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Bioactive constituents study of Pugionium cornutum L. Gaertn on intestinal motility
Fitoterapia 138 (2019) 104291
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Bioactive constituents study of Pugionium cornutum L. Gaertn on intestinal motility
T
Wenzhong Shia, Jingya Ruanb, Yuanqiang Guoc, Zhijuan Dingb, Jiejing Yana, Lu Qua, ⁎ ⁎ Chang Zhengb, Yi Zhanga,b, , Tao Wanga,b, a
Tianjin State Key Laboratory of Modern Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China Tianjin Key Laboratory of TCM Chemistry and Analysis, Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China c Tianjin Key Laboratory of Molecular Drug Research, College of Chemistry, Nankai University, Tianjin 300071, China b
ARTICLE INFO
ABSTRACT
Keywords: Traditional Mongolian Medicine Pugionium cornutum L. Gaertn Thiohydantoin derivatives Intestinal motility Mice isolated intestine tissue 5-(methylsulfinyl)-pentanenitrile
Thirty-one compounds were obtained and identified from the dried roots of Mongolian medicinal and edible plant, Pugionium cornutum L. Gaertn. Eight of them were new ones, and named as pugcornols A (1), B (2), C (3), and D (4), pugcornosides A (5), B (6), C (7), and D (8), respectively. Among them, 1–4 were rare naturally occurring thiohydantoin derivatives. Meanwhile, all the isolates were determined for their activities on isolated mice jejunum contraction, and ten of them increased height of the spontaneous contractions. Moreover, we partly clarified the mechanism of 5-(methylsulfinyl)-pentanenitrile (9), a characteristic compound in P. cornutum by using different kinds of inhibitor.
1. Introduction Mongolian medicine thinks that the body's normal physiological movement mainly depends on the substance decomposition in the stomach and liver, which suggests that protection of stomach or digestive function and treatment of liver diseases of the digestive system is the premise of prevention and treatment of various diseases [1]. Digestive system disease is one of the supermacy diseases in the Mongolian medicine, and it has accumulated rich clinical experience. It absorbs the theory essence of Tibetan medicine, Chinese medicine, and ancient Indian medicine, and developes into traditional ethnomedicine with unique theoretical system and clinical characteristics. As the first nutrition diet study monographs in China, “Yin Shan Zhengyao” integrats with the nutrition knowledge essence of Mongolian and Han nationalities, which refines the diet knowledge before Yuan dynasty, and emphasizes the importance of prevention and health care [2]. Meanwhile, how to govern sand has become a hot topic attention around the world along with the land desertification rising [3–5]. Psammophyte occupies very important position in the governance of desertification. As a kind of medicinal and edible plant, Pugionium cornutum L. Gaertn (PC) belongs to Cruciferae family, is widely distributed in the Badain Jaran Desert, Kubuqi Desert, Mu Us Desert, Horqin sandy land,
⁎
and Hulunbuir sandy land [6]. As the traditional food for Mongolian people for thousands of years, PC is consumed by the local people mostly after pickling, which owns unique flavor and high nutrition, and enjoys the reputation of desert ginseng [7]. As the traditional arenicolous Mongolian medicine, PC have accumulated rich experience and knowledge of promoting gastrointestinal motility and improving indigestion [8]. Moreover, as a well-known pioneer sandfixing plant, PC is very tolerant of drought and has a strong ability of resisting and fixing sand because of its well-developed root system and strong water absorption capacity. Based on the above-mentioned advantages, PC has drawn more and more attetion of the researchers around the world, and systemic research for it is more and more necessary. In this paper, we report the isolation and identification of constituents, along with their bioactivity evaluations on motility of mice isolated intestine tissue. 2. Results and discussion The 70% EtOH extract from the roots of PC was partitioned with H2O and EtOAc, the H2O layer partition was subjected to D101 macroporous resin CC and eluted with H2O, 95% EtOH, and acetone, successively. Then 95% EtOH eluate and EtOAc layer partition was isolated by silica gel, Sephadex LH-20 CC and preparative HPLC (pHPLC), respectively, to
Corresponding authors at: Tianjin State Key Laboratory of Modern Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China. E-mail addresses: [email protected] (Y. Zhang), [email protected] (T. Wang).
https://doi.org/10.1016/j.fitote.2019.104291 Received 14 July 2019; Received in revised form 3 August 2019; Accepted 5 August 2019 Available online 08 August 2019 0367-326X/ © 2019 Published by Elsevier B.V.
Fitoterapia 138 (2019) 104291
W. Shi, et al.
O
O S
OH
N
O N
N
S
O
O OH
O OH
OH 5
threo S
N
O
CH3OOC O OH HO
HO
HO
HO
OH
O OH
6
O
OH HO
HO OH
H
N
O OH
HO O OH
O
4
CH3OOC
OH
O
HO
HO OH
O
HO
HO
O S
3
HO
S
O
N
S
erythro OH
OCH3
N
2
HO
HO
O
N
S
1
HO
O S
OH
OH
O 7
O
O 8
Fig. 1. The new compounds 1–8 obtained from the roots of P. cornutum.
obtain eight new compounds, pugcornols A (1), B (2), C (3), D (4), pugcornosides A (5), B (6), C (7), D (8) (Fig. 1), along with twenty-three known isolates, 5-(methylsulfinyl)-pentanenitrile (5-MP) (9) [9], phenylethyl glucosinolate (10) [9], 2-phenylethyl-desulphoglucosinolate (11) [9], N-methoxyl-3-indolymethyldesulphoglucosinolate (12) [9], arvelexin (13) [9], cappariloside A (14) [9], L-tryptophan (15) [9], Lphenylalanine (16) [9], 3-phenylpropanamide (17) [9], adenosine (18) [9], (+)-1-hydroxyl pinoresinol-4-O-β-D-glucopyranoside (19) [10], (7R,8S,8′S)-lariciresinol-4,4′-bis-O-β-D-glucopyranoside (20) [11–13], (7R,8S,7′R,8′S)-3,3′-bisdemethylpinoresinol (21) [14,15], demethyl coniferin (22) [16], 2-phenylethyl-O-β-D-glucopyranoside (23) [17], phenethanol-β-D-gentiobioside (24) [18], β-sitosterol (25) [19], daucosterol (26) [19], staphylionoside D (27) [19], TgSSTg (28) [19], ethyl-α-Dfructofuranoside (29) [19], pinellic acid (30) [19], and Z-octadecyl caffeate (31) [19] (Fig. 2). Pugcornol A (1) was obtained as a colorless oil with negative optical rotation ([α]D25–31.3, MeOH). Its molecular formula was assigned as C10H14N2O2S on the basis of negative-ion HRESI-TOF-MS analysis (m/z 225.0708 [M – H]−; calcd for C10H13N2O2S, 225.0703), which suggested the unsaturation degree of it was five. The 1H, 13C NMR (Table 1) and DEPT spectroscopic data indicated that 1 had six methylenes, one methine, one oxygenated quaternary carbon [δC 92.1 (C5)] and two carbonyl groups [δC 171.9 (C-4), 185.0 (C-2)]. The correlations between δH 2.13, 2.28 (1H each, both m, H2-7) and δH [1.71 (1H, m), 1.93 (1H, ddd, J = 1.5, 7.0, 12.5 Hz), H2-6], [3.53 (1H, ddd, J = 3.5, 9.5, 12.0 Hz), 3.78 (1H, dt, J = 8.5, 12.0 Hz), H2-8] found in the 1H 1H COSY spectrum suggested the presence of “eCH2eCH2eCH2e” moiety. Meanwhile, the existence of n-butene group was clarified by the correlations between δH 2.35 (2H, m, H2-2′) and δH 3.71 (2H, t, J = 7.5 Hz, H2-1′), 5.76 (1H, ddt, J = 6.5, 13.5, 17.0 Hz, H-3′); δH 5.76 (H-3′) and δH [5.01 (1H, m), 5.05 (1H, ddd, J = 1.5, 3.0, 17.0 Hz), H2-4′]. The above mentioned NMR data accounted for three degrees of unsaturation, and the remaining two ones indicated the presence of a dicyclic ring system. Moreover, the longrange correlations from δH 1.71, 1.93 (H2-6), 2.13, 2.28 (H2-7), and 3.53, 3.78 (H2-8) to δC 92.1 (C-5) observed in its HMBC experiment established the partial structure of a five membered moiety (ring B) via C-5–N-1–C-8. Furthermore, the observed long-range correlations from δH 1.71, 1.93 (H2-6) to δC 171.9 (C-4); δH 3.53, 3.78 (H2-8) to δC 185.0 (C-2); δH 3.71 (H2-1′) to δC 171.9 (C-4), 185.0 (C-2) connected ring B and the n-butene group with ring A (thiohydantoin group) via C-2–N1–C-5–C-4 and C-1′–N-3 (Fig. 3), respectively. Finally, its absolute configuration, 5R, was further clarified by the time-dependent density functional theory (TDDFT) ECD calculation [20–23], which suggested the obtained ECD spectrum of 1 matched the experimental results closely (Fig. 4). (See Figs. 5 and 6.)
Pugcornol B (2), a colorless oil with negative optical rotation ([α]D25–39.3, MeOH). The molecular formula, C11H18N2O3S2, of 2 was established by negative-ion HRESI-TOF-MS (m/z 289.0693 [M – H]−; calcd for C11H17N2O3S2, 289.0686). The 1H, 13C NMR (Table 2) and DEPT spectra revealed the existence of one methyl, seven methylenes, along with three quaternary carbons. Moreover, the presence of “eCH2eCH2eCH2e” moiety was consolidated by the proton and proton correlations between δH 2.20, 2.40 (1H each, both m, H2-7) and δH [1.77 (1H, m), 2.05 (1H, br. dd, ca. J = 6, 13 Hz), H2-6], [3.61 (1H, ddd, J = 3.0, 9.5, 12.0 Hz), 3.93 (1H, dt, J = 7.5, 12.0 Hz), H2-8] observed in its 1H 1H COSY experiment. The long-range correlations from δH 1.77, 2.05 (H2-6) to δC 93.7 (C-5), 174.2 (C-4); δH 2.20, 2.40 (H2-7) to δC 93.7 (C-5); δH 3.61, 3.93 (H2-8) to δC 93.7 (C-5), 187.1 (C-2) displayed in its HMBC spectrum suggested rings A and B construction of 2 was the same as that of 1. The elemental composition of abovementioned moiety was C6H7N2O2S. According to the MS determination result (molecular formula, C11H18N2O3S2), we could deduce that there was a side chain with elemental composition C5H11OS in compound 2. The proton and proton correlations between δH 1.86 (2H, m, H2-2′) and δH 1.75, 1.83 (1H each, both m, H2-3′), 3.84 (2H, m, H2-1′); δH 1.75, 1.83 (H2-3′) and δH [2.80 (1H, ddd, J = 5.5, 8.0, 13.0 Hz), 2.89 (1H, dt, suggested the existence of J = 7.5, 13.0 Hz), H2-4′] “eCH2eCH2eCH2eCH2e” moiety, and the signal at δ 2.63 (H3-6′) indicated the presence of methyl group. Except for them, there only one sulfur atom and one oxygen atom were not assigned. According to the unsaturation degree (five) of it, the existence of sulfoxide group was elucidated. The observed long-range correlations from δH 3.84 (H2-1′) to δC 174.2 (C-4), 187.1 (C-2); δH 2.80, 2.89 (H2-4′) to δC 38.1 (C-6′); δH 2.63 (H3-6′) to δC 54.2 (C-4′) made the methyl, sulfoxide, “eCH2eCH2eCH2eCH2e” moiety, and ring A constructed together. Then, the planar structure of 2 was elucidated. The absolute configuration of 2 was identified as 5R, which was clarified by comparing the experimental spectrum with the computational ECD spectrum (Fig. 4) [20–23]. Both C-1′ and C-4′ dispalyed a pair of carbon signals (Table 2) with height ratio 1:1, which suggested pugcornol B (2) was the mixture of 5′R and 5′S isomer. The existence of 5′R and 5′S isomer for 2 maybe originated from sulfoxide group. Pugcornol C (3) was determined to possess the molecular formula, C12H20N2O3S2, by its quasi-molecular ion peak at m/z 327.0814 [M + Na]+ (calcd for C12H20N2O3S2Na, 327.0808) in the positive HRESI-TOF-MS experiment, which was 14 amu greater than that of 2. Moreover, the 1H and 13C NMR signals of 3 were coincident with those of 2 except for the methoxy signal [δH 3.19 (3H, s, 5-OCH3); δC 52.2 (5OCH3)]. Meanwhile, the chemical shift of C-5 of 3 [δC 98.7 (C-5)] shifted to lower field comparing with that of 2 [δC 93.7 (C-5)], which suggested C-5 was substituted by methoxyl, and it was verified by the 2
Fitoterapia 138 (2019) 104291
W. Shi, et al.
OR
S N
O O N S O O S
S O
N
OH
11
OH
CH3O
N
N
16 HO
O
OH O
OR2 H
R1O
O
2
19: Glc 20: Glc
HO OH 18
H
R
R1
NH2 17
15
OCH3
N
N
O
12
NH2
N H
OH
OH
NH2
NH2
COOH
HO
HO OH
10
N H
O OH
O OH
HO
N R 13: CH3 14: Glc
N
HO
HO O OH
COOH
N HO OCH3
S
HO
9
OH
O 21
H Glc
HO OH
O
-D-glucopyranosyl
Glc:
H O
HO
OH O
O
HO
HO
O OH OH HO
S O OH
OH S O HO
HO
OH COOH
OH 30
O
OH
OH OH
OH
O O HO
HO
HO
OH
O
OH
28
27 OH
24
23
HO
OH
OH OH
22
O OH
R 25: H 26: Glc
RO
HO
HO
HO
HO
O OH
O OH
O OH
OH
HO
O
O
HO
OH
29
31
Fig. 2. The known compounds 9–31 obtained from the roots of P. cornutum. Table 1 1 H and 13C NMR data for 1 in DMSO-d6. No. 2 4 5 6
δC 185.0 171.9 92.1 32.1
7
24.6
8
47.3
δH (J in Hz) – – – 1.71 (m) 1.93 (ddd, 1.5, 7.0, 12.5) 2.13 (m) 2.28 (m) 3.53 (ddd, 3.5, 9.5, 12.0)
No.
δC
1′ 2′ 3′
40.0 31.4 134.6
4′
117.0
5-OH
O
1' 3
2' 4'
δH (J in Hz)
N
3'
2
S
3.78 (dt, 8.5, 12.0) 3.71 (t, 7.5) 2.35 (m) 5.76 (ddt, 6.5, 13.5, 17.0) 5.01 (m)
OH
4 1
6
5
7
N
8
6'
O S 5'
3'
3
2'
5.05 (ddd, 1.5, 3.0, 17.0) 7.15 (br. s)
N
S 2 O
O S
OCH3
N S 3
OH
N
1 O S
O
1' 4'
N
O N N
O 4 1H 1H
COSY:
HMBC:
Fig. 3. The main 1H 1H COSY and HMBC correlations of 1–4.
3
Fitoterapia 138 (2019) 104291
W. Shi, et al.
Fig. 4. Calculated and experimental ECD spectra of 1–4.
OCH3 HO
1 7
9
3 4
HO O OH
O OH
HO
HO
5
3
OH
OH COSY:
O OH
1'
1H 1H
HMBC:
2
COSY:
1'
HO
HO OH 1 O 1''
7''
O
3
8''
HO OH 1 O 2
O
7
6 1H 1H
CH3OOC 6'
HO
HO
HO OH
OH
O OH
S
O OH
1''
HO
CH3OOC OH
O HO
O OH
1'
HO
OH
S
O HO
OCH3 HO
HMBC:
8
Fig. 6. The main 1H 1H COSY and HMBC correlations of 7 and 8.
Fig. 5. The main 1H 1H COSY and HMBC correlations of 5 and 6.
Comparing with 2, the signal of C-5 shifted to higher field significantly [δC 93.7 (C-5) for 2, δC 64.7 (C-5) for 4], which denoted that C-5 was one methine group. Meanwhile, the signal of C-2 also shifted to higher field [δC 187.1 (C-2) for 2, δC 162.5 (C-2) for 4], combining with the determination data of MS experiment, C-2 was speculated to be one carbonyl. Its planar structure was determined based on the key HMBC correlations from the following proton and carbon pairs: δH 4.18 (H-5) to δC 162.5 (C-2), 176.0 (C-4); δH 3.24, 3.60 (H2-8) to δC 162.5 (C-2); δH 3.51 (H2-1′) to δC 162.5 (C-2), 176.0 (C-4); δH 2.62 (H3-5′) to δC 54.0 (C-4′). Using the similar method [20–23], the absolute configuration of 5R was deduced for pugcornol D (4). Pugcornoside A (5) was obtained as a white powder with negative optical rotation ([α]D25–23.2, MeOH). Negative-ion HRESI-TOF-MS
long-range correlation from δH 3.19 (5-OCH3) to δC 98.7 (C-5). Furthermore, its ECD sepctrum (Fig. 4) was identical to that of 2, and 5R configuration was elucidated [20–23]. Finally, pugcornol C (3) was deduced to be a mixture of 5′R and 5′S isomer because C-3′, 4′ and 6′ showed a pair of carbon signals (Table 2) with height ratio 1:1. The molecular formula of pugcornol D (4) was deduced to be C11H18N2O3S (m/z 259.1113 [M + H]+; calcd for C11H19N2O3S, 259.1111) by the positive-ion HRESI-TOF-MS determination. The 1H, 13 C NMR (Table 3) spectra analysis suggested the presence of one methyl, seven methylenes, one methine, together with two quaternary carbons in 4. The 1H 1H COSY spectrum of 4 suggested the existence of two partial structures written in bold lines as shown in Fig. 3. 4
Fitoterapia 138 (2019) 104291
W. Shi, et al.
Table 2 1 H and 13C NMR data for 2 and 3 in CD3OD. No.
2
3
δC
δH (J in Hz)
δC
δH (J in Hz)
2 4 5 6
187.1 174.2 93.7 33.4
188.1 172.0 98.7 33.2
7 8
25.9 48.9
1′ 2′ 3′ 4′
41.66/41.67 27.6 20.7 54.17/54.20
6′ 5-OCH3
38.1 –
– – – 1.77 (m) 2.05 (br. d, ca. 6, 13) 2.20, 2.40 (both m) 3.61 (ddd, 3.0, 9.5, 12.0) 3.93 (dt, 7.5, 12.0) 3.84 (m) 1.86 (m) 1.75, 1.83 (both m) 2.80 (ddd, 5.5, 8.0, 13.0) 2.89 (dt, 7.5, 13.0) 2.63 (s) –
– – – 1.82 (m) 2.08 (ddd, 2.0, 7.0, 12.0) 2.18, 2.31 (both m) 3.56 (ddd, 3.5, 9.5, 12.0) 3.99 (dt, 8.0, 11.5) 3.88 (m) 1.85 (m) 1.78 (m) 2.80 (ddd, 6.0, 8.5, 13.5) 2.89 (ddd, 7.0, 8.0, 13.5) 2.62 (s) 3.19 (s)
δC
δH (J in Hz)
2 4 5 6
162.5 176.0 64.7 28.2
7 8
28.2 46.4
– – 4.18 1.67 2.21 2.09 3.24
(dd, 7.5, 8.5) (m) (m) (m) (ddd, 4.5, 8.0, 12.5)
No.
δC
1′ 2′ 3′ 4′
39.0 28.0 20.7 54.0
5′
38.1
δH (J in Hz) 3.60 3.51 1.76 1.76 2.79 2.88 2.62
(dt, 7.5, 12.5) (t, 6.0) (m, overlapped) (m, overlapped) (m) (m) (s)
Table 4 1 H and 13C NMR data for 5 and 6 in CD3OD. No.
5
6
δC
δH (J in Hz)
δC
δH (J in Hz)
1 2 3 4 5 6 7 8 9
135.4 116.3 148.3 146.5 118.4 120.3 84.8 53.1 63.1
136.1 116.7 148.3 146.6 118.6 120.8 85.3 53.1 63.3
1′ 2′ 3′ 4′ 5′ 6′
104.2 74.9 77.6 71.3 78.3 62.4
1″ 2″ 3″ 4″ 5″ 6″
86.5 74.6 79.4 71.3 82.0 62.8
7-OCH3
57.5
– 6.90 – – 7.17 6.82 4.49 3.31 3.65 3.74 4.78 3.50 3.48 3.42 3.42 3.72 3.90 4.40 3.19 3.33 3.32 3.24 3.65 3.85 3.25
– 6.90 – – 7.17 6.83 4.38 3.31 3.65 3.74 4.80 3.49 3.48 3.42 3.43 3.72 3.90 3.83 3.08 3.18 3.25 3.10 3.63 3.85 3.22
(d, 1.5) (d, 8.0) (dd, 1.5 8.0) (d, 5.5) (m) (m, overlapped) (dd, 7.0, 12.0) (d, 7.5) (dd, 7.5, 9.0) (dd, 9.0, 9.0) (m, overlapped) (m, overlapped) (dd, 5.0, 12.0) (dd, 2.0,12.0) (d, 9.5) (dd, 9.0, 9.5) (m, overlapped) (m, overlapped) (m) (m, overlapped) (dd, 3.0, 12.0) (s)
104.3 74.9 77.7 71.4 78.3 62.5 86.1 74.9 79.5 71.4 81.9 62.8 57.3
41.8 27.7 20.89/20.92 54.11/54.14 38.14/38.16 52.2
moiety was deduced by the correlations between δH 3.31 (1H, m, H-8) and δH [3.65 (1H, m, overlapped), 3.74 (1H, dd, J = 7.0, 12.0 Hz), H29], as well as the chemical shift of them. Its 13C NMR spectrum displayed twenty-two carbon signals. In addition to those assigned to above-mentioned groups, the other twelve signals was in the scope of 60–105. Among them, δH 4.78 (1H, d, J = 7.5 Hz, H-1′) combing with δC 62.4 (C-6′), 71.3 (C-4′), 74.9 (C-2′), 77.6 (C-3′), 78.3 (C-5′), 104.2 (C1′) suggested the presence of one 1-O-β-D-glucopyranosyl. Meanwhile, the existence of 1-thiol-β-D-glucopyranosyl was supported by δH 4.40 (1H, d, J = 9.5 Hz, H-1″) and δC 62.8 (C-6″), 71.3 (C-4″), 74.6 (C-2″), 79.4 (C-3″), 82.0 (C-5″), 86.5 (C-1″) [24,25]. Furthermore, the longrange correlations from δH 4.49 (H-7) to δC 57.5 (7-OCH3), 116.3 (C-2), 120.3 (C-6), 135.4 (C-1); δH 3.31 (H-8) to δC 135.4 (C-1); δH 4.78 (H-1′) to δC 146.5 (C-4); δH 4.40 (H-1″) to δC 53.1 (C-8); δH 3.25 (7-OCH3) to δC 84.8 (C-7) found in its HMBC experiment linked the above-mentiond moieties together. Finally, the relative configuration between C-7 and C-8 was determined as erythro according to the coupling constant of H-7 and H-8 (J = 5.5 Hz) [26]. Pugcornoside B (6) was also obtained as a white powder with negative optical rotation ([α]D25–52.6, MeOH). Its negative-ion HRESITOF-MS spectrum dispalyed the same molecular formula, C22H34O14S (m/z 553.1599 [M – H]−; calcd for C22H33O14S, 553.1597) as that of 5. The 1H, 13C NMR (Table 4) and various 2D NMR (1H 1H COSY, HSQC, HMBC) spectra suggested its planar structure was identical to that of 5. Moreover, the coupling constant of H-7 and H-8 (J = 7.5 Hz) suggested the relative configuration between C-7 and C-8 was threo [26]. Pugcornoside C (7) was obtained as a white powder with positive optical rotation ([α]D25 + 6.3, MeOH). Its molecular formula, C10H18O9 was deduced from the negative-ion HRESI-TOF-MS measurement (m/z 281.0878 [M–H]−; calcd for C10H17O9, 281.0878). Acid hydrolysis of 7 with 5% aqueous H2SO4-1,4-dioxane (1:1, v/v) afforded D-glucuronic acid, whose absolute configurations were determined by GC–MS analysis of their trimethysilyl thiazolidine derivatives [27]. The 1H, 13C NMR (Table 5) and 2D NMR (1H 1H COSY, HSQC, and HMBC) spectra suggested the presence of one methoxyl [δH 3.76 (3H, s, 6′-OCH3)], one α-D-glucuronic acid functional group [δ 4.85 (1H, d, J = 4.0 Hz, H-1′)], together with one glycerol moiety {δ [3.41 (1H, dd, J = 8.0, 11.0 Hz), 3.82 (1H, dd, J = 3.5, 11.0 Hz), H2-1], 3.57 (2H, t, J = 5.0 Hz, H2-3), 3.84 (1H, m, H-2)}. Finally, according to the long-rang correlations from δH 4.85 (H-1′) to δC 71.4 (C-3); δH 3.76 (6′-OCH3) to δC 172.0 (C6′), the above-mentioned fragments were linked with each other. Pugcornoside D (8) was also isolated as a white powder with positive optical rotation ([α]D25 + 28.1, MeOH). The molecular formula, C18H24O10, of 8 was determined from negative-ion HRESI-TOF-MS analysis (m/z 399.1295 [M–H]−; calcd for C18H23O10, 399.1297). The 1 H, 13C NMR (Table 5) and various 2D NMR (1H 1H COSY, HSQC,
Table 3 1 H and 13C NMR data for 4 in CD3OD. No.
25.8 49.3
(d, 2.0) (d, 8.5) (dd, 2.0, 8.5) (d, 7.5) (m) (dd, 5.5, 12.0) (dd, 4.5, 11.5) (d, 7.5) (dd, 7.5, 9.0) (dd, 9.0, 9.0) (m, overlapped) (m, overlapped) (dd, 3.0, 12.0) (dd, 2.0,12.0) (d, 9.5) (dd, 8.5, 9.5) (dd, 8.5, 9.0) (dd, 9.0, 9.5) (m) (dd, 5.5, 12.0) (dd, 3.0, 12.0) (s)
afforded [M – H]− peak at m/z 553.1605 (calcd for C22H33O14S, 553.1597), supporting molecular formula of C22H34O14S for 5. The 1H and 13C NMR (Table 4) spectra showed signals assignable to one ABXtype aromatic protons [δ 6.82 (1H, dd, J = 1.5, 8.0 Hz, H-6), 6.90 (1H, d, J = 1.5 Hz, H-2), 7.17 (1H, d, J = 8.0 Hz, H-5)] and one methoxy group [δ 3.25 (3H, s, 7-OCH3)]. The existence of “eCHeCHeCH2OH” 5
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and results in contraction of smooth muscle [28]. 5-MP is a characteristic compound in PC, in normal isolated jejunum tissue, it showed significant increasing effect on height of contractility at 50 μM. Further, atropine (the blocker of M-receptor), CuSO4 (the inhibitor of 5-HT receptor), and loperamide (the inhibitor of μ-opioid receptor) were used to clarify the mechanism on intestinal smooth muscle spontaneous contraction. Comparing the difference between groups treated or untreated with inhibitor, we partly clarified that the increasing effect of 5MP, at least, partly releated to M receptor and μ-opioid receptor, but not releated to 5-HT receptor. In summary, gastrointestinal prokinetic efficacy of the water extract from fresh and pickled PC had been reported by Li et al.. And they analyzed the contents of amino acids, vitamins, potassium, calcium, fat, and sugar in PC [8], too. But the secondary metabolites in PC, as well as their bioactivities have been not reported. In the process of investigating the active constituents on intestinal motility, we obtained four new rare thiohydantoin derivatives (1–4), two new glucosinolates (5, 6), two new others (7, 8), along with twenty-three known isolates (9–31) from the dried roots of Mongolian medicinal and edible plant. For thiohydantoin derivatives 1–4, only one paper reported them obtained from natural products [29] until now. These compounds are presumably derived by condensation of an isocyanate or isothiocyanate with the secondary amine group of proline, followed by acylation of the nascent amino group with the carboxylic group of proline. On the other hand, the main constituents in PC were mainly nitrogen- and/or sulfurcontaining heteroatom compounds as shown in Figs. 1 and 2. Our research suggested that nitrogen-containing heteroatom constituents were the active constituents on intestinal motility. The results will provide a scientific basis for guiding reasonable application of PC in the clinical Mongolian medicine. And which will be benefit for promoting the development of sand industry and constructing the green economy in China.
Table 5 1 H and 13CNMR data for 7 and 8 in CD3OD. No.
7
8
δC
δH (J in Hz)
δC
δH (J in Hz)
1
71.4 72.2 64.0
(dd, 8.0, 11.0) (dd, 3.5, 11.0) (m) (t, 5.0)
70.6
2 3
3.41 3.82 3.84 3.57
1′ 2′ 3′ 4′ 5′ 6′ 6′-OCH3 1″ 2″,6″ 3″,5″ 4″ 7″ 8″
101.2 73.3 74.5 73.3 73.0 172.0 52.8
4.85 3.45 3.66 3.53 4.12 – 3.76
(d, 4.0) (dd, 4.0, 10.0) (dd, 9.0, 10.0) (dd, 9.0, 9.5) (d, 9.5)
3.40 3.76 4.02 4.14 4.20 4.81 3.46 3.64 3.53 4.10 – 3.76 – 7.28 7.30 7.24 3.69 –
(s)
69.4 66.5 101.2 73.2 74.4 73.3 73.1 171.9 52.8 135.6 130.4 129.5 128.1 41.8 173.4
(dd, 6.5, 11.0) (m, overlapped) (m) (dd, 6.0, 11.5) (dd, 5.0, 11.5) (d, 3.5) (dd, 3.5, 9.5) (dd, 9.0, 9.5) (dd, 9.5, 9.5) (d, 10.0) (s) (m) (m) (m) (s)
HMBC) spectra suggested 8 had the same moiety, glycerin-1-O-D-glucuronic acid methyl ester, as that of 7. In addition, there was one benzene acetyl [δH 3.69 (2H, s, H2-7″), 7.24 (1H, m, H-4″), 7.28 (2H, m, H-2″,6″), 7.30 (2H, m, H-3″,5″); δC 173.4 (C-8″)] appeared in 8. And in the HMBC experiment, long-range correlations from δH 3.69 (H2–7″) to δC 130.4 (C-2″,6″), 135.6 (C-1″), 173.4 (C-8″); δH 4.14, 4.20 (H2-3) to δC 173.4 (C-8″); δH 3.76 (6′-OCH3) to δC 171.9 (C-6′) were observed. Consequently, the structure of pugcornoside D (8) was elucidated. As the traditional arenicolous Mongolian medicine, PC has accumulated rich experience and knowledge of promoting gastrointestinal motility and improving indigestion [8]. To clarify the material foundation of medicinal effectiveness of PC for gastrointestinal motility, the effects of all the fractions and isolated compounds from it were investigated on mice jejunum contractility. As results, 70% ethanol-water extract of PC (a), and 95% eluate of PC (e) fractions (200 μg/mL) (Fig. 7), as well as compounds 1, 2, 3, 8, 9, 12, 14, 17, 18, 24 (50 μM) significantly increased the height of isolated mice jejunum spontaneous contractions. However, all of them showed no significant changing on contraction frequency and tension at the same concentration. (See Figs. 8 and 9.) Impairment of gastrointestinal motility is one of the major reason in bowel disease, such as constipation and intestinal obstruction. M receptor, 5-HT receptor and opioid receptor play functional roles in intestinal smooth muscle contraction. Activation of these receptors lead increase in inositol 1,4,5-triphosphate and stimulates Ca2+ release from the endoplasmic reticulum, increases intracellular Ca2+ concentration,
3. Materials and methods 3.1. Phytochemistry study of PC 3.1.1. General UV and IR spectra were determined on a Varian Cary 50 UV–Vis and Varian 640-IR FT-IR spectrophotometer, respectively. Optical rotations were run on a Rudolph Autopol® IV automatic polarimeter. NMR spectra were obtained on a Bruker 500 MHz NMR spectrometer at 500 MHz for 1H and 125 MHz for 13C NMR (internal standard: TMS). Positive- and negative-ion HRESI-TOF-MS were recorded on an Agilent Technologies 6520 Accurate-Mass Q-Tof LC/MS spectrometer. CC were performed on macroporous resin D101 (Haiguang Chemical Co., Ltd., Tianjin, China), Silica gel (74–149 μm, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and Sephadex LH-20 (Ge
Fig. 7. Effects of fractions of P. cornutum on isolated jejunum contraction. N: normal group; a: 70% ethanol-water extract of P. cornutum; b: EtOAc layer partition of a; c: H2O layer partition of a; d: H2O eluate of c from D101 macroporous resin CC; e: 95% eluate of c from D101 macroporous resin CC; the final concentration of each fraction: 10 mg/mL; n = 6, ⁎P < .05, ⁎⁎P < .01, ⁎⁎⁎P < .001 vs. normal group. Data (mean ± SEM) were analyzed by ANOVA. 6
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Fig. 8. Effects of isolated compounds 1–31 from P. cornutum on isolated jejunum contraction. N: normal group; the final concentration of each compound: 50 μM; n = 6, ⁎P < .05, ⁎⁎P < .01, ⁎⁎⁎P < .001 vs. normal group. Data (mean ± SEM) were analyzed by ANOVA.
Healthcare Bio-Sciences, Uppsala, Sweden). pHPLC column, Cosmosil 5C18-MS-II (20 mm i.d. × 250 mm, 5 μm, Nakalai Tesque, Inc., Tokyo, Japan) were used to isolate compounds.
The H2O layer partition (1035.0 g) was dissolved in H2O, and subjected to D101 macroporous resin CC (H2O → 95% EtOH → Acetone) to afford H2O (980.0 g), 95% EtOH (44.1 g), and acetone (0.8 g) eluate, respectively. The 95% EtOH eluate (30.0 g, Fr. H) was isolated by ODS CC [MeOH-H2O (10:90 → 15:85 → 30:70 → 40:60 → 50:50 → 70:30 → 100:0, v/v)], and eight fractions (Fr. H1-Fr. H8) were yielded. Fraction H4 (9.0 g) was isolated by Sephadex LH-20 CC [MeOH-H2O (50:50, v/ v)] to obtain eight fractions (Fr. H4-1-Fr. H4-8). Fraction H4-2 (2.0 g) was separated by pHPLC [CH3CN-H2O (6:94 → 13:87 → 15:85, v/v)] to gain fifteen fractions (Fr. H4-2-1-Fr. H4-2-15). Fraction H4-2-3 (240.7 mg) was purified by pHPLC [MeOH-H2O (9:91, v/v)] to yield pugcornosides A (5, 11.2 mg) and B (6, 15.4 mg). Fraction H4-2-8 (149.9 mg) was prepared by pHPLC [MeOH-H2O (15:85, v/v)] to produce pugcornol D (4, 12.8 mg). Fraction H4-2-9 (148.2 mg) was isolated by pHPLC [CH3CN-H2O (9:91, v/v)], and (7R,8S,8′S)-lariciresinol4,4′-bis-O-β-D-glucopyranoside (20, 18.4 mg), and phenethanol-β-Dgentiobioside (24, 9.7 mg), together with staphylionoside D (27,
3.1.2. Plant material The roots of Pugionium cornutum L. Gaertn were collected from Alxa Youqi, Inner Mongolia Autonomous region, China, and identified by Dr. Li Tianxiang (Experiment Teaching Department, Tianjin University of Traditional Chinese Medicine). The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM. 3.1.3. Extraction and isolation The dried roots of PC (4.8 kg) were refluxed with 70% ethanolwater for three times. Evaporation of the solvent under pressure provided a 70% ethanol-water extract (1740.0 g), which was partitioned with H2O and EtOAc to gain H2O layer (1150.0 g) and EtOAc layer (153.0 g) partition, respectively. 7
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1-Fr. H6-13) were given. Fraction 6-6 (292.7 mg) was purified by pHPLC [CH3CN-H2O (15:85, v/v)] to yield 2-phenylethyl-O-β-D-glucopyranoside (23, 24.9 mg). Fraction H6–10 (271.0 mg) was isolated by pHPLC [MeOH-H2O (55:45, v/v)], and pugcornol C (3, 12.4 mg) was obtained. The EtOAc layer partition (100.0 g, Fr. E) was subjected to silica gel CC [CHCl3 → CHCl3-MeOH (100:3 → 100:5, v/v) → CHCl3-MeOH-H2O (10:3:1 → 7:3:1, lower layer, v/v/v) → MeOH]. As results, twelve fractions (Fr. E1-Fr. E12) were given. Fraction E8 (7.0 g) was subjected to ODS CC [MeOH-H2O (30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 90:10 → 100:0, v/v)] to give eleven fractions (Fr. E8-1-Fr. E811). Fraction E8-3 (175.0 mg) was separated by pHPLC [CH3CN-H2O (16:84, v/v)] to obtain (7R,8S,7′R,8′S)-3,3′-bisdemethylpinoresinol (21, 12.0 mg). Meanwhile, compounds 9–18, 25–31 were obtained by using the method discribed in references [9, 19]. 3.1.3.1. Pugcornol A (1). Colorless oil; [α]D25–31.3 (conc. 0.12, MeOH); CD (conc. 0.0015 M, MeOH) mdeg (λnm): −1.39 (235), −3.33 (256), −3.60 (269); CD (conc. 0.0015 M, CH3CN) mdeg (λnm): −1.21 (229), −2.83 (245), −4.52 (267); UV λmax (MeOH) nm (log ε): 269 (3.81); IR νmax (KBr) cm−1: 3364, 2943, 1750, 1424, 1347, 1225, 1124, 1061, 1025; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data see Table 1. HRESI-TOF-MS Negative-ion mode m/z 225.0708 [M – H]− (calcd for C10H13N2O2S, 225.0703). 3.1.3.2. Pugcornol B (2). Colorless oil; [α]D25–39.3 (conc. 0.12, MeOH); CD (conc. 0.0015 M, MeOH) mdeg (λnm): −1.30 (223), −5.34 (270); CD (conc. 0.0015 M, CH3CN) mdeg (λnm): −1.56 (223), −6.80 (252), −4.81 (272); UV λmax (MeOH) nm (log ε): 277 (3.96); IR νmax (KBr) cm−1: 3355, 2941, 1750, 1428, 1346, 1236, 1133, 1049, 1010; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data see Table 2. HRESI-TOF-MS Negative-ion mode m/z 289.0693 [M – H]− (calcd for C11H17N2O3S2, 289.0686). 3.1.3.3. Pugcornol C (3). Colorless oil; [α]D25–36.1 (conc. 0.14, MeOH); CD (conc. 0.0015 M, MeOH) mdeg (λnm): −0.34 (228), −2.60 (253), −2.99 (269); CD (conc. 0.0015 M, CH3CN) mdeg (λnm): −1.26 (231), −3.91 (250), −5.16 (267); UV λmax (MeOH) nm (log ε): 249 (3.50), 268 (3.58); IR νmax (KBr) cm−1: 3365, 2935, 1749, 1419, 1347, 1236, 1133, 1050, 1015; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data see Table 2. HRESI-TOF-MS Positive-ion mode m/z 327.0814 [M + Na]+ (calcd for C12H20N2O3S2Na, 327.0808).
Fig. 9. Mechanism research of 5-MP on isolated jejunum contraction height. N: normal group; 5-MP: 5-(methylsulfinyl)-pentanenitrile (compound 9); n = 6, ⁎ p < .05, ⁎⁎p < .01, ⁎⁎⁎p < .001 vs. normal group. Data (mean ± SEM) were analyzed by ANOVA.
3.1.3.4. Pugcornol D (4). Colorless oil; [α]D25–64.1 (conc. 0.13, MeOH); CD (conc. 0.0015 M, MeOH) mdeg (λnm): −35.54 (214), +14.06 (238), +0.12 (263); CD (conc. 0.0015 M, CH3CN) mdeg (λnm): −33.13 (212), 12.47 (238), +0.23 (262); UV λmax (MeOH) nm (log ε): 214 (3.44), 267 (2.38); IR νmax (KBr) cm−1: 3420, 2943, 1765, 1701, 1447, 1419, 1361, 1014; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data see Table 3. HRESI-TOF-MS Positive-ion mode m/z 259.1113 [M + H]+ (calcd for C11H19N2O3S, 259.1111).
6.2 mg) were given. Fraction H4-3 (1200.0 mg) was separated by pHPLC [MeOH-H2O (55:45, v/v)] to yield pugcornol C (3, 14.4 mg). Fraction H4-4 (530.0 mg) was subjected to pHPLC [MeOH-H2O (30:70, v/v)], and nine fractions (Fr. H4-4-1-Fr. H4-4-9) were obtained. Fraction H4-4-9 (62.8 mg) was separated by pHPLC [CH3CN-H2O (22:78, v/v)] to yield pugcornoside D (8, 8.5 mg). Fraction H4–5 (500.0 mg) was isolated by pHPLC [MeOH-H2O (20:80, v/v)] to gain fourteen fractions (Fr. H4-5-1-Fr. H4-5-14). Fraction H4-5-5 (14.7 mg) was subjected to silica gel CC [CHCl3-MeOH-H2O (7:3:1, lower layer, v/ v/v)] to obtain demethyl coniferin (22, 14.7 mg). Fraction H5 (5.0 g) was isolated by ODS CC [MeOH-H2O (10:90 → 20:80 → 30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 100:0, v/v)], and seven fractions (Fr. H5-1-Fr. H5-7) were produced. Fraction H5-4 (800.0 mg) was purified by pHPLC [CH3CN-H2O (16:84, v/v)] to obtain pugcornol B (2, 33.4 mg), and (+)-1-hydroxyl pinoresinol-4-O-β-D-glucopyranoside (19, 21.1 mg). Fraction H6 (4.8 g) was subjected by ODS CC [MeOH-H2O (10:90 → 20:80 → 30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 90:10 → 100: 0, v/v)], and thirteen fractions (Fr. H6-
3.1.3.5. Pugcornoside A (5). White powder; [α]D25–23.2 (conc. 0.11, MeOH); UV λmax (MeOH) nm (log ε): 223 (3.61), 277 (3.18); IR νmax (KBr) cm−1: 3365, 2922, 1646, 1558, 1507, 1457, 1277, 1070; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data see Table 4. HRESI-TOF-MS Negative-ion mode m/z 553.1605 [M – H]− (calcd for C22H33O14S, 553.1597). 3.1.3.6. Pugcornoside B (6). White powder; [α]D25–52.6 (conc. 0.13, MeOH); UV λmax (MeOH) nm (log ε): 220 (3.65), 277 (3.18); IR νmax (KBr) cm−1: 3366, 2918, 1600, 1507, 1456, 1436, 1278, 1045, 1072; 1 H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data see Table 4. HRESI-TOF-MS Negative-ion mode m/z 553.1599 [M – H]− (calcd for C22H33O14S, 553.1597). 8
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3.1.3.7. Pugcornoside C (7). White powder; [α]D25 + 6.3 (conc. 0.12， MeOH)；UV λmax (MeOH) nm (log ε): 266 (2.94); IR νmax (KBr) cm−1: 3386, 2391, 1734, 1652, 1107, 1044; 1H NMR (CD3OD, 500 MHz) and 13 C NMR (CD3OD, 125 MHz) data see Table 5. HRESI-TOF-MS Negative-ion mode m/z 281.0878 [M – H]− (calcd for C10H17O9, 281.0878).
0.1% and final concentration were 10 mg/mL for every fraction and 50 μM every compound, respectively. Intestine contractions were recorded for 1 min before and 4 min after drug additions using the Power Lab system and the Chart 7 software (AD instrument Ltd., New South Wales, Australia). Atropine (0.025 mg/mL), CuSO4 (0.012 mg/mL), and loperamide (0.004 mg/mL) were used for mechanism researches.
3.1.3.8. Pugcornoside D (8). White powder; [α]D25 + 28.1 (conc. 0.12, MeOH); UV λmax (MeOH) nm (log ε): 205 (3.72), 262 (2.77); IR νmax (KBr) cm−1: 3420, 2933, 1733, 1635, 1466, 1339, 1267, 1154, 1112, 1047; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data see Table 5. HRESI-TOF-MS Negative-ion mode m/z 399.1295 [M – H]− (calcd for C18H23O10, 399.1297).
3.2.4. Statistic analysis Values were expressed as means ± SD. All the grouped data were statistically analyzed with the SPSS 14.0 software. Significant differences between the normal group or control group were evaluated by one-way analysis of variance (ANOVA), and Tukey's studentized range test was used for post hoc evaluations. Least significant differences (P < .05) were used for statistical analyses.
3.1.4. Acid hydrolysis of 7 and 8 Solution of 7 and 8 (each 3.0 mg) in 5% aqueous H2SO4-1,4-dioxane were heated under reflux for 1 h, respectively. After cooling, the reaction mixture was neutralized with Amberlite IRA-400 (OH– form), removed by filtration, subjected to ODS CC (H2O), and the H2O eluate was reacted with L-cysteine methyl ester hydrochloride in pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), successively. Finally, the reaction product was elucidated by GC–MS analysis (GC conditions, column: RESTEK Rxi-5 ms, 30 m × 0.25 mm (i.d.) capillary column; column temperature: 230 °C; carrier gas: He), and D-glucuronic acid hydrolysate from 7 and 8 was identified by comparing it retention times (tR 13.4 min) with that of its authentic sample treated in the same way.
Declaration of Competing Interest None. Acknowledgments 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 (2018ZX09735-002). Appendix A. Supplementary data
3.1.5. Computations Using the similar methods as we described previously [20–23]: The ECD spectra for the optimized conformers were calculated at the CAMB3LYP/SVP level with a CPCM solvent model in acetonitrile, and the calculated ECD spectra of different conformers were simulated with a half bandwidth of −0.4 eV. The ECD curves were extracted by SpecDis 1.62 software. The overall ECD curves of all the compounds were weighed by Boltzmann distribution after UV correction.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2019.104291. References [1] Q. He, The experience of Mongolian medicine for the treatment of functional dyspepsia, Linchuang Yiyao Wenxian Zazhi 21 (4353) (2015) 4356. [2] R. Bu, Wild plants used as folk dietotherapy in Zhelimu Mongolians, Zhongguo Minzu Minjian Yiyao Zazhi 58 (2002) 296–299. [3] J.F. Reynolds, D.M. Smith, E.F. Lambin, B.L. Turner II, M. Mortimore, S.P. Batterbury, T.E. Downing, H. Dowlatabadi, R.J. Fernández, J.E. Herrick, E. Huber-Sannwald, H. Jiang, R. Leemans, T. Lynam, F.T. Maestre, M. Ayarza, B. Walker, Global desertification: building a science for dryland development, Science 316 (2007) 847–851. [4] S. Togashi, S. Obana, S. Watanabe, S. Horaguchi, M. Yashima, K. Inubushi, Collection screening and evaluation of terrestrial cyanobacterial strains for the bioreclamation of arid soils, Soil Microorg. 67 (2013) 3–9. [5] F. Wang, X. Pan, D. Wang, C. Shen, Q. Lu, Combating desertification in China: past, present and future, Land Use Policy 31 (2013) 311–313. [6] Q. Wang, R.J. Abbott, Q.S. Yu, K. Lin, J.Q. Liu, Pleistocene climate change and the origin of two desert plant species, Pugionium cornutum and Pugionium dolabratum (Brassicaceae), in northwest China, New Phytol. 199 (2013) 277–287. [7] G. Luo, Y. Chen, Study on nutrients evaluation and processing technology of Pugionium-a sandgrown wild vegetable, Shipin Kexue 25 (2004) 211–213. [8] H. Li, C. Li, C. Zhang, B. Chen, L. Hui, Y. Shen, Compositional and gastrointestinal prokinetic studies of Pugionium (L.), Food Chem. 186 (2015) 285–291. [9] P. Huang, W. Shi, L. Qu, C. Zheng, T. Wang, Y. Zhang, Isolation and identification of compounds containing nitrogen atoms from Pugionium cornutum, Zhongguo Yaowu Huaxue Zazhi 28 (2018) 318–322. [10] D.M. Wang, W.J. Pu, Y.H. Wang, Y.J. Zhang, S.S. Wang, A new isorhamnetin glycoside and other phenolic compounds from Callianthemum taipaicum, Molecules 17 (2012) 4595–4603. [11] Y.N. Yang, X.Y. Huang, Z.M. Feng, J.S. Jiang, P.C. Zhang, Hepatoprotective activity of twelve novel 7′-hydroxy lignan glucosides from Arctii fructus, J. Agric. Food Chem. 62 (2014) 9095–9102. [12] J. Lee, D. Lee, D.S. Jang, J.W. Nam, J.P. Kim, K.H. Park, M.S. Yang, E.K. Seo, Two new stereoisomers of tetrahydrofuranoid lignans from the flower buds of Magnolia fargesii, Chem. Pharm. Bull. 55 (2007) 137–139. [13] A.A. El Gamal, K. Takeya, H. Itokawa, A.F. Halim, M.M. Amer, H.E.A. Saad, Lignan bis-glucosides from Galium sinaicum, Phytochemistry 45 (1997) 597–600. [14] L. Xiong, C. Zhu, Y. Li, Y. Tian, S. Lin, S. Yuan, J. Hu, Q. Hou, N. Chen, Y. Yang, J. Shi, Lignans and neolignans from Sinocalamus affinis and their absolute configurations, J. Nat. Prod. 74 (2011) 1188–1200. [15] R. Waibel, G. Benirschke, M. Benirschke, H. Achenbach, Sesquineolignans and other constituents from the seeds of Joannesia princeps, Phytochemistry 62 (2003) 805–811. [16] Y. Zhou, Z. Zhou, P. Cao, X. Tan, Y. Ding, Studies the chemical constituents from
3.2. The effects of all fractions and isolated compounds from PC on mouseisolated jejunum contractility 3.2.1. Materials All fractions and compounds 1–31 were prepared by the method described in Section 3.1.3. Atropin, CuSO4, and loperamide were used for mechanism researches. 3.2.2. Animals Healthy male Kunming mice (22–25 g) were purchased from Beijing HFK Bioscience Co. Ltd. (Beijing, China; Certificate of Conformity: No. 11401300071030). All animal experiments were approved by the Science and Technological Committee and the Animal Use and Care Committee of TUTCM (No. 201712003). Mice were housed in a temperature controlled room at 24 ± 2 °C, relative humidity 60%. The mice were fed with standard diet (crude protein 16%, crude fat 4%, crude fiber 12%, and ash 8%) normally a week before the experiment to adapt to the environment. 3.2.3. Effects of all fractions and their compounds on mice jejunum contraction Mice were fasted for 18 h before being sacrificed for experiments, and about 1 cm of the jejunum was collected immediately. The Maxwell bath was filled with 10 mL of Tyrode's solution (one liter contains: NaCl 8.0 g, CaCl2 0.2 g, KCl 0.2 g, MgCl2 0.1 g, NaHCO3 1.0 g, KH2PO4 0.05 g, glucose 1.0 g, pH 7.4) and maintained at a constant temperature (37.0 ± 0.5 °C), and bubbled with 95% O2 and 5% CO2 gas. The jejunum was fixed on bottom hook in and the other end was connected to an isometric tension transducer. Samples in DMSO solution were added after 15 min equilibrate incubation, the final DMSO concentration was 9
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W. Shi, et al. Dichondra repens FORST, Tianran Chanwu Yanjiu Yu Kaifa 15 (13–14) (2003) 17. [17] B. Xu, Y. Jin, Y. Wang, J. Sun, X. Li, Chemical constituents from stems and leaves of Humulus scandens, Zhongcaoyao 45 (2014) 1228–1231. [18] L. Wang, J. Wang, C. Wang, S. Sun, B. Xu, L. Wu, Chemical constituents in the lipid lowering fraction of flos Helichrysum arenarium (III), Zhongguo Yaowu Huaxue Zazhi 22 (220–222) (2012) 226. [19] W. Shi, P. Huang, J. Ruan, S. Yang, T. Wang, Y. Zhang, Study on the constituents of traditional arenicolous Mongolian medicine Pugionium cornutum (L.) Gaerth, Tainjin Zhongyiyao Daxua Xuebao 28 (2019) 74–77. [20] X.C. Li, D. Ferreira, Y. Ding, Determination of absolute configuration of natural products: theoretical calculation of electronic circular dichroism as a tool, Curr. Org. Chem. 14 (2010) 1678–1697. [21] G. Mazzeo, E. Santoro, A. Andolfi, A. Cimmino, P. Troselj, A.G. Petrovic, S. Superchi, A. Evidente, N. Berova, Absolute configurations of fungal and plant metabolites by chiroptical methods. ORD, ECD, and VCD studies on phyllostin, scytolide, and oxysporone, J. Nat. Prod. 76 (2013) 588–599. [22] J. Xu, S. Li, X. Su, J. Ma, F. Liu, L. Tong, D. Lee, Y. Ohizumi, M. Tuerhong, Y. Guo, Diterpenoids from Callicarpa kwangtungensis and their NO inhibitory effects, Fitoterapia 113 (2016) 151–157. [23] Y. Zhang, Y. Guo, X. Li, J. Ruan, T. Wang, J. Li, L. Han, H. Yu, T. Wang, Rearranged
[24] [25] [26] [27] [28] [29]
10
oleanane type saponins, astraisoolesaponins A1-A3 and B, from the stems of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao, Tetrahedron 72 (2016) 7008–7013. C.F. Shaw, J. Eldridge, M.P. Cancro, 13C NMR studies of aurothioglucose: ligand exchange and redox disproportionation reactions, J. Inorg. Biochem. 14 (1981) 267–274. A.N. Ngane, M. Lavault, D. Séraphin, A. Landreau, P. Richomme, Three 1-thio-betaD-glucopyranosides from the seeds of Afrostyrax lepidophyllus Mildbr, Carbohydr. Res. 341 (2006) 2799–2802. X.X. Huang, S. Liu, L.L. Lou, Q.B. Liu, C.C. Zhou, L.Z. Li, Y. Peng, S.J. Song, Phenylpropanoids from Crataegus pinnatifida and their chemotaxonomic importance, Biochem. Syst. Ecol. 54 (2014) 208–212. Y. Zhang, X. Li, J. Ruan, Y. Dong, J. Hao, E. Liu, L. Han, X. Gao, T. Wang, Oleanane type saponins from the stem of Astragalus membranaceus (Fich.) Bge. var. mongholicus (Bge.) Hisao, Fitoterapia 109 (2016) 99–105. I.Y. Kuo, B.E. Ehrlich, Signaling in muscle contraction, Cold Spring Harb. Perspect. Biol. 7 (2015) a006023. M.Y. Yu, X.J. Qin, X.R. Peng, X. Wang, X.X. Tian, Z.R. Li, M.H. Qiu, Macathiohydantoins B-K, novel thiohydantoin derivatives from Lepidium meyenii, Tetrahedron 73 (2017) 4392–4397.
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Bioactive constituents study of Pugionium cornutum L. Gaertn on intestinal motility
T
Wenzhong Shia, Jingya Ruanb, Yuanqiang Guoc, Zhijuan Dingb, Jiejing Yana, Lu Qua, ⁎ ⁎ Chang Zhengb, Yi Zhanga,b, , Tao Wanga,b, a
Tianjin State Key Laboratory of Modern Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China Tianjin Key Laboratory of TCM Chemistry and Analysis, Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China c Tianjin Key Laboratory of Molecular Drug Research, College of Chemistry, Nankai University, Tianjin 300071, China b
ARTICLE INFO
ABSTRACT
Keywords: Traditional Mongolian Medicine Pugionium cornutum L. Gaertn Thiohydantoin derivatives Intestinal motility Mice isolated intestine tissue 5-(methylsulfinyl)-pentanenitrile
Thirty-one compounds were obtained and identified from the dried roots of Mongolian medicinal and edible plant, Pugionium cornutum L. Gaertn. Eight of them were new ones, and named as pugcornols A (1), B (2), C (3), and D (4), pugcornosides A (5), B (6), C (7), and D (8), respectively. Among them, 1–4 were rare naturally occurring thiohydantoin derivatives. Meanwhile, all the isolates were determined for their activities on isolated mice jejunum contraction, and ten of them increased height of the spontaneous contractions. Moreover, we partly clarified the mechanism of 5-(methylsulfinyl)-pentanenitrile (9), a characteristic compound in P. cornutum by using different kinds of inhibitor.
1. Introduction Mongolian medicine thinks that the body's normal physiological movement mainly depends on the substance decomposition in the stomach and liver, which suggests that protection of stomach or digestive function and treatment of liver diseases of the digestive system is the premise of prevention and treatment of various diseases [1]. Digestive system disease is one of the supermacy diseases in the Mongolian medicine, and it has accumulated rich clinical experience. It absorbs the theory essence of Tibetan medicine, Chinese medicine, and ancient Indian medicine, and developes into traditional ethnomedicine with unique theoretical system and clinical characteristics. As the first nutrition diet study monographs in China, “Yin Shan Zhengyao” integrats with the nutrition knowledge essence of Mongolian and Han nationalities, which refines the diet knowledge before Yuan dynasty, and emphasizes the importance of prevention and health care [2]. Meanwhile, how to govern sand has become a hot topic attention around the world along with the land desertification rising [3–5]. Psammophyte occupies very important position in the governance of desertification. As a kind of medicinal and edible plant, Pugionium cornutum L. Gaertn (PC) belongs to Cruciferae family, is widely distributed in the Badain Jaran Desert, Kubuqi Desert, Mu Us Desert, Horqin sandy land,
⁎
and Hulunbuir sandy land [6]. As the traditional food for Mongolian people for thousands of years, PC is consumed by the local people mostly after pickling, which owns unique flavor and high nutrition, and enjoys the reputation of desert ginseng [7]. As the traditional arenicolous Mongolian medicine, PC have accumulated rich experience and knowledge of promoting gastrointestinal motility and improving indigestion [8]. Moreover, as a well-known pioneer sandfixing plant, PC is very tolerant of drought and has a strong ability of resisting and fixing sand because of its well-developed root system and strong water absorption capacity. Based on the above-mentioned advantages, PC has drawn more and more attetion of the researchers around the world, and systemic research for it is more and more necessary. In this paper, we report the isolation and identification of constituents, along with their bioactivity evaluations on motility of mice isolated intestine tissue. 2. Results and discussion The 70% EtOH extract from the roots of PC was partitioned with H2O and EtOAc, the H2O layer partition was subjected to D101 macroporous resin CC and eluted with H2O, 95% EtOH, and acetone, successively. Then 95% EtOH eluate and EtOAc layer partition was isolated by silica gel, Sephadex LH-20 CC and preparative HPLC (pHPLC), respectively, to
Corresponding authors at: Tianjin State Key Laboratory of Modern Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China. E-mail addresses: [email protected] (Y. Zhang), [email protected] (T. Wang).
https://doi.org/10.1016/j.fitote.2019.104291 Received 14 July 2019; Received in revised form 3 August 2019; Accepted 5 August 2019 Available online 08 August 2019 0367-326X/ © 2019 Published by Elsevier B.V.
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O
O S
OH
N
O N
N
S
O
O OH
O OH
OH 5
threo S
N
O
CH3OOC O OH HO
HO
HO
HO
OH
O OH
6
O
OH HO
HO OH
H
N
O OH
HO O OH
O
4
CH3OOC
OH
O
HO
HO OH
O
HO
HO
O S
3
HO
S
O
N
S
erythro OH
OCH3
N
2
HO
HO
O
N
S
1
HO
O S
OH
OH
O 7
O
O 8
Fig. 1. The new compounds 1–8 obtained from the roots of P. cornutum.
obtain eight new compounds, pugcornols A (1), B (2), C (3), D (4), pugcornosides A (5), B (6), C (7), D (8) (Fig. 1), along with twenty-three known isolates, 5-(methylsulfinyl)-pentanenitrile (5-MP) (9) [9], phenylethyl glucosinolate (10) [9], 2-phenylethyl-desulphoglucosinolate (11) [9], N-methoxyl-3-indolymethyldesulphoglucosinolate (12) [9], arvelexin (13) [9], cappariloside A (14) [9], L-tryptophan (15) [9], Lphenylalanine (16) [9], 3-phenylpropanamide (17) [9], adenosine (18) [9], (+)-1-hydroxyl pinoresinol-4-O-β-D-glucopyranoside (19) [10], (7R,8S,8′S)-lariciresinol-4,4′-bis-O-β-D-glucopyranoside (20) [11–13], (7R,8S,7′R,8′S)-3,3′-bisdemethylpinoresinol (21) [14,15], demethyl coniferin (22) [16], 2-phenylethyl-O-β-D-glucopyranoside (23) [17], phenethanol-β-D-gentiobioside (24) [18], β-sitosterol (25) [19], daucosterol (26) [19], staphylionoside D (27) [19], TgSSTg (28) [19], ethyl-α-Dfructofuranoside (29) [19], pinellic acid (30) [19], and Z-octadecyl caffeate (31) [19] (Fig. 2). Pugcornol A (1) was obtained as a colorless oil with negative optical rotation ([α]D25–31.3, MeOH). Its molecular formula was assigned as C10H14N2O2S on the basis of negative-ion HRESI-TOF-MS analysis (m/z 225.0708 [M – H]−; calcd for C10H13N2O2S, 225.0703), which suggested the unsaturation degree of it was five. The 1H, 13C NMR (Table 1) and DEPT spectroscopic data indicated that 1 had six methylenes, one methine, one oxygenated quaternary carbon [δC 92.1 (C5)] and two carbonyl groups [δC 171.9 (C-4), 185.0 (C-2)]. The correlations between δH 2.13, 2.28 (1H each, both m, H2-7) and δH [1.71 (1H, m), 1.93 (1H, ddd, J = 1.5, 7.0, 12.5 Hz), H2-6], [3.53 (1H, ddd, J = 3.5, 9.5, 12.0 Hz), 3.78 (1H, dt, J = 8.5, 12.0 Hz), H2-8] found in the 1H 1H COSY spectrum suggested the presence of “eCH2eCH2eCH2e” moiety. Meanwhile, the existence of n-butene group was clarified by the correlations between δH 2.35 (2H, m, H2-2′) and δH 3.71 (2H, t, J = 7.5 Hz, H2-1′), 5.76 (1H, ddt, J = 6.5, 13.5, 17.0 Hz, H-3′); δH 5.76 (H-3′) and δH [5.01 (1H, m), 5.05 (1H, ddd, J = 1.5, 3.0, 17.0 Hz), H2-4′]. The above mentioned NMR data accounted for three degrees of unsaturation, and the remaining two ones indicated the presence of a dicyclic ring system. Moreover, the longrange correlations from δH 1.71, 1.93 (H2-6), 2.13, 2.28 (H2-7), and 3.53, 3.78 (H2-8) to δC 92.1 (C-5) observed in its HMBC experiment established the partial structure of a five membered moiety (ring B) via C-5–N-1–C-8. Furthermore, the observed long-range correlations from δH 1.71, 1.93 (H2-6) to δC 171.9 (C-4); δH 3.53, 3.78 (H2-8) to δC 185.0 (C-2); δH 3.71 (H2-1′) to δC 171.9 (C-4), 185.0 (C-2) connected ring B and the n-butene group with ring A (thiohydantoin group) via C-2–N1–C-5–C-4 and C-1′–N-3 (Fig. 3), respectively. Finally, its absolute configuration, 5R, was further clarified by the time-dependent density functional theory (TDDFT) ECD calculation [20–23], which suggested the obtained ECD spectrum of 1 matched the experimental results closely (Fig. 4). (See Figs. 5 and 6.)
Pugcornol B (2), a colorless oil with negative optical rotation ([α]D25–39.3, MeOH). The molecular formula, C11H18N2O3S2, of 2 was established by negative-ion HRESI-TOF-MS (m/z 289.0693 [M – H]−; calcd for C11H17N2O3S2, 289.0686). The 1H, 13C NMR (Table 2) and DEPT spectra revealed the existence of one methyl, seven methylenes, along with three quaternary carbons. Moreover, the presence of “eCH2eCH2eCH2e” moiety was consolidated by the proton and proton correlations between δH 2.20, 2.40 (1H each, both m, H2-7) and δH [1.77 (1H, m), 2.05 (1H, br. dd, ca. J = 6, 13 Hz), H2-6], [3.61 (1H, ddd, J = 3.0, 9.5, 12.0 Hz), 3.93 (1H, dt, J = 7.5, 12.0 Hz), H2-8] observed in its 1H 1H COSY experiment. The long-range correlations from δH 1.77, 2.05 (H2-6) to δC 93.7 (C-5), 174.2 (C-4); δH 2.20, 2.40 (H2-7) to δC 93.7 (C-5); δH 3.61, 3.93 (H2-8) to δC 93.7 (C-5), 187.1 (C-2) displayed in its HMBC spectrum suggested rings A and B construction of 2 was the same as that of 1. The elemental composition of abovementioned moiety was C6H7N2O2S. According to the MS determination result (molecular formula, C11H18N2O3S2), we could deduce that there was a side chain with elemental composition C5H11OS in compound 2. The proton and proton correlations between δH 1.86 (2H, m, H2-2′) and δH 1.75, 1.83 (1H each, both m, H2-3′), 3.84 (2H, m, H2-1′); δH 1.75, 1.83 (H2-3′) and δH [2.80 (1H, ddd, J = 5.5, 8.0, 13.0 Hz), 2.89 (1H, dt, suggested the existence of J = 7.5, 13.0 Hz), H2-4′] “eCH2eCH2eCH2eCH2e” moiety, and the signal at δ 2.63 (H3-6′) indicated the presence of methyl group. Except for them, there only one sulfur atom and one oxygen atom were not assigned. According to the unsaturation degree (five) of it, the existence of sulfoxide group was elucidated. The observed long-range correlations from δH 3.84 (H2-1′) to δC 174.2 (C-4), 187.1 (C-2); δH 2.80, 2.89 (H2-4′) to δC 38.1 (C-6′); δH 2.63 (H3-6′) to δC 54.2 (C-4′) made the methyl, sulfoxide, “eCH2eCH2eCH2eCH2e” moiety, and ring A constructed together. Then, the planar structure of 2 was elucidated. The absolute configuration of 2 was identified as 5R, which was clarified by comparing the experimental spectrum with the computational ECD spectrum (Fig. 4) [20–23]. Both C-1′ and C-4′ dispalyed a pair of carbon signals (Table 2) with height ratio 1:1, which suggested pugcornol B (2) was the mixture of 5′R and 5′S isomer. The existence of 5′R and 5′S isomer for 2 maybe originated from sulfoxide group. Pugcornol C (3) was determined to possess the molecular formula, C12H20N2O3S2, by its quasi-molecular ion peak at m/z 327.0814 [M + Na]+ (calcd for C12H20N2O3S2Na, 327.0808) in the positive HRESI-TOF-MS experiment, which was 14 amu greater than that of 2. Moreover, the 1H and 13C NMR signals of 3 were coincident with those of 2 except for the methoxy signal [δH 3.19 (3H, s, 5-OCH3); δC 52.2 (5OCH3)]. Meanwhile, the chemical shift of C-5 of 3 [δC 98.7 (C-5)] shifted to lower field comparing with that of 2 [δC 93.7 (C-5)], which suggested C-5 was substituted by methoxyl, and it was verified by the 2
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OR
S N
O O N S O O S
S O
N
OH
11
OH
CH3O
N
N
16 HO
O
OH O
OR2 H
R1O
O
2
19: Glc 20: Glc
HO OH 18
H
R
R1
NH2 17
15
OCH3
N
N
O
12
NH2
N H
OH
OH
NH2
NH2
COOH
HO
HO OH
10
N H
O OH
O OH
HO
N R 13: CH3 14: Glc
N
HO
HO O OH
COOH
N HO OCH3
S
HO
9
OH
O 21
H Glc
HO OH
O
-D-glucopyranosyl
Glc:
H O
HO
OH O
O
HO
HO
O OH OH HO
S O OH
OH S O HO
HO
OH COOH
OH 30
O
OH
OH OH
OH
O O HO
HO
HO
OH
O
OH
28
27 OH
24
23
HO
OH
OH OH
22
O OH
R 25: H 26: Glc
RO
HO
HO
HO
HO
O OH
O OH
O OH
OH
HO
O
O
HO
OH
29
31
Fig. 2. The known compounds 9–31 obtained from the roots of P. cornutum. Table 1 1 H and 13C NMR data for 1 in DMSO-d6. No. 2 4 5 6
δC 185.0 171.9 92.1 32.1
7
24.6
8
47.3
δH (J in Hz) – – – 1.71 (m) 1.93 (ddd, 1.5, 7.0, 12.5) 2.13 (m) 2.28 (m) 3.53 (ddd, 3.5, 9.5, 12.0)
No.
δC
1′ 2′ 3′
40.0 31.4 134.6
4′
117.0
5-OH
O
1' 3
2' 4'
δH (J in Hz)
N
3'
2
S
3.78 (dt, 8.5, 12.0) 3.71 (t, 7.5) 2.35 (m) 5.76 (ddt, 6.5, 13.5, 17.0) 5.01 (m)
OH
4 1
6
5
7
N
8
6'
O S 5'
3'
3
2'
5.05 (ddd, 1.5, 3.0, 17.0) 7.15 (br. s)
N
S 2 O
O S
OCH3
N S 3
OH
N
1 O S
O
1' 4'
N
O N N
O 4 1H 1H
COSY:
HMBC:
Fig. 3. The main 1H 1H COSY and HMBC correlations of 1–4.
3
Fitoterapia 138 (2019) 104291
W. Shi, et al.
Fig. 4. Calculated and experimental ECD spectra of 1–4.
OCH3 HO
1 7
9
3 4
HO O OH
O OH
HO
HO
5
3
OH
OH COSY:
O OH
1'
1H 1H
HMBC:
2
COSY:
1'
HO
HO OH 1 O 1''
7''
O
3
8''
HO OH 1 O 2
O
7
6 1H 1H
CH3OOC 6'
HO
HO
HO OH
OH
O OH
S
O OH
1''
HO
CH3OOC OH
O HO
O OH
1'
HO
OH
S
O HO
OCH3 HO
HMBC:
8
Fig. 6. The main 1H 1H COSY and HMBC correlations of 7 and 8.
Fig. 5. The main 1H 1H COSY and HMBC correlations of 5 and 6.
Comparing with 2, the signal of C-5 shifted to higher field significantly [δC 93.7 (C-5) for 2, δC 64.7 (C-5) for 4], which denoted that C-5 was one methine group. Meanwhile, the signal of C-2 also shifted to higher field [δC 187.1 (C-2) for 2, δC 162.5 (C-2) for 4], combining with the determination data of MS experiment, C-2 was speculated to be one carbonyl. Its planar structure was determined based on the key HMBC correlations from the following proton and carbon pairs: δH 4.18 (H-5) to δC 162.5 (C-2), 176.0 (C-4); δH 3.24, 3.60 (H2-8) to δC 162.5 (C-2); δH 3.51 (H2-1′) to δC 162.5 (C-2), 176.0 (C-4); δH 2.62 (H3-5′) to δC 54.0 (C-4′). Using the similar method [20–23], the absolute configuration of 5R was deduced for pugcornol D (4). Pugcornoside A (5) was obtained as a white powder with negative optical rotation ([α]D25–23.2, MeOH). Negative-ion HRESI-TOF-MS
long-range correlation from δH 3.19 (5-OCH3) to δC 98.7 (C-5). Furthermore, its ECD sepctrum (Fig. 4) was identical to that of 2, and 5R configuration was elucidated [20–23]. Finally, pugcornol C (3) was deduced to be a mixture of 5′R and 5′S isomer because C-3′, 4′ and 6′ showed a pair of carbon signals (Table 2) with height ratio 1:1. The molecular formula of pugcornol D (4) was deduced to be C11H18N2O3S (m/z 259.1113 [M + H]+; calcd for C11H19N2O3S, 259.1111) by the positive-ion HRESI-TOF-MS determination. The 1H, 13 C NMR (Table 3) spectra analysis suggested the presence of one methyl, seven methylenes, one methine, together with two quaternary carbons in 4. The 1H 1H COSY spectrum of 4 suggested the existence of two partial structures written in bold lines as shown in Fig. 3. 4
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Table 2 1 H and 13C NMR data for 2 and 3 in CD3OD. No.
2
3
δC
δH (J in Hz)
δC
δH (J in Hz)
2 4 5 6
187.1 174.2 93.7 33.4
188.1 172.0 98.7 33.2
7 8
25.9 48.9
1′ 2′ 3′ 4′
41.66/41.67 27.6 20.7 54.17/54.20
6′ 5-OCH3
38.1 –
– – – 1.77 (m) 2.05 (br. d, ca. 6, 13) 2.20, 2.40 (both m) 3.61 (ddd, 3.0, 9.5, 12.0) 3.93 (dt, 7.5, 12.0) 3.84 (m) 1.86 (m) 1.75, 1.83 (both m) 2.80 (ddd, 5.5, 8.0, 13.0) 2.89 (dt, 7.5, 13.0) 2.63 (s) –
– – – 1.82 (m) 2.08 (ddd, 2.0, 7.0, 12.0) 2.18, 2.31 (both m) 3.56 (ddd, 3.5, 9.5, 12.0) 3.99 (dt, 8.0, 11.5) 3.88 (m) 1.85 (m) 1.78 (m) 2.80 (ddd, 6.0, 8.5, 13.5) 2.89 (ddd, 7.0, 8.0, 13.5) 2.62 (s) 3.19 (s)
δC
δH (J in Hz)
2 4 5 6
162.5 176.0 64.7 28.2
7 8
28.2 46.4
– – 4.18 1.67 2.21 2.09 3.24
(dd, 7.5, 8.5) (m) (m) (m) (ddd, 4.5, 8.0, 12.5)
No.
δC
1′ 2′ 3′ 4′
39.0 28.0 20.7 54.0
5′
38.1
δH (J in Hz) 3.60 3.51 1.76 1.76 2.79 2.88 2.62
(dt, 7.5, 12.5) (t, 6.0) (m, overlapped) (m, overlapped) (m) (m) (s)
Table 4 1 H and 13C NMR data for 5 and 6 in CD3OD. No.
5
6
δC
δH (J in Hz)
δC
δH (J in Hz)
1 2 3 4 5 6 7 8 9
135.4 116.3 148.3 146.5 118.4 120.3 84.8 53.1 63.1
136.1 116.7 148.3 146.6 118.6 120.8 85.3 53.1 63.3
1′ 2′ 3′ 4′ 5′ 6′
104.2 74.9 77.6 71.3 78.3 62.4
1″ 2″ 3″ 4″ 5″ 6″
86.5 74.6 79.4 71.3 82.0 62.8
7-OCH3
57.5
– 6.90 – – 7.17 6.82 4.49 3.31 3.65 3.74 4.78 3.50 3.48 3.42 3.42 3.72 3.90 4.40 3.19 3.33 3.32 3.24 3.65 3.85 3.25
– 6.90 – – 7.17 6.83 4.38 3.31 3.65 3.74 4.80 3.49 3.48 3.42 3.43 3.72 3.90 3.83 3.08 3.18 3.25 3.10 3.63 3.85 3.22
(d, 1.5) (d, 8.0) (dd, 1.5 8.0) (d, 5.5) (m) (m, overlapped) (dd, 7.0, 12.0) (d, 7.5) (dd, 7.5, 9.0) (dd, 9.0, 9.0) (m, overlapped) (m, overlapped) (dd, 5.0, 12.0) (dd, 2.0,12.0) (d, 9.5) (dd, 9.0, 9.5) (m, overlapped) (m, overlapped) (m) (m, overlapped) (dd, 3.0, 12.0) (s)
104.3 74.9 77.7 71.4 78.3 62.5 86.1 74.9 79.5 71.4 81.9 62.8 57.3
41.8 27.7 20.89/20.92 54.11/54.14 38.14/38.16 52.2
moiety was deduced by the correlations between δH 3.31 (1H, m, H-8) and δH [3.65 (1H, m, overlapped), 3.74 (1H, dd, J = 7.0, 12.0 Hz), H29], as well as the chemical shift of them. Its 13C NMR spectrum displayed twenty-two carbon signals. In addition to those assigned to above-mentioned groups, the other twelve signals was in the scope of 60–105. Among them, δH 4.78 (1H, d, J = 7.5 Hz, H-1′) combing with δC 62.4 (C-6′), 71.3 (C-4′), 74.9 (C-2′), 77.6 (C-3′), 78.3 (C-5′), 104.2 (C1′) suggested the presence of one 1-O-β-D-glucopyranosyl. Meanwhile, the existence of 1-thiol-β-D-glucopyranosyl was supported by δH 4.40 (1H, d, J = 9.5 Hz, H-1″) and δC 62.8 (C-6″), 71.3 (C-4″), 74.6 (C-2″), 79.4 (C-3″), 82.0 (C-5″), 86.5 (C-1″) [24,25]. Furthermore, the longrange correlations from δH 4.49 (H-7) to δC 57.5 (7-OCH3), 116.3 (C-2), 120.3 (C-6), 135.4 (C-1); δH 3.31 (H-8) to δC 135.4 (C-1); δH 4.78 (H-1′) to δC 146.5 (C-4); δH 4.40 (H-1″) to δC 53.1 (C-8); δH 3.25 (7-OCH3) to δC 84.8 (C-7) found in its HMBC experiment linked the above-mentiond moieties together. Finally, the relative configuration between C-7 and C-8 was determined as erythro according to the coupling constant of H-7 and H-8 (J = 5.5 Hz) [26]. Pugcornoside B (6) was also obtained as a white powder with negative optical rotation ([α]D25–52.6, MeOH). Its negative-ion HRESITOF-MS spectrum dispalyed the same molecular formula, C22H34O14S (m/z 553.1599 [M – H]−; calcd for C22H33O14S, 553.1597) as that of 5. The 1H, 13C NMR (Table 4) and various 2D NMR (1H 1H COSY, HSQC, HMBC) spectra suggested its planar structure was identical to that of 5. Moreover, the coupling constant of H-7 and H-8 (J = 7.5 Hz) suggested the relative configuration between C-7 and C-8 was threo [26]. Pugcornoside C (7) was obtained as a white powder with positive optical rotation ([α]D25 + 6.3, MeOH). Its molecular formula, C10H18O9 was deduced from the negative-ion HRESI-TOF-MS measurement (m/z 281.0878 [M–H]−; calcd for C10H17O9, 281.0878). Acid hydrolysis of 7 with 5% aqueous H2SO4-1,4-dioxane (1:1, v/v) afforded D-glucuronic acid, whose absolute configurations were determined by GC–MS analysis of their trimethysilyl thiazolidine derivatives [27]. The 1H, 13C NMR (Table 5) and 2D NMR (1H 1H COSY, HSQC, and HMBC) spectra suggested the presence of one methoxyl [δH 3.76 (3H, s, 6′-OCH3)], one α-D-glucuronic acid functional group [δ 4.85 (1H, d, J = 4.0 Hz, H-1′)], together with one glycerol moiety {δ [3.41 (1H, dd, J = 8.0, 11.0 Hz), 3.82 (1H, dd, J = 3.5, 11.0 Hz), H2-1], 3.57 (2H, t, J = 5.0 Hz, H2-3), 3.84 (1H, m, H-2)}. Finally, according to the long-rang correlations from δH 4.85 (H-1′) to δC 71.4 (C-3); δH 3.76 (6′-OCH3) to δC 172.0 (C6′), the above-mentioned fragments were linked with each other. Pugcornoside D (8) was also isolated as a white powder with positive optical rotation ([α]D25 + 28.1, MeOH). The molecular formula, C18H24O10, of 8 was determined from negative-ion HRESI-TOF-MS analysis (m/z 399.1295 [M–H]−; calcd for C18H23O10, 399.1297). The 1 H, 13C NMR (Table 5) and various 2D NMR (1H 1H COSY, HSQC,
Table 3 1 H and 13C NMR data for 4 in CD3OD. No.
25.8 49.3
(d, 2.0) (d, 8.5) (dd, 2.0, 8.5) (d, 7.5) (m) (dd, 5.5, 12.0) (dd, 4.5, 11.5) (d, 7.5) (dd, 7.5, 9.0) (dd, 9.0, 9.0) (m, overlapped) (m, overlapped) (dd, 3.0, 12.0) (dd, 2.0,12.0) (d, 9.5) (dd, 8.5, 9.5) (dd, 8.5, 9.0) (dd, 9.0, 9.5) (m) (dd, 5.5, 12.0) (dd, 3.0, 12.0) (s)
afforded [M – H]− peak at m/z 553.1605 (calcd for C22H33O14S, 553.1597), supporting molecular formula of C22H34O14S for 5. The 1H and 13C NMR (Table 4) spectra showed signals assignable to one ABXtype aromatic protons [δ 6.82 (1H, dd, J = 1.5, 8.0 Hz, H-6), 6.90 (1H, d, J = 1.5 Hz, H-2), 7.17 (1H, d, J = 8.0 Hz, H-5)] and one methoxy group [δ 3.25 (3H, s, 7-OCH3)]. The existence of “eCHeCHeCH2OH” 5
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and results in contraction of smooth muscle [28]. 5-MP is a characteristic compound in PC, in normal isolated jejunum tissue, it showed significant increasing effect on height of contractility at 50 μM. Further, atropine (the blocker of M-receptor), CuSO4 (the inhibitor of 5-HT receptor), and loperamide (the inhibitor of μ-opioid receptor) were used to clarify the mechanism on intestinal smooth muscle spontaneous contraction. Comparing the difference between groups treated or untreated with inhibitor, we partly clarified that the increasing effect of 5MP, at least, partly releated to M receptor and μ-opioid receptor, but not releated to 5-HT receptor. In summary, gastrointestinal prokinetic efficacy of the water extract from fresh and pickled PC had been reported by Li et al.. And they analyzed the contents of amino acids, vitamins, potassium, calcium, fat, and sugar in PC [8], too. But the secondary metabolites in PC, as well as their bioactivities have been not reported. In the process of investigating the active constituents on intestinal motility, we obtained four new rare thiohydantoin derivatives (1–4), two new glucosinolates (5, 6), two new others (7, 8), along with twenty-three known isolates (9–31) from the dried roots of Mongolian medicinal and edible plant. For thiohydantoin derivatives 1–4, only one paper reported them obtained from natural products [29] until now. These compounds are presumably derived by condensation of an isocyanate or isothiocyanate with the secondary amine group of proline, followed by acylation of the nascent amino group with the carboxylic group of proline. On the other hand, the main constituents in PC were mainly nitrogen- and/or sulfurcontaining heteroatom compounds as shown in Figs. 1 and 2. Our research suggested that nitrogen-containing heteroatom constituents were the active constituents on intestinal motility. The results will provide a scientific basis for guiding reasonable application of PC in the clinical Mongolian medicine. And which will be benefit for promoting the development of sand industry and constructing the green economy in China.
Table 5 1 H and 13CNMR data for 7 and 8 in CD3OD. No.
7
8
δC
δH (J in Hz)
δC
δH (J in Hz)
1
71.4 72.2 64.0
(dd, 8.0, 11.0) (dd, 3.5, 11.0) (m) (t, 5.0)
70.6
2 3
3.41 3.82 3.84 3.57
1′ 2′ 3′ 4′ 5′ 6′ 6′-OCH3 1″ 2″,6″ 3″,5″ 4″ 7″ 8″
101.2 73.3 74.5 73.3 73.0 172.0 52.8
4.85 3.45 3.66 3.53 4.12 – 3.76
(d, 4.0) (dd, 4.0, 10.0) (dd, 9.0, 10.0) (dd, 9.0, 9.5) (d, 9.5)
3.40 3.76 4.02 4.14 4.20 4.81 3.46 3.64 3.53 4.10 – 3.76 – 7.28 7.30 7.24 3.69 –
(s)
69.4 66.5 101.2 73.2 74.4 73.3 73.1 171.9 52.8 135.6 130.4 129.5 128.1 41.8 173.4
(dd, 6.5, 11.0) (m, overlapped) (m) (dd, 6.0, 11.5) (dd, 5.0, 11.5) (d, 3.5) (dd, 3.5, 9.5) (dd, 9.0, 9.5) (dd, 9.5, 9.5) (d, 10.0) (s) (m) (m) (m) (s)
HMBC) spectra suggested 8 had the same moiety, glycerin-1-O-D-glucuronic acid methyl ester, as that of 7. In addition, there was one benzene acetyl [δH 3.69 (2H, s, H2-7″), 7.24 (1H, m, H-4″), 7.28 (2H, m, H-2″,6″), 7.30 (2H, m, H-3″,5″); δC 173.4 (C-8″)] appeared in 8. And in the HMBC experiment, long-range correlations from δH 3.69 (H2–7″) to δC 130.4 (C-2″,6″), 135.6 (C-1″), 173.4 (C-8″); δH 4.14, 4.20 (H2-3) to δC 173.4 (C-8″); δH 3.76 (6′-OCH3) to δC 171.9 (C-6′) were observed. Consequently, the structure of pugcornoside D (8) was elucidated. As the traditional arenicolous Mongolian medicine, PC has accumulated rich experience and knowledge of promoting gastrointestinal motility and improving indigestion [8]. To clarify the material foundation of medicinal effectiveness of PC for gastrointestinal motility, the effects of all the fractions and isolated compounds from it were investigated on mice jejunum contractility. As results, 70% ethanol-water extract of PC (a), and 95% eluate of PC (e) fractions (200 μg/mL) (Fig. 7), as well as compounds 1, 2, 3, 8, 9, 12, 14, 17, 18, 24 (50 μM) significantly increased the height of isolated mice jejunum spontaneous contractions. However, all of them showed no significant changing on contraction frequency and tension at the same concentration. (See Figs. 8 and 9.) Impairment of gastrointestinal motility is one of the major reason in bowel disease, such as constipation and intestinal obstruction. M receptor, 5-HT receptor and opioid receptor play functional roles in intestinal smooth muscle contraction. Activation of these receptors lead increase in inositol 1,4,5-triphosphate and stimulates Ca2+ release from the endoplasmic reticulum, increases intracellular Ca2+ concentration,
3. Materials and methods 3.1. Phytochemistry study of PC 3.1.1. General UV and IR spectra were determined on a Varian Cary 50 UV–Vis and Varian 640-IR FT-IR spectrophotometer, respectively. Optical rotations were run on a Rudolph Autopol® IV automatic polarimeter. NMR spectra were obtained on a Bruker 500 MHz NMR spectrometer at 500 MHz for 1H and 125 MHz for 13C NMR (internal standard: TMS). Positive- and negative-ion HRESI-TOF-MS were recorded on an Agilent Technologies 6520 Accurate-Mass Q-Tof LC/MS spectrometer. CC were performed on macroporous resin D101 (Haiguang Chemical Co., Ltd., Tianjin, China), Silica gel (74–149 μm, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and Sephadex LH-20 (Ge
Fig. 7. Effects of fractions of P. cornutum on isolated jejunum contraction. N: normal group; a: 70% ethanol-water extract of P. cornutum; b: EtOAc layer partition of a; c: H2O layer partition of a; d: H2O eluate of c from D101 macroporous resin CC; e: 95% eluate of c from D101 macroporous resin CC; the final concentration of each fraction: 10 mg/mL; n = 6, ⁎P < .05, ⁎⁎P < .01, ⁎⁎⁎P < .001 vs. normal group. Data (mean ± SEM) were analyzed by ANOVA. 6
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Fig. 8. Effects of isolated compounds 1–31 from P. cornutum on isolated jejunum contraction. N: normal group; the final concentration of each compound: 50 μM; n = 6, ⁎P < .05, ⁎⁎P < .01, ⁎⁎⁎P < .001 vs. normal group. Data (mean ± SEM) were analyzed by ANOVA.
Healthcare Bio-Sciences, Uppsala, Sweden). pHPLC column, Cosmosil 5C18-MS-II (20 mm i.d. × 250 mm, 5 μm, Nakalai Tesque, Inc., Tokyo, Japan) were used to isolate compounds.
The H2O layer partition (1035.0 g) was dissolved in H2O, and subjected to D101 macroporous resin CC (H2O → 95% EtOH → Acetone) to afford H2O (980.0 g), 95% EtOH (44.1 g), and acetone (0.8 g) eluate, respectively. The 95% EtOH eluate (30.0 g, Fr. H) was isolated by ODS CC [MeOH-H2O (10:90 → 15:85 → 30:70 → 40:60 → 50:50 → 70:30 → 100:0, v/v)], and eight fractions (Fr. H1-Fr. H8) were yielded. Fraction H4 (9.0 g) was isolated by Sephadex LH-20 CC [MeOH-H2O (50:50, v/ v)] to obtain eight fractions (Fr. H4-1-Fr. H4-8). Fraction H4-2 (2.0 g) was separated by pHPLC [CH3CN-H2O (6:94 → 13:87 → 15:85, v/v)] to gain fifteen fractions (Fr. H4-2-1-Fr. H4-2-15). Fraction H4-2-3 (240.7 mg) was purified by pHPLC [MeOH-H2O (9:91, v/v)] to yield pugcornosides A (5, 11.2 mg) and B (6, 15.4 mg). Fraction H4-2-8 (149.9 mg) was prepared by pHPLC [MeOH-H2O (15:85, v/v)] to produce pugcornol D (4, 12.8 mg). Fraction H4-2-9 (148.2 mg) was isolated by pHPLC [CH3CN-H2O (9:91, v/v)], and (7R,8S,8′S)-lariciresinol4,4′-bis-O-β-D-glucopyranoside (20, 18.4 mg), and phenethanol-β-Dgentiobioside (24, 9.7 mg), together with staphylionoside D (27,
3.1.2. Plant material The roots of Pugionium cornutum L. Gaertn were collected from Alxa Youqi, Inner Mongolia Autonomous region, China, and identified by Dr. Li Tianxiang (Experiment Teaching Department, Tianjin University of Traditional Chinese Medicine). The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM. 3.1.3. Extraction and isolation The dried roots of PC (4.8 kg) were refluxed with 70% ethanolwater for three times. Evaporation of the solvent under pressure provided a 70% ethanol-water extract (1740.0 g), which was partitioned with H2O and EtOAc to gain H2O layer (1150.0 g) and EtOAc layer (153.0 g) partition, respectively. 7
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1-Fr. H6-13) were given. Fraction 6-6 (292.7 mg) was purified by pHPLC [CH3CN-H2O (15:85, v/v)] to yield 2-phenylethyl-O-β-D-glucopyranoside (23, 24.9 mg). Fraction H6–10 (271.0 mg) was isolated by pHPLC [MeOH-H2O (55:45, v/v)], and pugcornol C (3, 12.4 mg) was obtained. The EtOAc layer partition (100.0 g, Fr. E) was subjected to silica gel CC [CHCl3 → CHCl3-MeOH (100:3 → 100:5, v/v) → CHCl3-MeOH-H2O (10:3:1 → 7:3:1, lower layer, v/v/v) → MeOH]. As results, twelve fractions (Fr. E1-Fr. E12) were given. Fraction E8 (7.0 g) was subjected to ODS CC [MeOH-H2O (30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 90:10 → 100:0, v/v)] to give eleven fractions (Fr. E8-1-Fr. E811). Fraction E8-3 (175.0 mg) was separated by pHPLC [CH3CN-H2O (16:84, v/v)] to obtain (7R,8S,7′R,8′S)-3,3′-bisdemethylpinoresinol (21, 12.0 mg). Meanwhile, compounds 9–18, 25–31 were obtained by using the method discribed in references [9, 19]. 3.1.3.1. Pugcornol A (1). Colorless oil; [α]D25–31.3 (conc. 0.12, MeOH); CD (conc. 0.0015 M, MeOH) mdeg (λnm): −1.39 (235), −3.33 (256), −3.60 (269); CD (conc. 0.0015 M, CH3CN) mdeg (λnm): −1.21 (229), −2.83 (245), −4.52 (267); UV λmax (MeOH) nm (log ε): 269 (3.81); IR νmax (KBr) cm−1: 3364, 2943, 1750, 1424, 1347, 1225, 1124, 1061, 1025; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data see Table 1. HRESI-TOF-MS Negative-ion mode m/z 225.0708 [M – H]− (calcd for C10H13N2O2S, 225.0703). 3.1.3.2. Pugcornol B (2). Colorless oil; [α]D25–39.3 (conc. 0.12, MeOH); CD (conc. 0.0015 M, MeOH) mdeg (λnm): −1.30 (223), −5.34 (270); CD (conc. 0.0015 M, CH3CN) mdeg (λnm): −1.56 (223), −6.80 (252), −4.81 (272); UV λmax (MeOH) nm (log ε): 277 (3.96); IR νmax (KBr) cm−1: 3355, 2941, 1750, 1428, 1346, 1236, 1133, 1049, 1010; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data see Table 2. HRESI-TOF-MS Negative-ion mode m/z 289.0693 [M – H]− (calcd for C11H17N2O3S2, 289.0686). 3.1.3.3. Pugcornol C (3). Colorless oil; [α]D25–36.1 (conc. 0.14, MeOH); CD (conc. 0.0015 M, MeOH) mdeg (λnm): −0.34 (228), −2.60 (253), −2.99 (269); CD (conc. 0.0015 M, CH3CN) mdeg (λnm): −1.26 (231), −3.91 (250), −5.16 (267); UV λmax (MeOH) nm (log ε): 249 (3.50), 268 (3.58); IR νmax (KBr) cm−1: 3365, 2935, 1749, 1419, 1347, 1236, 1133, 1050, 1015; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data see Table 2. HRESI-TOF-MS Positive-ion mode m/z 327.0814 [M + Na]+ (calcd for C12H20N2O3S2Na, 327.0808).
Fig. 9. Mechanism research of 5-MP on isolated jejunum contraction height. N: normal group; 5-MP: 5-(methylsulfinyl)-pentanenitrile (compound 9); n = 6, ⁎ p < .05, ⁎⁎p < .01, ⁎⁎⁎p < .001 vs. normal group. Data (mean ± SEM) were analyzed by ANOVA.
3.1.3.4. Pugcornol D (4). Colorless oil; [α]D25–64.1 (conc. 0.13, MeOH); CD (conc. 0.0015 M, MeOH) mdeg (λnm): −35.54 (214), +14.06 (238), +0.12 (263); CD (conc. 0.0015 M, CH3CN) mdeg (λnm): −33.13 (212), 12.47 (238), +0.23 (262); UV λmax (MeOH) nm (log ε): 214 (3.44), 267 (2.38); IR νmax (KBr) cm−1: 3420, 2943, 1765, 1701, 1447, 1419, 1361, 1014; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data see Table 3. HRESI-TOF-MS Positive-ion mode m/z 259.1113 [M + H]+ (calcd for C11H19N2O3S, 259.1111).
6.2 mg) were given. Fraction H4-3 (1200.0 mg) was separated by pHPLC [MeOH-H2O (55:45, v/v)] to yield pugcornol C (3, 14.4 mg). Fraction H4-4 (530.0 mg) was subjected to pHPLC [MeOH-H2O (30:70, v/v)], and nine fractions (Fr. H4-4-1-Fr. H4-4-9) were obtained. Fraction H4-4-9 (62.8 mg) was separated by pHPLC [CH3CN-H2O (22:78, v/v)] to yield pugcornoside D (8, 8.5 mg). Fraction H4–5 (500.0 mg) was isolated by pHPLC [MeOH-H2O (20:80, v/v)] to gain fourteen fractions (Fr. H4-5-1-Fr. H4-5-14). Fraction H4-5-5 (14.7 mg) was subjected to silica gel CC [CHCl3-MeOH-H2O (7:3:1, lower layer, v/ v/v)] to obtain demethyl coniferin (22, 14.7 mg). Fraction H5 (5.0 g) was isolated by ODS CC [MeOH-H2O (10:90 → 20:80 → 30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 100:0, v/v)], and seven fractions (Fr. H5-1-Fr. H5-7) were produced. Fraction H5-4 (800.0 mg) was purified by pHPLC [CH3CN-H2O (16:84, v/v)] to obtain pugcornol B (2, 33.4 mg), and (+)-1-hydroxyl pinoresinol-4-O-β-D-glucopyranoside (19, 21.1 mg). Fraction H6 (4.8 g) was subjected by ODS CC [MeOH-H2O (10:90 → 20:80 → 30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 90:10 → 100: 0, v/v)], and thirteen fractions (Fr. H6-
3.1.3.5. Pugcornoside A (5). White powder; [α]D25–23.2 (conc. 0.11, MeOH); UV λmax (MeOH) nm (log ε): 223 (3.61), 277 (3.18); IR νmax (KBr) cm−1: 3365, 2922, 1646, 1558, 1507, 1457, 1277, 1070; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data see Table 4. HRESI-TOF-MS Negative-ion mode m/z 553.1605 [M – H]− (calcd for C22H33O14S, 553.1597). 3.1.3.6. Pugcornoside B (6). White powder; [α]D25–52.6 (conc. 0.13, MeOH); UV λmax (MeOH) nm (log ε): 220 (3.65), 277 (3.18); IR νmax (KBr) cm−1: 3366, 2918, 1600, 1507, 1456, 1436, 1278, 1045, 1072; 1 H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data see Table 4. HRESI-TOF-MS Negative-ion mode m/z 553.1599 [M – H]− (calcd for C22H33O14S, 553.1597). 8
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3.1.3.7. Pugcornoside C (7). White powder; [α]D25 + 6.3 (conc. 0.12， MeOH)；UV λmax (MeOH) nm (log ε): 266 (2.94); IR νmax (KBr) cm−1: 3386, 2391, 1734, 1652, 1107, 1044; 1H NMR (CD3OD, 500 MHz) and 13 C NMR (CD3OD, 125 MHz) data see Table 5. HRESI-TOF-MS Negative-ion mode m/z 281.0878 [M – H]− (calcd for C10H17O9, 281.0878).
0.1% and final concentration were 10 mg/mL for every fraction and 50 μM every compound, respectively. Intestine contractions were recorded for 1 min before and 4 min after drug additions using the Power Lab system and the Chart 7 software (AD instrument Ltd., New South Wales, Australia). Atropine (0.025 mg/mL), CuSO4 (0.012 mg/mL), and loperamide (0.004 mg/mL) were used for mechanism researches.
3.1.3.8. Pugcornoside D (8). White powder; [α]D25 + 28.1 (conc. 0.12, MeOH); UV λmax (MeOH) nm (log ε): 205 (3.72), 262 (2.77); IR νmax (KBr) cm−1: 3420, 2933, 1733, 1635, 1466, 1339, 1267, 1154, 1112, 1047; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data see Table 5. HRESI-TOF-MS Negative-ion mode m/z 399.1295 [M – H]− (calcd for C18H23O10, 399.1297).
3.2.4. Statistic analysis Values were expressed as means ± SD. All the grouped data were statistically analyzed with the SPSS 14.0 software. Significant differences between the normal group or control group were evaluated by one-way analysis of variance (ANOVA), and Tukey's studentized range test was used for post hoc evaluations. Least significant differences (P < .05) were used for statistical analyses.
3.1.4. Acid hydrolysis of 7 and 8 Solution of 7 and 8 (each 3.0 mg) in 5% aqueous H2SO4-1,4-dioxane were heated under reflux for 1 h, respectively. After cooling, the reaction mixture was neutralized with Amberlite IRA-400 (OH– form), removed by filtration, subjected to ODS CC (H2O), and the H2O eluate was reacted with L-cysteine methyl ester hydrochloride in pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), successively. Finally, the reaction product was elucidated by GC–MS analysis (GC conditions, column: RESTEK Rxi-5 ms, 30 m × 0.25 mm (i.d.) capillary column; column temperature: 230 °C; carrier gas: He), and D-glucuronic acid hydrolysate from 7 and 8 was identified by comparing it retention times (tR 13.4 min) with that of its authentic sample treated in the same way.
Declaration of Competing Interest None. Acknowledgments 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 (2018ZX09735-002). Appendix A. Supplementary data
3.1.5. Computations Using the similar methods as we described previously [20–23]: The ECD spectra for the optimized conformers were calculated at the CAMB3LYP/SVP level with a CPCM solvent model in acetonitrile, and the calculated ECD spectra of different conformers were simulated with a half bandwidth of −0.4 eV. The ECD curves were extracted by SpecDis 1.62 software. The overall ECD curves of all the compounds were weighed by Boltzmann distribution after UV correction.
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3.2. The effects of all fractions and isolated compounds from PC on mouseisolated jejunum contractility 3.2.1. Materials All fractions and compounds 1–31 were prepared by the method described in Section 3.1.3. Atropin, CuSO4, and loperamide were used for mechanism researches. 3.2.2. Animals Healthy male Kunming mice (22–25 g) were purchased from Beijing HFK Bioscience Co. Ltd. (Beijing, China; Certificate of Conformity: No. 11401300071030). All animal experiments were approved by the Science and Technological Committee and the Animal Use and Care Committee of TUTCM (No. 201712003). Mice were housed in a temperature controlled room at 24 ± 2 °C, relative humidity 60%. The mice were fed with standard diet (crude protein 16%, crude fat 4%, crude fiber 12%, and ash 8%) normally a week before the experiment to adapt to the environment. 3.2.3. Effects of all fractions and their compounds on mice jejunum contraction Mice were fasted for 18 h before being sacrificed for experiments, and about 1 cm of the jejunum was collected immediately. The Maxwell bath was filled with 10 mL of Tyrode's solution (one liter contains: NaCl 8.0 g, CaCl2 0.2 g, KCl 0.2 g, MgCl2 0.1 g, NaHCO3 1.0 g, KH2PO4 0.05 g, glucose 1.0 g, pH 7.4) and maintained at a constant temperature (37.0 ± 0.5 °C), and bubbled with 95% O2 and 5% CO2 gas. The jejunum was fixed on bottom hook in and the other end was connected to an isometric tension transducer. Samples in DMSO solution were added after 15 min equilibrate incubation, the final DMSO concentration was 9
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