Article Cite This: J. Nat. Prod. 2018, 81, 1252−1259
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Structures and Biological Evaluation of Monoterpenoid Glycosides from the Roots of Paeonia lactif lora Rui Li,⊥ Jing-Fang Zhang,⊥ Yu-Zhuo Wu, Yan-Cheng Li, Gui-Yang Xia, Ling-Yan Wang, Bo-Lin Qiu, Min Ma, and Sheng Lin* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *
ABSTRACT: Fractionation of an aqueous extract of the air-dried roots of a traditional Chinese medicinal plant, Paeonia lactif lora, yielded the new monoterpenoid glycosides 1−10. Their structures were assigned via spectroscopic techniques, and the absolute configurations of 1, 4−6, and 8 were verified via chemical methods, specific rotation, and electronic circular dichroism data. Compounds 1−4 are rare compared to the reported cage-like paeoniflorin derivatives; that is, they comprised two monoterpenoidal moieties. In the in vitro assay, compounds 5, 8, and 9 showed weak inhibitions against lipopolysaccharideinduced nitric oxide production in RAW264.7 macrophages, with IC50 values of 64.8, 60.1, and 97.5 μM, respectively.
M
reported cage-like paeoniflorin derivatives by consisting of two monoterpenoidal moieties. Detailed herein are the isolation, structural elucidation, and bioactivity evaluation of the 10 new isolates.
onoterpene glycosides, with cage-like structures, are the characteristic chemotaxonomic markers of the Paeonia genus, which is one of the most important reasons to classify this genus of the Ranunculaceae family into an independent family (Paeoniaceae).1 The dried roots of Paeonia lactif lora and P. veitchii, known as the important crude drug “Chi-Shao” in traditional Chinese medicine (TCM), have been officially recorded in the Chinese Pharmacopoeia as a blood circulation, anti-inflammatory, and analgesic agent for the treatment of cardio-cerebrovascular diseases.1,2 The widespread utilization of “Chi-Shao” has drawn much attention to the bioactive constituents in P. lactif lora and P. veitchii root extracts,3 resulting in the characterization of paeoniflorin and other cage-like monoterpenoid analogues. Some analogues have been considered as the major active components of “Chi-Shao” due to their antihyperglycemic, anti-inflammatory, antioxidant, and other biological activities.1,4 Recently published findings also suggest paeoniflorin as a candidate for the treatment of Parkinson’s disease and Alzheimer’s disease.5 In a continuing effort to discover diverse chemical and biological constituents of TCM, this study aimed to identify novel bioactive molecules from the aqueous extract of P. lactif lora roots. Ten new compounds (1−10) were obtained, comprising four paeoniflorin analogues and six other monoterpene glycosides. Compounds 1−4 are rare compared to the © 2018 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION The molecular formula, C33H42O13, for compound 1 was established by the [M + Na]+ ion at m/z 669.2514 in the HRESIMS, consistent with an index of hydrogen deficiency of 13. The 1H and 13C NMR data of 1 showed features similar to paeoniflorin (Table 1).6 A series of COSY, HSQC, and HMBC correlations in the 2D NMR spectra confirmed the paeoniflorin moiety in 1. Compound 1 was subjected to acid hydrolysis followed by derivatization with trimethylsilyl-L-cysteine. Subsequent GC analysis identified D-glucose by comparison with a standard D-glucose sample.7 Comparison of the NMR data of 1 with those of paeoniflorin revealed the presence of an additional monoterpenoid ester or monoterpenoid acid moiety in 1, based on the NMR signals attributed to a methyl singlet [δH 1.27 (H3-9‴); δC 18.9 (C-9‴)], an oxygenated methylene [δH 3.57, 3.50 (each 1H, dd, J = 10.6, 7.7 Hz, H2-10‴); δC 67.7 (C-10‴)], three Received: January 28, 2018 Published: May 9, 2018 1252
DOI: 10.1021/acs.jnatprod.8b00087 J. Nat. Prod. 2018, 81, 1252−1259
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Chart 1
methines [δH 2.76 (ddd, J = 6.6, 6.0, 1.8 Hz, H-1‴) and 2.27, 2.63 (each 1H, m, H-2‴ and H-5‴); δC 42.8, 44.9, and 41.6 (C-1‴, C-2‴, and C-5‴)], three methylenes [δH 1.05−2.76 (6H, m, H2-3‴, H2-4‴, and H2-7‴); δC 19.6, 26.0, and 35.1 (C-3‴, C-4‴, and C-7‴)], a quaternary carbon at δC 52.1(C-6‴), and an ester carbonyl carbon at δC 180.0 (C-8‴). With 11 indices of hydrogen deficiency deduced from the paeoniflorin moiety and the carbonyl group, the monoterpenoid ester or monoterpenoid acid moiety is bicyclic. This partial structure was further elucidated by analysis of the 2D NMR data. The COSY spectrum of 1 showed correlations between H-1‴/H-2‴/H2-3‴/H2-4‴/H-5‴, H-2‴/H2-10‴, and H-5‴/H2-7‴/H-1‴ and a W-type long-range correlation of H-1‴/H-5‴. Finally, a 10-hydroxypinan-8-carbonyl moiety attached to C-6′ of the paeoniflorin unit via an ester bond was completed by the HMBC correlations from H2-10‴ to C-3‴ and C-1‴, from H3-9‴ to C-5‴ and C-1‴, from H-1‴ and C-8‴, from H-7b‴ to C-9‴, and from H2-6′ to C-8‴. Thus, compound 1 was identified as the 6′-O-10-hydroxypinan-8-carbonyl derivative of paeoniflorin. Alkaline hydrolysis of 1 afforded 10-hydroxypinan8-carboxylic acid (1a). Its structure was confirmed by MS and 1D and 2D NMR data (Figures S13−S18, Supporting Information). In the NOESY spectrum of 1 and 1a, the NOE correlation of H-3‴β and H-7‴β indicated that a typical boat cyclohexane ring
(C-1‴ to C-5‴ and C-7‴) was formed. H2-10‴ correlated with H3-9‴ and H-3‴α, whereas H-2‴ correlated with H-7‴β and H-3‴β, indicating that H3-9‴ and H2-10‴ (Figure 1) were cofacial with respect to the cyclohexane ring.8 The electronic circular dichroism (ECD) spectrum of compound 1a displayed a positive Cotton effect (CE) at 219 nm, as shown in Figure 2. Close agreement was observed with the calculated ECD data of (1R,2S,5R,6S)1a, exhibiting an intense positive CE at 219 nm (Figure 2). Therefore, the structure of compound 1 was defined as (−)-6′-O[(1R,2S,5R,6S)-10-hydroxypinan-8-oxo]paeoniflorin. The 2D NMR data analysis of compound 2 showed that its 2D structure was identical to that of 1. The 13C NMR spectrum of 2 showed that C-2‴, C-7‴, and C-9‴ were significantly shielded (7.2, 9.7, and 3.0 ppm, respectively) compared to those of 1. Additional differences were shown in the 1H NMR spectrum, especially for H2-7‴, H-9‴, and H2-10‴. The most significant variation was for H-7β‴, which was deshielded by 0.42 ppm versus H-7β‴ of 1. The H-7α‴ and H-9‴ resonances were shielded by 0.30 and 0.14 ppm, respectively, compared to those of 1. These observations, combined with the NOESY correlations of H3-9‴/H-2‴, H-3‴α/H-2‴, H-3‴β/H2-10‴, H-3‴β/ H-7‴β, and H-7‴β/H2-10‴ (Figure 1), revealed that compound 2 was the C-2‴ epimer of 1. On the basis of these data and 1253
DOI: 10.1021/acs.jnatprod.8b00087 J. Nat. Prod. 2018, 81, 1252−1259
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Table 1. NMR Data (δ) of Compounds 1−4 in Methanol-d4a 1 no. 1 2 3a 3b 4 5 6 7a 7b 8a 8b 9 10 1′ 2′ 3′ 4′ 5′ 6′a 6′b 1″ 2″/6″ 3″/5″ 4″ 7″ 1‴ 2‴ 3‴α 3‴β 4‴α 4‴β 5‴ 6‴ 7‴α 7‴β 8‴a 8‴b 9‴ 10‴a 10‴b
δH (mult, J, Hz)
2.12 d (12.6) 1.83 dd (12.6, 1.5) 2.61 dd (6.8, 1.8) 2.55 dd (10.8, 6.9) 1.91 d (10.8) 4.77 d (12.6) 4.74 d (12.6) 5.44 s 1.33 s 4.58 d (7.7) 3.23 dd (7.7, 9.0) 3.36 t (9.0, 9.0) 3.31 t (9.0, 9.0) 3.47 ddd (9.0, 6.0, 1.8) 4.62 dd (11.9, 1.8) 4.09 dd (11.9, 6.0) 8.07 dd (8.4, 1.2) 7.51 t (7.8) 7.64 t (7.8) 2.76 ddd (6.6, 6.0, 1.8) 2.27 m 1.51 m 2.00 m 1.96 m 2.05 m 2.63 m 2.12 m 1.05 d (9.6)
2 δC 89.6 87.2 44.7 106.4 44.1 72.3 23.3 61.8 102.4 19.9 100.2 75.1 78.0 71.7 75.6 65.0 131.4 130.8 129.8 134.6 168.1 42.8 44.9 19.6 26.0 41.6 52.1 35.1
δH (mult, J, Hz)
2.11 d (12.5) 1.84 dd (12.5, 1.5) 2.61 dd (6.8, 1.8) 2.54 dd (10.8, 6.9) 1.90 d (10.8) 4.77 d (12.6) 4.74 d (12.6) 5.44 s 1.33 s 4.58 d (7.7) 3.23 dd (7.7, 9.0) 3.34 d (9.0, 9.0) 3.30 t (9.0, 8.4) 3.47ddd (8.4, 6.0, 1.8) 4.61 dd (11.8, 1.8) 4.11 dd (11.8, 6.2) 8.07 dd (8.3, 1.1) 7.51 t (7.8) 7.64 t (7.8) 2.66 ddd (6.0, 5.4, 1.2) 2.16 m 1.66 m 1.28 m 1.83 m 1.87 m 2.58 m 1.82 m 1.47 d (9.6)
180.0 1.27 s 3.57 dd (10.6, 7.7) 3.50 dd (10.6, 7.7)
18.9 67.7
3 δC 89.6 87.2 44.7 106.4 44.1 72.3 23.3 61.8 102.5 19.9 100.2 75.1 78.0 71.7 75.6 65.1 131.4 130.8 129.8 134.6 168.1 42.1 37.7 19.0 24.0 40.9 52.6 25.4 180.2
1.13 s 3.41 dd (10.6, 7.7) 3.38 dd (10.6, 7.7)
15.9 66.5
δH (mult, J, Hz)
2.04 d (12.6) 1.75 dd, (12.6, 1.7) 2.53 dd (6.8, 1.6) 2.46 dd (10.7, 6.9) 1.81 d (10.7) 4.68 d (12.5) 4.64 d (12.5) 5.36 s 1.25 s 4.49 d (7.6) 3.13 dd (9.0, 7.6) 3.20 t (9.0) 3.18 t (9.0) 3.35 ddd (9.0, 6.0, 2.0) 4.45 dd (11.9, 1.7) 3.89 dd (11.9, 5.9) 7.99 d (7.2) 7.43 t (7.8) 7.55 t (7.8) 2.26 m 2.34 m 2.46 (1H, m) 1.16 (1H, m) 4.02 brd (9.0) 1.75 m 1.46 dt (10.3, 1.5) 1.61 dt (10.3, 2.0) 1.10 s 0.89 s
4 δC 89.5 87.2 44.8 106.4 44.1 72.3 23.3 61.8 102.5 19.9 100.2 75.1 78.0 71.8 75.4 64.3 131.4 130.8 129.8 134.6 168.1 56.1 41.5 35.3
δH (mult, J, Hz)
2.05 d (12.5) 1.81 brd (12.5) 2.60 d (6.7) 2.52 dd (10.8, 6.8) 1.82 d (10.8) 4.75 s 5.43 s 1.31 s 4.57 d (7.6) 3.24 dd (7.6, 8.6) 3.36 dd (9.6, 8.6) 3.27 dd (9.6, 8.6) 3.50 brdd (8.6, 7.3) 4.47 brd (11.9) 4.29 dd (11.9, 7.3)
δC 89.2 87.1 44.6 106.3 43.9 72.1 23.2 61.6 102.3 19.6 100.0 75.0 77.9 72.0 75.3 64.8
6.84 t (7.7)
131.2 130.7 129.6 134.4 167.9 169.2 128.6 144.5
70.4
2.24 m
24.6
57.7 39.0 34.3
1.62 m
41.8 73.6 145.9
32.6 22.9 175.0
8.07 dd (8.3, 1.1) 7.51 t (7.5) 7.64 t (7.5)
5.92 dd (17.4, 10.8) 5.24 dd (17.4, 1.5) 5.07 ddd (10.8, 2.2, 1.5) 1.28 s 1.85 s
112.5 27.9 12.5
a
NMR data (δ) were measured at 600 or 500 MHz for 1H NMR and at 150 or 125 MHz for 13C NMR. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1H−1H COSY, HSQC, and HMBC experiments.
Figure 1. COSY (bold lines), key HMBC (single arrows, 1H → 13C), and key NOESY (dashed double arrows) correlation of 1−4.
biosynthetic considerations,6b the structure of compound 2 was defined as (−)-6′-O-[(1R,2R,5R,6S)-10-hydroxypinan-8-oxo]paeoniflorin. Compound 3 exhibited similar NMR features compared to 1. The difference in the NMR spectra was that two of the methylenes (one oxygenated) of the 10-hydroxypinan-8-oxo
unit of 1 were replaced by a methyl [δH 0.89 (3H, s, H3-9‴); δC 22.9 (C-9‴)] and an oxygenated methine [δH 4.02 (1H, brd, J = 9.0 Hz, H-4‴); δC 70.4 (C-4‴)] in 3, respectively. These data suggested that a 4-hydroxypinan-10-oxo rather than a 10-hydroxypinan-8-oxo moiety was located at 6-CH2OH of the glucopyranosyl unit. This was affirmed by the 2D NMR data of 3, 1254
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the sequential positive and negative Cotton effect at 254 and 226 nm were indicative of positive exciton chirality (Figure S52, Supporting Information),13 confirming the same absolute configuration as mudanpioside F.12b Therefore, the structure of compound 5 was defined as (+)-(1S,5R,6R)-8-O-benzoylmudanpioside F. Compound 6 contained the same mudanpioside F and benzoyl moieties as 5. Comparison of the 1H NMR data of 6 and 5 revealed deshielded shifts of 0.78 and 0.83 ppm and shielded shifts of 1.08 and 0.76 ppm for H2-6′ and H2-8, respectively. These data showed that the benzoyl moiety was located at C-6′ in 6 instead of C-8 in 5. This conclusion was verified by the HMBC cross-peak of H2-6′/ C-7″. The NOESY correlation of H-7a with H2-8 was again noted, suggesting that compound 6 possessed the same relative configuration as 5. Similar ECD data (Figure S63, Supporting Information) for 6 and 5 revealed their identical absolute configurations. Therefore, the structure of compound 6 was defined as (+)-(1S,5R,6R)-6′-O-benzoylmudanpioside F. The molecular formula of compound 7 was found to be C23H28O10 based on the HRESIMS ion at m/z 487.1580 [M + Na]+. The NMR spectra of 7 were similar to those of floralabiflorin, a known monoterpenoid glucoside reported from the flowers of P. lactif lora and P. suf f ruticosa.14 The only difference was the replacement of a hydroxymethyl group by a methyl group at δH 1.32 (s, H3-8) in 7. This difference suggested that 7 was the 8-deoxy analogue of floralabiflorin. The COSY, HSQC, and HMBC data were consistent with the above deduction. The relative configuration of 7 is analogous to that of floralabiflorin based on the NOESY cross-peaks of H-7b/H3-8, H-7a/H-3a, and H-7a/H-4. Accordingly, the structure of compound 7 was identified as 8-deoxyfloralabiflorin. Compound 8, C23H28O11, was hypothesized to be the glucosidic analogue of paeoniflorigenone, as evidenced by direct comparison of their NMR spectra. The 1H and 13C NMR data of 8 and paeoniflorigenone were highly similar except that 8 showed a set of β-glucopyranosyl NMR signals.15 Enzymatic hydrolysis of 8 with snailase gave paeoniflorigenone (8a) and D-glucose.7 The deshielding of C-1 from δC 102.9 in 8a to 105.7 in 8 revealed the location of the β-D-glucopyranosyloxy group that was confirmed by the HMBC correlation of the anomeric proton (H-1′) and C-1. The configuration of 8 was established by its NOESY and ECD spectra similar to those of paeoniflorigenone (8a) (Figures S93 and S84, Supporting Information).15 Thus, the structure of compound 8 was identified as (+)-paeoniflorigenone-1-O-β-Dglucopyranoside. Compound 9 had the molecular formula C22H34O10 as indicated by the HRESIMS ion at m/z 481.2051 [M + Na]+. The NMR data of 9 were similar to those of the co-occurring cuminyl-β-D-glucopyranoside,16 but with the resonances of the α-rhamnopyranosyl group evident in the NMR spectra of 9. Compound 9 was assumed to be the α-rhamnopyranosyl derivative of cuminyl-β-D-glucopyranoside. The D-glucopyranosyl and L-rhamnopyranosyl moieties in 9 were assigned by acid hydrolysis of 9 using the published protocol.7 Comparison of the 13 C NMR signals of 9 and cuminyl-β-D-glucopyranoside allowed placement of the α-L-rhamnopyranosyl moiety at CH2-OH of the β-D-glucopyranosyl unit due to the characteristic 13C NMR deshielding of C-6′ (δC 68.1) in 9, which was corroborated by the key CH2-OH/C-1″ HMBC correlation. Thus, the structure of compound 9 was elucidated as (−)-cuminyl-[α-L-rhamnopyranosyl(1→ 6)]-β-D-glucopyranoside. Compound 10 was isolated as a white powder with a molecular formula of C22H36O11 as determined by m/z 499.2133 [M + Na]+.
Figure 2. Experimental ECD spectrum of 1a (black) and the calculated ECD spectra of (1R,2S,5R,6S)-1a (red) and (1S,2R,5S,6R)-1a (blue).
which showed COSY sequences of H-1‴/H-2‴/H2-3‴/H-4‴/ H-5‴/H2-7‴ and HMBC correlations from H3-8‴ and H3-9‴ to C-1‴, C-5‴, and C-6‴, from H-4‴, H-2‴, and H2-7‴ to C-6‴, and from H-1‴ and H2-6′ to C-10‴. The large trans-diaxial coupling (J = 9.0 Hz) between H-4‴ax and H-3‴ax established the β-orientation of OH-4‴. Likewise, the NOESY correlations of H-3‴β/H-7‴β, H-3‴β/H-2‴, and H-2‴/H-7‴β, in combination with the NOESY correlations of H-4‴/H3-9‴, H-4‴/H-3‴α, and H-7‴α/H3-8‴ (Figure 1), confirmed that 1 and 3 had the same relative configurations at C-1, C-2, and C-5. Based on these data and biosynthetic considerations,6b the structure of compound 3 was established as (−)-6′-O-[(1R,2S,4S,5R)-4-hydroxypinan10-oxo]paeoniflorin. Compound 4 had the same molecular formula as 1−3 and also contained the paeoniflorin moiety by comparison of the 1H and 13 C NMR data of 4 and 1−3. The major differences between 4 and 1−3 involved the monoterpene substituent located at 6-CH2OH of the glucopyranosyl unit. Analysis of the 2D NMR data with the COSY, HMBC, and NOESY correlations shown in Figure 1 established the (2E)-6-hydroxy-2,6-dimethylocta-2, 7-dienoyloxy moiety, which is an acyclic monoterpenoidal ester moiety in 4.9 Acid hydrolysis instead of alkaline hydrolysis of 4 was used to support this result (Figures S49 and S50, Supporting Information) and avoided the anticipated decomposition of (2E)-6-hydroxy-2,6-dimethylocta-2,7-dienoic acid under alkaline reaction conditions.9,10 Thus, the absolute configuration of (2E)6-hydroxy-2,6-dimethylocta-2,7-dienonic acid was assigned as S based on its [α]D value of +21.10,11 On the basis of these data, the structure of compound 4 was elucidated as (+)-6′-O-[(2E,6S)-6hydroxy-2,6-dimethylocta-2,7-dienoyl]paeoniflorin. The 1H and 13C NMR data of 5, C23H28O9, were highly similar to those of mudanpioside F, a monoterpene glucoside [(1S,5R,6R)1-(β-D-glucopyranosyloxy)-8-hydroxypin-2-en-4-one)] found in the root cortex of P. suf f ruticosa and the root of Cnidium silaifolium,12 except for the presence of a benzoyl group and a notable deshielding of H2-8 [δH 5.07, 4.57 (each 1H, d, J = 11.8 Hz)] and C-8 (δC 68.5)]. These observations indicated that the benzoyl group was located at C-8, which was verified by an HMBC correlation between H2-8 and C-7″. The NOESY correlation of H-7a with H2-8 revealed that these protons are spatially close. The sugar obtained by acid hydrolysis was established as 7 D-glucose. The absolute configuration of 5 was determined by its ECD spectrum. On the basis of the exciton chirality method, 1255
DOI: 10.1021/acs.jnatprod.8b00087 J. Nat. Prod. 2018, 81, 1252−1259
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Table 2. NMR Data (δ) for Compounds 5−10 in Methanol-d4a 5 no. 1 2 3a
δH (mult, J, Hz)
5.76 s
6 δC 84.6 176.0 122.0
δH (mult, J, Hz)
5.52 dd (1.8, 1.2)
7 δC 85.2 176.4 121.8
3b 4 5 6 7a
2.88 dd (7.0, 2.5)
204.3 48.9 62.9 44.6
7b
3.40 dd (9.6, 7.2) 2.62 d (9.6)
8a
5.07 d (11.8)
8b
4.57 d (11.8)
9 10 1′ 2′
1.24 s 2.16 s 4.61 d (7.7) 3.30 dd (7.8, 9.0) 3.44 t (9.0)
16.5 20.4 100.3 75.3
3.40 dd (9.0, 9.6) 3.30 ddd (9.6, 5.3, 2.2) 3.85 dd (11.9, 2.2) 3.66 dd (11.9, 5.3)
71.8
3′ 4′ 5′ 6′a 6′b 1″ 2″ 3″
8.05 dd (8.2, 1.1) 7.49 t (7.5)
4″ 5″ 6″ 7″
68.5
2.58 dd (7.1, 2.6) 3.23 dd (9.3, 7.2) 2.22 d (9.3) 3.99 d (12.1)
204.4 48.5 64.8 44.0
66.6
δH (mult, J, Hz)
2.22 ddd (13.8, 7.2, 1.2) 1.73 dd (13.8, 7.8) 3.87 t (7.2) 2.26 d (7.2)
2.55 dd (10.8, 7.1) 2.17 dd (10.8, 1.2) 1.32 s
8
9
10
δH (mult, J, Hz)
δC
δH (mult, J, Hz)
δH (mult, J, Hz)
7.35 d (8.0) 7.22 d (8.0)
136.1 129.6 127.3
5.76 s
2.91 d (17.4)
105.9 82.0 48.3
166.4 115.9 164.3
212.2 47.9
7.22 d (8.0)
149.7 127.3
2.23 m 2.20 m
42.0 27.1
44.6 34.0
7.35 d (8.0) 4.82 d (11.5)
129.6 69.8
5.12 m
124.1 133.5
77.9
2.44 m 2.57 br d (10.1) 2.55 br d (10.1) 4.27 dd (11.4, 6.1) 4.05 dd (11.4, 8.7) 5.52 s 1.31 s 4.85 d (7.7) 3.26 dd (9.0, 7.8) 3.41 t (9.0
δC 85.3 92.2 42.4
78.2 63.4
66.6 44.5 55.7 24.8
12.6
131.4 130.7
64.2
2.91 sep (6.9)
35.2
1.70 s
25.9
102.4 22.2 99.8 75.0
1.25 d (6.9) 1.25 d (6.9) 4.33 d (7.8) 3.25 dd (7.8, 8.5) 3.31 dd (9.0, 8.5) 3.36 t (9.0)
24.5 24.5 102.3 75.1
17.8 19.3 95.2 74.0
78.0
1.64 s 2.17 s 5.47 d (8.2) 3.35 dd (8.2, 9.0) 3.43 t (9.0)
71.6
3.66 t (9.0)
71.2
3.41 ddd (9.0, 6.2, 1.6) 4.02 dd (11.2, 1.6) 3.66 dd (11.2, 6.2) 4.82 d (1.7) 3.89 dd (3.3, 1.7) 3.72 dd (8.5,3.3) 3.41 t (8.5) 3.73 dt (8.5, 6.2) 1.30 d (6.2)
76.9
3.51 ddd (9.0, 5.4, 1.9) 3.96 dd (11.4, 1.9) 3.64 dd (11.4, 5.4) 4.72 d (1.5) 3.85 dd (3.3, 1.5) 3.37 dd (9.0, 3.3) 3.35 t (9.0) 3.68 dd (9.0, 6.2) 1.25 d (6.2)
77.7
78.2
1.35 s 4.55 d (7.7) 3.26 dd (9.0, 7.8) 3.42 t (9.0)
3.36 dd (9.0, 9.6) 3.61 ddd (9.6, 7.4, 2.5) 4.63 dd (11.7, 2.5) 4.49 dd (11.7, 7.4)
72.4
3.37 t (9.0)
72.0
3.28 t (9.0)
71.7
75.4
3.62 ddd (9.1, 6.6, 2.2) 4.71 dd (11.8, 2.2) 4.44 dd (11.8, 6.6)
75.1
3.33 ddd (9.0, 6.2, 2.3) 3.87 dd (11.9, 2.3) 3.62 dd (11.9,6.2)
78.2
129.8
7.61 t (7.5) 7.49 t (7.5)
134.6 129.8
8.05 dd (8.2, 1.1)
130.7 168.1
4.62 d (11.5)
15.9 20.1 99.8 74.9
7.99 dd (8.3, 1.2) 7.44 t (7.5)
131.4 130.7
179.6 20.5 99.9 75.4
2.91 m
1.07 s 1.99 s 4.58 d (7.8) 3.30 dd (7.8, 9.0) 3.45 t (9.0)
64.8
65.1
78.2
62.9
131.4 130.7
8.02 d (8.4)
131.3 130.7
129.9
7.48 t (7.8)
129.8
129.7
8.05 dd (8.2, 1.1) 7.49 t (7.5)
7.59 t (7.5) 7.44 t (7.5)
134.6 129.7
7.61 t (7.5) 7.49 t (7.5)
134.6 129.9
7.61 t (7.8) 7.48 t (7.8)
134.5 129.8
7.99 dd (8.3, 1.2)
130.7
8.05 dd (8.2, 1.1)
130.7
8.02 d (8.4)
130.7
167.2
δC
2.56 d (17.4)
3.81 d (12.1)
78.2
δC
167.8
68.1
102.9 72.2 72.4 74.0 69.8 18.1
78.1
67.8
102.3 72.1 72.4 74.0 69.8 18.0
167.8
a
NMR data (δ) were measured at 600 or 500 MHz for 1H NMR and at 150 or 125 MHz for 13C NMR. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1H−1H COSY, HSQC, and HMBC experiments.
deduction. Accordingly, compound 10 was defined as (E)-1oxogeranyl-O-[α-L-rhamnopyranosyl(1→6)]-β-D-glucopyranoside. Many of the paeoniflorin derivatives isolated from the Paeoniaceae family are known to inhibit the production of nitric oxide (NO), which is a multifunctional signaling molecule related to vascular and neurological functions.18 Thus, the inhibitory activity of the new isolates was screened against NO production in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. As shown in Table 3, compounds 5, 8, and 9 showed weak inhibitions against LPS-induced NO production in RAW264.7 macrophages, with IC50 values of 64.8, 60.1, and 97.5 μM, respectively. Dexamethasone was used as positive control with an IC50 value of 8.3 μM. At concentrations up to 100 μM, this group of compounds was not cytotoxic to LPS-induced RAW264.7
As in the case of 9, the diagnostic NMR resonances in 10 permitted identification of the α-L-rhamnopyranosyl(1→6)]-βD-glucopyranosyloxy disaccharide moiety. The E-geranyl moiety was readily recognized by the NMR signals attributed to two trisubstituted double bonds [δH 5.76 (1H, s, H-2), 5.12 (1H, m, H-6); δC 115.9, 164.3, 124.1, 133.5 (C-2, C-3, C-6, and C-7)], three olefinic methyl singlets [δH 1.70, 1.64, 2.17 (H3-8, H3-9, and H3-10); δC 25.9, 17.8, 19.3 (C-8, C-9, and C-10)], two methylenes [δH 2.23, 2.20 (each 2H, m, H2-4 and H2-5); δC 42.0, 27.1 (C-4 and C-5)], and an ester carbonyl carbon at δC 166.4 (C-1).17 The disaccharide chain attached to the E-geranyl moiety via an ester bond was rationalized by the distinctive deshielded chemical shift of the glucopyranosyl anomeric proton (δH 5.47) and the shieded chemical shift of the glucopyranosyl anomeric carbon (δC 95.2). Analysis of the 2D NMR data confirmed this 1256
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Table 3. Inhibitory Activity of 1−10 against LPS-Induced NO Production in RAW264.7 Macrophagesa inhibition (%)
a
compound
80 μM
60 μM
40 μM
20 μM
10 μM
IC50 (μM)
1 2 3 4 5 6 7 8 9 10 dexamethasoneb
44.59 ± 0.24 40.71 ± 0.31 40.75 ± 0.33 41.32 ± 0.52 61.73 ± 0.17 44.99 ± 0.18 39.43 ± 0.12 60.99 ± 0.18 52.38 ± 0.28 41.04 ± 0.17
29.30 ± 0.38 39.47 ± 0.48 38.71 ± 0.26 38.13 ± 0.18 45.87 ± 0.18 31.88 ± 0.22 34.82 ± 0.06 49.91 ± 0.28 38.66 ± 0.16 33.39 ± 0.12
25.76 ± 0.89 22.10 ± 0.24 27.49 ± 0.60 28.06 ± 0.54 25.98 ± 0.22 18.28 ± 0.29 28.17 ± 0.22 34.92 ± 0.42 31.14 ± 0.24 29.18 ± 0.34
22.81 ± 0.40 20.85 ± 0.50 23.34 ± 0.48 22.56 ± 0.42 19.33 ± 0.29 16.21 ± 0.29 24.43 ± 0.36 29.61 ± 0.10 23.65 ± 0.18 21.45 ± 0.70
16.51 ± 0.68 19.71 ± 0.54 8.58 ± 0.58 9.85 ± 0.39 25.72 ± 0.23 12.85 ± 0.16 19.09 ± 0.28 27.96 ± 0.53 22.16 ± 0.24 21.38 ± 0.39
>100 >100 >100 >100 64.8 >100 >100 60.1 97.5 >100 8.3
NO concentration of control group: 2.11 ± 0.09 μM, NO concentration of LPS-treated group: 42.25 ± 0.23 μM. bPositive control. 5 (tR 41.4 min, 6.7 mg). Fraction F14 (8.4 g) was subjected to silica gel column chromatography, using CHCl3−MeOH mixtures (15:1 → 1:1) for elution, resulting in subfractions F141−F146. Purification of F145 by RP C18 HPLC (C18 preparative column, 5 μm, 250 × 10 mm, 230 nm, MeCN−H2O, 33:67, 8.0 mL/min) afforded 1 (tR 30.2 min, 70 mg) and 2 (tR 46.1 min, 6.8 mg). Fraction F146 was separated by silica gel column chromatography eluting with CHCl3−MeOH mixtures (10:1), followed by RP p C18 HPLC (C18 preparative column, 5 μm, 250 × 10 mm, 210 nm, MeCN−H2O, 25:75, 8.0 mL/min) to yield 6 (tR 29.5 min, 3.4 mg). Separation of fraction F19 by silica gel column chromatography eluting with CHCl3−acetone−MeOH−HOAc mixtures (10:2:1:1) and RP C18 HPLC (C18 preparative column, 5 μm, 250 × 10 mm, 230 nm, MeOH−H2O, 55:45, 8.0 mL/min) yielded 9 (tR 33.6 min, 27.8 mg) and 10 (tR 36.3 min, 5.0 mg). Fraction E4 (8.0 g) was chromatographed via MPLC over reversedphase C18 silica gel using gradient elution (20−100% MeOH−H2O) to give fractions G1−G9 based on TLC analysis. Fraction G8 was subjected to silica gel column chromatography using a gradient CHCl3−MeOH solvent system (15:1 → 1:1), to give subfractions G81−G84. Fraction G82 was further separated by silica gel column chromatography again using CHCl3−MeOH (8:1) as solvent, to produce subfractions G82-1− G82-3. Using the same HPLC system as described above for the isolation of 6, fractions G82-2 and G82-3 gave 7 (tR 29.0 min, 3.0 mg), 8 (tR 35.7 min, 20.3 mg), and 3 (tR 51.0 min, 3.5 mg), respectively. (−)-6′-O-[(1R,2S,5R,6S)-10-Hydroxypinan-8-oxo]paeoniflorin (1): white powder; [α]20 D −25 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 201 (3.17), 229 (4.35), 273 (0.37) nm; IR (KBr) νmax 3415, 2944, 2877, 1719, 1602, 1452, 1384, 1345, 1315, 1275, 1178, 1111, 1076, 1053, 1011, 954, 922, 898, 855, 824, 799, 715, 687, 631 cm−1; 1H NMR (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data, see Table 1; HRESIMS m/z 669.2514 [M + Na]+ (calcd for C33H42O13Na, 669.2518). (−)-6′-O-[(1R,2R,5R,6S)-10-Hydroxypinan-8-oxo]paeoniflorin (2): white powder; [α]20 D −16 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 201 (3.15), 229 (4.44), 273 (0.35) nm; IR (KBr) νmax 3386, 2935, 2874, 1720, 1602, 1584, 1452, 1385, 1346, 1316, 1274, 1180, 1117, 1079, 1053, 1015, 955, 900, 881, 856, 824, 799, 716, 632, 577 cm−1; 1H NMR (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data, see Table 1; HRESIMS m/z 669.2518 [M + Na]+ (calcd for C33H42O13Na, 669.2518). (−)-6′-O-[(1R,2S,4S,5R)-4-Hydroxypinan-10-oxo]paeoniflorin (3): white powder; [α]20 D −22 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 201 (3.05), 229 (4.02), 273 (0.61) nm; IR (KBr) νmax 3416, 2966, 1723, 1602, 1584, 1451, 1386, 1347, 1315, 1276, 1230, 1197, 1177, 1079, 1047, 954, 899, 851, 822, 715, 686, 625, 578 cm−1; 1H NMR (methanold4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data, see Table 1; HRESIMS m/z 669.2530 [M + Na]+ (calcd for C33H42O13Na, 669.2518). (+)-6′-O-[(2E,6S)-6-Hydroxy-2,6-dimethylocta-2,7-dienoyl]paeoniflorin (4): white powder; [α]20 D +20 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (3.01), 225 (4.11), 275 (0.63) nm; IR (KBr) νmax 3394,
cells. The isolates were also tested for inhibitory activities of TNF-α secretion in mouse peritoneal macrophages,19 PTP1B (protein tyrosine phosphatase 1B),20 and acetaminopheninduced HepG2 cell injury9 and cytotoxic properties toward HCT-8 colon, A2780 ovary, BGC-823 stomach, Bel-7402 hepatoma, and A549 lung cell lines,21 but were inactive at 10 μM.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a Rudolph Research Autopol III automatic polarimeter. UV spectra were measured on a Cary 300 spectrometer. ECD spectra were recorded on a JASCO J-815 spectrometer. IR spectra were obtained on a Nicolet Impact 400 FT-IR spectrophotometer. The NMR experiments were conducted on a Bruker spectrometer (600 MHz for 1 H or 150 MHz for 13C) or a Varian INOVA spectrometer (500 MHz for 1 H or 125 MHz for 13C) equipped with an inverse detection probe. Residual solvent shifts for methanol-d4 were referenced to δH 3.31 and δC 49.15, respectively. ESIMS and HRESIMS data were acquired on a Q-Trap LC/MS/MS (Turbo ionspray source) and an Agilent 6520 Accurate-Mass Q-TOFL CMS spectrometer (Agilent Technologies, Ltd., Santa Clara, CA, USA), respectively. Column chromatography (CC) was run using MCI gel (CHP20P), silica gel (200−300 mesh, Qingdao Marine Chemical Inc., China), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala Sweden). HPLC separation was conducted on Waters HPLC equipment, namely, a Waters 600 pump, a Waters 600 controller, and Waters 2487 dual λ absorbance as well as GRACE semipreparative (250 × 10 mm) and preparative (250 × 19 mm) RP C18 (5 μm) columns. Plant Material. P. lactif lora roots were collected in Chifeng, Inner Mongolia Autonomous Region, People’s Republic of China, during September 2014 and identified by Prof. Min-Hui Li at Baotou Medical College. An herbarium specimen was deposited at the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Beijing 100050, People’s Republic of China (herbarium no. 2014-09-01). Extraction and Isolation. The air-dried P. lactiflora roots (50 kg) were ground and extracted using deionized water (150 L, 3 × 1 h) under ambient temperature and ultrasonication. The aqueous extracts were combined and subjected to a macroporous adsorbent resin (HPD-100, 30 kg) column (20 × 200 cm), eluting with H2O (50 L), 50% EtOH (150 L), and 95% EtOH (80 L), successively. The 50% EtOH fraction was concentrated and subjected to chromatography over MCI gel (CHP 20P, 10 L) with successive elution using H2O (30 L), 50% EtOH (80 L), 95% EtOH (30 L), and acetone (20 L), to afford fractions A−D. Fraction B was fractionated by Sephadex LH-20 column chromatography eluting with 50% MeOH to afford fractions E1−E8. Fraction E3 (42.8 g) was separated via MPLC over reversed-phase C18 silica gel using gradient elution (20−80% MeOH−H2O) to give subfractions F1−F19 based on TLC analysis. Fractions F12 (0.8 g) and F13 (1.3 g) were purified by RP C18 HPLC (C18 preparative column, 5 μm, 250 × 10 mm, 230 nm, MeCN−H2O, 35:65, 8.0 mL/min) to give 4 (tR 38.6 min, 15.0 mg) and 1257
DOI: 10.1021/acs.jnatprod.8b00087 J. Nat. Prod. 2018, 81, 1252−1259
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Acid Hydrolysis of 4. Compound 4 (8 mg) was hydrolyzed using 2 N HCl at 80 °C for 2 h. The reaction mixture was partitioned between EtOAc and H2O. The EtOAc fraction was concentrated and subjected to RP C18 HPLC (C18 preparative column, 5 μm, 250 × 10 mm, 230 nm, MeCN−H2O, 35:65) to give 4a (1.5 mg). (2E,6S)-6-Hydroxy-2,6-dimethylocta-2,7-dienoic acid (4a): color1 less solid; [α]20 D +21 (c 0.1, MeOH); H NMR (methanol-d4, 600 MHz) : 6.74 (1H, dt, J = 3.0, 1.8 Hz, H-3), 2.22 (2H, m, H2-4), 1.58 (2H, m, δH H2-5), 5.89 (1H, dd, J = 17.4, 10.8 Hz, H-7), 5.19 (1H, dd, J = 17.4, 1.8 Hz, H-8a), 5.03 (1H, d, J = 10.8 Hz, H-8b), 1.24 (3H, s, H3-9), 1.77 (3H, brs, H3-10); 13C NMR (methanol-d4, 150 MHz) δC 171.8 (C-1), 128.9 (C-2), 144.1 (C-3), 24.7 (C-4), 42.0 (C-5), 73.8 (C-6), 146.1 (C-7), 112.5 (C-8), 27.9 (C-9), 12.5 (C-10);10 negative-mode ESIMS m/z 183 [M − H]−. Enzymatic Hydrolysis of 8. Compound 8 (10.0 mg) was hydrolyzed with 15.0 mg of snailase (LJ0427B2011Z, Shanghai Sangon Biotech Co. Ltd.) in 2.5 mL of H2O at 37 °C for 12 h. The reaction mixture was extracted with EtOAc (3 × 3 mL). The aqueous phase of the hydrolysate was concentrated to dryness, and the sugar analysis was performed by the reported protocol.7 The EtOAc extract was chromatographed over silica gel, eluting with CHCl3−MeOH (50:1), to give 8a (3.5 mg). Paeoniflorigenone (8a). colorless solid; [α]20 D +7 (c 0.1, MeOH); 1 H NMR (methanol-d4, 500 MHz) δH 2.48 (1H, d, J = 18.0 Hz, H-3a), 2.69 (1H, d, J = 18.0 Hz, H-3b), 2.29 (1H, m, H-4), 2.17 (1H, dd, J = 2.0,13.0 Hz, H-6a), 2.27 (1H, dd, J = 2.0,13.0 Hz, H-6b), 1.18 (3H, s, H3-7), 2.77 (1H, br s, H-8), 5.34 (1H, s, H-9), 4.17, 3.96 (each 1H, m, H2-10), 7.91 (2H, m, H-2′, 6′), 7.37 (2H, m, H-3′, 5′), 7.50 (1H, m, H-4′); 13C NMR (methanol-d4, 125 MHz) δC: 80.2 (C-1), 102.9 (C-2), 48.3 (C-3), 44.5 (C-4), 216.7 (C-5), 35.7 (C-6), 22.1 (C-7), 47.9 (C-8), 101.0 (C-9), 64.3 (C-10), 131.3 (C-1′), 130.8 (C-2′, 6′), 129.8 (C-3′, 5′), 134.5 (C-4′), 167.8 (C-7′);15 negative-mode ESIMS m/z 317 [M − H]−. Calculation of ECD Data of 1a. See Supporting Information (Figures S1 and S2). Sugar Analysis. See ref 7. Inhibitory Assay of NO Production. See ref 19. Anti-inflammatory Activity Assay. See ref 20. PTP1B Inhibition Assay. See ref 21. Protective Effect of Acetaminophen-Induced HepG2 Cell Injury. See ref 9. Cytotoxicity Assay. See ref 22.
2974, 2928, 1710, 1646, 1602, 1452, 1386, 1346, 1316, 1279, 1230, 1179, 1109, 1077, 1055, 1012, 945, 923, 855, 824, 800, 738, 716, 631 cm−1; 1H NMR (methanol-d4, 600 MHz) and 13C NMR (methanold4, 150 MHz) data, see Table 1; HRESIMS m/z 669.2494 [M + Na]+ (calcd for C33H42O13Na, 669.2518). (+)-(1S,5R,6R)-8-O-Benzoylmudanpioside F (5): white powder; [α]20 D +56 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (3.92), 230 (4.68), 256 (2.27) nm; ECD (MeOH) 326 (Δε +0.79), 254 (Δε +4.22), 226 (Δε −6.37); IR (KBr) νmax 3377, 2974, 2927, 2880, 1780, 1716, 1677, 1601, 1579, 1451, 1412, 1316, 1274, 1229, 1209, 1171, 1078, 998, 980, 894, 857, 835, 805, 792, 715, 686, 657, 618 cm−1; 1H NMR (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) data, see Table 2; HRESIMS m/z 471.1639 [M + Na]+ (calcd for C23H28O9Na, 471.1626). (+)-(1S,5R,6R)-6′-O-Benzoylmudanpioside F (6): white powder; [α]20 D +48 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (4.01), 230 (4.83), 255 (2.49) nm; ECD (MeOH) 325 (Δε +1.11), 261 (Δε +4.28), 223 (Δε −7.01); IR (KBr) νmax 3318, 2967, 2921, 2850, 1775, 1720, 1677, 1574, 1414, 1320, 1278, 1228, 1179, 1116, 1078, 1025, 962, 878, 833, 799, 715, 654, 621 cm−1; 1H NMR (methanol-d4, 600 MHz) and 13 C NMR (methanol-d4, 150 MHz) data, see Table 2; HRESIMS m/z 471.1637 [M + Na]+ (calcd for C23H28O9Na, 471.1626). 8-Deoxyfloralabiflorin (7): white powder; [α]20 D −8 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (3.86), 229 (426), 273 (0.95) nm; IR (KBr) νmax 3412, 2974, 2932, 1748, 1722, 1602, 1584, 1452, 1383, 1319, 1282, 1176, 1079, 1033, 944, 924, 879, 851, 716, 672, 625 cm−1; 1H NMR (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data, see Table 2; HRESIMS m/z 487.1580 [M + Na]+ (calcd for C23H28O10Na, 487.1575). (+)-Paeoniflorigenone-1-O-β-D-glucopyranoside (8): white powder; [α]20 D +8 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (3.88), 228 (4.57), 271 (1.52) nm; ECD (MeOH) 296 (Δε −0.53), 222 (Δε −0.17); IR (KBr) νmax 3375, 2933, 1720, 1601, 1560, 1451, 1396, 1353, 1316, 1278, 1182, 1072, 1043, 962, 934, 899, 803, 772, 715, 687, 627 cm−1; 1H NMR (methanol-d4, 600 MHz) and 13C NMR (methanold4, 150 MHz) data, see Table 2; HRESIMS m/z 481.1707 [M + H]+ (calcd for C23H29O11, 481.1704). (−)-Cuminyl-[α-L-rhamnopyranosyl(1→6)]-β-D-glucopyranoside (9): white powder; [α]20 D −55 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (4.68), 263 (0.56) nm; IR (KBr) νmax 3406, 2960, 2929, 1646, 1515, 1457, 1419, 1364, 1316, 1268, 1134, 1064, 984, 915, 882, 836, 816, 671, 648, 618, 542 cm−1; 1H NMR (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) data, see Table 2; HRESIMS m/z 481.2051 [M + Na]+ (calcd for C22H34O10Na, 481.2044). (E)-1-Oxogeranyl-O-[α-L-rhamnopyranosyl(1→6)]-β-D-glucopyranoside (10): white powder; [α]20 D −55 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (2.98), 219 (3.51) nm; IR (KBr) νmax 3383, 2926, 2855, 1716, 1674, 1642, 1600, 1418, 1384, 1270, 1208, 1139, 1070, 986, 915, 838, 808, 722, 672, 622, 584 cm−1; 1H NMR (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data, see Table 2; HRESIMS m/z 499.2133 [M + Na]+ (calcd for C22H36O11Na, 499.2150). Alkaline Hydrolysis of 1. Compound 1 (5 mg) was hydrolyzed using 0.5 N NaOH (4 mL) at 30 °C for 2 h. The reaction was quenched by addition of 2 N HCl, and the reaction mixture was applied to a C18 solid phase extraction column and eluted with H2O (25 mL) and MeCN (25 mL) successively. The MeCN fractions were concentrated and separated by preparative TLC eluting with CHCl3−MeOH mixtures (20:1) to produce 1a (0.8 mg). (1R,2S,5R,6S)-10-Hydroxypinan-8-carboxylic acid (1a): white powder; [α]20 D −29 (c 0.2, MeOH); ECD (MeOH) 219 (Δε +0.7); 1 H NMR (methanol-d4, 600 MHz) δH 2.71 (1H, ddd, J = 6.0, 5.4, 1.8 Hz, H-1), 2.24 (1H, m, H-2), 1.92, 1.47 (each 1H, m, H2-3), 2.04, 1.96 (each 1H, m, H2-4), 2. 57 (1H, m, H-5), 2.15 (1H, m, H-7a), 1.00 (1H, d, J = 9.0 Hz, H-7b), 1.24 (3H, s, H3-9), 3.57, 3.47 (each 1H, dd, J = 10.8, 7.8, Hz, H2-10); 13C NMR (methanol-d4, 150 MHz) δC 42.7 (C-1), 44.9 (C-2), 19.3 (C-3), 26.1 (C-4), 41.5 (C-5), 52.0 (C-6), 34.8 (C-7), 185.0 (C-8), 19.0 (C-9), 67.6 (C-10); positive-mode ESIMS m/z 185 [M + H]+; negative-mode ESIMS m/z 183 [M − H]−; HRESIMS m/z 207.0989 [M + Na]+ (calcd for C10H16O3Na, 207.0992).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00087. 1D NMR spectra of compounds 4a and 8a and 1D and 2D NMR spectra of compounds 1−10 and 1a (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: 86-10-83154789. Fax: 86-10-63017757. E-mail: lsznn@ imm.ac.cn. ORCID
Sheng Lin: 0000-0002-9587-5664 Author Contributions ⊥
R. Li and J. F. Zhang contributed equally to this study.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (NNSFC; Nos. 81522050, 81773589), the National Science and Technology Project of 1258
DOI: 10.1021/acs.jnatprod.8b00087 J. Nat. Prod. 2018, 81, 1252−1259
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China (No. 2018zx09711001-001), and the CAMS Innovation Fund for Medical Science (CIFMS; No. 2017-I2M-3-010).
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DOI: 10.1021/acs.jnatprod.8b00087 J. Nat. Prod. 2018, 81, 1252−1259
