Waraluck Chaichompooa,
Pornchai Rojsitthisak*ab,
Wachirachai Pabuprapapc,
Yuttana Siriwattanasathienc,
Pathumwadee Yotmaneec,
Woraphot Haritakund and
Apichart Suksamrarnc
aDepartment of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand. E-mail: pornchai.r@chula.ac.th; Fax: +66-2-254-5195; Tel: +66-2-218-8310
bNatural Products for Aging and Chronic Diseases Research Unit, Chulalongkorn University, Bangkok 10330, Thailand
cDepartment of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand
dProgram in Chemical Technology, Faculty of Science and Technology, Suan Dusit University, Bangkok 10700, Thailand
First published on 15th June 2021
Eight new alkaloids, which are four new tetrahydroprotoberberine alkaloids, stephapierrines A–D (1–4), and four new aporphine alkaloids, stephapierrines E–H (5–8), together with three new naturally occurring alkaloids (9–11) and thirty-four known alkaloids (12–45) were isolated from the tubers of Stephania pierrei Diels. The structures of the new compounds were elucidated by spectroscopic analysis and physical properties. The structures of the known compounds were characterized by comparison of their spectroscopic data with those previously reported. Compound 42 exhibited the strongest acetylcholinesterase (AChE) inhibitory activity, which was more active than galanthamine, the reference drug. Compound 23 showed the highest butyrylcholinesterase (BuChE) inhibitory activity, which was also more active than galanthamine. Molecular docking studies are in good agreement with the experimental results.
Plant-derived natural products have long been and will continue to be extremely important as the most promising source of biologically active compounds. A great potential is expected for indigenous plants to be used as a source of new drugs. Among these natural products, alkaloids are considered to be the most promising candidates for the treatment of AD due to their complex nitrogen-containing structures.11 Many alkaloids that are active cholinesterase inhibitors have already been described in various families, for example, the Menispermaceae family.12
The genus Stephania belongs to the Menispermaceae family, a large family of approximately 65 genera and 350 species, distributed in temperate regions of the world. The plants of the genus Stephania are slender climbers with peltate and membranous leaves. The plants of this genus are commonly used in Asian folk medicine to treat a wide range of biological activities including malaria, fever, dysentery, and tuberculosis.13 At least 15 species in the genus Stephania have been found distributed throughout Thailand.14 Stephania pierrei Diels, known in Thai as Sabu-lueat, is a medicinal plant regularly used in traditional remedies and as herbal medicine to treat body oedema, migraine, and heart disease, which are primarily distributed in Cambodia, Thailand, and Vietnam.15 The reported data showed that several types of alkaloids had been isolated from this plant species, including aporphine, tetrahydroprotoberberine, tetrahydrobenzylisoquinoline, and miscellaneous compounds.16,17 However, the anti-cholinesterase activity of this plant has not been reported. From our preliminary investigation on phytochemicals with cholinesterase inhibitory activities from Thai medicinal plants, we found that the active crude extracts of the tubers of S. pierrei showed inhibitory activities on AChE and BuChE. Accordingly, we report herein the isolation, structure elucidation, and absolute configuration assignments of eight new alkaloids (1–8), and three new naturally occurring alkaloids (9–11), together with thirty-four known alkaloids (12–45) from the tuber extracts of S. pierrei. Most of the isolated compounds were also evaluated for their AChE and BuChE inhibitory activities. For further insight into the experimental results, in silico studies were performed.
Stephapierrine A (1) was assigned the molecular formula C21H23NO5 as deduced from the HR-ESI-TOF-MS at m/z 370.1633 [M + H]+ and NMR data. This alkaloid and all other isolated alkaloids gave positive orange coloration with Dragendorff's reagent. The IR spectrum indicated the presence of a hydroxy group (3380 cm−1), an acetoxy group (1761 cm−1) and aromatic rings (1608 and 1514 cm−1). The 1H NMR data (Table 1) showed two singlet methoxy signals at δH 3.79 and 3.85 (each 3H, s), and an acetoxy methyl proton signal at δH 2.30 (s), together with two ortho-coupled aromatic protons at δH 6.90 and 6.88 (each 1H, d, J = 8.8 Hz, H-11 and H-12) and two aromatic singlet protons at δH 6.78 and 6.58 (1H each, s, H-1 and H-4). The 13C NMR (Table 2) and DEPT spectra showed twenty-one carbon signals which included twelve aromatic carbons between δC 110.6 and 147.7, one carbonyl carbon (δC 169.1), three methyl carbons (δC 20.8, 55.9 and 60.6), four methylene carbons (δC 28.7, 36.0, 51.3 and 53.5), and one methine carbon (δC 58.8). The seventeen-carbon and one-nitrogen skeleton of 1 suggested a tetraoxygenated tetrahydroprotoberberine alkaloid core structure.45,46 This was further confirmed by the presence of two adjacent methylenes as multiplet signals at δH 2.68 and 3.18 (H-5) and δH 2.68 and 3.21 (H-6), methylene signals at δH 3.59 and 4.22 (each d, J = 15.7 Hz, H-8), methylene signals at δH 2.90 (dd, J = 16.4, 12.2 Hz) and 3.28 (dd, J = 16.4, 3.2 Hz) (H-13), together with a partially overlapping signal at δH 3.64 (H-13a). The COSY correlations of these protons are shown in Fig. 2. The structure of 1 was similar to that of stepholidine (12).21 The main difference between the 1H NMR spectrum of 12 and that of 1 was the presence of an acetoxy methyl signal at δH 2.30 (1H, s) which was proven to be at C-10 by HMBC correlations between COCH3 (δH 2.30) and COCH3 (δC 169.1), and C-10 (δC 141.0) (see Fig. 2). The HMBC correlations between the OH signal (δH 5.52) and C-1 (δC 111.2) and C-2 (δC 144.0) allowed the location of this phenolic function at C-2. The location of the methoxy groups at C-3 and C-9 were assigned according to the correlations of the methoxy signals at δH 3.85 and 3.79 with the carbons at δC 145.2 (C-3) and δC 147.7 (C-9), respectively. The structure of compound 1 was confirmed by key HMBC correlations from H-1 to C-3, C-4a and C-13a, H-4 to C-2 and C-5, H-8 to C-6, C-9, C-12a and C-13a, and H-13 to C-13b (see Fig. 2). The structure of compound 1 was further confirmed by the following key NOESY correlations: CH3O-3/H4, H-6/H-5 and H-8, CH3O-9/H-8, and H-13a/H-1 and H-13 (Fig. 3). The absolute configuration of C-13a of this class of alkaloids has been established, for example, by stereoselective asymmetric synthesis.47 The negative sign of specific optical rotation of 1 is consistent with an α-orientation of hydrogen at C-13a. The absolute configuration of 1 was confirmed by the electronic circular dichroism (ECD) spectrum (Fig. S234†), which was similar to those reported for tetrahydroprotoberberine alkaloids,48,49 with negative Cotton effects at 204 nm (Δε −10.69), 243 nm (Δε −50.55) and 287 nm (Δε −6.61). Thus, the absolute configuration of C-13a was established as S-configuration (Fig. 1). Accordingly, compound 1 was identified as 10-O-acetylstepholidine.
Position | 1b | 2b | 3b | 4c | 11d |
---|---|---|---|---|---|
a Assignments were based on 1H–1H COSY, HMQC, HMBC, and NOESY experiments; chemical shifts (δ) are given in ppm.b Recorded in CDCl3.c Recorded in CD3OD.d Recorded in DMSO-d6.e Overlapping signal.f Partially overlapping signal. | |||||
1 | 6.78 s | 6.89 s | 6.89 s | 6.92 s | 6.68 s |
4 | 6.58 s | 6.68 s | 6.79 s | 6.93 s | 6.63 s |
5 | 2.68 m | 2.71 m | 2.64f | 2.69f | 2.54f |
3.18 m | 3.14e | 3.08f | 3.07 m | 2.89 m | |
6 | 2.68 m | 2.63 m | 2.62f | 2.64f | 2.42f |
3.21 m | 3.15e | 3.15f | 3.20 m | 3.08f | |
8 | 3.59 d (15.7) | 3.51 d (15.7) | 3.54 d (15.9) | 3.35 d (15.6) | 3.35e |
4.22 d (15.7) | 4.17 d (15.7) | 4.18 d (15.9) | 4.19 d (15.6) | 4.02 d (15.7) | |
11 | 6.90 d (8.8) | 6.86 d (8.4) | 6.88 d (9.2) | 6.72 d (8.2) | 6.67 d (8.2) |
12 | 6.88 d (8.8) | 6.86 d (8.4) | 6.88 d (9.2) | 6.80 d (8.2) | 6.71 d (8.2) |
13 | 2.90 dd (16.4, 12.2) | 2.85 dd (16.1, 11.2) | 2.90 dd (16.1, 11.3) | 2.73 dd (16.1, 11.0) | 2.50 dd (15.7, 12.3) |
3.28 dd (16.4, 3.2) | 3.23 dd (16.1, 3.4) | 3.27 dd (16.1, 3.5) | 3.41 dd (16.1, 3.5) | 3.14 dd (15.7, 3.4) | |
13a | 3.64f | 3.55 dd (11.2, 3.4) | 3.60 dd (11.3, 3.5) | 3.58 dd (11.0, 3.5) | 3.34e |
2-OCH3 | 3.82 s | 3.86 s | |||
3-OCH3 | 3.85 s | 3.80 s | |||
9-OCH3 | 3.79 s | 3.79 s | 3.79 s | 3.80 s | 3.71 s |
10-OCH3 | 3.73 s | ||||
2-OCOCH3 | 2.29 s | ||||
3-OCOCH3 | 2.29 s | ||||
10-OCOCH3 | 2.30 s | 2.30 s | 2.31 s | ||
1-OH | 8.68 s | ||||
2-OH | 5.52 s | 9.06 s | |||
1′ | 4.88 d (7.5) | ||||
2′ | 3.45 dd (8.7, 7.5) | ||||
3′ | 3.48 t (8.7) | ||||
4′ | 3.38e | ||||
5′ | 3.38e | ||||
6′ | 3.68 dd (12.1, 5.2) | ||||
3.85 dd (12.1, 1.7) |
Position | 1b | 2b | 2b | 4c | 11d |
---|---|---|---|---|---|
a Assignments were based on 1H–1H COSY, HMQC, HMBC, and NOESY experiments; chemical shifts (δ) are given in ppm.b Recorded in CDCl3.c Recorded in CD3OD.d Recorded in DMSO-d6. | |||||
1 | 111.2 | 119.7 | 121.2 | 111.5 | 114.8 |
2 | 144.0 | 138.0 | 149.3 | 150.0 | 147.3 |
3 | 145.2 | 149.2 | 138.1 | 147.1 | 144.6 |
4 | 110.6 | 112.3 | 122.6 | 118.6 | 111.7 |
4a | 125.5 | 130.0 | 127.1 | 129.2 | 124.7 |
5 | 28.7 | 29.4 | 28.6 | 29.7 | 28.5 |
6 | 51.3 | 51.2 | 51.1 | 53.1 | 51.1 |
8 | 53.5 | 53.7 | 53.8 | 55.3 | 53.5 |
8a | 128.4 | 129.2 | 129.3 | 128.5 | 125.7 |
9 | 147.7 | 147.7 | 147.7 | 145.5 | 143.2 |
10 | 141.0 | 141.0 | 141.0 | 149.3 | 146.0 |
11 | 121.4 | 121.2 | 124.2 | 116.8 | 112.3 |
12 | 124.3 | 124.2 | 124.2 | 125.9 | 123.7 |
12a | 133.4 | 133.7 | 135.9 | 127.6 | 128.3 |
13 | 36.0 | 36.4 | 36.4 | 37.0 | 35.8 |
13a | 58.8 | 58.6 | 59.1 | 61.4 | 58.6 |
13b | 130.3 | 133.1 | 133.6 | 133.4 | 129.9 |
2-OCH3 | 56.0 | 57.5 | |||
3-OCH3 | 55.9 | 55.8 | |||
9-OCH3 | 60.6 | 60.6 | 60.6 | 60.9 | 59.2 |
10-OCH3 | 55.5 | ||||
20.8 | |||||
169.3 | |||||
20.8 | |||||
169.2 | |||||
20.8 | 20.6 | 20.6 | |||
169.1 | 169.2 | 169.2 | |||
1′ | 103.3 | ||||
2′ | 75.4 | ||||
3′ | 78.3 | ||||
4′ | 71.9 | ||||
5′ | 78.7 | ||||
6′ | 63.0 |
The molecular formula of stephapierrine B (2) was determined to be C23H25NO6 on the basis of HR-ESI-TOF-MS (m/z 412.1747 [M + H]+) and NMR data. The IR spectrum showed absorption bands of acetoxy groups (1753 cm−1) and aromatic rings (1623 and 1511 cm−1). The 1H and 13C NMR data (Tables 1 and 2) showed similar spectral features to those of compound 1. The significant difference was the presence of an additional acetoxy group at δH 2.29, and δC 20.8 and 169.3. The HMBC experiments showed correlations between AcO-2 (δH 2.29) and C-2 (δC 138.0), whereas H-1 (δH 6.89) correlated with C-2, C-3 (δC 149.2), C-4a (δC 130.0), C-13a (δC 58.6) and C-13b (δC 133.1) (see Fig. 2). It should be noted that the presence of the acetoxy group at the 2-position resulted in 0.11 and 8.5 ppm down-field shifts of H-1 and C-1 resonances, respectively, as compared with those of compound 1. These observations indicated that the acetoxy group should be located at the 2-position. Compound 2 showed the negative sign of specific optical rotation as that of compound 1, suggesting the same S-configuration or α-orientation of hydrogen at C-13a. This was confirmed by the similar ECD curve of 2 (Fig. S234†) that exhibited similar negative Cotton effects at 206 nm (Δε −14.21), 240 nm (Δε −63.97) and 286 nm (Δε −2.69), respectively. Compound 2 was therefore identified as 2,10-di-O-acetylstepholidine (see Fig. 1).
Stephapierrine C (3) was deduced to have the molecular formula C23H25NO6 by combined analysis of the HR-ESI-TOF-MS m/z 412.1748 [M + H]+ and NMR data. The IR spectrum displayed the absorption bands of acetoxy groups (1758 cm−1) and aromatic rings (1619 and 1512 cm−1). The 1H and 13C NMR data of 3 (Tables 1 and 2) were similar to those of compound 1. The main differences were the presence of an additional acetoxy group (δH 2.29, and δC 20.8 and 169.2) and the methoxy group should be placed at C-2 as was supported by the HMBC correlation between the CH3O-2 and C-2 (δC 149.3) (see Fig. 2). This was confirmed by the NOESY correlation between the CH3O-2 and H-1 (δH 6.89) (see Fig. 3). The location of the acetoxy group at C-3 was also supported by the HMBC correlation of the acetoxy methyl proton (δH 2.29) with C-3 (δC 138.1), together with the correlations of H-4 with C-2, C-3, C-4a (δC 127.1), C-5 (δC 28.6), and C-13b (δC 133.6). Compound 3 exhibited the same negative specific rotation and displayed a similar ECD spectrum as those of compound 1 (Fig. S234†), suggesting the same S-configuration at C-13a. Therefore, compound 3 was established as 3,10-di-O-acetyldiscretamine (see Fig. 1).
The molecular formula of stephapierrine D (4) was determined to be C25H31NO9 from the HR-ESI-TOF-MS (m/z 490.2056 [M + H]+) and NMR data. Its IR data indicated the presence of hydroxy groups (3314 cm−1) and aromatic rings (1611 and 1512 cm−1). The 1H and 13C NMR data of 4 (Tables 1 and 2) were similar to those of discretamine (13), which has also been isolated from S. pierrei in the present work. The significant differences were the presence of an anomeric proton signal at δH 4.88 (1H, d, J = 7.5 Hz, H-1′) along with signals of sugar residue at δH 3.38 (2H, overlapping, H-4′ and H-5′), 3.45 (1H, dd, J = 8.7 and 7.5 Hz, H-2′), 3.48 (1H, t, J = 8.7, H-3′), 3.68 (1H, dd, J = 12.1 and 5.2 Hz, Ha-6′), and 3.85 (1H, dd, J = 12.1 and 1.7 Hz, Hb-6′) in the 1H NMR spectrum, and from the carbon resonances at δC 103.3 (C-1′), 78.29 (C-3′ and C-5′), 75.4 (C-2′), 71.9 (C-4′), and 63.0 (C-6′) in the I3C NMR spectrum. The large coupling constant of the anomeric proton signal together with the remaining characteristic 1H and 13C signals of the sugar residue suggested this sugar moiety to be a β-glucoside.16,50
The anomeric proton signal showed correlation with the anomeric carbon signal at δC 103.3 (C-1′) in the HMQC spectra (Figs. S66–S68†). The sequence of the glucose unit connected to C-3 of the aglycone was deduced from the HMBC correlations of the anomeric H-1′ (δH 4.88) with the aglycone carbon signals at C-3 (δC 147.1), C-3′ (δC 78.3) and C-5′ (δC 78.7), indicating the attachment of β-D-glucose at C-3 (see Fig. 2). In addition, the 1H–1H COSY correlations, H-1′/H-2′, H-2′/H-3′, H-3′/H-4′, H-4′/H-5′, and H-5′/H-6′, were observed (see Fig. 2). Moreover, the NOESY correlations of H-1′ and H-4 (δH 6.93), H-3′ (δH 3.48) and H-5′ (δH 3.38) (Fig. 3) were also key interactions to support this glucoside structure. The β-D-glucosyl nature of this sugar residue was confirmed by enzymatic hydrolysis of 4 with β-glucosidase51 and the hydrolysis products were identified to be discretamine (13)22 and D-glucose by TLC comparison with authentic 13 and D-glucose. Furthermore, the location of the methoxy group at C-2 was supported by the HMBC correlation between the CH3O-2 signal and C-2 (δC 150.0) (see Fig. 2) and this was further confirmed by the NOESY correlation between the CH3O-2 and H-1 (δH 6.92) (see Fig. 3). As the substituent on tetrahydroprotoberberine molecule, including a glucose moiety, does not exert a considerable effect on the optical rotation and ECD spectrum,49,52 the same sign of optical rotation and similar ECD curve of compound 4 (Fig. S234†) when compared with those of compounds 1–3 has led to a conclusion that the absolute configuration at C-13a of 4 is S. On the basis of these findings, compound 4 is the glucoside analogue of discretamine (13).22 Compound 4 was therefore identified as discretamine 3-O-β-D-glucopyranoside (see Fig. 1).
The molecular formula of stephapierrine E (5) was deduced to be C18H18NO4 from HR-ESI-TOF-MS (m/z 312.1224 [M + Na]+) and NMR data. The IR spectrum exhibited the absorption bands of the hydroxy group (3383 cm−1) and aromatic rings (1603 and 1575 cm−1). The 1H NMR spectrum (Table 3) showed the characteristic signals of an aporphine alkaloid as a singlet N-methyl proton at δH 2.64 (3H, s, NCH3), a singlet aromatic proton at δH 6.50 (1H, s, H-3), two ortho-coupled aromatic protons at δH 7.46 (1H, d, J = 8.4 Hz, H-11) and 6.71 (1H, d, J = 8.4 Hz, H-10), one methine proton at δH 3.20 (1H, dd, J = 14.1 and 4.5 Hz, H-6a), one coupled methylene protons at δH 3.09 and 2.64 (2H, overlapping signal, H-5), and another coupled methylene protons at δH 3.14 and 2.68 (2H, overlapping signal, H-4). Additional methylene protons at the 7-position appeared at δH 3.69 (1H, dd, J = 14.5 and 4.5 Hz) and δH 2.19 (1H, dd, J = 14.5 and 14.1 Hz). In addition, the spectrum displayed resonances due to a methylenedioxy proton at δH 6.02 and 5.88 (each 1H, d, J = 1.1 Hz). The 13C NMR and DEPT-135 spectra (Table 4) revealed the presence of 18 signals which included one methylenedioxy carbon, three methylene carbons, four methine carbons, nine quaternary carbons, and one N-methyl carbon. The sixteen-carbon and one-nitrogen skeleton of 5 suggested an aporphine alkaloid core structure.41,53,54 The spectroscopic data for 5 was similar to those of crebanine (25),23 especially the substitution patterns on the molecule. The significant difference is the absence of two methoxy signals in the NMR spectra of 5 (Tables 3 and 4). The structure of compound 5 was further confirmed by HMBC and NOESY experiments (see Fig. 2 and 3). The absolute configuration of compound 5 was deduced by optical rotation experiments. The negative sign of specific optical rotation suggested that the absolute configuration of C-6a was R, or the hydrogen at the C-6a position is in the β-orientation.29 This was supported by the ECD spectrum (Fig. S235†). The structure of 5 was therefore elucidated as di-O-demethylcrebanine.
Position | 5b | 6b | 7b | 8c | 9b |
---|---|---|---|---|---|
a Assignments were based on 1H–1H COSY, HMQC, HMBC, and NOESY experiments; chemical shifts (δ) are given in ppm.b Recorded in CDCl3.c Recorded in CD3OD.d Overlapping signal.e Partially overlapping signal. | |||||
3 | 6.50 s | 7.08 s | 7.07 s | 6.91 s | |
4 | 2.68d | 2.94 dd (14.9, 2.5) | 7.76 d (5.2) | 7.82 s | 2.73e |
3.14d | 3.22e | 2.86e | |||
5 | 2.64d | 3.20e | 8.56 d (5.2) | 3.27e | |
3.09d | 3.59 brdd (12.6, 5.3) | 4.12 brdd (13.2, 2.0) | |||
6a | 3.20 dd (14.1, 4.5) | 4.11 dd (13.9, 4.3) | 4.97 dd (13.8, 3.8) | ||
7 | 2.19 dd (14.5, 14.1) | 2.87 dd (13.9, 13.8) | 2.79 t (13.8) | ||
3.69 dd (14.5, 4.5) | 3.03 dd (13.8, 4.3) | 2.96 dd (13.8, 3.8) | |||
8 | 7.31 dd (7.1, 1.2) | 8.03 s | 7.25–7.32e | ||
9 | 7.26 ddd (7.5, 7.1, 1.2) | 7.25–7.32e | |||
10 | 6.71 d (8.4) | 7.32 ddd (7.6, 7.5, 1.2) | 7.15 d (8.8) | 7.30 d (9.0) | 7.25–7.32e |
11 | 7.46 d (8.4) | 8.33 brd (7.6) | 8.19 d (8.8) | 8.66 d (9.0) | 8.30 brd (7.7) |
OCH2O | 5.88 d (1.1) | ||||
6.02 d (1.1) | 6.28 s | 6.40 s | |||
1-OCH3 | 3.72 s | 3.53 s | |||
3-OCH3 | 4.03 s | ||||
5-OCH3 | 3.82 s | ||||
8-OCH3 | 3.92 s | ||||
9-OCH3 | 4.03 s | ||||
2-OCOCH3 | 2.32 s | ||||
N–CH3 | 2.64 s | ||||
N–COCH3 | 2.21 s | ||||
1′ | 4.96 d (7.6) | ||||
2′ | 3.54 dd (8.9, 7.6) | ||||
3′ | 3.48 t (8.9) | ||||
4′ | 3.38 dd (8.9, 8.6) | ||||
5′ | 3.48 brdd (8.6, 6.0) | ||||
6′ | 3.69 dd (12.0, 6.0) | ||||
3.92 dd (12.0, 2.1) |
Position | 5b | 6b | 7b | 8c | 9b |
---|---|---|---|---|---|
a Assignments were based on 1H–1H COSY, HMQC, HMBC, and NOESY experiments; chemical shifts (δ) are given in ppm.b Recorded in CDCl3.c Recorded in CD3OD. | |||||
1 | 144.0 | 147.9 | 154.3 | 150.6 | 150.0 |
1a | 119.0 | 127.1 | 110.4 | 115.3 | 129.8 |
1b | 126.5 | 129.0 | 123.3 | 119.9 | 133.8 |
2 | 148.9 | 153.1 | 148.3 | 147.7 | 145.4 |
3 | 107.6 | 117.6 | 102.7 | 143.7 | 123.8 |
3a | 127.5 | 128.5 | 137.9 | 132.6 | 131.9 |
4 | 29.7 | 27.7 | 125.8 | 107.9 | 31.4 |
5 | 55.0 | 43.4 | 144.9 | 156.6 | 43.5 |
6a | 63.9 | 54.9 | 147.1 | 132.6 | 52.7 |
7 | 27.5 | 36.1 | 184.1 | 174.7 | 35.0 |
7a | 123.3 | 135.7 | 126.3 | 126.9 | 138.4 |
8 | 143.5 | 129.6 | 152.0 | 108.1 | 129.9 |
9 | 146.6 | 129.6 | 157.9 | 150.8 | 129.7 |
10 | 114.5 | 129.1 | 126.3 | 113.4 | 129.5 |
11 | 120.7 | 129.9 | 125.9 | 123.0 | 128.7 |
11a | 124.6 | 133.2 | 124.9 | 120.3 | 132.7 |
OCH2O | 102.4 | 104.4 | 102.8 | ||
1-OCH3 | 61.7 | 61.3 | |||
3-OCH3 | 61.3 | ||||
5-OCH3 | 56.2 | ||||
8-OCH3 | 61.7 | ||||
9-OCH3 | 56.2 | ||||
22.7 | |||||
171.4 | |||||
N–CH3 | 44.0 | ||||
21.1 | |||||
172.5 | |||||
1′ | 103.0 | ||||
2′ | 75.4 | ||||
3′ | 78.8 | ||||
4′ | 71.9 | ||||
5′ | 78.8 | ||||
6′ | 63.1 |
The molecular formula of stephapierrine F (6) was established as C23H27NO7 from the HR-ESI-TOF-MS (m/z 430.1856 [M + H]+) and NMR data. The IR spectrum indicated the presence of hydroxy groups (3311 cm−1) and aromatic rings (1593 cm−1). The 1H NMR data (Table 3) showed the presence of a methoxy signal at δH 3.72, four adjacent aromatic hydrogens δH 7.31 (1H, dd, J = 7.1, 1.2 Hz, H-8), 7.26 (1H, ddd, J = 7.5, 7.1, 1.2 Hz, H-9), 7.32 (1H, ddd, J = 7.6, 7.5, 1.2 Hz, H-10), and 8.33 (1H, brd, J = 7.6 Hz, H-11), which were ascribed to the hydrogens of the unsubstituted D ring of the aporphine alkaloid.54 In addition, the 1H NMR spectrum revealed the presence of a sugar unit with its anomeric proton appearing at δH 4.96 (1H, d, J = 7.6 Hz, H-1′). The remaining sugar proton signals H-2′ to H-6′ are in the region δH 3.38–3.92 exhibiting similar features to those of compound 4. The 13C NMR spectrum (Table 4) showed 23 carbon resonances, of which five methines and one methylene were assigned to the β-glucopyranose moiety, suggested that 6 was an aporphine glucoside. The existence of a β-D-glucosyl moiety was confirmed by enzymatic hydrolysis51 in a similar manner to compound 4. The sugar linkage was determined by the HMBC correlations of H-1′ with C-2 (δC 153.1), C-3′ (δC 78.8) and C-5′ (δC 78.8) (see Fig. 2).50 The HMBC correlation between the methoxy proton at δH 3.72 and a carbon resonance at δC 147.9 (C-1) confirmed that the location of the methoxy group was placed to C-1. Additionally, the structural assignment for compound 6 was supported by the analysis of its NOESY spectrum (see Fig. 3); the H-1′ signal displayed a key interaction with H-3, H-3′ and H-5′. Moreover, H-6a (δH 4.11) displayed key interactions with H-7 (δH 2.87 and 3.03) and H-8 (δH 7.31), whereas CH3O-1 (δH 3.72) showed correlation to H-11 (δH 8.33). The 1H and 13C NMR spectra were consistent with those of (−)-asimilobine-2-O-β-D-glucoside (33).16 However, the sign of specific optical rotation of 6 was opposite to that of 33. The reported specific optical rotation of 33 was −107 (c = 0.1, MeOH).16 It is therefore possible that these two alkaloids are C-6a enantiomers. The ECD of 6 has therefore been determined and it was found that its ECD curve exhibited opposite Cotton effects to those of compounds 5 (see Fig. S235†). This led to a conclusion that the absolute configuration at C-6a of 6 was S. It is normal for the aporphine alkaloids to exist at different chirality at the asymmetric carbon C-6a.57 Based on these data, the structure of compound 6 was established as (+)-asimilobine 2-O-β-D-glucopyranoside.
Stephapierrine G (7) was assigned the molecular formula C18H11NO5 as determined from the HR-ESI-TOF-MS at m/z 344.0523 [M + Na]+ and NMR data. The IR spectrum showed absorption bands of hydroxy groups (3276 cm−1) and a conjugated carbonyl group (1638 cm−1). The 13C NMR spectrum in combination with the DEPT-135 experiment (Table 4) indicated the presence of 18 carbon atoms belonging to eleven quaternary carbons, five methine carbons, one methylene carbon, and one methyl carbon. The 1H NMR spectrum (Table 3) revealed characteristic resonances for protons and carbons at C-4 and C-5 of an aporphine unit as a pair of doublet signals at δH 7.76 and 8.56 (each 1H, d, J = 5.2 Hz), which connected to δC 125.8 and 144.9, respectively. Two singlet signals at δH 7.07 (1H, s) and 6.28 (2H, s) attaching to carbons at δC 102.7, and 104.4, respectively, were assigned to proton signals at C-3 and methylenedioxy protons, respectively. Two doublets at δH 7.15 and 8.19 (each 1H, d, J = 8.8 Hz) were attributed to two ortho-coupled aromatic protons at C-10 and C-11, respectively. In addition, a singlet signal at δH 3.92 (3H, s) was assigned to the methoxy proton at C-8. This assignment was confirmed by the HMBC correlations of the CH3O-8 and H-10 with C-8 (δC 152.0). The correlations of H-11 with C-1a (δC 110.4), C-7a (δC 126.3), C-9 (δC 157.9), and C-11a (δC 124.9) suggested the location of the hydroxy group at C-9 (see Fig. 2). The carbonyl carbon resonance at δC 184.1 together with a high-field resonance of its adjacent carbon (δC 126.3, C-7a) supported that 7 was a 7-oxoaporphine scaffold.58,59 Furthermore, the NOESY correlations of H-3 with H-4 and H-4 with H-5 (Fig. 3) as well as additional key HMBC correlations (Fig. 2) confirmed the structure of compound 7 to be a 7-oxoaporphine analogue. The spectroscopic data suggested that 7 is closely related to oxocrebanine (36),35 which has also been isolated from S. pierrei in the present work. Comparison of the NMR data of compounds 7 and 36 revealed that 7 is 9-de-O-methyloxocrebanine. Thus, compound 7 was concluded to be 1,2-methylenedioxy-8-methoxy-9-hydroxyoxoaporphine.
The sodiated molecular ion of the HR-ESI-TOF-MS of stephapierrine H (8) at m/z 388.0772 and the NMR data have led to the assignment of the molecular formula C20H15NO6. The IR spectrum indicated the presence of a conjugated carbonyl group (1651 cm−1). The 1H and 13C NMR data (Tables 3 and 4) showed two ortho-coupled aromatic protons at δH 7.30 and 8.66 (each 1H, d, J = 9.0 Hz, H-10 and H-11),58 together with two singlet protons at δH 7.82 and 8.03 (each 1H, s, H-4, and H-8). The remaining signals were assignable to three methoxys at δH 3.82 (3H, s, CH3O-5) and 4.03 (6H, s, CH3O-3, and CH3O-9), together with a methylenedioxy signal at δH 6.40 (2H, s). The foregoing 1H NMR data, together with the 13C NMR data (Table 4) suggested 8 to be an oxoaporphine type alkaloid.46,58,59 The HMBC experiments (see Fig. 2) confirmed the structure of compound 8. The structure of 8 was also confirmed by the NOESY correlations (see Fig. 3). On the basis of these data, the structure of compound 9 was established as 1,2-methylenedioxy-3,5,9-trimethoxyoxoaporphine.
Compound 9 was assigned the molecular formula C21H21NO4 as determined from the HR-ESI-TOF-MS (m/z 374.1363 [M + Na]+). The IR spectrum exhibited absorption bands of the acetoxy group (1761 cm−1) and amide group (1636 cm−1). The 1H and 13C NMR spectra (Tables 3 and 4) suggested an aporphine alkaloid core structure.41,53,54 The COSY correlations of these protons are shown in Fig. 2. The 1H NMR data showed the typical resonance of the N-acetyl signal at δH 2.21. The aromatic protons showed four adjacent signals for H-8, H-9 and H-10 in the range δH 7.25–7.32 and at δH 8.30 (1H, brd, J = 7.7 Hz) for H-11, which were attributed to the hydrogens of the unsubstituted D ring of the aporphine alkaloid.54 These observations were confirmed by 1H–1H COSY and HMBC experiments (see Fig. 2). The significant down-field shift of H-11 is the characteristic of this class of alkaloids.55,56 Furthermore, an aromatic singlet signal at δH 6.91 (H-3), a methoxy signal at δH 3.53 and an acetoxy proton signal at δH 2.32 were observed. The 1H and 13C NMR spectra of 9 were in agreement with the structure of O,N-diacetylasimilobine (9),18 which has been synthesized from asimilobine (32).32 This was confirmed by the HMBC experiments (Fig. 2) and the NOESY experiment (Fig. 3). It is noteworthy that the H-11 down-field shift in 9 (δH 8.30) was more pronounced than that of compound 5 (δH 7.46), and this was due to the vicinity of the methylenedioxy group in 5.55,56 The negative sign of specific optical rotation of 9 suggested that the absolute configuration of C-6a was R, or the hydrogen at the C-6a position is in the β-orientation.29 This was supported by the ECD spectrum (Fig. S235†). Based on these data, the structure of compound 9 was identical to (−)-O,N-diacetylasimilobine.18 Compound 9 has been reported in the present work as a naturally occurring alkaloid for the first time.
The molecular formula of compound 10 was deduced to be C22H23NO5 from the HR-ESI-TOF-MS at m/z 382.1258 [M + H]+. The IR spectrum indicated the presence of an acetoxy group (1751 cm−1). The 1H and 13C NMR features of 10 (Table 5) were similar to those of 8-methoxyuvoriopsine (37),37 the significant difference of which was the presence of an N-acetyl group, and the absence of an N-methyl signal. The rest of the 1H and 13C NMR data were consistent with the core structure of both compounds. The structure of 10 was confirmed by HMBC and NOESY analyses (see Fig. 2 and 3, respectively). Compound 10 has been reported as a structurally modified analogue from crebanine (25) and was called N-acetamidesecocrebanine,19 though some of the reported NMR spectroscopic data do not match with our data. This compound has thus been reported in the present work as a naturally occurring alkaloid for the first time.
Position | 10b | |
---|---|---|
δH | δC | |
a Assignments were based on 1H–1H COSY, HMQC, HMBC, and NOESY experiments; chemical shifts (δ) are given in ppm.b Recorded in CDCl3. | ||
1 | 129.8 | |
2 | 7.06 s | 110.1 |
3 | 145.0 | |
4 | 141.9 | |
4a | 117.1 | |
4b | 127.3 | |
5 | 8.80 d (9.2) | 123.7 |
6 | 7.28 d (9.2) | 112.7 |
7 | 149.9 | |
8 | 143.2 | |
8a | 123.6 | |
9 | 7.92 d (9.6) | 118.6 |
10 | 7.81 d (9.6) | 122.9 |
10a | 125.1 | |
11 | 3.29 m | 31.4 |
12 | 3.14 m | 61.9 |
OCH2O | 6.19 s | 100.9 |
7-OCH3 | 4.00 s | 56.3 |
8-OCH3 | 3.98 s | 61.2 |
N–CH3 | 2.81 s | 46.9 |
2.09 s | 19.5 | |
169.7 |
Compound 11 was assigned the molecular formula C19H21NO4 as deduced from the HR-ESI-TOF-MS at m/z 328.1542 [M + H]+ and NMR data. The IR spectrum revealed the presence of hydroxy groups (3380 cm−1). The 1H and 13C NMR data (Tables 1 and 2) showed similar patterns to those of tetrahydropalmatine (14),23 except that the 1H NMR spectrum of 11 at the aromatic region was less well-defined. The NMR spectra of compound 11 showed only two methoxy signals at δH 3.71 (δC 59.2) and δH 3.73 (δC 55.5). Placement of the two methoxy groups at C-9 and C-10 was based on HMBC correlations of the CH3O-9 proton signal with C-9 (δC 143.2), CH3O-10 signal with C-10 (δC 146.0), H-8 and H-11 with C-9, and H-12 with C-10. This was further confirmed by NOESY correlations between CH3O-9 and H-8, and correlation between CH3O-10 and H-11. In addition, the locations of the hydroxy groups at C-2 and C-3 were deduced from HMBC correlations of H-1 with C-2 (δC 147.3) and C-3 (δC 144.6), and H-4 with C-2 and C-3 (Fig. 2). The positions of the hydroxy groups were confirmed by NOESY correlations (see Fig. 3). Compound 11 was therefore the 2,3-di-O-demethylated analogue of tetrahydropalmatine (14).23 Compound 11 was previously detected as one of the demethylated metabolites of compound 14 from rat's urine by ultra high-performance liquid chromatography–tandem mass spectrometric analysis.20 Thus, we report herein the spectroscopic data of 11, 2,3-didemethyltetrahydropalmatine, which was identified as a new naturally occurring alkaloid in the plant species.
Compounds | AChEb | BuChEc |
---|---|---|
IC50 (μM) | IC50 (μM) | |
a Data represent as IC50 values in μM ± S.D. of three independent experiments.b Acetylcholinesterase.c Butyrylcholinesterase.d Inactive at 0.1 mg mL−1.e Galanthamine was used as the reference drug. | ||
1 | 41.47 ± 0.77 | Inactived |
2 | 15.41 ± 0.54 | Inactived |
3 | 149.63 ± 1.33 | 63.12 ± 0.83 |
11 | Inactived | Inactived |
12 | Inactived | 221.16 ± 1.62 |
13 | 26.99 ± 0.58 | 109.33 ± 0.97 |
14 | 71.28 ± 1.90 | 65.35 ± 0.80 |
15 | 11.33 ± 0.15 | 13.52 ± 0.59 |
17 | 21.16 ± 0.40 | 234.34 ± 1.08 |
18 | 12.25 ± 0.44 | 31.46 ± 0.20 |
19 | 152.59 ± 0.81 | Inactived |
21 | 18.31 ± 1.55 | 32.82 ± 0.34 |
22 | 8.32 ± 0.12 | 2.85 ± 0.08 |
23 | 11.34 ± 0.20 | 2.80 ± 0.07 |
24 | 11.94 ± 0.39 | 16.58 ± 0.54 |
25 | 17.37 ± 0.22 | 10.51 ± 0.27 |
26 | 6.11 ± 0.38 | 26.41 ± 0.43 |
27 | 17.63 ± 0.67 | 7.42 ± 0.16 |
28 | 6.12 ± 0.63 | 5.87 ± 0.06 |
29 | 4.30 ± 0.28 | 22.47 ± 0.10 |
30 | 140.15 ± 0.83 | Inactived |
32 | 141.47 ± 0.82 | 10.08 ± 0.15 |
33 | Inactived | 175.55 ± 1.45 |
34 | 73.08 ± 0.33 | 13.60 ± 0.30 |
35 | 265.82 ± 0.80 | Inactived |
36 | Inactived | Inactived |
38 | 1.21 ± 0.09 | 3.34 ± 0.02 |
39 | 2.85 ± 0.24 | 3.26 ± 0.05 |
40 | 147.18 ± 0.71 | 20.32 ± 0.39 |
41 | 32.49 ± 0.52 | 14.11 ± 0.25 |
42 | 1.09 ± 0.02 | 5.57 ± 0.15 |
43 | Inactived | 150.57 ± 0.54 |
44 | 40.86 ± 0.67 | 20.46 ± 0.42 |
45 | 7.49 ± 0.66 | 7.83 ± 0.11 |
Galanthaminee | 1.21 ± 0.11 | 3.59 ± 0.07 |
For the BuChE inhibitory activities, compounds 23 and 22 displayed high inhibitory activity, with the IC50 values of 2.80 and 2.85 μM, respectively, which were approximately 1.3-fold more active than galanthamine (IC50 3.59 μM), followed by compounds 39 and 38, which also showed high activity of 3.26 and 3.34 μM, respectively, which were slightly more active than galanthamine. Compounds 27, 28, 42 and 45 showed high inhibitory activity with the IC50 values in the range 5.57–7.83 μM. Furthermore, compounds 15, 18, 21, 24–26, 29, 32, 34, 40, 41 and 44 exhibited moderate inhibitory activity with the IC50 values of 10.08–22.47 μM, followed by compounds 3, 12–14, 17, 33 and 43 that showed weak anti-BuChE activity with the IC50 values of 63.12–234.34 μM. Compounds 1, 2, 11, 19, 30, 35 and 36 were inactive to the test.
For the anti-BuChE activity, the aporphine alkaloids are also the most active group. Almost all protoberberine alkaloids are weakly active or inactive. SAR discussion was mainly on the aporphine alkaloids as well as that of the anti-AChE activity. In the aporphine group, compound 23 exhibited high BuChE inhibitory activity, followed by 22 (IC50 of 2.80 and 2.85 μM, respectively (see Table S1†)). The methoxy group on the 8-position seemed to enhance the activity. In contrast, the 9-methoxy group caused a 5.8-fold decrease in the activity of compound 24 (IC50 16.58 μM) when compared with compound 22. While the anti-AChE activity of the 8,9-dimethoxy analogue 25 was 2.8-fold less active than the 9,10-dimethoxy analogue 26, the anti-BuChE activity of 25 was 2.5-fold higher than that of 26 (Table 6). This implied that the oxygen functions at the 8-, 9- and 10-positions differently contributed to cholinesterase inhibitory activities of the aporphine alkaloids. It should also be noted that the presence of the 6-N-formyl group in the analogue 30 caused inactivity in this compound and the absence of the 6-N-methyl group in the analogue 32 did not cause a sharp decrease of anti-BuChE property as that occurred in the anti-AChE case since it exhibited anti-BuChE activity with IC50 of 10.08 μM. It should also be mentioned that, in going from the amino function in 25 to the corresponding N-oxide function in 43, a sharp decrease in BuChE inhibitory activity was observed. In the presence of an α-hydroxy group at the 7-position of the aporphine core structure, in contrast to most of the aporphine group, an approximately 2-fold decrease in inhibitory activity was observed for compounds 27, 28 and 29 (IC50 7.42, 5.87 and 22.47 μM, respectively). For the third group of the aporphine analogues, the dehydroaporphines, a slight decrease in activity was observed for compounds 38, 39, 40 and 41 (IC50 3.34, 3.26, 20.32 and 14.11 μM, respectively). However, the analogue 42 showed a 4.7-fold increase in anti-BuChE activity (IC50 5.57 μM). This implied that the more rigid dehydroaporphine skeleton and the number and position of the methoxy groups contributed to the anti-BuChE activity of this group of alkaloids.
Compound | Residue | Interaction | Distance (Å) | ΔGdocking (Kcal mol−1) | IC50 (μM) |
---|---|---|---|---|---|
AChE-inhibitor interaction | |||||
38 | F295 | Hydrogen bond | 2.82 | −9.57 | 1.21 |
W286 | Pi–Pi | 4.02, 4.92, 5.36 | |||
S293 | Pi–Pi | 3.63 | |||
F297 | Pi–Pi | 5.16 | |||
Y341 | Pi–Pi | 4.93 | |||
42 | R296 | Hydrogen bond | 1.70 | −8.95 | 1.09 |
W286 | Pi–Pi | 4.15, 3.71, 5.46, 4.13, 5.59, 5.56 | |||
Y124 | Pi–Pi | 5.78 | |||
S293 | Pi–Pi | 4.30 | |||
Galanthamine | G121 | Pi–σ | 3.76 | −7.91 | 1.21 |
F338 | Pi–Pi | 5.94 | |||
BuChE-inhibitor interaction | |||||
22 | W82 | Pi–Pi | 4.68, 5.40, 5.32, 5.35 | −8.07 | 2.85 |
Pi–σ | 3.89 | ||||
T120 | Pi–Pi | 3.36 | |||
23 | W82 | Pi–Pi | 4.95, 5.72, 4.38, 5.26 | −8.28 | 2.80 |
3.86, 5.26 | |||||
Pi–σ | 3.42, 3.87 | ||||
38 | W82 | Pi–Pi | 4.64, 5.52, 5.21, 5.42 | −8.01 | 3.34 |
T120 | Pi–σ | 3.37 | |||
39 | W82 | Pi–Pi | 5.88, 4.56, 5.31, 5.20 | −7.89 | 3.26 |
5.04 | |||||
Galanthamine | W82 | Pi–σ | 3.83 | −7.41 | 3.59 |
The active site of AChE and BuChE consists of five regions such as the catalytic triad (CT), the anionic site (AS), the acyl pocket (AC), the oxyanion hole (OAH) and the peripheral anionic site (PAS). Thus, the amino acids of S203, E334, H447, W86, Y133, F337, F338, of F295, F297, G121, G122, A204, Y72, D74, Y124, W286 and Y341 are considered as active residues of AChE,64 while W82, Y128, F330, F331, L286, V288, G116, G117, A199, D70 and Y332 are identified as the key residues of BuChE binding gorge.65
As can be seen from Fig. 4, the docking result shows that compounds 38 and 42 were well occupied in the active site of AChE, where compound 38 could form a hydrogen bond with residue F295 and hydrophobic interactions with W286, S293, F297 and Y341. In contrast, compound 42 formed a hydrogen bond with R296 and strong Pi–Pi interactions with W286, Y124 and S293. The binding free energies of compounds 38 and 42 were similarly found to be −9.57 and −8.95 kcal mol−1, respectively, which possessed higher binding affinity with the AChE binding site compared to galanthamine (ΔG of −7.91 kcal mol−1).
The best conformations of the docked complexes of BuChE and compounds 22, 23, 38 and 39 are shown in Fig. 5. The results showed that these alkaloids mainly interacted with BuChE by forming Pi–Pi interactions with the key residue W82. The free energy of binding for all complexes is similar, being about −8 kcal mol−1. Interestingly, the binding free energies of these alkaloids show good binding affinity with the BuChE binding site compared to galanthamine (−7.41 kcal mol−1).
The molecular docking results showed that, for the alkaloids isolated from the tubers of S. pierrei, compounds 38 and 42 as well as compounds 22, 23, 38 and 39 exhibited good binding affinity towards AChE and BuChE, respectively, which is in agreement with the experimental IC50 values.
The crude hexane extract (2.5 g) was fractionated by column chromatography, using a gradient solvent system of n-hexane, n-hexane–EtOAc, EtOAc, EtOAc–MeOH and MeOH with increasing amounts of the more polar solvent. The eluates were examined by TLC and 7 combined fractions (H1–H7) were obtained. Fraction H1 (280.0 mg) was column chromatographed eluting with a gradient system of n-hexane–CH2Cl2 (10:0.1 to 10:4) to give 5 subfractions (H1.1–H1.5). Subfraction H1.2 (23.0 mg) was rechromatographed over silica gel eluting with n-hexane–CH2Cl2 (10:0.5) to yield compound 38 (5.0 mg). Subfraction H1.3 (200 mg) was subjected to column chromatography eluting with n-hexane–CH2Cl2 (10:1) to afford compounds 39 (15.0 mg), 40 (1.6 mg), 41 (20.0 mg), and 42 (2.4 mg). Subfraction H1.4 (15.0 mg) was rechromatographed over silica gel and eluting with n-hexane–EtOAc (10:0.3) to give compound 30 (1.3 mg). Subfraction H1.5 (18.0 mg) was subjected to column chromatography eluting with n-hexane–CH2Cl2–EtOAc (9:9:0.2) to afford compound 8 (2.2 mg). Fraction H2 (850 mg) was subjected to silica column chromatography eluting with the isocratic condition of CH2Cl2–MeOH (10:0.1) to give 5 subfractions (H2.1–H2.5). Subfraction H2.1 (290 mg) was rechromatographed over silica gel eluting with n-hexane–EtOAc (6:4) to yield compounds 25 (240 mg), and 3 (9.0 mg). Subfraction H2.2, H2.3, and H2.4 were identified to be compounds 22 (7.6 mg), 24 (15.3 mg), and 23 (433.0 mg). Fraction H3 (550.0 mg) was subjected to silica column chromatography eluting with a gradient condition of CH2Cl2–MeOH (10:0.1 to 10:0.3) to give 2 subfractions (H3.1–H3.2). Subfraction H3.1 (350.0 mg) was chromatographed eluting with CH2Cl2–MeOH (10:0.1) to afford compound 2 (50.6 mg) and compound 36 (150.0 mg). Subfraction H3.2 (150.0 mg) was rechromatographed over silica gel eluting with CH2Cl2–MeOH (10:0.2) to yield compound 26 (68.0 mg). Fraction H4 (180.0 mg) was subjected to silica column chromatography eluting with the isocratic condition of CH2Cl2–MeOH (10:0.2) to give 4 subfractions (H4.1–H4.4). Subfraction H4.1 (75.0 mg) was chromatographed on Sephadex LH-20 column eluted with MeOH–CH2Cl2 (7:3) to yield compound 14 (15.0 mg). Subfraction H4.2 was identified to be compound 35 (5.5 mg). Subfraction H4.3 (15.0 mg) was chromatographed eluting with CH2Cl2–MeOH (10:0.3) to give compound 1 (2.5 mg). Subfraction H4.4 was identified to be compound 34 (2.0 mg). Fraction H5 (120.0 mg) was subjected to column chromatography eluting with the isocratic condition of CH2Cl2–MeOH (10:0.4) to give 3 subfractions (H5.1–H5.3). Subfraction H5.1 (25.0 mg) was chromatographed over silica gel eluting with CH2Cl2–MeOH (10:0.2), followed by column chromatography on Sephadex LH-20 eluted with MeOH to yield compound 7 (1.0 mg). Subfraction H5.3 (48.0 mg) was purified on a Sephadex LH-20 column eluted with MeOH to afford compound 17 (15.5 mg). Fraction H6 (80.0 mg) was chromatographed over silica gel and eluted under the isocratic condition of CH2Cl2–MeOH (10:0.8) to yield compounds 21 (12.0 mg), and 20 (2.5 mg). Subfraction H7 (125 mg) was chromatographed on Sephadex LH-20 column eluting with MeOH, followed by silica column chromatography, eluting with gradient solvent system of CH2Cl2–MeOH (10:1) to afford compound 19 (84.0 mg).
The crude EtOAc extract (3.0 g) was fractionated by column chromatography, using a gradient solvent system of EtOAc, EtOAc–MeOH and MeOH with increasing amounts of the more polar solvent. The eluates were examined by TLC and 3 combined fractions (E1–E3) were obtained. Fraction E1 (300.0 mg) was subjected to column chromatography eluting with an isocratic condition of CH2Cl2–MeOH (10:0.2) to give compounds 9 (1.5 mg), 31 (1.0 mg), 10 (1.2 mg), and 32 (3.2 mg). Fraction E2 (450.0 mg) was chromatographed on silica column eluting under isocratic condition of CH2Cl2–MeOH (10:0.5) to provide compounds 11 (3.4 mg), 13 (2.0 mg), and 12 (10.0 mg). Fraction E3 (150.0 mg) was subjected to silica column chromatography eluting under the isocratic condition of CH2Cl2–MeOH (10:1) to give compound 18 (5.0 mg).
The MeOH extract (4.2 g) was fractionated by column chromatography, using a gradient solvent system of EtOAc, EtOAc–MeOH, MeOH, and 5% water in MeOH with an increasing amount of the more polar solvent. The eluates were examined by TLC and 6 combined fractions (M1–M6) were obtained. Fraction M1 (450.0 mg) was subjected to column chromatography eluting under the isocratic condition of CH2Cl2–MeOH (10:1) to give 3 subfractions (M1.1–M1.3). Subfraction M1.2 (150.0 mg) was rechromatographed eluting with n-hexane–CH2Cl2–MeOH (40:50: 2) to afford compound 27 (30.0 mg). Subfraction M1.3 was identified to be compound 44 (29.1 mg). Fraction M2 (200.0 mg) was chromatographed over silica gel eluting with a gradient solvent system of CH2Cl2–MeOH (10:0.4) to give 4 subfractions (M2.1–M2.4). Subfraction M2.3 was identified to be compound 37 (3.9 mg).
Subfraction M2.4 (120.0 mg) was chromatographed eluting with the isocratic condition of CH2Cl2–MeOH (10:0.5) to yield compound 43 (20.6 mg). Fraction M3 (750.0 mg) was chromatographed on a silica gel column eluting under the isocratic condition of CH2Cl2–MeOH (10:0.5) to give 4 subfractions (M3.1–M3.4). Subfraction M3.2 was identified to be compound 45 (7.4 mg). Subfraction M3.3 (80.0 mg) was subjected to column chromatography eluting with CH2Cl2–MeOH (10:1) to afford compound 33 (9.0 mg). Subfraction M3.4 (30.0 mg) was rechromatographed over silica gel and eluted under the isocratic condition of CH2Cl2–MeOH (100:12) to yield compounds 16 (2.2 mg), and 15 (8.6 mg). Fraction M4 (150.0 mg) was purified by using a Sephadex LH-20 column eluting with MeOH, followed by silica column chromatography eluting with n-hexane–CH2Cl2–MeOH (40:50:10) to provide compound 4 (1.5 mg). Fraction M5 (280.0 mg) was chromatographed over silica gel eluting with an isocratic solvent system of CH2Cl2–EtOAc–MeOH (40:60:10) to give 4 subfractions (M5.1–M5.4). Subfraction M5.1 and M5.2 were identified as compounds 29 (8.8 mg) and 5 (1.3 mg), respectively. Subfraction M5.3 (80.0 mg) was rechromatographed over silica gel and eluted with CH2Cl2–EtOAc–MeOH (20:80:15) to give compound 28 (11.7 mg). Fraction M6 (80.0 mg) was chromatographed on Sephadex LH-20 column eluting with MeOH, followed by silica column chromatography, eluting with an isocratic solvent system of CH2Cl2–EtOAc–MeOH (20:60:20) to afford compound 6 (2.2 mg).
Compound 6 was similarly hydrolyzed under the same condition and the aglycone and the sugar were similarly analyzed.
Enzyme inhibitory activity assay (%) = [(absorbance of control − absorbance of sample)/ absorbance of control] × 100. |
The IC50 values were determined graphically from inhibition curves (inhibitor concentration vs. percent of inhibition) and each concentration was performed in triplicate.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ra03276c |
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