neo-Clerodane diterpenes from Ajuga decumbens and their inhibitory activities on LPS-induced NO production

Fitoterapia 83 (2012) 1409–1414

Contents lists available at SciVerse ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

neo-Clerodane diterpenes from Ajuga decumbens and their inhibitory activities on LPS-induced NO production Zhanping Sun a, b, Yushan Li b, Da-qing Jin c, Ping Guo b, Haibin Song d, Jing Xu a, Yuanqiang Guo a,⁎, Lei Zhang a a College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China b Shenyang Pharmaceutical University, Shenyang 110016, China c School of Medicine, Nankai University, Tianjin 300071, China d State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, China

a r t i c l e

i n f o

Article history: Received 7 May 2012 Accepted in revised form 3 August 2012 Available online 21 August 2012 Keywords: Ajuga decumbens neo-Clerodane diterpenes Inhibitory activities on NO production

a b s t r a c t A phytochemical investigation of the whole plants of Ajuga decumbens led to the isolation of three new (1, 2a, and 2b) and three known (3a−3c) neo-clerodane diterpenes. Their structures were elucidated by spectroscopic data analysis (IR, ESIMS, HR-ESIMS, 1D and 2D NMR), and the structure of 1 was confirmed by X-ray crystallography. The inhibitory activities on LPS-induced NO production of the six diterpenes were evaluated and compounds 2a, 2b, and 3a showed inhibitory effects. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Ajuga decumbens Thunb, belonging to the Labiatae family, is a herbaceous plant mainly distributed in the east, central south, and southwest of China [1]. The aerial parts of A. decumbens have been used as a folk medicine for its anti-inflammatory, antitussive, and expectorant effects [1]. Previous phytochemical investigations on this species revealed the presence of diterpenes [2–7], and iridoids [8], of which the main and characteristic constituents are diterpenes. In the course of our survey on biologically active substances in medicinal plants [9–12], much attention has been given to the occurrence of compounds with inhibitory effects of NO production, since these substances are expected to be useful as potential

⁎ Corresponding author. Tel./fax: +86 22 23502595. E-mail address: [email protected] (Y. Guo). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2012.08.003

anti-inflammatory agents. As a continuation of our work with the search for bioactive natural products, we investigated the chemical constituents of A. decumbens, which has been used for the treatment of inflammations [1]. As a result, three new (1, 2a, and 2b) and three known (3a–c) neo-clerodane diterpenes (Fig. 1) have been isolated from the whole plants of A. decumbens. Their structures were elucidated as (12S)-1α,19epoxy-6α,18-diacetoxy-4α,12-dihydroxy-neo-clerod-13-en-15, 16-olide (1), (12S)-6α,19-diacetoxy-18-chloro-4α-hydroxy-12tigloyloxy-neo-clerod-13-en-15,16-olide (2a), (12S,2′′S)-6α,19diacetoxy-18-chloro-4α-hydroxy-12-(2-methylbutanoyloxy)neo-clerod-13-en-15,16-olide (2b), ajuganipponin B (3a) [9], ajugamarin F4 (3b) [6] , and ajugamarin A1 (3c) [9] by spectroscopic methods (IR, ESIMS, HR-ESIMS, NMR, and X-ray crystallography). The inhibitory activities on LPS-induced NO production of these diterpenes were evaluated and compounds 2a, 2b, and 3a showed inhibitory effects. This paper herein describes the isolation and structure elucidation of these diterpenes and their inhibitory activities on LPS-induced NO production in murine microglial BV-2 cells.

1410

Z. Sun et al. / Fitoterapia 83 (2012) 1409–1414

13

12

OH

1 H

4

AcO

H

20

10

O

O

O

14

16

2

O

O

15 O

O

R1

OR

OR 2

H

O MeBu: (2S)-2methylbutanoyl

17

9 6

19

18 OH

1

OAc

Cl

OH

O

OAc OAc R

OAc OAc

R1

R2

2a

Tig

3a

H

Tig

2b

MeBu

3b

H

MeBu

3c OTig

O Tig: tigloyl

H

Fig. 1. Structures of 1, 2a, 2b, and 3a−3c from A. decumbens.

2. Experimental 2.1. General The optical rotations were measured in CH2Cl2 using a Rudolph Autopol IV automatic polarimeter. The IR spectra were recorded on a Bio-Rad FTS 6000 Fourier transform infrared (FTIR) spectrometer with KBr discs (DeFelsko Co. Ltd., CA). The ESIMS spectra were obtained on an LCQ-Advantage mass spectrometer (Finnigan Co., Ltd., San Jose, CA). HR-ESIMS spectra were recorded by an IonSpec 7.0T FTICR MS (IonSpec Co., Ltd., Lake Forest, CA). Melting points were determined with an XT-4 microscopic thermometer. 1D and 2D NMR data were recorded on a Bruker AV 400 instrument (400 MHz for 1H and 100 MHz for 13C) with TMS as an internal standard (Bruker BioSpin Co. Ltd., Switzerland). HPLC separations were performed on a CXTH system, equipped with a UV3000 detector at 210 nm (Beijing Chuangxintongheng Instruments Co. Ltd., China), and an YMC-pack J'Sphere ODS-M80 (250× 20 mm) column (YMC Co. Ltd., Japan). Silica gel was used for column chromatography (200–300 mesh, Qingdao Marine Chemical Group Co. Ltd., China). Chemical reagents for isolation were of analytical grade and purchased from Tianjin Yuanli Co. Ltd., China. X-ray crystallographic analysis was carried out on a Rigaku Saturn 944 CCD diffractometer equipped with a multilayer-monochromator and Cu Kα-radiation (λ = 1.54187 Å) (Rigaku Co. Ltd., Japan). The structure was solved by direct methods (SHELXL-97), expanded using Fourier techniques, and refined with full-matrix least-squares on F2 (SHELXL-97). Biological reagents were from Sigma Company. The murine microglial BV-2 cell line was from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (China). 2.2. Plant material The whole plants of A. decumbens were collected from Zhejiang Province, China, in Aug. 2008. The botanical identification was made by Dr. Yuanqiang Guo (College of Pharmacy, Nankai University, China), and a voucher specimen (No.

20080810) was deposited at the laboratory of the Research Department of Natural Medicine, College of Pharmacy, Nankai University, China. 2.3. Extraction and isolation The air-dried and powdered whole plants of A. decumbens (20 kg) were extracted with methanol three times (3× 50 L) under reflux. The organic solvent was evaporated under vacuum to afford a crude extract. The extract was suspended in H2O (1.5 L) and then partitioned successively with petroleum ether and ethyl acetate. The ethyl acetate soluble part (382.0 g) was subjected to silica gel column chromatography, using a gradient solvent system of 1−40% acetone in petroleum ether, to give 10 fractions (F1−F10) based on TLC analyses. F7 was separated by MPLC over C-18 eluting with a step gradient from 55% to 85% MeOH in H2O to give four subfractions (F7-1−F7-4). F7-2 was purified by HPLC (YMC-pack J'Sphere ODS-M80, 20× 250 mm, 68% MeOH in H2O) to afford compounds 3a (49.7 mg) and 3b (49.1 mg). F7-3 was purified to yield compounds 2a (33.3 mg) and 2b (32.0 mg), eluting with 75% MeOH in H2O. F9 and F10 were also separated by the same MPLC system, eluting with a step gradient from 55% to 85% MeOH in H2O, to give the respective subfractions F9-1−F9-4 and F10-1−F10-4, respectively. Fraction F9-1 was purified using the same HPLC system, eluting with 69% MeOH in H2O to yield compound 3c (54.6 mg). Compound 1 (29.4 mg) was obtained from F10-1, eluting with 62% MeOH in H2O with the above HPLC system. (12S)-1α,19-epoxy-6α,18-diacetoxy-4α,12-dihydroxy-neoclerod-13-en-15,16-olide (1). Colorless flakes (MeOH); mp 109−110 °C; [α]20D −4.6 (c 0.13, CH2Cl2); IR (KBr) νmax 3447, 2925, 1739, 1462, 1376, 1254, 1031 cm−1; 1H NMR (400 MHz CD3OD) and 13C NMR (100 MHz CD3OD) data, see Table 1; HR-ESIMS m/z 489.2094 [M+ Na]+ (calcd. for C24H34O9Na, 489.2101). X-ray crystal data of (12S)-1α,19-epoxy-6α,18-diacetoxy4α,12-dihydroxy-neo-clerod-13-en-15,16-olide (1). C24H34O9•2H2O, Mr = 502.54, monoclinic, space group P12(1)1, a = 12.6720(12) Å, b = 6.5440(8) Å, c = 16.0080(16)

Z. Sun et al. / Fitoterapia 83 (2012) 1409–1414

1411

Table 1 1 H and 13C NMR spectral data of compounds 1 (CD3OD), 2a (CDCl3), and 2b (CDCl3) a. 1 δH

Position 1α β 2α β 3α β 4 5 6 7α β 8 9 10 11a b 12 13 14 15 16a b 17 18a b 19a b 20 6-OAc 12-OR

18 or 19-OAc a

2a

4.35 1.87 1.47 2.05 1.75

δC m m m m m

5.61 1.61 1.64 1.83

dd (10.3, 7.2) m m m

2.78 1.77 1.54 4.77

s m m d (10.5)

5.99 s 4.98 br s 0.91 4.40 4.46 4.30 4.02 0.96 1′ 2′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴

d (6.6) d (10.8) d (10.8) d (9.5) d (9.5) s

1.96 s

79.3 31.7 31.0 77.2 54.8 72.1 33.6 34.8 38.3 52.1 45.4 66.0 176.4 114.5 178.0 72.8 15.7 67.6 71.1 18.0 172.5 21.4

2b

δH

δC

1.77 1.87 1.96 1.39 2.03 1.65

m m m m m m

5.03 1.71 1.75 1.72

dd (10.5, 5.0) m m m

1.39 2.17 1.68 5.99

m m m d (8.9)

5.95 s 4.84 4.74 0.87 3.87 3.79 4.94 4.63 0.84

d d d d d d d s

(17.6) (17.6) (5.9) (11.5) (11.5) (12.9) (12.9)

2.01 s

7.00 q (6.7) 1.87 d (6.7) 1.89 s 2.13 s

173.0 20.9

2.11 s

21.3

21.3 31.5 77.1 48.3 73.7 32.7 35.3 39.9 45.0 41.5 65.5 168.7 115.8 172.5 70.4 15.4 49.7 63.4 17.4 170.0 21.3 166.5 127.8 140.8 14.7 11.9 169.9 21.5

δH 1.71 1.80 1.91 1.36 2.02 1.63

δC m m m m m m

4.98 1.64 1.68 1.64

d (10.0, 4.9) m m m

1.38 2.10 1.54 5.66

m m d (16.1) d (7.7)

5.89 s 4.82 4.67 0.77 3.83

d (17.5) d (17.5) d (5.7) s

4.89 d (13.1) 4.58 d (13.1) 0.76 s 1.95 s 2.33 1.39 1.81 0.85 1.10

m m m t (7.4) d (6.6)

2.05 s

21.8 21.4 31.6 76.9 48.2 73.9 32.7 35.7 39.9 45.5 41.9 65.6 168.6 115.8 172.5 70.4 15.5 49.6 63.3 17.0 170.0 21.4 175.6 40.4 26.8 11.3 15.2 170.0 21.5

Proton coupling constants (J) in Hz are given in parentheses. Assignments of 1H NMR data are based on 1H–1H COSY, HMQC, and HMBC experiments.

Å, V =1230.5(2) Å 3, Z = 2, Dcalc = 1.356 g/cm3, crystal dimensions 0.22× 0.16 × 0.14 mm were used for measurements. The total number of reflections measured was 12,206, of which 4060 were unique and 3813 were observed, I > 2σ(I). Final indices: R1 = 0.0558, wR2 =0.1202 for observed reflections, and R1 = 0.0578, wR2 = 0.1222 for all reflections. (12S)-6α,19-diacetoxy-18-chloro-4α-hydroxy-12-tigloyloxyneo-clerod-13-en-15,16-olide (2a). Colorless flakes (MeOH); mp 71−73 °C; [α]20D −18.6 (c 0.14, CH2Cl2); IR (KBr) νmax 3469, 2926, 1738, 1458, 1375, 1247, 1128, 1077 cm−1; 1H NMR (400 MHz CDCl3) and 13C NMR (100 MHz CDCl3) data, see Table 1; HR-ESIMS m/z 567.2369 [M(35Cl)−H]−, 569.2344 [M(37Cl)−H]− (calcd. for C29H4035ClO9, 567.2361). (12S,2′′S)-6α,19-diacetoxy-18-chloro-4α-hydroxy-12-(2methylbutanoyloxy)-neo-clerod-13-en-15,16-olide (2b). White powder; [α] 20D −16.8 (c 0.19, CH2Cl2); IR (KBr) νmax 3496, 2927, 1736, 1461, 1374, 1242, 1142, 1037 cm−1; 1H NMR (400 MHz CDCl3) and 13C NMR (100 MHz CDCl3) data, see Table 1; HR-ESIMS m/z 593.2494 [M(35Cl) + Na] +,

595.2463 [M (37Cl)+ Na]+ (calcd. for C29H4335ClO9Na, 593.2493). 2.4. Bioassay for NO production Murine microglial BV-2 cells were cultured at 37 °C in DMEM supplemented with 10% (v/v) inactivated fetal bovine serum and 100 U/mL penicillin/streptomycin under a watersaturated atmosphere of 95% air and 5% CO2. The cells were seeded in 96-well culture plates (5 × 10 4 cells/well) and allowed to adhere for 24 h at 37 °C. The cells were incubated for 20 h with or without 0.4 μg/mL of LPS (Sigma Chemical Co., St. Louis, MO, U.S.A.) in the absence or presence of the test compounds. SMT was used as a positive control. As a parameter of NO synthesis, the nitrite concentration was measured by the Griess reaction using the supernatant of the BV-2 cells. Briefly, 50 μL of the cell culture supernatant was reacted with 50 μL of Griess reagent [1:1 mixture of 0.1% N-(1-naphtyl)ethylenediamine in H2O and 1% sulfanilamide in 5% phosphoric acid] in a 96 well plate and the absorbance

1412

Z. Sun et al. / Fitoterapia 83 (2012) 1409–1414

was read with a microplate reader (Thermo Fisher Scientific Inc. America) at 550 nm. The experiment was performed three times, and the IC50 values for the inhibition of NO production were determined on the basis of linear or nonlinear regression analysis of the concentration–response data curves. 3. Results and discussion Compound 1 was obtained as colorless flakes (MeOH). Its molecular formula was determined as C24H34O9 by HR-ESIMS (m/z 489.2094 [M+ Na]+, calcd. for C24H34O9Na, 489.2101). The 13C NMR spectrum of 1 exhibited 20 typical resonances (Table 1) for a neo-clerodane diterpene skeleton [2,9,10], which were classified into two methyls [δC 15.7 (C-17), and 18.0 (C-20)], seven methylenes [δC 31.7 (C-2), 31.0 (C-3), 33.6 (C-7), 45.4 (C-11), 72.8 (C-16), 67.6 (C-18), and 71.1 (C-19)], six methines [δC 79.3 (C-1), 72.1 (C-6), 34.8 (C-8), 52.1 (C-10), 66.0 (C-12), and 114.5 (C-14)], and five quaternary carbons [δC 77.2 (C-4), 54.8 (C-5), 38.3 (C-9), 176.4 (C-13), and 178.0 (C-15)]. Corresponding to the above carbon signals, the 1H NMR spectrum of 1 showed characteristic proton resonances of a neo-clerodane diterpene including one tertiary methyl group [δH 0.96 (3H, s, H3-20)], one secondary methyl group [δH 0.91 (3H, d, J =6.6 Hz, H3-17)], three oxygenated methylenes [δH 4.98 (2H, br s, H2-16), 4.40 and 4.46 (each 1H, d, J =10.8 Hz, H2-18), and 4.30 and 4.02 (each 1H, d, J = 9.5 Hz, H2-19)], three oxygenated methines [δH 4.35 (1H, m, H-1), 5.61 (1H, dd, J = 10.3, 7.2 Hz, H-6), and 4.77 (1H, d, J = 10.5 Hz, H-12)], and one olefinic proton at δH 5.99 (1H, s, H-14) (Table 1). In addition, the 1H and 13C NMR spectra revealed the presence of two acetoxy groups [δH 1.96 (3H, s, H3-2′), and δC 172.5 (C-1′) and 21.4 (C-2′); δH 2.13 (3 H, s, H3-2‴), and δC 173.0 (C-1‴) and 20.9 (C-2‴)] in compound 1. Further analyses of the HMQC, HMBC and 1H–1H COSY spectra (Fig. 2) confirmed the presence of the neo-clerodane skeleton and the two acetoxy groups. The positions of the two acetoxy groups were determined to be at C-6 and C-18 by the HMBC correlations (Fig. 2) of H-6 (δH 5.61) with the carbon signal at δC 172.5 and H2-18 (δH 4.40 and 4.46) with the carbon signal at δC 173.0. The long-range coupling of H-1 (δH 4.35) with C-19 (δC 71.1) in the HMBC spectrum indicated the presence of an oxygen bridge at C-1 and C-19,

Fig. 2. Key HMBC and 1H–1H COSY correlations of 1.

which is also supported by the HR-ESIMS. Thus, the planar structure with a neo-clerodane skeleton for 1 was established. The relative configuration of 1 was elucidated as follows. NOESY correlations (Fig. 3) observed for H-2β/H-10, H-2β/ H2-18, H-3α/H2-19, H-10/H2-18, H-6/H-8, H-10/H-8, H-6/ H-10, H-7α/H3-20 and H2-19/H3-20, suggested that the two six-membered rings were trans-fused and both existed in chair conformation, H-10 and H2-18 were β-axially oriented, H2-19 and H3-20 were α-axially oriented, and H3-17 α-equatorially oriented, respectively. Ultimately, in order to confirm the above assignments and the configuration, a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation was carried out [13]. A drawing of thermal ellipsoid representation is shown in Fig. 4, which suggested an S configuration for C-12. All of the above evidence confirmed the structure of 1 as (12S)-1α,19-epoxy-6α,18-diacetoxy4α,12-dihydroxy-neo-clerod-13- en-15,16-olide. Compound 2a was obtained as colorless flakes (MeOH), and the molecular formula was determined to be C29H41ClO9 by HR-ESIMS (m/z 567.2369 [M (35Cl) − H]−, 569.2344 [M (37Cl) − H]−, calcd. for C29H4035ClO9, 567.2361). Analyses of the 13C and 1H NMR spectra of compound 2a (Table 1), revealed that 2a had a characteristic neo-clerodane skeleton closely resembling those of ajugaciliatins isolated from A. ciliata [9]. Apart from the 20 carbon resonances for the clerodane diterpene skeleton, the 13C NMR spectrum of 2a also exhibited nine carbon resonances for the substituents. Based on the 1H and 13C NMR spectra and similar clerodane diterpenes [9], the substituents were deduced to be a tigloyloxy group and two acetoxy groups. The following HMQC and HMBC experiments confirmed the above conclusions. From the HMBC spectrum, the positions of the acyloxy groups were elucidated. The tigloyloxy group located at C-12 and the two acetoxy groups at C-6 and C-19 were determined by the HMBC correlations of H-12/C-1″, H-6/C-1′, and H2-19/C-1‴, respectively. The NOESY spectrum allowed the stereochemical features of 2a to be assigned (Fig. 3). NOESY correlations observed for H-2β/H-3α, H-3β/H2-18, H-10/H2-18, H-6/H-8, H-10/H-8, H-6/ H-10, H-7α/H3-20, and H2-19/H3-20, suggested that the two trans-fused six-membered rings existed in a twist-boat conformation and a chair conformation, and the C-4 hydroxy and the C-6 acetoxy groups were in α-positions. For the absolute configuration of C-12, considering the biosynthetic grounds, was determined to be S based on the absolute configuration of C-12 of compound 1 and the similar neoclerodane diterpenes isolated from the genus Ajuga, which all possess the same S configuration for C-12 [9]. Thus, compound 2a was determined as (12S)-6α,19-diacetoxy-18-chloro-4αhydroxy-12-tigloyloxy-neo-clerod-13-en-15,16-olide. Compound 2b possessed a molecular formula C29H43ClO9 on the basis of HR-ESIMS (m/z 593.2494 [M( 35Cl) + Na] +, 595.2463 [M(37Cl) + Na] +, calcd. for C29H4335ClO9Na, 593.2493). The 1H and 13C NMR spectra of 2b were very similar to those of compound 2a, which implied that compound 2b should also be a neo-clerodane diterpene. Close similarities of the chemical shifts from C-1 to C-20 in 2b with those in 2a (Table 1) suggested that compounds 2a and 2b had the same parent skeleton [9]. In addition to the 20 skeletal carbons, the 13C NMR spectrum revealed additional nine carbons for the substituents, which were defined as two acetoxy groups and one 2-methylbutanoyloxy group based on

Z. Sun et al. / Fitoterapia 83 (2012) 1409–1414

AcO H

H

HO H

8

H

CH3

10

1 4

H H Cl H 18 1 3

H

6

5

CH3

O

AcO 19

H

HH

R

10 H

8

4

19

7

6

H

HO H

CH3

9 5

2

H

H

H

H

H

R

H 18

H

H

H

HH

1413

AcO H

H

H

20CH 3

H

OAc 2a

1 Fig. 3. Key NOESY correlations of 1 and 2a.

the similar diterpenes [9]. The locations of three acyloxy groups were elucidated by the interpretation of the HMBC spectra as in the case of 2a. By the HMBC correlations of the protons H-6 (δH 4.98), H-12 (δH 5.66), and H2-19 (δH 4.89 and 4.58) with the corresponding carbonyl signals at δC 170.0, 175.6, and 170.0, the two acetoxy groups and one 2-methylbutanoyloxy group were attributed to C-6, C-19, and C-12, respectively. The same relative configuration to be inferred for compounds 2a and 2b was revealed by the careful comparison of the NOESY spectra of 2a and 2b. Based on the biosynthetic grounds, the absolute configuration of C-2″ (2-methylbutanoyloxy at C-12) was regarded as S as that in the known compound ajugamarin F4 (3b) isolated from the same plant, where the absolute configuration of 2-methylbutanoyloxy was elucidated as S in the literature [6]. The structure of compound 2b was elucidated therefore as (12S,2″S)-6α,19-diacetoxy-18-chloro-4α-hydroxy12-(2-methylbutanoyloxy)-neo-clerod-13-en-15,16-olide.

In order to explore the undiscovered and potential pharmacological activities of these diterpenes isolated from A. decumbens, all the isolates were evaluated for their inhibitory activities on LPS-induced NO production in murine microglial BV-2 cells by the Griess reaction as described [14]. 2-Methyl-2-thiopseudourea, sulfate (SMT) was used as a positive control (IC50 4.70 μM). Compounds 2a and 2b inhibited LPS-induced NO production in BV-2 cells dosedependently with IC50 values of 64.6 and 25.3 μM, respectively (Fig. 5). Compound 3a showed weak inhibitory effects only at the concentration 100 μM. Compounds 1, 3b, and 3c were inactive. MTT assay indicated that all the compounds had no significant cytotoxicity to the BV-2 cells at their effective concentration for the inhibition of NO production (data not shown). In conclusion, six neo-clerodane diterpenes, including three new (1, 2a, and 2b), and three known ones (3a−3c) have been isolated from A. decumbens. Their structures were elucidated

NO inhibition (%)

120

80

40

0 Conc. Comp.

Fig. 4. Thermal ellipsoid representation of 1.

10

30 2a

100

10

30 2b

100

100

30

µM

3a

Fig. 5. Inhibitory effects of compounds 2a, 2b, and 3a on LPS-induced NO production. BV-2 cells were treated with LPS alone or together with each compound at the concentrations indicated. After 20 h incubation, the supernatants were tested by Griess assay and the NO inhibitory rates were calculated. The experiment was performed three times, and the data are expressed as mean ± SD values. The inhibitory rate on NO production was calculated as follows: Inhibitory rate (%) = (1 − (LPS/sample − untreated) / (LPS − untreated)) × 100. ♦ Indicated positive control, SMT.

1414

Z. Sun et al. / Fitoterapia 83 (2012) 1409–1414

based on the analyses of IR, MS, and NMR spectra, and X-ray crystallography data, and the inhibitory effects on NO production were also evaluated. The presence of the bioactive neoclerodane diterpenes inhibiting NO production in A. decumbens may be responsible for the anti-inflammatory effects of the plant A. decumbens as a folk medicine described in the book “Dictionary of Chinese Materia Medica” [1]. Acknowledgments The project was supported by the Natural Science Foundation of China (No. 30970301). References [1] Nanjing University of Chinese Medicine. Dictionary of Chinese Materia Medica. Second ed. Shanghai: Shanghai Science and Technology Publishing House; 2006. p. 1035-7. [2] Huang XC, Qin S, Guo YW, Krohn K. Four new neo-clerodane diterpenoids from Ajuga decumbens. Helv Chim Acta 2008;91:628-34. [3] Amano T, Nishida R, Kuwahara Y. Ajugatakasins A and B, new diterpenoids from Ajuga decumbens, and feeding stimulative activity of related neoclerodane analogs toward the turnip sawfly. Biosci Biotechnol Biochem 1997;61:1518-22.

[4] Min Z, Mizuno M, Wang S, Iinuma M, Tanaka T. Two new neo-clerodane diterpenes in Ajuga decumbens. Chem Pharm Bull 1990;38:3167-8. [5] Min Z, Wang S, Zheng Q, Wu B, Mizuno M, Tanaka T, et al. Four new insect antifeedant neo-clerodane diterpenoids, ajugacumbins A, B, C and D, from Ajuga decumbens. Chem Pharm Bull 1989;37:2505-8. [6] Shimomura H, Sashida Y, Ogawa K. neo-Clerodane diterpenes from Ajuga decumbens. Chem Pharm Bull 1989;37:996-8. [7] Chen HM, Min ZD, Iinuma M, Tanaka T. Clerodane diterpenoids from Ajuga decumbens. Chem Pharm Bull 1995;43:2253-5. [8] Takeda Y, Tsuchida S, Fujita T. Four new iridoid glucoside p-coumaroyl esters from Ajuga decumbens. Phytochemistry 1987;26:2303-6. [9] Guo P, Li Y, Xu J, Liu C, Ma Y, Guo Y. Bioactive neo-Clerodane diterpenoids from the whole plants of Ajuga ciliata Bunge. J Nat Prod 2011;74:1575-83. [10] Guo P, Li Y, Xu J, Guo Y, Jin DQ, Gao J, et al. neo-Clerodane diterpenes from Ajuga ciliata Bunge and their neuroprotective activities. Fitoterapia 2011;82:1123-7. [11] Xu J, Jin D, Shi D, Ma Y, Yang B, Zhao P, et al. Sesquiterpenes from Vladimiria souliei and their inhibitory effects on NO production. Fitoterapia 2011;82:508-11. [12] Xu J, Jin DQ, Zhao P, Song X, Sun Z, Guo Y, et al. Sesquiterpenes inhibiting NO production from Celastrus orbiculatus. Fitoterapia 2012;83:1302-5. [13] Crystallographic data for compound 1 have been deposited in the Cambridge Crystallographic Data Centre (CCDC 880388). Copies of the data can be obtained, free of charge, on application to the director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: [email protected]). [14] Schmidt HHHW, Kelm M. In: Feelish M, Stamler J, editors. Methods in nitric oxide research. New York: John Wiley & Sons Ltd.; 1996. p. 491-7.