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Rare noriridoids from the roots of Andrographis paniculata
Phytochemistry 77 (2012) 275–279
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Rare noriridoids from the roots of Andrographis paniculata Chong Xu a, Gui-Xin Chou a,b, Chang-Hong Wang a, Zheng-Tao Wang a,b,⇑ a The MOE Key Laboratory for Standardization of Chinese Medicines, and Shanghai Key Laboratory of Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, PR China b Shanghai R&D Center for Standardization of Chinese Medicines (SCSCM), Shanghai 201203, PR China
a r t i c l e
i n f o
Article history: Received 30 March 2011 Received in revised form 6 October 2011 Available online 16 February 2012 Keywords: Andrographis paniculata Acanthaceae Iridoid Noriridoids Andrographidoids A–E
a b s t r a c t The rare noriridoids, Andrographidoids A–E (1–5), along with a known iridoid curvifloruside F (6), were isolated from roots of Andrographis paniculata. All noriridoids were aglycones and 1–4 had (semi-) acetal structures located at C-3 but not at C-1. Their structures were established by a series of 1D and 2D NMR analyses. The antibacterial activity of these iridoids was also assessed using the microtitre plate broth dilution method. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Andrographis paniculata Nees (Acanthaceae) is a conventional medicinal herb in China. Its aerial parts are used to treat inflammation, cold, fever and diarrhea. Previous research showed that the crude extract, as well as its main bioactive components, the entlabdane diterpenoids, possess a wide spectrum of bioactivities, such as antibacterial (Singha et al., 2003), anti-inflammatory (Liu et al., 2008), antimalarial (Dua et al., 2004), antithrombotic (Thisoda et al., 2006), antitumor (Yang et al., 2009; Zhou et al., 2006), immunostimulatory (Puri et al., 1993) and heptoprotection (Kapil et al., 1993) properties. However, research on root tissues have been few although these showed interesting results. For example, four xanthones having anti-malarial activity were isolated from the roots of A. paniculata and one of these showed substantial antiplasmodial activity (Dua et al., 2004). Meanwhile, the chloroform extract of A. paniculata roots also exhibited significant antidiabetic and nephroprotective activities (Rao, 2006). In our continuing studies on A. paniculata (Liu et al., 2007, 2008; Ji et al., 2007, 2009; Shen et al., 2009; Xu et al., 2010), herein is the chemical components of the roots were systemically investigated and reported the isolation and identification of five new noriridoids andrographidoids A–E (1–5) together with a known iridoid curvifloruside F. The antibacterial activity of these iridoids was assayed using the microtitre plate broth dilution method.
The ethanol extract of A. paniculata roots was suspended in H2O and partitioned with petroleum ether, dichloromethane, ethyl acetate and n-butanol successively. The dichloromethane extract was subjected to sequential column chromatography over silica gel, Sephadex LH-20, and followed by either prep. TLC or recrystallization to yield the five new noriridoids andrographidoids A–E (1–5) and a known iridoid curvifloruside F (6) (Lai et al., 2009). Andrographidoid A (1), obtained as pale yellow oil, displayed a quasi molecular ion peak at m/z 371.1471 in the HR-ESI–MS (attributed to the [M+Na]+, calcd for C19H24O6Na, 371.1471). Together with NMR Spectroscopic data, its molecular formula was established as C19H24O6, X = 8. The 1H NMR spectrum showed the presence of a cinnamyl group: a mono substituted phenyl (d 7.50, 2H and 7.38, 3H), a trans-double bond (d 6.35, 1H, d, J = 15.9 Hz and 7.58, 1H, d, J = 16.0 Hz) and their corresponding signals were also observed in the 13C NMR spectrum, together with an ester carbonyl resonance at d 166.4. Excluding a methoxyl (3.43, 3H, s) and a methyl (1.68, 3H, s), there were eight carbons left to form a bicyclic structure. According to analysis of the HSQC and COSY spectra, three pairs of CH–CH2 groups were present and separated by two nonprotonated carbons and an oxygen atom. Together with consideration of the HMBC spectrum, these detailed analyses indicated a reduced jioglutolide-like (Morota et al., 1989) iridoid structure for 1. However, compared with the lactone group in jioglutolide, this was replaced in 1 with a methyl acetal functionality. In the HMBC spectrum, the methoxyl protons (d 3.43, 3H) had only one cross-peak with the C-3 carbon atom (d 97.7). Moreover,
⇑ Corresponding author at: Shanghai R&D Center for Standardization of Chinese Medicines (SCSCM), Shanghai 201203, PR China. Tel.: +86 21 51322507; fax: +86 21 51322519. E-mail addresses: [email protected], [email protected] (Z.-T. Wang). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.12.020
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the carbonyl group of the cinnamyl moiety had no cross-peaks with any hydrogen in the bicyclic group thus indicating it must be attached to a non-protonated carbon. Based on the chemical shifts (d 76.6, 84.4), the cinnamate should be located at C-8 (d 84.4). Therefore, the C-5 (d 76.6) must be oxygenated with a hydroxyl group. According to the literature (Morota et al., 1989; Kouno et al., 1994; Lu et al., 1997; Yang et al., 2006), there was only one known configuration of this lactone-type iridoid as regards the cyclopentane ring and the six membered ring. Therefore, H-9 and 5-OH should be b-oriented. From analysis of the NOESY spectrum, interactions between H-9 and H-6 as well as H-9 and H-1 were observed, indicating that H-6 was b-oriented. As a result the corresponding methyl group functionality therefore was a-oriented. Meanwhile, to H-1a showed a strong NOE correlation with the methyl, and the H-1b showed an interaction with the methoxyl group at C-3 thereby suggesting that H-3 (d, J = 2.2 Hz) must be aoriented. Also the small coupling constant of H-3 (d 4.92, d, J = 2.2 Hz), as well as the couplings between H-9 (d 2.17, d, J = 4.3 Hz) and 2 H-1 (d 4.34, d, J = 13.0 and d 4.01, dd, J = 12.9, 5.0) suggested that the 6-membered ring of 1 was in a boat conformation, which indicated H-3 must be equatorial and that the 3-OMe group was b-oriented. Detailed analysis of the NMR spectroscopic data are shown in Table 1. On the basis of these data, andrographidoid A (1) was established as (3S,4aS,5S,7S,7aS)-4a,5dihydroxy-3-methoxy-7-methyloctahydrocyclopenta[c]pyran-7-yl cinnamate (Fig. 1). Andrographidoid B (2) was isolated as pale yellow oil. Following consideration of the HR-ESI–MS (m/z 371.1469, [M+Na]+, calcd for C19H24O6Na, 371.1471) and the NMR spectroscopic data, it had the same molecular formula as compound 1. Moreover the spectroscopic data were similar as well, indicating that 2 was a stereoisomer of 1, although there was one difference in the NOESY spectrum. Since the correlation between H-6 (d 4.27) and H-9 (d 2.29) was absent, this could mean that the hydroxyl at C-6 was b-oriented. Meanwhile, the H-3 had a large coupling constant J = 8.0 Hz indicating it must be axial. Therefore, the structure of 2 was concluded to be the C-3 and C-6 epimer of 1 (the optical rotational value also proved this), with the 6-membered ring in a chair conformation. Thus andrographidoid B (2) was elucidated as
(3R,4aS,5R,7S,7aS)-4a,5-dihydroxy-3-methoxy-7-methyloctahydrocyclopenta[c]pyran-7-yl cinnamate (Fig. 1). Andrographidoid C (3) was obtained as pale yellow oil. It also had the same molecular formula as compounds 1 and 2 (HR-ESI– MS m/z 371.1473 [M+Na]+, calcd for C19H24O6Na, 371.1471). While the spectroscopic data were similar to 2 (see Table 1), absence of NOE correlations between H-9 and methyl at C-10 as well as H-9 and H-6 (dd, J = 7.0, 2.5 Hz, unlike H-6 (dd, J = 11.2, 8.1 Hz) of compound 1 indicated that the methyl and H-6 must be a-oriented. Furthermore, since the H-3 signal showed a NOE interaction with the methyl group at C-10, this suggested that H-3 must be a-oriented. This was confirmed from the coupling constant of H-3 (d, J = 4.5 Hz) which indicates that the 6-membered ring possessed a boat conformation as for compound 1. Therefore, the structure of 3 was established as (3S,4aS,5R,7S,7aS)-4a,5-dihydroxy-3-methoxy-7methyloctahydrocyclopenta[c]pyran-7-yl cinnamate (Fig. 1). Andrographidoid D (4) was obtained as pale yellow oil. Its molecular formula, C18H22O6, was established on the basis of its HR-ESI–MS (m/z 357.1412 [M+Na]+, calcd for C18H22O6Na, 357.1314) and was supported by analysis of the NMR spectroscopic data. These data were quite similar to those of compounds 1–3, except for the absence of the methoxyl group in 1H NMR and 13C NMR spectrum. Meanwhile, the coupling constant of H-3 (J = 3.6 Hz), as well as the NOESY spectrum gave the same configuration of the 6-membered ring as 1: H-6 was a-oriented (d, J = 3.7 Hz) and had a NOE interaction with H-4a; a cross-peak of H-3 (d, J = 3.6 Hz) and H-4a was observed. Therefore, a b-oriented hydroxyl group was located at C-3 of compound 4. Its structure was thus formulated as (3S,4aS,5R,7S,7aS)-3,4a,5-trihydroxy-7methyloctahydrocyclopenta[c]pyran-7-yl cinnamate (Fig. 1). Andrographidoid E (5) was isolated and recrystallized from methanol as colorless prisms. HR-ESI–MS gave a quasimolecular ion peak at m/z 207.0631 [M+Na]+ (calcd for C9H12O4Na, 207.0633), which together with analysis of the NMR spectroscopic data, group its formula as C9H12O4. The HSQC spectrum showed three CH2, one oxygenated CH (d 88.4) and a methyl group, respectively. Except for the double bond and ester signals (d 136.9, 137.4 and 176.3), the 13C NMR spectroscopic data were similar to the core skeleton of compounds 1–4. This indicated that the hydroxyl
Table 1 NMR spectroscopic data for andrographidoids A (1) and B (2) (500 MHz for 1H NMR, 125 MHz for No.
1
3 4 5 6 7 8 9 10 OMe C@O
a b 10 20 30 40 50 60 a
4.34 4.01 4.92 1.82 1.74
d (13.0) dd (13.0, 5.0) d (2.2) d (15.0) oa
4.28 dd (11.2, 8.1) 2.61 dd (14.7, 8.0) 1.72 dd (14.7, 3.0) 2.17 br.d (4.3) 1.68 s 3.43 s 6.35 d (16.0) 7.58 d (16.0) 7.50 7.38 7.38 7.38 7.50
C NMR, CDCl3). 2
d (J in Hz) 1
13
m m m m m
‘‘o’’ denotes overlapping signals.
HMBC (H ? C)
d (J in Hz)
54.4
3, 5, 8, 9
97.7 31.3
1, 5 3, 5, 6, 9
4.26 3.90 4.65 1.90 1.70
76.6 75.5 44.3
5 5, 6, 8, 9, 10
4.26 oa 2.67 dd (14.5, 7.5) 1.79 dd (14.5, 9.5)
5, 8 7, 8, 9 3
2.29 t (5.0) 1.66 s 3.48 s
dC
84.4 51.1 22.6 55.2 166.4 119.3 144.4 134.4 128.0 128.9 130.2 128.9 128.0
dd dd dd dd dd
(12.5, 5.0) (12.5, 5.5) (8.0, 3.5) (14.5, 2.5) (14.5, 7.5)
6.35 d (16.0) 7.59 d (16.0) 7.50 7.37 7.37 7.37 7.50
m m m m m
dC
HMBC (H ? C)
60.6
3, 5, 8, 9
99.4 34.8
3, 5, 6
80.0 78.1 45.7
4, 5 5, 6, 8, 9, 10
85.9 51.1 22.3 55.9 166.5 119.2 144.7 134.3 128.1 128.9 130.4 128.9 128.1
4, 5, 8 7, 8, 9 3
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Fig. 1. Structures of five new noriridoids.
Fig. 2. Dd of the Mosher esters (Dd = dR dS). (A) In CDCl3; (B) in C5D5N.
group at C-3 must be oxidized to form a lactone moiety as jioglutolide which matched the ester signal at d 176.3. Additionally, the double bond should be formed by dehydration of one of the hydroxyls. Indeed, while the two carbons of the double bond were non-protonated, and the absence of the C-9 signal in the 13C NMR spectrum suggested that dehydration must occur between C-5 and C-9. A detailed analysis of the HMBC spectrum further verified this deduction. H-1, H-4, H-6, H-7 and H-10 (methyl protons) signals showed HMBC cross-peaks with the resonance of C-9 (d 136.9). A
cross-peak correlation was also observed between H-4 (d 3.35 and 3.85) and C-5 (d 137.4). In the NOESY spectrum, H-6 showed a NOE interaction with the methyl group at C-8. Thus, andrographidoid E (5) was deduced as 5,9-didehydrojioglutolide (Fig. 1). More detailed 2D NMR analyses are given in Table 3. In order to determine the absolute stereostructure of 5, a Mosher’s reaction was carried out. Two samples of the compound were mixed respectively with (S)-(+) and (R)-()-alpha-methoxyalpha-(trifluromethyl)phenylacetyl chloride in anhydrous pyridine
Table 2 NMR spectroscopic data for andrographidoids C (3) and D (4) (500 MHz for 1H NMR, 125 MHz for No.
3
3 4 5 6 7 8 9 10 OMe C@O
a b 10 20 30 40 50 60
4.04 3.88 5.11 2.25 2.05
dd (10.0, 6.0) t (10.0) d (4.5) dd (14.0, 4.5) d (14.0)
4.40 dd (7.0, 2.5) 2.47 m
2.74 dd (9.0, 6.0) 1.47 s 3.38 s 6.36 d (16.0) 7.61 d (16.0) 7.51 7.38 7.38 7.38 7.51
C NMR, CDCl3). 4
d (J in Hz) 1
13
m m m m m
dC 60.1 105.3 43.4 87.0 87.8 43.8 87.3 57.8 20.4 54.5 165.8 119.0 144.8 134.3 128.1 128.9 130.3 128.9 128.1
HMBC (H ? C)
d (J in Hz)
5
3.93 3.83 5.33 2.33 2.23
4, 5, 6, OCH3 3, 5, 6
6, 8, 9, 10
1, 4, 5, 8, 10 7, 8, 9 3
d (12.2) dd (12.2, 4.5) d (3.6) d (11.8) dd (11.8, 3.7)
4.05 d (3.7) 2.90 dd (15.0, 3.8) 2.13 d (15.0) 2.74 d (4.2) 1.73 s
6.38 d (16.0) 7.67 d (16.0) 7.52 7.40 7.40 7.40 7.52
m t-like t-like t-like m
dC
HMBC (H ? C)
58.0
3, 5, 8, 9
100.0 36.0
1, 5 3, 5, 6, 9
84.7 84.9 42.9
7, 8, 9 5, 6, 8, 9, 10
92.3 57.2 25.9
5, 8 7, 8, 9
167.2 118.9 145.6 134.1 128.2 128.9 130.6 128.9 128.2
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Table 3 NMR spectroscopic data for andrographidoid E (5) (500 MHz for 1H NMR, 125 MHz for 13 C NMR, pyr-d5). No.
5 d (J in Hz)
1 3 4 5 6 7 8 9 10
4.77 d (12.5) 4.70 d (12.5) 3.85 d (17.5) 3.35 d (18.0) 5.07 dd (6.5, 1.0) 2.78 dd (18.0, 6.0) 2.46 d (17.5)
1.69 s
dC 55.8
HMBC (H ? C) 8, 9
176.3 41.1
3, 5, 6, 9
137.4 88.4 42.6
7, 8, 9 6, 8, 9, 10
89.2 136.9 14.1
1, 7, 8, 9
under an argon atmosphere and stirred overnight at room temperature. The target (S) and (R) esters were purified by prep. TLC and the corresponding 1H NMR spectroscopic data of each proton were compared (Dd = dR dS). However, the absolute stereostructure of compound 5 was not obtained (see Fig. 2). As the chemical structures of andrographidoid A–E are similar to villosol and patriscabrol, which were reported to possess a significant antibacterial activity (Yang et al., 2006), their potential antibacterial activity was assayed. However, none showed any inhibitory activity (MIC > 100 lg/ml). The bacteria including: Escherichiacoli, Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa and Bacillus subtilis; Gentamycin, Chloramphenicol and Ciprofloxacin were used as positive controls.
4.3. Extraction and isolation Dried and powdered roots of A. paniculata (5 kg) were extracted with EtOH–H2O (4:1, v/v, 5 30 L) at room temperature for 3 days and filtered. The corresponding combined filtrates were evaporated, then partitioned between water and petroleum ether, dichloromethane, ethyl acetate and n-butanol successively. The CH2Cl2 extract (47 g) was subjected to silica gel column chromatography (CC) (1 kg, 100–200 mesh) and eluted with petroleum ether (60–90 °C)–EtOAc (10:1, 5:1, 2:1, 0:1) and finally MeOH to afford 14 fractions. Fraction IX was further purified by repeated CC (silica gel, 300–400 mesh, CH2Cl2–EtOAc (20:1, 10:1, 5:1, 2:1)) and prep. TLC (layer thickness: 0.4–0.5 mm; sample amount, about 20 mg; observed under UV 254 nm to locate the bands; recovery solvent: EtOAc; CH2Cl2–EtOAc (2:1) as a mobile phase) to yield compounds 1 (70 mg), 2 (10 mg), 3 (8 mg) and 4 (15 mg), respectively. Fraction XI was also subjected to sequential Sephadex LH-20 CC (MeOH), silica gel CC (300–400 mesh, eluting with CH2Cl2–acetone in gradient (from 10:1 to 5:1)), and prep. TLC (mobile phase CH2 Cl2–MeOH (10:1), Rf = 0.5) to yield compound 5 (50 mg). Repeated chromatography of fraction XIV followed by recrystallization in methanol resulted in the isolation of curvifloruside F (1.8 g). 4.3.1. Andrographidoid A (1) Pale yellow oil; ½a24 D 111 (c 0.20, CHCl3); IR (KBr) mmax 3448, 2931, 2848, 1706, 1637, 1577, 1450, 1059 cm1; for 1H NMR and 13 C NMR spectroscopic data, see Table 1; HR-ESI–MS m/z 371.1471 [M+Na]+ (calcd for C19H24O6Na, 371.1471). 4.3.2. Andrographidoid B (2) Pale yellow oil; ½a24 D -7 (c 0.26, CHCl3); IR (KBr) mmax 3421, 2917, 2850, 1706, 1635, 1577, 1550, 1448, 1070 cm1; for 1H NMR and 13 C NMR spectroscopic data, see Table 1; HR-ESI–MS m/z 371.1469 [M+Na]+ (calcd for C19H24O6Na, 371.1471).
3. Concluding remarks Previous studies on A. paniculata have mostly focused on the aerial parts but seldom on the roots. This present study demonstrated that the roots of A. paniculata are a potential source of chemically diverse natural products. It is noteworthy that the epimers of reductive noriridolactone were isolated and identified for the first time in this genus.
4. Experimental
4.3.3. Andrographidoid C (3) Pale yellow oil; ½a24 D -35 (c 0.18, CHCl3); IR (KBr) mmax 3413, 2927, 1706, 1637, 1577, 1438, 1082 cm1; for 1H NMR and 13C NMR spectroscopic data, see Table 2; HR-ESI–MS m/z 371.1473 [M+Na]+ (calcd for C19H24O6Na, 371.1471). 4.3.4. Andrographidoid D (4) Pale yellow oil; ½a24 D 25 (c 0.24, CHCl3); IR (KBr) mmax 3423, 2929, 1702, 1635, 1577, 1496, 1450, 1105 cm1; for 1H NMR and 13C NMR spectroscopic data, see Table 2; HR-ESI–MS m/z 357.1312 [M+Na]+ (calcd for C18H22O6Na, 357.1314).
4.1. General experimental procedures Melting points were measured with a BÜCHI Melting Point B540, whereas optical rotations was obtained with a PerkinElmer341 polarimeter. FT-IR spectra were recorded as KBr pellets using a Nicolet Magan 750 spectrometer, and NMR spectra were acquired on a Bruker 500 ultrashield instrument. ESI–MS and HRESI–MS were determined using a Thermo Finnigan Survyor LCQ DECA XP Plus spectrometer and Waters ACQUITY™ Synapt G2 quadrupole time-of-flight (Q/TOF) tandem mass spectrometry, respectively.
4.2. Plant materials Roots of A. paniculata were purchased in Linquan, Anhui province, China, in May 2008, and were identified by associate professor Li-Hong Wu. A voucher specimen (No. cxlg-051225) is deposited at the Herbarium of the Institute of Chinese Materia Medica, Shanghai University of TCM.
4.3.5. Andrographidoid E (5) Colorless prisms (methanol); mp > 410 °C (fusion); ½a24 D 18 (c 0.455, MeOH); IR (KBr) mmax 3421, 2919, 1762, 1672 cm1; for 1H NMR and 13C NMR spectroscopic data, see Table 3; HR-ESI–MS m/z 207.0631 [M+Na]+ (calcd for C9H12O4Na, 207.0633). 4.4. Mosher’s reaction of compound 5 Compound 5 (3.3 mg) was dissolved in anhydrous pyridine and mixed with 10 ll (S)-(+)-alpha-methoxy-alpha-(trifluromethyl)phenylacetyl chloride (purchased from Alfa Aesar) under Ar and stirred overnight at room temperature. The reaction mixture was evaporated in vacuo and subjected to preparative TLC (10 10 cm) with, CH2Cl2–EtOAc (2:1) as mobile phase. Target (S)-ester 4.1 mg was obtained. The same procedure was applied to (R)-()-alpha-methoxy-alpha-(trifluromethyl)phenylacetyl chlo ride with 5 (3.5 mg), another (R)-ester (3.2 mg) was obtained. (R)-ester: 1H NMR (400 MHz, CDCl3): d 5.0545 (1H, d, J = 12.7 Hz, H-1a), 4.8432 (1H, d, J = 12.6 Hz, H-1b), 2.7680 (2H, s,
C. Xu et al. / Phytochemistry 77 (2012) 275–279
H-4), 4.7401 (1H, d, J = 5.7 Hz, H-6), 2.9312 (1H, dd, J = 18.5, 5.9 Hz, H-7a), 2.4981 (1H, d, J = 18.6 Hz, H-7b), 1.8259 (3H, s, H-10), 3.5411 (3H, s, OCH3), 7.5035 (2H, m, phenyl H), 7.4535 (3H, m, phenyl H). (400 MHz, pyr-d5): d 5.3201 (1H, d, J = 12.3 Hz, H-1a), 5.1813 (1H, d, J = 12.4 Hz, H-1b), 3.0797 (1H, d, J = 18.0 Hz, H-4a), 3.1990 (1H, d, J = 18.1 Hz, H-4b), 4.9456 (1H, dd, J = 6.4, 1.3 Hz, H-6), 2.5491 (1H, dd, J = 18.4, 6.4 Hz, H-7a), 2.3282 (1H, d, J = 18.4 Hz, H-7b), 1.5884 (3H, s, H-10), 3.5974 (3H, s, OCH3), 7.7384 (2H, d, J = 7.2 Hz, phenyl H), 7.3955 (3H, m, phenyl H). ESIMS: m/z 418.3 [M+NH4]+, 423.4 [M+Na]+, 445.1 [M+HCOO]. (S)-ester: 1H NMR (400 MHz, CDCl3): d 4.9826 (1H, d, J = 12.8 Hz, H-1a), 4.9099 (1H, d, J = 12.8 Hz, H-1b), 2.6897 (1H, d, J = 18.2 Hz, H-4a), 2.7486 (1H, d, J = 17.0 Hz, H-4b), 4.7569 (1H, d, J = 5.6 Hz, H-6), 2.9262 (1H, dd, J = 18.8, 5.7 Hz, H-7a), 2.4739 (1H, d, J = 18.6 Hz, H-7b), 1.7429 (3H, s, H-10), 3.5545 (3H, s, OCH3), 7.4821 (2H, m, phenyl H), 7.4263 (3H, m, phenyl H). (400 MHz, pyr-d5): d 5.3450 (1H, d, J = 12.3 Hz, H-1a), 5.2181 (1H, d, J = 12.4 Hz, H-1b), 3.1477 (1H, d, J = 18.1 Hz, H-4a), 3.2166 (1H, d, J = 18.1 Hz, H-4b), 4.9815 (1H, overlapped, H-6), 2.6025 (1H, dd, J = 18.2, 6.5 Hz, H-7a), 2.3280 (1H, d, J = 17.8 Hz, H-7b), 1.6276 (3H, s, H-10), 3.5900 (3H, s, OCH3), 7.7318 (2H, d, J = 7.3 Hz, phenyl H), 7.3819 (3H, m, phenyl H). ESIMS: m/z 418.4 [M+NH4]+, 423.3 [M+Na]+, 445.2 [M+HCOO]. 4.5. Antibacterial assay The microtitre plate broth dilution method was applied. The bacteria used were E. coli (ATCC 25922), S. aureus (ATCC 29213), S. epidermidis (ATCC 26069), P. aeruginosa (ATCC 27853) and B. subtilis (provided by Huashan Hospital, Shanghai, PR China). The minimal inhibitory concentration (MIC) was evaluated using 96-well microplates. Strong activity: MIC < 25 lg/ml; Low activity: 25 lg/ ml < MIC < 100 lg/ml; Not active: MIC > 100 lg/ml. Procedure: 100 ll MH media were added in each well. The first well was mixed with a certain amount of compound solution (1 mg/ml, in MH media) and 50 ll of this mixture was pipetted to the second well. Then 50 ll of the mixture from the 2nd well was pipetted to the third well, and performing the same diluted procedure to give a gradient concentration (from 100 lg/ml to 0.024 lg/ml). The well containing MH media only was used as a control. Bacteria solutions 50 ll (1 107 CFU/ml) was added to each well and incubated at 35 °C for 18 h. Afterwards, 0.1% resazurin solution (10 ll) was added and these were incubated for another 2 h. The lowest concentration in the well which turned blue was he MIC. Acknowledgements The authors are grateful to the Nano-tech Foundation of Shanghai Science and Technology Development (0952nm05200 awarded to Professor Chang-Hong Wang) and the Program for Changjiang Scholars and Innovative Research Team in University of China (IRT1071) for financial support of this study.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2011.12.020.
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Rare noriridoids from the roots of Andrographis paniculata Chong Xu a, Gui-Xin Chou a,b, Chang-Hong Wang a, Zheng-Tao Wang a,b,⇑ a The MOE Key Laboratory for Standardization of Chinese Medicines, and Shanghai Key Laboratory of Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, PR China b Shanghai R&D Center for Standardization of Chinese Medicines (SCSCM), Shanghai 201203, PR China
a r t i c l e
i n f o
Article history: Received 30 March 2011 Received in revised form 6 October 2011 Available online 16 February 2012 Keywords: Andrographis paniculata Acanthaceae Iridoid Noriridoids Andrographidoids A–E
a b s t r a c t The rare noriridoids, Andrographidoids A–E (1–5), along with a known iridoid curvifloruside F (6), were isolated from roots of Andrographis paniculata. All noriridoids were aglycones and 1–4 had (semi-) acetal structures located at C-3 but not at C-1. Their structures were established by a series of 1D and 2D NMR analyses. The antibacterial activity of these iridoids was also assessed using the microtitre plate broth dilution method. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Andrographis paniculata Nees (Acanthaceae) is a conventional medicinal herb in China. Its aerial parts are used to treat inflammation, cold, fever and diarrhea. Previous research showed that the crude extract, as well as its main bioactive components, the entlabdane diterpenoids, possess a wide spectrum of bioactivities, such as antibacterial (Singha et al., 2003), anti-inflammatory (Liu et al., 2008), antimalarial (Dua et al., 2004), antithrombotic (Thisoda et al., 2006), antitumor (Yang et al., 2009; Zhou et al., 2006), immunostimulatory (Puri et al., 1993) and heptoprotection (Kapil et al., 1993) properties. However, research on root tissues have been few although these showed interesting results. For example, four xanthones having anti-malarial activity were isolated from the roots of A. paniculata and one of these showed substantial antiplasmodial activity (Dua et al., 2004). Meanwhile, the chloroform extract of A. paniculata roots also exhibited significant antidiabetic and nephroprotective activities (Rao, 2006). In our continuing studies on A. paniculata (Liu et al., 2007, 2008; Ji et al., 2007, 2009; Shen et al., 2009; Xu et al., 2010), herein is the chemical components of the roots were systemically investigated and reported the isolation and identification of five new noriridoids andrographidoids A–E (1–5) together with a known iridoid curvifloruside F. The antibacterial activity of these iridoids was assayed using the microtitre plate broth dilution method.
The ethanol extract of A. paniculata roots was suspended in H2O and partitioned with petroleum ether, dichloromethane, ethyl acetate and n-butanol successively. The dichloromethane extract was subjected to sequential column chromatography over silica gel, Sephadex LH-20, and followed by either prep. TLC or recrystallization to yield the five new noriridoids andrographidoids A–E (1–5) and a known iridoid curvifloruside F (6) (Lai et al., 2009). Andrographidoid A (1), obtained as pale yellow oil, displayed a quasi molecular ion peak at m/z 371.1471 in the HR-ESI–MS (attributed to the [M+Na]+, calcd for C19H24O6Na, 371.1471). Together with NMR Spectroscopic data, its molecular formula was established as C19H24O6, X = 8. The 1H NMR spectrum showed the presence of a cinnamyl group: a mono substituted phenyl (d 7.50, 2H and 7.38, 3H), a trans-double bond (d 6.35, 1H, d, J = 15.9 Hz and 7.58, 1H, d, J = 16.0 Hz) and their corresponding signals were also observed in the 13C NMR spectrum, together with an ester carbonyl resonance at d 166.4. Excluding a methoxyl (3.43, 3H, s) and a methyl (1.68, 3H, s), there were eight carbons left to form a bicyclic structure. According to analysis of the HSQC and COSY spectra, three pairs of CH–CH2 groups were present and separated by two nonprotonated carbons and an oxygen atom. Together with consideration of the HMBC spectrum, these detailed analyses indicated a reduced jioglutolide-like (Morota et al., 1989) iridoid structure for 1. However, compared with the lactone group in jioglutolide, this was replaced in 1 with a methyl acetal functionality. In the HMBC spectrum, the methoxyl protons (d 3.43, 3H) had only one cross-peak with the C-3 carbon atom (d 97.7). Moreover,
⇑ Corresponding author at: Shanghai R&D Center for Standardization of Chinese Medicines (SCSCM), Shanghai 201203, PR China. Tel.: +86 21 51322507; fax: +86 21 51322519. E-mail addresses: [email protected], [email protected] (Z.-T. Wang). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.12.020
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the carbonyl group of the cinnamyl moiety had no cross-peaks with any hydrogen in the bicyclic group thus indicating it must be attached to a non-protonated carbon. Based on the chemical shifts (d 76.6, 84.4), the cinnamate should be located at C-8 (d 84.4). Therefore, the C-5 (d 76.6) must be oxygenated with a hydroxyl group. According to the literature (Morota et al., 1989; Kouno et al., 1994; Lu et al., 1997; Yang et al., 2006), there was only one known configuration of this lactone-type iridoid as regards the cyclopentane ring and the six membered ring. Therefore, H-9 and 5-OH should be b-oriented. From analysis of the NOESY spectrum, interactions between H-9 and H-6 as well as H-9 and H-1 were observed, indicating that H-6 was b-oriented. As a result the corresponding methyl group functionality therefore was a-oriented. Meanwhile, to H-1a showed a strong NOE correlation with the methyl, and the H-1b showed an interaction with the methoxyl group at C-3 thereby suggesting that H-3 (d, J = 2.2 Hz) must be aoriented. Also the small coupling constant of H-3 (d 4.92, d, J = 2.2 Hz), as well as the couplings between H-9 (d 2.17, d, J = 4.3 Hz) and 2 H-1 (d 4.34, d, J = 13.0 and d 4.01, dd, J = 12.9, 5.0) suggested that the 6-membered ring of 1 was in a boat conformation, which indicated H-3 must be equatorial and that the 3-OMe group was b-oriented. Detailed analysis of the NMR spectroscopic data are shown in Table 1. On the basis of these data, andrographidoid A (1) was established as (3S,4aS,5S,7S,7aS)-4a,5dihydroxy-3-methoxy-7-methyloctahydrocyclopenta[c]pyran-7-yl cinnamate (Fig. 1). Andrographidoid B (2) was isolated as pale yellow oil. Following consideration of the HR-ESI–MS (m/z 371.1469, [M+Na]+, calcd for C19H24O6Na, 371.1471) and the NMR spectroscopic data, it had the same molecular formula as compound 1. Moreover the spectroscopic data were similar as well, indicating that 2 was a stereoisomer of 1, although there was one difference in the NOESY spectrum. Since the correlation between H-6 (d 4.27) and H-9 (d 2.29) was absent, this could mean that the hydroxyl at C-6 was b-oriented. Meanwhile, the H-3 had a large coupling constant J = 8.0 Hz indicating it must be axial. Therefore, the structure of 2 was concluded to be the C-3 and C-6 epimer of 1 (the optical rotational value also proved this), with the 6-membered ring in a chair conformation. Thus andrographidoid B (2) was elucidated as
(3R,4aS,5R,7S,7aS)-4a,5-dihydroxy-3-methoxy-7-methyloctahydrocyclopenta[c]pyran-7-yl cinnamate (Fig. 1). Andrographidoid C (3) was obtained as pale yellow oil. It also had the same molecular formula as compounds 1 and 2 (HR-ESI– MS m/z 371.1473 [M+Na]+, calcd for C19H24O6Na, 371.1471). While the spectroscopic data were similar to 2 (see Table 1), absence of NOE correlations between H-9 and methyl at C-10 as well as H-9 and H-6 (dd, J = 7.0, 2.5 Hz, unlike H-6 (dd, J = 11.2, 8.1 Hz) of compound 1 indicated that the methyl and H-6 must be a-oriented. Furthermore, since the H-3 signal showed a NOE interaction with the methyl group at C-10, this suggested that H-3 must be a-oriented. This was confirmed from the coupling constant of H-3 (d, J = 4.5 Hz) which indicates that the 6-membered ring possessed a boat conformation as for compound 1. Therefore, the structure of 3 was established as (3S,4aS,5R,7S,7aS)-4a,5-dihydroxy-3-methoxy-7methyloctahydrocyclopenta[c]pyran-7-yl cinnamate (Fig. 1). Andrographidoid D (4) was obtained as pale yellow oil. Its molecular formula, C18H22O6, was established on the basis of its HR-ESI–MS (m/z 357.1412 [M+Na]+, calcd for C18H22O6Na, 357.1314) and was supported by analysis of the NMR spectroscopic data. These data were quite similar to those of compounds 1–3, except for the absence of the methoxyl group in 1H NMR and 13C NMR spectrum. Meanwhile, the coupling constant of H-3 (J = 3.6 Hz), as well as the NOESY spectrum gave the same configuration of the 6-membered ring as 1: H-6 was a-oriented (d, J = 3.7 Hz) and had a NOE interaction with H-4a; a cross-peak of H-3 (d, J = 3.6 Hz) and H-4a was observed. Therefore, a b-oriented hydroxyl group was located at C-3 of compound 4. Its structure was thus formulated as (3S,4aS,5R,7S,7aS)-3,4a,5-trihydroxy-7methyloctahydrocyclopenta[c]pyran-7-yl cinnamate (Fig. 1). Andrographidoid E (5) was isolated and recrystallized from methanol as colorless prisms. HR-ESI–MS gave a quasimolecular ion peak at m/z 207.0631 [M+Na]+ (calcd for C9H12O4Na, 207.0633), which together with analysis of the NMR spectroscopic data, group its formula as C9H12O4. The HSQC spectrum showed three CH2, one oxygenated CH (d 88.4) and a methyl group, respectively. Except for the double bond and ester signals (d 136.9, 137.4 and 176.3), the 13C NMR spectroscopic data were similar to the core skeleton of compounds 1–4. This indicated that the hydroxyl
Table 1 NMR spectroscopic data for andrographidoids A (1) and B (2) (500 MHz for 1H NMR, 125 MHz for No.
1
3 4 5 6 7 8 9 10 OMe C@O
a b 10 20 30 40 50 60 a
4.34 4.01 4.92 1.82 1.74
d (13.0) dd (13.0, 5.0) d (2.2) d (15.0) oa
4.28 dd (11.2, 8.1) 2.61 dd (14.7, 8.0) 1.72 dd (14.7, 3.0) 2.17 br.d (4.3) 1.68 s 3.43 s 6.35 d (16.0) 7.58 d (16.0) 7.50 7.38 7.38 7.38 7.50
C NMR, CDCl3). 2
d (J in Hz) 1
13
m m m m m
‘‘o’’ denotes overlapping signals.
HMBC (H ? C)
d (J in Hz)
54.4
3, 5, 8, 9
97.7 31.3
1, 5 3, 5, 6, 9
4.26 3.90 4.65 1.90 1.70
76.6 75.5 44.3
5 5, 6, 8, 9, 10
4.26 oa 2.67 dd (14.5, 7.5) 1.79 dd (14.5, 9.5)
5, 8 7, 8, 9 3
2.29 t (5.0) 1.66 s 3.48 s
dC
84.4 51.1 22.6 55.2 166.4 119.3 144.4 134.4 128.0 128.9 130.2 128.9 128.0
dd dd dd dd dd
(12.5, 5.0) (12.5, 5.5) (8.0, 3.5) (14.5, 2.5) (14.5, 7.5)
6.35 d (16.0) 7.59 d (16.0) 7.50 7.37 7.37 7.37 7.50
m m m m m
dC
HMBC (H ? C)
60.6
3, 5, 8, 9
99.4 34.8
3, 5, 6
80.0 78.1 45.7
4, 5 5, 6, 8, 9, 10
85.9 51.1 22.3 55.9 166.5 119.2 144.7 134.3 128.1 128.9 130.4 128.9 128.1
4, 5, 8 7, 8, 9 3
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Fig. 1. Structures of five new noriridoids.
Fig. 2. Dd of the Mosher esters (Dd = dR dS). (A) In CDCl3; (B) in C5D5N.
group at C-3 must be oxidized to form a lactone moiety as jioglutolide which matched the ester signal at d 176.3. Additionally, the double bond should be formed by dehydration of one of the hydroxyls. Indeed, while the two carbons of the double bond were non-protonated, and the absence of the C-9 signal in the 13C NMR spectrum suggested that dehydration must occur between C-5 and C-9. A detailed analysis of the HMBC spectrum further verified this deduction. H-1, H-4, H-6, H-7 and H-10 (methyl protons) signals showed HMBC cross-peaks with the resonance of C-9 (d 136.9). A
cross-peak correlation was also observed between H-4 (d 3.35 and 3.85) and C-5 (d 137.4). In the NOESY spectrum, H-6 showed a NOE interaction with the methyl group at C-8. Thus, andrographidoid E (5) was deduced as 5,9-didehydrojioglutolide (Fig. 1). More detailed 2D NMR analyses are given in Table 3. In order to determine the absolute stereostructure of 5, a Mosher’s reaction was carried out. Two samples of the compound were mixed respectively with (S)-(+) and (R)-()-alpha-methoxyalpha-(trifluromethyl)phenylacetyl chloride in anhydrous pyridine
Table 2 NMR spectroscopic data for andrographidoids C (3) and D (4) (500 MHz for 1H NMR, 125 MHz for No.
3
3 4 5 6 7 8 9 10 OMe C@O
a b 10 20 30 40 50 60
4.04 3.88 5.11 2.25 2.05
dd (10.0, 6.0) t (10.0) d (4.5) dd (14.0, 4.5) d (14.0)
4.40 dd (7.0, 2.5) 2.47 m
2.74 dd (9.0, 6.0) 1.47 s 3.38 s 6.36 d (16.0) 7.61 d (16.0) 7.51 7.38 7.38 7.38 7.51
C NMR, CDCl3). 4
d (J in Hz) 1
13
m m m m m
dC 60.1 105.3 43.4 87.0 87.8 43.8 87.3 57.8 20.4 54.5 165.8 119.0 144.8 134.3 128.1 128.9 130.3 128.9 128.1
HMBC (H ? C)
d (J in Hz)
5
3.93 3.83 5.33 2.33 2.23
4, 5, 6, OCH3 3, 5, 6
6, 8, 9, 10
1, 4, 5, 8, 10 7, 8, 9 3
d (12.2) dd (12.2, 4.5) d (3.6) d (11.8) dd (11.8, 3.7)
4.05 d (3.7) 2.90 dd (15.0, 3.8) 2.13 d (15.0) 2.74 d (4.2) 1.73 s
6.38 d (16.0) 7.67 d (16.0) 7.52 7.40 7.40 7.40 7.52
m t-like t-like t-like m
dC
HMBC (H ? C)
58.0
3, 5, 8, 9
100.0 36.0
1, 5 3, 5, 6, 9
84.7 84.9 42.9
7, 8, 9 5, 6, 8, 9, 10
92.3 57.2 25.9
5, 8 7, 8, 9
167.2 118.9 145.6 134.1 128.2 128.9 130.6 128.9 128.2
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Table 3 NMR spectroscopic data for andrographidoid E (5) (500 MHz for 1H NMR, 125 MHz for 13 C NMR, pyr-d5). No.
5 d (J in Hz)
1 3 4 5 6 7 8 9 10
4.77 d (12.5) 4.70 d (12.5) 3.85 d (17.5) 3.35 d (18.0) 5.07 dd (6.5, 1.0) 2.78 dd (18.0, 6.0) 2.46 d (17.5)
1.69 s
dC 55.8
HMBC (H ? C) 8, 9
176.3 41.1
3, 5, 6, 9
137.4 88.4 42.6
7, 8, 9 6, 8, 9, 10
89.2 136.9 14.1
1, 7, 8, 9
under an argon atmosphere and stirred overnight at room temperature. The target (S) and (R) esters were purified by prep. TLC and the corresponding 1H NMR spectroscopic data of each proton were compared (Dd = dR dS). However, the absolute stereostructure of compound 5 was not obtained (see Fig. 2). As the chemical structures of andrographidoid A–E are similar to villosol and patriscabrol, which were reported to possess a significant antibacterial activity (Yang et al., 2006), their potential antibacterial activity was assayed. However, none showed any inhibitory activity (MIC > 100 lg/ml). The bacteria including: Escherichiacoli, Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa and Bacillus subtilis; Gentamycin, Chloramphenicol and Ciprofloxacin were used as positive controls.
4.3. Extraction and isolation Dried and powdered roots of A. paniculata (5 kg) were extracted with EtOH–H2O (4:1, v/v, 5 30 L) at room temperature for 3 days and filtered. The corresponding combined filtrates were evaporated, then partitioned between water and petroleum ether, dichloromethane, ethyl acetate and n-butanol successively. The CH2Cl2 extract (47 g) was subjected to silica gel column chromatography (CC) (1 kg, 100–200 mesh) and eluted with petroleum ether (60–90 °C)–EtOAc (10:1, 5:1, 2:1, 0:1) and finally MeOH to afford 14 fractions. Fraction IX was further purified by repeated CC (silica gel, 300–400 mesh, CH2Cl2–EtOAc (20:1, 10:1, 5:1, 2:1)) and prep. TLC (layer thickness: 0.4–0.5 mm; sample amount, about 20 mg; observed under UV 254 nm to locate the bands; recovery solvent: EtOAc; CH2Cl2–EtOAc (2:1) as a mobile phase) to yield compounds 1 (70 mg), 2 (10 mg), 3 (8 mg) and 4 (15 mg), respectively. Fraction XI was also subjected to sequential Sephadex LH-20 CC (MeOH), silica gel CC (300–400 mesh, eluting with CH2Cl2–acetone in gradient (from 10:1 to 5:1)), and prep. TLC (mobile phase CH2 Cl2–MeOH (10:1), Rf = 0.5) to yield compound 5 (50 mg). Repeated chromatography of fraction XIV followed by recrystallization in methanol resulted in the isolation of curvifloruside F (1.8 g). 4.3.1. Andrographidoid A (1) Pale yellow oil; ½a24 D 111 (c 0.20, CHCl3); IR (KBr) mmax 3448, 2931, 2848, 1706, 1637, 1577, 1450, 1059 cm1; for 1H NMR and 13 C NMR spectroscopic data, see Table 1; HR-ESI–MS m/z 371.1471 [M+Na]+ (calcd for C19H24O6Na, 371.1471). 4.3.2. Andrographidoid B (2) Pale yellow oil; ½a24 D -7 (c 0.26, CHCl3); IR (KBr) mmax 3421, 2917, 2850, 1706, 1635, 1577, 1550, 1448, 1070 cm1; for 1H NMR and 13 C NMR spectroscopic data, see Table 1; HR-ESI–MS m/z 371.1469 [M+Na]+ (calcd for C19H24O6Na, 371.1471).
3. Concluding remarks Previous studies on A. paniculata have mostly focused on the aerial parts but seldom on the roots. This present study demonstrated that the roots of A. paniculata are a potential source of chemically diverse natural products. It is noteworthy that the epimers of reductive noriridolactone were isolated and identified for the first time in this genus.
4. Experimental
4.3.3. Andrographidoid C (3) Pale yellow oil; ½a24 D -35 (c 0.18, CHCl3); IR (KBr) mmax 3413, 2927, 1706, 1637, 1577, 1438, 1082 cm1; for 1H NMR and 13C NMR spectroscopic data, see Table 2; HR-ESI–MS m/z 371.1473 [M+Na]+ (calcd for C19H24O6Na, 371.1471). 4.3.4. Andrographidoid D (4) Pale yellow oil; ½a24 D 25 (c 0.24, CHCl3); IR (KBr) mmax 3423, 2929, 1702, 1635, 1577, 1496, 1450, 1105 cm1; for 1H NMR and 13C NMR spectroscopic data, see Table 2; HR-ESI–MS m/z 357.1312 [M+Na]+ (calcd for C18H22O6Na, 357.1314).
4.1. General experimental procedures Melting points were measured with a BÜCHI Melting Point B540, whereas optical rotations was obtained with a PerkinElmer341 polarimeter. FT-IR spectra were recorded as KBr pellets using a Nicolet Magan 750 spectrometer, and NMR spectra were acquired on a Bruker 500 ultrashield instrument. ESI–MS and HRESI–MS were determined using a Thermo Finnigan Survyor LCQ DECA XP Plus spectrometer and Waters ACQUITY™ Synapt G2 quadrupole time-of-flight (Q/TOF) tandem mass spectrometry, respectively.
4.2. Plant materials Roots of A. paniculata were purchased in Linquan, Anhui province, China, in May 2008, and were identified by associate professor Li-Hong Wu. A voucher specimen (No. cxlg-051225) is deposited at the Herbarium of the Institute of Chinese Materia Medica, Shanghai University of TCM.
4.3.5. Andrographidoid E (5) Colorless prisms (methanol); mp > 410 °C (fusion); ½a24 D 18 (c 0.455, MeOH); IR (KBr) mmax 3421, 2919, 1762, 1672 cm1; for 1H NMR and 13C NMR spectroscopic data, see Table 3; HR-ESI–MS m/z 207.0631 [M+Na]+ (calcd for C9H12O4Na, 207.0633). 4.4. Mosher’s reaction of compound 5 Compound 5 (3.3 mg) was dissolved in anhydrous pyridine and mixed with 10 ll (S)-(+)-alpha-methoxy-alpha-(trifluromethyl)phenylacetyl chloride (purchased from Alfa Aesar) under Ar and stirred overnight at room temperature. The reaction mixture was evaporated in vacuo and subjected to preparative TLC (10 10 cm) with, CH2Cl2–EtOAc (2:1) as mobile phase. Target (S)-ester 4.1 mg was obtained. The same procedure was applied to (R)-()-alpha-methoxy-alpha-(trifluromethyl)phenylacetyl chlo ride with 5 (3.5 mg), another (R)-ester (3.2 mg) was obtained. (R)-ester: 1H NMR (400 MHz, CDCl3): d 5.0545 (1H, d, J = 12.7 Hz, H-1a), 4.8432 (1H, d, J = 12.6 Hz, H-1b), 2.7680 (2H, s,
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H-4), 4.7401 (1H, d, J = 5.7 Hz, H-6), 2.9312 (1H, dd, J = 18.5, 5.9 Hz, H-7a), 2.4981 (1H, d, J = 18.6 Hz, H-7b), 1.8259 (3H, s, H-10), 3.5411 (3H, s, OCH3), 7.5035 (2H, m, phenyl H), 7.4535 (3H, m, phenyl H). (400 MHz, pyr-d5): d 5.3201 (1H, d, J = 12.3 Hz, H-1a), 5.1813 (1H, d, J = 12.4 Hz, H-1b), 3.0797 (1H, d, J = 18.0 Hz, H-4a), 3.1990 (1H, d, J = 18.1 Hz, H-4b), 4.9456 (1H, dd, J = 6.4, 1.3 Hz, H-6), 2.5491 (1H, dd, J = 18.4, 6.4 Hz, H-7a), 2.3282 (1H, d, J = 18.4 Hz, H-7b), 1.5884 (3H, s, H-10), 3.5974 (3H, s, OCH3), 7.7384 (2H, d, J = 7.2 Hz, phenyl H), 7.3955 (3H, m, phenyl H). ESIMS: m/z 418.3 [M+NH4]+, 423.4 [M+Na]+, 445.1 [M+HCOO]. (S)-ester: 1H NMR (400 MHz, CDCl3): d 4.9826 (1H, d, J = 12.8 Hz, H-1a), 4.9099 (1H, d, J = 12.8 Hz, H-1b), 2.6897 (1H, d, J = 18.2 Hz, H-4a), 2.7486 (1H, d, J = 17.0 Hz, H-4b), 4.7569 (1H, d, J = 5.6 Hz, H-6), 2.9262 (1H, dd, J = 18.8, 5.7 Hz, H-7a), 2.4739 (1H, d, J = 18.6 Hz, H-7b), 1.7429 (3H, s, H-10), 3.5545 (3H, s, OCH3), 7.4821 (2H, m, phenyl H), 7.4263 (3H, m, phenyl H). (400 MHz, pyr-d5): d 5.3450 (1H, d, J = 12.3 Hz, H-1a), 5.2181 (1H, d, J = 12.4 Hz, H-1b), 3.1477 (1H, d, J = 18.1 Hz, H-4a), 3.2166 (1H, d, J = 18.1 Hz, H-4b), 4.9815 (1H, overlapped, H-6), 2.6025 (1H, dd, J = 18.2, 6.5 Hz, H-7a), 2.3280 (1H, d, J = 17.8 Hz, H-7b), 1.6276 (3H, s, H-10), 3.5900 (3H, s, OCH3), 7.7318 (2H, d, J = 7.3 Hz, phenyl H), 7.3819 (3H, m, phenyl H). ESIMS: m/z 418.4 [M+NH4]+, 423.3 [M+Na]+, 445.2 [M+HCOO]. 4.5. Antibacterial assay The microtitre plate broth dilution method was applied. The bacteria used were E. coli (ATCC 25922), S. aureus (ATCC 29213), S. epidermidis (ATCC 26069), P. aeruginosa (ATCC 27853) and B. subtilis (provided by Huashan Hospital, Shanghai, PR China). The minimal inhibitory concentration (MIC) was evaluated using 96-well microplates. Strong activity: MIC < 25 lg/ml; Low activity: 25 lg/ ml < MIC < 100 lg/ml; Not active: MIC > 100 lg/ml. Procedure: 100 ll MH media were added in each well. The first well was mixed with a certain amount of compound solution (1 mg/ml, in MH media) and 50 ll of this mixture was pipetted to the second well. Then 50 ll of the mixture from the 2nd well was pipetted to the third well, and performing the same diluted procedure to give a gradient concentration (from 100 lg/ml to 0.024 lg/ml). The well containing MH media only was used as a control. Bacteria solutions 50 ll (1 107 CFU/ml) was added to each well and incubated at 35 °C for 18 h. Afterwards, 0.1% resazurin solution (10 ll) was added and these were incubated for another 2 h. The lowest concentration in the well which turned blue was he MIC. Acknowledgements The authors are grateful to the Nano-tech Foundation of Shanghai Science and Technology Development (0952nm05200 awarded to Professor Chang-Hong Wang) and the Program for Changjiang Scholars and Innovative Research Team in University of China (IRT1071) for financial support of this study.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2011.12.020.
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