Bioactive Constituents from the Whole Plants of Gentianella Acuta (Michx.) Hulten

molecules

Article Bioactive Constituents from the Whole Plants of Gentianella acuta (Michx.) Hulten

Zhijuan Ding 1,2, Yanxia Liu 1,2, Jingya Ruan 1,2, Shengcai Yang 1,2, Haiyang Yu 1,2, Meiling Chen 1,2, Yi Zhang 1,2,* and Tao Wang 1,2,*

1 Tianjin State Key Laboratory of Modern Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China; [email protected] (Z.D.); [email protected] (Y.L.); [email protected] (J.R.); [email protected] (S.Y.); [email protected] (H.Y.); [email protected] (M.C.) 2 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 * Correspondence: [email protected] (Y.Z.); [email protected] (T.W.); Tel./Fax: +86-22-5959-6163 (Y.Z. & T.W.)

Received: 6 July 2017; Accepted: 4 August 2017; Published: 6 August 2017

Abstract: As a Mongolian native medicine and Ewenki folk medicinal plant, Gentianella acuta has been widely used for the treatment of diarrhea, hepatitis, arrhythmia, and coronary heart disease. In the course of investigating efficacy compounds to treat diarrhea using a mouse isolated intestine tissue model, we found 70% EtOH extract of G. acuta whole plants had an inhibitory effect on intestine contraction tension. Here, nineteen constituents, including five new compounds, named as gentiiridosides A (1), B (2), gentilignanoside A (3), (1R)-2,2,3-trimethyl-4- hydroxymethylcyclopent-3-ene-1-methyl-O-β-D-glucopyranoside (4), and (3Z)-3-hexene- 1,5-diol 1-O-α-L-arabinopyranosyl(1→6)-β-D-glucopyranoside (5) were obtained from it. The structures of them were elucidated by chemical and spectroscopic methods. Furthermore, the inhibitory effects on motility of mouse isolated intestine tissue of the above mentioned compounds and other thirteen iridoid- and secoiridoid-type monoterpenes (7–10, 13–16, 18, 19, 21, 22, and 25) previously obtained in the plant were analyzed. As results, new compound 5, some secoiridoid-type monoterpenes 7, 10, 12–14, 16, and 17, as well as 7-O-90-type lignans 31 and 32 displayed significant inhibitory effect on contraction tension at 40 µM.

Keywords: Gentianella acuta (Michx.) Hulten; lignan; iridoid- and secoiridoid-type monoterpene; intestine motility; mouse isolated intestine tissue

1. Introduction Gentianella acuta (Michx.) Hulten belongs to the family Gentianaceae, distributed mainly in the north of China, Mongolia plateau, Siberia, and Far East areas of Russia [1]. As a Mongolian native medicine and Ewenki folk medicinal plant, G. acuta has been widely used for the treatment of diarrhea, hepatitis, arrhythmia, coronary heart disease, jaundice, fever, and headache [1–3]. In the course of investigating efﬁcacy compounds to treat diarrhea using a mouse isolated intestine tissue model, we found that a 70% EtOH extract of G. acuta whole plants had an inhibitory effect on intestine contraction tension. Moreover, eighteen xanthones had been obtained and their inhibitory effects on the model were assayed. As results, some xanthones were found to have a signiﬁcant reducing effect on intestine contraction tension [4]. Additionally, for xanthones, monoterpenes, and lignans were elucidated to be main constituents in the plant in our continuing study, among them, the spectroscopy data of thirteen iridoid- and secoiridoid-type monoterpenes had been reported [5,6] by us. Here,

Molecules 2017, 22, 1309; doi:10.3390/molecules22081309 www.mdpi.com/journal/molecules Molecules 2017, 22, 1309 2 of 13

Molecules 2017, 22, 1309 2 of 13 we obtained other nineteen constituents, including ﬁve new compounds (1–5) (Figure1) and fourteen inhibitory effect on motility of mouse isolated intestine tissue, too? In this paper, we describe the knowninhibitor onesy (effect6, 11, on12 ,motility17, 20, 23 of, 24mouse, 26– 32isolated) (Figure intestine2). Do theytissue, have too? an In inhibitory this paper, effect we describe on motility the of isolation and structure elucidation of them, along with evaluations of their inhibitory effects on mouse isolated intestine tissue, too? In this paper, we describe the isolation and structure elucidation intestine contraction tension. of them, along with evaluations of their inhibitory effects on intestine contraction tension.

FigureFigure 1. 1.The The new new compoundscompounds 1–5 obtained from from the the whole whole plant plant of of G.G. acuta acuta. .

FigureFigure 2. 2.The The known known compoundscompounds (6–32) obtained from from the the whole whole plant plant of of G.G. acuta acuta. .

Molecules 2017, 22, 1309 3 of 13

2. Results and Discussion During the course of our continuous studies on bioactive constituents from a 95% EtOH eluate of D101 CC and CHCl3 layer [4–6], obtained from the whole plants of G. acuta, nineteen constituents, including five new compounds, named as gentiiridosides A (2), B (2), gentilignanoside A (3), (1R)-2,2,3-trimethyl-4-hydroxymethylcyclopent-3-ene-1-methyl-O-β-D-glucopyranoside (4), and (3Z)-3-hexene-1,5-diol 1-O-α-L-arabinopyranosyl(1→6)-β-D-glucopyranoside (5) (Figure1), together with fourteen known ones, 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one (6)[7] (E)-aldosecologanin (11)[8], 5α-carboxystrictosidine (12)[9], trifloroside (17)[10], gentiopicroside (20)[11], loganin (23)[12], 8-epiloganin (24)[13], swertiaside (26)[14], (7R,8S)-erythro-4,7,9,90- tetrahydroxy-3,30-dimethoxy-8-O-40-neolignan (27)[15,16], (7S,8R)-dehydrodiconiferyl alcohol 0 (28)[17–19], plucheoside D3 (29)[17,18,20], (7S,8R)-9 -methoxy-dehydrodiconiferyl alcohol 4-O-β- 0 D-glucopyranoside (30)[17,18,21], (–)-berchemol (31)[22,23], and berchemol-4 -O-β-D-glucoside (32)[22,24] (Figure2), were further obtained. Among the known isolates, 6, 11, 12, 17, 23, and 27–32 were isolated from the genus firstly. This paper will elucidate the isolation and structure of the new compounds. Meanwhile, the effects of the abovementioned compounds and previously-isolated thirteen iridoid- and secoiridoid-type monoterpenes, secologanol (7)[5], secologanin [6](8), secologanoside (9)[5], secoxyloganin (10)[5], sweroside (13)[6], swertiapunimarin (14)[5], deacetylcentapicrin (15)[6], decentapicrin A (16)[6], 0 swertiamarin (18)[5], eustomoside (19)[5], 6 -O-β-D-glucopyranosyl gentiopicroside (21)[5], loganic acid (22)[6], 7-ketologanin (25)[6] (Figure2) on the motility of mouse isolated intestine tissue were determined. 25 Gentiiridoside A (1) was isolated as a white powder with negative optical rotation [[α]D −90.0◦ (c 0.14, MeOH)]. Negative high resolution electrospray ionization-time of flight-mass spectra − (HRESI-TOF-MS) afforded [M – H] at m/z 777.2261 (calcd for C36H41O19, 777.2248), supporting a molecular formula of C36H42O19 for 1. The absorption bands showed in the infrared (IR) spectrum suggested the presence of hydroxyl (3372 cm−1), α,β-unsaturated carbonyl (1712 cm−1), aromatic ring (1635, 1588, 1486 cm−1), and O-glycosidic linkage (1078 cm−1). The sugars in 1 were found to be D-glucose by acid hydrolysis with 1 M HCl [4]. The 1H, 13C-nuclear magnetic resonance (NMR) spectra (Table1) and various two-dimensional (2D) NMR spectra, including 1H 1H chemical-shift correlation spectroscopy (1H 1H COSY), heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC) displayed signals assignable to two 3-hydroxy benzoyl {[δ 7.42 (1H, br. d, ca. J = 8 Hz, H-400), 7.48 (1H, dd, J = 7.5, 7.5 Hz, H-500), 7.84 (1H, br. d, ca. J = 8 Hz, H-600), 7.89 (1H, br. s, 00 00 0000 0000 H-2 ); δC 166.1 (C-7 )]; [δ 7.45 (1H, br. d, ca. J = 8 Hz, H-4 ), 7.54 (1H, dd, J = 7.5, 7.5 Hz, H-5 ), 7.82 0000 0000 0000 (1H, br. s, H-2 ), 7.88 (1H, br. d, ca. J = 8 Hz, H-6 ); δC 166.8 (C-7 )]}, and two β-D-glucopyranosyl [δ 4.69 (1H, d, J = 7.5 Hz, H-10), 5.02 (1H, d, J = 8.0 Hz, H-1000)]. On the other hand, thirty-six carbon signals were shown in its 13C-NMR spectrum, in addition to twenty-six carbon signals occupied by the abovementioned fragments, the other 10 carbon signals, together with the relative proton signals [δ 5.50 (1H, d, J = 3.0 Hz, H-1), 7.46 (1H, s, H-3)] indicated the aglycon of 1 was iridoid. As shown in Figure3, the 1H 1H COSY experiment on 1 suggested the existence of five partial structures. Furthermore, in the HMBC experiment, long-range correlations from δH 5.50 (H-1) to δC 152.4 (C-3); δH 7.46 (H-3) to 00 0 δC 32.5 (C-5), 96.2 (C-1), 112.8 (C-4), 170.9 (C-11); δH 4.96 (H-7) to δC 166.1 (C-7 ); δH 4.69 (H-1 ) to 00 00 00 000 00 δC 96.2 (C-1); δH 7.89 (H-2 ), 7.84 (H-6 ) to δC 166.1 (C-7 ); δH 5.02 (H-1 ) to δC 159.2 (C-3 ); δH 000 0000 0000 0000 0000 3.51 (H-2 ) to δC 166.8 (C-7 ); δH 7.82 (H-2 ), 7.88 (H-6 ) to δC 166.8 (C-7 ) were observed. Therefore, the planar structure of 1 was constructed. The relative configuration of 1 was determined by a nuclear Overhauser effect spectroscopy (NOESY) experiment, and NOE correlations were observed 1 13 between H-1 and H-8; H-5 and H-7; H3-10 and H-7, H-9. The H and C-NMR sepctra of 1 were found very similar to those of swertiaside (26)[14], except that a 2-(3-hydroxybenzoyl)-β-D-glucopyranosyl appeared at the 300-position in 1. Consequently, the structure of gentiiridoside A (1) was determined. Molecules 2017, 22, 1309 4 of 13

1 13 Molecules 2017, 22, 1309 Table 1. H and C-NMR data for 1 in CD3OD. 4 of 13

No. δC δH (J in Hz) No. δC δH (J in Hz) 1 13 1 96.2 Table5.50 1. (d,H and3.0) C-NMR data3 for′′ 1 in159.2 CD3OD. - 3 152.4 7.46 (s) 4′′ 123.5 7.42 (br. d, ca. 8)

4 No. 112.8δ C δH (J in- Hz) No. 5′′ δC131.0 δH 7.48(J in (dd, Hz) 7.5, 7.5) 5 132.5 96.2 3.05 5.50 (q (d,like, 3.0) ca. 7) 300 6′′ 159.2125.1 7.84 - (br. d, ca. 8) 6 338.0 152.4 2.00 7.46 (m) (s) 400 7′′ 123.5166.1 7.42 (br. d, ca. 8)- 00 4 112.82.55 (ddd, 6.5, - 7.0, 13.5) 5 1′′′ 131.0102.3 7.48 (dd,5.02 7.5, 7.5)(d, 8.0) 00 7 583.7 32.5 4.96 3.05 (ddd, (q like,4.5, ca7.0,. 7) 10.5) 6 2′′′ 125.177.9 7.843.51 (br. (m, d, ca overlapped). 8) 6 38.0 2.00 (m) 700 166.1 - 8 43.0 2.55 (ddd,2.12 6.5, (m) 7.0, 13.5) 1000 3′′′ 102.374.8 5.023.53 (d, (m, 8.0) overlapped) 9 748.7 83.7 4.962.00 (ddd, (m, overlapped) 4.5, 7.0, 10.5) 2000 4′′′ 77.971.4 3.51 (m,3.32 overlapped) (m, overlapped) 10 818.4 43.0 1.23 2.12 (d, (m) 7.0) 3000 5′′′ 74.878.1 3.53 (m,3.33 overlapped) (m, overlapped) 000 11 9170.9 48.7 2.00 (m, overlapped)- 4 6′′′ 71.462.6 3.32 (m,3.66 overlapped) (dd, 4.0, 11.5) 10 18.4 1.23 (d, 7.0) 5000 78.1 3.33 (m, overlapped) 1′ 100.2 4.69 (d, 7.5) 3.84 (br. d, ca. 12) 11 170.9 - 6000 62.6 3.66 (dd, 4.0, 11.5) 2′ 10 74.6100.2 3.22 4.69 (dd, (d, 7.5, 7.5) 8.5) 1′′′′ 133.1 3.84 (br. d, ca. 12)- 3′ 20 77.874.6 3.40 3.22 (dd, 7.5,8.5, 8.5) 8.5) 10000 2′′′′ 133.1123.9 - 7.82 (br. s) 0 0000 4′ 3 71.377.8 3.46 3.40 (dd, 8.5,8.5, 8.5) 8.5) 2 3′′′′ 123.9152.3 7.82 (br. s) - 0 0000 5′ 4 78.271.3 3.51 3.46 (m, (dd, overlapped) 8.5, 8.5) 3 4′′′′ 152.3127.7 7.45 - (br. d, ca. 8) 50 78.2 3.51 (m, overlapped) 40000 127.7 7.45 (br. d, ca. 8) 6′ 60 62.462.4 3.75 3.75 (dd, 5.0,5.0, 11.5) 11.5) 50000 5′′′′ 130.8130.8 7.54 (dd,7.54 7.5, (dd, 7.5) 7.5, 7.5) 3.923.92 (br. d,d,ca ca.. 12) 12) 60000 6′′′′ 128.1128.1 7.88 (br.7.88 d, (br.ca. 8) d, ca. 8) 1′′ 100 131.7131.7 -- 70000 7′′′′ 166.8166.8 - - 00 2′′ 2 119.3119.3 7.89 (br.(br. s)s)

Figure 3. The main 1H 11HH COSY, COSY, HMBC correlations of 1 and 2, and N NOEOE correlations of 1.

Gentiiridoside B (2) was obtained as a white powder with negative optical rotation [[α]25 −36.7° (c Gentiiridoside B (2) was obtained as a white powder with negative optical rotation [[αD]25 −36.7◦ 0.12, MeOH)]. HRESI-TOF-MS exhibited a molecular ion peak at m/z 679.1023 [M – H]−, andD revealed (c 0.12, MeOH)]. HRESI-TOF-MS exhibited a molecular ion peak at m/z 679.1023 [M – H]−, and the molecular formula C28H40O18 (calcd for C28H39O18, 679.1033) for it. Acid hydrolysis of 2 with 1 M revealed the molecular formula C28H40O18 (calcd for C28H39O18, 679.1033) for it. Acid hydrolysis of 2 HCl afforded D-glucose, whose absolute configuration was determined by HPLC analysis [4]. The 1H with 1 M HCl afforded D-glucose, whose absolute conﬁguration was determined by HPLC analysis [4]. and 13C-NMR (Table 2) spectra of 2 indicated the presence of two β-D-glucopyranosyl [δ 4.27 (1H, d, J The 1H and 13C-NMR (Table2) spectra of 2 indicated the presence of two β-D-glucopyranosyl [δ 4.27 = 7.5 Hz, H-1′′), 4.49 (1H,00 d, J = 8.0 Hz, H-1′)], and one0 α-D-glucopyranosyl [δ 4.91 (1H, d, J = 3.5 Hz, (1H, d, J = 7.5 Hz, H-1 ), 4.49 (1H, d, J = 8.0 Hz, H-1 )], and one α-D-glucopyranosyl [δ 4.91 (1H, d, H-1′′′)]. Twenty-eight carbon signals were displayed in its 13C-NMR spectrum, except for the above J = 3.5 Hz, H-1000)]. Twenty-eight carbon signals were displayed in its 13C-NMR spectrum, except for mentiond moieties, the other ten signals as well as their relative 1H-NMR signals [δ 5.21 (2H, m, the above mentiond moieties, the other ten signals as well as their relative 1H-NMR signals [δ 5.21 (2H, H2-10), 5.59 (1H, d, J = 3.0 Hz, H-1), 5.72 (1H, ddd, J = 6.5, 10.5, 17.5 Hz, H-8), 7.41 (1H, s, H-3)] m, H2-10), 5.59 (1H, d, J = 3.0 Hz, H-1), 5.72 (1H, ddd, J = 6.5, 10.5, 17.5 Hz, H-8), 7.41 (1H, s, H-3)] suggested the aglycon of 2 was the same as that of gentiopicroside (20) [11]. Finally, the long-range suggested the aglycon of 2 was the same as that of gentiopicroside (20)[11]. Finally, the long-range correlations from δH 5.59 (H-1) to δC 124.9 (C-5), 148.8 (C-3); δH 7.41 (H-3) to δC 96.4 (C-1), 103.2 (C-4), correlations from δH 5.59 (H-1) to δC 124.9 (C-5), 148.8 (C-3); δH 7.41 (H-3) to δC 96.4 (C-1), 103.2 (C-4), 124.9 (C-5), 162.7 (C-11); δH 5.65 (H-6) to δC 44.3 (C-9), 103.2 (C-4); δH 4.97, 5.04 (H2-7) to δC 124.9 (C-5), 124.9 (C-5), 162.7 (C-11); δH 5.65 (H-6) to δC 44.3 (C-9), 103.2 (C-4); δH 4.97, 5.04 (H2-7) to δC 124.9 (C-5), 162.7 (C-11); δH 5.72 (H-8) to δC 96.4 (C-1), 124.9 (C-5); δH 3.31 (H-9) to δC 103.2 (C-4), 116.1 (C-6), 124.9 162.7 (C-11); δH 5.72 (H-8) to δC 96.4 (C-1), 124.9 (C-5); δH 3.31 (H-9) to δC 103.2 (C-4), 116.1 (C-6), 124.9 (C-5); δH 4.49 (H-1′) to δC 96.4 (C-1); δH 4.27 (H-1′′) to δC 76.7 (C-1′); δH 4.91 (H-1′′′) to δC 69.9 (C-1′′) (C-5); δ 4.49 (H-10) to δ 96.4 (C-1); δ 4.27 (H-100) to δ 76.7 (C-10); δ 4.91 (H-1000) to δ 69.9 (C-100) were observedH in the HMBCC spectrum.H Then, the structureC of gentiiridosideH B (2) was clarified.C were observed in the HMBC spectrum. Then, the structure of gentiiridoside B (2) was clariﬁed.

Molecules 2017, 22, 1309 5 of 13

Table 2. 1H and 13C-NMR data for 2 in DMSO-d6. Molecules 2017, 22, 1309 5 of 13 No. δC δH (J in Hz) No. δC δH (J in Hz) 1 96.4 5.59 (d, 3.0) 6′ 61.0 3.44 (m, overlapped) 1 13 3 148.8 Table 2. 7.41H and (s) C-NMR data for 2 in DMSO- d6. 3.68 (br. d, ca. 12) 4 103.2 - 1′′ 96.8 4.27 (d, 7.5) No. δ δ (J in Hz) No. δ δ (J in Hz) 5 124.9 C H - 2′′ C 74.7 H 2.90 (dd, 7.5, 8.5) 0 6 116.11 96.4 5.595.65 (d, (m) 3.0) 6 361.0′′ 76.6 3.44 (m, overlapped)3.12 (m, overlapped) 3 148.8 7.41 (s) 3.68 (br. d, ca. 12) 7 69.14 103.24.97 (dd, -3.0, 18.0) 100 496.8′′ 70.2 4.27 (d,3.05 7.5) (m, overlapped) 5 124.95.04 (br. -d, ca. 18) 200 574.7′′ 76.7 2.90 (dd,3.03 7.5, 8.5) (m, overlapped) 6 116.1 5.65 (m) 300 76.6 3.12 (m, overlapped) 8 134.07 69.15.72 (ddd, 4.97 (dd, 6.5, 3.0, 10.5, 18.0) 17.5) 400 670.2′′ 61.1 3.05 (m, overlapped)3.44 (m, overlapped) 9 44.3 5.043.31 (br. d,(m)ca. 18) 500 76.7 3.03 (m, overlapped)3.66 (br. d, ca. 12) 00 10 117.98 134.0 5.72 (ddd,5.21 6.5, (m) 10.5, 17.5) 6 161.1′′′ 92.1 3.44 (m, overlapped)4.91 (d, 3.5) 9 44.3 3.31 (m) 3.66 (br. d, ca. 12) 11 162.710 117.9 5.21- (m) 1000 292.1′′′ 72.3 4.91 (d,3.12 3.5) (m, overlapped) 1′ 98.711 162.74.49 (d, - 8.0) 2000 372.3′′′ 72.7 3.12 (m, overlapped)3.42 (m, overlapped) 0 000 2′ 73.01 98.72.95 4.49(dd, (d, 8.0, 8.0) 8.5) 3 472.7′′′ 70.5 3.42 (m, overlapped)3.05 (m, overlapped) 20 73.0 2.95 (dd, 8.0, 8.5) 4000 70.5 3.05 (m, overlapped) 3′ 76.730 76.73.15 3.15 (m, (m, overlapped) overlapped) 5000 571.9′′′ 71.9 3.57 (m) 3.57 (m) 4′ 69.940 69.93.03 3.03 (m, (m, overlapped) overlapped) 6000 661.1′′′ 61.1 3.44 (m, overlapped)3.44 (m, overlapped) 0 5′ 77.35 77.33.15 3.15 (m, (m, overlapped) overlapped)

Gentilignanoside A (3) was obtained as a white powder that exhibited negative optical rotation 25Gentilignanoside A (3) was obtained as a white powder that exhibited negative optical rotation [[α]25D −36.0◦° (c 0.10, MeOH)]. The molecular formula, C27H34O13, of 3 was determined from [[α]D −36.0 (c 0.10, MeOH)]. The molecular formula, C27H34O13, of 3 was determined from Q-TOF-ESI-MS analysis (m/z 567.2083 [M – H]−−, calcd for C27H33O13, 567.2083). Its IR spectrum Q-TOF-ESI-MS analysis (m/z 567.2083 [M – H] , calcd for C27H33O13, 567.2083). Its IR spectrum showed absorption bands due to hydroxyl (3368 cm−1), aromatic ring (1613, 1513, 1463 cm−1), and showed absorption bands due to hydroxyl (3368 cm−1), aromatic ring (1613, 1513, 1463 cm−1), and O-glycosidic linkage (1073 cm−1). The 1H, 13C-NMR spectra (Table 3) and kinds of 2D NMR spectra O-glycosidic linkage (1073 cm−1). The 1H, 13C-NMR spectra (Table3) and kinds of 2D NMR spectra (1H 1H COSY, HSQC, HMBC) showed signals ascribable to one ABX-type aromatic protons [δ 6.77 (1H 1H COSY, HSQC, HMBC) showed signals ascribable to one ABX-type aromatic protons [δ 6.77 (1H, (1H, br. d, ca. J = 8 Hz, H-6′), 6.90 (1H, br. s, H-2′), 7.10 (1H, d, J = 8.0 Hz, H-5′)], one br. d, ca. J = 8 Hz, H-60), 6.90 (1H, br. s, H-20), 7.10 (1H, d, J = 8.0 Hz, H-50)], one 1,3,4,5-symmetrical 1,3,4,5-symmetrical substituted phenyl group [δ 6.64 (2H, s, H-2,6)], two methylene bearing oxygen substituted phenyl group [δ 6.64 (2H, s, H-2,6)], two methylene bearing oxygen funtion {δ [3.64, 3.80 funtion {δ [3.64, 3.80 (1H each, both m, overlapped, H2-9)], [3.64 (1H, m, overlapped), 4.06 (1H, dd,0 J = (1H each, both m, overlapped, H2-9)], [3.64 (1H, m, overlapped), 4.06 (1H, dd, J = 7.5, 7.5 Hz), H2-9 ]]}, 7.5, 7.5 Hz), H2-9′]]}, three methoxyl [δ 3.84 (6H, s,0 3,5-OCH3), 3.86 (3H, s, 3′-OCH3)], and one three methoxyl [δ 3.84 (6H, s, 3,5-OCH3), 3.86 (3H, s, 3 -OCH3)], and one β-D-glucopyranosyl [δ 4.88 β-D-glucopyranosyl [δ 4.88 (1H, d, J = 7.5 Hz, H-1′′)]. The planar structure of 3 was constructed by the (1H, d, J = 7.5 Hz, H-100)]. The planar structure of 3 was constructed by the assignment of the 1H assignment of the 1H 1H COSY and HMBC experiments as shown in Figure 4. The 1H 1H COSY 1H COSY and HMBC experiments as shown in Figure4. The 1H 1H COSY experiment indicated the experiment indicated the presence of three partial moieties. On the other hand, in the HMBC presence of three partial moieties. On the other hand, in the HMBC experiment, long-range correlations experiment, long-range correlations were found from the following proton and carbon pairs: δH 6.64 were found from the following proton and carbon pairs: δH 6.64 (H-2,6) to δC 129.9 (C-1), 136.2 (C-4), (H-2,6) to δC 129.9 (C-1), 136.2 (C-4), 148.90 (C-3,5); δH 4.84 (H-7) to δC 51.8 (C-8′), 64.6 (C-9), 106.2 148.9 (C-3,5); δH 4.84 (H-7) to δC 51.8 (C-8 ), 64.6 (C-9), 106.2 (C-2,6), 129.9 (C-1); δH 3.64, 3.80 (H2-9) (C-2,6), 129.9 (C0 -1); δH 3.64, 3.80 (H2-9) to δC 51.8 (C0-8′), 83.3 (C-8), 85.80 (C-7); δH 06.90 (H-2′) to0 δC 35.1 to δC 51.8 (C-8 ), 83.3 (C-8), 85.8 (C-7); δH 6.90 (H-2 ) to δC 35.1 (C-7 ), 122.4 (C-6 ), 146.5 (C-4 ), 150.9 (C-70′), 122.4 (C-6′), 0146.5 (C-4′), 150.90 (C-3′); δH 7.100 (H-5′) to δ0C 136.9 (C-1′), 146.50 (C-4′), 150.90 (C-3′); (C-3 ); δH 7.10 (H-5 ) to δC 136.9 (C-1 ), 146.5 (C-4 ), 150.9 (C-3 ); δH 6.77 (H-6 ) to δC 35.1 (C-7 ), 114.4 δH 6.770 (H-6′) to 0 δC 35.1 (C-7′), 114.4 0(C-2′), 146.5 (C-04′); δH 2.54, 3.130 (H2-7′) to0 δC 72.0 (C0-9′), 114.4 (C-2 ), 146.5 (C-4 ); δH 2.54, 3.13 (H2-7 ) to δC 72.0 (C-9 ), 114.4 (C-2 ), 122.4 (C-6 ), 136.9 (C-1 ); δH 2.59 (C-2′0), 122.4 (C-6′), 136.90 (C-1′); δH 2.59 (H0-8′) to δC 136.9 (C0 -1′); δH 3.64, 4.06 (H2-9′) to δC 35.1 (C-7′), (H-8 ) to δC 136.9 (C-1 ); δH 3.64, 4.06 (H2-9 ) to δC 35.1 (C-7 ), 83.3 (C-8), 85.8 (C-7); δH 3.84 (3,5-OCH3) 83.3 (C-8), 85.8 (C-7); δH 3.84 0(3,5-OCH3) to δC 148.9 (C0-3,5); δH 3.86 (300′-OCH3) to δC 150.90 (C-3′); δH 4.88 to δC 148.9 (C-3,5); δH 3.86 (3 -OCH3) to δC 150.9 (C-3 ); δH 4.88 (H-1 ) to δC 146.5 (C-4 ). Furthermore, (H-1′′) to δC 146.5 (C-4′). Furthermore, the relative configuration of 3 was determined by the NOE the relative conﬁguration of 3 was determined by the NOE correlations between δH 4.84 (H-7) and δH correlations between δH 4.84 (H-7) and δH 3.64, 3.80 (H2-9); δH 3.64, 3.80 (H2-9) and δH 2.54, 3.13 (H2-7′) 3.64, 3.80 (H -9); δ 3.64, 3.80 (H -9) and δ 2.54, 3.13 (H -70) observed in its NOESY spectrum. Finally, observed in 2its NOESYH spectrum.2 Finally,H 3 showed negative2 Cotton effect at 278 and 232 nm, which 3 showed negative Cotton effect at 278 and 232 nm, which indicated the absolute conﬁguration of it indicated the absolute configuration of it was 7R,8S,8′S [22]. was 7R,8S,80S [22].

1 1 Figure 4. The main H H COSY, HMBC, and NOE correlations of 3. Molecules 2017, 22, 1309 6 of 13

Figure 4. The main 1H 1H COSY, HMBC, and NOE correlations of 3.

Table 3. 1H and 13C-NMR data for 3 in CD3OD Molecules 2017, 22, 1309 6 of 13 No. δC δH (J in Hz) No. δC δH (J in Hz) 1 129.9 - 7′ 35.1 2.54 (dd, 12.5, 12.5) Table 3. 1H and 13C-NMR data for 3 in CD OD. 2,6 106.2 6.64 (s) 3 3.13 (br. d, ca. 13) 3,5 148.9 - 8′ 51.8 2.59 (m) No. δC δH (J in Hz) No. δC δH (J in Hz) 4 136.2 - 9′ 72.0 3.64 (m, overlapped) 1 129.9 - 70 35.1 2.54 (dd, 12.5, 12.5) 7 2,685.8 106.2 4.84 6.64 (s) 3.13 (br.4.06 d, ca (dd,. 13) 7.5, 7.5) 8 3,583.3 148.9 -- 3,5 80 -OCH3 51.856.8 2.59 (m) 3.84 (s) 0 9 464.6 136.2 3.64 (m, overlapped) - 93′-OCH3 72.056.83.64 (m, overlapped)3.86 (s) 785.8 3.80 (m, 4.84 overlapped) (s) 1′′ 103.1 4.06 (dd, 7.5,4.88 7.5) (d, 7.5) 8 83.3 - 3,5-OCH3 56.8 3.84 (s) 1′ 136.9 - 0 2′′ 75.0 3.49 (dd, 7.5, 8.5) 9 64.6 3.64 (m, overlapped) 3 -OCH3 56.8 3.86 (s) 2′ 114.4 3.80 (m,6.90 overlapped) (br. s) 100 3′′ 103.177.9 4.88 (d, 7.5)3.45 (m) 3′ 10 150.9136.9 -- 200 4′′ 75.071.4 3.49 (dd,3.40 7.5, (m, 8.5) overlapped) 0 00 4′ 2 146.5114.4 6.90 (br.- s) 3 5′′ 77.978.2 3.453.40 (m) (m, overlapped) 0 00 5′ 3 118.3150.9 7.10 (d, - 8.0) 4 6′′ 71.462.63.40 (m, overlapped)3.68 (dd, 5.0, 11.5) 40 146.5 - 500 78.2 3.40 (m, overlapped) 6′ 50 122.4118.3 6.77 7.10 (br. (d, d, 8.0) ca. 8) 600 62.6 3.68 (dd,3.89 5.0, (m, 11.5) overlapped) 60 122.4 6.77 (br. d, ca. 8) 3.89 (m, overlapped) (1R)-2,2,3-Trimethyl-4-hydroxymethylcyclopent-3-ene-1-methyl-O-β-D-glucopyranoside (4), was obtained as a white powder. It had the molecular formula C16H28O7, determined by negative-ion HRESI(1R)-2,2,3-Trimethyl-4-hydroxymethylcyclopent-3-ene-1-methyl-O--TOF-MS (m/z 377.1814 [M + COOH]−, calcd for C17H29O9, 377.1817β-D).-glucopyranoside Its IR spectrum showed (4), was absorptionobtained as ban a whiteds at powder. 3367, 1636 It had, and the 1076 molecular cm−1 ascribable formula Cto16 H hydroxyl,28O7, determined olefin, and by negative-ionO-glycosidic − linkageHRESI-TOF-MSs, respectively. (m/z 377.1814 It was reated [M + COOH] with 1 M, calcdHCl to for give C17 HD-29glucoseO9, 377.1817). [4]. The Its 1H, IR 13 spectrumC-NMR (Table showed 4) −1 spectraabsorption showed bands signals at 3367, assignable 1636, and 1076to three cm methylascribable [δ 0.87, to hydroxyl,1.09, 1.56 olefin,(3H each, and Oall-glycosidic s, 2β, 2α, linkages,3-CH3)], tworespectively. methylene It waswith reated oxygen with function 1 M HCl {[δ to3.66 give (1H,D-glucose m, overlapped), [4]. The 13.95H, 13 (1H,C-NMR dd, J (Table = 6.5,4 )11.0 spectra Hz), 1showed-CH2OH], signals 4.07 (2H, assignable d, J = to9.0 three Hz, 4 methyl-CH2OH)}, [δ 0.87, one 1.09,β-D-glucopyranosyl 1.56 (3H each, all[δ s,4.26 2β (1H,, 2α, d, 3-CH J = 37.5)], twoHz, Hmethylene-1′)], one withmethylene oxygen [ functionδ 2.09 (1H, {[δ dd,3.66 J (1H, = 8.0, m, 9.0 overlapped), Hz), 2.49 (1H, 3.95 (1H,dd, J dd, = 8.0,J = 6.5,8.0 Hz), 11.0 Hz),H2-5], 1-C togetherH2OH], 0 with4.07 (2H, one d, methineJ = 9.0 [ Hz,δ 2.15 4-C (1H,H2OH)}, m, overlapped, one β-D-glucopyranosyl H-1)]. The 1H [1δH4.26 COSY (1H, experiment d, J = 7.5 Hz, suggested H-1 )], one the presencemethylene of [ δtwo2.09 partial (1H, dd, fragmentsJ = 8.0, 9.0 shown Hz), 2.49 in bold (1H, lines dd, J (Fig= 8.0,ure 8.0 5). Hz), Then, H2 -5],the togetherplanar structur with onee of methine 4 was 1 1 further[δ 2.15 (1H, elucidated m, overlapped, by the long H-1)].-range The correlationsH H COSY fromexperiment δH 0.87 suggested (2β-CH3)the to presenceδC 27.2 (2α of- twoCH3 partial), 49.0 (Cfragments-1), 49.3 shown(C-2), 143.5 in bold (C- lines3); δH (Figure 1.09 (2α5).-CH Then,3) to the δC planar20.1 (2β structure-CH3), 49.0 of (C4 was-1), 49.3 further (C-2), elucidated 143.5 (C by-3); theδH 1.56long-range (3-CH3) correlations to δC 49.3 (C from-2), 133.1δH 0.87 (C (2-4),β -CH143.53) (C to-δ3);C 27.2δH 4.07 (2α -CH(4-C3H),2OH) 49.0 (C-1),to δC 36.6 49.3 (C (C-2),-5), 133.1 143.5 (C (C-3);-4), 143.5δH 1.09 (C (2-3);α-CH δH 2.15,3) to 2.48δC 20.1 (H2 (2-5)β -CHto δC3 ),49.3 49.0 (C (C-1),-2), 72.1 49.3 (1 (C-2),-CH2OH), 143.5 133.1 (C-3); (CδH-4),1.56 143.5 (3-CH (C3-)3); to δδHC 4.2649.3 ( (C-2),H-1′) 133.1to δC 72.1 (C-4), (1 143.5-CH2OH) (C-3); observedδH 4.07 (4-C in theH2 OH)HMBC to δ sCpe36.6ctrum. (C-5), To 133.1 determine (C-4), 143.5the sterostru (C-3); δHcture2.15, of 2.48 it, (H4 was2-5) 0 treatedto δC 49.3 (C-2), 72.1with (1-CH 2OH),β-glucosidase 133.1 (C-4), 143.5, (C-3);toδ H 4.26 (H-1 give) to δC 72.1 the (1-CH 2OH) observed aglycon, (in1R the)-2,2,3 HMBC-trimethyl spectrum.-4-hydroxymethylcyclopent To determine the sterostructure-3-ene-1-methanol of it, 4 was (4a) treated, which with wasβ -glucosidase, obtained with to negativegive the aglycon, optical rotation (1R)-2,2,3-trimethyl-4-hydroxymethylcyclopent-3-ene-1-methanol ([α]D −6.5°, CHCl3) and had only one chiral carbon. Using (4 a), the which the sam wase ◦ methodobtained reported with negative in literatures optical rotation [25,26 ([],α c]omparedD −6.5 , CHCl optical3) and rotation had only of 4a one with chiral that carbon. of its Using simialr the compound,the same method (–)-(R reported)-γ-necrodol in literatures ([α]D − [21.225,26°, ], CHCl compared3) [27 optical], the rotation absolute of configuration4a with that of of its simialr4 was ◦ elucidatedcompound, to (–)-( beR 1)-Rγ.-necrodol Finally, the ([α ]chemicalD −21.2 ,shift CHCl of3)[ two27], methyl the absolute at the configuration 2-position was of 4 determinedwas elucidated by NOEto be 1correlationsR. Finally, the displayed chemical in shift NOESY of two experiment methyl at the. On 2-position the basis was of above determined mentioned by NOE evidences, correlations the structuredisplayed in NOESY experiment. of On the4 basis of abovewas mentioned evidences, identified the structure of 4 was as (1identifiedR)-2,2,3- astrimethyl (1R)-2,2,3-trimethyl-4-hydroxymethylcyclopent-3-ene-1-methyl--4-hydroxymethylcyclopent-3-ene-1-methyl-O-β-D-glucopyranoside.O-β-D-glucopyranoside.

Figure 5. The main 1H 1H COSY, HMBC correlations of 4 and 4a. Figure 5. The main 1H 1H COSY, HMBC correlations of 4 and 4a.

Molecules 2017, 22, 1309 7 of 13

1 13 Table 4. H and C-NMR data for 4 and 4a in CD3OD.

4 4a

No. δC δH (J in Hz) δC δH (J in Hz) 1 49.0 2.15 (m, overlapped) 51.7 2.00 (m) 2 49.3 - 49.0 - 3 143.5 - 143.7 - 4 133.1 - 133.2 - 5α 36.6 2.15 (dd, 8.0, 9.0) 36.5 2.09 (dd, 8.0, 9.0) 5β 2.48 (dd, 8.0, 8.0) 2.49 (dd, 8.0, 8.0) 1-CH2OH 72.1 3.66 (m, overlapped) 64.2 3.54 (dd, 8.5, 11.0) 3.95 (dd, 6.5, 11.0) 3.72 (dd, 6.5, 11.0) 2α-CH3 27.2 1.09 (s) 27.3 1.08 (s) 2β-CH3 20.1 0.87 (s) 19.9 0.85 (s) 3-CH3 9.4 1.56 (s) 9.4 1.56 (s) 4.06, 4.10 (both d, 4-CH OH 59.4 4.07 (m) 59.4 2 12.0) 10 104.5 4.26 (d, 7.5) 20 75.2 3.17 (dd, 7.5, 8.0) 30 78.3 3.34 (dd, 8.0, 8.0) 40 71.7 3.29 (m, overlapped) 50 78.0 3.29 (m, overlapped) 60 62.8 3.66 (m, overlapped) 3.87 (br. d, ca. 12)

(3Z)-3-Hexene-1,5-diol 1-O-α-L-arabinopyranosyl(1→6)-β-D-glucopyranoside (5) was obtained 25 ◦ as a white powder with negative optical rotation [[α]D −14.5 (c 0.11, MeOH)]. Its molecular − formula, C17H30O11 (m/z 455.1773 [M + COOH] ; calcd for C18H31O13, 455.1770), was recorded by Q-TOF-ESI-MS. Furthermore, using acid hydrolysis and HPLC analysis, the presence of D-glucose and L-arabinose in 5 was revealed [4]. The 1H, 13C-NMR spectra (Table5) and 2D NMR ( 1H 1H COSY, HSQC, HMBC) spectra indicated the presence of two oleﬁnic protons [δ 5.45, 5.47 (1H each, both m, H-3 and 4)], one methoxyl [δ 1.20 (3H, d, J = 6.0 Hz, H3-6)], one β-D-glucopyranosyl [δ 4.27 (1H, d, 0 00 J = 7.5 Hz, H-1 )], along with one α-L-arabinopyranosyl [δ 4.31 (1H, d, J = 6.5 Hz, H-1 )]. The planar structure of 5 was constructed on the basis of 1H 1H COSY and HMBC experiments. Namely, the 1H 1H COSY experiment suggested the existence of three partial structures, as shown as bold lines in Figure6. 0 Meanwhile, in its HMBC spectrum, long-rang correlations from δH 4.27 (H-1 ) to δC 70.4 (C-1); δH 4.31 00 0 (H-1 ) to δC 69.6 (C-6 ) were observed. Finally, the NOE correlation between δH 2.39, 2.46 (H2-2), and δH 4.61 (H-5); 5.45 (H-3) and δH 5.47 (H-5) found in the NOESY spectrum indicated the conﬁguration in the 3-position was Z. Consequently, the structure of 5 was elucidated to be (3Z)-3-hexene-1,5-diol 1-O-α-L-arabinopyranosyl(1→6)-β-D-glucopyranoside.

1 13 Table 5. H and C-NMR data for 5 in CD3OD.

No. δC δH (J in Hz) No. δC δH (J in Hz) 3.61 (dd, 6.5, 11.5) 40 71.7 3.33 (dd, 8.0, 8.0) 1 70.4 3.86 (m, overlapped) 50 76.9 3.43 (m) 2.39 (m) 60 69.6 3.72 (dd, 5.5, 11.0) 2 29.3 2.46 (m) 4.09 (dd, 2.0, 11.0) 3 127.4 5.45 (m) 100 105.2 4.31 (d, 6.5) 4 137.0 5.47 (m) 200 72.4 3.58 (dd, 6.5, 9.0) 5 64.4 4.61 (m) 300 74.2 3.52 (dd, 3.5, 9.0) 6 23.9 1.20 (d, 6.0) 400 69.5 3.80 (m) 10 104.4 4.27 (d, 7.5) 500 66.7 3.53 (dd, 2.0, 12.5) 20 75.1 3.17 (dd, 7.5, 8.0) 3.86 (m, overlapped) 30 78.0 3.34 (dd, 8.0, 8.0) Molecules 2017, 22, 1309 8 of 13 Molecules 2017, 22, 1309 8 of 13

1 1 Figure 6. TheThe main main 1HH 1HH COSY, COSY, HMBC HMBC,, and and NOE NOE correlations correlations of of 55..

Furthermore,Furthermore, inhibitory effectseffects ofof all all fractions fractions obtained obtained from from 70% 70% EtOH EtOH extract extract of G.of acutaG. acutanda and the theabovementioned abovementioned isolates isolates on motilityon motility of mouse of mouse isolated isolated intestine intestine tissue tissue were were determined determined by usingby using the thesame same method method as reported as reported previously previously [4,28]. [ As4,28 results,]. As results, all of the all test of samplesthe test showed samples no showed significant no significantchanging on changing isolated on intestinal isolated tissueintestinal contraction tissue contraction frequency, frequency while 70%, w EtOHhile 70% extract EtOH of extractG. acut ,of 95% G. acutEtOH, 95% eluate EtOH from elu D101ate from macroporous D101 macroporous resin CC, resin CHCl CC,3 layer, CHCl as3 well layer, as as compounds well as compounds5, 7, 10, 12 5–,14 7,, 1016,, 1217–, 1431,, 16 and, 1732, 31displayed, and 32 displayed significant significant inhibitory inhibitory effects on contractioneffects on contraction tension (Table tension6). (Table 6). FromFrom the whole pplantslants of G. acutacutaa, two two types of monoterpenes,monoterpenes, iridoid iridoid-- ( (11,, 2222––2626)) and and secoiridoidsecoiridoid-type-type (7 (7––2121)) monoterpenes monoterpenes were were obtained. obtained. Structure-activity Structure-activity relationship relationship analysis revealedanalysis revealedthat iridoid-type that iridoid monoterpenes-type monoterpenes showed no showed significant no significant effect on contraction effect on contraction tension. However, tension. However,secoiridoid-type secoiridoid monoterpenes,-type monoterpenes, such as 7, 10 such, 12 –as14 ,716, 10, 17, 12had–14 strong, 16, 17 inhibitory had strong effect. inhibitory Furthermore, effect. Furthermorewhen an olefin, when functional an olefin existed function betweenal existed the 5- between and 6-positions the 5- and (2, 620-position, 21), ors H-5 (2, 20 was, 21 substituted), or H-5 was by substitutedhydroxyl (18 by, 19 hydroxyl), the bioactivity (18, 19), the disappeared. bioactivity disappeared. Meanwhile,Meanwhile, comparing comparing the the inhibitory inhibitory effect effect on contractionon contraction tension tension of 8- O of-4 0 8-(-O27-),4′- 7- (O27-4),0 -( 728-O–-304′-) (with28–30 those) with of those 7-O-9 of0-type 7-O-9 (31′-type, 32) ( lignan,31, 32) lignan, we found we thatfound 7-O that-90-type 7-O-9 (′31-type, 32 )(31 lignan, 32) lignan displayed displayed strong stronginhibitory inhibitory bioactivity bioactivity on contraction on contraction tension. tension.

TableTable 6. Inhibitory 6. Inhibitory effects effects of fractions of fractions and compounds and compounds 1–32 on motility1–32 on of mouse motility isolated of mouse intestine isolated tissue. intestine tissue. Intestine Motility (%) Intestine Motility (%)

Relative Tension Relative Frequency Relative Tension Relative Frequency Intestine Motility (%) Intestine Motility (%) N 100.0 ± 4.0 100.0 ± 7.3 14 90.1 ± 2.6 * 102.1 ± 1.9 P 74.1Relative ± 9.3 * Tension82.7 Relative ± 5.3 Frequency * 15 88.9 Relative ± 5.0 Tension Relative104.7 Frequency ± 2.3 A N83.1 ± 100.03.4 * ± 4.097.5 100.0 ± 3.6± 7.3 16 14 80.5 ±90.1 6.1 ±* 2.6 * 102.1 ± ± 1.95.8 ± ± 15 ± ± B P93.9 ± 74.1 4.2 9.3 *103.2 82.7 ± 3.1 5.3 * 17 64.9 ± 88.97.1 ** 5.0 104.794.3 ± 2.02.3 A 83.1 ± 3.4 * 97.5 ± 3.6 16 80.5 ± 6.1 * 102.1 ± 5.8 C B74.5 ± 3.8 93.9 ***± 4.299.3 103.2 ± 5.3± 3.1 18 17 88.264.9 ± 4.9± 7.1 ** 94.386.4 ±± 2.08.8 D C78.4 ± 74.53.5 ***± 3.8 ***103.2 99.3 ± 4.4± 5.3 19 18 93.7 ±88.2 7.1 ± 4.9100.3 86.4 ± ±8.8 2.4 1 D93.2 78.4± 3.5± 3.5 ***102.0 103.2 ± 3.7± 4.4 20 19 85.8 ±93.7 5.0 ± 7.1 100.398.2 ± 1.42.4 2 1 88.2 ± 93.23.4 ± 3.597.7 102.0 ± 4.9± 3.7 21 20 86.8 ±85.8 4.6 ± 5.0102.2 98.2 ± ±1.4 3.2 2 88.2 ± 3.4 97.7 ± 4.9 21 86.8 ± 4.6 102.2 ± 3.2 3 3 88.8 ± 88.85.1 ± 5.1113.0 113.0 ± 14.0± 14.0 22 22 89.6 ±89.6 4.4 ± 4.4 112.0 ± ± 8.1 4 4 100.4 ±100.4 2.0 ± 2.0100.4 100.4 ± 2.0± 2.0 23 23 91.2 ±91.2 2.7 ± 2.7 103.6 ± ± 7.4 5 5 81.6 ± 81.62.9 **± 2.9 **98.5 98.5± 0.9± 0.9 24 24 91.0 ±91.0 3.5 ± 3.5 101.0 ± ± 4.4 ± ± ± ± 6 6 89.5 ± 89.57.0 7.0100.9 100.9 ± 5.5 5.5 25 25 91.0 ±91.0 3.8 3.8 102.1 ± 2.6 7 82.9 ± 7.0 * 100.8 ± 2.6 26 84.8 ± 4.7 105.9 ± 1.6 7 82.9 ± 7.0 * 100.8 ± 2.6 26 84.8 ± 4.7 105.9 ± 1.6 8 86.6 ± 4.6 85.2 ± 3.0 27 77.4 ± 7.4 100.8 ± 5.6 8 9 86.6 ± 91.84.6 ± 3.385.2 100.7 ± 3.0± 2.3 27 28 77.4 76.7± 7.4± 10.1100.8 97.4 ± ±3.5 5.6 9 10 91.8 ±75.5 3.3± 6.4 **100.7 99.7 ± 2.3± 1.4 28 29 76.7 ±90.4 10.1± 9.0 102.697.4 ± 3.51.0 10 11 75.5 ± 6.488.9 **± 5.099.7 98.4± 1.4± 3.3 29 30 90.4 ±91.0 9.0 ± 6.3102.6 95.9 ± ±3.3 1.0 ± ± ± ± 11 12 88.9 ±75.7 5.0 9.1 *98.4 96.6± 3.3 4.6 30 31 91.079.1 ± 6.3 3.7 ** 102.395.9 ± 3.35.2 13 84.9 ± 4.0 * 100.3 ± 1.4 32 82.4 ± 4.7 * 92.2 ± 1.3 12 75.7 ± 9.1 * 96.6 ± 4.6 31 79.1 ± 3.7 ** 102.3 ± 5.2 ± 13Values are84.9 the ± means 4.0 * standard error100.3 of ± measurement,1.4 32 significantly82.4 different ± 4.7 * from the control92.2 group, ± 1.3 * p < 0.05, ** p < 0.01, *** p < 0.001, n = 6. Normal (N): isolated intestine tissue; Positive control (P): Loperamide hydrochloride, Valuesfinal concentration are the means was 10 ±µ M. stand A: G.ard acuta error70% of EtOH measurement, extract; B: H significantly2O eluate from different D101 resin from for extract; the control C: 95% group,EtOH eluate * p < from0.05, D101** p < resin 0.01, for *** extract; p < 0.001, D: CHCl n =3 6.layer Normal for extract, (N): isolated and their intestine final concentration tissue; Positive was 100 controlµg/mL. Compounds 1–32: final concentration was 40 µM. Tension and frequency of normal group was set as 100%, relative (P): Loperamide hydrochloride, final concentration was 10 μM. A: G. acuta 70% EtOH extract; B: H2O tension, and frequency were calculated as: (sample/normal) × 100%. eluate from D101 resin for extract; C: 95% EtOH eluate from D101 resin for extract; D: CHCl3 layer for extract, and their final concentration was 100 μg/mL. Compounds 1–32: final concentration was 40 μM. Tension and frequency of normal group was set as 100%, relative tension, and frequency were calculated as: (sample/normal) × 100%.

Molecules 2017, 22, 1309 9 of 13

3. Experimental

3.1. General Physical data was obtained by using the following instruments: UV and IR spectra were determined on a Varian Cary 50 UV-VIS (Varian, Inc., Hubbardsdon, MA, USA) and Varian 640-IR FT-IR spectrophotometer (Varian Australia Pty Ltd., Mulgrave, Australia), respectively. Optical rotations were obtained on a Rudolph Autopol® IV automatic polarimeter (l = 50 mm) (Rudolph Research Analytical, 55 Newburgh Road, Hackettstown, NJ, 07840 USA). NMR spectra were run on a Bruker 500 MHz NMR spectrometer (Bruker BioSpin AG Industriestrasse 26 CH-8117, Fällanden, Switzerland) at 500 MHz for 1H and 125 MHz for 13CNMR (internal standard: TMS). Negative-ion HRESI-TOF-MS were recorded on an Agilent 6520 Accurate-Mass Q-Tof LC/MS spectrometer (Agilent Corp., Santa Clara, CA, USA). Column chromatographies (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 Healthcare Bio-Sciences, Uppsala, Sweden). Preparative high-performance liquid chromatography (PHPLC) column, cosmosil 5C18-MS-II (20 mm i.d. × 250 mm, Nakalai Tesque, Inc., Tokyo, Japan) were used to isolate the compounds.

3.2. Plant Material The whole plants of Gentianella acuta (Michx.) Hulten were collected from Alxa Youqi, Inner Mongolia Autonomous region, China in September 2013, 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.

3.3. Extraction and Isolation The whole plants of G. acuta (3.0 kg) were cut and refluxed with 70% ethanol–water. Then the 70% EtOH extract (868.5 g) was partitioned in a CHCl3–H2O mixture (1:1, v/v). The H2O layer (670.0 g) was subjected to D101 macroporous resin CC (H2O → 95% EtOH → acetone). As a result, H2O (332.4 g), 95% EtOH (294.9 g), and acetone (5.1 g) eluates were obtained. The 95% EtOH eluate (200.0 g) was subjected to silica gel CC [CHCl3 → CHCl3-MeOH (100:1 → 100:5, v/v) → CHCl3–MeOH–H2O (10:3:1 → 7:3:1 → 6:4:1, v/v/v, lower layer)] to give 16 fractions (Fr. 1–Fr. 16). Fraction 7 (25.7 g) was centrifugated (MeOH), and two fractions (Fr. 7-1–Fr. 7-2) were yielded. Fraction 7-2 (9.8 g) was separated by PHPLC [CH3CN–H2O (18:82 → 35:65 → 42:58, v/v) + 1% HAc] to give 27 fractions (Fr. 7-2-1–Fr. 7-2-27). Fraction 7-2-2 (70.6 mg) was purified by PHPLC [MeOH–H2O (22:78, v/v)], and 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one (6, 5.9 mg) was given. Fraction 7-2-8 (46.3 mg) was isolated by PHPLC [CH3CN-H2O (20:80, v/v) + 1% HAc] to gain (7R,8S)-erythro-4,7,9,90-tetrahydroxy-3,30-dimethoxy-8-O-40-neolignan (27, 6.0 mg). Fraction 9 (15.0 g) was subjected to Sephadex LH-20 CC [CHCl3–MeOH (1:1, v/v)] to yield seven fractions (Fr. 9-1–Fr. 9-7). Fraction 9-4 (7.3 g) was separated by PHPLC [CH3CN–H2O (22:78 → 30:70 → 45:55, v/v) + 1% HAc], as a result, 12 fractions (Fr. 9-4-1–Fr. 9-4-12) were obtained. Fraction 9-4-2 (962.8 mg) was purified by PHPLC [MeOH–H2O (23:77, v/v) + 1% HAc] to give gentiiridoside B (2, 14.8 mg). Fraction 9-4-9 (277.1 mg) was centrifuged (MeOH), and two fractions (Fr. 9-4-9-1–Fr. 9-4-9-2) were gained. Fraction 9-4-9-2 (199.7 mg) was isolated by PHPLC [MeOH–H2O (45:55, v/v) + 1% HAc] to yield plucheoside D3 (29, 8.8 mg). Fraction 11 (20.0 g) was subjected to PHPLC [CH3CN–H2O (15:85 → 25:75 → 42:58, v/v) + 1% HAc], and 29 fractions (Fr. 11-1–Fr. 11-29) were given. Fraction 11-5 (631.3 mg) was separated by PHPLC [CH3CN–H2O (10:90, v/v) + 1% HAc], and 8-epiloganin (24, 29.7 mg) was yielded. Fraction 11-6 (388.0 mg) was isolated by PHPLC [CH3CN–H2O (11:89, v/v) + 1% HAc] to yield gentilignanoside A (3, 9.2 mg), loganin 0 (23, 143.5 mg), and berchemol-4 -O-β-D-glucoside (32, 84.5 mg). Fraction 11-12 (660.5 mg) was centrifugated (MeOH) and further purified by PHPLC [MeOH–H2O (35:65, v/v) + 1% HAc] to give (1R)-2,2,3-trimethyl-4-hydroxymethylcyclopent-3-ene-1-methyl-O-β-D-glucopyranoside (4, 11.5 mg). Molecules 2017, 22, 1309 10 of 13

Fraction 11-17 (790.7 mg) was isolated by PHPLC [MeOH–H2O (42:58, v/v) + 1% HAc] to obtain six fractions (Fr. 11-17-1–Fr. 11-17-6). Fraction 11-17-1 (97.1 mg) was purified by PHPLC [CH3CN-H2O (24:76, v/v) + 1% HAc] to gain swertiaside (26, 79.0 mg). Fraction 11-21 (376.1 mg) was separated by 0 PHPLC [MeOH–H2O (42:58, v/v) + 1% HAc] to afford (7S,8R)-9 -methoxy-dehydrodiconiferyl alcohol 4-O-β-D-glucopyranoside (30, 17.4 mg). Fraction 13 (20.0 g) was subjected to PHPLC [MeOH-H2O (35:65 → 45:55 → 55:45, v/v) + 1% HAc], and 20 fractions (Fr. 13-1–Fr. 13-20) were yielded. Fraction 13-11 (1.9 g) was centrifuged (MeOH) to obtain two fractions (Fr. 13-11-1–Fr. 13-11-2). Fraction 13-11-1 (1.0 g) was isolated by PHPLC [CH3CN–H2O (16:84, v/v) + 1% HAc], as a result, eleven fractions (Fr. 13-11-1-1–Fr. 13-11-1-11) were given. Fraction 13-11-1-9 (117.1 mg) were purified by Sephadex LH-20 CC (MeOH) and PHPLC [MeOH–H2O (35:65, v/v) + 1% HAc] to afford 5α-carboxystrictosidine (12, 26.2 mg). Fraction 13-12 (501.4 mg) was isolated by PHPLC [CH3CN–H2O (18:82, v/v) + 1% HAc] and to yield (E)-aldosecologanin (11, 38.2 mg). Fraction 13-17 (504.0 mg) was purified by PHPLC [CH3CN–H2O (22:78, v/v) + 1% HAc] to yield gentiiridoside A (1, 265.0 mg). Fraction 14 (15.3 g) was subjected to PHPLC [CH3CN–H2O (15:85 → 25:75, v/v) + 1% HAc], and 20 fractions (Fr. 14-1–Fr. 14-20) were obtained. Fraction 14-1 (1.4 g) was separated by PHPLC [CH3CN–H2O (9:91, v/v) + 1% HAc] to gain 14 fractions (Fr. 14-1-1–Fr. 14-1-14). Fraction 14-1-2 (32.1 mg) was purified by Sephadex LH-20 CC (MeOH) and finally by PHPLC [CH3CN–H2O (7:93, v/v)] to give (3Z)-3-hexene-1,5-diol 1-O-α-L-arabinopyranosyl(1→6)-β-D-glucopyranoside (5, 2.8 mg). The CHCl3 layer (50.0 g, Fr. C) was subjected to Silica gel CC [CHCl3–MeOH (100:2 → 100:3 → 100:5, v/v) → CHCl3–MeOH–H2O (10:3:1, v/v/v, lower layer) → MeOH], and eight fractions (Fr. C-1–Fr. C-8) were yielded. Fraction C-5 (1.1 g) was separated by Sephadex LH-20 CC [MeOH–CH2Cl2 (1:1, v/v)] to gain three fractions (Fr. C-5-1–Fr. C-5-3). Fraction C-5-2 (110.0 mg) was purified by PHPLC [MeOH-H2O (45:55, v/v)] to afford (7S,8R)-dehydrodiconiferyl alcohol (28, 13.3 mg) and (–)-berchemol (31, 14.6 mg). Fraction C-7 (9.0 g) was isolated by ODS CC [MeOH–H2O (20:80 → 30:70 → 40:60 → 50:50, v/v) → MeOH], and eight fractions (Fr. C-7-1–Fr. C-7-8) were obtained. Fraction C-7-2 (351.4 mg) was purified by PHPLC [MeOH–H2O (32:68, v/v) + 1% HAc] to give gentiopicroside (20, 15.9 mg). Fraction C-7-6 (486.4 g) was subjected to Sephadex LH-20 CC [MeOH–CH2Cl2 (1:1, v/v)] and PHPLC [MeOH–H2O (50:50, v/v) + 1% HAc] to afford trifloroside (17, 16.0 mg). Compounds 7–10, 13–16, 18, 19, 21, 22 and 25 were obtained and identified by using the method reported previously [2,3].

25 ◦ Gentiiridoside A (1): White powder; [α]D −90.0 (c 0.14, MeOH); UV λmax (MeOH) nm (log ε): 229 (4.46), 285 (3.53); IR νmax (KBr): 3372, 2929, 1712, 1635, 1588, 1486, 1372, 1264, 1201, 1154, 1078, 1017, −1 1 13 902, 873 cm ; H-NMR (CD3OD, 500 MHz) and C-NMR (CD3OD, 125 MHz) data, see Table1. − HRESI-TOF-MS negative-ion mode m/z 777.2261 [M – H] (calcd for C36H41O19, 777.2248). 25 ◦ Gentiiridoside B (2): White powder; [α]D −36.7 (c 0.12, MeOH); UV λmax (MeOH) nm (log ε): 231 (4.26, sh), 273 (4.08, sh); IR νmax (KBr) 3357, 2924, 1705, 1609, 1518, 1457, 1418, 1375, 1272, 1209, 1074, −1 1 13 1024 cm ; H-NMR (DMSO-d6, 500 MHz) and C-NMR (DMSO-d6, 125 MHz) data see Table2. – HRESI-TOF-MS negative-ion mode m/z 679.1023 [M – H] (calcd for C28H39O18, 679.1033). 25 ◦ Gentilignanoside A (3): White powder; [α]D −36.0 (c 0.10, MeOH); CD (c 0.0018 M, MeOH) mdeg (λnm): −3.8 (278), −16.9 (232), −27.6 (206); UV λmax (MeOH) nm (log ε): 226 (4.32), 275 (3.73); IR −1 1 νmax (KBr): 3368, 2937, 1613, 1514, 1463, 1425, 1324, 1266, 1224, 1158, 1115, 1073, 1026 cm ; H-NMR 13 (CD3OD, 500 MHz) and C-NMR (CD3OD, 125 MHz) data see Table3. HRESI-TOF-MS negative-ion − − mode m/z 679.1023 [M – H] m/z 567.2083 [M – H] (calcd for C27H33O13, 567.2083).

(1R)-2,2,3-Trimethyl-4-hydroxymethylcyclopent-3-ene-1-methyl-O-β-D-glucopyranoside (4): White powder; 25 ◦ −1 [α]D −43.9 (c 0.12, MeOH); IR νmax (KBr): 3367, 2927, 1636, 1576, 1436, 1286, 1161, 1076, 1038 cm . 1 13 H-NMR (CD3OD, 500 MHz) and C-NMR (CD3OD, 125 MHz) data see Table4. HRESI-TOF-MS – negative-ion mode m/z 377.1814 [M + COOH] (calcd for C17H29O9, 377.1817). Molecules 2017, 22, 1309 11 of 13

25 (3Z)-3-Hexene-1,5-diol 1-O-α-L-arabinopyranosyl(1→6)-β-D-glucopyranoside (5): White powder; [α]D ◦ −1 −14.5 (c 0.11, MeOH); IR νmax (KBr): 3364, 2966, 2920, 1593, 1419, 1370, 1258, 1166, 1047, 1009 cm ; 1 13 H-NMR (CD3OD, 500 MHz) and C-NMR (CD3OD, 125 MHz) data see Table5. HRESI-TOF-MS – negative-ion mode m/z 455.1773 [M + COOH] (calcd for C18H31O13, 455.1770).

Enzymatic Hydrolysis of 4 A solution of 4 (6.0 mg) in H2O (2.0 mL) was reacted with β-glucosidase (6.0 mg, Almond, Sigma-Aldrich, Co. 3050 Spruce Street, St. Louis, MO, 63103 USA) at 37 ◦C for 2.5 h. Then the reaction mixture was extracted with EtOAc. And the residue from EtOAc solvent was subjected to Silica gel CC [CHCl3–MeOH (100:5, v/v)], as a result, the aglycon, (1R)-2,2,3-trimethyl-4-hydroxymethylcyclopent-3-ene-1-methanol (4a, 2.8 mg, 93.33%) was yielded.

25 ◦ (1R)-2,2,3-Trimethyl-4-hydroxymethylcyclopent-3-ene-1-methanol (4a): White powder; [α]D −6.5 (c 0.09, CHCl3); IR νmax (KBr): 3318, 2953, 2925, 2867, 1717, 1576, 1462, 1437, 1380, 1240, 1179, 1118, −1 1 13 1087, 999 cm ; H-NMR (CD3OD, 500 MHz) and C-NMR (CD3OD, 125 MHz) data see Table4. − HRESI-TOF-MS negative-ion mode m/z 170.1423 [M – H] (calcd for C10H17O2, 170.1433). Acid Hydrolysis of 1–5 The solution of compounds 1–5 (each 2.0 mg) in 1 M HCl (1.0 mL) was treated by using the same method as described in reference [4]: They were heated under reﬂux for 3 h. The reaction mixture was then analyzed by CH3CN–H2O (75:25, v/v; ﬂow rate 0.7 mL/min). As results, D-glucose was detected from the aqueous phase of 1–5, and L-arabinose was found from that of 5 by comparison of its retention time and optical rotation with those of the authentic sample, D-glucose (tR 16.8 min (positive)) and L-arabinose (tR 13.1 min (positive)), respectively.

3.4. Inhibitory Effects of Fractions and Compounds 1–32 on the Motility of Mouse Isolated Intestine Tissue Inhibitory effects of fractions and compounds 1–32 on motility of mice isolated intestine tissue were determined by using the similar method as we reported previously [4,28]: Mice were fasted for 12 h before experiments, intestinal tissue were 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 intestinal tissue 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 to equilibrate incubation, the final DMSO concentration was 0.1%, and the final concentration of fractions and compounds was 100 µg/mL and 40 µM, respectively. The mean tension and frequency of intestine muscle contractions were recorded for 1 min before and 4 min after drug additions using isolated tissue bath systems (Radnoti Glass Technology Inc., Monrovia, CA, 159901A, USA). Loperamide hydrochloride (Xi’an Janssen Pharmaceutical Ltd., Xi’an, China) was used as a positive control, and the final concentration was 10 µM. Values are expressed as mean ± S.D. All the grouped data were statistically performed with SPSS 11.0. Significant differences between means were evaluated by one-way analysis of variance (ANOVA) and Tukey’s Studentized range test was used for post hoc evaluations. p < 0.05 was considered to indicate statistical significance.

4. Conclusions In summary, nineteen constituents, including five new ones, gentiiridosides A (1), B (2), gentilignanoside A (3), (1R)-2,2,3-trimethyl-4-hydroxymethylcyclopent-3-ene-1-methyl-O-β-D- glucopyranoside (4), and (3Z)-3-hexene-1,5-diol 1-O-α-L-arabinopyranosyl(1→6)-β-D-glucopyranoside (5) were obtained from the whole plants of G. acuta in our on-going program of screening the phytochemical and bioactive constituents. Among the known isolates, 6, 11, 12, 17, 23, and 27–32 were isolated from the genus firstly. The structures of them were elucidated by chemical and spectroscopic methods. Furthermore, the inhibitory effects on motility of mouse isolated intestine tissue of the above mentioned compounds and other thirteen isolates (7–10, 13–16, 18, 19, 21, 22 and 25) previously Molecules 2017, 22, 1309 12 of 13 obtained in the plant were analyzed. Resultingly, 5, 7, 10, 12, 14, 16, 17, 31, and 32 displayed significant inhibitory effects on contraction tension. On the other hand, structure-activity relationship analysis revealed that the inhibitory effect of secoiridoid-type monoterpenes was stronger than that of iridoid-type monoterpenes, and 7-O-90-type lignan displayed stronger inhibitory bioactivity than 8-O-40- and 7-O-40-type lignan. Our previous [4–6] and present research results suggested that the chemical constituents in G. acuta were xanthones, monoterpenes, lignans, and phenolic acids. Among them, xanthones and part of the monoterpenes were major bioactivity substances of G. acuta. These results suggested that G. acuta and its constituents have potential value in discovering new medicines for abnormal intestinal motility.

Acknowledgments: This work was financially supported by programs for National Natural Science Foundation of China (21405113, 81673688), Important Drug Development Fund, Ministry of Science and Technology of China (2017ZX09305002-002). Author Contributions: Yi Zhang and Tao Wang designed the research and wrote the manuscript; Zhijuan Ding and Yanxia Liu performed the experimental work; Jingya Ruan and Shengcai Yang retrieved the literature; and Haiyang Yu and Meiling Chen perfected the language. All authors discussed, edited, and approved the final version. Conflicts of Interest: The authors declare no conflicts of interest.

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Sample Availability: Samples of all the compounds are available from the authors.

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