Three New Abietane-Type Diterpenoids from Callicarpa macrophylla Vahl.

Three new abietane-type diterpenoids, named callicapoic acid M3 (1), callicapoic acid M4 (2) and callicapoic acid M5 (3), were isolated from the Callicarpa macrophylla Vahl. Their structures were established by spectroscopic techniques (IR, UV, MS, 1D and 2D NMR). All the isolated three compounds were evaluated for inhibitory activity on NO production in LPS-activated RAW 264.7 macrophage cells by using MTT assays. Compounds 1, 2 and 3 showed potent inhibitory activity, with inhibition rates of 34.47–40.13%.


Introduction
The genus Callicarpa belongs to the family Verbenaceae, with about 190 species widely distributed throughout the tropical and subtropical regions of Asia and Oceanica and parts of America [1,2]. Many Callicarpa species are used in Chinese folk medicine for various indications [2]. Compounds that represent a variety of different classes have been reported, including diterpenoids, triterpenoids, flavonoids, phenolic acids, volatile oils and so on [2][3][4][5]. Currently, Callicarpa macrophylla Vahl. is used as a folk medicine in China's Yunnan Province, were the root, the stem, and the leaves are all used in medicine. Callicarpa macrophylla Vahl. has a bitter taste, slightly acrid and flat, with clinical actions that eliminate stasis to activate blood circulation and stop bleeding, and it also has detumescence and analgesic actions [6].

Introduction
The genus Callicarpa belongs to the family Verbenaceae, with about 190 species widely distributed throughout the tropical and subtropical regions of Asia and Oceanica and parts of America [1,2]. Many Callicarpa species are used in Chinese folk medicine for various indications [2]. Compounds that represent a variety of different classes have been reported, including diterpenoids, triterpenoids, flavonoids, phenolic acids, volatile oils and so on [2][3][4][5]. Currently, Callicarpa macrophylla Vahl. is used as a folk medicine in China's Yunnan Province, were the root, the stem, and the leaves are all used in medicine. Callicarpa macrophylla Vahl. has a bitter taste, slightly acrid and flat, with clinical actions that eliminate stasis to activate blood circulation and stop bleeding, and it also has detumescence and analgesic actions [6]. Past phytochemical studies on Callicarpa macrophylla Vahl. Have revealed the presence of pentacyclic triterpenes, sterols [6], and diterpenes [7,8]. Additionally, some of the literature has reported that diterpenoid compounds in Callicarpa showed potent anti-inflammatory activities [9,10]. Being interested in finding more biologically active substances from this folk medicine, we further Past phytochemical studies on Callicarpa macrophylla Vahl. Have revealed the presence of pentacyclic triterpenes, sterols [6], and diterpenes [7,8]. Additionally, some of the literature has reported that diterpenoid compounds in Callicarpa showed potent anti-inflammatory activities [9,10]. Being interested in finding more biologically active substances from this folk medicine, we further undertook an investigation to explore its phytochemical composition. As a result, three new abietane-type diterpenoids 1-3, named callicapoic acids M 3-5 ( Figure 1) were isolated from the dried whole herb of Callicarpa macrophylla Vahl. This paper deals with their structural elucidation and anti-inflammatory activity against RAW 264.7 macrophage cells line determined by means of MTT assays.

Results and Discussion
Compound 1 (callicapoic acid M3), obtained as white amorphous powder (from acetone), gave the molecular formula C 20 H 26 O 3 , as deduced from the HR-ESI-MS peak at m/z 315.1958 [M + H] + . Its IR spectrum showed hydroxyl (3401 cm −1 ) and carboxyl group (1700 cm −1 ) absorptions, and the UV spectrum showed the presence of an aromatic moiety with maxima at 211 and 250 nm. The 1 H-NMR spectrum of 1 (  [11][12][13]. The 13 C-NMR spectrum of 1 (Table 1) confirmed the presence of a benzene ring, in addition to two methyl, six methylene, one methine, one carboxyl, two olefinic carbon signals, and two quaternary carbon signals, as well as an additional methylene carbon (δ C 71.4) attached to an oxygen function. From this information, compound 1 was inferred to be an abietane-type diterpene by comparison with the literature identification data of similar typical abietanes, like the triptobenzenes A-K isolated from T. wilfordiivar. Regelii [14] and T. hypoglaucum [15].
Callicapoic acid M4 (2) was isolated as a white amorphous powder (from acetone). Its molecular formula was established as C 20 H 28 O 3 by the HR-ESI-MS signal at m/z 339.1927 [M + Na] + . The 1D (Table 1) and 2D NMR data of 2 showed the presence of an abietane-type diterpene skeleton, which indicated its structure be similar to that of 1, except for a side chain isopropyl group, suggested by the appearance of characteristic resonances at δ H 1.21 (3H, s), 1.22 (3H, s), and 2.82 (1H, m); and δ C 24.0, 24.0 and 33.4. In the HMBC spectrum (Figure 2), the methine proton signal (δ H 2.82) of the isopropyl group correlated with C-12 (δ 124.1), C-13 (δ 145.8) and C-14 (δ 126.8). In turn, the proton signals at δ H 6.99 (H-12) and 6.87 (H-14) correlated with the methine signal at δ C 33.4 (C-15). From these observations, the location of the side chain of isopropyl group at C-13 was inferred. The relative configuration of 2 was established by a NOESY experiment, and 4-CH 2 OH, 10-CH 3 were found to be the same as those of 1. Accordingly, compound 2 was identified as 18-hydroxy-8,11,13-abietatetraen-19-oic acid ( Figure 1) and named callicapoic acid M4 (2). Compound 2 is the C-4 epimer of a known compound described in the literature [16].    (Table 1) and 2D NMR data of 3 clearly revealed the presence of an abietane-type diterpene skeleton, and also indicated its structure be similar to that of 1 except for the two side chains. The differences between them were that the isopropenyl was replaced by an acetyl at C-13, and the hydroxyl at C-18 was acetylated. The first difference had characteristic resonances at δH 2.56 (3H, s); and δC 26.7, 198.3 which implied the presence of an acetyl, while HMBC correlations from protons of methyl at δH 2.56 to C-13 (δ 134.8) suggested that acetyl was connected to C-13. The second difference in the characteristic resonances at δH 2.06 (3H, s); and δC 20.9, 171.0 also implied the presence of an acetyl, HMBC correlations from  (Table 1) and 2D NMR data of 3 clearly revealed the presence of an abietane-type diterpene skeleton, and also indicated its structure be similar to that of 1 except for the two side chains. The differences between them were that the isopropenyl was replaced by an acetyl at C-13, and the hydroxyl at C-18 was acetylated. The first difference had characteristic resonances at δ H 2.56 (3H, s); and δ C 26.7, 198.3 which implied the presence of an acetyl, while HMBC correlations from protons of methyl at δ H 2.56 to C-13 (δ 134.8) suggested that acetyl was connected to C-13. The second difference in the characteristic resonances at δ H 2.06 (3H, s); and δ C 20.9, 171.0 also implied the presence of an acetyl, HMBC correlations from the methylene protons at δ H 4.10 and 4.49 (H 2 -18) to C-21 (δ 171.0) suggested that the C-18 hydroxyl was acetylated. The relative configuration of 10-CH 3 was established as β-oriented, 4-CH 2 O-was confirmed to be α-oriented by a NOESY experiment, the same as those of 1 and 2. Therefore, compound 3 was thus identified as 15-acetyl-19-carbethoxy-8,11,13-abietatetraen-18-oic acid (Figure 1).
Nitric oxide (NO) plays an important role in the inflammatory process [17].The inhibition of NO release may be effective as a therapeutic agent in the inflammatory diseases [18]. Therefore, compounds 1 to 3 were tested for the inhibitory activity against the production of NO in RAW 264.7 stimulated by lipopolysaccharide (LPS). The anti-inflammatory assay was carried out according to the procedure described previously. The results were summarized in Table 2 and indicated that all the three compounds could significantly inhibit NO introduction in LPS-activated RAW 264.7 macrophage cells. a The three compounds were tested in the same value as 50 µM. b p < 0.01, significantly different from LPS model group. Data were presented as mean ± SD of three independent experiments.

General Procedures
Optical rotations were determined on a 241 MC polarimeter (Perkin-Elmer, Waltham, MA, USA). UV spectra were obtained on Perkin Elmer Lambda 35 UV/VIS Spectrometer. IR spectra were recorded on a IFS 55 spectrophotometer (Bruker, Billerica, MA, USA). The NMR data were recorded on a Bruker AV-600 spectrometer. The HR-ESI-MS data were obtained on a LCT Premier XE time-of-flying mass spectrometer (Waters, Milford, MA, USA). Chromatography was performed on silica gel (200-300 mesh; Qingdao Marine Chemical Group Co. Ltd., Qingdao, China) and ODS (30-50 µm; Tianjin Mical Reagent Co., Tianjin, China). Prep. HPLC was performed on a system comprised of a L-7110 pump and a L-7420 UV spectrophotometric detector set at 203 nm (Hitachi, Tokyo, Japan). A YMC C 18 reversed-phase column (5 µm, 10 × 250 mm; flow rate 2.0 mL/min) was used.

Exaction and Isolation
Dried whole herb of Callicarpa macrophylla Vahl. (4.5 kg) was extracted with 50 L 95% EtOH (×3) under reflux conditions for three hours to give a crude extract, which was suspended in H 2 O and successively extracted with petroleum ether (PE), CHCl 3 and EtOAc to yield a PE-soluble fraction (17.2 g), a CHCl 3 -soluble fraction (261.7 g) and an EtOAc-soluble fraction (55.3 g). A part of the CHCl 3 -soluble fraction (70 g) was subjected to column chromatography (CC, silica gel, gradient of PE-acetone 100:1-0:100) to afford 46 fractions (numbered

Anti-Inflammatory Assay
The anti-inflammatory activities of compounds 1 to 3 were evaluated using LPS-induced RAW 264.7 cells. RAW 264.7 macrophages cells (8 × 10 4 cells/well) were suspended in 100 µL of DMEM supplemented with 10% fetal bovine serum, and precultured in 96-well microplates and 5% CO 2 in air for 12 h at 37 • C, then test compounds (50 µmol/L) were cultured, and were treated with or without 1 µg/mL LPS for 24 h. NO production in each well was assessed by measuring the accumulation of nitrite in the culture medium using Griess reagent. Cytotoxicity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay. Briefly, after 24 h incubation with test compounds, MTT (20 µL, 5 mg/mL in PBS) solution was added to the wells. After 4 h of culturing, the medium was removed and DMSO 100 µL/well was then added to dissolve the formazan produced in the cells. The optical density of the formazan solution was measured with a microplate reader at 490 nm. Z,Z'-6,6',7,3'α-diligustilide was used as positive control. Each test compound was dissolved in dimethyl sulfoxide (DMSO), and the solution was added to the medium (final DMSO concentration was 0.1%). MTT experiments were repeated three times. The NO inhibitory ratio (%) was calculated by the following formula: NO inhibitory ratio (%) = (A 570, LPS − A 570, sample )/A 570, LPS × 100