Exploring Chemical Basis of Toxicity Reduction for Processed Roots of Stellera chamaejasme L. Using Ultraperformance Liquid Chromatography–Triple Quadrupole Tandem Mass Spectrometry

Some herbal medicines are treated with various processing methods to ensure their safe and effective use. However, chemical basis of toxicity reducing for most herbal medicines remains unclear. Stellera chamaejasme L., particularly the root (ruixianglangdu), is toxic. Thus, ruixianglangdu is commonly processed with vinegar or milk to reduce toxicity. Here, with help of multiple-ion monitoring (MIM)-based metabolomics, we comprehensively capture chemical information of ruixianglangdu. Then, 33 differential components between crude drugs and processed products were identified or tentatively characterized by multiple-ion monitoring–information dependent acquiring–enhanced product ion (MIM-IDA-EPI), whose level changed after being processed by vinegar or milk. It was found that flavonoids especially biflavonones could be the important chemical basis of toxicity reduction for processed ruixianglangdu. In addition, some coumarins and lignanoids could also play a role in reducing toxicity. It is believed that MIM-based metabolomics method was valuable for exploring chemical basis of toxicity reduction for processing. The data is critical to further study the mechanism of toxicity reducing for processed ruixianglangdu.


Introduction
Processing of herbal medicinal materials is a pharmaceutical technique based on traditional Chinese medicine (TCM) theory, which aimed to enhance the efficacy and/or reduce the toxicity of crude drugs [1]. For herbal medicines, many processing approaches could be used to reduce the toxicity of crude drugs based on different clinical experience. However, chemical basis of toxicity reduction for most herbal medicines remains unclear. Because of multiple components in herbal medicines, exploring chemical basis is extensively dampened by comprehensively and accurately capture information in complex multicomponent samples.
Stellera chamaejasme L. is a perennial herb belonging to the Thymelaeaceae family of flowering plants, native to Russia, China, and Mongolia [2]. Ruixianglangdu, roots of Stellera chamaejasme L. (ruixianglangdu), is one of the TCM most commonly used as a remedy for furuncle carbuncle and tuberculosis of the lymph nodes, with varied biological activity, including antiviral, antitumor, antibacterial, and antiinflammatory activities [3][4][5]. Ruixianglangdu could poison or even kill the cattle if ingested by mistake [6]. By now, some processing methods have been used to attenuate toxicity of ruixianglangdu. Processing with milk is the special processing method of Mongolian medicine [7]. Ruixianglangdu treated with an optimum amount of vinegar is the processing

Reagents and Chemicals.
Neochamaejasmin A was purchased from Shanghai Jianglai Biological Technology Co., Ltd. (Shanghai, China). Neochamaejasmin B, isochamaejasmin, and daphnoretin were purchased from Chengdu Chroma-Biotechnology Co., Ltd. (Sichuan, China). The purity of the four standards was > 95%. Glutamic acid, tyrosine, isoleucine, leucine, phenylalanine, and tryptophan were purchased from the National Institutes for Food and Drug Control (Beijing, China). HPLC-grade acetonitrile from Honeywell (Morris, America) and formic acid (HPLC grade) from Sigma-Aldrich (Steinheim, Germany) were purchased. Rice vinegar and fresh milk were obtained from Foshan Haitian Flavoring & Food Co. Ltd. (China) and Yili (Inner Mongolia Yili Industrial Group Co., Ltd., China), respectively. Ultrapure water was prepared using a Milli-Q SP system (Millipore, Bedford, MA, USA).

Plant Materials and Sample Preparation.
Ruixianglangdu was purchased in Anguo (Hebei, China) and identified using morphological and histological methods by Dr. Guihua Cui, which was deposited at Institute of Chinese Materia Medica. Raw ruixianglangdu was processed by vinegar or milk to obtain 3 processed products. 350 g of raw ruixianglangdu was mixed with rice vinegar or fresh milk, left to soak for 6 h, then boiled for 30 min, and eventually dried. Ultimately, 6 batches of samples were obtained for subsequent analysis.
According to the usage of TCM, the raw or processed products (50 g) were boiled for 30 min with 500 mL water, and most solvent was removed through boiling. Then the whole supernatant was collected, diluted to 100 mL with water, and passed through a 0.22 mm filter. The filtrate was stored at 4 ∘ C in a refrigerator before the LC-MS analysis.

LC-MS Analysis.
A Waters ACQUITY UPLC-QTRAP6 5500 mass spectrometer was used to perform multiple-ion monitoring-information dependent acquiring-enhanced product ion (MIM-IDA-EPI) and MIM. A Waters ACQUITY UPLC (Waters, USA) equipped with a binary solvent delivery system, an autosampler, and a column compartment was used. Detection was performed using a QTRAP6 5500 system from Applied Biosystems/MDS Sciex (Applied Biosystems, Foster City, CA, USA), a hybrid triple quadrupole linear ion trap mass spectrometer. The instrument was operated using an electrospray ionization source (ESI). The ion spray voltage was set to 5.5 kV, and the turbo spray temperature was maintained at 550 ∘ C. Nebulizer gas (gas 1) and heater gas (gas 2) were set at 50 and 50 psi, respectively. The curtain gas was kept at 35 psi and the interface heater was on. Nitrogen was used as nebulizer and auxiliary gas. Stepwise MIM in positive and negative scan was adopted, with 3 scans ranging from m/z 51 to m/z 350, from m/z 351 to m/z 650, and from m/z 651 to m/z 950, respectively, with mass step 1.0 Da and dwell time 5 msec. Three hundred MIM transitions in a single run were obtained. The declustering potential (DP) and collision energy (CE) of each MIM were set at 60 V and 5 eV, respectively. For obtaining fragmentation ions, all the ions exceeding 5000 cps were used to trigger the acquisition of EPI spectra. The MS scan function was controlled with the Analyst software (versions 1.6.2) from Applied Biosystems/MDS Sciex.
The chromatographic separation was performed on a Waters HSS C 18 column (2.1 × 100 mm, 1.8 m) with the column temperature set at 40 ∘ C. The mobile phase consisted of water containing 0.1% formic acid (A) and only acetonitrile (B), with a linear gradient elution at a flow rate of 0.3mL/min. The gradient program was as follows: 99-70% A (0-10 min); 70-55% A (10-25 min); 1% A (25-30 min); 99% A (30-33 min). The volume of the sample injection was 1 L. AB, Umea, Sweden). Furthermore, an independent T-test (p<0.01) (Microsoft Office Excel 2010) was used to determine if the change in processing was statistically different at the univariate analysis level. Furthermore, an independent Ttest (p<0.01) (Microsoft Office Excel 2010) was used to analyze the differential component between raw and milkprocessed ruixianglangdu. Meanwhile, the differential component between raw and vinegar-processed ruixianglangdu was also analyzed with T-test. Components with statistical difference were marked with asterisk.

Metabolic Profiling of Ruixianglangdu.
Global profiling of stepwise scan MIM in positive and negative mode was adopted to analyze 6 batches of raw, vinegar-processed, and milk-processed ruixianglangdu, respectively. The chromatogram of ruixianglangdu is shown in Figure 1. On the chromatogram, the ion peaks were easy to detect and their peak areas could be calculated by Analyst software. The ion peaks in the scan range from m/z 651 to m/z 950 was relatively less than those in the scan range from m/z 51 to m/z 650, and most components were eluted before 13 min. It was suggested that most of the compounds are hydrophilic with higher polarity. A total of 1175 ions with signal-to-noise ratio (S/N) > 10 were obtained, including 563 ions in positive mode and 612 ions in negative mode. The resultant 2D matrices, including paired m/z-retention time, sample names, and peak areas of 1175 ions, were analyzed by PCA to visualize general clustering and trends. As shown in Figure 2, an obvious separation trend can be observed between ruixianglangdu and its processed products. And the 2 kinds of processed products were also obviously separated in the PCA.
Total flavonoids obtained from Stellera chamaejasme have showed strong toxicity to mice and rabbit [30]. Chamaechromone (no. 8) and isochamaejasmin (no. 12) were major components in ruixianglangdu with cytotoxicity to canine kidney MDCK cells [31,32]. Coumarin daphnoretin (no. 19) was found to be a cytotoxic constituent of Dirca occidentalis (Thymelaeaceae) [33]. Based on these changing components and literature, for the processed ruixianglangdu, flavonoids especially biflavonones could be the important chemical basis of toxicity reduction. In addition, some coumarins and lignanoids that decreased after processing could also play a role in reducing toxicity.
Compared with milk-processed ruixianglangdu groups, the 10 components involving 8 flavonoids had a lower level in the vinegar-processed ruixianglangdu group. It was inferred that processing method with milk could be better for toxicity reduction of ruixianglangdu. According to literature, the mortality of zebrafish embryo was higher after treating with vinegar-processed ruixianglangdu than milk-processed ruixianglangdu [34], which was consistent with our inference.

Conclusions
With the help of MIM-based metabolomics, this study illustrated the chemical basis of toxicity reducing for processed ruixianglangdu. Flavonoids especially biflavonones could be the important chemical basis of toxicity reducing. In addition, some coumarins and lignanoids that decreased after processing could also play a role in reducing toxicity. Compared with vinegar processing, processing method with milk could be better for toxicity reduction of ruixianglangdu. It is believed that MIM-based metabolomics method was valuable for exploring chemical basis of toxicity reduction for processed TCM. The data is critical to further study the toxicity reduction mechanism of processed ruixianglangdu.

Data Availability
The data supporting the findings of this study are available within the article. Raw data and additional information of this study are available from the corresponding author on request.