Article pubs.acs.org/jnp
Anti-inflammatory Hydrolyzable Tannins from Myricaria bracteata Jia-Bao Liu,† Ya-Si Ding,† Ying Zhang,†,‡ Jia-Bao Chen,† Bao-Song Cui,† Jin-Ye Bai,† Ming-Bao Lin,† Qi Hou,† Pei-Cheng Zhang,† and Shuai Li*,† †
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ Forensic Medical Examination & Identification Center of Beijing Public Security Bureau, Key Laboratory of Forensic Toxicology, Ministry of Public Security, Beijing 100192, People’s Republic of China S Supporting Information *
ABSTRACT: Twelve hydrolyzable tannins were obtained from the twigs of Myricaria bracteata, including two new hellinoyl-type dimers, bracteatinins D1 (1) and D2 (2); a new hellinoyl-type trimer, bracteatinin T1 (3); two known monomers, nilotinin M4 (4) and 1,3di-O-galloyl-4,6-O-(aS)-hexahydroxydiphenoyl-β-D-glucose (5); six known dimers, tamarixinin A (6), nilotinin D8 (7), hirtellins A (10), B (9), and E (8), and isohirtellin C (11); and a known trimer, hirtellin T3 (12). The structures of the tannins were elucidated by spectroscopic data analysis and comparisons to known tannins. All compounds were evaluated as free radical scavengers using 1,1-diphenyl-2-picrylhydrazyl and hydroxy radicals and compared to the activity of BHT and Trolox. Compound 6 showed a significant anti-inflammatory effect on croton oil-induced ear edema in mice (200 mg/kg, inhibition rate 69.8%) and on collagen-induced arthritis in DBA/1 mice (20 mg/kg, inhibition rate 46.0% at day 57).
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RESULTS AND DISCUSSION An extract of the dried twigs of M. bracteata was subjected to Diaion HP 20 column chromatography. The EtOH/H2O eluate was subjected to a combination of chromatographic steps on Toyopearl HW-40 and MCI-gel CHP-20P gels, followed mainly by HPLC purification to afford three new hellinoyl-type tannins, bracteatinins D1 (1), D2 (2), and T1 (3). Additionally, nine known tannins were obtained from the genus Myricaria for the first time, and their structures were identified by spectroscopic data analysis and comparison to literature values. Furthermore, the 13C NMR spectroscopic assignments of hirtellin E (8) and hirtellin T3 (12) are reported for the first time. Structure Elucidation of Dimeric Hellinoyl-Type Ellagitannins. Bracteatinin D1 (1) was isolated as an off-white, amorphous powder. It was presumed to be a dimeric ellagitanin, as established from the following spectroscopic features. The molecular formula of 1 was determined to be C61H46O40 by 13C NMR spectroscopic data and HRESIMS analyses (m/z 1418.1563 [M − H]−, calcd for C61H44O40, 1418.1565). The 1 H NMR data (Table 1) of 1 revealed two distinct patterns of proton resonances (1.9:1) for the sugar and phenolic moieties, suggesting that the anomeric position of the sugar moiety was not acylated. The 13C NMR data (Table 2) also showed two anomeric carbons at δC 90.9 (α-anomer) and 96.1 (β-anomer),
Myricaria bracteata Royle (Tamaricaceae) is a traditional Tibetan herb named “Wenbu”, which has been used to treat several diseases, in particular rheumatism and arthritis.1 The herb is widely distributed in Asia and Europe, especially in Qinghai, Tibet, and the Gansu Province of China.2 Although previous studies have reported some pharmacological properties of extracts, such as anti-inflammatory,3 antibiosis,4 cellular immunity,5 antifatigue,6 and antioxidant7 activities, the active constituents have not been identified. Previous phytochemical investigations on Myricaria Desv led to the isolation of flavonoids,8−13 phenolic acids,8−10,12,13 triterpenoids, sterols,14−16 and long-chain fatty alcohols.17 Although these ingredients have anti-inflammatory and antioxidant activities,18−20 our further investigation showed that the hydrophilic portion of M. bracteata, which is rich in polyphenols, mainly hydrolyzable tannins, has significant anti-inflammatory activity. As a group of natural products, tannins show marked antiinflammatory, antiviral, antimicrobial, immunomodulatory, antitumor, and hepatic protective activities.21−26 This report describes the isolation and characterization of two new hellinoyltype dimers, bracteatinins D1 (1) and D2 (2), and a new hellinoyl-type trimer, bracteatinin T1 (3), along with two known monomers, nilotinin M4 (4)27 and 1,3-di-O-galloyl-4,6-O-(aS)hexahydroxydiphenoyl-β-D-glucose (5),28 six known dimers, tamarixinin A (6),29 nilotinin D8 (7),27 hirtellins A (10),29 B (9),30 and E (8),31 and isohirtellin C (11),27 and a known trimer, hirtellin T3 (12).32 We also report the anti-inflammatory and free radical scavenging activity of these tannins. © 2015 American Chemical Society and American Society of Pharmacognosy
Received: November 28, 2014 Published: April 28, 2015 1015
DOI: 10.1021/np500953e J. Nat. Prod. 2015, 78, 1015−1025
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Table 1. 1H NMR Spectroscopic Data for the Glucosidic Protons of 1, 2, 3, 7, 8, and 12 (600 MHz, Acetone-d6/D2O, 9:1, J in Hz) 1a
2b
α-anomer glucose-1 1 2 3 4 5
5.46 d (3.6) 5.26 dd (9.6, 3.6) 5.76 t (9.6) 5.00 m 4.64 dd (9.6, 6.0)
6
β-anomer
α-anomer
β-anomer
7
5.10 d (7.8) 5.27 m
5.42 d (3.6) 5.09 m
5.08 d (7.2) 5.40 m
6.19 d (8.4) 5.65 dd (8.4, 9.6)
6.08 d (7.8) 5.44 dd (7.8, 9.6)
5.99 d (8.0) 5.70 t (8.0)
6.02 d (8.5) 5.72 t (8.5)
5.41 t (9.6) 5.01 m 4.20 dd (9.6, 6.0) 5.22 m
5.73 t (9.6) 3.77 m 4.03 m
5.44 t (9.6) 3.72 m 3.60 m
5.69 t (9.6) 3.86 overlapped 3.85 overlapped
5.57 t (9.6) 5.10 t (9.6) 4.42 m
3.92 dd (1.8, 12.6)
5.30 overlapped
5.59 t (10.0) 5.12 t (10.0) 4.42 dd (6.5, 10.0) 5.29 m
3.76 m
5.80 t (9.6) 5.14 t (9.6) 4.52 dd (6.6, 10.2) 5.31 dd (6.6, 13.2) 3.87 br d (13.2)
3.74 overlapped
3.78 overlapped
3.86 overlapped
5.80h d (8.4) 5.78h d (8.4) 5.39i m 5.41i m j 5.74 d (9.6) 5.77j d (9.6) 5.05 m 4.45 m
5.54 d (8.4) 5.19 dd (8.4, 9.6) 5.49 t (9.6) 3.85 overlapped 3.73 overlapped
5.63 d (8.4) 5.38 dd (8.4, 10.2) 5.65 t (10.2) 5.20 t (10.2) 4.38 ddd (1.8, 7.2, 10.2) 5.34 dd (7.2, 13.8)
5.62 t (8.4) 5.37 dd (8.4, 9.6) 5.68 t (9.6) 5.14 t (9.6) 4.34 m
5.62 t (8.5) 5.36 t (10.0) 5.69 t (10.0) 5.14 t (10.0) 4.34 dd (8.0, 10.0) 5.25 dd (4.5, 13.0) 3.95 overlapped
3.77 m
3.77 m glucose-2 1 2 3 4 5
5.64c d (8.4) 5.21d m 5.50e d (8.4) 3.84f overlapped 3.70g m
6
5.62c d (8.4) 5.19d t (10.2) 5.52e d (8.4) 3.82f overlapped 3.72g m
3.90 m
5.29 m
3.79 m
3.83 m
4.07 dd (2.4, 12.6) 3.88 dd (5.4, 12.6)
8
3
5.34 dd (7.2, 13.2) 4.05 overlapped
4.16 dd (1.8, 13.8)
glucose-3 1 2 3 4 5
5.42 d (8.4) 5.33 m 5.32 m 3.86 overlapped 3.62 overlapped
6 a
12
3.88 overlapped 3.77 overlapped
Ratio of α- and β-anomers = 1.9:1. bRatio of α- and β-anomers = 1:1.
c−j
5.44 d (8.5) 5.54 t (10.0) 5.66 t (10.0) 5.13 t (10.0) 4.26, dd (10.0, 6.5) 5.31 overlapped 3.84 overlapped
Interchangeable.
Table 2. 13C NMR Spectroscopic Data for the Glucosyl Moieties of 1, 2, 3, 7, 8, and 12 (151 MHz, Acetone-d6/D2O, 9:1) 1 glucose-1 1 2 3 4 5 6 glucose-2 1 2 3 4 5 6 glucose-3 1 2 3 4 5 6
2
α-anomer
β-anomer
α-anomer
β-anomer
7
8
3
12
90.9 73.8 71.4 71.5 66.9 63.4
96.1 72.1 73.6 71.3 71.7 63.5
90.7 70.7 70.65 69.69 72.7 61.7
95.5 76.7 73.8 69.65 77.3 61.8
93.30 71.3 73.1 70.9 72.8 63.1
92.9 71.6 74.2 69.2 77.9 61.5
93.5 71.2 73.0 70.4 72.6 63.1
93.4 71.3 73.0 70.6 72.7 63.1
93.3 70.7 76.3 68.6 77.8
93.4 70.6 76.4 68.5 77.8
93.74 70.7 73.7 72.55 72.7
93.72 70.58 73.8 72.51 72.6
93.34 70.7 76.5 69.0 77.7 61.5
93.4 70.3 75.9 70.8 72.3 63.1
93.5 70.5 74.1 70.5 72.2 62.8
93.6 71.1 74.0 70.5 72.3 63.0
93.1 76.3 71.3 68.6 78.1 61.2
93.5 71.4 73.5 70.6 72.8 62.9
60.9
62.8
which indicated that compound 1 exists as an equilibrium mixture of α- and β-anomers in solution.33 Although the 1H
NMR spectrum of 1 was complex, the typical aromatic proton signals indicated the presence of a hellinoyl group [a pair of one1016
DOI: 10.1021/np500953e J. Nat. Prod. 2015, 78, 1015−1025
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Figure 1. Structures of 1, 2, 6−8, 6a, and 6b. The arrows (H→C) indicate important HMBC correlations.
proton singlets at δH 7.58, 7.54 (each s), 6.692, 6.685 (each s), two mutually coupled doublets at δH 7.00, 6.94 (each d, J = 1.8 Hz), 5.93, 5.92 (each d, J = 1.8 Hz),27 a hexahydroxydiphenoyl (HHDP) unit [δH 6.59, 6.58 (each s) and 6.52, 6.51 (each s)],34 and two galloyl groups [6.98 (2H, s), 6.86, 6.85 (each s)].34 In the aliphatic region of the spectrum, the two seven-spin proton
systems (δH 5.80−3.70), assignable to the protons of two glucose cores with large coupling constants (Table 1), were distinguished by a 1H−1H COSY experiment, indicating the presence of glucopyranose cores in the 4C1 conformation. The anomeric protons of glucose-1 [δH 5.46 (d, J = 3.6 Hz) and 5.10 (d, J = 7.8 Hz), 1H in total], [5.64 (d, J = 8.4 Hz) and 5.62 (d, J = 8.4 Hz), 1017
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Table 3. 13C NMR Spectroscopic Data for the Aromatic Moieties of 1, 2, 7, and 8 (151 MHz, Acetone-d6/D2O, 9:1) 1a,b hellinoyl C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-1′ C-2′ C-3′ C-4′ C-5′ C-6′ C-7′ C-1″ C-2″ C-3″ C-4″ C-5″ C-6″ C-7″ HHDP C-1 C-2, 2′ C-3 C-4, 4′ C-5 C-6, 6′ C-7 C-1′ C-3′ C-5′ C-7′ galloyl C-1 C-2, 6 C-3, 5 C-4 C-7 a
α- and β-Anomers. Interchangeable.
c,d
2a,b
7
119.8 120.0 120.0 121.1 107.8 110.0 147.8 148.0 148.07 148.04 139.5d 139.4d 140.0c 139.9c 146.75 146.83 145.6 145.7 111.0 111.2 107.6 107.8 165.0 165.2 164.8 165.0 111.4 111.5 108.02 108.03 142.4 142.3 142.6 142.5 139.7d 139.8d 139.6c 139.7c 139.10d 139.06d 139.0c 138.9c 141.61 141.60 141.83 141.82 108.9 108.7 163.7d 163.6d 163.57c 163.60c 113.8 114.5 114.1 114.7 145.0d 144.7d 143.9c 143.8c d d c 140.7 140.3 140.6 140.5c d d c 139.7 139.8 139.6 139.7c 143.40 143.36 143.23 143.16 118.6 118.4 118.7 118.6 164.6 164.3 164.0 163.6 115.59 115.63 115.6 115.7 125.6, 125.9, 126.0 (2C in total) 125.8, 125.5 (2C in total) 107.7 107.8 108.02 108.04 145.07, 145.06, 145.09, 145.07, 145.04 (2C in total) 145.06 (2C in total) 136.2 136.4 144.32, 144.30, 144.3, 144.2 144.25 (2C in total) (2C in total) 168.6 168.5 168.2 168.1 115.67 115.71 115.8 115.7 107.6 107.7 107.84 107.87 136.3 136.3 167.73 167.75 167.71 167.74 120.4, 120.0, 119.8, 119.5 121.1, 120.9, 119.9, 119.8 (2C in total) (2C in total) 110.30, 110.2, 109.87, 109.85 110.00, 109.93, 109.91 (4C in total) (4C in total) 145.65, 145.63, 145.4, 145.33 145.67, 145.65, 145.61 (4C in total) (4C in total) 139.12, 139.10, 139.06, 139.02 139.28, 139.26, 138.91, (2C in total) 138.82 (2C in total) 167.24, 167.22, 166.71, 166.73 167.1, 167.0, 166.99 (2C in total) (2C in total) b
The
13
8
119.7 111.7 147.5 139.6 146.7 111.7 164.8 111.4 142.2 139.9 139.2 141.8 108.7 163.5 114.7 144.3 140.7 139.7 143.1 118.8 164.2 115.7 125.9, 126.0 (2C in total) 108.4 145.0, 145.1 (2C in total)
119.4 107.3 147.6 139.9 146.9 107.5 164.8 111.3 142.3 139.7 139.2 141.8 108.9 163.5 114.1 144.4 140.7 139.7 143.2 119.0 164.0 115.7 125.5, 126.0 (2C in total) 107.8 145.1, 145.2 (2C in total)
136.4 144.2, 144.3 (2C in total)
136.3 144.37, 144.40 (2C in total)
168.4 115.8 107.5 136.4 167.7 118.8, 119.8, 120.5 (3C in total)
168.4 115.8 107.7 136.5 167.8 118.8, 119.7, 120.6 (3C in total)
109.9, 110.0, 110.4 (2C each)
110.0, 110.3, 110.5 (2C each)
145.4, 145.6, 145.7 (2C each)
145.5, 145.6, 145.8 (2C each)
139.0, 139.2, 139.4 (3C in total)
139.2, 139.3, 139.6 (3C in total)
165.1, 166.9, 167.7 (3C in total)
165.1, 166.5, 167.3 (3C in total)
C NMR assignments for this compound were achieved based on HSQC and comparison with 7 and 8.
HMBC spectrum showed correlations between the galloyl proton signals [δH 6.98 (2H, s), 6.86, 6.85 (each s)] and the signals of H-3 of glucose-1 (δH 5.76, 5.41) and H-3 of glucose-2 (δH 5.52, 5.50) to the respective carbonyl carbon peaks (δC 167.24, 167.22) and (δC 166.71, 166.73). The OH-1 of glucose-1 and the OH-4 and OH-6 of glucose-2 were unacylated, as indicated by shielding of the corresponding proton signals; the remaining hydroxy groups of the glucose cores, i.e., at C-1 and C2 of glucose-2 and C-2 of glucose-1, were also O-acylated, as judged by the deshielding of the corresponding proton signals (Table 1). Consequently, the galloyl units of the hellinoyl moiety should be placed at O-1 and O-2 of glucose-2 and O-2 of glucose1. This attachment mode of the hellinoyl moiety was also substantiated by the HMBC correlations between two sets of meta-coupled proton signals of the hellinoyl G-1-ring [δH 7.00,
1H in total] and the two large coupling constants (J1, 2 = 8.4, 8.4 Hz) of the anomeric proton signals indicated the existence of the two β-glucopyranose cores in the 4C1 conformation. The large and small coupling constants (Jβ‑anomer1,2 = 7.8 Hz, Jα‑anomer1,2 = 3.6 Hz) of the anomeric protons indicated the presence of the α- and β-anomers, both in the 4C1 conformation. Compared to 7, H-1 of the glucose-1 moiety of 1 resonated at a higher field [δH 5.46 (d, J = 3.6 Hz, H-1, α-anomer); 5.10 (d, J = 7.8 Hz, H-1, β-anomer)] relative to that of 7 [δH 6.19 (d, J = 8.4 Hz)], indicating the presence of an unsubstituted C-1 hydroxy group. An HHDP unit was located at C-4/C-6 of glucose-1, as indicated by the large chemical shift difference (ΔδH 1.45) between the C-6 gemproton signals (δH 5.22 and 3.77). Correspondingly, the 4- and 6hydroxy groups of glucose-2 were unacylated because of the upfield shifts (δH 3.79, 3.90) of the glucose-2 H-4 and H-6.34 The 1018
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6.94 (each d, J = 1.8 Hz), 5.93, 5.92 (each d, J = 1.8 Hz)] and the H-1 signal of glucose-2 (δH 5.64, 5.62) via a set of carbonyl carbon signals (δC 165.2, 165.0) and the correlations between the proton signal of the hellinoyl G-2-ring [δH 6.692, 6.685 (each s)] and the H-2 signal of glucose-2 (δH 5.21, 5.19) via a set of carbonyl carbon signals (δC 163.7, 163.6); however, the proton signal [δH 7.58, 7.54 (each s)] of the hellinoyl G-3-ring showed HMBC correlations of the glucose-1 H-2 signal (δH 5.27, 5.26) to a set of carbonyl carbon signals (δC 164.6, 164.3) (Figure 1). The (aS)-absolute configuration of the HHDP groups in 1 was assigned based on the positive Cotton effect at 233 nm in the electronic circular dichroism (ECD) spectrum.35 The characteristics of the NMR spectrum of 1 are similar to those of nilotinin D8 (7)27 but lack a hydrogen, a carbonyl, and six aromatic carbon signals of the galloyl moiety compared with that of 7. Regioselective degalloylation of 7 by tannase yielded 1 and confirmed the structure of 1 as bracteatinin D1. Bracteatinin D2 (2) was isolated as an off-white, amorphous powder. Its molecular formula was the same as that of 1, determined as C61H46O40 by 13C NMR spectroscopic and HRESIMS analyses, 1418.1569 [M − H] − (calcd for C61H44O40, 1418.1565). The 1H and 13C NMR spectra showed two sets of signals and a peak area ratio of 1:1. The 1H NMR spectroscopic characteristics are similar to those of bracteatinin D1 (1). The aromatic proton signals were consistent with a hellinoyl group [δH 7.63, 7.58 (each s), 6.71, 6.70 (each s), 7.06, 7.05 (each d, J = 1.8 Hz), 6.05, 6.04 (each d, J = 1.8 Hz)], an HHDP group [δH 6.61, 6.59 (each s), 6.48, 6.47 (each s)], and two galloyl groups [7.05 (2H, s), 6.88, 6.87 (each s)]. Therefore, we speculate that 2 is an isomer of 1. Compared to 1, H-4 (δH 5.05, m) and H-6a (δH 5.29, m) of the glucose-2 moiety of 2 resonated at a higher field relative to H-4 [δH 3.84 (overlapped, α-anomer)] and H-6a (δH 3.90, m) of glucose-2 of 1, which were assigned on the basis of 1H−1H COSY and HSQC correlations. The resonances of two 4C1 glucopyranose cores were evident in the aliphatic region of the 1H NMR spectrum, and their sets of proton signals were differentiated by the 1H−1H COSY experiment (Table 1). Compared to 1, the HHDP unit was connected to glucose-2 at C-4/C-6 instead of glucose-1, as indicated by the large chemical shift difference (ΔδH 1.46) between the C-6 gem-proton signals (δH 5.29 and 3.83). Correspondingly, the C-4 and C-6 hydroxy groups of glucose-1 were unacylated because of the shielding (δH 3.72−3.77) of the glucose-1 H-4 and H-6 resonances. The presence of the acyl units was confirmed based on their aromatic and ester carbonyl carbon resonances in the 13C NMR spectrum (Table 3). The structure of 2 was thus assigned to be an HHDP regioisomer of 1. The HMBC correlations of 2 (Figure 1) agreed with the assigned structure. The (aS)-absolute configuration of the HHDP groups in 2 was assigned based on the positive Cotton effect at 232 nm in the ECD spectrum.35 Subsequently, regioselective degalloylation of 8 by tannase yielded 2 and was found to be bracteatinin D2. Tamarixinin A (6) was isolated as a major tannin from the antiinflammatory active fraction of M. bracteata and showed a moderate anti-inflammatory effect in vivo. It was also a mixture of α- and β-glucopyranose anomers of the hydrolyzable tannin, which was first isolated from Tamarix pakistanica.29 However, the proton signals of the two anomers of glucose-1 were overlapped in the 1H NMR spectrum. To further substantiate the structure of the isomers, 6 was methylated and the mixture was separated to afford 6a and 6b. The complete proton and carbon assignments of 6a and 6b were also achieved on the basis of 1D
and 2D NMR experiments. The detailed assignments of 6a and 6b are shown in the Experimental Section. Hirtellin E (8) was first isolated as an off-white, amorphous powder from Reaumuria hirtella.27 However, its 13C NMR assignments were not reported. Its 1H NMR spectroscopic data (see Experiment Section and Table 1) agreed well with literature values. The locations of the acyl groups were substantiated by 1 H−1H COSY data listed in Table 1 and the HMBC correlations shown in Figure 1. The complete assignment of the carbon atoms of 8 is listed in Tables 2 and 3. Structure Elucidation of Trimeric Hellinoyl-Type Ellagitannins. Bracteatinin T1 (3) was isolated as an offwhite, amorphous powder. Its molecular formula, C109H78O70, was established from 13C NMR data and a prominent ion peak at m/z 2506.2526 [M − H]− (calcd for C109H76O70, 2506.2544) by HRESIMS analyses, which suggested the compound was a trimeric ellagitanin. The aromatic proton signals exhibited two meta-coupled doublets [δH 7.22, 6.41 (each 1H, d, J = 1.8 Hz)] and a proton singlet at δH 7.02, which are characteristic of a dehydrodigalloyl moiety (DHDG),27 in addition to the other two meta-coupled doublets [δH 6.83, 5.76 (each 1H, d, J = 1.8 Hz)] and two proton singlets at δH 7.52 and 6.61 that were attributed to a hellinoyl unit. Four singlets [δH 6.45, 6.53, 6.60, and 6.67 (each 1H, s)] indicated the presence of two HHDP groups. The four singlets [δH 6.76, 6.89, 7.00, 7.03 (each 2H, s)] in the aromatic region indicated the presence of four galloyl groups. The 1H NMR spectrum also showed three seven-spin systems assignable to the protons of three glucose cores (Table 1). Among these signals, three broad signals (δH 5.99, 5.62, and 5.42) were assigned to anomeric protons of the glucose-1, 2, and 3 moieties on the basis of HSQC correlations with anomeric carbon peaks [δC 93.5 (2C) and 93.1], respectively. Despite the overlapping of these anomeric proton signals, the 4 C 1 conformations of the glucose cores were implied from the large coupling constants of the remaining proton signals (Table1). The OH-6 and OH-4 of glucose-3 were unacylated, as indicated by the relatively shielded H-4 and H-6 (δH 3.86, 3.88, and 3.77) resonances of glucose-3. The remaining hydroxy groups of the glucose cores were O-acylated, as judged by the deshielded corresponding proton signals (Table 1). The 13C NMR spectrum of 3 showed carbon peaks (Table 2) consistent with the presence of these acyl moieties and the glucose cores. The locations of the galloyl units were assigned to C-3 of each of the glucose cores and C-1 of glucose-3, as determined from the HMBC correlations of the galloyl proton signals (δH 6.76, 6.89, 7.00, and 7.03) with the H-3 signals (δH 5.68, 5.57, and 5.32) of the three glucose cores and H-1 (δH 5.42) of glucose-3 through common carbonyl carbon peaks (δC 166.3, 167.1, 165.0, and 167.2). Two HHDP units at C-4/C-6 of the glucose-1 and glucose-2 cores were demonstrated by the large chemical shift difference (ΔδH 1.52 and 1.29) between the C-6 methylene proton signals (δH 5.30/3.78 and 5.34/4.05) of the glucosyl moieties.32 The location of the HHDP units was further confirmed by HMBC correlations among the HHDP proton signals (δH 6.45, 6.53, 6.60, and 6.67) and signals of H-4 (δH 5.10 and 5.14) and H-6 (δH 5.30 and 5.34) of the glucose-1 and glucose-2 cores through common ester carbonyl carbon peaks (δC 167.6, 167.7, 168.3, and 168.5). The galloyl parts of the hellinoyl moiety should be placed at O-1 and O-2 of glucose-1 and O-2 of glucose-2. This attachment mode of the hellinoyl moiety was also substantiated by the HMBC correlations between two meta-coupled proton signals [δH 6.83 (1H, d, J = 1.8 Hz), 5.76 (1H, d, J = 1.8 Hz)] of the hellinoyl G-1-ring and 1019
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the H-1 signal (δH 5.99) of glucose-1 via a carbonyl carbon signal (δC 165.1) and a correlation between the proton signal [δH 6.61 (1H, s)] of the hellinoyl G-2-ring and the H-2 signal (δH 5.70) of glucose-1 via a carbonyl carbon signal (δC 163.7). However, the proton signal [δH 7.52 (1H, s)] of the hellinoyl G-3-ring showed an HMBC correlation with the H-2 signal (δH 5.37) of glucose-2 through a carbonyl carbon signal (δC 165.6). The HMBC correlations among DHDG H-2′, 6′ signals [δH 7.22, 6.41 (each 1H, d, J = 1.8 Hz)] to the H-1 signal (δH 5.62) of glucose-2 via a carbonyl carbon peak (δC 164.5), and DHDG H-2 [δH 7.02 (1H, s)] to the H-2 signal (δH 5.33) of glucose-3 via a carbonyl carbon peak (δC 164.6) confirmed that the galloyl units of the DHDG moiety should be placed at O-1 of glucose-2 and O-2 of glucose3. Thus, the characteristics of the NMR spectra are similar to those of hirtellin T3 (12),32 minus an HHDP unit, as the Mr value of 3 is 302 units lower than that of 12. The assignments of the carbon resonances of 3 (Table 2) were determined on the basis of HSQC data and comparison with the corresponding signals of the analogue 12. The (aS)-absolute configuration of the HHDP groups in 3 was assigned based on the positive Cotton effect at 234 nm in the ECD spectrum. On the basis of the above spectroscopic analysis, the structure of bracteatinin T1 was formulated as shown in 3 (Figure 2). Hirtellin T3 (12) was first isolated as an off-white, amorphous powder from R. hirtella.32 Herein, we identified the structure of 12 on the basis of a comparison of its 1H NMR spectroscopic data (see Experimental Section and Table 1) with those in the literature. However, the chemical shifts of the assigned H-6 and H-6′ (δH 7.71 and 7.57) resonances of the hellinoyl moiety were unusually high compared to those of the related hellinoyl-type tannins. In this present study, the 1H NMR spectrum of 12 was revised on the basis of HSQC and HMBC correlations. We also present the full assignment of carbon atoms of 12 and substantiated the acyl group locations by 1H−1H COSY data listed in Table 1 and the HMBC correlations shown in Figure 2. Structure Elucidation of Monomeric Hydrolyzable Ellagitannins. 1,3-Di-O-galloyl-4,6-O-(aS)-hexahydroxydiphenoyl-β- D-glucose (5) was first isolated as an off-white, amorphous powder from the leaves of Acacia raddiana, and its structure was previously assigned on the basis of chemical manipulation and spectroscopic data.21 However, the reported chemical shift of the glucose H-2 signal was much higher [δH 4.70 (t, J = 9 Hz)] than that of the corresponding signal of the unsubstituted glucose. Therefore, we performed extensive NMR spectroscopic experiments including 1H and 13C NMR, 1H−1H COSY, HSQC, and HMBC to verify the assignments for 5. However, the proton signal at δH 4.70, which was previously assigned to the glucose H-2, was not observed, and an alternative proton signal (δH 3.96) in our experiment was assigned here to the glucose H-2 on the basis of 1H−1H COSY and HSQC correlations. Since the NMR data were recorded in DMSO-d6, the resonance at δH 4.70 was likely a 2-OH proton signal of glucose, and the H-2 signal overlapped with the solvent peak. The remaining proton signals have been corrected, and the complete assignment of the carbon resonances of 5 and the substitution site of acyl group were determined by 1H−1H COSY data and HMBC correlations shown in Figure 3. Antioxidant Activities of Tannins. The antioxidant activity of the hydrolyzable tannins was tested due to the presence of phenolic hydroxy groups in their structures. In our experiment, tannins suppressed the content of the lipoperoxidation product malondialdehyde (MDA) in mitochondria induced by Fe2+cysteine. Compounds 6, 9, and 10 showed moderate activity,
Figure 2. Structures of 3 and 12. The arrows (H→C) indicate important HMBC correlations.
with IC50 values of 18.4, 48.8, and 15.6 μM, respectively. Additionally, the hydrolyzable tannins were evaluated for their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxy free radicals. Compounds examined here showed moderate to high scavenging rates of DPPH and hydroxy free radicals (Table 5). Compounds 1 and 8 showed the highest scavenging activities for the hydroxy free radical, with IC50 values of 15.8 and 16.3 μM, respectively. These values are higher than those of BHT and similar to those of gallic acid. Compounds 2, 4, 1020
DOI: 10.1021/np500953e J. Nat. Prod. 2015, 78, 1015−1025
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Figure 3. Structures of known tannins 4, 5, and 9−11.
To explain the mechanism of anti-inflammatory effects, 6 was tested in a cell-based assay at various doses for its ability to suppress inflammatory cytokines in lipopolysaccharide (LPS)induced murine macrophages compared with dexamethasone. Compound 6 only marginally inhibited nitric oxide (NO), TNFα, and IL-6 production (Figure. 4); some of the other compounds showed similar inhibitory effects on NO production (Supporting Information Figure S1). However, the viability rates of LPS-induced murine macrophages that were detected by the MTT assay increased significantly. Previous studies have shown that casuarinin, casuarictin, pedunculagin, and nobotannin, four hydrolyzable tannins isolated from Melastoma dodecandrum, exhibited inhibitory effects on NO production,37 suggesting that the presence of an HHDP group at C-2 and C-3 of the glucose core led to inhibitory effects. The results shown in Figure 4 did not factor dose−effect relationships, therefore hydrolyzable tannins lacking HHDP groups at C-2 and C-3 of the glucose core cannot inhibit the production of NO and inflammatory cytokines to produce an anti-inflammatory effect.
5, 7, 9, and 10 showed moderate scavenging activities, with IC50 values ranging from 32.6 to 39.7 μM. Scavenging activities of DPPH free radicals were also examined. All tested compounds showed higher activity than BHT, Trolox, or gallic acid. Anti-inflammatory Activity of Tannins. Free radicals are thought to be involved in numerous human disorders including atherosclerosis, arthritis, cancer, and immune complex-induced injury.36 Combining this with the traditional pharmaceutical performance, the isolated compounds were screened for potential anti-inflammatory effects using croton oil-induced ear edema, carrageenan-induced paw edema, and collagen-induced arthritis (CIA) experiments. Because of the large dosage needed for the in vivo test, the major dimeric tannin 6 (0.14% of the crude plant weight) was selected. Compound 6 showed a dosedependent anti-inflammatory effect in the inhibition of ear swelling and a moderate suppressive effect in paw edema, especially blocking the progression and development of CIA in DBA/1 mice (Table 6). 1021
DOI: 10.1021/np500953e J. Nat. Prod. 2015, 78, 1015−1025
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Table 4. 13C NMR Spectroscopic Data for the Aromatic Moieties of 3 and 12 hellinoyl C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-1′ C-2′ C-3′ C-4′ C-5′ C-6′ C-7′ C-1″ C-2″ C-3″ C-4″ C-5″ C-6″ C-7″ DHDG C-1 C-2 C-3 C-4 a
3a
12b
119.3 111.7 147.6 139.8 146.6 107.5 165.1 111.1 142.4 139.7 139.3 141.7 108.6 163.7 115.6 144.3 140.3 139.7 143.2 118.8 165.6 119.64 107.5 147.90 140.3
119.2 111.9 147.5 139.8 146.6 107.3 165.1 111.0 142.4 139.8 139.3 141.8 108.6 163.5 115.6 144.3 140.2 139.7 143.2 118.9 164.5 119.6 107.7 147.8 140.2
C-5 C-6 C-7 C-1′ C-2′ C-3′ C-4′ C-5′ C-6′ C-7′ HHDP C-1, 1′ C-2, 2′ C-3, 3′ C-4, 4′ C-5, 5′ C-6, 6′ HHDP-1-C-7 HHDP-1-C-7′ HHDP-2 (3)-C-7 HHDP-2 (3)-C-7′ galloyl C-1 C-2, 6 C-3, 5 C-4 C-7
3a
12b
146.0 112.8 164.6c 113.9 136.9 140.0 140.3 143.2 110.1 164.5c 115.6, 115.7, 115.8 (4C in total) 125.5, 125.78, 125.82 (4C in total) 107.6, 107.9, 108.2, 108.6 (4C in total) 145.0, 145.1 (4C in total) 136.2, 136.4 (4C in total) 144.3 (4C in total) 168.3d 167.6e 168.5d 167.7e 119.3, 119.6, 119.7, 120.2 109.8, 109.9, 110.1, 110.2 (each 2C) 145.5, 145.6, 145.7, 145.8 (each 2C) 138.7, 139.3, 139.4, 139.7 165.0, 166.3, 167.1, 167.2
146.0 112.7 164.5f 113.1 136.8 140.1 140.2 143.3 110.1 165.4f 115.5, 115.6, 115.7, 115.8, 115.9 (6C in total) 125.3, 125.5, 125.9 (6C in total) 107.7, 107.9, 108.3, 108.6 (6C in total) 145.0, 145.1 (6C in total) 136.2−136.8 (6C in total) 144.2−144.5 (6C in total) 168.3g 167.8h 168.4g (2C in total) 167.6h (2C in total) 119.3, 119.6, 119.7, 119.8 110.0, 110.1, 110.3 (8C in total) 145.1, 145.6, 145.7, 145.8 (each 2C) 138.7, 139.3, 139.4, 139.8 164.5, 166.2, 166.8, 167.1
Data were recorded at 151 MHz. bData were recorded at 125 MHz.
c−h
Interchangeable.
Table 5. Scavenging Rate Values of •OH and DPPH Free Radicals hydroxy free radicals
DPPH free radicals
IC50 (μM)
IC50 (μM)
sample BHT Trolox gallic acid 1 2 3 4 5 6 7 8 9 10 11 12 6a 6b
22.63 76.73 15.66 15.80 36.81 47.59 37.96 34.62 41.52 32.55 16.27 39.72 38.49 42.91 55.10 >100 >100
Nevertheless, it is still not clear how 6 elicits anti-inflammatory activities in vivo. Oxidative stress may activate a variety of transcription factors, which can lead to the expression of genes for production of inflammatory cytokines and chemokines. Expression of these genes can lead to chronic inflammation, which in turn can mediate a wide spectrum of diseases, including rheumatoid arthritis, cancer, and inflammatory joint disease.38 It is possible that the ability of 6 to eliminate free radicals mitigates the inflammatory reaction. The methylated products of 6, 6a and 6b, were obtained and evaluated to determine their ability to scavenge DPPH and hydroxy free radicals and their antiinflammatory effect in croton oil-induced ear edema. These results confirmed our hypothesis that 6a and 6b, without free phenolic hydroxy groups, exhibit neither radical scavenging effects nor anti-inflammatory activities (Tables 5 and 7). Thus, our results show that compound 6 has significant antiinflammatory activities with the following features: (1) therapeutic effects in CIA mice; (2) more effective for inhibiting edema in vivo; (3) more potent for scavenging free radicals; and (4) less toxic to murine macrophages. On the basis of these characteristics, 6 appears to be a major bioactive constituent of M. bracteata to exert an anti-inflammatory effect in traditional Tibetan usage.
7.08 8.00 7.23 2.40 4.73 3.06 3.16 5.89 3.57 4.67 4.44 4.10 4.61 4.43 4.23 >100 >100
■
Table 6. Anti-inflammatory Effects of 6 croton oil-induced ear edema doses (sc mg/kg) inhibition rates (%) p valuea a
carrageenaninduced paw edema
collageninduced arthritis
50.0
100.0
200.0
50.0
20.0
34.4
39.0
69.8
25.0
46.0 (day 57)
**
**
***
*
*
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO P-2000 polarimeter, and UV spectra with a JASCO V-650 spectrophotometer. ECD spectra were measured on a JASCO J-815 spectrometer. IR spectra were recorded on a Nicolet 5700 spectrometer by an FT-IR microscope transmission method. The 1H and 13C NMR spectra were recorded on VNS-600, INOVA-500, and Bruker AV500-III spectrometers. Chemical shifts are given in δ (ppm) values relative to those of the solvent signal [acetone-d6 (δH 2.04; δC 29.8)] on the TMS scale. The standard pulse sequences programmed
p < 0.001, ***; p < 0.01, **; p < 0.05, *. 1022
DOI: 10.1021/np500953e J. Nat. Prod. 2015, 78, 1015−1025
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Figure 4. Effect on nitric oxide (NO), TNF-α, and IL-6 production of 6. HPLC with MeOH−1% HOAc/H2O (23:77, v/v) to give bracteatinin T1 (3) (97 mg), tamarixinin A (6) (1.1 g), hirtellin B (9) (373 mg), and isohirtellin C (11) (140 mg). The MeOH/H2O (3:7, v/v) eluate (0.9 g) was further separated by preparative HPLC with MeOH−1% HOAc/ H2O (23:77, v/v) to give hirtellin E (8) (40 mg), hirtellin A (10) (19 mg), and hirtellin T3 (12) (180 mg). The residue after 95% EtOH extraction was extracted with H2O. The homogenate was applied to a Diaion HP-20 column, eluted with H2O, aqueous EtOH (20% → 40%), and 95% EtOH, successively, to give the corresponding H2O, EtOH/ H2O (2:8, v/v) (70.5 g), EtOH/H2O (4:6, v/v) (15.2 g), and 95% EtOH (1.5 g) fractions. The EtOH/H2O (2:8, v/v) fraction (25 g) was subjected to a Toyopearl HW-40 (2.6 i.d. × 46 cm) column and eluted with MeOH/H2O (5:5 → 6:4 → 7:3, v/v), MeOH/H2O/acetone (7:2:1 → 6:2:2 → 5:2:3, v/v/v), and acetone. The MeOH/H2O (6:4, v/ v) eluate (3.2 g) was subjected to an MCI-gel CHP-20P (1.3 i.d. × 46 cm) column and eluted with H2O, MeOH/H2O (2:8 → 3:7 → 4:6, v/v), and aqueous MeOH. The MeOH/H2O (2:8, v/v) eluate (0.9 g) was further separated by preparative HPLC with MeOH−1% HOAc/H2O (15:85, v/v) to give bracteatinin D2 (1) (98 mg), bracteatinin D1 (2) (120 mg), nilotinin M4 (4) (43 mg), 1,3-di-O-galloyl-4, 6-O-(aS)hexahydroxydiphenoyl-β-D-glucose (5) (51 mg), and nilotinin D8 (7) (44 mg). Bracteatinin D1 (1): off-white, amorphous powder; [α]20 D +78 (c 1, MeOH); UV (MeOH) λmax (log ε) 217 (5.27), 295 (5.08); ECD (MeOH) [θ] (nm) +2.3 × 105 (233), −3.1 × 104 (263), +2.6 × 104 (289); 1H NMR (acetone-d6/D2O, 9:1, 600 MHz) δH 7.58, 7.54 (1H in total, each s, hellinoyl H-6″), 7.00, 6.94 (1H in total, each d, J = 1.8 Hz, hellinoyl H-6), 6.98 [2H, s, galloyl-2 (H-2/H-6)], 6.86, 6.85 [2H in total, each s, galloyl-1 (H-2/H-6)], 6.692, 6.685 (1H in total, each s, hellinoyl H-6′), 6.59, 6.58 (1H in total, each s, HHDP H-3), 6.52, 6.51 (1H in total, each s, HHDP H-3′), 5.93, 5.92 (1H in total, each d, J = 1.8 Hz, hellinoyl H-2), and glucose protons (Table 1); 13C NMR spectroscopic assignments, see Tables 2 and 3; ESIMS m/z 1417 ([M − H]−); HRESIMS m/z 1418.1563 [M − H]− (calcd for C61H46O40, 1418.1565). Bracteatinin D2 (2): off-white, amorphous powder; [α]20 D +89 (c 1, MeOH); UV (MeOH) λmax (log ε) 220 (5.15), 299 (4.74); ECD (MeOH) [θ] (nm) +1.8 × 105 (232), −1.7 × 104 (263), +2.8 × 104 (291); 1H NMR (acetone-d6/D2O, 9:1, 600 MHz) δH 7.63, 7.58 (1H in
Table 7. Anti-inflammatory Effects in Croton Oil-Induced Ear Edema of 6, 6a, and 6b doses (sc mg/kg) inhibition rates (%) p valuea a
6
6a
6b
200.0 76.9 ***
200.0 −0.7
200.0 2.1
p < 0.001, ***.
into the instrument were used for each 2D measurement. HRESIMS was performed using an Agilent 1100 series LC/MSD ion trap mass spectrometer. Analytical reversed-phase HPLC was performed on a COSMOSIL 5C18-PAQ Waters column (4.6 i.d. × 250 mm) eluted with H2O containing 2% HOAc/MeCN (flow rate, 1 mL/min; 280 nm UV detection) at room temperature. Preparative reversed-phase HPLC was performed on a COSMOSIL 5C18-PAQ Waters column (250 × 10 mm, 5 μm) using H2O containing 1% HOAc/MeOH (flow rate, 2 mL/min; 280 nm UV detection) at room temperature. Plant Material. Twigs of M. bracteata were collected at the Chaidamu basin of Qinghai Province, People’s Republic of China, in August 2008, and identified by Professor Ma Lin, Institute of Materia Medica, Chinese Academy of Medical Science, and Peking Union Medical College. A voucher specimen (ID-S-2428) is deposited in the herbarium, in the same department. Extraction and Isolation. The EtOH/H2O (95:5, v/v, 84 L) homogenate (420.3 g) of M. bracteata dried twigs (14 kg) was extracted with petroleum ether, EtOAc, and H2O. The aqueous layer was applied to a Diaion HP-20 column, eluted with H2O, aqueous EtOH (30% → 50%), and 95% EtOH, successively, to give the corresponding H2O (56 g), EtOH/H2O (3:7, v/v) (141 g), EtOH/H2O (5:5, v/v) (10 g), and 95% EtOH (5 g) fractions. The EtOH/H2O (3:7, v/v) fraction (28 g) was subjected to a Toyopearl HW-40 (2.6 i.d. × 46 cm) column, eluted with MeOH/H2O (5:5 → 6:4 → 7:3, v/v), MeOH−H2O/acetone (7:2:1 → 6:2:2 → 5:2:3, v/v/v), and acetone, successively. The MeOH/ H2O/acetone (5:2:3, v/v/v) eluate (8.4 g) was subjected to an MCI-gel CHP-20P (1.3 i.d. × 46 cm) column and eluted with H2O, MeOH/H2O (2:8 → 2.5:7.5 → 3:7 → 4:6, v/v), and aqueous MeOH. The MeOH/ H2O (2.5:7.5, v/v) eluate (3.9 g) was further separated by preparative 1023
DOI: 10.1021/np500953e J. Nat. Prod. 2015, 78, 1015−1025
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13.2 Hz, glucose-2, H-6a), 3.95 (1H, overlapped, glucose-2, H-6b); 13C NMR (acetone-d6, 150 MHz) δ galloyl, 124.3, 124.6 (C × 2, C-1), 107.5, 107.6 (C × 4, C-2/6), 153.5, 153.6 (C × 4, C-3/5), 143.3, 143.2 (C × 2, C-4), 166.4, 165.7 (C × 2, C-7); HHDP, 122.4, 122.7, 122.8 (C × 4, C-2, 2′), 106.4, 106.2 (C × 4, C-3, 3′), 153.78, 153.81, 153.84, 153.86 (C × 4, C-4, 4), 144.5, 144.7, 144.8, 144.9 (C × 4, C-5, 5′), 167.8 (HHDP-1 C7), 167.2 (HHDP-1 C-7), 167.6 (HHDP-2 C-7), 167.0 (HHDP-2 C-7); hellinoyl, 124.6 (C-1), 107.9 (C-2), 153.5 (C-3), 143.2 (C-4), 153.3 (C5), 110.1 (C-6), 163.6 (C-7), 114.3 (C-1′), 144.5 (C-2′), 143.2 (C-3′), 148.4 (C-4′), 149.9 (C-5′), 109.2 (C-6′), 162.7 (C-7′), 120.1 (C-1″), 144.8 (C-2″), 144.5 (C-3″), 148.4 (C-4″), 149.9 (C-5″), 121.6 (C-6″), 163.8 (C-7″); glucose-1, 97.9 (C-1), 72.2 (C-2), 72.0 (C-3), 71.4 (C-4), 66.9 (C-5), 63.3 (C-6); glucose-2, 93.1 (C-1), 71.3 (C-2), 74.9 (C-3), 70.9 (C-4), 72.2 (C-5), 63.6 (C-6). 1,3-Di-O-galloyl-4,6-O-(aS)-hexahydroxydiphenoyl-β-D-glucose (5): off-white, amorphous powder; [α]20 D +85 (c 1, MeOH); ESIMS m/z 785 [M − H]−; 1H NMR (acetone-d6/D2O, 9:1, 600 MHz) δH 7.16 [2H, s, galloyl-1 (H-2/H-6)], 7.01 [2H, s, galloyl-2 (H-2/H-6)], 6.58 (1H, s, HHDP H-3), 6.45 (1H, s, HHDP H-3′), 5.83 (1H, d, J = 7.8 Hz, glucose H-1), 3.96 (1H, t, J = 9.6 Hz, glucose H-2), 5.43 (1H, t, J = 9.6 Hz, glucose H-3), 5.01 (1H, t, J = 9.6 Hz, glucose H-4), 4.30 (1H, dd, J = 6.6, 9.6 Hz, glucose H-5), 5.24 (1H, dd, J = 13.2, 6.6 Hz, glucose H-6a), 3.79 (1H, d, J = 13.2 Hz, glucose H-6b); 13C NMR (acetone-d6/D2O, 9:1, 150 MHz) δC galloyl-1, 125.5 (C-1), 110.0 (C-2, 6), 139.7 (C-4), 145.9 (C-3, 5), 165.7 (C-7); galloyl-2, 125.9 (C-1), 110.1 (C-2, 6), 139.0 (C4), 144.6 (C-3, 5), 167.2 (C-7); HHDP, 115.7 (C-1), 115.8 (C-1′), 125.5, 125.9 (C-2, 2′), 107.7 (C-3), 107.6 (C-3′), 144.3 (C-4, 4′), 136.2 (C-5), 136.4 (C-5′), 145.0 (C-6, 6′), 168.4 (C-7), 167.9 (C-7′); glucose, 93.1 (C-1), 71.9 (C-2), 75.6 (C-3), 70.6 (C-4), 72.5 (C-5), 63.1 (C-6). Hirtellin T3 (12): off-white, amorphous powder; [α]20 D +80 (c 1, MeOH); ESIMS m/z 2806 [M − H]−; 1H NMR (acetone-d6/D2O, 9:1, 500 MHz) δ 7.53 (1H, s, hellinoyl H-6″), 7.25 (1H, d, J = 2.0 Hz, DHDG H-6), 7.04 (1H, s, DHDG H-6′), 7.01 [2H, s, galloyl-4 (H-2/H-6)], 6.95 [2H, s, galloyl-3 (H-2/H-6)], 6.91 [2H, s, galloyl-2 (H-2/H-6)], 6.87 (1H, d, J = 2.0 Hz, hellinoyl H-6), 6.77 [2H, s, galloyl-1 (H-2/H-6)], 6.69 (1H, s, HHDP-1 H-3), 6.65 (1H, s, hellinoyl H-6′), 6.62 (1H, s, HHDP-3 H-3), 6.59 (1H, s, HHDP-2 H-3), 6.54 (1H, s, HHDP-3 H3′), 6.51 (1H, s, HHDP-1 H-3′), 6.46(1H, s, HHDP-2 H-3′), 6.44 (1H, d, J = 2.0 Hz, DHDG H-2), 5.74 (1H, d, J = 2.0 Hz, hellinoyl H-2) and glucose protons (Table 1); 13C NMR spectroscopic assignments, see Tables 2 and 4. Tannase Hydrolysis of Hirtellin E (7) and Nilotinin D8 (8).33 An aqueous solution (6 mL) of 8 (6 mg, tR 21.840 min on RP-HPLC) was incubated with 200 μL of tannase (Sigma Aldrich Trading Co., Shanghai, China) solution (3 mg/100 mL citrate buffer of pH 5.5), at 37 °C for 6 h. The reaction mixture was acidified with 0.1 N HCl and subjected to MCI-gel CHP-20P CC (1.1 i.d. × 11 cm), eluted with aqueous MeOH (10% → 15% → 25% → 35%) and 100% MeOH. The 25% aqueous MeOH eluate yielded the degalloylated derivative. This was identified as 1 (0.9 mg) by chromatography on reversed-phase HPLC (tR 12.000, 12.437 min) and 1H NMR spectroscopic comparison with the samples isolated from the plant. An aqueous solution (3 mL) of 7 (6 mg, tR 17.988 min on reversed-phase HPLC) was incubated with 200 μL of tannase solution at 37 °C for 6 h. The reaction mixture was treated similarly to that of 7. The degalloylated derivative was identified as 2 (0.3 mg) by chromatography on RP-HPLC (tR 11.365, 13.720 min) and by 1H NMR spectral comparison with a sample isolated from the plant. Anti-inflammatory Assay. The evaluation of croton oil-induced ear edema in mice,39,40 carrageenan-induced paw edema,41 and collageninduced arthritis42 for the hydrolyzable tannins was carried out according to established methods. The biological evaluation of NO inhibition for the hydrolyzable tannins was carried out according to established methods.43 Antioxidant Assay. Inhibitory activity against lipid peroxidation in rat liver microsomes,44,45 DPPH radical-scavenging activity,46 and hydroxy radical-scavenging activity47 of tannins were measured by the methods referred to in the literature.
total, each s, hellinoyl H-6″), 7.06, 7.05 (1H in total, each d, J = 1.8 Hz, hellinoyl H-6), 7.05 [2H, s, galloyl-2 (H-2/H-6)], 6.88, 6.87 [2H in total, each s, galloyl-1 (H-2/H-6)], 6.71, 6.70 (1H in total, each s, hellinoyl H-6′), 6.61, 6.59 (1H in total, each s, HHDP H-3), 6.48, 6.47 (1H in total, each s, HHDP H-3′), 6.05, 6.04 (1H in total, each d, J = 1.8 Hz, hellinoyl H-2), and glucose protons (Table 1); 13C NMR spectroscopic assignments, see Tables 2 and 3; ESIMS m/z 1417 ([M − H]−); HRESIMS m/z 1418.1569 [M − H]− (calcd for C61H46O40, 1418.1565). Bracteatinin T1 (3): off-white, amorphous powder; [α]20 D +36 (c 1, MeOH); UV (MeOH) λmax (log ε) 214 (5.06), 297 (4.88); ECD (MeOH) [θ] (nm) +4.2 × 104 (234), −4.5 × 103 (262), +5.3 × 103 (290); 1H NMR (acetone-d6/D2O, 9:1, 600 MHz) δH 7.52 (1H, s, hellinoyl H-6″), 7.22 (1H, d, J = 1.8 Hz, DHDG H-6), 7.03 [2H, s, galloyl-4 (H-2/H-6)], 7.02 (1H, s, DHDG H-6′), 7.00 [2H, s, galloyl-3 (H-2/H-6)], 6.89 [2H, s, galloyl-2 (H-2/H-6)], 6.83 (1H, d, J = 1.8 Hz, hellinoyl H-6), 6.76 [2H, s, galloyl-1 (H-2/H-6)], 6.67 (1H, s, HHDP-1 H-3), 6.60 (1H, s, HHDP-2 H-3), 6.61 (1H, s, hellinoyl H-6′), 6.53 (1H, s, HHDP-1 H-3′), 6.45 (1H, s, HHDP-2 H-3′), 6.41 (1H, d, J = 1.8 Hz, DHDG H-2), 5.76 (1H, d, J = 1.8 Hz, hellinoyl H-2) and glucose protons (Table 1); 13C NMR spectroscopic assignments, see Tables 2 and 4; ESIMS m/z 2506 [M − H]−; HRESIMS m/z 2506.2526 [M − H]− (calcd for C109H78O70, 2506.2544). Methylation of Tamarixinin A (6). A mixture of tamarixinin A (6) (40 mg), anhydrous K2CO3 (200 mg), and dimethyl sulfate (200 μL) in dry acetone (5 mL) was stirred overnight at room temperature and refluxed for 4 h. After removal of the inorganic material by centrifugation, the supernatant was concentrated and subjected to preparative TLC (PF254, CHCl3/MeOH, 40:1) to yield two octacosamethyl derivatives (6a (11 mg) and 6b (12 mg)). 6a: light yellow, amorphous powder; ESIMS m/z 2084.6 [M − H]−; 1 H NMR (acetone-d6, 600 MHz) δ 7.69 (1H, s, hellinoyl H-6″), 7.16 (1H, d, J = 2.0 Hz, hellinoyl H-6), 7.13 [2H, s, galloyl-2 (H-2/H-6)], 7.05 [2H, s, galloyl-1 (H-2/H-6)], 6.96 (1H, s, HHDP-2 H-3), 6.95 (1H, s, HHDP-1 H-3), 6.90 (1H, s, hellinoyl H-6′), 6.80 (1H, s, HHDP-2 H3′), 6.79 (1H, s, HHDP-1 H-3′), 6.13 (1H, d, J = 2.0 Hz, hellinoyl H-2). 4.89 (1H, d, J = 7.8 Hz, glucose-1, H-1), 5.43 (1H, t, J = 9.6 Hz, glucose1, H-2), 5.84 (1H, t, J = 9.6 Hz, glucose-1, H-3), 5.23 (1H, t, J = 9.6 Hz, glucose-1, H-4), 4.48 (1H, ddd, J = 9.6, 7.2, 1.8 Hz, glucose-1, H-5), 5.32 (1H, d, J = 13.2 Hz, glucose-1, H-6a), 4.00 (1H, d, J = 13.2 Hz, glucose-1, H-6b), 5.84 (1H, t, J = 8.4 Hz, glucose-2, H-1), 5.47 (1H, t, J = 9.6 Hz, glucose-2, H-2), 5.49 (1H, t, J = 9.6 Hz, glucose-2, H-3), 5.12 (1H, t, J = 9.6 Hz, glucose-2, H-4), 4.34 (1H, dd, J = 9.6, 6.0 Hz, glucose-2, H-5), 5.32 (1H, dd, J = 13.2, 6.6 Hz, glucose-2, H-6a), 4.09 (1H, br d, J = 13.2 Hz, glucose-2, H-6b); 13C NMR (acetone-d6, 150 MHz) δ galloyl, 120.8, 121.4 (C × 2, C-1), 107.5, 107.9 (C × 4, C-2/6), 153.2, 153.6 (C × 4, C3/5), 143.4, 145.5 (C × 2, C-4), 163.3, 165.7 (C × 2, C-7); HHDP, 122.5, 122.7, 122.9 (4 × C, C-2, 2′), 106.3, 106.2 (4 × C, C-3, 3′), 153.7, 153.8, 153.9 (4 × C, C-4, 4), 144.7, 144.8 (4 × C, C-5, 5′), 167.8 (HHDP-1 C-7), 167.2 (HHDP-1 C-7), 167.6 (HHDP-2 C-7), 166.5 (HHDP-2 C-7); hellinoyl: 124.4 (C-1), 107.9 (C-2), 154.6 (C-3), 143.4 (C-4), 153.2 (C-5), 109.7 (C-6), 163.8 (C-7), 114.5 (C-1′), 144.8 (C2′), 143.4 (C-3′), 145.5 (C-4′), 148.6 (C-5′), 109.2 (C-6′), 163.3 (C7′), 120.8 (C-1″), 149.3 (C-2″), 144.7 (C-3″), 148.3 (C-4″), 149.9 (C5″), 121.4 (C-6″), 162.7 (C-7″); glucose-1, 102.7 (C-1), 72.7 (C-2), 75.0 (C-3), 70.9 (C-4), 72.1 (C-5), 63.6 (C-6); glucose-2, 93.2 (C-1), 71.5 (C-2), 74.4 (C-3), 71.3 (C-4), 71.4 (C-5), 63.4 (C-6). 6b: light yellow, amorphous powder; ESIMS m/z 2084.6 [M − H]−; 1 H NMR (acetone-d6, 600 MHz) δ 7.73 (1H, s, hellinoyl H-6″), 7.14 [2H, s, galloyl-2 (H-2/H-6)], 7.12 (1H, d, J = 2.4 Hz, hellinoyl H-6), 7.05 [2H, s, galloyl-1 (H-2/H-6)], 6.96 (1H, s, HHDP-1 H-3), 6.93 (1H, s, HHDP-2 H-3), 6.91 (1H, s, hellinoyl H-6′), 6.78 (1H, s, HHDP-1 H3′), 6.77 (1H, s, HHDP-2 H-3′), 6.19 (1H, d, J = 2.4 Hz, hellinoyl H-2). 5.23 (1H, d, J = 3.6 Hz, glucose-1, H-1), 5.44 (1H, dd, J = 9.6, 3.6 Hz, glucose-1, H-2), 5.75 (1H, t, J = 9.6 Hz, glucose-1, H-3), 5.14 (1H, t, J = 9.6 Hz, glucose-1, H-4), 4.51 (1H, m, glucose-1, H-5), 5.33 (1H, dd, J = 13.2, 6.6 Hz, glucose-1, H-6a), 4.08 (1H, overlapped, glucose-1, H-6b), 5.84 (1H, d, J = 8.4 Hz, glucose-2, H-1), 5.48 (1H, dd, J = 9.6, 8.4 Hz, glucose-2, H-2), 5.84 (1H, t, J = 9.6 Hz, glucose-2, H-3), 5.21 (1H, t, J = 9.6 Hz, glucose-2, H-4), 4.50 (1H, m, glucose-2, H-5), 5.29 (1H, br d, J = 1024
DOI: 10.1021/np500953e J. Nat. Prod. 2015, 78, 1015−1025
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ASSOCIATED CONTENT
S Supporting Information *
1
H NMR spectra, 13C NMR spectra, HMBC spectra, HSQC spectra, H−H COSY spectra, and HRESIMS data for compounds 1−12 and the detailed experimental conditions for anti-inflammation and antioxidation are available free of charge via the Internet at http://pubs.acs.org.
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[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported financially by National Natural Science Foundation of China (No. 21072233).
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DOI: 10.1021/np500953e J. Nat. Prod. 2015, 78, 1015−1025
