Phytochemistry 77 (2012) 275–279

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Phytochemistry

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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 Chinab 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 a b s t r a c t

Article history:Received 30 March 2011Received in revised form 6 October 2011Available online 16 February 2012

Keywords:Andrographis paniculataAcanthaceaeIridoidNoriridoidsAndrographidoids A–E

0031-9422/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.phytochem.2011.12.020

⇑ Corresponding author at: Shanghai R&D Center foMedicines (SCSCM), Shanghai 201203, PR China. Tel.: +51322519.

E-mail addresses: wangzht@shutcm.edu.cn, wangzh

The rare noriridoids, Andrographidoids A–E (1–5), along with a known iridoid curvifloruside F (6), wereisolated from roots of Andrographis paniculata. All noriridoids were aglycones and 1–4 had (semi-) acetalstructures located at C-3 but not at C-1. Their structures were established by a series of 1D and 2D NMRanalyses. The antibacterial activity of these iridoids was also assessed using the microtitre plate brothdilution method.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Andrographis paniculata Nees (Acanthaceae) is a conventionalmedicinal herb in China. Its aerial parts are used to treat inflamma-tion, cold, fever and diarrhea. Previous research showed that thecrude extract, as well as its main bioactive components, the ent-labdane diterpenoids, possess a wide spectrum of bioactivities,such as antibacterial (Singha et al., 2003), anti-inflammatory (Liuet 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 tissueshave been few although these showed interesting results. Forexample, four xanthones having anti-malarial activity were iso-lated from the roots of A. paniculata and one of these showed sub-stantial antiplasmodial activity (Dua et al., 2004). Meanwhile, thechloroform extract of A. paniculata roots also exhibited significantantidiabetic and nephroprotective activities (Rao, 2006). In ourcontinuing studies on A. paniculata (Liu et al., 2007, 2008; Ji etal., 2007, 2009; Shen et al., 2009; Xu et al., 2010), herein is thechemical components of the roots were systemically investigatedand reported the isolation and identification of five new noririd-oids andrographidoids A–E (1–5) together with a known iridoidcurvifloruside F. The antibacterial activity of these iridoids wasassayed using the microtitre plate broth dilution method.

ll rights reserved.

r Standardization of Chinese86 21 51322507; fax: +86 21

t@hotmail.com (Z.-T. Wang).

2. Results and discussion

The ethanol extract of A. paniculata roots was suspended in H2Oand partitioned with petroleum ether, dichloromethane, ethyl ace-tate and n-butanol successively. The dichloromethane extract wassubjected to sequential column chromatography over silica gel,Sephadex LH-20, and followed by either prep. TLC or recrystalliza-tion 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 aquasi molecular ion peak at m/z 371.1471 in the HR-ESI–MS(attributed to the [M+Na]+, calcd for C19H24O6Na, 371.1471). To-gether with NMR Spectroscopic data, its molecular formula wasestablished as C19H24O6, X = 8. The 1H NMR spectrum showedthe presence of a cinnamyl group: a mono substituted phenyl (d7.50, 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 sig-nals were also observed in the 13C NMR spectrum, together with anester 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 toform a bicyclic structure. According to analysis of the HSQC andCOSY spectra, three pairs of CH–CH2 groups were present andseparated by two nonprotonated carbons and an oxygen atom.Together with consideration of the HMBC spectrum, these detailedanalyses indicated a reduced jioglutolide-like (Morota et al., 1989)iridoid structure for 1. However, compared with the lactone groupin jioglutolide, this was replaced in 1 with a methyl acetalfunctionality.

In the HMBC spectrum, the methoxyl protons (d 3.43, 3H) hadonly one cross-peak with the C-3 carbon atom (d 97.7). Moreover,

276 C. Xu et al. / Phytochemistry 77 (2012) 275–279

the carbonyl group of the cinnamyl moiety had no cross-peakswith any hydrogen in the bicyclic group thus indicating it mustbe attached to a non-protonated carbon. Based on the chemicalshifts (d 76.6, 84.4), the cinnamate should be located at C-8 (d84.4). Therefore, the C-5 (d 76.6) must be oxygenated with a hydro-xyl group. According to the literature (Morota et al., 1989; Kounoet al., 1994; Lu et al., 1997; Yang et al., 2006), there was onlyone known configuration of this lactone-type iridoid as regardsthe cyclopentane ring and the six membered ring. Therefore, H-9and 5-OH should be b-oriented. From analysis of the NOESY spec-trum, interactions between H-9 and H-6 as well as H-9 and H-1were observed, indicating that H-6 was b-oriented. As a resultthe corresponding methyl group functionality therefore was a-ori-ented. Meanwhile, to H-1a showed a strong NOE correlation withthe methyl, and the H-1b showed an interaction with the methoxylgroup at C-3 thereby suggesting that H-3 (d, J = 2.2 Hz) must be a-oriented. 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 boatconformation, which indicated H-3 must be equatorial and thatthe 3-OMe group was b-oriented. Detailed analysis of the NMRspectroscopic data are shown in Table 1. On the basis of these data,andrographidoid A (1) was established as (3S,4aS,5S,7S,7aS)-4a,5-dihydroxy-3-methoxy-7-methyloctahydrocyclopenta[c]pyran-7-ylcinnamate (Fig. 1).

Andrographidoid B (2) was isolated as pale yellow oil. Followingconsideration of the HR-ESI–MS (m/z 371.1469, [M+Na]+, calcd forC19H24O6Na, 371.1471) and the NMR spectroscopic data, it hadthe same molecular formula as compound 1. Moreover the spectro-scopic data were similar as well, indicating that 2 was a stereoiso-mer of 1, although there was one difference in the NOESYspectrum. Since the correlation between H-6 (d 4.27) and H-9 (d2.29) was absent, this could mean that the hydroxyl at C-6 wasb-oriented. Meanwhile, the H-3 had a large coupling constantJ = 8.0 Hz indicating it must be axial. Therefore, the structure of 2was concluded to be the C-3 and C-6 epimer of 1 (the optical rota-tional value also proved this), with the 6-membered ring in a chairconformation. Thus andrographidoid B (2) was elucidated as

Table 1NMR spectroscopic data for andrographidoids A (1) and B (2) (500 MHz for 1H NMR, 125

No. 1

d (J in Hz) dC HMBC (H ? C)

1 4.34 d (13.0)4.01 dd (13.0, 5.0)

54.4 3, 5, 8, 9

3 4.92 d (2.2) 97.7 1, 54 1.82 d (15.0)

1.74 oa31.3 3, 5, 6, 9

5 76.66 4.28 dd (11.2, 8.1) 75.5 57 2.61 dd (14.7, 8.0)

1.72 dd (14.7, 3.0)44.3 5, 6, 8, 9, 10

8 84.49 2.17 br.d (4.3) 51.1 5, 810 1.68 s 22.6 7, 8, 9OMe 3.43 s 55.2 3C@O 166.4a 6.35 d (16.0) 119.3b 7.58 d (16.0) 144.410 134.420 7.50 m 128.030 7.38 m 128.940 7.38 m 130.250 7.38 m 128.960 7.50 m 128.0

a ‘‘o’’ denotes overlapping signals.

(3R,4aS,5R,7S,7aS)-4a,5-dihydroxy-3-methoxy-7-methyloctahy-drocyclopenta[c]pyran-7-yl cinnamate (Fig. 1).

Andrographidoid C (3) was obtained as pale yellow oil. It alsohad the same molecular formula as compounds 1 and 2 (HR-ESI–MS m/z 371.1473 [M+Na]+, calcd for C19H24O6Na, 371.1471). Whilethe spectroscopic data were similar to 2 (see Table 1), absence ofNOE correlations between H-9 and methyl at C-10 as well as H-9and H-6 (dd, J = 7.0, 2.5 Hz, unlike H-6 (dd, J = 11.2, 8.1 Hz) of com-pound 1 indicated that the methyl and H-6 must be a-oriented. Fur-thermore, since the H-3 signal showed a NOE interaction with themethyl 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 confor-mation as for compound 1. Therefore, the structure of 3 wasestablished as (3S,4aS,5R,7S,7aS)-4a,5-dihydroxy-3-methoxy-7-methyloctahydrocyclopenta[c]pyran-7-yl cinnamate (Fig. 1).

Andrographidoid D (4) was obtained as pale yellow oil. Itsmolecular formula, C18H22O6, was established on the basis of itsHR-ESI–MS (m/z 357.1412 [M+Na]+, calcd for C18H22O6Na,357.1314) and was supported by analysis of the NMR spectro-scopic data. These data were quite similar to those of compounds1–3, except for the absence of the methoxyl group in 1H NMRand 13C NMR spectrum. Meanwhile, the coupling constant of H-3(J = 3.6 Hz), as well as the NOESY spectrum gave the same configu-ration 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 ofH-3 (d, J = 3.6 Hz) and H-4a was observed. Therefore, a b-orientedhydroxyl group was located at C-3 of compound 4. Its structurewas thus formulated as (3S,4aS,5R,7S,7aS)-3,4a,5-trihydroxy-7-methyloctahydrocyclopenta[c]pyran-7-yl cinnamate (Fig. 1).

Andrographidoid E (5) was isolated and recrystallized frommethanol as colorless prisms. HR-ESI–MS gave a quasimolecularion peak at m/z 207.0631 [M+Na]+ (calcd for C9H12O4Na,207.0633), which together with analysis of the NMR spectroscopicdata, group its formula as C9H12O4. The HSQC spectrum showedthree CH2, one oxygenated CH (d 88.4) and a methyl group, respec-tively. Except for the double bond and ester signals (d 136.9, 137.4and 176.3), the 13C NMR spectroscopic data were similar to thecore skeleton of compounds 1–4. This indicated that the hydroxyl

MHz for 13C NMR, CDCl3).

2

d (J in Hz) dC HMBC (H ? C)

4.26 dd (12.5, 5.0)3.90 dd (12.5, 5.5)

60.6 3, 5, 8, 9

4.65 dd (8.0, 3.5) 99.41.90 dd (14.5, 2.5)1.70 dd (14.5, 7.5)

34.8 3, 5, 6

80.04.26 oa 78.1 4, 52.67 dd (14.5, 7.5)1.79 dd (14.5, 9.5)

45.7 5, 6, 8, 9, 10

85.92.29 t (5.0) 51.1 4, 5, 81.66 s 22.3 7, 8, 93.48 s 55.9 3

166.56.35 d (16.0) 119.27.59 d (16.0) 144.7

134.37.50 m 128.17.37 m 128.97.37 m 130.47.37 m 128.97.50 m 128.1

Fig. 1. Structures of five new noriridoids.

Fig. 2. Dd of the Mosher esters (Dd = dR � dS). (A) In CDCl3; (B) in C5D5N.

C. Xu et al. / Phytochemistry 77 (2012) 275–279 277

group at C-3 must be oxidized to form a lactone moiety as jiogluto-lide which matched the ester signal at d 176.3. Additionally, thedouble bond should be formed by dehydration of one of thehydroxyls. Indeed, while the two carbons of the double bond werenon-protonated, and the absence of the C-9 signal in the 13C NMRspectrum suggested that dehydration must occur between C-5 andC-9. A detailed analysis of the HMBC spectrum further verified thisdeduction. H-1, H-4, H-6, H-7 and H-10 (methyl protons) signalsshowed HMBC cross-peaks with the resonance of C-9 (d 136.9). A

Table 2NMR spectroscopic data for andrographidoids C (3) and D (4) (500 MHz for 1H NMR, 125

No. 3

d (J in Hz) dC HMBC (H ? C)

1 4.04 dd (10.0, 6.0)3.88 t (10.0)

60.1 5

3 5.11 d (4.5) 105.3 4, 5, 6, OCH3

4 2.25 dd (14.0, 4.5)2.05 d (14.0)

43.4 3, 5, 6

5 87.06 4.40 dd (7.0, 2.5) 87.87 2.47 m 43.8 6, 8, 9, 10

8 87.39 2.74 dd (9.0, 6.0) 57.8 1, 4, 5, 8, 1010 1.47 s 20.4 7, 8, 9OMe 3.38 s 54.5 3C@O 165.8a 6.36 d (16.0) 119.0b 7.61 d (16.0) 144.810 134.320 7.51 m 128.130 7.38 m 128.940 7.38 m 130.350 7.38 m 128.960 7.51 m 128.1

cross-peak correlation was also observed between H-4 (d 3.35and 3.85) and C-5 (d 137.4). In the NOESY spectrum, H-6 showeda NOE interaction with the methyl group at C-8. Thus, andrograp-hidoid 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, aMosher’s reaction was carried out. Two samples of the compoundwere mixed respectively with (S)-(+) and (R)-(�)-alpha-methoxy-alpha-(trifluromethyl)phenylacetyl chloride in anhydrous pyridine

MHz for 13C NMR, CDCl3).

4

d (J in Hz) dC HMBC (H ? C)

3.93 d (12.2)3.83 dd (12.2, 4.5)

58.0 3, 5, 8, 9

5.33 d (3.6) 100.0 1, 52.33 d (11.8)2.23 dd (11.8, 3.7)

36.0 3, 5, 6, 9

84.74.05 d (3.7) 84.9 7, 8, 92.90 dd (15.0, 3.8)2.13 d (15.0)

42.9 5, 6, 8, 9, 10

92.32.74 d (4.2) 57.2 5, 81.73 s 25.9 7, 8, 9

167.26.38 d (16.0) 118.97.67 d (16.0) 145.6

134.17.52 m 128.27.40 t-like 128.97.40 t-like 130.67.40 t-like 128.97.52 m 128.2

Table 3NMR spectroscopic data for andrographidoid E (5) (500 MHz for 1H NMR, 125 MHz for13C NMR, pyr-d5).

No. 5

d (J in Hz) dC HMBC (H ? C)

1 4.77 d (12.5)4.70 d (12.5)

55.8 8, 9

3 176.34 3.85 d (17.5)

3.35 d (18.0)41.1 3, 5, 6, 9

5 137.46 5.07 dd (6.5, 1.0) 88.4 7, 8, 97 2.78 dd (18.0, 6.0)

2.46 d (17.5)42.6 6, 8, 9, 10

8 89.29 136.9

10 1.69 s 14.1 1, 7, 8, 9

278 C. Xu et al. / Phytochemistry 77 (2012) 275–279

under an argon atmosphere and stirred overnight at room temper-ature. The target (S) and (R) esters were purified by prep. TLC andthe corresponding 1H NMR spectroscopic data of each proton werecompared (Dd = dR � dS). However, the absolute stereostructure ofcompound 5 was not obtained (see Fig. 2).

As the chemical structures of andrographidoid A–E are similarto villosol and patriscabrol, which were reported to possess a sig-nificant antibacterial activity (Yang et al., 2006), their potentialantibacterial activity was assayed. However, none showed anyinhibitory activity (MIC > 100 lg/ml). The bacteria including: Esch-erichiacoli, Staphylococcus aureus, S. epidermidis, Pseudomonas aeru-ginosa and Bacillus subtilis; Gentamycin, Chloramphenicol andCiprofloxacin were used as positive controls.

3. Concluding remarks

Previous studies on A. paniculata have mostly focused on theaerial parts but seldom on the roots. This present study demon-strated that the roots of A. paniculata are a potential source ofchemically diverse natural products. It is noteworthy that the epi-mers of reductive noriridolactone were isolated and identified forthe first time in this genus.

4. Experimental

4.1. General experimental procedures

Melting points were measured with a BÜCHI Melting Point B-540, whereas optical rotations was obtained with a PerkinElmer-341 polarimeter. FT-IR spectra were recorded as KBr pellets usinga Nicolet Magan 750 spectrometer, and NMR spectra were ac-quired on a Bruker 500 ultrashield instrument. ESI–MS and HR-ESI–MS were determined using a Thermo Finnigan Survyor LCQDECA XP Plus spectrometer and Waters ACQUITY™ Synapt G2quadrupole time-of-flight (Q/TOF) tandem mass spectrometry,respectively.

4.2. Plant materials

Roots of A. paniculata were purchased in Linquan, Anhui prov-ince, China, in May 2008, and were identified by associate profes-sor Li-Hong Wu. A voucher specimen (No. cxlg-051225) isdeposited at the Herbarium of the Institute of Chinese MateriaMedica, Shanghai University of TCM.

4.3. Extraction and isolation

Dried and powdered roots of A. paniculata (5 kg) were extractedwith EtOH–H2O (4:1, v/v, 5 � 30 L) at room temperature for 3 daysand filtered. The corresponding combined filtrates were evapo-rated, then partitioned between water and petroleum ether,dichloromethane, ethyl acetate and n-butanol successively.

The CH2Cl2 extract (47 g) was subjected to silica gel columnchromatography (CC) (1 kg, 100–200 mesh) and eluted with petro-leum ether (60–90 �C)–EtOAc (10:1, 5:1, 2:1, 0:1) and finally MeOHto afford 14 fractions. Fraction IX was further purified by repeatedCC (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, about20 mg; observed under UV 254 nm to locate the bands; recoverysolvent: EtOAc; CH2Cl2–EtOAc (2:1) as a mobile phase) to yield com-pounds 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–acetonein 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). Repeatedchromatography of fraction XIV followed by recrystallization inmethanol resulted in the isolation of curvifloruside F (1.8 g).

4.3.1. Andrographidoid A (1)Pale yellow oil; ½a�24

D 111 (c 0.20, CHCl3); IR (KBr) mmax 3448,2931, 2848, 1706, 1637, 1577, 1450, 1059 cm�1; for 1H NMR and13C NMR spectroscopic data, see Table 1; HR-ESI–MS m/z371.1471 [M+Na]+ (calcd for C19H24O6Na, 371.1471).

4.3.2. Andrographidoid B (2)Pale yellow oil; ½a�24

D -7 (c 0.26, CHCl3); IR (KBr) mmax 3421, 2917,2850, 1706, 1635, 1577, 1550, 1448, 1070 cm�1; for 1H NMR and13C NMR spectroscopic data, see Table 1; HR-ESI–MS m/z371.1469 [M+Na]+ (calcd for C19H24O6Na, 371.1471).

4.3.3. Andrographidoid C (3)Pale yellow oil; ½a�24

D -35 (c 0.18, CHCl3); IR (KBr) mmax 3413,2927, 1706, 1637, 1577, 1438, 1082 cm�1; for 1H NMR and 13CNMR 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; ½a�24

D 25 (c 0.24, CHCl3); IR (KBr) mmax 3423, 2929,1702, 1635, 1577, 1496, 1450, 1105 cm�1; for 1H NMR and 13CNMR spectroscopic data, see Table 2; HR-ESI–MS m/z 357.1312[M+Na]+ (calcd for C18H22O6Na, 357.1314).

4.3.5. Andrographidoid E (5)Colorless prisms (methanol); mp > 410 �C (fusion); ½a�24

D 18 (c0.455, MeOH); IR (KBr) mmax 3421, 2919, 1762, 1672 cm�1; for 1HNMR and 13C NMR spectroscopic data, see Table 3; HR-ESI–MSm/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 andmixed with 10 ll (S)-(+)-alpha-methoxy-alpha-(triflurometh-yl)phenylacetyl chloride (purchased from Alfa Aesar) under Arand stirred overnight at room temperature. The reaction mixturewas 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 appliedto (R)-(�)-alpha-methoxy-alpha-(trifluromethyl)phenylacetyl chloride 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) 275–279 279

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, phe-nyl 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, phenylH). (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. Thebacteria used were E. coli (ATCC 25922), S. aureus (ATCC 29213),S. epidermidis (ATCC 26069), P. aeruginosa (ATCC 27853) and B. sub-tilis (provided by Huashan Hospital, Shanghai, PR China). The min-imal inhibitory concentration (MIC) was evaluated using 96-wellmicroplates. 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 wasmixed with a certain amount of compound solution (1 mg/ml, inMH media) and 50 ll of this mixture was pipetted to the secondwell. Then 50 ll of the mixture from the 2nd well was pipettedto the third well, and performing the same diluted procedure togive a gradient concentration (from 100 lg/ml to 0.024 lg/ml).The well containing MH media only was used as a control. Bacteriasolutions 50 ll (1 � 107 CFU/ml) was added to each well and incu-bated at 35 �C for 18 h. Afterwards, 0.1% resazurin solution (10 ll)was added and these were incubated for another 2 h. The lowestconcentration in the well which turned blue was he MIC.

Acknowledgements

The authors are grateful to the Nano-tech Foundation of Shang-hai Science and Technology Development (0952nm05200 awardedto Professor Chang-Hong Wang) and the Program for ChangjiangScholars and Innovative Research Team in University of China(IRT1071) for financial support of this study.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.phytochem.2011.12.020.

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