Phytochemistry and Comprehensive Chemical Profiling Study of Flavonoids and Phenolic Acids in the Aerial Parts of Allium Mongolicum Regel and Their Intestinal Motility Evaluation

To clarify whether flavonoids and phenols in Allium mongolicum Regel have the effect of improving gastrointestinal function and analyze its quality, this study was designed to isolate and identify them from the aerial parts of A. mongolicum by using various chromatographic and spectrophotometric methods, a bioassay on motility of mouse isolated intestine tissue, as well as qualitative analysis using liquid chromatography/mass spectrometry (LC-MS) analysis. As a result, 31 flavonoids and phenolic acids were obtained and identified, including six new flavonoid glycosides, mongoflavonosides A1 (1), A2 (2), A3 (3), A4 (4), B1 (5), B2 (6), and four new phenolic acid glycosides, mongophenosides A1 (7), A2 (8), A3 (9), B (10). Among them, eleven flavonoids and three phenolic acids showed significant increase in the height of mouse small intestinal muscle. It was a first systematic bioactive constituents’ study for A. mongolicum on gastrointestinal tract. Furthermore, according to the retention time (tR) and the exact mass-to-charge ratio (m/z), thirty-one compounds were unambiguously identified by comparing to the standard references by using LC-MS. Then, on the basis of generalized rules of MS/MS fragmentation pattern, chromatographic behaviors, as well as biosynthetic laws of the 31 isolates, five flavonoid glycosides and one phenolic acid glycoside were tentatively speculated. On the basis of the study, a fast analysis method for flavonoids and phenolic acids in A. mongolicum was established.


Results and Discussion
The fresh, aerial parts of A. mongolicum (17.8 kg) was successively heated under reflux with 95% EtOH for 3 h and 50% EtOH for 2 h one time each to obtain dry extract of A. mongolicum aerial parts (AM, 515.0 g). Then 470.1 g of it was partitioned with EtOAc/H 2 O (1:1, 8L/8L) to yield EtOAc layer dry extract (AME, 64.9 g) and H 2 O layer dry extract (AMH, 381.0 g).
Then, AM, AMH, and AME were tested for frequency and height by using a tissue perfusion method. As results, AM and AMH showed significant increase in the contraction amplitude of mouse small intestinal muscle at 200 µg/mL (relative height for AM: 137.4 ± 11.8%* and AMH: 121.8 ± 1.0%**, respectively), but had no significant effect on frequency (relative frequency for AM: 95.2 ± 2.8% and AMH: 100.1 ± 1.9%, respectively). While AME displayed no significant effect on both of them (relative height: 127.9 ± 20.8%; relative frequency: 100.0 ± 9.18%).

Identification of Compounds
The molecular formula of mongoflavonoside A 4 (4) was measured to be C 39 26 , 931.23501). Its acid hydrolysis product was derived to obtain trimethylsilane thiazolidine derivatives, then the existence of d-glucuronic acid, d-glucose, and l-rhamnose were clarified by GC analysis [27]. Its 1 H, 13 C-NMR (Table 1)  6 -COCH 3 to 6 -COCH 3 ; H 2 -6 to 6 -COCH 3 were observed. Finally, after treating 5 with 1 M HCl, d-glucose was detected from its acid hydrolysis product [28]. Consequently, the structure of 5 was identified, and named as mongoflavonoside B 1 Figure 4 were determined by proton and proton correlations observed in its 1 H 1 H COSY experiment. The planar structure of 5 was finally elucidated according to the long-range correlations from H-1 to C-9; H-1 to C-2 ( Figure 4) found in HMBC experiment, and the structure of 7 was named as mongophenoside A 1 . in 8. Meanwhile, C-6 of it was found to significantly shift to lower field (δ C 67.7 for 8; 60.3 for 7) comparing with 7, which suggested C-6 was substituted by the β-d-glucopyranosyl. In the HMBC spectrum, the long-range correlations from H-1 to C-6 ; H-1 to C-2 ; H-1 to C-9 ( Figure 4) were observed. Moreover, treated 8 with 1 M HCl, d-glucose was yielded [28]. Consequently, the structure of mongophenoside A 2 (8) was elucidated.

Inhibitory Effects of Obtained Compounds 1-31 on the Motility of Mouse Isolated Intestine Tissue
Moreover, the obtained constituents of 1-31 were tested for frequency and height by using a tissue perfusion method [30]. Through tissue perfusion experiments, it was found that all compounds displayed no effect on isolated intestinal tissue contraction frequency (Table 3). While almost all isolates exhibited the tendency of increasing the contraction amplitude of mouse small intestinal muscle though only flavonoids 3, 4, 11-15, 21-23, and 26, as well as phenolic acids 7, 29, and 30 showed significant difference comparing with normal group.

Qualitative Analysis
As an important edible medicinal plant for Mongolian people, A. mongolicum has made a great contribution to the development of the local economy, yet there is a lack of analysis of its quality until now.
Our systematic phytochemistry isolation results indicated the main constituents of AM were flavonoids and phenolic acids. The aglycones in the plant mainly included quercetin, kaempferol, as well as isorhamnetin for flavonoid glycosides; while coumaric acid, caffeic acid, and ferulic acid for phenolic acid glycosides. The sugars consisted of β-d-glucopyranoside (Glc), α-d-glucopyranoside (α-Glc), β-d-glucuronic acid (Glu), and α-l-rhamnopyranoside (Rha). While α-Glc was only found in phenolic acid glycosides, Glu and Rha substituted only for flavonoid glycosides.
As for flavonoids, 3-, 7-, and 4 -OH of quercetin, kaempferol, and isorhamnetin were easily substituted by various glycosyls to format O-glycosides. Among them, 7-and 4 -OH was substituted by monosaccharose such as Glc and Glu, while Glu was found to only link with their 7-position. Meanwhile, 3-OH was with a high degree of glycosylation, having one to three sugar moieties, and all of the glycosyl groups directly linked to flavonoid was Glc group, then its 2-, 4-, or 6-position was substituted by another Glc continuously; moreover, its 6-position could also be replaced by Rha [to form rutinosyl (Rut)] or acetyl group ( Figure 5).
Herein, on the basis of above-mentioned phytochemistry study, a fast analysis method for flavonoids and phenolic acids in AM was established by LC-MS on an ESI-Q-Orbitrap MS in negative ion mode (Figure 7). According to the chromatographic retention time (t R ) and the exact mass-to-charge ratio (m/z), 31 compounds (1-31) were unambiguously identified by comparing to the standard references. Meanwhile, the rules of the MS/MS fragmentation pattern and chromatographic elution order have been generalized. Then, five flavonoid glycosides (32-36) and one phenolic acid glycoside (37) were tentatively speculated. Among them, 36 was a potential new compound (Table S1, Figure 8). On the other hand, the carboxyl of obtained phenolic acids from AM was easily substituted by sugar moiety such as Glc(1→2)Glc-, α-Glc(1→2)Glc-, or Glc(1→6)Glc(1→2)Glc-on their 9-position, while 4-OH of them was only substituted by monosaccharose, Glc ( Figure 6). Herein, on the basis of above-mentioned phytochemistry study, a fast analysis method for flavonoids and phenolic acids in AM was established by LC-MS on an ESI-Q-Orbitrap MS in negative ion mode (Figure 7). According to the chromatographic retention time (tR) and the exact mass-to-charge ratio (m/z), 31 compounds (1-31) were unambiguously identified by comparing to the standard references. Meanwhile, the rules of the MS/MS fragmentation pattern and chromatographic elution order have been generalized. Then, five flavonoid glycosides (32-36) and one phenolic acid glycoside (37) were tentatively speculated. Among them, 36 was a potential new compound (Table S1, Figure 8). On the other hand, the carboxyl of obtained phenolic acids from AM was easily substituted by sugar moiety such as Glc(1→2)Glc-, α-Glc(1→2)Glc-, or Glc(1→6)Glc(1→2)Glc-on their 9-position, while 4-OH of them was only substituted by monosaccharose, Glc ( Figure 6). Herein, on the basis of above-mentioned phytochemistry study, a fast analysis method for flavonoids and phenolic acids in AM was established by LC-MS on an ESI-Q-Orbitrap MS in negative ion mode (Figure 7). According to the chromatographic retention time (tR) and the exact mass-to-charge ratio (m/z), 31 compounds (1-31) were unambiguously identified by comparing to the standard references. Meanwhile, the rules of the MS/MS fragmentation pattern and chromatographic elution order have been generalized. Then, five flavonoid glycosides (32-36) and one phenolic acid glycoside (37) were tentatively speculated. Among them, 36 was a potential new compound (Table S1, Figure 8).

Structural Elucidation of Flavonoids
Peaks 3 -6 , 9 , 10 , 13 , 16 , 21 , 23 -27 and 29 -37 were identified by comparison with reference standards (Table S1, Figure 7). Figure 9 and Figure S74 (Table  S1). Therefore, their ionic strength could be used to quickly determine whether the C-4' position of the aglycone was replaced by sugar.    (Table S1). Therefore, their ionic strength could be used to quickly determine whether the C-4 position of the aglycone was replaced by sugar.  .02880 suggested the aglycone of it was isorhamnetin and the substituted sugar moieties included one Glu, one Glc, and one Rha. According to the biosynthesis laws summarized above, peak 28' was deduced to be isorhamnetin-3-O-rutinosyl-7-O-β-D-glucuronide (37) (Table S1, Figure  S75).  .02880 suggested the aglycone of it was isorhamnetin and the substituted sugar moieties included one Glu, one Glc, and one Rha. According to the biosynthesis laws summarized above, peak 28' was deduced to be isorhamnetin-3-O-rutinosyl-7-O-β-D-glucuronide (37) (Table S1, Figure  S75). .02880 suggested the aglycone of it was isorhamnetin and the substituted sugar moieties included one Glu, one Glc, and one Rha. According to the biosynthesis laws summarized above, peak 28 was deduced to be isorhamnetin-3-O-rutinosyl-7-O-β-d-glucuronide (37) (Table S1, Figure S75).

Structural Elucidation of Phenolic Acids
Peaks 1 , 2 , 7 , 12 , and 17 -20 were identified unequivocally by comparing with reference standards (Table S1, Figure 7). As what have been mentioned above, the aglycones of phenolic acid glycosides included coumaric acid, caffeic acid, and ferulic acid. It was well known that the characteristic ions of coumaroyl, caffeoyl, and feruloyl were at m/z 163. Peaks 1', 2', 7', 12', and 17'−20' were identified unequivocally by comparing with reference standards (Table S1, Figure 7). As what have been mentioned above, the aglycones of phenolic acid glycosides included coumaric acid, caffeic acid, and ferulic acid. It was well known that the characteristic ions of coumaroyl, caffeoyl, and feruloyl were at m/z 163.03897 (   Meanwhile, the phenomenon of the neutral loss 120 Da on the basis of [M − H] − were only found in β-d-glucopyranosyl(1→2)-β-d-glucopyranosyl substituted phenolic acid glycosides 7 (peak 7 ), 28 (peak 12 ), and 29 (peak 17 ) ( Figure 13 and Figure S76), which could be used to distinguish the type of substituted sugar moieties.
The molecular formula of peak 15 (m/z 487.14313) was C 21 H 28 O 13 . Its MS/MS fragment ion peaks displayed at m/z 367.10297, 163.03888, and 145.02829, which was similar to those of peak 12 (Table S1, Figure 14). According to the above-mentioned chromatographic retention behavior, we could deduce that peak 15 was not p-hydroxycinnamic acid-9-O-α-d-glucopyranosyl(1→2)-β-d-glucopyranoside. As Han et al. reported, the t R of cis-phenylpropane glycoside was longer than that of trans one when they were analysed by HPLC with the acetonitrile-water system [24]. Consequently, peak 15 was tentatively presumed to be cis-p-hydroxycinnamate sophorose (35).
The molecular formula of peak 15' (m/z 487.14313) was C21H28O13. Its MS/MS fragment ion peaks displayed at m/z 367.10297, 163.03888, and 145.02829, which was similar to those of peak 12' (Table S1, Figure 14). According to the above-mentioned chromatographic retention behavior, we could deduce that peak 15' was not p-hydroxycinnamic acid-9-O-α-D-glucopyranosyl(1→2)-β-D-glucopyranoside. As Han et al. reported, the tR of cis-phenylpropane glycoside was longer than that of trans one when they were analysed by HPLC with the acetonitrile-water system [24]. Consequently, peak 15' was tentatively presumed to be cis-p-hydroxycinnamate sophorose (35).

Materials and Methods
University of TCM, China). The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM.

Extraction and Isolation
See supporting information.

Materials and Methods for Bioassay
The activities of compounds 1-31 were tested for frequency and height by using tissue perfusion method reported before [30]. Samples in DMSO solution were added after 15 min equilibrate incubation; the final DMSO concentration was 0.1% and final concentration of samples were 50 µM. Mosapride citrate dihydrate (Xi'an Janssen Pharmaceutical Ltd., Xi'an, China), final concentration was 200 µg/mL. Data were analyzed by SPSS 22.0 software. All values were expressed as mean ± S.D. A p-value of 0.05 was considered to indicate statistical significance. One-way analysis of variance (ANOVA) and Tukey's studentized range test were used for the evaluation of the significant differences between means and post hoc, respectively.

Materials
The isolated 31 compounds including 24 flavonoids, and 7 phenolic acids were used for reference standards. Their purities were > 98%.

Preparation of Standard Solutions
Standard test solutions of the above-mentioned standard references were prepared in MeOH at a final concentration of 1 µg/mL approximately. All stock solutions were stored at 4 • C in darkness and brought to room temperature before use.

Preparation of the Aerial Parts of A. mongolicum Extract Test Solutions
A. mongolicum extract (AM) was prepared by using the same method as described in "Extraction and Isolation" section. The AM was dissolved with MeOH and filtered with 0.22 µm microporous membrane to get test stock solution at a final concentration of 30 mg/mL, which was stored at 4 • C in darkness and brought to room temperature before use.

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 (N 2 ) 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 (N 2 ) 40 L/h, auxiliary gas (N 2 ) 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.

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 A 1 (1), A 2 (2), A 3 (3), A 4 (4), B 1 (5), B 2 (6), four new phenolic acid glycosides, mongophenosides A 1 (7), A 2