Skip to main content
Download PDF

UHPLC-QQQ-MS/MS assay for the quantification of dianthrones as potential toxic markers of Polygonum multiflorum Thunb: applications for the standardization of traditional Chinese medicines (TCMs) with endogenous toxicity

Chinese Medicine volume 16, Article number: 51 (2021) Cite this article

Abstract

Background

The raw and processed roots of Polygonum multiflorum Thunb (PM) are commonly used in clinical practice to treat diverse diseases; however, reports of hepatotoxicity induced by Polygoni Multiflori Radix (PMR) and Polygoni Multiflori Radix Praeparata (PMRP) have emerged worldwide. Thus, it is necessary for researchers to explore methods to improve quality standards to ensure their quality and treatment effects.

Methods

In the present study, an ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method was optimized and validated for the determination of dianthrones in PMR and PMRP using bianthronyl as the internal standard. Chromatographic separation with a gradient mobile phase [A: acetonitrile and B: water containing 0.1% formic acid (v/v)] at a flow rate of 0.25 mL/min was achieved on an Agilent ZORBAX SB-C18 column (2.1 mm × 50 mm, 1.8 μm). The triple quadrupole mass spectrometer (TQMS) was operated in negative ionization mode with multiple reaction monitoring for the quantitative analysis of six dianthrones. Moreover, compounds 5 and 6 were further evaluated for their cytotoxicity in HepaRG cells by CCK-8 assay.

Results

The UHPLC-QQQ-MS/MS method was first developed to simultaneously determine six dianthrones in PMR and PMRP, namely, polygonumnolides C1–C4 (1–4), trans-emodin dianthrones (5), and cis-emodin dianthrones (6). The contents of 1–6 in 90 batches of PMR were in the ranges of 0.027–19.04, 0.022–13.86, 0.073–15.53, 0.034–23.35, 0.38–83.67 and 0.29–67.00 µg/g, respectively. The contents of 1–6 in 86 batches of commercial PMRP were in the ranges of 0.020–13.03, 0.051–8.94, 0.022–7.23, 0.030–12.75, 0.098–28.54 and 0.14–27.79 µg/g, respectively. Compounds 1–4 were almost completely eliminated after reasonable processing for 24 h and the contents of compounds 5 and 6 significantly decreased. Additionally, compounds 5 and 6 showed inhibitory activity in HepaRG cells with IC50 values of 10.98 and 15.45 μM, respectively. Furthermore, a systematic five-step strategy to standardize TCMs with endogenous toxicity was proposed for the first time, which involved the establishment of determination methods, the identification of potentially toxic markers, the standardization of processing methods, the development of limit standards and a risk–benefit assessment.

Conclusion

The results of the cytotoxicity evaluation of the dianthrones indicated that trans-emodin dianthrones (5) and cis-emodin dianthrones (6) could be selected as toxic markers of PMRP. Taking PMR and PMRP as examples, we hope this study provides insight into the standardization and internationalization of endogenous toxic TCMs, with the main purpose of improving public health by scientifically using TCMs to treat diverse complex diseases in the future.

Introduction

Polygonum multiflorum Thunb, including Polygoni Multiflori Radix (PMR) and PMR Praeparata (PMRP), is a commonly used TCMs used to treat various diseases in China and is also popular in many other countries [1, 2]. PMR has many common indications, including detoxification, elimination of carbuncles, malaria prevention, and relaxation of the bowel, while PMRP is well known as a tonic medicine for blackening of the hair, nourishing the liver and kidney, haematopoiesis, and so on [3,4,5]. However, since the 1990s, a significant number of adverse hepatotoxic reactions have occurred in China, South Korea, Japan, England, Canada, and other countries from the use of these medicines [6,7,8]. The chemical composition of PMR can be significantly altered by processing, and its hepatotoxicity can be minimized accordingly. Some studies have shown that processing could result in a decrease in certain compounds, such as 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucopyranoside (THSG), emodin-8-O-β-d-glucoside, catechin, epicatechin, and physcion-8-O-β-d-glucopyranoside; however, these compounds did not disappear [2, 9, 10]. These studies demonstrated that there may be no direct link between the above mentioned compounds and PMR-induced liver injury.

Our previous work on PMR toxicity showed that the dianthrones that were first isolated from PMR by our team could have potential hepatotoxicity, and there are many minor dianthrones in PMR [11,12,13,14,15,16,17]. Moreover, dianthrones can increase the content of Fe3+ ions and degrade easily when heated [18, 19]. These features are very similar to those of the hepatotoxic components of PMR [20, 21]. However, to the best of our knowledge, there have been no reports on which types of dianthrones are toxicity markers of PMRP or the mechanisms to decrease the toxicity of these TCMs. Therefore, in this study, an effective and sensitive UHPLC-QQQ-MS/MS method was established, and the qualitative analysis of six dianthrones was presented. The excellent selectivity and sensitivity achieved for these target compounds in multi-reaction monitoring (MRM) mode allowed for satisfactory confirmation and quantitation [22]. In addition, the proposed UHPLC-QQQ-MS/MS method was successfully used for dianthrone determination in PMR and PMRP. To the best of our knowledge, this work is the most comprehensive study on the contents of dianthrones in PMR and PMRP. The results showed that there is a strong correlation between dianthrones and PMR-induced liver damage, and trans-emodin dianthrones (5) and cis-emodin dianthrones (6) could be chosen as potential toxicity markers of PMRP. Furthermore, a systematic five-step strategy to standardize TCMs with endogenous toxicity was proposed for the first time, which involved the establishment of determination methods, the identification of toxic markers, the standardization of the processing method, the development of limit standards and a risk–benefit assessment. Taking PMR as an example, it is hoped that these findings will improve the standardization and internationalization of endogenous toxic TCMs and provide indispensable evidence for ensuring safe and effective clinical treatment in the future.

In the past several decades, many human liver cell lines have been used for in vitro screening tests to evaluate hepatotoxic drugs and other compounds. The HepaRG cell line has been proven to be suitable human hepatocytes for the assessment of hepatotoxicity in vitro [23]. HepaRG cells were identified from a human hepatocellular carcinoma cell line infected with the hepatitis B virus and isolated for the first time from non-neoplastic tissue in women with chronic hepatitis C virus infection [24]. HepaRG cells are derived from highly proliferating progenitor cells, which differentiate into both biliary and hepatocellular cells in 2% dimethyl sulfoxide (DMSO) [25]. Compared with HepG2 cells and the others, HepaRG cells, which are similar to human primary hepatocytes, are capable of expressing phase I drug metabolic CYP enzymes, phase II drug metabolic enzymes, transporters, and the nuclear receptor specificity of liver functions [26]. Therefore, in this study, HepaRG cells were selected to evaluate the toxicity of trans-emodin dianthrones (5), and cis-emodin dianthrones (6) to hepatocytes in vitro.

Materials and methods

Reagents and materials

HPLC-grade acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid was purchased from Merck Inc. (Darmstadt, Germany). Ethanol was of analytical grade and purchased from Shanghai Chemical Reagent Co. (Shanghai, China). Water was purified with a Milli-Q water purification apparatus (Millipore, Billerica, MA, USA). The immortalized hepatic cell line HepaRG was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The following reagents were also used in this study: RPMI 1640 culture medium (Biological Industries, Israel), foetal bovine serum (Biosera, France), penicillin (Targetmol, China), staurosporine (STSP; Targetmol, China), 0.25% trypsin–EDTA (Wisent, Canada), CCK-8 reagent (Targetmol, China), DMSO (Sinopharm, China), and a Victor Nivo multi-mode plate reader (PerkinElmer, China).

Polygonum multiflorum samples were authenticated by Associate Professors Ji Zhang and Jian-Bo Yang (Research and Inspection Center of TCM and Ethnomedicine, National Institutes for Food and Drug Control, State Food and Drug Administration) in accordance with the Chinese Pharmacopoeia (edition 2015, volume 1) [3]. A voucher sample of PMR (No. 20191001) was collected from Deqing County, Guangdong Province, China and deposited at the TCM and Ethnomedicine Research and Inspection Center, National Institutes for Food and Drug Control, State Food and Drug Administration, Beijing, China.

The chemical compounds polygonumnolide C4 (1), polygonumnolide C3 (2), polygonumnolide C1 (3), polygonumnolide C2 (4), trans-emodin dianthrones (5), and cis-emodin dianthrones (6) were isolated and purified. The structures of the six dianthrones (1–6) were confirmed by UV, MS, 1H NMR and 13C NMR analyses, which have been reported in the literature [13,14,15]. The purity of these compounds was greater than 98.0% (as determined by HPLC). The internal standard (IS) (Bianthronyl) was purchased from Moving Your Chemistry Forward (Shanghai, China). Figure 1 shows the structures of the six dianthrones and one IS. All solvents and samples were filtered through 0.22 μm filters before UHPLC injection.

Fig. 1
figure 1

Chemical structure of six dianthrones (1–6) and one internal standard (IS)

Full size image

Apparatus

The UHPLC-MS/MS instrument consisted of an Agilent 1200 series UHPLC system equipped with an Agilent 6410B TQMS/MS system (Agilent Technologies, Santa Clara, CA, USA). Chromatographic analyses were performed using an Agilent 1200 series UHPLC system (Agilent Technologies, Santa Clara, CA, USA) consisting of a quaternary pump, an online degasser, an auto plate-sampler, and a thermostatically controlled column compartment. Chromatographic separation was carried out at 30 °C on an Agilent ZORBAX SB-C18 column (2.1 mm × 50 mm, 1.8 μm). Separation was achieved with a gradient of mobile phases consisting of acetonitrile (A) and water containing 0.1% formic acid (v/v) (B) at a flow rate of 0.25 mL/min. The gradient was programmed as follows: 0–8 min, maintenance at 37% A; 8–10 min, linear change to 60% A; 10–12 min, linear change to 78% A; 12–20 min, linear change to 90% A; 20–22 min, linear change to 37% A; and 22–30 min, maintenance at 37% A. The column temperature was maintained at 30 °C. The injection volume was 2.0 μL.

All MS experiments were conducted using an ESI source in negative ion electrospray mode with a 6410B TQMS (Agilent, USA). The optimal MS conditions were as follows: drying gas temperature, 300 °C; drying gas flow rate, 10 L/min; nebulizer gas pressure, 30 psi; sheath gas temperature, 300 °C; sheath gas flow, 11 L/min and capillary voltage, 4.0 kV. Detection was carried out in MRM mode. All data were processed using MassHunter Workstation software (V.7.0 Quantitative Analysis; Agilent, USA).

Preparation of standard solutions

Standard stock solutions for the six dianthrones, namely, polygonumnolide C4 (1), polygonumnolide C3 (2), polygonumnolide C1 (3), polygonumnolide C2 (4), trans-emodin dianthrones (5) and cis-emodin dianthrones (6), were prepared in 70% ethanol. Accordingly, a standard mixture solution was obtained by precisely mixing the six stock solutions with 70% ethanol so that the concentrations were 0.210 (1), 0.214 (2), 0.283 (3), 0.280 (4), 0.318 (5) and 0.280 (6) μg/mL. The mixture solutions were further diluted to generate standard solutions in different concentration ranges. The calibration curves were generated with at least six appropriate concentrations. Bianthronyl (IS) was prepared in DMSO/methanol (v/v, 2:1) at a concentration of 100.44 μg/mL. All standards solutions were stored at 4 °C.

Sample preparation

Polygoni multiflori radix (PMR)

Ninety batches of PMR (PMR-01–PMR-90) were collected from different provinces of China, as shown in Table 1.

Table 1 Sample collection information in the present study
Full size table

Polygoni Multiflori Radix Praeparata (PMRP)

PMRP can improve the efficacy and reduce the hepatotoxicity of PMRP after processing. PMRP could be extracted from PMR using the method from the Chinese Pharmacopoeia (2020 edition) [3] and traditional methods [27]. Eighty-six batches of PMR (PMRP-01–PMRP-86) were collected from different provinces of China, as shown in Table 1.

The water-steaming method was as follows: A sample (PMR-49) was collected for examination at different points and labelled PMRP-S0h, S2h, S4h, S6h, S8h, S10h, S12h, S16h, S20h, or S24h. In addition, ten samples of PMRP-(S0h–S24h) were successfully obtained. Moreover, 15 batches of crude PMR (300 g) were infiltrated by distilled water and steamed at 100 °C for 0, 12, or 24 h. These processed products were then dried in the sunlight. Finally, 45 samples of PMRP-(SZ01-0h, SZ01-12h, and SZ01-24h and SZ15-0h, SZ15-12h, and SZ15-24h) were successfully obtained.

Sample analysis

An aliquot of 1.0 g of PMR or PMRP (filtered through a no. 3 sieve) was weighed into a stoppered conical flask, 50 mL of accurately measured ethanol–water (7:3, v/v) was added followed by weighing and ultrasonication (power, 100 W; frequency, 40 kHz) for 30 min. The solution was cooled and weighed again, the loss of weight was replenished with ethanol–water (7:3, v/v) and the solution was mixed well. This extract was then filtered through a 0.22 μm syringe filter. The filtrate was used as the test solution and analysed with UHPLC-QQQ-MS/MS according to the above procedure.

Cytotoxic effects of dianthrone exposure in HepaRG cells

HepaRG cells were maintained in RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin and streptomycin at 37 °C with 5% CO2. The effects of the toxic dianthrone markers on HepaRG cell viability were determined using a CCK-8 assay. According to the experimental operation requirements, the day before detection, HepaRG cells were inoculated in 384-well cell plates at a density of 1000 cells/well with 40 μL of cell suspension inoculated in each well. The cell plates were placed in an incubator at 37 °C with 5% CO2 for overnight incubation. On the day of the experiment, 10 μL of compound working solution (0.064, 0.32, 1.6, 8, 40, 200 or 1000 μg/mL) was added to each well, and the plate was incubated at 37 °C in a 5% CO2 incubator in the dark for 72 h. After incubation, 5 μL of CCK-8 reagent was added to each cell well followed by incubation for 4 h. The absorbance at 450 nm was measured on the NIVO instrument, and the inhibition rate was calculated by the following formula:

$${\text{Inhibition}}\;{\text{ratio}}\left( \% \right) = \left( {{\text{Ods}}\, - \,{\text{OD}}_{{{\text{NC}}}} } \right)/\left( {{\text{OD}}_{{{\text{STSP}}}} \, - \,{\text{OD}}_{{{\text{NC}}}} } \right)\, \times \,100\%$$

where Ods is the absorbance of the sample solution (cell + medium + compound to be tested), ODNC is the absorbance of the negative control (cell + medium + DMSO), and ODSTSP is the absorbance of the positive control (cell + medium + 10 μM STSP). According to the inhibition ratios of the compounds, the IC50 values (the concentration corresponding to 50% of the maximum inhibition response) were calculated from the dose–response curves using GraphPad. All tests were conducted in triplicate, and the mean values were finally obtained.

Results and discussion

Optimization of the extraction method

PMR (No. 20191001) was used to optimize the extraction process. Optimization was successfully completed using a three-step approach, described as follows. Step 1. Optimization of the extraction solvent system: the first step in the preparation of the sample solution was to select a suitable extraction solvent because of its paramount role in achieving good recovery. Five solutions, H2O and 30%, 50%, 70%, and 95% ethanol (v/v in water), were systematically compared by virtue of the peak areas of the six dianthrones in PMR. As a result, 70% ethanol exhibited the highest extraction efficiency among the tested solvents, as shown in Fig. 2A. Hence, 70% aqueous ethanol was selected as the best extraction solvent for this study. Step 2. Optimization of solvent volume: extractant volume may be another factor that could affect extraction efficiency. This study aimed to obtain the minimum volume of extractant required to achieve the highest extraction efficiency. Four different volumes of 70% ethanol (25, 50, 100 and 150 mL) were systematically studied. From Fig. 2B, the peak areas of the six dianthrones increased with increasing volume of 70% ethanol. However, there was no significant difference among the results of four different volumes of 70% ethanol. Therefore, 50 mL of 70% ethanol was eventually selected as the optimized volume for environmentally friendly reasons. Step 3. Optimization of ultrasonication time: in this study, an ultrasonic process was used to extract the six dianthrones from PMR. From Fig. 2C, there was no significant difference among ultrasonication times of 15, 30, and 45 min. Accordingly, 30 min was selected as the best extraction time to save energy.

Fig. 2
figure 2

Optimization of different parameters of the method of sample solution, A type of extractant, B volume of extractant and C ultrasound time

Full size image

In conclusion, the optimal sample preparation method was found to be the extraction of a 1.0 g sample with 50 mL of 70% ethanol in an ultrasonic water bath for 30 min.

Optimization of UHPLC-QQQ-MS/MS conditions

The chromatographic conditions, especially the composition of the mobile phase, were optimized to achieve the best possible resolution and symmetric peaks of the seven compounds within a suitable run time. Over the course of the tests, four mobile phases were examined, i.e., methanol-, acetonitrile-, and methanol–water containing 0.1% formic acid (v/v), and acetonitrile–water containing 0.1% formic acid (v/v), in different ratios. The acetonitrile–water containing 0.1% formic acid (v/v) combination had the lowest pressure, best baseline stability, and highest ionization efficiency among those tested and was eventually selected as the mobile phase. Both positive and negative ion modes were also tested for MS analysis. The seven compounds showed cleaner mass spectral backgrounds and higher sensitivities in negative mode than in positive mode. The parameters of fragmented voltage and collision energy were optimized to obtain the richest relative abundance of parent ions and outputs for the optimization of MRM conditions. In addition, the MRM transitions and parameters of these seven dianthrone compounds are shown in Table 2. Other parameters, such as dry gas flow rate, temperature, nebulizer, and capillary voltage were set to 10.0 L/min, 300 °C, 15 psi, and 4000 V, respectively. The production mass spectra and proposed fragmentation pathways of 1–6 and the IS are also shown in Fig. 3. These seven dianthrones (1–6 and IS) identify cleavage of the C10–C10′ bond to yield anthrone-free radicals in the MS/MS product ion spectra. The MS/MS product ion spectra of 1–6 were reported in the article [12].

Table 2 Parameters of dianthrones of 6 analytes and 1 internal standard in MRM analysis
Full size table
Fig. 3
figure 3

Product ion mass spectra and proposed fragmentation pathway of six dianthrones (1–6) and one internal standard (IS)

Full size image

Method validation

Specificity

The peaks of the six dianthrones and the IS presented good separation without interference peaks based on the chromatographic and MS conditions mentioned above. The typical MRM chromatograms for a blank test sample, a mixed standard solution and a sample of P. multiflorum are shown in Fig. 4A–C. This result showed that the method is highly selective.

Fig. 4
figure 4figure 4

Typical multiple reaction monitoring (MRM) chromatograms for a blank test sample (A), a mixed standard solution (B) and a sample of P. multiflorum (C) (1. polygonumnolide C4; 2. polygonumnolide C3; 3. polygonumnolide C1; 4. polygonumnolide C2; 5. trans-emodin dianthrones; 6. cis-emodin dianthrones; IS. Bianthronyl)

Full size image

Linearity range, limits of detection (LODs) and limits of quantification (LOQs)

The developed UHPLC-QQQ-MS/MS method was further validated in accordance with the guidelines of the Validation of the Quality Standard of TCMs (Chinese Pharmacopoeia, 2015, volume 1) [3]. Table 3 lists the linear calibration curve with R2, linearity range, LOD, and LOQ values. All calibration curves showed good linear regression (r2 ≥ 0.9965) within the tested ranges; the LOD (S/N = 3) and the LOQ (S/N = 10) for the six dianthrones were in the ranges of 0.3–0.4 ng/mL and 0.7–1.1 ng/mL, respectively, showing high sensitivity.

Table 3 Regression equation, LOD and LOQ of the six dianthrones
Full size table

Precision

The precision of the method was evaluated based on intra- and inter-day precision. The intra-day precision was tested with mixed standard solutions in 1 day. The standard solutions are examined in triplicate on three consecutive days for inter-day precision. The corresponding % RSD values were calculated. The RSDs for the intra-day (n = 6) and inter-day (n = 3) assays were less than 2.73% and 4.63%, respectively (see Table 4).

Table 4 Stability, repeatability, precision and recovery of 6 compounds
Full size table

Stability and repeatability

The stability was measured using a sample solution and performed at 0, 2, 4, 8, 12, and 24 h after preparation and storage at room temperature. Six independent sample solutions were prepared and analysed to measure the repeatability. The concentration of each solution was determined by calibration curves produced on the same day. The RSDs for stability were less than 3.95% within 24 h. Moreover, the RSDs for repeatability were less than 3.30% (see Table 4). The results of the stability and repeatability tests show that all analytes are stable within the duration of the whole analysis and that the test method is sufficiently effective for conventional analysis.

Recovery

The recovery tests were carried out by adding a known number of mixed standards to a certain amount of the six dianthrones. Six replicates were performed for the test. The recoveries were calculated using the following equation: recovery (%) = (total amount detected − amount original)/amount spiked × 100%. Table 4 also shows that the analytical method developed for the six dianthrone compounds has a good recovery rate ranging from 104.38 to 150.04%, and the RSDs were less than 9.70%. Therefore, the UHPLC-QQQ-MS/MS method is precise, accurate, sensitive, and reliable enough for the simultaneous and quantitative determination of the six minor potential hepatotoxic compounds in PMR and PMRP.

Quantification of the 6 dianthrones in different batches of PMR and PMRP

Comparing the UHPLC retention times and m/z values of the six dianthrones with those of the reference compounds, the identification of the target peaks was successful by the UHPLC-QQQ-MS/MS method. The content of each analyte was determined using the respective calibration curves with the IS method. The developed method was successfully applied to analyse the contents of the six dianthrones in PMR and PMRP.

Quantification of the 6 dianthrones in 90 batches of PMR

The developed and validated UHPLC-QQQ-MS/MS method was subsequently applied to evaluate the six dianthrones from 90 batches of PMR, and the quantification results are summarized in Table 5. The contents of 1, 2, 3, 4, 5 and 6 were in the ranges of 0.027–19.04, 0.022–13.86, 0.073–15.53, 0.034–23.35, 0.38–83.67 and 0.29–67.00 µg/g, respectively. The total contents of 1–6 ranged from 1.39 to 171.45 µg/g. There were distinct differences in the contents of 1–6 in the 90 batches of PMR. Interestingly, the contents of 5 and 6 in PMR extracted with 70% ethanol were remarkably higher than those of 1–4. The average content order in the 90 batches of PMR was 5 > 6 > 1 > 4 > 3 > 2. According to previous studies [17], dianthrones may be the potential hepatotoxic components in PMR, and 5 and 6 are more toxic than 1–4. Therefore, 5 and 6 could be used as potential toxicity markers of PMRP.

Table 5 Contents of 6 dianthrones in 90 batches of Polygoni Multiflori Radix (PMR)
Full size table

Quantification of the 6 dianthrones in different batches of PMRP (the water-steaming method)

The developed and validated UHPLC-QQQ-MS/MS method was subsequently applied to identify the six dianthrones in 10 samples of PMRP (PMRP-S0h, PMRP-S2h, PMRP-S4h, PMRP-S6h, PMRP-S8h, PMRP-S10h, PMRP-S12h, PMRP-S16h, PMRP-S20h, and PMRP-S24h) using the water-steaming method, and the quantification results are summarized in Table 6. The contents of 1, 2, 3, 4, 5 and 6 were in the ranges of 0.18–2.09, 0.58–3.67, 0.26–2.04, 0.71–4.07, 0.25–7.20 and 0.22–6.11 µg/g, respectively. The total contents of 1–6 ranged from 2.20 to 20.74 µg/g.

Table 6 Contents of 6 dianthrones in 10 samples of Polygoni Multiflori Radix Praeparata (PMRP) with the water steaming method
Full size table

Additionally, the developed and validated UHPLC-QQQ-MS/MS method was subsequently applied to determine the contents of the six dianthrones in 45 samples of PMRP after 0, 12, and 24 h of processing using the water-steaming method, and the quantification results are summarized in Table 7. The total contents of 1–6 were found to have decreased significantly. Compounds 1 and 3 could be detected in 5 samples after 12 h of processing, Compound 1 could not be detected in 15 samples, and compound 2 could not be detected in 6 samples after 12 h of processing. However, compounds 5 and 6 could be detected in all samples after 12 h of processing, with the contents of 5 ranging from 0.17 to 0.78 µg/g and those of 6 ranging from 0.14 to 0.73 µg/g Finally, after 24 h of processing, the contents of the six dianthrones all decreased by more than 80%.

Table 7 Contents of 6 compounds in 45 samples of Polygoni Multiflori Radix Praeparata (PMRP) with the water steaming method
Full size table

Quantification of the 6 dianthrones in 86 batches of commercial PMRP

The developed and validated UHPLC-QQQ-MS/MS method was subsequently applied to determine the contents of the six dianthrones in 86 batches of commercial PMRP, and the quantification results are summarized in Table 8. The contents of 1, 2, 3, 4, 5 and 6 were in the ranges of 0.020–13.03, 0.051–8.94, 0.022–7.23, 0.030–12.75, 0.098–28.54 and 0.14–27.79 µg/g, respectively. The total contents of 1–6 ranged from 0.35 to 65.27 µg/g. There were distinct differences in the contents of compounds 1–6 in the 86 batches of commercial PMRP. Interestingly, the contents of 5 and 6 in the PMR sample extracted with 70% ethanol were remarkably higher than those of 1–4. The average content order in the 86 batches of PMR was 5 > 6 > 4 > 2 > 1 > 3.

Table 8 Contents of 6 dianthrones in 86 batches of Polygoni Multiflori Radix Praeparata (PMRP)
Full size table

In above-mentioned experiments, the quality control (QC) samples consist of standard solutions of different concentrations and they were injected every 24 h. According to the literature [9], the best technology to process PMR was to steam for 24 h to eliminate the potential hepatotoxicity of PM. Further analysis was performed by focusing on the 45 samples of PMRP using the water-steaming method, since this processing technology is the most commonly used and has been recommended by the Chinese Pharmacopoeia. The contents of 5 and 6 decreased from 17.52 to 0.78 µg/g and 13.11 to 0.73 µg/g, respectively. The possible limit of the total contents of 5 and 6 could be no more than 1.51 µg/g in PMRP. If this possible limit is used to evaluate different PMRP samples on the market, more than 65% of the 86 commercial PMRP samples exceeded this limit. Therefore, it is noteworthy that there are problems with the processing methods of commercial PMRP.

Cytotoxicity evaluation of dianthrones in HepaRG cells

The two potentially toxic compounds, 5 and 6, were evaluated for their cytotoxicity in HepaRG cells by CCK-8 assay. According to the concentration-HepaRG cell inhibition rate curves drawn at different concentrations of the compounds, the IC50 values of each compound in the HepaRG cell model were determined. The IC50 values of compounds 5 and 6 were 5.60 μg/mL and 7.88 μg/mL, respectively. These values corresponded to 10.98 μM and 15.45 μM, respectively. The results suggested that compounds 5 and 6 had strong hepatocellular toxicity and could be used as potential toxicity markers.

Discussion

TCMs with endogenous toxicity have a relatively narrow treatment window. If they are used improperly in the clinic, severe adverse reactions may occur. Attention has been directed towards TCMs with endogenous toxicity because of the negative effects and serious risks they cause to humans. Therefore, it is essential to develop a system to standardize TCMs with endogenous toxicity to guide the clinical use of TCMs. For the first time, in the present study, a systematic five-step strategy standardize TCMs with endogenous toxicity was proposed and involved the establishment of determination methods, the determination of toxic markers, the standardization of the processing method, the development of limit standards and a risk–benefit assessment (Fig. 5).

Fig. 5
figure 5

A systematic five-step strategy for standardization of endogenous toxic TCMs

Full size image

First, determination methods are expected to be developed to isolate and identify endogenous toxic chemicals in TCMs. The present study innovatively established a UHPLC-QQQ-MS/MS technique to simultaneously detect six dianthrones in PMR and PMRP. UHPLC-QQQ-MS/MS techniques are widely used for applications in chromatography–MS analysis. The method developed herein could not only provide rapid and improved chromatographic separation and a shorter chromatographic run time but can also provide higher sensitivity and selectivity, which are ultimately helpful for determining the contents of dianthrones in PMR and PMRP.

Second, determining toxic markers and clarifying the mechanism could decrease the toxicity of TCMs. Interestingly, this study showed that dianthrones are widely distributed in PMR and demonstrated that these compounds, especially trans-emodin dianthrones (5) and cis-emodin dianthrones (6), could be selected as potential toxic markers of PMRP [11, 17]. The possible degradation process of the 6 dianthrones (1–6) in PMRP are as follows. Free dianthrones (5 and 6) may undergo glycosidation and be further converted into the combined dianthrones 1–4. On the other hand, the C10–\({\text{C}}_{{10}} ^{\prime }\) dianthrone bond could be easily cleaved under heating conditions. These dianthrones could be converted into anthrones and then further oxidized into anthracenols. Anthracenols may be further oxidized into anthraquinones, such as emodin and emodin-8-O-glucopyranoside, which may undergo methylation. Finally, the combined anthraquinones could be converted into free anthraquinones originating from the loss of the glucoside unit. Accordingly, the postulated degradation process of dianthrones in PMRP was speculated as Scheme 1. The contents of dianthrones may decrease significantly after reasonable processing. Therefore, this study could provide a theoretical basis to explore the mechanism of decreasing the toxicity of PMRP.

Scheme 1
scheme 1

Hypothetical degradation process of dianthrones (1–6) in PMRP

Full size image

Third, standardization of the processing method is of great significance. Taking P. multiflorum preparations (PMPs) as an example, this study illustrated the relationship between different solvent extracts and the contents of dianthrones in PMRs and PMRPs for the first time. Different extracts using ethanol at different concentrations as an extracting agent could significantly influenced the hepatotoxicity of PMR, as reported in the references [28,29,30]. Therefore, five different concentrations of aqueous ethanol were chosen to evaluate the extraction efficiency of the dianthrones in this study. The results showed that 70% ethanol exhibited the highest extraction efficiency among the tested solvents. Interestingly, these results were consistent with our previous research, which also showed that the toxicity of the 70% ethanol extract was considered to be higher than that of other extracts, such as the H2O extract and 30% ethanol extract [11]. Furthermore, the present study showed that after extraction by the water-steaming method, the total contents of the 6 types of dianthrones decreased by more than 80%. Therefore, the extraction method of PMR is closely related to the contents of dianthrones. In addition, our study demonstrated that dianthrones were potential toxic markers of PMR, indicating that the extraction method is of significance for the potential toxicity of PMR and PMRP. Based on the results of this study and previous studies, it was suggested to pre-treat the PMR with the water-steaming method for 24 h. On the other hand, extraction with 70% ethanol is not encouraged. Overall, in the interest of public health, the standardization of pre-treatment methods is recommended to minimize the toxicity of TCMs with endogenous toxicity.

Fourth, considering public confidence in the safe use of TCMs and TCM preparations, the development of a scientific and practical limit standard for TCMs with endogenous toxicity is beneficial and urgently needed. Taking P. multiflorum preparations (PMPs) as an example, there are more than 300 Chinese patent medicines (CPMs) containing PMRs and PMRPs in the Chinese Pharmacopoeia and Drug Standard of the Ministry of Public Health of the People’s Republic of China [3, 31]. It has been reported that many PMPs, such as Yangxue Shengfa capsules, show certain hepatotoxicity [31,32,33]. However, to the best of our knowledge, there is no regulatory standard for PMR or PMP. Therefore, it is necessary to determine the limit standards for these dianthrones in PMR or PMP to guarantee medicinal safety of TCMs in the future. An appropriate method to formulate limit standards is the key. A scientific and practical limit standard should be based on the toxicological characteristics of the contained chemicals, the amount of TCM or TCM preparation ingested by the consumer, body weight, and safety factors. The following formula to calculate the maximum theoretical limit is recommended: L = AWδ/M (1) where L is the maximum theoretical limit, W is the body weight (70 kg), and M is the daily ingestion rate of the TCM or TCM preparation (g/day), which could be based on the consumption rate in the Pharmacopoeia of the People’s Republic of China (PPRC), and δ is a safety factor, accounting for the contribution of dietary supplements as a component of daily food intake. According to the judgement of the National Science Foundation (NSF), δ could be 10. A is the acceptable daily intake (ADI), which is defined as the estimated amount of a chemical to which a person can be exposed on a daily basis over a lifetime without suffering a detectable deleterious effect. For some endogenous toxic chemicals, such as pyrrolizidine alkaloids, ADI values have been set by organizations involving the World Health Organization (WHO) and European Food Safety Authority (EFSA), as references. However, for other endogenous toxic chemicals, such as the dianthrones in PMR or PMP, the ADI should be determined under the guidance of Good Laboratory Practice (GLP). In future studies, we will make great efforts to determine the crucial parameter ADI, especially the ADI for trans-emodin dianthrones (5) and cis-emodin dianthrones (6), based on which the maximum theoretical limit could be acquired. A practical maximum theoretical limit is the basis of a practical limit standard, and other factors involved in economic development, human cognition, and even history and culture, are recommended to be considered to maintain a balance between public safety and economic progress.

Finally, it is necessary to establish a benefit and risk assessment model of TCMs with endogenous toxicity to comprehensively evaluate the benefits and risks of TCMs and ensure both their safety and effectiveness. The evaluation of the risk–benefit ratio is determined by many factors and involves the establishment of a value tree of the risk–benefit ratio. The value tree is composed of the characteristics of the disease, clinical efficacy of the TCM, adverse reactions caused by the TCM, etc. On the basis of the severity, duration and incidence of the adverse reactions caused by TCMs with endogenous toxicity, these indexes should be weighed to obtain the estimated risk–benefit ratio. Moreover, it is paramount to build a very large mass spectral database to identify endogenous toxic chemicals, including dianthrones, as well as accumulate a wider range of extensive health risk assessment data on these endogenous toxic chemicals.

Conclusions

In the present study, a rapid, sensitive, precise, and reliable UHPLC-QQQ-MS/MS method was developed for the simultaneous determination of six dianthrones in PMR and PMRP for the first time. The results indicated that trans-emodin dianthrones (5) and cis-emodin dianthrones (6) could be considered as potentially toxic markers of PMRP. Furthermore, taking PMR as an example, a systematic five-step strategy to promote the standardization of TCMs with endogenous toxicity was proposed for the first time, covering the research gap in this field. The systematic strategy consisted of the following steps: the establishment of determination methods, the identification of toxic markers, the standardization of the processing method, the development of limit standards and a risk–benefit assessment. Taking PMR and PMRP as examples, we hope this study provides insight into the standardization and internationalization of endogenous toxic TCMs and is conducive to improving the quality standard of these endogenous toxic TCMs and ensuring safe and effective clinical treatment.

Availability of data and materials

The research data generated from this study are included within the article.

References

  1. Li CY, Niu M, Bai ZF, Zhang CG, Zhao YL, Li RY, Tu C, Li HF, Jing J, Meng YK, Ma ZJ, Feng WW, Tang JF, Zhu Y, Li JJ, Shang XY, Zou ZS, Xiao XH, Wang JB. Screening for main components associated with the idiosyncratic hepatotoxicity of a tonic herb, Polygonum multiflorum. Front Med. 2017;11:253–65.

    Article  Google Scholar 

  2. Huang J, Zhang JP, Bai JQ, Wei MJ, Zhang J, Huang ZH, Qu GH, Xu W, Qiu XH. Chemical profiles and metabolite study of raw and processed Polygoni multoflori radix in rats by UPLC-LTQ-Orbitrap MSn spectrometry. Chin J Nat Med. 2018;16:0375–400.

    Google Scholar 

  3. Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China. Beijing: China Medical Science and Technology Press; 2020. p. 183–4.

    Google Scholar 

  4. Lin LF, Ni BR, Lin HM, Zhang M, Li XC, Yin XB, Qu CH, Ni J. Traditional usages, botany, phytochemistry, pharmacology and toxicology of Polygonum multiflorum thumb.: a review. J Ethnopharmacol. 2015;159:158–83.

    Article  CAS  Google Scholar 

  5. Liu Y, Wang Q, Yang JB, Guo XX, Liu WX, Ma SC, Li SP. Polygonum multiflorum Thunb.: a review on chemical analysis, processingn mechanism, quality evaluation, and hepatotoxicity. Front Pharmacol. 2018;9:364.

    Article  Google Scholar 

  6. Lei X, Chen J, Ren JT, Li Y, Zhai JB, Mu W, Zhang L, Zheng WK, Tian GH, Shang HC. Liver damage associated with Polygonum multiflorum thumb.: a systematic review of case reports and case series. Evid Based Compl Altern Med. 2015. https://doi.org/10.1155/2015/459749.

    Article  Google Scholar 

  7. Park GJ, Mann SP, Ngu MC. Acute hepatitis induced by Shou-Wu-Pian, a herbal product derived from Polygonum multiflorum. J Gastroenol Hepatol. 2001;16:115–7.

    Article  CAS  Google Scholar 

  8. Yu HL, Yu DM, Song HB, Ren JT. Literature study on adverse drug reactions and analysis of risk factors of Polygonum multiflorum Thunb and its common preparations. Chin J PV. 2018;15:470–5.

    Google Scholar 

  9. Han LF, Wang P, Wang YL, Zhao QY, Zheng F, Dou ZY, Yang WZ, Hu LM, Liu CX. Rapid discovery of the potential toxic compounds in Polygonum multiflorum by UHPLC/Q-Orbitrap-MS-based metabolomics and correlation analysis. Front Pharmacol. 2019;10:329.

    Article  CAS  Google Scholar 

  10. Han LF, Wu B, Pan GX, Wang YF, Song XB, Gao XM. UPLC-PDA analysis for simultaneous quantification of four active compounds in crude and processed rhizome of Polygonum multiflorum Thunb. Chromatographia. 2009;70:657–9.

    Article  CAS  Google Scholar 

  11. Yang JB, Li WF, Liu Y, Wang Q, Cheng XL, Wei F, Wang AG, Jin HT, Ma SC. Acute toxicity screening of different extractions, components and constituents of Polygonum multiflorum thunb, on zebrafish (Danio rerio) embryos in vivo. Biomed Pharmacother. 2018;99:205–13.

    Article  CAS  Google Scholar 

  12. Yang JB, Liu Y, Wang Q, Ma SC, Wang AG, Cheng XL, Wei F. Characterization and identification of the chemical constituents of Polygonum multiflorum Thunb, by high-performance liquid chromatography coupled with ultraviolet detection and linear ion trap FT-ICR hybrid mass spectrometry. J Pharm Biomed. 2019;172:149–66.

    Article  CAS  Google Scholar 

  13. Yang JB, Li L, Dai Z, Wu Y, Geng XC, Li B, Ma SC, Wang AG, Su YY. Polygonumnolides C1–C4, minor dianthrone glycosides from the roots of Polygonum multiflorum Thunb. J Asian Nat Prod Res. 2016;18:813–22.

    Article  CAS  Google Scholar 

  14. Yang JB, Tian JY, Dai Z, Ye F, Ma SC, Wang AG. a-Glucosidase inhibitors extracted from the roots of Polygonum multiflorum Thunb. Fitoterapia. 2017;117:65–70.

    Article  CAS  Google Scholar 

  15. Yang JB, Yan Z, Ren J, Dai Z, Ma SC, Wang AG, Su YY. Polygonumnolides A1–B3, minor dianthrone derivatives from the roots of Polygonum multiflorum Thunb. Arch Pharm Res. 2018;41:617–24.

    Article  CAS  Google Scholar 

  16. Wang Q, Wang YD, Li Y, Wen BY, Dai Z, Ma SC, Zhang YJ. Identification and characterization of the structure-activity relationships involved in UGT1A1 inhibition by anthraquinone and dianthrone constituents of Polygonum multiflorum. Sci Rep. 2017;7:17592.

    Article  Google Scholar 

  17. Li HY, Yang JB, Li WF, Qiu CX, Wang ST, Song YF, Gao HY, Liu Y, Wang Q, Wang Y, Cheng XL, Wei F, Jin HT, Ma SC. In vivo hepatotoxicity screening of different extracts, components, and constituents of Polygoni multiflori Thunb. in zebrafish (Danio rerio) larvae. Biomed Pharmacother. 2020;131:110524.

    Article  CAS  Google Scholar 

  18. Mai LP, Guéritte F, Dumontet V, Tri MV, Hill B, Thoison O, Guénard D, Sévenet T. Cytotoxicity of rhamnosylanthraquinones and rhamnosyl anthrones from Rhamnus nepalensis. J Nat Prod. 2001;64:1162–8.

    Article  CAS  Google Scholar 

  19. Ha F, Li RM, Zhang LL, Yan XJ. Transformation relationship of four sennoside components in Rhei Radix et Rhizoma under the heating condition. Chin Tradit Pat Med. 2012;34:2169–73.

    CAS  Google Scholar 

  20. Song HB, Du XX, Guo XX, Ren JT, Yang L, Pang Y. Safety and risk factor analysis on Polygoni multiflora radix base on ancient traditional Chinese medicine literatures. China J Chin Mater Med. 2015;40:985–8.

    Google Scholar 

  21. Zhang L, Bai ZF, Li CY, Huhuang WY, Sha MC, Liu ZX, He Q, Li YM, Liu YP, Xiao XH, Wang JB. Study on idiosyncratic liver injury and content of cis-2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside in radix Polygoni multiflori Preparata. Acta Pharm Sin B. 2017;52:1041–7.

    Google Scholar 

  22. Chan CO, Xie XJ, Wan SW, Zhou GL, Yuen ACY, Mok DKW, Chen SB. Qualitative and quantitative analysis of sesquiterpene lactones in Centipeda minima by UPLC-Orbitrap-MS & UPLC-QQQ-MS. J Pharm Biomed. 2019;174:360–6.

    Article  CAS  Google Scholar 

  23. Saito J, Okamura A, Takeuchi K, Hanioka K, Okada A, Ohata T. High content analysis assay for prediction of human hepatotoxicity in HepaRG and HepG2 cells. Toxicol In Vitro. 2016;33:63–70.

    Article  CAS  Google Scholar 

  24. Gripon P, Rumin S, Urban S. Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci USA. 2002;99:15655–60.

    Article  CAS  Google Scholar 

  25. Rebelo SP, Costa R, Estrada M, Shevchenko V, Brito C, Alves PM. HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism. Arch Toxicol. 2015;89:1347–58.

    Article  CAS  Google Scholar 

  26. Jackson JP, Li L, Chamberlain ED, Wang H, Ferguson SS. Contextualizing hepatocyte functionality of cryopreserved HepaRG cell cultures. Drug Metab Dispos. 2016;44:1463–79.

    Article  CAS  Google Scholar 

  27. Ge CL, Cheng G, Yu JP, Xu DJ. The influence of chemical components of Polygonum multiflorum after the nine-time repeat of the steaming and sun-drying traditional processing. Chin Med J Res Prac. 2017;31:43–7.

    Google Scholar 

  28. Lv Y, Wang JB, Ji Y, Zhao YL, Ma ZJ, Li Q, Zou ZS, Liang QS, Teng GJ, Yan D, Zhang P, Xiao XH, Yu CY. Influence of extracting solvent on hepatocytes toxicity of Polygonum multiflorum. Chin J Exp Tradit Med Formulae. 2013;19:268–72.

    Google Scholar 

  29. Lin LF. Study on components and mechanism of liver injury induced by polygonum multiflorum. Beijing: Beijing University of Chinese Medicine; 2016.

    Google Scholar 

  30. Xiao R. Study on the basis and quality control of hepatotoxicity of polygonum multiflorum. Hunan: Hunan University of Chinese Medicine; 2018.

    Google Scholar 

  31. Yang Q, Li XY, Zhao XM, Sun R. System analysis on adverse drug reaction of Chinese patent medicine containing Polygoni multiflori Radix. Chin Tradit Herb Drugs. 2017;48:1878–87.

    Google Scholar 

  32. Zhu Y, Liu SH, Wang JB, Song HB, Li YG, He TT, Ma X, Wang ZX, Wang LP, Zhou K, Bai YF, Zou ZS, Xiao XH. Clinical analysis of drug-induced liver injury caused by Polygonum multiflorum and its preparations. Chin J Integr Tradit West Med. 2015;35:1442–7.

    Google Scholar 

  33. Xu N, Shi HY, Li XY, Zhang LL, Sun R. Progress and cause analysis on cause of adverse reactions of Polygoni multiflori radix preparations. Chin J Exp Tradit Med Formulae. 2017;23:208–14.

    Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This project was financially supported by the National Natural Science Foundation of China (Nos. 81903807, 81773874 and 81703665) and the National Major Scientific and Technological Special Project for “Significant New Drugs Development” (2018ZX09735006).

Author information

Author notes

  1. Jian-Bo Yang and Yun-Fei Song contributed equally to this work

Authors and Affiliations

  1. Institute for Control of Chinese Traditional Medicine and Ethnic Medicine, National Institutes for Food and Drug Control, Beijing, 100050, China

    Jian-Bo Yang, Yun-Fei Song, Hui-Yu Gao, Qi Wang, Ying Wang, Xian-Long Cheng, Tian-Tian Zuo, Xiao-Wen Hu, Feng Wei & Shuang-Cheng Ma

  2. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 102488, China

    Yue Liu

  3. Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China

    Hong-Tao Jin & Shu-Ting Wang

Authors
  1. Jian-Bo Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yun-Fei Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yue Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hui-Yu Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Qi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ying Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xian-Long Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tian-Tian Zuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Xiao-Wen Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Feng Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Hong-Tao Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Shu-Ting Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Shuang-Cheng Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JBY, FW and SCM designed the study. JBY, YFS and TTZ drafted the manuscript. FW and SCM revised the manuscript. HYG, YL and XWH were responsible for collecting the samples. QW, YW and XLC provided technical support and advice for the study. HTJ and STW were responsible for the pharmacological experiments. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Tian-Tian Zuo, Feng Wei or Shuang-Cheng Ma.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, JB., Song, YF., Liu, Y. et al. UHPLC-QQQ-MS/MS assay for the quantification of dianthrones as potential toxic markers of Polygonum multiflorum Thunb: applications for the standardization of traditional Chinese medicines (TCMs) with endogenous toxicity. Chin Med 16, 51 (2021). https://doi.org/10.1186/s13020-021-00463-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13020-021-00463-w

Keywords

Chinese Medicine

ISSN: 1749-8546

Contact us