Investigation of the Chemical Changes from Crude and Processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma Herbal Pair Extracts by Using Q Exactive High-Performance Benchtop Quadrupole-Orbitrap LC-MS/MS

The Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair is mainly used for regulating the functions of liver and spleen, benefiting qi, and nourishing blood. However, the bioactive compounds for the pharmacological activities of the crude and processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair extracts are still unclear to date. In the present study, Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS was applied to identify the complicated components from crude and processed Paeoniae Radix Alba, crude and processed Atractylodis Macrocephalae Rhizoma, and their crude and processed herbal pair extracts. 123 and 101 compounds were identified in crude and processed Paeoniae Radix Alba samples, respectively. Meanwhile, 32 and 26 compounds were identified in crude and processed Atractylodis Macrocephalae Rhizoma samples, respectively. In the crude and processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair extracts, co-decoction could significantly change the chemical composition of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma in solution. The developed method may provide a scientific foundation for deeply elucidating the processing and compatibility mechanism of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma.


Introduction
Traditional Chinese medicine (TCM) processing is regarded as a pharmaceutical technology based on TCM theory, the requirements of different syndrome treatment, the quality nature of medicine, and different demands of clinical dispensing and preparations [1]. It is one of the characteristics in application of TCM. The compatible components of prescription are composed of prepared Chinese crude drugs after TCM processing.
The prescription compatibility and TCM processing are not only two major features of clinical medication in TCM, but are also critical to distinguish TCM from natural medicine. The research on structural features, compatible effect, and material basis of the herbal pair is the important support in the study of the prescription compatibility since the herbal pair is the minimum unit in prescription of TCM [2,3]. They play a guidance and significant role in reveal of the compatibility rule and the scientific connotation. The herbal pair compatibility theory can explain the relationship of the prescription compatibility to some extent. The research on the relationship between the herbal pair compatibility and the prescription compatibility contributes to the elucidation of the prescription compatibility mechanism and the action mechanism of treatment. There are many herbal pairs commonly used in the clinical practice of TCM, such as the herbal pairs of Paeonia Lactiflora-Liquorice, Ginseng-Aconite, and Aconite-Rhizome Zingiberis [4,5] besides the 2 Evidence-Based Complementary and Alternative Medicine   Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair frequently used in all China dynasties [6,7]. Paeoniae Radix Alba nourishes blood and liver, and Atractylodis Macrocephalae Rhizoma helps invigorate spleen and eliminate dampness [8][9][10][11][12]. Thus, the compatibility of these two medicines could help achieve the goal of purging wood from the earth, regulating the functions of liver and spleen, benefiting qi, and nourishing blood [13][14][15]. Although the compositions of these two medicines have been extensively studied, the appropriate processing method of them, such as frying, which is believed by the practitioners of traditional medicine to have the effects for enhancing the efficacy of the medicine, and their underlying compatibility mechanism are still under investigation.
The objective of this study is to investigate the qualitative, preprocessing, and postprocessing changes in the composition and compatibility of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma by using Q Exactive hybrid quadrupole-Orbitrap mass spectrometer combined with high-performance quadrupole precursor selection with high-resolution and accurate-mass Orbitrap detection. The work could serve as a theoretical basis for the development of medicines from Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma, and the reasonable clinical medication. Furthermore, it provides new insights into the investigation of the herbal pair and for the study of the appropriate processing method for Chinese herbal medicines and their underlying compatibility mechanism.     were prepared. A total of 2.0 g of each sample powder was accurately weighed and transferred into a 50 mL round bottom flask with 20 mL of 70% methanol aqueous solution (v/v) and refluxed in a 80 ∘ C water bath for 1 h. The filtrate was collected after filtration and the residue was then refluxed with 20 mL of 70% methanol aqueous solution in a 80 ∘ C water bath for 1 h, the filtrate was collected again after filtration and the residue was removed. Finally, the combined filtrates were treated by rotary evaporation concentration and the resultant residue was dissolved and transferred into a 25 mL volumetric flask with 70% methanol aqueous solution to make it up to a final concentration of 0.08 g⋅mL −1 . All solutions were stored at 4 ∘ C and filtered through a 0.22 m filter membrane before injection into the HPLC system.

Liquid Chromatography and Mass Spectrometry.
Analyses were performed by using Dionex UltiMate 3000 HPLC system (Dionex, Sunnyvale, CA, USA) with a diode array detector. Detection wavelengths were set at 255 nm. A Thermo Scientific Hypersil Gold C 18 column (100 mm × 2.1 mm, 1.9 m) was used with a flow rate of 0.35 mL⋅min −1 . The injection volume was 5 L, and the column temperature was maintained at 30 ∘ C. The sample separation was performed according to the previous reports with minor modification [16][17][18]. The mobile phase was composed of (a) aqueous formic acid (0.1%, v/v) and (b) acetonitrile under following gradient elution: 10-55% B from 0 to 40 min, 55-90% B from 40 to 51 min, 90% B from 51 to 56 min, 90-10% B from 56 to 56.1 min, and 10% B from 56.1 to 60 min. Mass spectrometry was performed on a Q Exactive high-resolution benchtop quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, USA) using a heated electrospray ionization (HESI-II) source for ionization of the target compounds in positive and negative ion modes. The key parameters were as follows: ionization voltage, +3.0 kV/−2.8 kV; sheath gas pressure, 35 arbitrary units; auxiliary gas, 10 arbitrary units; heat temperature, 300 ∘ C; and capillary temperature, 300 ∘ C. For

Identification of the Main Components in Crude and
Processed Paeoniae Radix Alba. Tentative identification of the main compounds in crude and processed Paeoniae Radix Alba samples was generated based on elemental composition data determined from accurate mass measurements and comparison with the literature data. The total ion chromatograms of crude and processed Paeoniae Radix Alba samples obtained from both positive and negative ion modes were shown in Figure 1. In the preliminary study, the Q Exactive mass spectrometer was confirmed to be highly selective and sensitive. Under the present chromatographic and MS    conditions, 123 and 101 compounds were identified in crude and processed Paeoniae Radix Alba samples, respectively. Compounds 16,30,31,42,45,58,59,61,62,63,64,75,78,80,87,90,91,94,95,103,112, and 120 were not detected in processed Paeoniae Radix Alba sample. Meanwhile, the ESI-MS data of crude and processed samples demonstrated that the peak areas of components 8, 113, and 122 varied significantly, and their amounts were dramatically increased in processed sample. The results were shown in Table 1.
From ESI-MS information, it was found that the sensitivities for all kinds of components in Paeoniae Radix Alba were high in both positive and negative ion modes. On the basis of the elemental compositions of fragment ions, peak 1 was assigned as albiflorin. Peaks 2 and 3 were therefore identified as paeoniflorin, and 1, 2, 3, 4, 6-penta-O-galloyl-beta-Dglucopyranose with above mentioned method. The mass spectra and proposed fragmentations of albiflorin, paeoniflorin, and 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucopyranose were shown in Figure 2. Figure 3 showed the total ion chromatograms of crude and processed Atractylodis Macrocephalae Rhizoma samples obtained from both positive and negative ion modes. 32 and 26 compounds were identified in crude and processed Atractylodis Macrocephalae Rhizoma samples, respectively. Compounds 2, 4, 13, 14, 17, and 29 were not detected in processed Atractylodis Macrocephalae Rhizoma sample. Moreover, the amounts of compounds 3, 7, 9, 10, 21, 23, and 27 were substantially decreased, and the amounts of compounds 8, 18, and 22 were increased in processed sample compared with crude one. The results were shown in Table 2

Analysis of the Chemical Changes of Atractylodis Macrocephalae Rhizoma after Compatibility with Paeoniae Radix
Alba. For crude Atractylodis Macrocephalae Rhizoma, the relative contents of compounds 17, 18, and 25 were increased clearly except those of compounds 6, 23, and 30 decreased considerably and six compounds including protocatechuic acid isomer I, protocatechuic acid isomer II, atracetylentriol, 12-methylbutyryl-14-acetyl-2E, 8EZ, 10E-atractylentriol, 12methylbutyryl-14-acetyl-2E, 8EZ, 10E-atractylentriol isomer, and linoleic acid isomer were lost after its compatibility with crude Paeoniae Radix Alba. For processed Atractylodis Macrocephalae Rhizoma, compounds 9, 20, 26, 27, and 30 were not found except the relative contents of compounds 5, 6, and 8 were decreased while those of compounds 15, 19, 21, and 31 were increased after its compatibility with processed Paeoniae Radix Alba. Furthermore, compound 4 (protocatechuic acid isomer II) was not found in processed Atractylodis Macrocephalae Rhizoma but could be detected in processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair by using Exact Finder and MassFrontier softwares. The above results illustrated that Paeoniae Radix Alba significantly changed the components of Atractylodis Macrocephalae Rhizoma in solution when they decocted together. The corresponding results were presented in Figure 5 and Table 2.

Conclusions
Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS is a powerful tool for discriminating the chemical changes between single herbal and codecocting medicines. In our present study, the Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS based on chemical profiling approach to investigate and evaluate chemical changes from crude and processed Paeoniae Radix Alba, crude and processed Atractylodis Macrocephalae Rhizoma, and their crude and processed herbal pair extracts was proposed. The results showed that processing and compatibility of TCM could significantly change the chemical composition of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma. The developed