Authors:
Chun ChenDepartment of Pharmacy, Third Affiliated Hospital of Naval Medical University, Shanghai, China
Jiangxi University of Chinese Medicine, Nanchang, China

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Lei LvDepartment of Pharmacy, Third Affiliated Hospital of Naval Medical University, Shanghai, China

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Yueying HuangDepartment of Pharmacy, Third Affiliated Hospital of Naval Medical University, Shanghai, China

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Mingzhu GaoDepartment of Pharmacy, Third Affiliated Hospital of Naval Medical University, Shanghai, China
Jiangxi University of Chinese Medicine, Nanchang, China

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Xue JiangDepartment of Pharmacy, Third Affiliated Hospital of Naval Medical University, Shanghai, China
Jiangxi University of Chinese Medicine, Nanchang, China

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Xiaoying GeDepartment of Pharmacy, Third Affiliated Hospital of Naval Medical University, Shanghai, China
Jiangxi University of Chinese Medicine, Nanchang, China

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Dan ZhengDepartment of Pharmacy, Third Affiliated Hospital of Naval Medical University, Shanghai, China
Jiangxi University of Chinese Medicine, Nanchang, China

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Leilei BaoDepartment of Pharmacy, Third Affiliated Hospital of Naval Medical University, Shanghai, China
Jiangxi University of Chinese Medicine, Nanchang, China

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https://orcid.org/0000-0003-4403-4598
Open access

Abstract

Rationale

The bark of Eucommia ulmoides and the roots of Achyranthes bidentata are commonly used in traditional Chinese medicine, and their pairing appears in many traditional Chinese medicine formulas as a recognized compatible unit. However, the changes and interactions of the main components of these two formulas when paired remain unclear, and there is currently no standard or method for their quality control and assessment of pharmacological effects.

Methods

An optimized ultra-high-performance liquid chromatography triple-quadrupole mass spectrometry (UHPLC-MS/MS) method was established for the simultaneous identification of 10 components in E. ulmoides and A. bidentata using in vitro and in vivo models. Tributyltin methacrylate was the internal standard solution, and the blood samples were treated by an organic solvent precipitation method. Gradient elution was conducted on a C18 column at 25 °C with 0.1% formic acid water:acetonitrile as the mobile phase at a flow rate of 0.5 mL min−1. Dynamic multiple response monitoring was performed in negative-ion mode using an Agilent Jet Stream electrospray ionization ion source.

Results

In negative-ion detection mode, eucommiol exhibited a good response, and the isomers ginsenoside Ro and achyranthoside C could also be well separated. The developed method accurately detected the five components with a low blood content. Compared to controls, the levels of ginsenoside Ro, chikusetsusaponin Ⅳa, and achyranthoside C increased; the contents of geniposidic acid and pinoresinol diglucoside were unchanged; and the levels of eucommiol, geniposide, β-ecdysterone, genipin, and achyranthoside D decreased in vitro. In vivo, the contents of geniposidic acid, geniposide, pinoresinol diglucoside, and β-ecdysterone were reduced; the contents of eucommiol and ginsenoside Ro were unchanged; and those of achyranthoside D, chikusetsusaponin Ⅳa, and achyranthoside C increased compared to the corresponding levels in the internal control.

Conclusions

A method for the quality control of the E. ulmoides-A. bidentata drug pair was established for the first time and the main components in 10 drug pairs could be determined simultaneously in vitro and in vivo. These findings show that the E. ulmoides and A. bidentata drug pair cause a compositional change, providing new ideas for the development of this combination to improve clinical efficacy.

Abstract

Rationale

The bark of Eucommia ulmoides and the roots of Achyranthes bidentata are commonly used in traditional Chinese medicine, and their pairing appears in many traditional Chinese medicine formulas as a recognized compatible unit. However, the changes and interactions of the main components of these two formulas when paired remain unclear, and there is currently no standard or method for their quality control and assessment of pharmacological effects.

Methods

An optimized ultra-high-performance liquid chromatography triple-quadrupole mass spectrometry (UHPLC-MS/MS) method was established for the simultaneous identification of 10 components in E. ulmoides and A. bidentata using in vitro and in vivo models. Tributyltin methacrylate was the internal standard solution, and the blood samples were treated by an organic solvent precipitation method. Gradient elution was conducted on a C18 column at 25 °C with 0.1% formic acid water:acetonitrile as the mobile phase at a flow rate of 0.5 mL min−1. Dynamic multiple response monitoring was performed in negative-ion mode using an Agilent Jet Stream electrospray ionization ion source.

Results

In negative-ion detection mode, eucommiol exhibited a good response, and the isomers ginsenoside Ro and achyranthoside C could also be well separated. The developed method accurately detected the five components with a low blood content. Compared to controls, the levels of ginsenoside Ro, chikusetsusaponin Ⅳa, and achyranthoside C increased; the contents of geniposidic acid and pinoresinol diglucoside were unchanged; and the levels of eucommiol, geniposide, β-ecdysterone, genipin, and achyranthoside D decreased in vitro. In vivo, the contents of geniposidic acid, geniposide, pinoresinol diglucoside, and β-ecdysterone were reduced; the contents of eucommiol and ginsenoside Ro were unchanged; and those of achyranthoside D, chikusetsusaponin Ⅳa, and achyranthoside C increased compared to the corresponding levels in the internal control.

Conclusions

A method for the quality control of the E. ulmoides-A. bidentata drug pair was established for the first time and the main components in 10 drug pairs could be determined simultaneously in vitro and in vivo. These findings show that the E. ulmoides and A. bidentata drug pair cause a compositional change, providing new ideas for the development of this combination to improve clinical efficacy.

1 Introduction

In traditional Chinese medicine (TCM), a drug pair is a fixed compatibility unit between a single drug and a formulation. Drug pairs have developed from the application of a single drug to the formulation of multiple drugs that enrich patient treatment. The emergence of a drug pair improves the effect and efficacy of drugs, expands the scope of their application, and sets the foundation for the composition of prescriptions [1]. Eucommia ulmoides and Achyranthes bidentata are recorded as a common drug pair in The couplet medicines of the traditional Chinese medicine encyclopedia [2]. Both E. ulmoides and A. bidentata are used to tonify separately the liver and kidney, and to strengthen muscles and bones. Their combination is considered not only to nourish “vitality” and enrich the blood but also to enhance the curative effect of pain relief.

E. ulmoides and A. bidentata have demonstrated anti-inflammatory, antitumor, antioxidant, and other pharmacological activities [3, 4]. Several methods have been used to identify the main components of E. ulmoides and A. bidentata in the blood to date. Wu et al. evaluated seven components in E. ulmoides by gradient elution of high-performance liquid chromatography (HPLC) fingerprints in vitro [5]; however, the detection time was very long and three wavelength conversion conditions were required for analysis. Fu et al. determined 26 types of saponins in A. bidentata using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-TOF/MS) and explored their possible pharmacological mechanisms using a network pharmacology approach [6]. Hu et al. developed an in vivo method to simultaneously measure five components of the Eucommia cortex extract in blood samples after oral administration [7]. There are numerous relevant studies on A. bidentata saponins metabolism [8, 9] and tissue quantitative studies on a characteristic sterone [10]. However, few of the two main ingredients in A. bidentata have been evaluated together. Furthermore, changes in the content of the main components of E. ulmoides and A. bidentata as a drug pair have not been evaluated in vivo or in vitro.

In this study, we selected components from E. ulmoides and A. bidentata that are required by the pharmacopoeia and could be used as criteria for evaluating qualified medicinal materials. Eucommiol is a unique ingredient in E. ulmoides and one of its main water-soluble components, which has suggested sedation, hypnosis [11], and collagen synthesis-promotion [12] effects. Establishing a standard to determine the content of eucommiol could help to expand the pharmacodynamic potential for its clinical use. E. ulmoides also contain the lignans genipin, geniposidic acid, and geniposide. Although studies have been conducted on the transformation and pharmacological activity of these three lignans, it is also worth exploring whether their relative content changes and if these three components interact in vivo and in vitro [13, 14].

A. bidentata is enriched in β-ecdysterone, which is used to qualify for the drug. Total saponins in A. bidentata can promote osteogenic differentiation of bone marrow stromal cells through stimulation of extracellular regulated protein kinases and mitogen-activated protein kinase (MAPK) signaling pathways [15]. Previous studies have combined total saponins with geniposide to explore their effect on the fibroblast cytoskeleton through the MAPK signaling pathway [16]. If specific saponins can be quantitatively analyzed from blood samples, their active forms can be characterized, which would facilitate the discovery of specific monomer combinations that play a role in drug-pair compatibility.

In this study, 10 representative components were selected through basic research of drug-pair chemicals and a related literature review, including representative compounds with reported pharmacological activity in both E. ulmoides and A. bidentata [17–20], and unique components of E. ulmoides and A. bidentata with no previously reported quality control (QC) standards. Thus, there is a need to optimize existing methods to develop a suitable method that can detect the above selected components equally sensitive to lower sample concentrations. Therefore, we developed an efficient UHPLC tandem mass spectrometry (MS/MS) method to evaluate the changes in these 10 main components in vitro and investigate the medicinal compatibility of E. ulmoides and A. bidentata. We also evaluated the absorption of this drug pair in vivo by detecting these components in blood samples from rats after administration. Therefore, this study provides a foundation and a new method for the QC of novel drug pairs, along with a comprehensive analysis of the unique pharmacological activities produced by this drug pair.

2 Material and methods

2.1 Samples and regents

Biological reference standards for β-ecdysterone (LOT: T02A9F57640), ginsenoside Ro (LOT: A09GB144447), achyranthoside C (LOT: J18GB155125), achyranthoside D (LOT: N06GB166738), chikusetsusaponin Ⅳa (LOT: C19D10S106246), pinoresinol diglucoside (LOT: C19M11S109808), geniposide (LOT: S14J10Y79596), genipin (LOT: J21J9T66173), and eucommiol (LOT: J20GB155124) were purchased from Shanghai Yuanye Biotechnology Co., Ltd., and geniposidic acid (LOT: MUST-21062003) was purchased from Chengdu Manst Biotechnology Co., Ltd. Acetonitrile and formic acid were of MS grade, and water was self-made in the laboratory, and other reagents were analytically pure. E. ulmoides (Sichuan, China LOT: 191,231) and A. bidentata (Henan, China LOT: 210,326) were purchased from Shanghai Hongqiao TCM Yinpian Co., Ltd. and identified as acceptable by Professor Zheng Chengjian of the Department of TCM Identification, School of Pharmacy, Naval Medical University.

2.2 Animals and blood collection

Sprague-Dawley (SD) rats (200 ± 20 g) of specific pathogen-free grade were supplied by Shanghai Regen Biological Co., Ltd. (experimental animal production license number SCXK2019-0002). Fifteen SD rats were kept in the animal room of the School of Pharmacy of Naval Medical University, under a fixed room temperature of 20 °C and a 12-h light/dark cycle. After one week of acclimatization to the laboratory environment, blood samples were collected by orbital blood collection into an Eppendorf (EP) tube precoated with sodium heparin. Blank plasma was obtained after centrifugation (13,800 × g for 5 min) of the blood samples and stored at −80 °C until analysis. Guide concerning the Care and Use of Laboratory Animals, and the experiments were carried out with the approval of the Animal Experimentation Ethics Committee of Naval Medical University.

The 15 rats were randomly divided into E. ulmoides, A. bidentata, or drug-pair groups (n = 5 per group). The rats were administered 1 g mL−1 and 2 g mL−1 of the crude drug concentration solution intragastrically, depending on their weight. Approximately 240 g of E. ulmoides or A. bidentata powder were weighed and incubated overnight in 2,400 mL of pure water. After boiling and maintenance for 1 h, pour out the filtrate. This step was repeated three times, and the filtrates were combined, concentrated, and dissolved with water to the desired concentration. After 3 days, the plasma concentration reached steady state and blood samples were collected 0.5 and 1 h after administration on the third day. The operation was the same as blank blood samples. The blood samples obtained at these two time points were mixed, which in a 1:1 ratio (n = 5 per group).

2.3 Instrument and UHPLC-MS method

We used the Agilent 1,290 Series Ultra Performance Liquid Chromatography platform (Agilent, Santa Clara, CA, USA) platform. Chromatographic separation was conducted on a SHISEIDO MG-C18 column (100 mm × 3.0 mm, 3.0 μm) with mobile phases comprising 0.1% formic acid in water (A) and acetonitrile (B) by gradient elution (0–6 min: 5%–30% B; 6–13 min: 30%–70% B; 13–16 min, 70% B). The post time was 3 min, the flow rate was 0.5 mL min−1, the column temperature was 25 °C, and the injection volume was 2 μL. The Agilent 6470 Series Triple Quad Mass Spectrometry system was used for MS, with mass spectrometric detection in dynamic multiple response monitoring mode equipped with a JetStream electrospray interface source (Agilent) in negative-ion mode. The MS operating conditions were optimized as follows: gas temperature: 325 °C, gas flow rate: 10 L min−1, atomizer pressure: 40 psi, sheath gas temperature: 350 °C, sheath gas flow rate: 11 L min−1, capillary voltage: 3,500 V, nozzle voltage: 1,500 V, and collision gas: nitrogen. Other MS parameters are shown in Table 1.

Table 1.

Mass spectrometry parameters for 10 components in E. ulmoides and A. bidentata

CompoundtR/minFragmented ion pair m/zCollision Energy/eVFragmentor/V
Eucommiol1.87233.1→233.1170
Geniposidic acid2.91373.11→210.99127
Geniposide4.71433.14→2259113
Pinoresinol diglucoside4.84681.24→3572580
β-ecdysterone5.81525.31→479.117107
Genipin5.94225.07→101979
Ginsenoside Ro9.15955.49→955.49180
Achyranthoside D9.611117.5→1117.5180
Chikusetsusaponin Ⅳa9.81793.44→631.24980
Achyranthoside C10.14955.45→955.45180
TBTM(IS)10.89269.09→169.917108

2.4 Preparation of solutions

2.4.1 Standard solution

We measured 1 mg of eucommiol, geniposidic acid, geniposide, pinoresinol diglucoside, β-ecdysterone, genipin, ginsenoside Ro, achyranthoside D, chikusetsusaponin Ⅳa, and achyranthoside C in 1.5-mL brown EP tubes, which were then solubilized in methanol to obtain a stock solution concentration of 1 mg mL−1. These stocks were diluted in methanol to a working concentration of 4 μg mL−1 geniposidic acid,geniposide, genipin or 20 μg mL−1 eucommiol, pinoresinol diglucoside, β-ecdysterone, ginsenoside Ro, achyranthoside D, chikusetsusaponin Ⅳa, or achyranthoside C, and stored at 4 °C until further use.

2.4.2 Standard experimental blood and QC samples

Single standard solutions of 1 mg mL−1 eucommiol, geniposidic acid, geniposide, pinoresinol diglucoside, β-ecdysterone, genipin, ginsenoside Ro, achyranthoside D, chikusetsusaponin Ⅳa, and achyranthoside C were prepared, respectively. A 20 μL aliquot of each solution was added to 800 μL methanol to prepare 20 μg mL−1 mixed solutions, which were then serially diluted to prepare 10,000, 2,000, 1,000, 400, 200, 100, and 20 ng mL−1 mixed solutions. Subsequently, 50 μL of each solution was added to 950 μL of a blank blood sample. Standard blood samples containing 1,000, 500, 100, 50, 20, 10, 5, and 1 ng mL−1 concentrations were obtained by blending the blank blood samples. QC sample solutions at concentrations of 1, 2, 50, and 800 ng mL−1 were prepared using the same method.

2.5 Sample pretreatment

2.5.1 Test solution

One gram of E. ulmoides powder and A. bidentata powder were separately dissolved in 100 mL pure water in a beaker and allowed to stand overnight, before being boiled in an electric furnace for 1 h. Pour out the filtrate. This operation was repeated three times, the filtrate was combined and concentrated before being transferred to the measuring cylinder to fill the volume and mixed well to a final concentration of 0.02 g mL−1 to obtain the drug-pair test solution. The method of preparation of single-drug test solutions was consistent with that of the drug-pair solution; 0.01 g mL−1E. ulmoides and A. bidentata were used in these experiments.

2.5.2 Blood sample pretreatment

A 200 μL plasma sample was added to four times its volume of acetonitrile to precipitate the protein, followed by vortexing to mix the solution and centrifugation at 13,800 × g for 5 min. The solvent was evaporated with a solvent concentrator for 1 h before adding 50 ng mL−1 of tributyltin methacrylate as the internal standard solution. After mixing and centrifugation at 13,800 × g for 5 min, the supernatant was collected for later use.

2.6 Method validation

2.6.1 In vitro method validation

The method described in Section 2.3 was used for the methodological verification in vitro and the application of the method to medicinal materials was investigated by comparing the chromatogram of blank, standard, E. ulmoides, A. bidentata, and drug-pair solutions. By mixing 200, 500, 1,000, 2,000, 5,000, and 10,000 ng mL−1 solutions of eucommiol, pinoresinol diglucoside, β-ecdysterone, ginsenoside Ro, achyranthoside D, chikusetsusaponin Ⅳa, and achyranthoside C were obtained. In addition, solutions of 40, 100, 200, 400, 1,000, and 2,000 ng mL−1 of geniposidic acid, geniposide, and genipin were obtained. Standard curves for these components were constructed and the precision and repeatability of the calculations were measured by the fourth concentration point of the mixed standard solution for three consecutive measurements and six parallel measurements, respectively. The recovery rate was extracted by adding the drug pair to the mixed standard solution, followed by preparing the drug-pair solution and comparing the sample after mixing with the theoretical quantity. The stability of E. ulmoides, A. bidentata, and drug-pair solutions was evaluated by sampling single and drug-pair solutions at six time points over 24 h. In addition, the column temperature, flow rate, and initial ratio of the mobile phase were altered to record the changes in peak area and retention time to investigate the durability of the method. Finally, the standard sample was injected into the MS machine to determine any changes in the content of the components of single-drug and drug-pair solutions. The relative standard deviation (RSD) of the durability peak area was within 5% and the RSD of the retention time was within 10%. The RSD of other tests should be within 2%.

2.6.2 In vivo method validation

After the blood samples were processed as described in Section 2.5.2, in vivo methodological verification was conducted by UHPLC-MS/MS, as described in Section 2.3. By comparing the selectivity of blank blood samples; QC blood samples; and blood samples after E. ulmoides, A. bidentata, and drug-pair administration; as well as standard blood samples containing 1,000, 500, 100, 50, 20, 10, 5, and 1 ng mL−1 of single components, the calibration curves were created by plotting the peak area ratios of the analyte to the internal standard (y) against the concentration of the analyte (x) with 1/x weighting, with a strong linear relationship (R ≥ 0.9959) obtained for each compound. Precision and accuracy were determined using QC samples with low, medium, and high concentrations six times and for three consecutive days. The matrix effect was investigated using 2 ng mL−1 of the low-concentration QC sample (LQC) and 800 ng mL−1 of the high-concentration QC sample (HQC) by detecting the response of the extracted blank matrix with different rats and the response of the tested substance in methanol solution. The extraction recovery was determined with 2 ng mL−1 LQC, 50 ng mL−1 of the middle-concentration QC sample (MQC), and 800 ng mL−1 HQC. The extraction recovery was calculated by detecting the response of the QC sample and the blank matrix with the tested substance. The stability of the compound was investigated under various storage conditions after sample preparation. After the upper limit of quantification was determined, a blank blood sample was used to investigate the residual monomer. Finally, the blood samples of each group after drug administration were measured by injection; the RSD of all results should be <15%.

2.7 Experimental design and data analysis

Initially, the databases of TCMSP (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform https://old.tcmsp-e.com/tcmsp.php) and Batman-TCM (Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine http://bionet.ncpsb.org/batman-tcm/) were searched to identify candidate compounds E. ulmoides and A. bidentata. After the database was established according to chemical structure classification, HPLC-TOF/MS was used to detect the compound components in the purchased medicinal materials. After detection, numerous components were detected at high concentrations in the in vitro samples. Additional relevant articles [5–8] were retrieved to determine the most suitable initial method for identifying the 10 candidate compounds. After optimization, these methods were applied for validation of the in vitro methodology and to determine the concentration of the crude drugs during the initial determination of total compounds associated with the medicine solutions, which were used to make suitable compound concentration ranges according to the curve. Because the in vitro method showed a good response, this method was then applied to the in vivo condition and used to establish a detection system that would be suitable for both in vivo and in vitro models. For the components that changed under in vitro conditions, we adopted content correction measures using standard solutions to compensate for differences in in vitro compatibility following an established method to investigate the changes of 10 components with higher species content in vivo, so that the total content of the drug-pair and single-drug solutions remained the same. We used IBM SPSS Statistics 21 analysis software to perform Student's t-tests on all data to evaluate whether there were significant differences between conditions.

3 Results and discussion

3.1 Method development

3.1.1 Optimization of the UHPLC-MS/MS method

The commonly used mobile-phase systems include methanol-glacial acetic acid [21], acetonitrile-water [22], and methanol-water [23]. We selected an acetonitrile-0.1% formic acid aqueous solution as the mobile phase so each component could be well eluted. In the negative ion mode, the mobile phase contains trace amounts of formic acid, which contributes to the improvements of the peak shape. In addition, the common ion effect of formic acid can increase the ionization efficiency of the substance to be measured to some extent. To further ensure a good elution of each component, the running time was shortened by adjusting the gradient from 25 to 16 min, so each component had a high separation and a good peak shape. Achyranthoside C and ginsenoside Ro are isomers of each other. By optimizing the gradient elution time of the first 13 min, the peak time interval of the two was 1 min, which enables their clear separation so they can be reliably identified and quantified. In the preliminary experiment, eucommiol was found to show a good response in positive-ion detection mode, whereas the other nine components showed a better response in negative-ion mode. We would consider the method of positive and negative switching to detect all components; however, this would affect the stability of the test result. Therefore, we optimized the multiple reaction monitoring parameters so the instrument could detect all components efficiently and accurately only in negative-ion mode. This optimization resulted in a good response, high separation, and good peak shape for each component. Furthermore, all the tested compounds could be eluted in 16 min. Because low sample concentrations are difficult to detect using conventional methods, we used a special ion source with a sheath gas. Using Agilent Jet Stream technology increased the response of the analyte on electrospray ionization, and the lower limit of quantitation (LLOQ) was as low as 1 ng mL−1. Therefore, the optimal multiple reaction monitoring conditions were finally obtained.

3.1.2 Selection of a preparation method of TCM test solutions

Because the study of the medicinal ingredient content itself has clinical value, when a drug enters the clinical stage, the extraction of medicinal materials must be nontoxic, safe, and effective. Therefore, in this study, we only considered clinically available solutions as the tested extraction solutions. TCM extraction methods is a collective name for a class of methods that use some preparative processes to extract the active ingredients of Chinese medicine. We used a TCM extraction method for producing test sample water decoctions, which can release numerous hydrophilic components into the medicinal solution. In the drug-pair preparation process, ethanol was used to remove polysaccharides after crude extraction in water; however, the use of ethanol does not meet the clinical requirements for medicinal materials. Despite the presence of polysaccharides, the drug solutions did not adhere to the sampler or ion source due to the suitable crude drug concentration; therefore, the drug was directly decocted and then combined with the concentrated filtrate. When considering the medicinal materials of the bark of E. ulmoides and the roots of A. bidentata, direct decoction was not conducive to the dissolution of the effective ingredients. Thus, a mixture of the two was prepared as described in the literature [24, 25] to develop an appropriate extraction process by breaking the herbs into a certain size. These filtrates were concentrated and then diluted in water to obtain the test solution with a known concentration.

3.2 In vitro method validation

3.2.1 Specificity

An appropriate amount of methanol solution was used as a blank control and reference solution of E. ulmoides, A. bidentata, and the drug-pair test solution to evaluate the optimal instrumental setting and conditions for content analysis. The chromatogram showed that each compound had a good response under the optimized conditions with no obvious miscellaneous peaks, which confirmed that the method was highly specific for the 10 target components (Fig. 1). The number of theoretical plates of all measured components was >3,000, the separation degree was >1.5, and the tailing factor was between 0.95 and 1.05. These parameters indicated that the system had good applicability to the chromatographic column.

Fig. 1.
Fig. 1.

Extracted Ion Chromatography (EIC) of blank (A), E. ulmoides (B), A. bidentata (C), drug pair (D), and reference (E) test solutions

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01090

3.2.2 Linearity

The mixed reference solution was prepared and used in a 1:2:2.5:2:2: 2.5 cascade dilution mode for sample determination. The 10 compounds tested were analyzed and the limit of quantitation (LOQ) was calculated with a signal-to-noise ratio of 3; see Table 2 for details.

Table 2.

Linear investigation of 10 components from E. ulmoidesA. bidentata drug pair solutions

CompoundRegression equationRange (ng mL−1)RLOQLOD
(ng mL−1)(ng mL−1)
Eucommioly = 0.0141*x + 0.1133200∼10,0000.999952.5
Geniposidic acidy = 0.0355*x + 0.001840∼2,0000.999910.5
Geniposidey = 0.0631*x − 0.002840∼2,0000.999910.5
Pinoresinol diglucosidey = 0.0434*x − 0.0311200∼10,0000.99990.50.25
β-ecdysteroney = 0.0527*x + 0.1094200∼10,0000.99950.50.25
Genipiny = 0.0577*x − 0.001540∼2,0000.999910.5
Ginsenoside Roy = 0.2826*x + 0.6165200∼10,0000.99970.50.25
Achyranthoside Dy = 0.0245*x + 0.2036200∼10,0000.99990.50.25
Chikusetsusaponin Ⅳay = 0.0358*x + 0.1516200∼10,0000.99960.50.25
Achyranthoside Cy = 0.0271*x + 0.2023200∼10,0000.99950.50.25

3.3.3 Precision

The mixed control product solution at the fourth concentration point was measured three times a day for three consecutive days to calculate the RSD for all components. The intra- and inter-day precisions were 0.23–1.95% and 0.06–1.96%, respectively, according to the concentration of the 10 components, using the QQQ quantitative analysis software, indicating that the precision of the instrument was good.

3.3.4 Assay repeatability

Six drug-pair samples were prepared in parallel and injected into the system, followed by analysis with QQQ quantitative analysis software. The contents of eucommiol, geniposidic acid, geniposide, pinoresinol diglucoside, β-ecdysterone, genipin, ginsenoside Ro, achyranthoside D, chikusetsusaponin Ⅳa, and achyranthoside C were 4477.51 ng mL−1 (RSD = 1.41%), 1840.19 ng mL−1 (RSD = 0.49%), 665.68 ng mL−1 (RSD = 0.92%), 9981.70 ng mL−1 (RSD = 0.17%), 5079.33 ng mL−1 (RSD = 0.95%), 1.65 ng mL−1 (RSD = 1.83%), 7305.99 ng mL−1 (RSD = 1.92%), 1152.58 ng mL−1 (RSD = 1.86%), 5717.31 ng mL−1 (RSD = 0.79%), and 4164.34 ng mL−1 (RSD = 1.78%), respectively. Therefore, the RSDs were below 2.0% (n = 6), indicating that the method has good repeatability and could quantify the complex composition of the drug-pair treatments.

3.3.5 Recovery

Six 0.5 g samples for E. ulmoides and A. bidentata were taken, which were compared with the mixed reference solutions in quantitative QQQ analysis to obtain the concentration of each compound and calculate its recovery. The results are shown in Table 3.

Table 3.

Recovery of the 10 candidate components from E. ulmoidesA. bidentata drug pair treatment samples

CompoundOriginal (μg)Added (μg)Found (μg)Recovery (%)Average (%)RSD (%)
Eucommiol375.1100273.1101.96100.791.73
373.9100273.1100.8
379.3100279.999.44
377.6100275102.54
386.3100288.398.01
386.3100284.4101.97
Geniposidic acid101.62082.893.6192.691.98
99.42080.395.48
103.12084.493.47
100.1208290.4
103.82085.591.48
119.420101.191.73
Geniposide54.32033.4104.57101.651.61
54.52034.4100.18
542033.7101.32
54.52034.1102.1
53.52033.5100.08
53.82033.5101.66
Pinoresinol diglucoside555.1100454.5100.5999.960.43
566100466.299.81
558.9100458.999.92
570.3100470.1100.24
560.1100460.899.29
569100469.199.91
β-ecdysterone346.1100246.599.4999.680.33
346100245.8100.16
359.8100260.199.63
353.8100254.299.53
356100255.999.99
356.6100257.399.27
Genipin0.22020.3100.59103.531.58
0.22020.8102.78
0.22020.7104.45
0.22020.7104.69
0.22020.7103.72
0.12020.7104.96
Ginsenoside Ro520.5100422.697.997.461.89
530.9100433.297.58
552.1100452.199.98
542.7100447.395.27
543100447.695.39
541.6100442.998.67
Achyranthoside D158.310058.699.6699.830.42
153.410053.799.64
159.110059.799.4
151.110050.5100.57
156.310056.2100.06
167.810068.199.65
Chikusetsusaponin Ⅳa334.5100232.7101.86100.991.26
347.4100244.4102.99
344100243.9100.02
325.8100226.199.7
344100243.9100.13
322.6100221.3101.24
Achyranthoside C300.8100200.6100.22100.211.64
310.5100213.397.14
312.1100210102.09
316.6100216.3100.3
317.1100216.5100.6
334.8100233.9100.89

3.3.6 Stability

E. ulmoides and A. bidentata were prepared as a drug pair for compositional analysis. After being left to rest at room temperature for 0, 2, 4, 8, 12, and 24 h, the samples were injected into the MS machine and the peak area was recorded. The RSD of eucommiol, geniposidic acid, geniposide, pinoresinol diglucoside, β-ecdysterone, genipin, ginsenoside Ro, achyranthoside D, chikusetsusaponin Ⅳa, and achyranthoside C was 1.21%, 1.49%, 1.88%, 0.44%, 1.02%, 1.28%, 1.44%, 1.85%, 1.99%, and 0.75%, respectively, which were all <2.0%, indicating that the test product had good stability within 24 h at room temperature. These results suggested that the experimental sample had stable elution under the optimized conditions.

3.3.7 Durability

E. ulmoides and A. bidentata were used to prepare drug-pair test solutions. The peak area and retention time RSDs were all lower than 4.47% and 0.54% after changing the column temperature and were below 6.26% and 3.59% after changing the flow rate, respectively. After changing the initial acetonitrile ratio, the peak area and retention time RSDs were all less than 3.93% and 9.8%, respectively, which indicated that the method had good durability.

3.4 In vitro sample content determination

E. ulmoides and A. bidentata were prepared as single test solutions and a drug pair, and injected into the MS machine for analysis using QQQ quantitative analysis software to calculate the content of each compound under each condition (Table 4).

Table 4.

The content of E. ulmoides, A. bidentata, and drug pair solutions

CompoundContents in E. ulmoide (μg/g)Contents in A. bidentata (μg/g)Contents in drug pair (μg/g)Content change
Eucommiol561.04410.04
Geniposidic acid179.06162.13
Geniposide108.3461.29
Pinoresinol diglucoside992.35911.44
β-ecdysterone654.75508.52
Genipin34.910.18
Ginsenoside Ro489.46770.99
Achyranthoside D206.57162.02
Chikusetsusaponin Ⅳa414.74628.65
Achyranthoside C266.99411.63

↗: Up, ↘: Down, →: Almost unchanged, —: Undected.

As shown in Table 4, the contents of eight components changed to a certain extent after the drug-pair combination compared to those of single-drug solutions. Among them, the contents of ginsenoside Ro, chikusetsusaponin Ⅳa, and achyranthoside C in A. bidentata increased significantly after drug pairing. Ginsenoside Ro is an oleanolic acid-type saponin found in A. bidentata, which has been shown to inhibit apoptosis and inflammation of rat chondrocytes by inhibiting the NF-κB signaling pathway [26], and could improve obesity and insulin resistance induced by a high-fat diet in mice by activating Takeda G-protein-coupled receptor 5 (TGR5) [17]. Another study also found that the ginsenoside Ro content increased when used in a combined ginseng decoction with Trogopteroum feces or Raphani semen, suggesting that the combined decoction promoted the dissolution of ginsenoside Ro [27].

In the combination of E. ulmoides and A. bidentata decoction, the pH environment of the solution changed. Previous studies have shown that chikusetsusaponin Ⅳa can inhibit adipose homeostasis induced by a high-fiber diet and has anti-adipose tissue inflammation effects [18]. Furthermore, chyranthoside C can inhibit reactive oxygen species and cell apoptosis [19]. Apparently, the dissolving out of these two ingredients can be affected by the pH environment of the solution. The contents of geniposidic acid and pinoresinol diglucoside decreased after the combination of E. ulmoides and A. bidentata, although the difference was small compared to the content of the individual drugs. The content of these components remained almost unchanged from that of the single drugs after the E. ulmoides and A. bidentata solutions were combined as a drug pair. The dissolution of eucommiol, β-ecdysterone, and achyranthoside D decreased slightly in the drug pair compared to single-drug solutions, which may have been caused by the change in the final pH of the test solutions. The geniposide and genipin showed an obvious decrease; these two components are iridoids, and genipin is made from geniposide via β-glucosidase hydrolysis. Thus, as the geniposide content decreased, the genipin content was also reduced. Besides the pH affecting the compounds dissolved in the drug solution, geniposide and genipin may react with other substances within the solution that cause them to precipitate in water. Therefore, the filtration of the drug solutions may have separated these compounds from the final medicinal solutions tested. In vivo and in vitro tests have confirmed that genipin is hepatotoxic [20]; thus, reducing the genipin content may help reduce hepatotoxicity and improve the side effect of liver injury induced by TCM drugs observed in the clinical use of E. ulmoides. However, further verification is needed to determine whether genipin is transformed into a new compound during drug pairing.

3.5 In vivo method validation

3.5.1 Selectivity

Analysis of blank blood, QC samples and blood from animals treated with E. ulmoides, A. bidentata, or drug-pair solutions showed each compound has a good response under the test conditions with no obvious miscellaneous peaks, which confirmed that the method is highly selective (Fig. 2).

Fig. 2.
Fig. 2.

Extracted Ion Chromatography (EIC) of blank blood sample (A), quality control sample (B), blood containing E. ulmoides (C), blood containing A. bidentata (D), and blood containing drug pair (E)

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01090

3.5.2 Standard curve and LLOQ

Standard blood samples were obtained by mixing a standard solution with blank blood samples, which were used to determine the concentration ranges. The standard curves of these components were evaluated by linear regression analysis with 1/x weighting. The LLOQ was determined by the signal-to-noise ratio of >10, as shown in Table 5.

Table 5.

Standard curves and LLOQ of blood samples containing 10 components

CompoundRegression equationRange (ng mL−1)RLLOQ
(ng mL−1)
Eucommioly = 0.0111*x + 0.00161∼1,0000.99891
Geniposidic acidy = 0.1034*x + 0.00031∼1,0000.99931
Geniposidey = 0.1323*x + 0.00141∼1,0000.99961
Pinoresinol diglucosidey = 0.0976*x + 0.00041∼1,0000.99991
β-ecdysteroney = 0.1415*x + 0.00091∼1,0000.99941
Genipiny = 0.0183*x − 0.000041∼1,0000.99821
Ginsenoside Roy = 0.4152*x + 0.01991∼1,0000.99961
Achyranthoside Dy = 0.0350*x + 0.00621∼1,0000.99591
Chikusetsusaponin Ⅳay = 0.0930*x + 0.00331∼1,0000.99811
Achyranthoside Cy = 0.0530*x + 0.00251∼1,0000.99961

3.5.3 Precision and accuracy

Three known concentrations of each drug solution were used as QC samples, with six samples at each concentration point prepared in parallel for the analysis of the experimental samples. Blood samples were pretreated and continuously injected for three days for analysis. The intra-day and inter-day precision RSD and relative error (RE) of the 10 compounds were less than 19.61% and 19.46%, respectively, which met the methodological requirements. Furthermore, these results showed that the performance of the instrument is stable.

3.5.4 Matrix effect

Six blank blood samples from different sources were mixed with acetonitrile in a 1:4 ratio without an internal standard solution. The blank supernatant was mixed with the standard mixed solution prepared in methanol to obtain a solution with a 2 ng mL−1 LOQ and 800 ng mL−1 HOQ. Mixed standard solutions of the same concentration without blood matrix were also prepared. Six parallel samples were calculated for each concentration of the matrix factor and internal standard matrix factor using the compound and internal standard responses obtained after injection analysis, and the normalized matrix factor was calculated. The coefficient of variation obtained was less than 15% and the matrix had little influence on the accuracy of the method (Table 6).

Table 6.

Experimental results of matrix effects on drug pair (n = 6)

CompoundConcentrationMatrix factorIS matrix factorNormalized matrix factorRSD
(ng ml−1)(%)(%)(%)(%)
Eucommiol292.6995.3597.211.46
80077.5595.6381.061.73
Geniposidic acid2112.8495.35118.416.80
80090.9695.6394.960.38
Geniposide2106.3995.35111.549.74
80088.7295.6392.630.61
Pinoresinol diglucoside2107.7695.35112.905.80
80091.5895.6395.590.52
β-ecdysterone2103.4995.35108.269.64
80091.8395.6395.870.96
Genipin264.1795.3566.9211.06
80087.5595.6390.843.57
Ginsenoside Ro2207.7095.35215.336.84
800109.0295.63113.810.64
Achyranthoside D290.8495.3595.241.77
80080.8595.6383.283.21
Chikusetsusaponin Ⅳa2183.8695.35187.7315.43
800103.6895.63108.240.88
Achyranthoside C294.7395.3599.375.00
80099.3795.6394.102.95

3.5.5 Extraction recovery

Blank blood samples from the same rat were taken to obtain the blank supernatant, which was then mixed with a standard solution prepared in methanol to obtain 2 ng mL−1 of LOQ, 50 ng mL−1 of MOQ, and 800 ng mL−1 of HOQ. Three concentrations of QC samples were prepared with six parallel samples for each concentration. After injection analysis, extraction recoveries were calculated, and the RSDs were all less than 15% (Table 7).

Table 7.

Experimental results of extraction recovery (n = 6)

CompoundConcentration (ng ml−1)Extraction recovery (%)RSD (%)
Eucommiol281.1210.34
5086.485.80
80090.643.79
Geniposidic acid280.559.71
5089.816.90
80091.964.07
Geniposide280.2110.40
5099.174.25
80097.292.74
Pinoresinol diglucoside283.955.28
5084.143.38
80083.872.96
β-ecdysterone284.3210.43
50102.883.72
80099.343.15
Genipin282.837.39
5079.444.30
80090.3410.38
Ginsenoside Ro283.467.90
5081.575.55
80080.494.13
Achyranthoside D288.276.74
5082.0410.29
80080.156.99
Chikusetsusaponin Ⅳa280.476.74
5089.9510.29
80090.286.99
Achyranthoside C280.2311.27
5082.3414.29
80091.374.85

3.5.6 Stability

The stock solutions of these components and the internal standard were stored stably at 4 °C for 30 days, and the REs were less than 10%. Three QC samples at low and high concentrations were incubated at room temperature for 3 h, at 4 °C for 24 h, put through a freeze-thaw cycle three times, and at 80 °C for 30 days. The sample determination results are shown in Table 8. The RSDs and REs were less than 14.78% and 17.94%, respectively.

Table 8.

Experimental results of stability (n = 3)

CompoundConcentrationRoom temperature 3hAutosampler (24h, 4 °C)Three freeze/thaw cycleLong term (30 day, −80 °C)
(ng ml−1)RSDRERSDRERSDRERSDRE
(%)(%)(%)(%)(%)(%)(%)(%)
Eucommiol22.668.177.47−4.2811.129.876.675.84
8003.75−8.410.632.810.629.293.14−3.47
Geniposidic acid214.783.165.92−2.132.91−8.324.78−6.13
8000.951.281.24−3.730.62−7.552.95−10.83
Geniposide25.7610.4610.258.684.94−9.8713.7513.86
8000.588.060.571.460.855.530.5714.07
Pinoresinol diglucoside23.156.676.746.892.43−12.5213.81−6.68
8002.393.522.372.640.72−1.872.76−5.60
β-ecdysterone24.22−2.878.170.594.03−14.678.8017.94
8001.118.751.654.840.94−3.250.863.75
Genipin210.8810.919.29−1.0210.31−9.8910.40−13.53
8001.7114.531.636.130.901.171.28−8.72
Ginsenoside Ro212.089.541.281.944.07−2.044.277.83
8000.5611.471.320.840.51−10.150.676.89
Achyranthoside D27.424.583.5911.489.331.238.32−2.13
8001.95−2.191.066.401.830.970.77−4.92
Chikusetsusaponin Ⅳa29.9514.465.367.303.38−0.735.389.32
8000.8413.610.756.400.562.531.278.68
Achyranthoside C29.576.8412.317.3013.498.572.7914.82
8001.01−1.290.770.612.533.400.877.12

3.5.7 Residual

Blank blood samples were injected according to the upper limit of injection quantity, which showed that the peak area at the retention time of each analyte was less than 20% of the LLOQ, whereas the peak area at the retention time for the internal standard was less than 5% of the actual internal standard. These results indicated that the method resulted in almost no residue and did not affect the determination of components.

3.6 Determination of blood sample content

Blood samples were collected and pretreated to obtain three groups of sample solutions. The samples were injected into the MS machine and QQQ quantitative analysis software was used to calculate the content of each compound in single and drug-pair combinations (Table 9).

Table 9.

Determination of sample content in the blood of rats (n = 5)

CompoundContents in Eucommia ulmoides (ng/g)Contents in Achyranthes bidentata (ng/g)Contents in drug pair (ng/g)Content change
Eucommiol224.37 ± 26.96205.93 ± 26.34
Geniposidic acid152.73 ± 21.5026.42 ± 3.95**
Geniposide13.30 ± 1.921.40 ± 0.21**
Pinoresinol diglucoside7.42 ± 0.784.74 ± 0.65**
β-ecdysterone1.77 ± 0.251.19 ± 0.16**
Genipin
Ginsenoside Ro5.77 ± 0.345.95 ± 0.79
Achyranthoside D12.12 ± 1.1122.46 ± 3.22**
Chikusetsusaponin Ⅳa1.08 ± 0.161.38 ± 0.20*
Achyranthoside C1.05 ± 0.042.29 ± 0.34**

*P < 0.05, **P < 0.01 compare with single drug group.

↗: Up, ↘: Down, →: Almost unchanged, —: Undected.

Among the 10 components investigated, all but genipin were present in the prototypical form in the rat plasma (Table 9). Genipin levels were already extremely low in vitro, indicating that incompatibility did not affect genipin absorption. Compared to those in the single-drug treatment groups, after lavage, plasma concentrations of achyranthoside C and achyranthoside D increased after drug mixing. The contents of geniposidic acid and pinoresinol diglucoside showed almost no change in vitro, whereas in the in vivo microenvironment, drug mixing accelerated the absorption of both compounds, resulting in faster elimination from the body. Drug accumulation can cause toxicity and side effects, and mixing single drugs into a paired solution could prevent absorption to a certain extent, resulting in an overall reduction in the blood drug concentration. However, our results suggest that after drug-pair mixing, geniposide may be transformed into other forms with off-target biological effects, or the mixing can directly reduce the time to peak geniposide blood concentrations.

The β-ecdysterone in vitro tests showed a general reduction in the drug-pair samples; however, in the in vivo condition, compared with the single-drug samples, the blood content of β-ecdysterone was similar. β-Ecdysterone is a component of A. bidentata, which has been shown to alleviate osteoarthritis [28], and its adsorption is promoted in vivo when used in a drug pair, thus enhancing the curative effect of osteoarthritis.

As a highly specific component of E. ulmoides, the in vivo eucommiol absorption properties were consistent with the results of the single drug sample, and were the only components that showed nearly no change in the drug-pair samples compared to the single-drug samples. Ginsenoside Ro exhibited a rising trend after mixing in vitro, whereas there was no significant difference between the single-drug groups and drug pairing in vivo. This suggests that internal environment may have also contributed to the production of metabolites with other biological activities because of drug pairing.

Although most drugs are administered orally and absorbed through the intestine to further exert their efficacy, the saponins in A. bidentata are difficult to absorb in the intestine because of their large molecular weight and polarity [29], and they are also highly susceptible to the influence of the intestinal flora. Under the action of various metabolic enzymes such as hydrolase and oxidoreductase, secondary glycosides, or aglycones are generated from these saponins. This study suggests that the compatibility of a drug pair may improve the absorption of saponins to a certain extent, enabling them to play a bioactive role as prototypes. We found that the content of some drug components increased in vitro when combined as a drug pair, whereas there was little difference in the component content when mixed drug pairs were administered in vivo. It is essential to use an orthogonal design of the compatibility ratio of E. ulmoides and A. bidentata to find the optimal drug ratio and improve the absorption of these saponins in vivo and to further explore the pharmacokinetic behaviors.

The metabolism of relevant active components by intestinal flora after drug pairing in the blood should also be studied to identify metabolites that are more easily absorbed after conversion of the associated compounds, which would lay the foundation for the study of the efficacy of TCM. In addition, in this study, the content of single TCM drugs changed after drug pairing, with a single drug sometimes showing the complete opposite effect in vivo and in vitro, which may indicate that a drug pair can exert a greater potency potential.

4 Conclusion

To our knowledge, this is the first study to establish a method for the in vitro determination of the content of 10 components in E. ulmoides and A. bidentata drug-pair decoctions. In our method, low-content components in a drug pair can be detected even when using only a negative-ion detection mode, and the ginsenoside Ro and achyranthoside C, which are mutual isomers, can be well separated. We tested whether the 10 components tested were observed at quantifiable levels in their prototype forms in the blood of rats administered single and paired drug solutions. Our results provide a quality standard evaluation method for E. ulmoides and A. bidentata, as well as providing information on the possible causes of observed changes in component content in vitro and in vivo after drug pairing. Therefore, this work provides a basis for a MS method that can promote the further clinical application and research of TCM drug-pair treatments.

Author contributions

Chun Chen was responsible for sample analysis, data processing, and manuscript writing. Lei Lv provided experimental guidance and development of mass spectrometry methods. Yueying Huang created the tables and figures. Mingzhu Gao was in charge of the literature review. Xue Jiang participated in the pretreatment of samples. Xiaoying Ge collected and sorted the samples. Dan Zheng analyzed some of the data. Leilei Bao provided project sources, writing guidance, and supervision.

Declaration of interest statement

The authors state that they have no known competing interests or personal ties related to the subject of this article.

Funding

This study was supported by the Shanghai Natural Fund [grant number 19ZR1456500] and Innovation and Generation of Military Medical Support Capability [grant number 20WQ011].

Acknowledgements

The authors thank Editage (www.editage.cn) for English language editing.

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    Lin, F.; Wang, Y-H.; Wan, L.; Yang, R-P. Study on the quality control of eucommiae cortex by multi-components quantitation by one marker method and fingerprint. Chin. J. Exp. Traditional Med. Formulae 2012, 18(13), 7882. https://doi.org/10.13422/j.cnki.syfjx.2012.13.027.

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    Zhang, M.; Zhao, H.; Zhou, S. Content determination of β-ecdysterone and oleanolic acid in Achyranthes bidentata blume by HPLC and their fingerprints. Shandong Sci. 2015, 28(05), 16. https://doi.org/10.3976/j.issn.1002-4026.2015.05.001.

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    Zhang, X.-H.; Xu, X.-X.; Xu, T. Ginsenoside Ro suppresses interleukin-1β-induced apoptosis and inflammation in rat chondrocytes by inhibiting NF-ΚB. Chin. J. Nat. Medicines 2015, 13(4), 283289. https://doi.org/10.1016/S1875-5364(15)30015-7.

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    Zhang, X.; Wang, L.; Song, F.; Liu, Z.; Liu, S. Study on the variation of chemical constituents during combination of ginseng with Trogopteroum feces and semen Raphani by high performance liquid chromatography mass spectrome try. Chin. J. Anal. Chem. 2007, 35(4), 559563. https://doi.org/10.3321/j.issn:0253-3820.2007.04.021.

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    Chen, M.-Y.; Shao, L.; Zhang, W.; Wang, C.-Z.; Zhou, H.-H.; Huang, W.-H.; Yuan, C.-S. Metabolic analysis of panax notoginseng saponins with gut microbiota-mediated biotransformation by HPLC-DAD-Q-TOF-MS/MS. J. Pharm. Biomed. Anal. 2018, 150, 199207. https://doi.org/10.1016/j.jpba.2017.12.011.

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    Li, S. Effects of Geniposide, Achyranthes Bidentata Saponions and Their Combination on the Expression of Filamentous Actin in Fibroblast-like Synovicytes of Adjuvant Arthritis Rats; Anhui University of Traditional Chinese Medicine: Anhui AH, 2017. https://kns.cnki.net/kcms/detail/detail.aspx?FileName=1017199133.nh&DbName=CMFD2018.

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    Jiang, L.; Li, W.; Zhuang, T.; Yu, J.; Sun, S.; Ju, Z.; Wang, Z.; Ding, L.; Yang, L. Ginsenoside Ro ameliorates high-fat diet–induced obesity and insulin resistance in mice via activation of the G protein–coupled bile acid receptor 5 pathway. J. Pharmacol. Exp. Ther. 2021, 377(3), 441451. https://doi.org/10.1124/jpet.120.000435.

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    Yuan, C.; Liu, C.; Wang, T.; He, Y.; Zhou, Z.; Dun, Y.; Zhao, H.; Ren, D.; Wang, J.; Zhang, C.; Yuan, D. Chikusetsu saponin IVa ameliorates high fat diet-induced inflammation in adipose tissue of mice through inhibition of NLRP3 inflammasome activation and NF-ΚB signaling. Oncotarget 2017, 8(19), 3102331040. https://doi.org/10.18632/oncotarget.16052.

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    Wang, S.; Zeng, M.; Li, B.; Kan, Y.; Zhang, B.; Zheng, X.; Feng, W. Raw and salt-processed Achyranthes bidentata attenuate LPS-induced acute kidney injury by inhibiting ROS and apoptosis via an estrogen-like pathway. Biomed. Pharmacother. 2020, 129, 110403. https://doi.org/10.1016/j.biopha.2020.110403.

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    Lin, F.; Wang, Y-H.; Wan, L.; Yang, R-P. Study on the quality control of eucommiae cortex by multi-components quantitation by one marker method and fingerprint. Chin. J. Exp. Traditional Med. Formulae 2012, 18(13), 7882. https://doi.org/10.13422/j.cnki.syfjx.2012.13.027.

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    • Export Citation
  • 22.

    Zhang, M.; Zhao, H.; Zhou, S. Content determination of β-ecdysterone and oleanolic acid in Achyranthes bidentata blume by HPLC and their fingerprints. Shandong Sci. 2015, 28(05), 16. https://doi.org/10.3976/j.issn.1002-4026.2015.05.001.

    • Search Google Scholar
    • Export Citation
  • 23.

    Zhang, C.; Liang, S.; Zhang, G. Determination of ecdysterone in Achyranthes bidentata from different locations. Chin. J. Pharm. 2001, 10, 5253. https://kns.cnki.net/kcms/detail/detail.aspx?FileName=ZGYX200110018&DbName=CJFQ2001.

    • Search Google Scholar
    • Export Citation
  • 24.

    Xie, G.; Jiang, N.; Wang, S.; Qi, R.; Wang, L.; Zhao, P.; Liang, L.; Yu, B. Eucommia ulmoides oliv. Bark aqueous extract inhibits osteoarthritis in a rat model of osteoarthritis. J. Ethnopharmacol. 2015, 162, 148154. https://doi.org/10.1016/j.jep.2014.12.061.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Wang, Y.; Zhang, M.; Zha, L.; Guo, Y. Research on the extraction process of polysaccharides in cyathulae radix and Achyranthes bidentatae radix. J. Changchun Normal Univ. 2016, 35(10), 7781. https://kns.cnki.net/kcms/detail/detail.aspx?FileName=CCSS201610018&DbName=CJFQ2016.

    • Search Google Scholar
    • Export Citation
  • 26.

    Zhang, X.-H.; Xu, X.-X.; Xu, T. Ginsenoside Ro suppresses interleukin-1β-induced apoptosis and inflammation in rat chondrocytes by inhibiting NF-ΚB. Chin. J. Nat. Medicines 2015, 13(4), 283289. https://doi.org/10.1016/S1875-5364(15)30015-7.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Zhang, X.; Wang, L.; Song, F.; Liu, Z.; Liu, S. Study on the variation of chemical constituents during combination of ginseng with Trogopteroum feces and semen Raphani by high performance liquid chromatography mass spectrome try. Chin. J. Anal. Chem. 2007, 35(4), 559563. https://doi.org/10.3321/j.issn:0253-3820.2007.04.021.

    • Search Google Scholar
    • Export Citation
  • 28.

    Tang, Y.; Mo, Y.; Xin, D.; Zeng, L.; Yue, Z.; Xu, C. β-Ecdysterone alleviates osteoarthritis by activating autophagy in chondrocytes through regulating PI3K/AKT/MTOR signal pathway. Am. J. Transl Res. 2020, 12(11), 71747186, PMCID: PMC7724317.

    • Search Google Scholar
    • Export Citation
  • 29.

    Chen, M.-Y.; Shao, L.; Zhang, W.; Wang, C.-Z.; Zhou, H.-H.; Huang, W.-H.; Yuan, C.-S. Metabolic analysis of panax notoginseng saponins with gut microbiota-mediated biotransformation by HPLC-DAD-Q-TOF-MS/MS. J. Pharm. Biomed. Anal. 2018, 150, 199207. https://doi.org/10.1016/j.jpba.2017.12.011.

    • Crossref
    • Search Google Scholar
    • Export Citation
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Senior editors

Editor(s)-in-Chief: Kowalska, Teresa

Editor(s)-in-Chief: Sajewicz, Mieczyslaw

Editors(s)

  • Danica Agbaba (University of Belgrade, Belgrade, Serbia)
  • Ivana Stanimirova-Daszykowska (University of Silesia, Katowice, Poland)
  • Monika Waksmundzka-Hajnos (Medical University of Lublin, Lublin, Poland)

Editorial Board

  • R. Bhushan (The Indian Institute of Technology, Roorkee, India)
  • J. Bojarski (Jagiellonian University, Kraków, Poland)
  • B. Chankvetadze (State University of Tbilisi, Tbilisi, Georgia)
  • M. Daszykowski (University of Silesia, Katowice, Poland)
  • T.H. Dzido (Medical University of Lublin, Lublin, Poland)
  • A. Felinger (University of Pécs, Pécs, Hungary)
  • K. Glowniak (Medical University of Lublin, Lublin, Poland)
  • B. Glód (Siedlce University of Natural Sciences and Humanities, Siedlce, Poland)
  • A. Gumieniczek (Medical University of Lublin, Lublin, Poland)
  • U. Hubicka (Jagiellonian University, Kraków, Poland)
  • K. Kaczmarski (Rzeszow University of Technology, Rzeszów, Poland)
  • H. Kalász (Semmelweis University, Budapest, Hungary)
  • K. Karljiković Rajić (University of Belgrade, Belgrade, Serbia)
  • I. Klebovich (Semmelweis University, Budapest, Hungary)
  • A. Koch (Private Pharmacy, Hamburg, Germany)
  • Ł. Komsta (Medical University of Lublin, Lublin, Poland)
  • P. Kus (Univerity of Silesia, Katowice, Poland)
  • D. Mangelings (Free University of Brussels, Brussels, Belgium)
  • E. Mincsovics (Corvinus University of Budapest, Budapest, Hungary)
  • Á. M. Móricz (Centre for Agricultural Research, Budapest, Hungary)
  • G. Morlock (Giessen University, Giessen, Germany)
  • A. Petruczynik (Medical University of Lublin, Lublin, Poland)
  • R. Skibiński (Medical University of Lublin, Lublin, Poland)
  • B. Spangenberg (Offenburg University of Applied Sciences, Germany)
  • T. Tuzimski (Medical University of Lublin, Lublin, Poland)
  • Y. Vander Heyden (Free University of Brussels, Brussels, Belgium)
  • A. Voelkel (Poznań University of Technology, Poznań, Poland)
  • B. Walczak (University of Silesia, Katowice, Poland)
  • W. Wasiak (Adam Mickiewicz University, Poznań, Poland)
  • I.G. Zenkevich (St. Petersburg State University, St. Petersburg, Russian Federation)

 

KOWALSKA, TERESA
E-mail: kowalska@us.edu.pl

SAJEWICZ, MIECZYSLAW
E-mail:msajewic@us.edu.pl

Indexing and Abstracting Services:

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2021  
Web of Science  
Total Cites
WoS
652
Journal Impact Factor 2,011
Rank by Impact Factor Chemistry, Analytical 66/87
Impact Factor
without
Journal Self Cites
1,789
5 Year
Impact Factor
1,350
Journal Citation Indicator 0,40
Rank by Journal Citation Indicator Chemistry, Analytical 72/99
Scimago  
Scimago
H-index
29
Scimago
Journal Rank
0,27
Scimago Quartile Score Chemistry (miscellaneous) (Q3)
Scopus  
Scopus
Cite Score
2,8
Scopus
CIte Score Rank
General Chemistry 210/409 (Q3)
Scopus
SNIP
0,586

2020
 
Total Cites
650
WoS
Journal
Impact Factor
1,639
Rank by
Chemistry, Analytical 71/83 (Q4)
Impact Factor
 
Impact Factor
1,412
without
Journal Self Cites
5 Year
1,301
Impact Factor
Journal
0,34
Citation Indicator
 
Rank by Journal
Chemistry, Analytical 75/93 (Q4)
Citation Indicator
 
Citable
45
Items
Total
43
Articles
Total
2
Reviews
Scimago
28
H-index
Scimago
0,316
Journal Rank
Scimago
Chemistry (miscellaneous) Q3
Quartile Score
 
Scopus
393/181=2,2
Scite Score
 
Scopus
General Chemistry 215/398 (Q3)
Scite Score Rank
 
Scopus
0,560
SNIP
 
Days from
58
submission
 
to acceptance
 
Days from
68
acceptance
 
to publication
 
Acceptance
51%
Rate

2019  
Total Cites
WoS
495
Impact Factor 1,418
Impact Factor
without
Journal Self Cites
1,374
5 Year
Impact Factor
0,936
Immediacy
Index
0,460
Citable
Items
50
Total
Articles
50
Total
Reviews
0
Cited
Half-Life
6,2
Citing
Half-Life
8,3
Eigenfactor
Score
0,00048
Article Influence
Score
0,164
% Articles
in
Citable Items
100,00
Normalized
Eigenfactor
0,05895
Average
IF
Percentile
20,349
Scimago
H-index
26
Scimago
Journal Rank
0,255
Scopus
Scite Score
226/167=1,4
Scopus
Scite Score Rank
Chemistry (miscellaneous) 240/398 (Q3)
Scopus
SNIP
0,494
Acceptance
Rate
41%

 

Acta Chromatographica
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Acta Chromatographica
Language English
Size A4
Year of
Foundation
1992
Volumes
per Year
1
Issues
per Year
4
Founder Institute of Chemistry, University of Silesia
Founder's
Address
PL-40-007 Katowice, Poland, Bankowa 12
Publisher Akadémiai Kiadó
Publisher's
Address
H-1117 Budapest, Hungary 1516 Budapest, PO Box 245.
Responsible
Publisher
Chief Executive Officer, Akadémiai Kiadó
ISSN 2083-5736 (Online)

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