3.3.4. ESI-Q-Orbitrap MS and Automatic Components Extraction

For tandem mass spectrometry analysis, a Thermo ESI-Q-Orbitrap MS mass spectrometer was connected to the UltiMate 3000 UHPLC instrument via ESI interface. Ultra-high purity nitrogen (N2) was used as the collision gas and the sheath/auxiliary gas. The ESI source parameters were set as follows: ion spray voltage 3.2 kV, capillary temperature 350 ◦C, ion source heater temperature 300 ◦C, sheath gas (N2) 40 L/h, auxiliary gas (N2) 10 L/min, and a normalized collision energy (NCE) of −35 V was used. The Orbitrap analyzer scanned the mass range from *m*/*z* 150 to 1500 in negative ion mode. Monitoring time was 0–17 min. Detection was obtained by full mass-dd mass mode. The MS data were recorded in both profile and centroid formats. Data recording and processing were performed using the Xcalibur 4.0 software (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The accuracy error threshold was fixed at 5 ppm.

Software-aided, automatic background subtraction and components extraction technique was used to generate a peak list containing all the components profiled from the aerial part of *A. mongolicum*. Sieve v2.2 SP2 (Thermo Fisher Scientific) was used for the automatic components extraction: time range, 1–17 min; BP minimum count, 10,000; BP minimum scans, 5; Background SN, 3; MZ Step, 10; and Frame, >1.

#### **4. Conclusions**

This paper displayed a study—the first of its kind—focused on the systematic bioactive constituents of the aerial parts of *A. mongolicum* in the gastrointestinal tract. As a result, AM and AMH showed a significant increase in the contraction amplitude of mouse small intestinal muscles, which indicated they might have therapeutic effects on constipation. During this process, we made several achievements:

The first comprehensive phytochemistry investigation was developed for AM by using various spectral and chromatographic methods: six new flavonoid glycosides, mongoflavonosides A1 (**1**), A2 (**2**), A3 (**3**), A4 (**4**), B1 (**5**), B2 (**6**), four new phenolic acid glycosides, mongophenosides A1 (**7**), A2 (**8**), A3 (**9**), B (**10**), as well as 21 known compounds were yielded. They were mainly flavonoids and phenolic acids.

The flavonoids and phenolic acids were clarified for the first time to be the main bioactive constituents of *A. mongolicum* on gastrointestinal tract: flavonoids **3**, **4**, **11**–**15**, **21**–**23**, and **26**, as well as phenolic acids **7**, **29**, and **30** showed significant increase in the height of mouse small intestinal muscle.

Furthermore, a fast analysis method for flavonoids and phenolic acids in the aerial parts of *A. mongolicum* was established for the first time by using LC-MS. According to *tR* and *m*/*z*, 31 compounds (**1**–**31**) were unambiguously identified by comparing to the standard references. Then, on the basis of generalized rules of MS/MS fragmentation pattern, chromatographic behaviors, as well as their biosynthetic laws, five flavonoid glycosides (**32**–**36**) and one phenolic acid glycoside (**37**) were tentatively speculated. Among them, peak **36** was a potential new one. Thus, the first evidence for quality control of *A. mongolicum* has been duly provided.

**Supplementary Materials:** Supplementary data (The NMR and HRESIMS spectra of compounds **1**–**10**, as well as extraction and isolation process) associated with this article can be found in the online version.

**Author Contributions:** Y.Z. (Yi Zhang) and T.W. designed the research and wrote the manuscript; Y.D., J.R., and Z.D. performed the experimental work; W.Z. and H.J. corrected the data and reviewed the literatures; M.H. and Y.Z. (Ying Zhang) perfected the language. All authors discussed, edited, and approved the final version. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Program for National Natural Science Foundation of China (81673688), and the Important Drug Development Fund, Ministry of Science and Technology of China (2018ZX09711001-009-010, 2018ZX09735-002).

**Conflicts of Interest:** The authors declare no conflict of interest.
