**4. Discussion**

The objective of the current study is the development of UPLC-Q exactive mass spectrometer methodology to allow qualitative screening of geographical origin traceability in *G. straminea*. Firstly, the chemical profiles of *G. straminea* were determined with a UPLC-Q exactive mass spectrometer, from which 43 compounds were identified by comparing the retention times and mass spectrometry. Meanwhile, a pair of isomers (loganin and secologanol) was identified by mass spectrometry based on their fragmentation pathway. Although Wu, et al. had identified 30 constituents in *G. straminea* with LC-MS [9], the result was also conducive to have a comprehensive understanding of the constituents of *G. straminea*.

Secondly, 42 samples from different habitats were determined by a UPLC-Q exactive mass spectrometer and the data were assayed with multivariate statistical analysis. Gentiopicroside, vitexin, swertiamarin, gentiobiose, sweroside, 2-metho-xyanofinic acide, loganic acide, and 1β,2α,3α,24 tetrahydroxyursa-12,20(30)-dien-28-oic acid were identified as characteristic compounds to identify the geographical origin of the herb. Notably, according to the importance of these characteristic compounds, gentiopicroside was explored as the most characteristic marker to distinguish the geographical origin of *G. straminea.* Additionally, the result also confirmed the rationality of gentiopicroside as the biomarker to determine the quality of *G. straminea.* Moreover, the result indicated that samples from Gansu province would be the most suitable choice for traditional prescriptions and preparations.

It should be emphasized that, according to the Chinese Pharmacopoeia, samples from Gansu province have been shown to have higher gentiopicroside and loganic acide amounts of some compounds than others. However, samples from Sichuan province showed a higher content of swertiamarin, and pharmacological research has revealed that this characteristic compound possesses anti-diabetic and anti-hyperlipidemic effects [28], and inhibits liver fibrosis [29]. Additionally, samples from Qinghai province have shown a higher content of sweroside, which exhibited a hepatoprotective effect [30], protective effects on osteoporosis [31], and aconitine-induced cardiac toxicity effects [32]. In view of the above reasons, it remains a challenge to estimate the herb quality of different populations. Since the herb exhibits various clinical uses in traditional prescriptions, further research should be conducted to better understand its geographical origin and its associated the clinical uses.

#### **5. Materials and Methods**

#### *5.1. Plant Materials, Reagents, and Chemicals*

Forty-two wild herbs of *G. straminea* were collected around the Qinghai-Tibet plateau in Qinghai, Sichuan, and Gansu provinces during the flowering period (the locations of the samples are provided in Table 2), individuals 10 m apart from each other were sampled randomly throughout the entire range of each location. The herbs were authenticated by Professor Yi Zhang (Chengdu University of Traditional Chinese Medicine, Chengdu, China). The samples were carefully divided into roots, leaves, and inflorescences parts, and dried in the shade. The voucher samples were deposited in the College of Ethnic Medicine (Chengdu University of Traditional Chinese Medicine, Chengdu, China) and the Qinghai Key Laboratory of Qinghai-Tibet Plateau Biological Resources (Northwest Onstitute of Plateau Biology, Chinese Academy of Science, Xining, China).


**Table 2.** Populations of *G. straminea* from different geographical origin.

Gentiopicrin (CAS:20831-76-9), loganic acid (CAS: 22255-40-9), swertamarin (CAS: 1738839-5); loganin (CAS: 18524-94-2), vitexin (CAS: 3681-93-4), sweroside (CAS: 14215-86-2), and 6- -*O*-β-dglucopyranosylgentiopicroside (CAS: 115713-06-9) were purchased from Biopurify Phytochemicals Ltd. (Chengdu, China). The purity of all of the standards is higher than 98% (determined by HPLC), and were confirmed by the 1H-NMR spectra to those in the literature [14–20].

HPLC-grade methanol and formic acid were purchased from Merck (Darmstadt, Germany) and Tedia (Fairfield, OH, USA). Deionized water was prepared using a Millipore water treatment system (Bedford, MA, USA). Lipopolysaccharide (LPS, *Escherichia coli* 055: B5) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The Cell Counting Kit-8 was purchased from Dojindo (Kyushu Japan). Griess reagents and dimethyl sulfoxide (DMSO) were purchased from Beyotime (Shanghai, China). All other reagents were of analytical grade.
