Toxins, Vol. 14, Pages 879: Comparative Analysis of Aristolochic Acids in Aristolochia Medicinal Herbs and Evaluation of Their Toxicities

1. IntroductionAristolochic acids (AAs) are a group of nitrophenanthrene carboxylic acids mainly produced by plants of the Aristolochia and Asarum genera in the Aristolochiaceae family [1,2,3]. Currently, over 180 AAs analogues have been discovered, and AAΙ, AAΙΙ, AAΙΙΙa (AA C) and AAΙVa (AA D) are the most common in the Aristolochia genus [4]. It is worth noting that AAs are naturally occurring toxic compounds, and they could cause irreversible aristolochic acid nephropathy (AAN), as well as known toxicities including hepatotoxicity, nephrotoxicity, genotoxicity and carcinogenicity [5,6,7,8,9]. Toxicological studies have shown that metabolites of AAs could form AA–DNA adducts with DNA in target organs, and subsequently induce characteristic mutations of A-T transversion, which may be the reason of carcinogenesis [10]. Although AAΙ is considered to be the most toxic component to produce hepatotoxicity and nephrotoxicity, both AAΙ and AAΙΙ have been reported to be responsible for genotoxicity [4,11,12]. Due to the risk of human exposure to these toxic compounds, AAs have attracted increasing clinical attention, and have become a research hotspot worldwide [13,14,15].Considering these noxious toxicities, the International Agency for Research on Cancer classified AAs as Group I carcinogens [16]. In addition, the Chinese Pharmacopoeia has continuously excluded four AA-containing herbal medicines of the Aristolochia genus since 2003 [17]. Although AA-containing herbs with abundant varieties, e.g., A. debilis, A. contorta, and A. fangchi, find limited use in China, many herbal materials and their derived prescriptions that contain AAs are still available on the market, and are in use due to clinical needs [18]. In 2017, the China Food and Drug Administration (CDFA) announced 24 herbal species, and more than 40 Chinese patent medicines derived from the Aristolochia genus are still in clinical use. Some AA analogues with lower content, such as aristolactam BΙ, BΙΙ and aristolochic acid ΙΙΙ a, have demonstrated their genotoxicity and cytotoxicity [4]. Therefore, further investigation on the use of AA limits is necessary.As a result, the objectives of our study were to analyze AA compositions and their contents (Figure 1) of three representative herbal medicines from Aristolochia genera, A. mollissima Hance (AMH), A. debilis Sieb. et Zucc (ADS) and A. cinnabaria C.Y.Cheng (ACY), and to evaluate their toxicities against hepatic tumor cells. A. mollissima Hance (called Xun-Gu-Feng in China) is a common anti-rheumatic and analgesia medicinal herb in China [19,20,21], and mainly contains aristolochic acids Ι (AAΙ), ΙΙ (AAΙΙ), ΙΙΙa (AA C) and aristolactam Ι (AL–Ι) [22,23]. A. debilis Sieb.etZucc (Tian-Xian-Teng) is used in the form of the aerial part of the plant. It can be used to relieve abdominal pain and rheumatic arthralgia [24]. A. cinnabaria C.Y.Cheng (Zhu-Sha-Lian) is the root of A. cinnabaria C.Y.Chengtj.L.wu, and has positive effects for the treatment of enteritis, dysentery and sore throat, etc. [25,26,27].

Although the main toxic aristolochic acids Ι and ΙΙ have been found in the Aristolochia genus, the toxicity of their analogues and their specific presence in the Aristolochia plant are uncertain. Importantly, medicinal herbs containing AAs are still commonly used in certain Chinese patent drugs, such as Shedan Chuanbei powder, Duzhong Zhuanggu capsule and Hewei jiangni capsule, etc. Therefore, it is necessary to conduct a thorough study on the existence of AAs in Aristolochia herbs, in order to reasonably control AAs in such medicinal herbs and related Chinese patent drugs.

Figure 1. Chemical structures of AAs in the Aristolochiacea family.

Figure 1. Chemical structures of AAs in the Aristolochiacea family.

Toxins 14 00879 g001 3. Conclusions

In this study, UPLC-QTOF-MS/MS was used to establish a qualitative and quantitative method for the analysis of AA components of Aristolochia herbs. A comparative analysis of AAs was applied to different medicinal herbs in Aristolochia family. Over forty AA analogues, including AAs and ALs types, were identified from each studied herb, whereby AAΙ was the principle component in both AMH and ACY. However, AAΙVa (AA D) was the main component in ADS. It is worth mentioning that ACY possesses large amounts of AAsΙ, ΙΙ, ΙΙΙ and ΙVa, which should be the most toxic among the three medicinal herbs. Chemometric results indicate that more than 14 AAs have statistical significance for differentiating the three herbal samples from different origins. The toxicity of AAs and the herbal extracts were evaluated by MTT method and Comet assay in HepG2 cell line. The compound of AL-BΙΙ exhibited the most potent toxicity, and the herbal extract of AMH also had strong toxicity. Similarly, comet assay showed that the DNA damage caused by AMH was greater than that caused by ADS. Thus, the quantitative results are consistent with the experimental data on toxicity, suggesting that long-term exposure to high levels of AMH and ADS would cause a risk of liver injury. The dosages of AMH and ADS should be paid more attention and limited in the clinical application. Our findings will have important significance for the safety control of such AA-containing herbs and their related prescriptions.

4. Materials and Methods 4.1. Chemicals and Materials

UPLC-QTOF-MS/MS data were obtained on a Waters Vion QTOF/MS (Waters Micromass, Manchester, UK) in positive electrospray ionization mode, for which the software MassFragment TM 1.9.4.053 was used. An AUW120 electronic analytical balance (Shimadzu Company, Kyoto, Japan), a Dy6c electrophoresis instrument power supply (Beijing Liuyi Instrument Factory, Beijing, China), and a Nikon Eclipse 80i fluorescence microscope (Nippon Nikon Company, Tokyo, Japan) were used for the Comet assay.

Eight standard compounds were purchased from Chengdu Pusi Bio-Technology Co., Ltd. (Chengdu, China), including AAΙ (PS010645), AAΙΙ (PS010031), AA C (PS010029), AA D (PS010039), AL-Ι (PS010658), AL-BΙΙ (BBP01017), AL-FΙ (BBP01020) and indomethacin (9AEUSEHD). The purity of each compound was above 98%. LC-MS grade solvents of methanol and acetonitrile were purchased from Fisher Scientific, and deionized water was obtained from Watsons (Guangzhou, China). All other chemicals and reagents were of analytical grade and were purchased from Beijing Chemical Works (Beijing, China). DMEM medium, fetal bovine serum, and trypsin were purchased from cytiva Co., Ltd. (Guangzhou, China). Comet assay kit was purchased from ELK Biotechnology Co., Ltd. (Wuhan, China). Human hepatocellular carcinoma cells HepG2 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

AMH were purchased from Hubei and Shandong provinces, and ADS was purchased from Hubei and Guangxi provinces in China. Their botanical species of the medicinal samples were identified by Professor Pan Yingni. The medicinal samples were deposited in the China Academy of Traditional Chinese Medicine and the Chinese Medicine Research Institute.

4.2. Preparation of Standard and Sample Solutions

The stock solutions were prepared separately by dissolving the reference compounds in methanol to obtain solutions of AAΙ, AAΙΙ, AA C, AA D, AL-Ι, AL-BΙΙ, and AL-FΙ, respectively. Then, fix the volume in a volumetric flask and prepare the mixed reference substance solution with the corresponding mass concentrations of 140.00, 90.00, 20.00, 30.00, 10.00, 0.20, and 9.00 μg/mL, respectively, and store at 4 °C for later use. The stock solution was prepared by dissolving the internal standard substance in methanol to obtain a solution of indomethacin (50.00 μg/mL), which was stored at 4 °C for later use.

A total of 5.0 g powdered sample was extracted with 100 mL 75% ethanol for 2 h under reflux. The organic solvent was removed under reduced pressure to yield the extract. Then the extract was filled to a 25 mL volumetric flask with methanol to get the test solution. Then, 180 μL of the test solution and 20 μL of the internal standard solution were mixed and successively filtered through a 0.22 μm membrane filter before injection. Each sample preparation and injection were repeated. The solution was stored at 4 °C before quantitative analysis.

For the qualitative experiment, the above ethanol sample was further extracted with petroleum ether and ethyl acetate. The ethyl acetate layer was concentrated and dissolved in methanol to make a solution of 0.5 mg/mL. Finally, the solution was filtered through a 0.22 μm membrane filter before injection. The preparation and injection of each sample was repeated. The solution was stored at 4 °C.

4.3. UPLC-QTOF-MS Conditions

Ultraperformance liquid chromatography–tandem mass spectrometry was performed using Waters UHPLC system, coupled to a Q-TOF MS/MS spectrometer (Waters, Milford, CT, USA). The chromatographic separation was carried out on a Waters Acquity ACQUITY UPLC-BEH C18 column (2.1 mm × 100 mm, 1.7 μm) with the flow rate of 0.3 mL/min. The column was set at 35 °C, and the injection volume was 1 μL. The mobile phase was composed of 0.1% formic acid aqueous solution (A)–acetonitrile (B). The gradient elution conditions were set as follows: 0–2 min, 10% B; 2–7 min, 10–30% B; 7–12 min, 30–35% B; 12–15 min, 35–50% B; 15–18 min, 50–95% B; 18–22 min, 10% B.

For MS detection, a Q-TOF MS spectrometer was fit with electrospray ionization (ESI) in positive ionization mode at full scan mode ranged m/z 50–1500. The MS parameters were as follows: cone voltage 30 V, capillary voltage 3.0 kV, cone gas flow rate (N2) 50 L/h, desolvation gas flow rate 800 L/h, ion source temperature 120 °C, desolvation gas temperature 450 °C, and spectrum acquisition frequency 0.2 s.

All the data were analyzed using UNIFITM1.9.4.053, Progenesis QI, simca14.0 and GraphPad Prism 7. The mass spectrum parameters of quantitative compounds are shown in Table 5. 4.4. Method Validation for Quantification 4.4.1. Calibration Curves, Limits of Detection and Quantification

Using the peak area Y (the ratio of the peak area of the reference to that of the internal standard) versus the mass concentration X (the ratio of the reference to the internal standard), the standard curve was drawn and calculated to obtain a linear regression equation and a linear range. The limits of detection (LODs) and limits of quantification (LOQs) of the samples were estimated at signal-to-noise ratios (S/N) of 3 and 10, respectively.

4.4.2. Precision, Stability, Repeatability and Recovery

To verify the precision, Sample X03 was injected six times consecutively and the RSD values of peak areas were calculated. To verify the stability, Sample X03 was analyzed at 0, 2, 4, 8, 12, 16, 20, and 24 h, respectively. To verify the repeatability, we prepared six test samples and performed six replicate analyses. The variability is expressed as RSD (%). The recovery experiment was determined by the standard addition method. The controls of seven components equal to the content in sample X03 were added precisely. The recoveries were calculated based on the difference in mass of these standards before and after addition.

4.5. Multivariate Statistical Analysis and Comparison

The data files for the qualitative components and peak areas of 16 batches of AMH (from Hubei and Shandong), 16 batches of ADS (from Hubei and Guangxi) and 21 batches of ACY (from Sichuan and Yunnan) were exported using Progenesis QI software and then imported to simca14. 0 software for PCA principal component analysis. The supervised pattern analysis of OPLS-DA was performed according to the different origins of the same medicinal material and three different medicinal herbs. The compounds that contributed significantly to the isolation between groups were initially screened (VIP value > 1) according to variable importance in projection (VIP), and potential differentially labeled compounds were screened based on independent sample t-test (p < 0.05 for statistically significant difference).

4.6. Cytotoxicity Assay and Comet Assay 4.6.1. Cytotoxicity Assay

The cytotoxicity of the compounds to HepG2 was determined using the MTT method. First, the cells were transferred from the culture dish to a centrifuge tube, and centrifuged for 5 min (at 1000 r/min). After discarding the supernatant, the tumor cells were cultured in a complete medium containing 15% fetal bovine serum at 37 C, 5% CO2, and saturated humidity. When the cells grew logarithmically, they were spread onto plates, and the cell density was adjusted to 8.0 × 104 cells/mL before the cell sap was added into a 96-well plate (200 µL per well). The cells grew adherent for 24 h. After 24 h, 2 µL of two standards, two extracts of medicinal herbs were added to each plate well and incubated for 72 h. Adriamycin was used as the positive control, and the blank control contained 2 µL DMSO. After incubation, 20 μL of 5 mg/mL MTT solution was added, and the plates were incubated for 4 h. The supernatant liquid was removed, and the cells were disrupted with 200 µL of DMSO for 10 min. After the blue crystalline substance was fully dissolved, the absorbance value was measured on a microplate reader with the detection wavelength of 562 nm and the reference wavelength of 630 nm. The IC50 value of each compound was calculated.

4.6.2. Comet AssayCells in the logarithmic growth phase were inoculated in 6-well plates, and the cell density was adjusted to 2.5 × 105 cells /mL, 2 mL per well. After 24 h, the experimental group was combined with AAⅠ (50 μM), AAII (131.5 μM), extracts of the AMH (10 mg/mL) and ADS (93 mg/mL), the positive control group was combined with ADM (3.4 μM), and the blank control group were added with the same volume of cell culture solution. After 48 h of culture, the cells were collected. DNA damage was detected according to the operating instructions of comet assay kit. The experiment was repeated 3 times, and 50 cells were selected at each dose in each experiment and analyzed by Comet Assay software (CASP, http://casplab.com/, accessed on 5 October 2022). The mean values of tail DNA percentage, tail length, and tail moment of three experiments were used to express DNA damage. Results one-way ANOVA was used to test the data between groups, and the difference was statistically significant when p

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