animal Center, Chongqing Medical University. Before experiments, food was not given for 14 h and water was available at any time. All experimental procedures acclimatized were approved by the Institutional Animal Ethical Committee of Chongqing University and were conducted according to the Guide for the Care and Use of Laboratory Animal of the National Institute of Health (Publication No. 80–23, revised 1996). 2.3. Establishment of online Ussing chamber-SPE–HPLC procedure The schematic diagram and digital photo of online platform was shown in Fig. 1 and Fig. S1, respectively. Intestinal absorption test was carried out by a two-channel Ussing chamber model (KengTech, China). Sampling process of SPE was actualized by a peristaltic pump (Lab2015/MC4, Shenchen, China). SPE was implemented by a mini chromatographic hollow column (10 × 10 mm). Eluting process of SPE was carried out by a binary high pressure chromatographic pump (900P, Chuanyi, China). HPLC analysis was carried out on a Shimadzu 10A Series HPLC system, connected to a ODS column (250 × 4.6 mm, 5 μm). The flow path switching of online system is realized by the conversion of the manual six-way valve (Shimadzu, Japan). The online method comprises four steps: intestinal absorption, loading, eluting and detection. 2.3.1. Intestinal absorption by Ussing chamber model Tyrode’s solution comprised 8.0 g NaCl, 0.20 g KCl, 0.13 g MgSO4 , 0.065 g NaH2 PO4 ·2H2 O, 1.1 g d-glucose, 1.0 g NaHCO3 , 0.26 g CaCl2 ·2H2 O, and 10 0 0 mL water at pH 7.4. The extract of HQ and HZ was prepared into 10 mg/mL with Tyrode’s solution, respectively. Rats were anesthetized with 1.5% (w/v) pentobarbital sodium aqueous solution (60 mg/kg) by intraperitoneal administration after fasting for 14 h. The small intestine was excised and flushed with Tyrode’s solution at room temperature. Segments (length of 2.0 cm) were cut open and mounted on the specimen holder of Ussing chamber. Donor chamber and receiving chamber was added with 4.0 mL of Tyrode’s solution, respectively. After a preincubation
Please cite this article as: D. Wang, J. Zeng and W. Xiang et al., Online coupling of the Ussing chamber, solid-phase extraction and high-performance liquid chromatography for screening and analysis of active constituents of traditional Chinese medicines, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460480
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Fig. 1. The schematic diagram of online system. (A) Ussing chamber model, (B) Loading and enrichment of SPE, (C) Eluting of SPE and loading of HPLC, (D) Detection of HPLC.
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period of 10 min, Tyrode’s solution was removed from the donor chamber and 4.0 mL sample solution was added into the donor chamber. The small intestine was maintained at 37 °C by thermostatic water bath and oxygenated with O2 /CO2 (95%/5%) by a gas cylinder. When the absorption time reached 90 min, the solution in the receiving chamber was removed by a peristaltic pump. The intestinal absorption process and the digital photo of Ussing chamber was shown in Fig. 1A and Fig. S1B, respectively.
2.5. Application of online Ussing chamber-SPE–HPLC to real samples
2.3.2. Loading of SPE For loading of SPE, 4.0 mL of intestinal absorption solution was moved from the receiving chamber and poured into a C18 (grade of 74 μm, mass of 500 mg) SPE column (Fig. S1C) by a peristaltic pump with flow rate at 7.0 mL/min. At this step, the six-way valve (valve 1) was in the state of ’Laod’. The schematic diagram of SPE loading and enrichment process of components were shown in Fig. 1B.
2.5.2. Application of online Ussing chamber-SPE–HPLC to TCMs 40 mg extract of HQ and HZ was dispersed into 4.0 mL Tyrode’s solution as the intestinal absorption sample, respectively. 4.0 mL of two TCMs sample solution was added into the donor chamber of the Ussing system, respectively. Then, the potential active components in TCMs were screened and detected by online Ussing chamber-SPE–HPLC system. The specific steps are the same as those in Section 2.3. The components of two TCMs screened before and after were analyzed and identified by UPLC-MS/MS. Moreover, to compare with the online system, the components of two TCMs were also studied by offline method. In offline research, the components of HQ and HZ were screened by the same method with online Ussing chamber model with the lack of the online sampling. With the manual sampling, the samples were volatilized, redissolved by methanol to original volume and centrifuged to obtain supernatants. Finally, the supernatants were analyzed by HPLC and UPLC–MS/MS.
2.3.3. Eluting of SPE and loading of HLPC For this step, the valve 1 was switched to ‘Inject’ and the valve 2 was in the state of ‘Load’. The eluting solvent was transported by a binary high pressure chromatographic pump and flowed through the SPE column. The eluting condition was set to be methanolacetic acid (8:2, v/v) with flow rate at 1 mL/min for 0.82 min. Meanwhile, the active ingredients were eluted from the SPE column and transported to the quantitative ring of the HPLC system. The schematic diagram of SPE eluting and HPLC loading was shown in Fig. 1C. And the digital photo of the apparatus of loading and eluting process of SPE was shown in Fig. S1D. 2.3.4. Detection of HLPC For this step, the valve 2 was switched to ’Inject’, and components in the samples were analyzed by HPLC. HPLC analysis was carried out on a Shimadzu 10A Series HPLC system, connected to a ODS column (250 × 4.6 mm, 5 μm). The schematic diagram of detection process of HPLC was shown in Fig. 1D. The digital photo of the apparatus of loading and detection process of HPLC was shown in Fig. S1E. 2.4. Evaluation of the method performance Rutin is a common flavonoid with nuclear parent of flavonoids and exists in many TCMs. It was selected as the detected target to evaluate the online method. The present method was evaluated by a series of experiments about the calibration curve, limit of detection (LOD) and limit of quantification (LOQ), linear range, recovery and reproducibility of HPLC. This calibration curve was established in matrix by spiking rutin standard into the blank intestinal absorption, and it was made with twelve concentration levels of rutin (0.125 μg/mL–400 μg/mL) and three tests per sample. The LOD and LOQ were calculated as three and ten times the standard deviation of the area of the peaks detected, respectively. In order to test the precision of method, the intra-day precision was performed by analyzing a spiked rutin sample five times in one day at three different concentration levels and the inter-day precision was executed for six days at three different concentration levels. Further, the recovery test was used to evaluate the repeatability and accuracy of the online system method. The recoveries were calculated by spiking rutin standard solution into the blank intestinal liquid at three levels and the samples were analyzed by online HPLC. In addition, selectivity evaluation was established by comparing chromatograms of blank solvent with that of sample spiked with rutin in solvent. Finally, robustness study was conducted by deliberate changes in mobile phase composition and flow rate.
2.5.1. Preparation of TCMs extract 50 g of HQ and HZ was reflux extracted in 400 mL ethanol– water (7:3, v/v) at 90 °C for 1 h, repeating extraction three times, respectively. The extract solution was filtered and evaporated, then dried under vacuum at 40 °C. The extract of TCMs were conserved in a desiccator at room temperature for further experiment.
2.6. Comparison of the online system with the method of gavage in rats for screening active components of HQ and HZ To evaluate the reliability of the online system, the intestinal absorption of HQ and HZ by the gavage in living rats was detected. The extract of HQ and HZ was dissolved in 0.5% carboxy methyl cellulose sodium (CMC–Na) and orally administered to rats (n = 3) at a dosage of 5.0 g/kg, respectively. After the drug administration for 60 min, rats were anesthetized with 1.5% (w/v) pentobarbital sodium aqueous solution (60 mg/kg) by intraperitoneal administration. The blood samples were collected from the abdominal vein and centrifuged at 10,0 0 0 rpm for 10 min to obtain plasma samples. Afterwards, 750 μL plasma samples were added into a 5 mL centrifuge tube and extracted with 3 mL methanol-ethyl acetate (1:1, v/v) for three times, by which the potential active components were extracted and the protein was precipitated. Extraction was performed by vortexing for 2 min and centrifuging at 10,0 0 0 rpm for 10 min. The supernatants were combined and volatilized to dryness by nitrogen at 37 °C. Then the dried residue was dissolved in 200 μL methanol and centrifuged at 10,000 rpm for 10 min. The supernatants were detected by HPLC and UPLC–MS/MS for analysis. 2.7. HPLC analysis HPLC analysis was carried out on a Shimadzu 10A Series HPLC system, connected to a ODS column (250 × 4.6 mm, 5 μm). The mobile phase consisted of methanol (solvent A) and 0.1% formic acid-aqueous solution (solvent B). For HQ, the gradient elution process was as followed: 0–15 min, 20% A-40% A; 15–50 min, 40% A60% A; 50–65 min, 60% A-95% A; 65–70 min, 95% A; 70–80 min, 95% A-20% A; 80–90 min, 20% A. For HZ, the gradient elution process was as followed: 0–10 min, 20% A-32% A; 10–35 min, 32% A-50% A; 35–55 min, 50% A-70% A; 55–60 min, 70% A-80% A, 60–70 min, 80% A; 70–80 min, 80% A-20% A; 80–90 min, 20% A. The column temperature in HPLC analysis of two TCMs was maintained at 30 °C and the injection volume was 20 μL. The detection wavelength applied in all sections was 254 nm.
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2.8. UPLC–MS/MS analysis UPLC–MS/MS analysis was based on a LCMS-8060 series UPLC– MS/MS system (Shimadzu, Japan) which consists of a Nexera X2 UPLC system and a triple quadruple mass spectrometer. UPLC analysis methods of HQ and HZ were the same as those of online HPLC analysis in Section 2.7. High resolution mass spectrometry (MS) detections of two TCMs were performed on a triple quadruple mass spectrometer (Shimadzu, Japan). The conditions of MS/MS spectra were as followed: ion source: electronic electrospray ionization (ESI) source; ionization ways: positive or negative ion scanning; quality scan range (m/z): 100–1000; auxiliary gas (GS1 and GS2): 60 and 60 psi, respectively; air curtain air pressure (CUR): 35 psi; ion source temperature: 300 °C; ion spraying voltage (ISVF): 2500 v; the parent ion collision energy (CE): 35; the child away from the collision energy (CE): 35. 3. Results and discussion 3.1. Online system setup 3.1.1. Intestinal absorption model The common in vitro intestinal absorption models include the everted rat gut sac model, Ussing chamber model and Caco-2 cell model, these models have been used to study the intestinal absorption of oral drugs [14,16–19]. However, the absorption solution of the everted rat gut sac model is difficult to be loaded online because the diameter of small intestine of rats is too small. Thus, everted rat gut sac model is not suitable for online system. The process of culturing Caco-2 cell is time consuming, and it takes at least 50 days to complete the model. In addition, Caco-2 cell model has the same problem as the everted rat gut sac model for the online system. The absorption solution of Caco-2 cell model is not enough for loading online. The Ussing chamber model can overcome these shortcomings. It is not only easy to operate and time-saving, but also meets the requirements of online loading. The device of Ussing system has a hollow metal plate that can be poured with thermostatic water to provide a temperature environment (37 °C) for the small intestine in vitro. The spiracles on the Ussing chamber can be connected with a mixture of 95% O2 and 5% CO2 to provide a gas environment for the small intestine in vitro. In addition, Ussing chamber model has been successfully applied to study the absorption of TCMs [17]. Therefore, Ussing chamber model is an ideal intestinal absorption model for online screening of potential active ingredients in TCMs. The schematic diagram of Ussing chamber was shown in Fig. 1A. The components of TCMs are very complex, and there are differences in the absorption rate of different components. In order to ensure the effectiveness of intestinal absorption study, the intestinal absorption time of HQ and HZ was investigated by Ussing chamber model in terms of absorption kinetics and the activity of in vitro small intestines. For the absorption kinetics, it was found that the composition of the absorption solution was stable after absorption for 60 min while the content of components absorbed increased along with the absorption time. However, the small intestine will lose its activity if it is in vitro for long time. Generally, cells in the small intestine deformed severely and lost their function after 3 h in vitro, which limits the extension of absorption
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time. In study of intestinal absorption by Ussing chamber model, 90 min was usually selected as an effective absorption time [26,27]. Thus, the intestinal absorption time of the online system was determined to be 90 min. 3.1.2. Online enrichment of potential active components by SPE column Due to the species diversity and content differentia of components in TCMs, the components in intestinal absorption samples of TCMs often needed to be purified and enriched before detection. SPE is the predominant technique to enrich components of TCMs. The filling material of SPE is an important factor to affect the effect of enrichment. C18 is one of the most widely used adsorbent in SPE. Due to the effect of C18 long chain, the weak polar interaction between C18 and substance is stronger than polar interaction. Thus, C18 was usually applied to enrich the weak and medium polar compounds like flavonoids. And C18 filler has no reservation to salt, which can be used to desalinate for some small molecular samples. Thus, in this work, C18 filling was used for online enrichment of flavonoids in TCMs. When the intestinal absorption solution flowed through the SPE column, the weak polar substance (flavonoids) in the buffer solution would be retained by the C18, and the salt flowed with the water. After determining the filling of SPE, the specifications of SPE column were also discussed. The amounts of filling determined by the size of SPE column will affect the enrichment effect on components. The SPE columns in specifications of 2.1 × 10 mm and 10 × 10 mm were applied to enrich rutin online. The enrichment multiple was used to evaluate the enrichment effect of SPE column, which was defined as the chromatographic peak area ratio of rutin after and before online enrichment. As shown in Table 1, the enrichment multiple of SPE column was higher than that of 2.1 × 10 mm SPE column. The results indicated that SPE column (10 × 10 mm) filled with 500 mg C18 possessed the excellent enrichment effect on flavonoids. Therefore, the SPE column in specifications of 10 × 10 mm was considered as the optimum SPE column to enrich the active flavonoids online. 3.1.3. Online eluting of potential active components from the SPE column Considering the complexity of ingredients in TCMs, ingredients should be eluted simultaneously from the SPE column as many as possible. The interaction between C18 and flavonoids is the weak polar interaction. The high polar solvents can destroy the interaction of C18 with flavonoids and elute the components from SPE column. In the present investigation, six kinds of elution solvents were selected to elute rutin online, including ultrapure water, methanol and methanol-acetic acid (9:1, 8:2, 7:3 and 6:4, v/v). After online elution, the concentration of rutin in different eluents was shown in Fig. 2A. It was obviously that the concentration of rutin in methanol-acetic acid was higher than that in ultrapure water and methanol, indicating that methanol-acetic acid can effectively elute rutin from SPE column. The concentration of rutin in eluting solvent increased along with the proportion of acetic acid until the volume ratio of acetic acid and methanol reached 2:8. Thus, methanol-acetic acid with the volume ratio in 8:2 was considered as the optimum elution. Further, the concentration of rutin in methanol-acetic acid (8:2, v/v) was the 5.57 times of that in initial sample.
Table 1 The enrichment multiple of rutin by SPE columns in two specifications. (n = 3). Specification of SPE column
Filling
Quality of filling (mg)
Enrichment multiple of rutin
2.1 × 10 mm 10 × 10 mm
C18 C18
10.0 500
0.73 5.8
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Fig. 2. Optimization of online system. (A) The concentration of rutin in different eluting solvents. (B) The concentration of rutin in eluents at different elution time. (C) The concentration of rutin in eluents with different flow rates of eluents. Table 2 Linear regression data and precision of rutin in HPLC analysis by online system. Analyte
Linear regression Calibration curves
Rutin a
y = 7081x+5085
Precision a
Correlation coefficient
Linear range (μg/mL)
LOD (μg/mL)
LOQ (μg/mL)
Intra-day RSD (%)
Inter-day RSD (%)
0.9947
0.125–368
0.0500
0.125
2.5
3.8
y and x stand for the peak area (mAU) and the concentration (μg/mL) of the analyte, respectively.
Online eluting time was also investigated systematically. A total of twelve elution time points (0.1–1 min) were evaluated and optimized (Fig. 2B). The concentration of rutin in eluent at 0.82 min was the highest, and it was 5.81 times of that in initial sample. Therefore, the optimum time of elution was confirmed at 0.82 min with the 1 mL/min of methanol-acetic acid (8:2, v/v). Flow rates of eluting solution also had an effect on the elution efficiency. The pressure exerted on the SPE column was different by eluents at different flow rates. The elution efficiency of eluent at five different flow rates (0.3–5 mL/min) was discussed under the same volume of eluent. As shown in Fig. 2C, the eluent performed the highest elution efficiency with the flow rate at 1 mL/min. To sum up, the elution process was methanol-acetic acid (8:2, v/v) at 1 mL/min to elute for 0.82 min. All above results indicated that this online part can enrich and elute rutin (flavonoids) effectively for further analysis. 3.2. Method validation The present method was evaluated by a series of experiments about the linear range, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) of HPLC (Table 2). The calibration curve was obtained by the determination of rutin ranged from 0.125 to 368 μg/mL, and the regression equation was y = 7081x + 5085 with the R2 value of 0.9947. As shown in Table S1, the intra-day precision was performed by analyzing a spiked
Table 3 Recovery values (%) obtained for samples of intestinal absorption with the analyte at three different concentration levels. (n = 3). Analyte
Cadd (μg/mL)
Cfound (μg/mL)
Recovery (%)
RSD (%)
Rutin
1.63 11.0 55.9
1.53 10.3 52.1
94.0 93.7 93.2
11 7.3 3.1
rutin sample five times in one day at three different levels. And the inter-day precision was executed for six days at three different concentration levels of rutin sample (Table S2). The RSD of intraday precision and inter-day precision was 2.5% and 3.8%, respectively. Moreover, the LOD and LOQ detected as 3 and 10 times of the signal to noise ratio was 0.0500 μg/mL and 0.125 μg/mL, respectively. The recovery test was used to evaluate the repeatability and accuracy of the online Ussing chamber-SPE–HPLC method. The recoveries were calculated by spiking rutin standard into the blank intestinal absorption samples at three levels and the samples were analyzed by online system. As shown in Table 3, the recoveries ranged from 93.2% to 94.0% with the RSD of 3.1%−11%. In addition, selectivity evaluation was established by comparing chromatogram of blank solvent with that of sample spiked with rutin in solvent. No interfering peaks were observed at the retention time of analyte in Fig. S2. Finally, robustness study was conducted by deliberate changes in mobile phase composition and flow rate. The results indicated that there was no significant variation in RSD and
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Fig. 3. (A) The HPLC chromatograms of the extract of HQ. The UPLC–MS/MS spectra of (B) scutellarin, (C) dihydrobaicalein-7-O-glucuronide, (D) baicalin, (E) chrysin-7-Oglucuronide, (F) wogonoside, (G) baicalein, (H) dihydrowogonin, (I) wogonin, (J) chrysin, (K) oroxylin A.
retention time of rutin (Table S3). All above results showed that the proposed method was sufficiently accurate, selective, and sensitive for determination of rutin (flavonoids) in samples. 3.3. Application of online Ussing chamber-SPE–HPLC to screen the potential active components in TCMs The online system has been applied to screen the potential active flavonoids of HQ and HZ. Both HQ and HZ are indigenous medicinal plants in China and listed in the Chinese Pharmacopoeia as the typical Chinese medicines. Flavonoids are the main active components of both HQ [28] and HZ [29]. Ten flavonoids in HQ have been identified by UPLC-MS/MS, including scutellarin, dihydrobaicalein-7-O-glucuronide, baicalin, chrysin7-O-glucuronide, wogonoside, baicalein, dihydrowogonin, wogonin, chrysin and oroxylin A (Fig. 3). As shown in Fig. 4B–E, four flavonoids (catechin, polydatin, resveratrol, luteolin -7-glucuronide) were identified in HZ. Thus, a total of fourteen flavonoids were analyzed in two TCMs, and twelve flavonoids of them were screened
by online system. The HPLC chromatograms of these twelve potential active flavonoids screened online in two TCMs were shown in Fig. 5. According to previous researches [30–34], baicalin, wogonoside, baicalein and oroxylin A, polydatin and resveratrol can indeed be absorbed by organisms in multicomponent system and have certain pharmacological activities. These six flavonoids also have been screened by the online system, it indicated that the online system is credible to screen potential active components from the complex matrix of TCMs. However, the analysis of another six flavonoids (scutellarin, dihydrobaicalein-7-O-glucuronide, chrysin-7-O-glucuronide, dihydrowogonin, chrysin, and luteolin-7glucuronide) as the absorbed active components in multicomponent system was very limited, even not identified by some methods [32,33]. It is noteworthy that scutellarin, dihydrobaicalein-7O-glucuronide, chrysin-7-O-glucuronide, dihydrowogonin, chrysin, and luteolin-7-glucuronide were successfully screened and analyzed by online system in multicomponent system of TCMs. Some of them possess many pharmacological activities [35,36], such as hepatoprotective and nephroprotective activities of chrysin. The
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Fig. 4. (A) The HPLC chromatograms of the extract of HZ. The UPLC–MS/MS spectra of (B) catechin, (C) polydatin, (D) resveratrol, (E) luteolin-7-glucuronide, (F) anthraglycoside B, (G) emodin, (H) physcion, (I) citreorosein.
Fig. 5. The HPLC chromatograms of the potential active constituents screened by offline method and online system in HQ and HZ. (A) The HPLC chromatograms of (1) scutellarin, (2) dihydrobaicalein-7-O-glucuronide, (3) baicalin, (4) chrysin-7-O-glucuronide, (5) wogonoside, (6) baicalein, (7) dihydrowogonin, (9) chrysin and (10) oroxylin A in HQ. (B) The HPLC chromatograms of (2) polydatin, (3) resveratrol, (4) luteolin-7-glucuronide, (5) anthraglycoside B, (6) emodin and (8) citreorosein in HZ.
results showed the online system could screen potential flavonoid active ingredients reliably and comprehensively. Moreover, in addition to flavonoids, four anthraquinones in HZ were identified by UPLC–MS/MS (Fig. 4F–I). As shown in Fig. 5B, anthraglycoside B, emodin and citreorosein were screened as the potential active anthraquinones by the online Ussing chamber-SPE–HPLC platform. This indicated that online platform is not only suitable for
screening of flavonoids, but also for screening of anthraquinones active ingredients. The anthraquinones absorbed by Ussing chamber model were also enriched by SPE column. Therefore, the platform will be suitable to screen most kinds of ingredients as long as the proper filling of SPE are selected. Although wogonin, catechin and physcion have some pharmacological activities in vitro [37–39], they were not absorbed by intestinal absorption model in
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multicomponent system. This may be due to the interaction among the different components of TCMs, and the reason may also be that the contents of wogonin, catechin and physcion in HQ and HZ were too low to be absorbed. Furthermore, to evaluate reliability and validity of the online system, the intestinal absorption and detection of HQ and HZ were also carried out offline by Ussing model, HPLC and UPLC–MS/MS analysis. The components absorbed were same by online and offline methods according to the results of UPLC–MS/MS, indicating that the online Ussing chamber model had the same effect as that of offline. The absorption function of Ussing chamber model was not affected after installing to online system. The HPLC chromatograms for online and offline detection of intestinal absorption samples of HQ and HZ were shown in Fig. 5. Despite the high sensitivity of HPLC, there were no the chromatographic peaks of wogonoside and citreorosein by offline analysis with the trace samples. By the online analysis, these components can not only be detected, but also some can be analyzed at quantization. In addition, chromatographic peak area of these components detected by online was 1.3–14 times of that detected by offline detection, suggesting the online SPE possessed the excellent enrichment ability. All the above results showed that the online system was able to screen, enrich and detect the potential active components of HQ and HZ in multicomponent system.
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proposed method was practical, accurate and sensitive for determination of flavonoids in samples. Finally, the online system was applied in preliminary screening of potential active flavonoids of HQ and HZ. A total of 14 flavonoids were identified by UPLC–MS/MS, and 12 of them were screened as the potential active constituents by online Ussing chamber-SPE–HPLC platform. It was showed that the online system possessed the ability to screen the potential active flavonoids of TCMs in multicomponent system. And three anthraquinones in HZ were screened and analyzed by online platform. The platform would be suitable to screen most kinds of ingredients as long as the appropriate filler was selected for online SPE process. Moreover, in comparison with offline method and gavage in rats, the online system can screen the active components from TCMs more accurately and completely. The results showed that the online system was reliable and sufficiently accurate for screening and determination of the potential active constituents of TCMs in multicomponent system. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment
3.4. Comparison of the online system with the method of gavage in rats for screening active components of HQ and HZ To evaluate the reliability of the online system, the intestinal absorption of HQ and HZ by the gavage in living rats was detected. The chromatograms of the components of HQ and HZ in rats’ plasma at 60 min were shown in Fig. S3. For the intestinal absorption of the HQ extract, the results of Ussing chamber and the gavage in rats were the same. It indicated that the components of HQ screened by the online Ussing chamber-SPE–HPLC platform were the same as that really absorbed by the animals. This further proved the online system was reliable to screen the potential active components of TCMs. In the study of intestinal absorption of HZ, one component (citreorosein) absorbed by the gavage in rats was less than that by the Ussing chamber model. According to the report, the citreorosein in HZ possessed strong anti-inflammatory effect [40]. However, there was no detection of citreorosein in rats’ plasma sample. Although the gavage in living animals is the most classic method to study the intestinal absorption of the components in TCMs by oral administration, this method will lose the trace components in the complex processing of plasma samples. Comparing with the method of gavage in living animals, online system can screen the potential active components more completely, and it is easily to operate the online system with no complex processing of samples. Therefore, the results further prove the reliability of the online system to screen the potential active components of TCMs in multicomponent system. 4. Conclusions In the present work, a semi-automated platform was established successfully for preliminary screening of potential active flavonoids of TCMs in multicomponent system. Online coupling of the Ussing chamber model, SPE and HPLC was founded for the first time. The Ussing chamber with rats’ intestinal canal was used to absorb the components of TCMs. A mini chromatographic column filled with C18 was used as a SPE column to enrich the flavonoids online. Furthermore, the specifications of SPE column, eluting solvent, elution time and flow rate of eluent were discussed for the optimization of online system. Under the optimal conditions, the
The present work was financially supported by grants from the Collaborative Innovation Center of Modern Technology and Industrial Development of Jiangxi Traditional Minority Medicine, China (no. JXXT2018001). Supplementary material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2019.460480. References [1] Y. Sun, S. Shi, Y. Li, Q. Wang, Development of quantitative structure-activity relationship models to predict potential nephrotoxic ingredients in traditional Chinese medicines, Food Chem. Toxicol. 128 (2019) 163–170. [2] L. Teng, Q. Zu, G. Li, T. Yu, K.M. Job, X. Yang, L. Di, C.M. Sherwin, E.Y. Enioutina, Herbal medicines: challenges in the modern world. Part 3. China and Japan, Expert Rev. Clin. Pharmacol. 9 (2016) 1225–1233. [3] J.L. Tang, S.Y. Zhan, E. Ernst, Review of randomised controlled trials of traditional Chinese medicine, Brit. Med. J. 319 (1999) 160–161. [4] A.L. Harvey, Natural products in drug discovery, Drug Discov. Today 13 (2008) 894–901. [5] J. Fu, Y. Lv, Q. Jia, Y. Lin, S. Han, Dual-mixed/CMC model for screening target components from traditional Chinese medicines simultaneously acting on EGFR & FGFR4 receptors, Talanta 192 (2019) 248–254. [6] X.Y. Wang, X.F. Chen, Y.Q. Gu, Y. Cao, Y.F. Yuan, Z.Y. Hong, Y.F. Chai, Progress of cell membrane chromatography and its application in screening active ingredients of traditional Chinese medicine, Chinese J. Anal. Chem. 46 (2018) 1695–1702. [7] O.U. Oghale, M. Idu, Phytochemistry, anti-asthmatic and antioxidant activities of Anchomanes difformis (Blume) Engl. leaf extract, Asian Pac. J. of Trop. Biomed. 6 (2016) 225–231. [8] Y.Q. Cai, J.H. Hu, J. Qin, T. Sun, X.L. Li, Rhododendron molle (Ericaceae): phytochemistry, pharmacology, and toxicology, Chin. J. Nat. Medicines 16 (2018) 401–410. [9] B.M. Zhang, Z.B. Wang, P. Xin, Q.H. Wang, H. Bu, H.X. Kuang, Phytochemistry and pharmacology of genus Ephedra, Chin. J. Nat. Med. 16 (2018) 811–828. [10] P. Jiang, D.G. Nagle, Y. Zhou, W. Zhang, Theories and methods for the evaluation of the pharmacodynamic material basis of traditional Chinese medicine, in: W.D. Zhang (Ed.), Systems Biology and Its Application in TCM Formulas Research, China, 2018, pp. 19–30. [11] C.S. Liu, X. Liang, X.H. Wei, F.L. Chen, Q.F. Tang, X.M. Tan, Comparative pharmacokinetics of major bioactive components from Puerariae Radix-Gastrodiae Rhizome extracts and their intestinal absorption in rats, J. Chromatogr. B 1105 (2019) 38–46. [12] Z. Zuo, L. Zhang, L. Zhou, Q. Chang, M. Chow, Intestinal absorption of hawthorn flavonoids-in vitro, in situ and in vivo correlations, Life Sci 79 (2006) 2455–2462.
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[13] R.M. Hernández, A.R. Gascón, M.B. Calvo, C. Caramella, U. Conte, A. Domínguez-Gil, J.L. Pedraz, Correlation of ‘in vitro’ release and ‘in vivo’ absorption characteristics of four salbutamol sulphate formulations, Int. J. Pharm. 139 (1996) 45–52. [14] J.M. Yang, S.P.P. Ip, H.K.J. Yeung, C.T. Che, HPLC-MS analysis of Schisandra lignans and their metabolites in Caco-2 cell monolayer and rat everted gut sac models and in rat plasma, Acta Pharm. Sin. B 1 (2011) 46–55. [15] M. Mauro, R.A. De Grandis, M.L. Campos, A. Bauermeister, R.G. Peccinini, F.R. Pavan, N.P. Lopes, N.V. De Moraes, Acid diterpenes from Copaiba oleoresin (Copaifera langsdorffii): chemical and plasma stability and intestinal permeability using Caco-2 cells, J. Ethnopharmacol. 235 (2019) 183–189. [16] D. Scherbl, S. Muentnich, E. Richling, In vitro absorption studies of chlorogenic acids from coffee using the Ussing chamber model, Food Res. Int. 63 (2014) 456–463. [17] J. Wang, G. Aierken, X. Li, L. Li, X. Mao, Testing the absorption of the extracts of Coreopsis tinctoria Nutt. in the intestinal canal in rats using an Ussing chamber, J. Ethnopharmacol. 186 (2016) 73–83. [18] H. Yang, B. Zhai, Y. Fan, J. Wang, J. Sun, Y. Shi, D. Guo, Intestinal absorption mechanisms of araloside a in situ single-pass intestinal perfusion and in vitro Caco-2 cell model, Biomed. Pharmacother. 106 (2018) 1563–1569. [19] M. Zorzan, D. Collazuol, G. Ribaudo, A. Ongaro, C. Scaroni, G. Zagotto, D. Armanini, S. Barollo, F. Galeotti, N. Volpi, M. Redaelli, R. Pezzani, Biological effects and potential mechanisms of action of Pistacia lentiscus Chios mastic extract in Caco-2 cell model, J. Funct. Foods 54 (2019) 92–97. [20] K. Georgi, K.S. Boos, Multidimensional on-line SPE for undisturbed LC–MS–MS analysis of basic drugs in biofluids, Chromatographia 63 (2006) 523–531. [21] J. Li, Y. Cai, Y. Shi, S. Mou, G. Jiang, Analysis of phthalates via HPLC-UV in environmental water samples after concentration by solid-phase extraction using ionic liquid mixed hemimicelles, Talanta 74 (2008) 498–504. [22] X. Gao, B. Yang, Z. Tang, X. Luo, F. Wang, H. Xu, X. Cai, Determination of phthalates released from paper packaging materials by solid-phase extraction-high-performance liquid chromatography, J. Chromatogr. Sci. 52 (2014) 383–389. [23] D. Salazar-Beltran, L. Hinojosa-Reyes, E. Ruiz-Ruiz, A. Hernandez-Ramirez, J. Luis Guzman-Mar, Determination of phthalates in bottled water by automated on-line solid phase extraction coupled to liquid chromatography with uv detection, Talanta 168 (2017) 291–297. [24] X. Jiang, J. Zhou, Q. Lin, G. Gong, H. Sun, W. Liu, Q. Guo, F. Feng, W. Qu, Anti-angiogenic and anticancer effects of baicalein derivatives based on transgenic zebrafish model, Bioorg. Med. Chem. 26 (2018) 4481–4492. [25] Z. Chen, Q. Wei, G. Hong, D. Chen, J. Liang, W. He, M.H. Chen, Polydatin induces bone marrow stromal cells migration by activation of ERK1/2, Biomed. Pharmacother. 82 (2016) 49–53. [26] C. Mendes, G.C. Meirelles, M.A.S. Silva, G. Ponchel, Intestinal permeability determinants of norfloxacin in Ussing chamber model, Eur. J. Pharm. Sci. 121 (2018) 236–242. [27] S. Kondo, M. Miyake, Simultaneous prediction of intestinal absorption and metabolism using the mini-Ussing chamber system, J. Pharm. Sci. 108 (2019) 763–769.
[28] O.N. Seo, G.S. Kim, Y.H. Kim, S. Park, S.W. Jeong, S.J. Lee, J.S. Jin, S.C. Shin, Determination of polyphenol components of Korean Scutellaria baicalensis Georgi using liquid chromatography–tandem mass spectrometry: contribution to overall antioxidant activity, J. Funct. Foods 5 (2013) 1741–1750. [29] G.S. Qian, S.Y. Leung, G.H. Lu, K.S.Y. Leung, Differentiation of Rhizoma et Radix Polygoni Cuspidati from closely related herbs by HPLC fingerprinting, Chem. Pharm. Bull. 54 (2006) 1179–1186. [30] Y.K. Fong, C.R. Li, S.K. Wo, S. Wang, L. Zhou, L. Zhang, G. Lin, Z. Zuo, In vitro and in situ evaluation of herb-drug interactions during intestinal metabolism and absorption of baicalein, J. Ethnopharmacol. 141 (2012) 742–753. [31] X.L. Liang, Z.G. Liao, J.Y. Zhu, G.W. Zhao, M. Yang, R.L. Yin, Y.C. Cao, J. Zhang, L.J. Zhao, The absorption characterization effects and mechanism of Radix Angelicae dahuricae extracts on baicalin in Radix Scutellariae using in vivo and in vitro absorption models, J. Ethnopharmacol. 139 (2012) 52–57. [32] C. Li, S.Y. Fong, Q. Mei, G. Lin, Z. Zuo, Influence of mefenamic acid on the intestinal absorption and metabolism of three bioactive flavones in Radix Scutellariae and potential pharmacological impact, Pharm. Biol. 52 (2014) 291– 297. [33] S. Xing, M. Wang, Y. Peng, D. Chen, X. Li, Simulated gastrointestinal tract metabolism and pharmacological activities of water extract of Scutellaria baicalensis roots, J. Ethnopharmacol. 152 (2014) 183–189. [34] J. Fu, S. Wu, M. Wang, Y. Tian, Z. Zhang, R. Song, Intestinal metabolism of Polygonum cuspidatum in vitro and in vivo, Biomed. Chromatogr. 32 (2018) e4190. [35] R.B. Pingili, A.K. Pawar, S.R. Challa, T. Kodali, S. Koppula, V. Toleti, A comprehensive review on hepatoprotective and nephroprotective activities of chrysin against various drugs and toxic agents, Chem. Biol. Interact. 308 (2019) 51– 60. [36] C.Y. Li, Q. Wang, X. Wang, G. Li, S. Shen, X. Wei, Scutellarin inhibits the invasive potential of malignant melanoma cells through the suppression epithelial-mesenchymal transition and angiogenesis via the PI3K/Akt/mTOR signaling pathway, Eur. J. Pharmacol. 858 (2019) 172463. [37] D.L. Huynh, N. Sharma, A. Kumar Singh, S. Singh Sodhi, J.J. Zhang, R.K. Mongre, M. Ghosh, N. Kim, Y. Ho Park, D. Kee Jeong, Anti-tumor activity of wogonin, an extract from Scutellaria baicalensis, through regulating different signaling pathways, Chin. J. Nat. Med. 15 (2017) 15–40. [38] P. Pheomphun, C. Treesubsuntorn, P. Thiravetyan, Effect of exogenous catechin on alleviating O3 stress: the role of catechin-quinone in lipid peroxidation, salicylic acid, chlorophyll content, and antioxidant enzymes of Zamioculcas zamiifolia, Ecotoxicol. Environ. Saf. 180 (2019) 374–383. [39] X.P. Pan, C. Wang, Y. Li, L.H. Huang, Physcion induces apoptosis through triggering endoplasmic reticulum stress in hepatocellular carcinoma, Biomed. Pharmacother. 99 (2018) 894–903. [40] Y. Lu, S.J. Suh, X. Li, S.L. Hwang, Y. Li, K. Hwangbo, S.J. Park, M. Murakami, S.H. Lee, Y. Jahng, J.K. Son, C.H. Kim, H.W. Chang, Citreorosein, a naturally occurring anthraquinone derivative isolated from Polygoni cuspidati radix, attenuates cyclooxygenase-2-dependent prostaglandin D2 generation by blocking Akt and JNK pathways in mouse bone marrow-derived mast cells, Food Chem. Toxicol. 50 (2012) 913–919.
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Online coupling of the Ussing chamber, solid-phase extraction and high-performance liquid chromatography for screening and analysis of active constituents of traditional Chinese medicines Dandan Wang a,1, Jinxiang Zeng b,1, Wei Xiang a, Manni Yin a, Guoyue Zhong b,∗, Zhining Xia a,∗ a
School of Pharmaceutical Sciences, Chongqing University, Chongqing, 401331, China Research Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang, 330004, China
b
a r t i c l e
i n f o
Article history: Received 17 June 2019 Revised 18 August 2019 Accepted 22 August 2019 Available online xxx Keywords: Traditional Chinese medicines Active constituents Flavonoid Ussing chamber-SPE–HPLC Online screening Intestinal absorption
a b s t r a c t A semi-automated online platform was established successfully for preliminary screening of potential active flavonoids of traditional Chinese medicines (TCMs) in multicomponent system. Online coupling of the in vitro intestinal absorption model, solid phase extraction (SPE) and high-performance liquid chromatography (HPLC) was actualized at the first time. The Ussing chamber model was selected to absorb the constituents of TCMs. A mini chromatographic column filled with C18 was used as a SPE column for online enrichment of flavonoids. HPLC was applied to analyze the constituents screened by platform. With the use of rutin as a model flavonoid, the specifications of SPE column, eluting solvent, elution time and flow rate of eluent were systematically investigated to optimize online system. Under the optimal conditions, the linear range of rutin was 0.125–368 μg/mL with the correlation coefficient (R2 ) greater than 0.9947. The limit of detection (LOD) was as low as 0.0500 μg/mL and the limit of quantification (LOQ) was 0.125 μg/mL. The intra-day relative standard deviation (RSD) and inter-day RSD was 2.5% and 3.8%, respectively. The recoveries of rutin in the intestinal absorption samples ranged from 93.2% to 94.0%. Finally, the online system was applied to screen the potential active flavonoids of Scutellaria baicalensis Georgi (Huangqin, HQ) and Polygoni Cuspidati Rhizoma et Radix (Huzhang, HZ). A total of 14 flavonoids of these two TCMs were identified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS), and 12 flavonoids of them were screened as the potential active components by online Ussing chamber-SPE–HPLC. In comparison with offline method and gavage in rats, the online system can screen the active constituents from TCMs more accurately and completely. The results demonstrated that the online system was reliable and sufficiently accurate for screening and determination of the potential active flavonoids of TCMs in multicomponent system. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Traditional Chinese medicines (TCMs) possess a long history and remarkable curative effect. TCMs have been widely applied in prevention and treatment of diseases, relief of anxiety, weight loss and etc. [1]. In virtue of their powerful medicinal properties, TCMs have been spread beyond China and Asia, with increasing worldwide acceptance [2]. However, the unclear therapeutic basis of most TCMs has become a bottleneck to restrict the development
∗
1
Corresponding authors. E-mail addresses: [email protected] (G. Zhong), [email protected] (Z. Xia). These authors contributed equally to this work.
of TCMs. Bioactive components are the basis of TCMs pharmacodynamics and the important source of new drug development [3–5]. Screening of active constituents is an indispensable part in clarifying the therapeutic material basis of TCMs and searching for active ingredients producing pharmacodynamic effects in TCMs [6]. Hence, it is very important for the modernization research of TCMs to screen the active constituents from TCMs rapidly and effectively. The classical study method of active ingredients of TCMs is to extract total components, extract a single component, and then determine its active ingredients through pharmacological experiments [7–9]. Although this method based on phytochemistry can screen the active constituents from TCMs, it has complicated steps, high labor intensity and low probability of success. Moreover, the chemical constituents of TCMs are complex and diverse. The
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pharmacodynamic material basis of TCMs does not simply consider the additive effects of all the components but treats the TCMs as a potentially synergistic combination of its various active components [10]. In the complex matrixes of TCMs, some components have pharmacological activity in vitro, but they may be transformed and lose their function in vivo, and some components even cannot be absorbed by organisms [11]. Therefore, it is urgent to develop a universal, effective, quick and practicable method or platform for preliminary screening of the active components of TCMs in multicomponent system. Oral administration is an important route of administration for TCMs, and the small intestine is the main site for absorption of oral drugs. [12]. Only the components absorbed into the body have the opportunity to play a pharmacological role and can be defined as the real active ingredients. The ingredients that can be absorbed will be quickly and effectively screened by intestinal absorption models. In recent years, intestinal absorption models such as in vivo animal model [11,13], everted rat gut sac model [14], in situ single-pass intestinal perfusion [15], Ussing chamber model [16,17] and cell model [18,19] have been widely used to study the absorption kinetics, absorption mechanism, effective absorption sites, factors affecting absorption and bioavailability of active ingredients. In particularly, the Ussing chamber model has been used to study the potential active components in complex matrix of TCMs, and these components were detected by highperformance liquid chromatography-mass spectrometry (HPLC–MS) [17]. Regrettably, the processing of intestinal absorbent sample is complicated, samples need to be removed protein, centrifuged, enriched and re-dissolved before monitoring and analysis. Despite the high sensitivity of HPLC–MS, the analysis of highly complex samples like the intestinal absorbent samples of TCMs is often error prone due to the species diversity and content differentia of components in TCMs [20]. To avoid this problem, the components in intestinal absorption samples of TCMs will be purified and enriched before detection [18]. Solid phase extraction (SPE) is the predominant technique to enrich the components, which has been widely combined with HPLC for enrichment, clean-up and detection of samples. However, most of the SPE methods reported are used in offline mode [21,22]. The major problems of offline SPE are risks of sample loss and contamination [23]. Thus, online coupling of the intestinal absorption model, SPE and HPLC detection will bring advantages for screening active constituents from TCMs. On the other hand, flavonoids are a large group of polyphenols that occur ubiquitously in TCMs. Flavonoids have attracted substantial attention for their various biological activities. Baicalein, a well-known naturally occurring flavonoid, is the main constituent of Scutellaria baicalensis Georgi (Huangqin, HQ). Baicalein possesses many pharmacological and biological properties, such as anti-inflammatory, anti-cancer, anti-neurotoxic and anti-angiogenic [24]. Polydatin is a main flavonoid of Polygoni Cuspidati Rhizoma et Radix (Huzhang, HZ) with the pharmacological and biological properties of anti-inflammatory, anti-oxidative and anti-cancer [25]. However, the biopotency of flavonoids is not only determined by its activity, but also by absorption, distribution, metabolism, and excretion [24]. For oral drugs, only the components absorbed by organisms can give full play to therapeutic effect. Therefore, it is significant to screen the potential active flavonoids from TCMs by intestinal absorption model. In present work, a semi-automated online platform was established successfully for preliminary screening of potential active flavonoids of TCMs in multicomponent system. The Ussing chamber model as the intestinal absorption model was applied to absorb and screened the potential active components of TCMs. A mini-hollow chromatographic column filled with C18 was used as a SPE column. Furthermore, the experimental conditions of online enrichment and elution of flavonoids were systematically investi-
gated. Finally, the online system was applied in preliminary screening of potential active flavonoids of Scutellaria baicalensis Georgi (Huangqin, HQ) and Polygoni Cuspidati Rhizoma et Radix (Huzhang, HZ). Potential active components screened by online system of two TCMs were identified by UPLC-MS/MS analysis. Moreover, the accuracy and reliability of the online screening system was compared with those of offline method and gavage in rats. 2. Materials and methods 2.1. Reagents and materials Scutellaria baicalensis Georgi (Huangqin, HQ) and Polygoni Cuspidati Rhizoma et Radix (Huzhang, HZ) were obtained from Lotus pond traditional Chinese medicine market in Chengdu (Sichuan, China), authenticated by professor Zhining Xia (Chongqing University). Rutin (purity >96%) was obtained from Adamas Reagent Co., Ltd. (Shanghai, China). Sodium chloride (NaCl), potassium chloride (KCl), calcium chloride dihydrate (CaCl2 ·2H2 O), sodium dihydrogen phosphate dihydrate (NaH2 PO4 ·2H2 O), magnesium sulfate anhydrous (MgSO4 ), sodium hydrogen carbonate (NaHCO3 ) and dglucose were obtained from Aladdin Reagents (Shanghai, China). Methanol and formic acid were of chromatographic grade and obtained from Kermel Reagent (Tianjin, China). All solutions used for HPLC were filtered through a 0.22 μm membrane filter to use. 2.2. Animals The male Sprague-Dawley rats (200–250 g) were purchased from the Animal Center, Chongqing Medical University. Before experiments, food was not given for 14 h and water was available at any time. All experimental procedures acclimatized were approved by the Institutional Animal Ethical Committee of Chongqing University and were conducted according to the Guide for the Care and Use of Laboratory Animal of the National Institute of Health (Publication No. 80–23, revised 1996). 2.3. Establishment of online Ussing chamber-SPE–HPLC procedure The schematic diagram and digital photo of online platform was shown in Fig. 1 and Fig. S1, respectively. Intestinal absorption test was carried out by a two-channel Ussing chamber model (KengTech, China). Sampling process of SPE was actualized by a peristaltic pump (Lab2015/MC4, Shenchen, China). SPE was implemented by a mini chromatographic hollow column (10 × 10 mm). Eluting process of SPE was carried out by a binary high pressure chromatographic pump (900P, Chuanyi, China). HPLC analysis was carried out on a Shimadzu 10A Series HPLC system, connected to a ODS column (250 × 4.6 mm, 5 μm). The flow path switching of online system is realized by the conversion of the manual six-way valve (Shimadzu, Japan). The online method comprises four steps: intestinal absorption, loading, eluting and detection. 2.3.1. Intestinal absorption by Ussing chamber model Tyrode’s solution comprised 8.0 g NaCl, 0.20 g KCl, 0.13 g MgSO4 , 0.065 g NaH2 PO4 ·2H2 O, 1.1 g d-glucose, 1.0 g NaHCO3 , 0.26 g CaCl2 ·2H2 O, and 10 0 0 mL water at pH 7.4. The extract of HQ and HZ was prepared into 10 mg/mL with Tyrode’s solution, respectively. Rats were anesthetized with 1.5% (w/v) pentobarbital sodium aqueous solution (60 mg/kg) by intraperitoneal administration after fasting for 14 h. The small intestine was excised and flushed with Tyrode’s solution at room temperature. Segments (length of 2.0 cm) were cut open and mounted on the specimen holder of Ussing chamber. Donor chamber and receiving chamber was added with 4.0 mL of Tyrode’s solution, respectively. After a preincubation
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Fig. 1. The schematic diagram of online system. (A) Ussing chamber model, (B) Loading and enrichment of SPE, (C) Eluting of SPE and loading of HPLC, (D) Detection of HPLC.
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period of 10 min, Tyrode’s solution was removed from the donor chamber and 4.0 mL sample solution was added into the donor chamber. The small intestine was maintained at 37 °C by thermostatic water bath and oxygenated with O2 /CO2 (95%/5%) by a gas cylinder. When the absorption time reached 90 min, the solution in the receiving chamber was removed by a peristaltic pump. The intestinal absorption process and the digital photo of Ussing chamber was shown in Fig. 1A and Fig. S1B, respectively.
2.5. Application of online Ussing chamber-SPE–HPLC to real samples
2.3.2. Loading of SPE For loading of SPE, 4.0 mL of intestinal absorption solution was moved from the receiving chamber and poured into a C18 (grade of 74 μm, mass of 500 mg) SPE column (Fig. S1C) by a peristaltic pump with flow rate at 7.0 mL/min. At this step, the six-way valve (valve 1) was in the state of ’Laod’. The schematic diagram of SPE loading and enrichment process of components were shown in Fig. 1B.
2.5.2. Application of online Ussing chamber-SPE–HPLC to TCMs 40 mg extract of HQ and HZ was dispersed into 4.0 mL Tyrode’s solution as the intestinal absorption sample, respectively. 4.0 mL of two TCMs sample solution was added into the donor chamber of the Ussing system, respectively. Then, the potential active components in TCMs were screened and detected by online Ussing chamber-SPE–HPLC system. The specific steps are the same as those in Section 2.3. The components of two TCMs screened before and after were analyzed and identified by UPLC-MS/MS. Moreover, to compare with the online system, the components of two TCMs were also studied by offline method. In offline research, the components of HQ and HZ were screened by the same method with online Ussing chamber model with the lack of the online sampling. With the manual sampling, the samples were volatilized, redissolved by methanol to original volume and centrifuged to obtain supernatants. Finally, the supernatants were analyzed by HPLC and UPLC–MS/MS.
2.3.3. Eluting of SPE and loading of HLPC For this step, the valve 1 was switched to ‘Inject’ and the valve 2 was in the state of ‘Load’. The eluting solvent was transported by a binary high pressure chromatographic pump and flowed through the SPE column. The eluting condition was set to be methanolacetic acid (8:2, v/v) with flow rate at 1 mL/min for 0.82 min. Meanwhile, the active ingredients were eluted from the SPE column and transported to the quantitative ring of the HPLC system. The schematic diagram of SPE eluting and HPLC loading was shown in Fig. 1C. And the digital photo of the apparatus of loading and eluting process of SPE was shown in Fig. S1D. 2.3.4. Detection of HLPC For this step, the valve 2 was switched to ’Inject’, and components in the samples were analyzed by HPLC. HPLC analysis was carried out on a Shimadzu 10A Series HPLC system, connected to a ODS column (250 × 4.6 mm, 5 μm). The schematic diagram of detection process of HPLC was shown in Fig. 1D. The digital photo of the apparatus of loading and detection process of HPLC was shown in Fig. S1E. 2.4. Evaluation of the method performance Rutin is a common flavonoid with nuclear parent of flavonoids and exists in many TCMs. It was selected as the detected target to evaluate the online method. The present method was evaluated by a series of experiments about the calibration curve, limit of detection (LOD) and limit of quantification (LOQ), linear range, recovery and reproducibility of HPLC. This calibration curve was established in matrix by spiking rutin standard into the blank intestinal absorption, and it was made with twelve concentration levels of rutin (0.125 μg/mL–400 μg/mL) and three tests per sample. The LOD and LOQ were calculated as three and ten times the standard deviation of the area of the peaks detected, respectively. In order to test the precision of method, the intra-day precision was performed by analyzing a spiked rutin sample five times in one day at three different concentration levels and the inter-day precision was executed for six days at three different concentration levels. Further, the recovery test was used to evaluate the repeatability and accuracy of the online system method. The recoveries were calculated by spiking rutin standard solution into the blank intestinal liquid at three levels and the samples were analyzed by online HPLC. In addition, selectivity evaluation was established by comparing chromatograms of blank solvent with that of sample spiked with rutin in solvent. Finally, robustness study was conducted by deliberate changes in mobile phase composition and flow rate.
2.5.1. Preparation of TCMs extract 50 g of HQ and HZ was reflux extracted in 400 mL ethanol– water (7:3, v/v) at 90 °C for 1 h, repeating extraction three times, respectively. The extract solution was filtered and evaporated, then dried under vacuum at 40 °C. The extract of TCMs were conserved in a desiccator at room temperature for further experiment.
2.6. Comparison of the online system with the method of gavage in rats for screening active components of HQ and HZ To evaluate the reliability of the online system, the intestinal absorption of HQ and HZ by the gavage in living rats was detected. The extract of HQ and HZ was dissolved in 0.5% carboxy methyl cellulose sodium (CMC–Na) and orally administered to rats (n = 3) at a dosage of 5.0 g/kg, respectively. After the drug administration for 60 min, rats were anesthetized with 1.5% (w/v) pentobarbital sodium aqueous solution (60 mg/kg) by intraperitoneal administration. The blood samples were collected from the abdominal vein and centrifuged at 10,0 0 0 rpm for 10 min to obtain plasma samples. Afterwards, 750 μL plasma samples were added into a 5 mL centrifuge tube and extracted with 3 mL methanol-ethyl acetate (1:1, v/v) for three times, by which the potential active components were extracted and the protein was precipitated. Extraction was performed by vortexing for 2 min and centrifuging at 10,0 0 0 rpm for 10 min. The supernatants were combined and volatilized to dryness by nitrogen at 37 °C. Then the dried residue was dissolved in 200 μL methanol and centrifuged at 10,000 rpm for 10 min. The supernatants were detected by HPLC and UPLC–MS/MS for analysis. 2.7. HPLC analysis HPLC analysis was carried out on a Shimadzu 10A Series HPLC system, connected to a ODS column (250 × 4.6 mm, 5 μm). The mobile phase consisted of methanol (solvent A) and 0.1% formic acid-aqueous solution (solvent B). For HQ, the gradient elution process was as followed: 0–15 min, 20% A-40% A; 15–50 min, 40% A60% A; 50–65 min, 60% A-95% A; 65–70 min, 95% A; 70–80 min, 95% A-20% A; 80–90 min, 20% A. For HZ, the gradient elution process was as followed: 0–10 min, 20% A-32% A; 10–35 min, 32% A-50% A; 35–55 min, 50% A-70% A; 55–60 min, 70% A-80% A, 60–70 min, 80% A; 70–80 min, 80% A-20% A; 80–90 min, 20% A. The column temperature in HPLC analysis of two TCMs was maintained at 30 °C and the injection volume was 20 μL. The detection wavelength applied in all sections was 254 nm.
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2.8. UPLC–MS/MS analysis UPLC–MS/MS analysis was based on a LCMS-8060 series UPLC– MS/MS system (Shimadzu, Japan) which consists of a Nexera X2 UPLC system and a triple quadruple mass spectrometer. UPLC analysis methods of HQ and HZ were the same as those of online HPLC analysis in Section 2.7. High resolution mass spectrometry (MS) detections of two TCMs were performed on a triple quadruple mass spectrometer (Shimadzu, Japan). The conditions of MS/MS spectra were as followed: ion source: electronic electrospray ionization (ESI) source; ionization ways: positive or negative ion scanning; quality scan range (m/z): 100–1000; auxiliary gas (GS1 and GS2): 60 and 60 psi, respectively; air curtain air pressure (CUR): 35 psi; ion source temperature: 300 °C; ion spraying voltage (ISVF): 2500 v; the parent ion collision energy (CE): 35; the child away from the collision energy (CE): 35. 3. Results and discussion 3.1. Online system setup 3.1.1. Intestinal absorption model The common in vitro intestinal absorption models include the everted rat gut sac model, Ussing chamber model and Caco-2 cell model, these models have been used to study the intestinal absorption of oral drugs [14,16–19]. However, the absorption solution of the everted rat gut sac model is difficult to be loaded online because the diameter of small intestine of rats is too small. Thus, everted rat gut sac model is not suitable for online system. The process of culturing Caco-2 cell is time consuming, and it takes at least 50 days to complete the model. In addition, Caco-2 cell model has the same problem as the everted rat gut sac model for the online system. The absorption solution of Caco-2 cell model is not enough for loading online. The Ussing chamber model can overcome these shortcomings. It is not only easy to operate and time-saving, but also meets the requirements of online loading. The device of Ussing system has a hollow metal plate that can be poured with thermostatic water to provide a temperature environment (37 °C) for the small intestine in vitro. The spiracles on the Ussing chamber can be connected with a mixture of 95% O2 and 5% CO2 to provide a gas environment for the small intestine in vitro. In addition, Ussing chamber model has been successfully applied to study the absorption of TCMs [17]. Therefore, Ussing chamber model is an ideal intestinal absorption model for online screening of potential active ingredients in TCMs. The schematic diagram of Ussing chamber was shown in Fig. 1A. The components of TCMs are very complex, and there are differences in the absorption rate of different components. In order to ensure the effectiveness of intestinal absorption study, the intestinal absorption time of HQ and HZ was investigated by Ussing chamber model in terms of absorption kinetics and the activity of in vitro small intestines. For the absorption kinetics, it was found that the composition of the absorption solution was stable after absorption for 60 min while the content of components absorbed increased along with the absorption time. However, the small intestine will lose its activity if it is in vitro for long time. Generally, cells in the small intestine deformed severely and lost their function after 3 h in vitro, which limits the extension of absorption
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time. In study of intestinal absorption by Ussing chamber model, 90 min was usually selected as an effective absorption time [26,27]. Thus, the intestinal absorption time of the online system was determined to be 90 min. 3.1.2. Online enrichment of potential active components by SPE column Due to the species diversity and content differentia of components in TCMs, the components in intestinal absorption samples of TCMs often needed to be purified and enriched before detection. SPE is the predominant technique to enrich components of TCMs. The filling material of SPE is an important factor to affect the effect of enrichment. C18 is one of the most widely used adsorbent in SPE. Due to the effect of C18 long chain, the weak polar interaction between C18 and substance is stronger than polar interaction. Thus, C18 was usually applied to enrich the weak and medium polar compounds like flavonoids. And C18 filler has no reservation to salt, which can be used to desalinate for some small molecular samples. Thus, in this work, C18 filling was used for online enrichment of flavonoids in TCMs. When the intestinal absorption solution flowed through the SPE column, the weak polar substance (flavonoids) in the buffer solution would be retained by the C18, and the salt flowed with the water. After determining the filling of SPE, the specifications of SPE column were also discussed. The amounts of filling determined by the size of SPE column will affect the enrichment effect on components. The SPE columns in specifications of 2.1 × 10 mm and 10 × 10 mm were applied to enrich rutin online. The enrichment multiple was used to evaluate the enrichment effect of SPE column, which was defined as the chromatographic peak area ratio of rutin after and before online enrichment. As shown in Table 1, the enrichment multiple of SPE column was higher than that of 2.1 × 10 mm SPE column. The results indicated that SPE column (10 × 10 mm) filled with 500 mg C18 possessed the excellent enrichment effect on flavonoids. Therefore, the SPE column in specifications of 10 × 10 mm was considered as the optimum SPE column to enrich the active flavonoids online. 3.1.3. Online eluting of potential active components from the SPE column Considering the complexity of ingredients in TCMs, ingredients should be eluted simultaneously from the SPE column as many as possible. The interaction between C18 and flavonoids is the weak polar interaction. The high polar solvents can destroy the interaction of C18 with flavonoids and elute the components from SPE column. In the present investigation, six kinds of elution solvents were selected to elute rutin online, including ultrapure water, methanol and methanol-acetic acid (9:1, 8:2, 7:3 and 6:4, v/v). After online elution, the concentration of rutin in different eluents was shown in Fig. 2A. It was obviously that the concentration of rutin in methanol-acetic acid was higher than that in ultrapure water and methanol, indicating that methanol-acetic acid can effectively elute rutin from SPE column. The concentration of rutin in eluting solvent increased along with the proportion of acetic acid until the volume ratio of acetic acid and methanol reached 2:8. Thus, methanol-acetic acid with the volume ratio in 8:2 was considered as the optimum elution. Further, the concentration of rutin in methanol-acetic acid (8:2, v/v) was the 5.57 times of that in initial sample.
Table 1 The enrichment multiple of rutin by SPE columns in two specifications. (n = 3). Specification of SPE column
Filling
Quality of filling (mg)
Enrichment multiple of rutin
2.1 × 10 mm 10 × 10 mm
C18 C18
10.0 500
0.73 5.8
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Fig. 2. Optimization of online system. (A) The concentration of rutin in different eluting solvents. (B) The concentration of rutin in eluents at different elution time. (C) The concentration of rutin in eluents with different flow rates of eluents. Table 2 Linear regression data and precision of rutin in HPLC analysis by online system. Analyte
Linear regression Calibration curves
Rutin a
y = 7081x+5085
Precision a
Correlation coefficient
Linear range (μg/mL)
LOD (μg/mL)
LOQ (μg/mL)
Intra-day RSD (%)
Inter-day RSD (%)
0.9947
0.125–368
0.0500
0.125
2.5
3.8
y and x stand for the peak area (mAU) and the concentration (μg/mL) of the analyte, respectively.
Online eluting time was also investigated systematically. A total of twelve elution time points (0.1–1 min) were evaluated and optimized (Fig. 2B). The concentration of rutin in eluent at 0.82 min was the highest, and it was 5.81 times of that in initial sample. Therefore, the optimum time of elution was confirmed at 0.82 min with the 1 mL/min of methanol-acetic acid (8:2, v/v). Flow rates of eluting solution also had an effect on the elution efficiency. The pressure exerted on the SPE column was different by eluents at different flow rates. The elution efficiency of eluent at five different flow rates (0.3–5 mL/min) was discussed under the same volume of eluent. As shown in Fig. 2C, the eluent performed the highest elution efficiency with the flow rate at 1 mL/min. To sum up, the elution process was methanol-acetic acid (8:2, v/v) at 1 mL/min to elute for 0.82 min. All above results indicated that this online part can enrich and elute rutin (flavonoids) effectively for further analysis. 3.2. Method validation The present method was evaluated by a series of experiments about the linear range, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) of HPLC (Table 2). The calibration curve was obtained by the determination of rutin ranged from 0.125 to 368 μg/mL, and the regression equation was y = 7081x + 5085 with the R2 value of 0.9947. As shown in Table S1, the intra-day precision was performed by analyzing a spiked
Table 3 Recovery values (%) obtained for samples of intestinal absorption with the analyte at three different concentration levels. (n = 3). Analyte
Cadd (μg/mL)
Cfound (μg/mL)
Recovery (%)
RSD (%)
Rutin
1.63 11.0 55.9
1.53 10.3 52.1
94.0 93.7 93.2
11 7.3 3.1
rutin sample five times in one day at three different levels. And the inter-day precision was executed for six days at three different concentration levels of rutin sample (Table S2). The RSD of intraday precision and inter-day precision was 2.5% and 3.8%, respectively. Moreover, the LOD and LOQ detected as 3 and 10 times of the signal to noise ratio was 0.0500 μg/mL and 0.125 μg/mL, respectively. The recovery test was used to evaluate the repeatability and accuracy of the online Ussing chamber-SPE–HPLC method. The recoveries were calculated by spiking rutin standard into the blank intestinal absorption samples at three levels and the samples were analyzed by online system. As shown in Table 3, the recoveries ranged from 93.2% to 94.0% with the RSD of 3.1%−11%. In addition, selectivity evaluation was established by comparing chromatogram of blank solvent with that of sample spiked with rutin in solvent. No interfering peaks were observed at the retention time of analyte in Fig. S2. Finally, robustness study was conducted by deliberate changes in mobile phase composition and flow rate. The results indicated that there was no significant variation in RSD and
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Fig. 3. (A) The HPLC chromatograms of the extract of HQ. The UPLC–MS/MS spectra of (B) scutellarin, (C) dihydrobaicalein-7-O-glucuronide, (D) baicalin, (E) chrysin-7-Oglucuronide, (F) wogonoside, (G) baicalein, (H) dihydrowogonin, (I) wogonin, (J) chrysin, (K) oroxylin A.
retention time of rutin (Table S3). All above results showed that the proposed method was sufficiently accurate, selective, and sensitive for determination of rutin (flavonoids) in samples. 3.3. Application of online Ussing chamber-SPE–HPLC to screen the potential active components in TCMs The online system has been applied to screen the potential active flavonoids of HQ and HZ. Both HQ and HZ are indigenous medicinal plants in China and listed in the Chinese Pharmacopoeia as the typical Chinese medicines. Flavonoids are the main active components of both HQ [28] and HZ [29]. Ten flavonoids in HQ have been identified by UPLC-MS/MS, including scutellarin, dihydrobaicalein-7-O-glucuronide, baicalin, chrysin7-O-glucuronide, wogonoside, baicalein, dihydrowogonin, wogonin, chrysin and oroxylin A (Fig. 3). As shown in Fig. 4B–E, four flavonoids (catechin, polydatin, resveratrol, luteolin -7-glucuronide) were identified in HZ. Thus, a total of fourteen flavonoids were analyzed in two TCMs, and twelve flavonoids of them were screened
by online system. The HPLC chromatograms of these twelve potential active flavonoids screened online in two TCMs were shown in Fig. 5. According to previous researches [30–34], baicalin, wogonoside, baicalein and oroxylin A, polydatin and resveratrol can indeed be absorbed by organisms in multicomponent system and have certain pharmacological activities. These six flavonoids also have been screened by the online system, it indicated that the online system is credible to screen potential active components from the complex matrix of TCMs. However, the analysis of another six flavonoids (scutellarin, dihydrobaicalein-7-O-glucuronide, chrysin-7-O-glucuronide, dihydrowogonin, chrysin, and luteolin-7glucuronide) as the absorbed active components in multicomponent system was very limited, even not identified by some methods [32,33]. It is noteworthy that scutellarin, dihydrobaicalein-7O-glucuronide, chrysin-7-O-glucuronide, dihydrowogonin, chrysin, and luteolin-7-glucuronide were successfully screened and analyzed by online system in multicomponent system of TCMs. Some of them possess many pharmacological activities [35,36], such as hepatoprotective and nephroprotective activities of chrysin. The
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Fig. 4. (A) The HPLC chromatograms of the extract of HZ. The UPLC–MS/MS spectra of (B) catechin, (C) polydatin, (D) resveratrol, (E) luteolin-7-glucuronide, (F) anthraglycoside B, (G) emodin, (H) physcion, (I) citreorosein.
Fig. 5. The HPLC chromatograms of the potential active constituents screened by offline method and online system in HQ and HZ. (A) The HPLC chromatograms of (1) scutellarin, (2) dihydrobaicalein-7-O-glucuronide, (3) baicalin, (4) chrysin-7-O-glucuronide, (5) wogonoside, (6) baicalein, (7) dihydrowogonin, (9) chrysin and (10) oroxylin A in HQ. (B) The HPLC chromatograms of (2) polydatin, (3) resveratrol, (4) luteolin-7-glucuronide, (5) anthraglycoside B, (6) emodin and (8) citreorosein in HZ.
results showed the online system could screen potential flavonoid active ingredients reliably and comprehensively. Moreover, in addition to flavonoids, four anthraquinones in HZ were identified by UPLC–MS/MS (Fig. 4F–I). As shown in Fig. 5B, anthraglycoside B, emodin and citreorosein were screened as the potential active anthraquinones by the online Ussing chamber-SPE–HPLC platform. This indicated that online platform is not only suitable for
screening of flavonoids, but also for screening of anthraquinones active ingredients. The anthraquinones absorbed by Ussing chamber model were also enriched by SPE column. Therefore, the platform will be suitable to screen most kinds of ingredients as long as the proper filling of SPE are selected. Although wogonin, catechin and physcion have some pharmacological activities in vitro [37–39], they were not absorbed by intestinal absorption model in
Please cite this article as: D. Wang, J. Zeng and W. Xiang et al., Online coupling of the Ussing chamber, solid-phase extraction and high-performance liquid chromatography for screening and analysis of active constituents of traditional Chinese medicines, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460480
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multicomponent system. This may be due to the interaction among the different components of TCMs, and the reason may also be that the contents of wogonin, catechin and physcion in HQ and HZ were too low to be absorbed. Furthermore, to evaluate reliability and validity of the online system, the intestinal absorption and detection of HQ and HZ were also carried out offline by Ussing model, HPLC and UPLC–MS/MS analysis. The components absorbed were same by online and offline methods according to the results of UPLC–MS/MS, indicating that the online Ussing chamber model had the same effect as that of offline. The absorption function of Ussing chamber model was not affected after installing to online system. The HPLC chromatograms for online and offline detection of intestinal absorption samples of HQ and HZ were shown in Fig. 5. Despite the high sensitivity of HPLC, there were no the chromatographic peaks of wogonoside and citreorosein by offline analysis with the trace samples. By the online analysis, these components can not only be detected, but also some can be analyzed at quantization. In addition, chromatographic peak area of these components detected by online was 1.3–14 times of that detected by offline detection, suggesting the online SPE possessed the excellent enrichment ability. All the above results showed that the online system was able to screen, enrich and detect the potential active components of HQ and HZ in multicomponent system.
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proposed method was practical, accurate and sensitive for determination of flavonoids in samples. Finally, the online system was applied in preliminary screening of potential active flavonoids of HQ and HZ. A total of 14 flavonoids were identified by UPLC–MS/MS, and 12 of them were screened as the potential active constituents by online Ussing chamber-SPE–HPLC platform. It was showed that the online system possessed the ability to screen the potential active flavonoids of TCMs in multicomponent system. And three anthraquinones in HZ were screened and analyzed by online platform. The platform would be suitable to screen most kinds of ingredients as long as the appropriate filler was selected for online SPE process. Moreover, in comparison with offline method and gavage in rats, the online system can screen the active components from TCMs more accurately and completely. The results showed that the online system was reliable and sufficiently accurate for screening and determination of the potential active constituents of TCMs in multicomponent system. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment
3.4. Comparison of the online system with the method of gavage in rats for screening active components of HQ and HZ To evaluate the reliability of the online system, the intestinal absorption of HQ and HZ by the gavage in living rats was detected. The chromatograms of the components of HQ and HZ in rats’ plasma at 60 min were shown in Fig. S3. For the intestinal absorption of the HQ extract, the results of Ussing chamber and the gavage in rats were the same. It indicated that the components of HQ screened by the online Ussing chamber-SPE–HPLC platform were the same as that really absorbed by the animals. This further proved the online system was reliable to screen the potential active components of TCMs. In the study of intestinal absorption of HZ, one component (citreorosein) absorbed by the gavage in rats was less than that by the Ussing chamber model. According to the report, the citreorosein in HZ possessed strong anti-inflammatory effect [40]. However, there was no detection of citreorosein in rats’ plasma sample. Although the gavage in living animals is the most classic method to study the intestinal absorption of the components in TCMs by oral administration, this method will lose the trace components in the complex processing of plasma samples. Comparing with the method of gavage in living animals, online system can screen the potential active components more completely, and it is easily to operate the online system with no complex processing of samples. Therefore, the results further prove the reliability of the online system to screen the potential active components of TCMs in multicomponent system. 4. Conclusions In the present work, a semi-automated platform was established successfully for preliminary screening of potential active flavonoids of TCMs in multicomponent system. Online coupling of the Ussing chamber model, SPE and HPLC was founded for the first time. The Ussing chamber with rats’ intestinal canal was used to absorb the components of TCMs. A mini chromatographic column filled with C18 was used as a SPE column to enrich the flavonoids online. Furthermore, the specifications of SPE column, eluting solvent, elution time and flow rate of eluent were discussed for the optimization of online system. Under the optimal conditions, the
The present work was financially supported by grants from the Collaborative Innovation Center of Modern Technology and Industrial Development of Jiangxi Traditional Minority Medicine, China (no. JXXT2018001). Supplementary material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2019.460480. References [1] Y. Sun, S. Shi, Y. Li, Q. Wang, Development of quantitative structure-activity relationship models to predict potential nephrotoxic ingredients in traditional Chinese medicines, Food Chem. Toxicol. 128 (2019) 163–170. [2] L. Teng, Q. Zu, G. Li, T. Yu, K.M. Job, X. Yang, L. Di, C.M. Sherwin, E.Y. Enioutina, Herbal medicines: challenges in the modern world. Part 3. China and Japan, Expert Rev. Clin. Pharmacol. 9 (2016) 1225–1233. [3] J.L. Tang, S.Y. Zhan, E. Ernst, Review of randomised controlled trials of traditional Chinese medicine, Brit. Med. J. 319 (1999) 160–161. [4] A.L. Harvey, Natural products in drug discovery, Drug Discov. Today 13 (2008) 894–901. [5] J. Fu, Y. Lv, Q. Jia, Y. Lin, S. Han, Dual-mixed/CMC model for screening target components from traditional Chinese medicines simultaneously acting on EGFR & FGFR4 receptors, Talanta 192 (2019) 248–254. [6] X.Y. Wang, X.F. Chen, Y.Q. Gu, Y. Cao, Y.F. Yuan, Z.Y. Hong, Y.F. Chai, Progress of cell membrane chromatography and its application in screening active ingredients of traditional Chinese medicine, Chinese J. Anal. Chem. 46 (2018) 1695–1702. [7] O.U. Oghale, M. Idu, Phytochemistry, anti-asthmatic and antioxidant activities of Anchomanes difformis (Blume) Engl. leaf extract, Asian Pac. J. of Trop. Biomed. 6 (2016) 225–231. [8] Y.Q. Cai, J.H. Hu, J. Qin, T. Sun, X.L. Li, Rhododendron molle (Ericaceae): phytochemistry, pharmacology, and toxicology, Chin. J. Nat. Medicines 16 (2018) 401–410. [9] B.M. Zhang, Z.B. Wang, P. Xin, Q.H. Wang, H. Bu, H.X. Kuang, Phytochemistry and pharmacology of genus Ephedra, Chin. J. Nat. Med. 16 (2018) 811–828. [10] P. Jiang, D.G. Nagle, Y. Zhou, W. Zhang, Theories and methods for the evaluation of the pharmacodynamic material basis of traditional Chinese medicine, in: W.D. Zhang (Ed.), Systems Biology and Its Application in TCM Formulas Research, China, 2018, pp. 19–30. [11] C.S. Liu, X. Liang, X.H. Wei, F.L. Chen, Q.F. Tang, X.M. Tan, Comparative pharmacokinetics of major bioactive components from Puerariae Radix-Gastrodiae Rhizome extracts and their intestinal absorption in rats, J. Chromatogr. B 1105 (2019) 38–46. [12] Z. Zuo, L. Zhang, L. Zhou, Q. Chang, M. Chow, Intestinal absorption of hawthorn flavonoids-in vitro, in situ and in vivo correlations, Life Sci 79 (2006) 2455–2462.
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JID: CHROMA 10
ARTICLE IN PRESS
[m5G;September 14, 2019;12:50]
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[13] R.M. Hernández, A.R. Gascón, M.B. Calvo, C. Caramella, U. Conte, A. Domínguez-Gil, J.L. Pedraz, Correlation of ‘in vitro’ release and ‘in vivo’ absorption characteristics of four salbutamol sulphate formulations, Int. J. Pharm. 139 (1996) 45–52. [14] J.M. Yang, S.P.P. Ip, H.K.J. Yeung, C.T. Che, HPLC-MS analysis of Schisandra lignans and their metabolites in Caco-2 cell monolayer and rat everted gut sac models and in rat plasma, Acta Pharm. Sin. B 1 (2011) 46–55. [15] M. Mauro, R.A. De Grandis, M.L. Campos, A. Bauermeister, R.G. Peccinini, F.R. Pavan, N.P. Lopes, N.V. De Moraes, Acid diterpenes from Copaiba oleoresin (Copaifera langsdorffii): chemical and plasma stability and intestinal permeability using Caco-2 cells, J. Ethnopharmacol. 235 (2019) 183–189. [16] D. Scherbl, S. Muentnich, E. Richling, In vitro absorption studies of chlorogenic acids from coffee using the Ussing chamber model, Food Res. Int. 63 (2014) 456–463. [17] J. Wang, G. Aierken, X. Li, L. Li, X. Mao, Testing the absorption of the extracts of Coreopsis tinctoria Nutt. in the intestinal canal in rats using an Ussing chamber, J. Ethnopharmacol. 186 (2016) 73–83. [18] H. Yang, B. Zhai, Y. Fan, J. Wang, J. Sun, Y. Shi, D. Guo, Intestinal absorption mechanisms of araloside a in situ single-pass intestinal perfusion and in vitro Caco-2 cell model, Biomed. Pharmacother. 106 (2018) 1563–1569. [19] M. Zorzan, D. Collazuol, G. Ribaudo, A. Ongaro, C. Scaroni, G. Zagotto, D. Armanini, S. Barollo, F. Galeotti, N. Volpi, M. Redaelli, R. Pezzani, Biological effects and potential mechanisms of action of Pistacia lentiscus Chios mastic extract in Caco-2 cell model, J. Funct. Foods 54 (2019) 92–97. [20] K. Georgi, K.S. Boos, Multidimensional on-line SPE for undisturbed LC–MS–MS analysis of basic drugs in biofluids, Chromatographia 63 (2006) 523–531. [21] J. Li, Y. Cai, Y. Shi, S. Mou, G. Jiang, Analysis of phthalates via HPLC-UV in environmental water samples after concentration by solid-phase extraction using ionic liquid mixed hemimicelles, Talanta 74 (2008) 498–504. [22] X. Gao, B. Yang, Z. Tang, X. Luo, F. Wang, H. Xu, X. Cai, Determination of phthalates released from paper packaging materials by solid-phase extraction-high-performance liquid chromatography, J. Chromatogr. Sci. 52 (2014) 383–389. [23] D. Salazar-Beltran, L. Hinojosa-Reyes, E. Ruiz-Ruiz, A. Hernandez-Ramirez, J. Luis Guzman-Mar, Determination of phthalates in bottled water by automated on-line solid phase extraction coupled to liquid chromatography with uv detection, Talanta 168 (2017) 291–297. [24] X. Jiang, J. Zhou, Q. Lin, G. Gong, H. Sun, W. Liu, Q. Guo, F. Feng, W. Qu, Anti-angiogenic and anticancer effects of baicalein derivatives based on transgenic zebrafish model, Bioorg. Med. Chem. 26 (2018) 4481–4492. [25] Z. Chen, Q. Wei, G. Hong, D. Chen, J. Liang, W. He, M.H. Chen, Polydatin induces bone marrow stromal cells migration by activation of ERK1/2, Biomed. Pharmacother. 82 (2016) 49–53. [26] C. Mendes, G.C. Meirelles, M.A.S. Silva, G. Ponchel, Intestinal permeability determinants of norfloxacin in Ussing chamber model, Eur. J. Pharm. Sci. 121 (2018) 236–242. [27] S. Kondo, M. Miyake, Simultaneous prediction of intestinal absorption and metabolism using the mini-Ussing chamber system, J. Pharm. Sci. 108 (2019) 763–769.
[28] O.N. Seo, G.S. Kim, Y.H. Kim, S. Park, S.W. Jeong, S.J. Lee, J.S. Jin, S.C. Shin, Determination of polyphenol components of Korean Scutellaria baicalensis Georgi using liquid chromatography–tandem mass spectrometry: contribution to overall antioxidant activity, J. Funct. Foods 5 (2013) 1741–1750. [29] G.S. Qian, S.Y. Leung, G.H. Lu, K.S.Y. Leung, Differentiation of Rhizoma et Radix Polygoni Cuspidati from closely related herbs by HPLC fingerprinting, Chem. Pharm. Bull. 54 (2006) 1179–1186. [30] Y.K. Fong, C.R. Li, S.K. Wo, S. Wang, L. Zhou, L. Zhang, G. Lin, Z. Zuo, In vitro and in situ evaluation of herb-drug interactions during intestinal metabolism and absorption of baicalein, J. Ethnopharmacol. 141 (2012) 742–753. [31] X.L. Liang, Z.G. Liao, J.Y. Zhu, G.W. Zhao, M. Yang, R.L. Yin, Y.C. Cao, J. Zhang, L.J. Zhao, The absorption characterization effects and mechanism of Radix Angelicae dahuricae extracts on baicalin in Radix Scutellariae using in vivo and in vitro absorption models, J. Ethnopharmacol. 139 (2012) 52–57. [32] C. Li, S.Y. Fong, Q. Mei, G. Lin, Z. Zuo, Influence of mefenamic acid on the intestinal absorption and metabolism of three bioactive flavones in Radix Scutellariae and potential pharmacological impact, Pharm. Biol. 52 (2014) 291– 297. [33] S. Xing, M. Wang, Y. Peng, D. Chen, X. Li, Simulated gastrointestinal tract metabolism and pharmacological activities of water extract of Scutellaria baicalensis roots, J. Ethnopharmacol. 152 (2014) 183–189. [34] J. Fu, S. Wu, M. Wang, Y. Tian, Z. Zhang, R. Song, Intestinal metabolism of Polygonum cuspidatum in vitro and in vivo, Biomed. Chromatogr. 32 (2018) e4190. [35] R.B. Pingili, A.K. Pawar, S.R. Challa, T. Kodali, S. Koppula, V. Toleti, A comprehensive review on hepatoprotective and nephroprotective activities of chrysin against various drugs and toxic agents, Chem. Biol. Interact. 308 (2019) 51– 60. [36] C.Y. Li, Q. Wang, X. Wang, G. Li, S. Shen, X. Wei, Scutellarin inhibits the invasive potential of malignant melanoma cells through the suppression epithelial-mesenchymal transition and angiogenesis via the PI3K/Akt/mTOR signaling pathway, Eur. J. Pharmacol. 858 (2019) 172463. [37] D.L. Huynh, N. Sharma, A. Kumar Singh, S. Singh Sodhi, J.J. Zhang, R.K. Mongre, M. Ghosh, N. Kim, Y. Ho Park, D. Kee Jeong, Anti-tumor activity of wogonin, an extract from Scutellaria baicalensis, through regulating different signaling pathways, Chin. J. Nat. Med. 15 (2017) 15–40. [38] P. Pheomphun, C. Treesubsuntorn, P. Thiravetyan, Effect of exogenous catechin on alleviating O3 stress: the role of catechin-quinone in lipid peroxidation, salicylic acid, chlorophyll content, and antioxidant enzymes of Zamioculcas zamiifolia, Ecotoxicol. Environ. Saf. 180 (2019) 374–383. [39] X.P. Pan, C. Wang, Y. Li, L.H. Huang, Physcion induces apoptosis through triggering endoplasmic reticulum stress in hepatocellular carcinoma, Biomed. Pharmacother. 99 (2018) 894–903. [40] Y. Lu, S.J. Suh, X. Li, S.L. Hwang, Y. Li, K. Hwangbo, S.J. Park, M. Murakami, S.H. Lee, Y. Jahng, J.K. Son, C.H. Kim, H.W. Chang, Citreorosein, a naturally occurring anthraquinone derivative isolated from Polygoni cuspidati radix, attenuates cyclooxygenase-2-dependent prostaglandin D2 generation by blocking Akt and JNK pathways in mouse bone marrow-derived mast cells, Food Chem. Toxicol. 50 (2012) 913–919.
Please cite this article as: D. Wang, J. Zeng and W. Xiang et al., Online coupling of the Ussing chamber, solid-phase extraction and high-performance liquid chromatography for screening and analysis of active constituents of traditional Chinese medicines, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460480