**2. 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/H2O (1:1, 8L/8L) to yield EtOAc layer dry extract (AME, 64.9 g) and H2O 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%).

Therefore, AMH was further fractionated by column chromatography (CC) and purified by HPLC to afford six new flavonoid glycosides, named as mongoflavonosides A1–A4 (**1**–**4**), B1 (**5**), B2 (**6**), four new phenolic acid glycosides, named as mongophenosides A1–A3 (**7**–**9**), B (**10**) (Figure 1), along with known compounds, kaempferol-3,7,4- -tri-*O*-β-glucoside (**18**) [19], quercetin-3-*O*-β-d-rutinoside-7-*O*-β-d-glucuronide (**25**) [20], quercetin-3,7,4- -tri-*O*-glucoside (**26**) [21], isorhamnetin 3-*O*-β-d-glucopyranoside (**27**) [22], as well as three phenols, *trans*-*p*-hydroxycinnamate sophorose (**28**) [23], tuberonoid A (**29**) [24], trans-caffeic acid (**30**) [25], and benzyl-*O*-β-d-glucopyranoside (**31**) [26] (Figure 2). Among the known isolates, **28** and **30** were obtained from the *Allium* genus for the first time; **18**, **25**–**27**, **29**, and **31** were firstly found from the plant. Furthermore, the improved effects on the motility of the mouse isolated intestine tissue of the above-mentioned compounds as well as our previously reported flavonoids, kaempferol-3-*O*-β-d-glucopyranoside (**11**), kaempferol-3-*O*-β-d-glucopyranosyl(1→4)-β-d-glucopyranoside (**12**), kaempferol-3-*O*-β-d- rutinoside (**13**), kaemperol-3-*O*-β-d-glucopyranosyl(1→4)[α-l-rhamanopyranosyl(1→6)]-β-d-glucopyranoside (**14**), kaempferol-3-*O*-rutinoside-7-*O*-glucuronide (**15**), kaempferol-3-rutinoside-4- -glucopyranoside (**16**), kaempferol-3-*O*-gentiobioside-4- -*O*-glucopyranoside (**17**) [17], isoquercetin (**19**), quercetin-3-*O*-(6---*O*-acetyl)-β-d-glucopyranoside (**20**), quercetin-3-*O*-β-d-glucopyranosyl(1→4)-β-d-glucopyranoside (**21**), rutin (**22**), quercetin-3,4- -di-*O*-β-d-glucopyranoside (**23**), quercetin 3-*O*-(6---*O*-α-l-rhamnopyranosyl)-β-dglucopyranoside-7-*O*-β-d-glucopyranoside (**24**) [18] (Figure 2) were reported here. Then, qualitative analysis for the aerial parts of *A. mongolicum* by using LC-MS spectrometry technology was developed.

**Figure 1.** The new compounds obtained from the aerial parts of *A. mongolicum* (**1**–**10**).

89

**Figure 2.** The known compounds obtained from the aerial parts of *A. mongolicum* (**11**–**31**).

#### *2.1. Identification of Compounds*

Mongoflavonoside A1 (**1**) was isolated as a yellow powder with negative optical rotation ([α]D<sup>25</sup> <sup>−</sup>54.0, H2O). Its molecular formula was deduced to be C33H38O22 by the negative-ion Electron Spray Ionization-Quadrupole-Orbitrap-Mass Spectrometry (ESI-Q-Orbitrap MS) analysis (*m*/*z* 785.17883 [M − H]−, calculated for C33H37O22, 785.17710). The IR spectrum displayed the absorption bands assignable to hydroxyl (3354 cm<sup>−</sup>1), carbonyl (1716 cm−1), α,β-unsaturated ketone carbonyl (1652 cm<sup>−</sup>1), aromatic ring (1601, 1507, 1457 cm−1), and ether functions (1072 cm−1), respectively. The 1H and 13C-NMR (Table 1) spectra suggested that **1** was a flavonoid glycoside with a kaempferol aglycone [δ 6.48 (1H, br. s, H-6), 6.88 (1H, br. s, H-8), 7.19 (2H, d, *J* = 9.0 Hz, H-3- ,5- ), 8.16 (2H, d, *J* = 9.0 Hz, H-2- ,6- )] and three glycosyl groups [δ 5.05 (1H, d, *J* = 7.0 Hz, H-1----), 5.26 (1H, d, *J* = 6.5 Hz, H-1---), 5.50 (1H, d, *J* = 7.0 Hz, H-1--)]. Acid hydrolysis of **1** with 5% aqueous H2SO4 solution–1,4-dioxane (1:1, *v*/*v*) under 110 ◦C for 2 h to afford d-glucuronic acid and d-glucose, whose absolute configurations were determined by GC-MS analysis of their trimethysilyl thiazolidine derivatives [27]. Meanwhile, correlations were observed between the following proton and carbon pairs in its HSQC-TOCSY spectrum: H-1- and C-1--–C-5--; δ<sup>H</sup> 3.35, 3.58 (H2-6--) and C-4--–C-6--; H-1-- and C-1---–C-4---; δ<sup>H</sup> 3.98 (H-5---) and C-2---–C-5---; H-1--- and C-2----–C-4----; δ<sup>H</sup> 3.50 (H2-6----) and C-4----–C-6----. Combining with the correlations displayed in its 1H 1H COSY and HSQC spectrum, the NMR data of three glycosyls were assigned in detail. Finally, according to the long-range correlations from H-1- to C-3; H-1--- to C-7; H-1--- to C-4 showed in its HMBC experiment (Figure 3), the connections between glycosyl groups and aglycone were determined. On the basis of above-mentioned evidence, the structure of mongoflavonoside A1 (**1**) was identified.


**Table 1.** 13C-NMR data for **1**–**6** in DMSO-*d*6.

**Figure 3.** Key 1H 1H COSY and HMBC correlations of **1**–**6**.

Mongoflavonoside A2 (**2**) is a yellow powder with negative optical rotation ([α]D<sup>25</sup> <sup>−</sup>26.0, MeOH). Its ESI-Q-Orbitrap MS spectrum showed a peak at *m*/*z* 771.19971 [M − H]<sup>−</sup> (calculated for C33H39O21, 771.19783), and its molecular formula was deduced to be C33H40O21. After hydrolyzing with 1 M HCl,

the product was analyzed by using HPLC with an optical rotation detector. As a result, d-glucose was detected [28]. The 1H and 13C-NMR (Table 1) spectra suggested that compound **2** had the same aglycone, kaempferol [δ 6.21 (1H, br. s, H-6), 6.44 (1H, br. s, H-8), 7.17 (2H, d, *J* = 9.0 Hz, H-3- ,5- ), 8.11 (2H, d, *J* = 9.0 Hz, H-2- ,6- )] as that of **1**. In addition, there were three β-d-glucopyranosyl moieties [δ 4.27 (1H, d, *J* = 8.0 Hz, H-1---), 5.03 (1H, d, *J* = 7.5 Hz, H-1-----), 5.51 (1H, d, *J* = 8.0 Hz, H-1--)]. To solve the overlapping problem of three β-d-glucopyranosyl groups, the HSQC-TOCSY experiment was performed. The correlations between C-1- and δ<sup>H</sup> 3.26 (H-2--), 3.39 (H-4--), 3.41 (H-3--), 5.51 (H-1--); δ<sup>H</sup> 3.51, 3.63 (H2-6--) and C-4--–C-6--; H-1-- and C-1---–C-4---; δ<sup>H</sup> 3.42, 3.71 (H2-6---) and C-4---–C-6---; H-1---- and C-1-----–C-4-----; δ<sup>H</sup> 3.50, 3.71 (H2-6-----) and C-4-----–C-6---- were found in it. Moreover, the HMBC displayed the long-range correlations from H-1- to C-3; H-1-- to C-4--; H-1---- to C-4- (Figure 3). Consequently, the structure of mongoflavonoside A2 (**2**) was elucidated to be kaempferol 3-*O*-β-d-glucopyranosyl(1→4)-β-d-glucopyranosyl-4- -*O*-β-d-glucopyranoside.

Mongoflavonoside A3 (**3**) exhibited negative optical rotation ([α]D<sup>25</sup> <sup>−</sup>64.7, H2O). Its molecular formula was revealed to be C39H48O27 by negative ESI-Q-Orbitrap MS analysis (*m*/*z* 947.23242 [M − H]<sup>−</sup>, calculated for C39H47O27, 947.22992). The 1H, 13C-NMR (Table 1) along with various 2D NMR (1H 1H COSY, HSQC, HMBC, and HSQC-TOCSY) spectra denoted that **3** had the same moiety, kaempferol 3-*O*-β-d-glucopyranosyl(1→4)-β-d-glucopyranosyl-4- -*O*-β-d-glucopyranosyl [δ 4.26 (1H, d, *J* = 7.0 Hz, H-1---), 5.04 (1H, d, *J* = 7.0 Hz, H-1-----), 5.53 (1H, d, *J* = 7.5 Hz, H-1--), 6.46 (1H, br. s, H-6), 6.85 (1H, br. s, H-8), 7.18 (2H, d, *J* = 8.5 Hz, H-3- ,5- ), 8.14 (2H, d, *J* = 8.5 Hz, H-2- ,6- )] as that of **2**. Meanwhile, one more β-d-glucuropyranosyl [δ 5.12 (1H, d, *J* = 7.0 Hz, H-1----)] appeared in **3**. On the other hand, the proton signals at H-6 and H-8 and the carbon signal at C-7 shifted to the lower field in comparison with those of **2**, which suggested that the β-d-glucuropyranosyl linked with 7-position of kaempferol. It was clarified by the long-range correlation from H-1--- to C-7 (Figure 3). Then, the structure of mongoflavonoside A3 (**3**) was determined.

The molecular formula of mongoflavonoside A4 (**4**) was measured to be C39H48O26 by negative ESI-Q-Orbitrap MS analysis (*m*/*z* 931.23785 [M − H]<sup>−</sup>, (calculated for C39H47O26, 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 1H, 13C-NMR (Table 1) and 2D NMR spectra indicated that **4** had the same moiety, kaempferol 3-*O*-β-d-glucopyranosyl(1→4)[α-l-rhamanopyranosyl(1→6)]-β-d-glucopyranosyl [<sup>δ</sup> 4.14 (1H, d, *J* = 7.5 Hz, H-1---), 4.40 (1H, br. s, H-1----), 5.23 (1H, d, *J* = 7.5 Hz, H-1--), 6.37 (1H, br. s, H-6), 6.73 (1H, br. s, H-8), 6.88 (2H, d, *J* = 9.0 Hz, H-3- ,5- ), 8.00 (2H, d, *J* = 9.0 Hz, H-2- ,6- )] as that of kaempferol 3-*O*-β-d-glucopyranosyl(1→4)[α-l-rhamanopyranosyl(1→6)]-β-d-glucopyranoside [29]. In addition, one β-d-glucuropyranosyl [δ<sup>H</sup> 5.16 (1H, d, *J* = 7.0 Hz, H-1-----); δ<sup>C</sup> 71.6 (C-4-----), 72.8 (C-2-----), 73.7 (C-5-----), 76.2 (C-3-----), 98.7 (C-1-----), 171.7 (C-6-----)] appeared in **4**. The long-range correlation observation from H-1---- to C-7 (Figure 3) in its HMBC spectrum suggested the β-d-glucuropyranosyl connected with C-7 of kaempferol 3-*O*-β-d-glucopyranosyl(1→4)[α-l-rhamanopyranosyl(1→6)]-β-d-glucopyranosyl. Then, the structure of mongoflavonoside A4 (**4**) was constructed.

Mongoflavonoside B1 (**5**) was isolated as a yellow powder and showed negative optical rotation ([α]D<sup>25</sup> <sup>−</sup>12.0, MeOH). The molecular formula, C29H32O18 of **<sup>5</sup>** was determined from ESI-Q-Orbitrap MS (*m*/*z* 667.15228 [M − H]<sup>−</sup>; calculated for C29H31O18, 667.15049) analysis. Its IR spectrum exhibited characteristic absorptions of hydroxyl (3362 cm<sup>−</sup>1), ester carbonyl (1721 cm−1), α,β-unsaturated ketone carbonyl (1654 cm<sup>−</sup>1), aromatic ring (1605, 1507, 1448 cm−1), and ether functions (1070 cm−1). The 1H and 13C-NMR spectra displayed signals of a quercetin moiety [δ 6.18 (1H, br. s, H-6), 6.39 (1H, br. s, H-8), 6.83 (1H, d, *J* = 8.5 Hz, H-5- ), 7.50 (1H, dd, *J* = 2.0, 8.5 Hz, H-6- ), 7.51 (1H, d, *J* = 2.0 Hz, H-2- )], two β-d-glucopyranosyl groups [δ 4.21 (1H, d, *J* = 8.0 Hz, H-1---), 5.40 (1H, d, *J* = 8.0 Hz, H-1--)], along with an acetyl [δ<sup>H</sup> 1.71 (3H, s, 6---COC*H*3); δ<sup>C</sup> 19.9 (6---CO*C*H3), 169.6 (6---*C*OCH3)]. As shown in Figure 3, the 1H 1H COSY experiment on **5** indicated the presence of partial structures written in bold lines. Moreover, in the HMBC spectrum, the long-range correlations from H-1- to C-3; H-1-- to C-4--; 6---COC*H*<sup>3</sup> to 6---*C*OCH3; H2-6- to 6---*C*OCH3 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 B1.

The molecular formula, C33H38O23 of **6** was measured on ESI-Q-Orbitrap MS (*m*/*z* 801.17407 [M <sup>−</sup> H]−, calculated for C33H37O23, 801.17201) analysis. The 1H, 13C NMR (Table 1) and 2D NMR (1H 1H COSY, HSQC, HMBC, HSQC-TOCSY) spectra suggested **<sup>6</sup>** had the same glycosyl moieties with **<sup>1</sup>**: two β-d-glucopyranosyls [δ 4.88 (1H, d, *J* = 7.0 Hz, H-1----), 5.52 (1H, d, *J* = 7.0 Hz, H-1--)], and one β-d-glucuropyranosyl [5.16 (1H, d, *J* = 7.0 Hz, H-1---)]. Meanwhile, **6** possessed the same aglycone, quercetin [δ 6.45 (1H, br. s, H-6), 6.85 (1H, br. s, H-8), 7.23 (1H, d, *J* = 8.5 Hz, H-5- ), 7.64 (1H, d, *J* = 1.5, 8.5 Hz, H-6- ), 7.69 (1H, d, *J* = 1.5 Hz, H-2- )] as that of **5**. Finally, the connectivities of glycosyl moieties with aglycone were determined by the correlations from H-1- to C-3; H-1-- to C-7; H-1--- to C-4- (Figure 3) showed in its HMBC spectrum.

Mongophenoside A1 (**7**) was obtained as a white powder with negative optical rotation ([α]D<sup>25</sup> −21.0, MeOH). ESI-Q-Orbitrap MS of **7** exhibited quasimolecular ion peak at *m*/*z* 503.14151 [M − H]<sup>−</sup> (calculated for C21H27O14, 503.13953), and its molecular formula was deduced to be C21H28O14. The IR spectrum of it showed absorption bands ascribable to hydroxyl (3362 cm−1), α,β-unsaturated ester carbonyl (1709 cm−1), aromatic ring (1601, 1521, 1447 cm−1), and ether function (1074 cm−1). Acid hydrolysis of **7** liberated d-glucose, which was identified by HPLC analysis [28]. Its 1H, 13C NMR (Table 2) spectra indicated the existence of one *trans*-caffeoyl [δ<sup>H</sup> 6.27 (1H, d, *J* = 16.0 Hz, H-8), 6.75 (1H, d, *J* = 7.5 Hz, H-5), 7.01 (1H, br. d, ca. *J* = 8 Hz, H-6), 7.06 (1H, br. s, H-2), 7.55 (1H, d, *J* = 16.0 Hz, H-7); δ<sup>C</sup> 113.3 (C-8), 146.2 (C-7), 164.9 (C-9)], along with two β-d-glucopyranosyl groups [δ 4.42 (1H, d, *J* = 8.0 Hz, H-1--), 5.56 (1H, d, *J* = 8.0 Hz, H-1- )]. Meanwhile, the partial structures written in bold lines shown in Figure 4 were determined by proton and proton correlations observed in its 1H 1H 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 A1.



Determined in *<sup>a</sup>* DMSO-*d*6, *<sup>b</sup>* CD3OD.

Mongophenoside A2 (**8**), a white powder, showed negative optical rotation ([α]D<sup>25</sup> <sup>−</sup>14.5, MeOH). ESI-Q-Orbitrap MS analysis suggested its molecular formula was C27H38O19 (665.19427 [M − H]−; calculated for C27H37O19, 665.19236). The 1H and 13C-NMR (Table 2) spectra indicated **8** possessed the same moiety, trans-caffeic acid-9-*O*-β-d-glucopyranosyl(1→2)-β-d-glucopyranosyl [<sup>δ</sup> 4.42 (1H, d, *J* = 7.5 Hz, H-1--), 5.56 (1H, d, *J* = 7.0 Hz, H-1- ), 6.27 (1H, d, *J* = 16.0 Hz, H-8), 6.76 (1H, d, *J* = 7.5 Hz, H-5), 7.02 (1H, br. d, ca. *J* = 8 Hz, H-6), 7.06 (1H, br. s, H-2), 7.55 (1H, d, *J* = 16.0 Hz, H-7)] as that of **7**. Except for that, one more β-d-glucopyranosyl [δ 4.17 (1H, d, *J* = 7.5 Hz, H-1---)] appeared

in **8**. Meanwhile, C-6 of it was found to significantly shift to lower field (δ<sup>C</sup> 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 A2 (**8**) was elucidated.

The ESI-Q-Orbitrap MS spectrum of mongophenoside A3 (**9**) displayed the same molecular formula, C27H38O19 (*m*/*z* 665.19452 [M − H]−; calculated for C27H37O19, 665.19236) as that of **8**. Meanwhile, the 1H, 13C NMR (Table 2) and 2D NMR (1H 1H COSY, HSQC, HMBC, HSQC-TOCSY) spectra suggested they had same functional groups as following: trans-caffeic acid aglycone [δ 6.45 (1H, d, *J* = 16.0 Hz, H-8), 7.19 (1H, br. s, H-2), 7.12 (2H, m, H-5 and H-6), 7.61 (1H, d, *J* = 16.0 Hz, H-7)] and three β-d-glucopyranosyl groups [δ 4.43 (1H, d, *J* = 7.5 Hz, H-1---), 4.80 (1H, d, *J* = 7.5 Hz, H-1- ), 5.57 (1H, d, *J* = 7.0 Hz, H-1--)]. Finally, the connectivities of the above-mentioned groups were clarified by the long-range correlations from H-1 to C-4; H-1- to C-9; H-1-- to C-2-- (Figure 4), as shown in its HMBC experiment.

Mongophenoside B (**10**) was obtained as a white powder with positive optical rotation ([α]D<sup>25</sup> +8.0, MeOH). Its ESI-Q-Orbitrap MS spectrum showed the negative ion peak at *m*/*z* 517.15668 [M − H]<sup>−</sup> (calculated for C22H29O14, 517.15518), which indicated the molecular formula of it was C22H30O14. Acid hydrolysis **10** with 1 M HCl, d-glucose was liberated [28]. The 1H, 13C NMR (Table 2) and 2D NMR spectra of it suggested the existence of one trans-feruloyl [δ 6.34 (1H, d, *J* = 16.0 Hz, H-8), 6.80 (1H, d, *J* = 8.0 Hz, H-5), 7.06 (1H, br. d, ca. *J* = 8 Hz, H-6), 7.17 (1H, br. s, H-2), 7.60 (1H, d, *J* = 16.0 Hz, H-7), 3.88 (3H, s, 3-OC*H*3)], one β-d-glucopyranosyl [δ 4.48 (1H, d, *J* = 8.0 Hz, H-1- )], together with one α-d-glucopyranosyl [δ 5.10 (1H, *J* = 3.5 Hz, H-1--)]. Moreover, the long-range correlations from H-1 to C-9; H-1- to C-2 were observed in its HMBC experiment. On the basis of above-mentioned evidence, the structure of mongophenoside B (**10**) was identified as trans-ferulic acid-9-*O*-α-d-glucopyranosyl(1→2)-β-d-glucopyranoside.

**Figure 4.** Key 1H 1H COSY and HMBC correlations of **7**–**10**.
