Fitoterapia 105 (2015) 61–65
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Four new phenolic acid with unusual bicycle [2.2.2] octane moiety from Clerodendranthus spicatus and their anti-inflammatory activity Guo-Xu Ma a,1, Xiao-Po Zhang b,1, Peng-Fei Li a, Zhong-Hao Sun a, Nai-Liang Zhu a, Yin-Di Zhu a, Jun-Shan Yang a, De-Li Chen c,⁎, Hai-Feng Wu a,⁎, Xu-Dong Xu a,⁎ a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100193, China b School of Pharmaceutical Science, Hainan Medical University, Haikou 571101, China c Hainan Branch Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Wanning 571533, China
a r t i c l e
i n f o
Article history: Received 25 March 2015 Received in revised form 7 June 2015 Accepted 8 June 2015 Available online 12 June 2015 Keywords: Clerodendranthus spicatus Phenolic acids Anti-inflammatory
a b s t r a c t Four new phenolic acids, clerodens A–D (1–4) possessing an unusual bicycle [2.2.2] octane moiety were isolated from the whole plants of Clerodendranthus spicatus. Their structures were elucidated by extensive spectroscopic methods, including NMR, MS, and ECD data. All isolates were evaluated for their anti-inflammatory activities on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7, and compound 4 showed significant inhibitory activities with IC50 value of 6.8 μM. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Clerodendranthus spicatus (Thunb.) C.Y. Wu (syn. Orthosiphon stamineus Benth.) known as “Kumis kucing” in Southeast Asia, is a popular herbal medicine extensively used for the treatment of nephritis and cystitis [1,2]. The leaves and stems of this plant cultivated in Southern China are used by the name of “Shen Cha” as a diuretic tea in Chinese folk medicine for the treatment of acute and chronic nephritis, urinary lithiasis, cystitis and rheumatism [3–5]. Previous chemical and pharmacological studies on the ethanol extract of this species resulted in the separation of triterpenes [6], diterpenes [7,8], flavonoids [9] and phenolic acids [10,11] with broad bioactivities including anti-inflammatory [7, 12], diuresis [13], immunoregulation [14], and antidiabetic [15,16]. However, the composition of the water-soluble of C. spicatus has been little investigated, and as a part of our ongoing program toward the discovery of novel bioactive constituents from this plant, four new phenolic acids, clerodens A–D with an unusual bicycle [2.2.2] octane moiety (1–4) (Fig. 1), were isolated from the aqueous extract of C. spicatus. In this paper, we report the structure elucidation of the new phenolic
⁎ Corresponding authors. E-mail addresses: [email protected] (D.-L. Chen), [email protected] (H.-F. Wu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.fitote.2015.06.010 0367-326X/© 2015 Elsevier B.V. All rights reserved.
acid derivatives and their inhibitory activities against LPS-induced NO production in macrophages.
2. Experimental 2.1. General experimental procedures Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter. UV and IR spectra were recorded on Shimadzu UV2550 and FTIR-8400S spectrometers, respectively. ECD spectra were obtained using a JASCO J-815 spectropolarimeter. NMR spectra were obtained with a Bruker AV III 600 NMR spectrometer (chemical shift values are presented as δ values with TMS as the internal standard). HRESIMS spectra were performed on a LTQ-Obitrap XL spectrometer. Preparative HPLC was performed on a Lumtech K-1001 analytic LC equipped with two pumps of K-501, a UV detector of K-2600, and an YMC Pack C18 column (250 mm × 10 mm, i.d., 5 μM, YMC Co. Ltd., Japan) eluted with CH3OH– H2O at a flow rate of 2 mL/min. C18 reversed-phase silica gel (40–63 μM, Merk, Darmstadt, Germany), Toyopearl HW-40C (50–100 μM, Tosoh Corp., Tokyo, Japan), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), MCI gel (CHP 20P, 75–150 μM, Mitsubishi Chemical Corporation, Tokyo, Japan) and silica gel (100–200 and 300–400 mesh, Qingdao Marine Chemical plant, Qingdao, People's Republic of China) were used for column chromatography. And pre-coated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, People's Republic
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G.-X. Ma et al. / Fitoterapia 105 (2015) 61–65
Fig. 1. Structures of compounds 1–4.
of China) were used for TLC. All solvents used were of analytical grade (Beijing Chemical Works). 2.2. Plant material The whole plants of C. spicatus were collected in Jinghong, Yunnan Province, People's Republic of China, in September 2012, and were authenticated by Prof. Jing-Quan Yuan. A voucher specimen (CS-21209) has been deposited at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences. 2.3. Extraction and isolation The ground powder of air-dried whole plants of C. spicatus (15 kg) was decocted with boiled water (50 L × 2 h × 3) and the solution was then precipitated in 70% ethanol and filtered to yield the supernatant. The supernatant was concentrated under reduced pressure to yield an extract. The extract was subjected to D101 macro porous resin column chromatography using ethanol–water gradient elution, and the eluted fraction with 30–95% ethanol–water elution combined after concentration yielded the total phenolic acids of C. spicatus (TPC, 260 g). TPC fraction was further separated by MCI-gel column chromatography with methanol–water gradient elution, giving seven fractions (A–G). Fraction D (11.4 g) was fractionated on HW-40C column chromatography elution with MeOH–H2O (20:80; 30:70; 40:60; 60:40; 100:0) giving five subfractions (FrD1–D5). Fraction D2 (673 mg) was chromatographed by semi-preparative HPLC using MeOH–H2O (26: 74, v/v) to yield compound 2 (4.3 mg, tR = 25.3 min) and 3 (3.8 mg, tR = 26.6 min). Fraction D3 (124 mg) was purified through semi-preparative HPLC elution using a MeOH–H2O (35: 65, v/v) system to give compound 4 (2.1 mg, t R = 22.4 min). Subfraction D4 (245 mg) was chromatographed by successive preparative and semipreparative HPLC to yield 1 (6.8 mg, tR = 28.1 min). 2.3.1. Cleroden A (1) C29H28O12, white amorphous powder; [α]20 D + 237.6 (c = 0.17, MeOH); IR (KBr) νmax: 3353, 1713, 1606, 1527 cm−1; UV (MeOH) λmax (log ε): 275 (3.28) nm; for 1H NMR (600 MHz, CD3OD) and 13C-APT
(150 MHz, CD3OD) spectroscopic data see Tables 1 and 2; HRESIMS m/z: 567.1478 [M–H]− (calcd for C29H27O12, 567.1508).
2.3.2. Cleroden B (2) C37H34O15, white amorphous powder; [α]20 D + 145.7 (c = 0.12, MeOH); IR (KBr) νmax: 3425, 1634, 1610, 1575 cm− 1; UV (MeOH) λmax (log ε): 277 (3.45) nm; for 1H NMR (600 MHz, CD3OD) and 13CAPT (150 MHz, CD3OD) spectroscopic data see Tables 1 and 2; HRESIMS m/z: 717.1799 [M–H]− (calcd for C37H33O15, 717.1825).
2.3.3. Cleroden C (3) C37H34O15, white amorphous powder; [α]20 D + 174.1 (c = 0.14, MeOH); IR (KBr) νmax: 3355, 1723, 1589, 1515 cm− 1; UV (MeOH) λmax (log ε): 275 (3.25) nm; for 1H NMR (600 MHz, CD3OD) and 13CAPT (150 MHz, CD3OD) spectroscopic data see Tables 1 and 2; HRESIMS m/z: 717.1795 [M–H]− (calcd for C37H33O15, 717.1825).
2.3.4. Cleroden D (4) C36H32O15, white amorphous powder; [α]20 D + 145.6 (c = 0.09, MeOH); IR (KBr) νmax: 3407, 1634, 1606, 1515 cm− 1; UV (MeOH) λmax (log ε): 275 (3.02) nm; for 1H NMR (600 MHz, CD3OD) and 13CAPT (150 MHz, CD3OD) spectroscopic data see Tables 1 and 2; HRESIMS m/z: 703.1638 [M–H]− (calcd for C36H31O15, 703.1668).
2.4. Assay for inhibitory ability against LPS-induced NO production in RAW 264.7 macrophages RAW 264.7 macrophages were seeded in 24-well plates (105 cells/well). The cells were co-incubated with drugs and LPS (1 μg/mL) for 24 h. The amount of NO was assessed by determining the nitrite concentration in the cultured RAW 264.7 macrophage supernatants with Griess reagent. Aliquots of supernatants (100 μL) were incubated, in-sequence, with 50 μL of 1% sulfanilamide and 50 μL of 0.1% naphthylethylenediamine in 2.5% phosphoric acid solution. The absorbance was recorded on a microplate reader at a wavelength of 570 nm.
G.-X. Ma et al. / Fitoterapia 105 (2015) 61–65 Table 1 1 H NMR spectroscopic data (600 MHz, in CD3OD) for compounds 1–4 (δH in ppm, J in Hz). No
1
2 3 5 6 7 8 9 10 2′ 3′ 5′ 6′ 7′
3.78, m 3.71, d (3.6) 3.16, dd (6.0, 2.4) 6.72, d (6.0) 3.46, dd (7.2, 2.4) 3.51, dd (7.2, 1.8) 7.34, d (15.6) 6.14, d (15.6) 6.74, d (1.8)
8′ 2″ 5″ 6″
2
3.74, m 3.65, d (3.6) 3.10, d (5.4) 6.63, d (5.4) 3.26, (6.6) 3.60, d (6.6) 7.23, d (15.6) 6.18, d (15.6) 7.07, d (8.4) 6.73, d (8.4) 6.67, d (8.4) 6.73, d (8.4) 6.62, dd (7.8, 1.8) 7.07, d (8.4) 2.97, dd 3.06, dd (14.4, 9.0) (14.4, 7.8) 3.12, dd 3.12, dd (14.4, 3.6) (14.4, 4.8) 5.19, dd 5.20, dd (9.0, 3.6) (7.8, 4.8) 6.72, d (1.8) 6.76, d (1.8) 6.68, d (7.8) 6.68, d (7.8) 6.59, dd (7.8, 1.8) 6.58, dd (7.8, 1.8)
2‴ 5‴ 6‴ 7‴
8‴ 9′-OCH3 9‴-OCH3 12-OCH2CH3 4.03, m 12-OCH2CH3 1.12, t (7.2)
6.60, d (1.2) 6.59, d (7.8) 6.34, dd (7.8, 1.2) 2.83, dd (14.4, 9.6) 3.00, dd (12.6, 4.2) 5.02, dd (9.6, 4.2) 3.69, s
63
Table 2 13 C NMR spectroscopic data (150 MHz, in CD3OD) for compounds 1–4.
3
4
No
1
2
3
4
3.76, m 3.66, (3.0) 3.12, d (6.0) 6.61, d (6.0) 3.34, d (7.2) 3.55, d (6.0) 7.24, d (15.6) 6.19, d (15.6) 7.10, d (7.8) 6.69, d (7.8) 6.69, d (7.8) 7.10, d (7.8) 3.03, dd (14.4, 4.8) 3.05, dd (14.4, 9.0) 5.19, dd (9.0, 4.8) 6.76, d (1.8) 6.68, d (7.8) 6.56, dd (7.8, 1.8) 6.76, d (1.2) 6.57, d (7.8) 6.34, dd (7.8, 1.2) 2.81, dd (14.4, 9.6) 3.02, dd (12.6, 4.2) 5.03, dd (9.6, 4.2)
3.75, m 3.76, d (3.6) 3.09, d (4.8) 6.52, d (6.0) 3.30, d (6.0) 3.56, d (6.0) 7.13, d (15.6) 6.14, d (15.6) 7.12, d (7.8) 6.73, d (7.8) 6.73, d (7.8) 7.12, d (7.8) 3.06, dd (14.4, 4.8) 3.15, dd (14.4, 9.0) 5.10, dd (9.0, 4.8) 6.77, d (1.8) 6.59, d (7.8) 6.34, dd (7.8, 1.8) 6.60, d (1.2) 6.67, d (7.8) 6.60, dd (7.8, 1.2) 2.82, dd (14.4, 9.6) 3.03, dd (12.6, 4.2) 5.06, dd (9.6, 4.2)
1 2 3 4 5 6 7 8 9 10 11 12 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ 9′-OCH3 9‴-OCH3 12-OCH2CH3 12-OCH2CH3
141.9, C 44.6, CH 70.7, CH, 209.5, C 58.1, CH 137.7, CH 49.9, CH 43.9, CH, 143.4, CH 119.0, CH 168.0, C 175.5, C 129.6, C 117.6, CH 146.3, C 145.3, C 116.5, CH 122.0, CH 38.1, CH2 75.4, CH 173.0, C 133.8, C 116.9, CH 145.5, C 145.3, C 116.5, CH 121.8, CH
142.5, C 44.5, CH 70.9, CH 209.5, C 58.1, CH 137.2, CH 50.4, CH 43.9, CH 143.7, CH 119.0, CH 168.1, C 175.2, C 128.2, C 131.7, CH 116.5, CH 157.7, C 116.5, CH 131.7, CH 38.1, CH2 75.1, CH 172.2, C 134.0, C 116.9, CH 146.3, C 145.6, C 116.6, CH 121.2, CH 129.9, C 117.7, CH 146.2, C 145.2, C 116.6, CH 122.0, CH 37.8, CH2 76.1a, CH 171.4b, C 52.9, CH3
142.2, C 44.6, CH 70.9, CH 209.5, C 57.9, CH 136.8, CH 50.2, CH 43.7, CH 142.7, CH 120.0, CH 168.5, C 174.9, C 128.7, C 131.7, CH 116.2, CH 157.3, C 116.2, CH 131.7, CH 38.4, CH2 77.0a, CH 172.6b, C 133.8, C 116.8, CH 146.3, C 145.6, C 116.6, CH 121.1, CH 129.9, C 117.7, CH 146.2, C 145.5, C 116.6, CH 122.0, CH 37.8, CH2 75.9, CH 171.6, C
142.7, C 44.8, CH 69.4, CH 209.8, C 57.9, CH 136.1, CH 50.0, CH 44.0, CH 142.4, CH 120.3, CH 168.4b, C 175.1, C 130.6, C 131.7, CH 116.4, CH 157.3, C 116.4, CH 131.7, CH 38.7, CH2 77.0a, CH 172.5b, C 134.2, C 116.9, CH 146.1, C 145.0, C 116.6, CH 122.0, CH 130.2, C 117.7, CH 146.2, C 145.4, C 116.6, CH 121.2, CH 38.6, CH2 77.0a, CH 171.5b, C
3.54, s
3. Results and discussion
a b
Compound 1 was obtained as a yellowish amorphous powder with [α]25 D + 237.6 (c 0.17, MeOH) and gave a greenish blue coloration characteristic of catechol with 2% ethanolic FeCl3 on TLC [17]. The UV and IR spectra showed absorption of phenyl (275 nm; 1606, 1527 cm−1), OH (3353 cm−1), and carbonyl groups (1713 cm−1). The HRESIMS spectrum showed a pseudomolecular ion at mnz 567.1478 [M–H]− in the negative ion mode (Calcd for: 567.1508, C29H27O12), from which in conjunction with NMR data the molecular formula was established as C29H27O12, compatible with eleven degrees of unsaturation. The 1H NMR and 13C APT spectra of 1 (Tables 1 and 2) displayed the presence of one propanoic acid moiety at δH 6.74 (1H, d, J = 1.8 Hz, H-2′), 6.67 (1H, d, J = 8.4 Hz, H-5′), 6.62 (1H, dd, J = 8.4, 1.8 Hz, H-6′), 2.97 (1H, dd, J = 14.4, 9.0 Hz, H-7a′), 3.12 (1H, dd, J = 14.4, 3.6 Hz, H-7b′), 5.19 (1H, dd, J = 9.0, 3.6 Hz, H-8′); δC 129.6 (C-1′), 117.6 (C-2′), 146.3 (C-3′), 145.3 (C-4′), 116.5 (C-5′), 122.0 (C-6′), 38.1 (C-7′), 75.4 (C-8′), 173.0 (C-9′) [18]. The proton signals at δH 6.72 (1H, d, J = 1.8 Hz, H-2″), 6.68 (1H, d, J = 8.4 Hz, H-5″), 6.59 (1H, dd, J = 8.4, 1.8 Hz, H-6″) and carbon signals at δC 133.8 (C-1″), 116.9 (C-2″), 145.5 (C-3″), 145.3 (C-4″), 116.5 (C-5″), 121.8 (C-6″) indicated the existence of one ABXspin aromatic system. The trans-double bond signals at δH 6.14 (1H, d, J = 15.6 Hz, H-10), 7.34 (1H, d, J = 15.6 Hz, H-9) together with the carbons at δC 119.0 (C-10), 143.4 (C-9), 168.0 (C-11) exhibited the presence of an α,β-unsaturated carbonyl moiety. The protons at δH 4.03 (2H, m), 1.12 (3H, t, J = 7.2 Hz) indicated the existence of a carbethoxy group. Furthermore, except for one carbonyl carbon at δC 175.5 (C-12), the 13C APT spectrum displayed another eight carbons at δC 141.9 (C-1), 44.6 (C-2), 70.7 (C-3), 209.5 (C-4), 58.1 (C-5), 137.7 (C-6), 49.9 (C-7), 43.9 (C-8), compatible with the protons at δH 3.78 (1H, m, H-2), 3.71
52.9, CH3 62.6, CH2 14.8, CH3
The signals were observed in the HSQC experiment. The signal were observed in the HMBC experiment.
(1H, d, J = 3.6 Hz, H-3), 3.16, (1H, dd, J = 6.0, 2.4 Hz, H-5), 6.72, (1H, d, J = 6.0 Hz, H-6), 3.46, (1H, dd, J = 7.2, 2.4 Hz, H-7), 3.51, (1H, dd, J = 7.2, 1.8 Hz, H-8). In the 1H–1H COSY spectrum (Fig. 2), the correlations from H-1 to H2 and H-8, H-8 to H-7, H-7 to H-5, and H-5 to H-6 together with the HMBC correlations (Fig. 2) between H-2/6 and C-1, H-3/5 and C-4 displayed the existence of an unusual bicycle-[2.2.2]-oct-5-en-4-one ring moiety which was established by eight carbons from C-1 to C-8. Moreover, the HMBC correlations from H-7 to C-1″ and C-2″/6″, H-8 to C-12, and H-9 to C-1 indicated that the ABX-spin aromatic ring was located at C-7, the carbethoxy was attached at C-8, and the α,βunsaturated carbonyl moiety was connected with C-1. Finally, the propanoid acid moiety was linked to C-11 on the basis of the correlations of H-8′ with C-11. The relative stereochemistry of 1 was determined by the NOE experiment. Irradiation of δH 3.51 (H-8) caused a NOE enhancement of the signal at δH 3.16 (H-5), indicating that the C-12 was in an exo position. The peak at δH 3.78 (H-2) showed NOE correlations with the peaks at δH 3.71 (H-3) and at δH 3.46 (H-7), which established the hydroxyl group and the phenyl group in an endo position, respectively. Irradiation of δH 7.34 (H-9) did not increase the intensity of δH 6.14 (H-10), which indicated that the double bond geometry between C-9 and C-10 was in the E configuration. The absolute configuration of 1 was determined by application of CD method. Compound 1 showed a split CD curve with a negative Cotton effect at 270 nm followed by positive Cotton effect at
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G.-X. Ma et al. / Fitoterapia 105 (2015) 61–65
Fig. 2. Key 1H–1H COSY (bond) and HMBC (arrows) correlations for compound 1.
318 nm, which is in good agreement with the λ max of the 3,4dihydroxyphenyl (270 nm) and β,γ-unsaturated ketone (318 nm) (Fig. 3). Due to the existing of carbonyl-carbon-π system as an inherent dissymmetric chromophore in 1, the absolute configuration was successfully determined as (2S, 3S, 5S, 7S, 8R) by applying the detailed octant rule [19]. Thus, the structure of 1 was determined, and named as cleroden A. Compound 1 contains an unusual bicycle [2.2.2] octane moiety representing a new class of phenolic acid. Compound 2 was obtained as a yellowish amorphous powder with an [α]25 D + 145.7 (c 0.12, MeOH) and assigned as C37H33O15 on the basis of its negative HRESIMS result (m/z 717.1799 [M–H]−). The NMR (Tables 1 and 2) and IR spectroscopic data for this compound was analogous to those of 1, except for the appearance of a 3-(3,4dihydroxyphenyl) lactic acid moiety at δH 6.73 (2H, d, J = 8.4 Hz), 7.07 (2H, d, J = 8.4 Hz), 3.06 (1H, dd, J = 14.4, 7.8 Hz), 3.12 (1H, dd, J = 14.4, 4.8 Hz), 5.20 (1H, dd, J = 7.8, 4.8 Hz), 3.69 (3H, s); δC 128.2, 131.7 (overlapped), 116.5 (overlapped), 157.7, 38.1, 75.1, and 172.2.
In the HMBC spectrum, the long-range correlation between δH 5.20 (1H, dd, J = 7.8, 4.8 Hz, H-8′) and C-11 (δC 168.1) indicated that the additional lactic acid moiety was located at C-11. Similarly, the propanoid acid moiety was attached to C-12 on the basis of correlation of H-8‴ with C-12. The relative configuration and absolute configuration of 2 were consistent to 1 by comparison with their NOESY and CD spectra data. Therefore, compound 2 was determined as cleroden B. Compound 3 exhibited the molecular formula C37H33O15 according to HRESIMS (m/z 717.1795 [M–H]−). The 1H NMR and 13C APT data were closely related to those of 2 (Tables 1 and 2). The differences were in the position of the methoxy group which was placed at C-9′ in 2, but at C-9‴ in 3. The HMBC correlations from δH 3.54 (3H, s) to C-9‴ (δC 171.6) confirmed the locations of methoxy at C-9‴. Taking together with the NOESY and CD spectra data, compound 3 was established as shown and named as cleroden C. Compound 4 exhibited the molecular formula C36H31O15 according to the negative HRESIMS (m/z 703.1638 [M–H]−). The 1H and 13C APT spectra (Tables 1 and 2) of 4 were comparable with those of 2. The differences were shown that the methoxy group in 2 disappeared in 4 which was fully supported by the molecular formula C36H31O15 above. The absolute configuration of 4 was determined by NOESY spectrum and CD method. Accordingly, the structure of compound 4 was established as cleroden D. Compounds 1–4 represent novel metabolites of the shikimate pathway. The bicycle [2.2.2] octane moiety of clerodens A–D strongly suggested a biogenesis as shown in Scheme 1. After the loss of a proton and electron, the caffeic acid molecule was oxidated and formed an intermediate A with a diene carrying three electron-attracting groups. The intermediate combined with the dienophile bearing an electrondonating 3,4-dihydroxyphenyl group lead to the acceleration of the Diels–Alder reaction. Then, the final esterification reaction generated compounds 1–4. Considering this medicinal herb as therapeutical agent of nephritis and cystitis, the isolated compounds (1–4) were studied for their antiinflammatory activities on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7. The results showed that compounds 1–3 showed moderate inhibitory activities with IC50 values ranging from 12.4 to 18.9 μM, in particular, compound 4 displayed significant inhibitory activities with IC50 value of 6.8 μM (Table 3). From the
Fig. 3. CD spectrum of compound 1, recorded in MeOH.
G.-X. Ma et al. / Fitoterapia 105 (2015) 61–65
65
Scheme 1. The proposed biogenetic pathways for the new compounds 1–4.
Table 3 Inhibitory activity of compounds 1–4 on LPS-induced NO production in RAW 264.7 macrophages. a
Compounds
IC50 (μM)
1 2 3 4 Aminoguanidineb
18.9 ± 1.2 14.7 ± 0.48 12.4 ± 1.5 6.8 ± 0.92 1.53 ± 0.23
a b
Value present mean ± SD of triplicate experiments. Positive control substance.
biological results, it can be inferred that the number of phenolic hydroxyl groups in the structure may enhance the anti-inflammatory activity.
Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.
Acknowledgments This work was financially supported by the technological large platform for comprehensive research and development of new drugs in the Twelfth Five-Year “Significant New Drugs Created” Science and Technology Major Projects (No. 2012ZX09301-002-001-032), National Natural Science Foundation of China (No. 30973626), the Science and Technology Grant of Guangxi Province (No. 0639039) and special purpose of basic scientific research operation grant for Commonweal Academy and Institute of Central Authorities (No. YZ-1-24), and innovation capacity-building in Guangxi Science and Technology Agency (0443002-2).
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Four new phenolic acid with unusual bicycle [2.2.2] octane moiety from Clerodendranthus spicatus and their anti-inflammatory activity Guo-Xu Ma a,1, Xiao-Po Zhang b,1, Peng-Fei Li a, Zhong-Hao Sun a, Nai-Liang Zhu a, Yin-Di Zhu a, Jun-Shan Yang a, De-Li Chen c,⁎, Hai-Feng Wu a,⁎, Xu-Dong Xu a,⁎ a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100193, China b School of Pharmaceutical Science, Hainan Medical University, Haikou 571101, China c Hainan Branch Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Wanning 571533, China
a r t i c l e
i n f o
Article history: Received 25 March 2015 Received in revised form 7 June 2015 Accepted 8 June 2015 Available online 12 June 2015 Keywords: Clerodendranthus spicatus Phenolic acids Anti-inflammatory
a b s t r a c t Four new phenolic acids, clerodens A–D (1–4) possessing an unusual bicycle [2.2.2] octane moiety were isolated from the whole plants of Clerodendranthus spicatus. Their structures were elucidated by extensive spectroscopic methods, including NMR, MS, and ECD data. All isolates were evaluated for their anti-inflammatory activities on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7, and compound 4 showed significant inhibitory activities with IC50 value of 6.8 μM. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Clerodendranthus spicatus (Thunb.) C.Y. Wu (syn. Orthosiphon stamineus Benth.) known as “Kumis kucing” in Southeast Asia, is a popular herbal medicine extensively used for the treatment of nephritis and cystitis [1,2]. The leaves and stems of this plant cultivated in Southern China are used by the name of “Shen Cha” as a diuretic tea in Chinese folk medicine for the treatment of acute and chronic nephritis, urinary lithiasis, cystitis and rheumatism [3–5]. Previous chemical and pharmacological studies on the ethanol extract of this species resulted in the separation of triterpenes [6], diterpenes [7,8], flavonoids [9] and phenolic acids [10,11] with broad bioactivities including anti-inflammatory [7, 12], diuresis [13], immunoregulation [14], and antidiabetic [15,16]. However, the composition of the water-soluble of C. spicatus has been little investigated, and as a part of our ongoing program toward the discovery of novel bioactive constituents from this plant, four new phenolic acids, clerodens A–D with an unusual bicycle [2.2.2] octane moiety (1–4) (Fig. 1), were isolated from the aqueous extract of C. spicatus. In this paper, we report the structure elucidation of the new phenolic
⁎ Corresponding authors. E-mail addresses: [email protected] (D.-L. Chen), [email protected] (H.-F. Wu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.fitote.2015.06.010 0367-326X/© 2015 Elsevier B.V. All rights reserved.
acid derivatives and their inhibitory activities against LPS-induced NO production in macrophages.
2. Experimental 2.1. General experimental procedures Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter. UV and IR spectra were recorded on Shimadzu UV2550 and FTIR-8400S spectrometers, respectively. ECD spectra were obtained using a JASCO J-815 spectropolarimeter. NMR spectra were obtained with a Bruker AV III 600 NMR spectrometer (chemical shift values are presented as δ values with TMS as the internal standard). HRESIMS spectra were performed on a LTQ-Obitrap XL spectrometer. Preparative HPLC was performed on a Lumtech K-1001 analytic LC equipped with two pumps of K-501, a UV detector of K-2600, and an YMC Pack C18 column (250 mm × 10 mm, i.d., 5 μM, YMC Co. Ltd., Japan) eluted with CH3OH– H2O at a flow rate of 2 mL/min. C18 reversed-phase silica gel (40–63 μM, Merk, Darmstadt, Germany), Toyopearl HW-40C (50–100 μM, Tosoh Corp., Tokyo, Japan), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), MCI gel (CHP 20P, 75–150 μM, Mitsubishi Chemical Corporation, Tokyo, Japan) and silica gel (100–200 and 300–400 mesh, Qingdao Marine Chemical plant, Qingdao, People's Republic of China) were used for column chromatography. And pre-coated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, People's Republic
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G.-X. Ma et al. / Fitoterapia 105 (2015) 61–65
Fig. 1. Structures of compounds 1–4.
of China) were used for TLC. All solvents used were of analytical grade (Beijing Chemical Works). 2.2. Plant material The whole plants of C. spicatus were collected in Jinghong, Yunnan Province, People's Republic of China, in September 2012, and were authenticated by Prof. Jing-Quan Yuan. A voucher specimen (CS-21209) has been deposited at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences. 2.3. Extraction and isolation The ground powder of air-dried whole plants of C. spicatus (15 kg) was decocted with boiled water (50 L × 2 h × 3) and the solution was then precipitated in 70% ethanol and filtered to yield the supernatant. The supernatant was concentrated under reduced pressure to yield an extract. The extract was subjected to D101 macro porous resin column chromatography using ethanol–water gradient elution, and the eluted fraction with 30–95% ethanol–water elution combined after concentration yielded the total phenolic acids of C. spicatus (TPC, 260 g). TPC fraction was further separated by MCI-gel column chromatography with methanol–water gradient elution, giving seven fractions (A–G). Fraction D (11.4 g) was fractionated on HW-40C column chromatography elution with MeOH–H2O (20:80; 30:70; 40:60; 60:40; 100:0) giving five subfractions (FrD1–D5). Fraction D2 (673 mg) was chromatographed by semi-preparative HPLC using MeOH–H2O (26: 74, v/v) to yield compound 2 (4.3 mg, tR = 25.3 min) and 3 (3.8 mg, tR = 26.6 min). Fraction D3 (124 mg) was purified through semi-preparative HPLC elution using a MeOH–H2O (35: 65, v/v) system to give compound 4 (2.1 mg, t R = 22.4 min). Subfraction D4 (245 mg) was chromatographed by successive preparative and semipreparative HPLC to yield 1 (6.8 mg, tR = 28.1 min). 2.3.1. Cleroden A (1) C29H28O12, white amorphous powder; [α]20 D + 237.6 (c = 0.17, MeOH); IR (KBr) νmax: 3353, 1713, 1606, 1527 cm−1; UV (MeOH) λmax (log ε): 275 (3.28) nm; for 1H NMR (600 MHz, CD3OD) and 13C-APT
(150 MHz, CD3OD) spectroscopic data see Tables 1 and 2; HRESIMS m/z: 567.1478 [M–H]− (calcd for C29H27O12, 567.1508).
2.3.2. Cleroden B (2) C37H34O15, white amorphous powder; [α]20 D + 145.7 (c = 0.12, MeOH); IR (KBr) νmax: 3425, 1634, 1610, 1575 cm− 1; UV (MeOH) λmax (log ε): 277 (3.45) nm; for 1H NMR (600 MHz, CD3OD) and 13CAPT (150 MHz, CD3OD) spectroscopic data see Tables 1 and 2; HRESIMS m/z: 717.1799 [M–H]− (calcd for C37H33O15, 717.1825).
2.3.3. Cleroden C (3) C37H34O15, white amorphous powder; [α]20 D + 174.1 (c = 0.14, MeOH); IR (KBr) νmax: 3355, 1723, 1589, 1515 cm− 1; UV (MeOH) λmax (log ε): 275 (3.25) nm; for 1H NMR (600 MHz, CD3OD) and 13CAPT (150 MHz, CD3OD) spectroscopic data see Tables 1 and 2; HRESIMS m/z: 717.1795 [M–H]− (calcd for C37H33O15, 717.1825).
2.3.4. Cleroden D (4) C36H32O15, white amorphous powder; [α]20 D + 145.6 (c = 0.09, MeOH); IR (KBr) νmax: 3407, 1634, 1606, 1515 cm− 1; UV (MeOH) λmax (log ε): 275 (3.02) nm; for 1H NMR (600 MHz, CD3OD) and 13CAPT (150 MHz, CD3OD) spectroscopic data see Tables 1 and 2; HRESIMS m/z: 703.1638 [M–H]− (calcd for C36H31O15, 703.1668).
2.4. Assay for inhibitory ability against LPS-induced NO production in RAW 264.7 macrophages RAW 264.7 macrophages were seeded in 24-well plates (105 cells/well). The cells were co-incubated with drugs and LPS (1 μg/mL) for 24 h. The amount of NO was assessed by determining the nitrite concentration in the cultured RAW 264.7 macrophage supernatants with Griess reagent. Aliquots of supernatants (100 μL) were incubated, in-sequence, with 50 μL of 1% sulfanilamide and 50 μL of 0.1% naphthylethylenediamine in 2.5% phosphoric acid solution. The absorbance was recorded on a microplate reader at a wavelength of 570 nm.
G.-X. Ma et al. / Fitoterapia 105 (2015) 61–65 Table 1 1 H NMR spectroscopic data (600 MHz, in CD3OD) for compounds 1–4 (δH in ppm, J in Hz). No
1
2 3 5 6 7 8 9 10 2′ 3′ 5′ 6′ 7′
3.78, m 3.71, d (3.6) 3.16, dd (6.0, 2.4) 6.72, d (6.0) 3.46, dd (7.2, 2.4) 3.51, dd (7.2, 1.8) 7.34, d (15.6) 6.14, d (15.6) 6.74, d (1.8)
8′ 2″ 5″ 6″
2
3.74, m 3.65, d (3.6) 3.10, d (5.4) 6.63, d (5.4) 3.26, (6.6) 3.60, d (6.6) 7.23, d (15.6) 6.18, d (15.6) 7.07, d (8.4) 6.73, d (8.4) 6.67, d (8.4) 6.73, d (8.4) 6.62, dd (7.8, 1.8) 7.07, d (8.4) 2.97, dd 3.06, dd (14.4, 9.0) (14.4, 7.8) 3.12, dd 3.12, dd (14.4, 3.6) (14.4, 4.8) 5.19, dd 5.20, dd (9.0, 3.6) (7.8, 4.8) 6.72, d (1.8) 6.76, d (1.8) 6.68, d (7.8) 6.68, d (7.8) 6.59, dd (7.8, 1.8) 6.58, dd (7.8, 1.8)
2‴ 5‴ 6‴ 7‴
8‴ 9′-OCH3 9‴-OCH3 12-OCH2CH3 4.03, m 12-OCH2CH3 1.12, t (7.2)
6.60, d (1.2) 6.59, d (7.8) 6.34, dd (7.8, 1.2) 2.83, dd (14.4, 9.6) 3.00, dd (12.6, 4.2) 5.02, dd (9.6, 4.2) 3.69, s
63
Table 2 13 C NMR spectroscopic data (150 MHz, in CD3OD) for compounds 1–4.
3
4
No
1
2
3
4
3.76, m 3.66, (3.0) 3.12, d (6.0) 6.61, d (6.0) 3.34, d (7.2) 3.55, d (6.0) 7.24, d (15.6) 6.19, d (15.6) 7.10, d (7.8) 6.69, d (7.8) 6.69, d (7.8) 7.10, d (7.8) 3.03, dd (14.4, 4.8) 3.05, dd (14.4, 9.0) 5.19, dd (9.0, 4.8) 6.76, d (1.8) 6.68, d (7.8) 6.56, dd (7.8, 1.8) 6.76, d (1.2) 6.57, d (7.8) 6.34, dd (7.8, 1.2) 2.81, dd (14.4, 9.6) 3.02, dd (12.6, 4.2) 5.03, dd (9.6, 4.2)
3.75, m 3.76, d (3.6) 3.09, d (4.8) 6.52, d (6.0) 3.30, d (6.0) 3.56, d (6.0) 7.13, d (15.6) 6.14, d (15.6) 7.12, d (7.8) 6.73, d (7.8) 6.73, d (7.8) 7.12, d (7.8) 3.06, dd (14.4, 4.8) 3.15, dd (14.4, 9.0) 5.10, dd (9.0, 4.8) 6.77, d (1.8) 6.59, d (7.8) 6.34, dd (7.8, 1.8) 6.60, d (1.2) 6.67, d (7.8) 6.60, dd (7.8, 1.2) 2.82, dd (14.4, 9.6) 3.03, dd (12.6, 4.2) 5.06, dd (9.6, 4.2)
1 2 3 4 5 6 7 8 9 10 11 12 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ 9′-OCH3 9‴-OCH3 12-OCH2CH3 12-OCH2CH3
141.9, C 44.6, CH 70.7, CH, 209.5, C 58.1, CH 137.7, CH 49.9, CH 43.9, CH, 143.4, CH 119.0, CH 168.0, C 175.5, C 129.6, C 117.6, CH 146.3, C 145.3, C 116.5, CH 122.0, CH 38.1, CH2 75.4, CH 173.0, C 133.8, C 116.9, CH 145.5, C 145.3, C 116.5, CH 121.8, CH
142.5, C 44.5, CH 70.9, CH 209.5, C 58.1, CH 137.2, CH 50.4, CH 43.9, CH 143.7, CH 119.0, CH 168.1, C 175.2, C 128.2, C 131.7, CH 116.5, CH 157.7, C 116.5, CH 131.7, CH 38.1, CH2 75.1, CH 172.2, C 134.0, C 116.9, CH 146.3, C 145.6, C 116.6, CH 121.2, CH 129.9, C 117.7, CH 146.2, C 145.2, C 116.6, CH 122.0, CH 37.8, CH2 76.1a, CH 171.4b, C 52.9, CH3
142.2, C 44.6, CH 70.9, CH 209.5, C 57.9, CH 136.8, CH 50.2, CH 43.7, CH 142.7, CH 120.0, CH 168.5, C 174.9, C 128.7, C 131.7, CH 116.2, CH 157.3, C 116.2, CH 131.7, CH 38.4, CH2 77.0a, CH 172.6b, C 133.8, C 116.8, CH 146.3, C 145.6, C 116.6, CH 121.1, CH 129.9, C 117.7, CH 146.2, C 145.5, C 116.6, CH 122.0, CH 37.8, CH2 75.9, CH 171.6, C
142.7, C 44.8, CH 69.4, CH 209.8, C 57.9, CH 136.1, CH 50.0, CH 44.0, CH 142.4, CH 120.3, CH 168.4b, C 175.1, C 130.6, C 131.7, CH 116.4, CH 157.3, C 116.4, CH 131.7, CH 38.7, CH2 77.0a, CH 172.5b, C 134.2, C 116.9, CH 146.1, C 145.0, C 116.6, CH 122.0, CH 130.2, C 117.7, CH 146.2, C 145.4, C 116.6, CH 121.2, CH 38.6, CH2 77.0a, CH 171.5b, C
3.54, s
3. Results and discussion
a b
Compound 1 was obtained as a yellowish amorphous powder with [α]25 D + 237.6 (c 0.17, MeOH) and gave a greenish blue coloration characteristic of catechol with 2% ethanolic FeCl3 on TLC [17]. The UV and IR spectra showed absorption of phenyl (275 nm; 1606, 1527 cm−1), OH (3353 cm−1), and carbonyl groups (1713 cm−1). The HRESIMS spectrum showed a pseudomolecular ion at mnz 567.1478 [M–H]− in the negative ion mode (Calcd for: 567.1508, C29H27O12), from which in conjunction with NMR data the molecular formula was established as C29H27O12, compatible with eleven degrees of unsaturation. The 1H NMR and 13C APT spectra of 1 (Tables 1 and 2) displayed the presence of one propanoic acid moiety at δH 6.74 (1H, d, J = 1.8 Hz, H-2′), 6.67 (1H, d, J = 8.4 Hz, H-5′), 6.62 (1H, dd, J = 8.4, 1.8 Hz, H-6′), 2.97 (1H, dd, J = 14.4, 9.0 Hz, H-7a′), 3.12 (1H, dd, J = 14.4, 3.6 Hz, H-7b′), 5.19 (1H, dd, J = 9.0, 3.6 Hz, H-8′); δC 129.6 (C-1′), 117.6 (C-2′), 146.3 (C-3′), 145.3 (C-4′), 116.5 (C-5′), 122.0 (C-6′), 38.1 (C-7′), 75.4 (C-8′), 173.0 (C-9′) [18]. The proton signals at δH 6.72 (1H, d, J = 1.8 Hz, H-2″), 6.68 (1H, d, J = 8.4 Hz, H-5″), 6.59 (1H, dd, J = 8.4, 1.8 Hz, H-6″) and carbon signals at δC 133.8 (C-1″), 116.9 (C-2″), 145.5 (C-3″), 145.3 (C-4″), 116.5 (C-5″), 121.8 (C-6″) indicated the existence of one ABXspin aromatic system. The trans-double bond signals at δH 6.14 (1H, d, J = 15.6 Hz, H-10), 7.34 (1H, d, J = 15.6 Hz, H-9) together with the carbons at δC 119.0 (C-10), 143.4 (C-9), 168.0 (C-11) exhibited the presence of an α,β-unsaturated carbonyl moiety. The protons at δH 4.03 (2H, m), 1.12 (3H, t, J = 7.2 Hz) indicated the existence of a carbethoxy group. Furthermore, except for one carbonyl carbon at δC 175.5 (C-12), the 13C APT spectrum displayed another eight carbons at δC 141.9 (C-1), 44.6 (C-2), 70.7 (C-3), 209.5 (C-4), 58.1 (C-5), 137.7 (C-6), 49.9 (C-7), 43.9 (C-8), compatible with the protons at δH 3.78 (1H, m, H-2), 3.71
52.9, CH3 62.6, CH2 14.8, CH3
The signals were observed in the HSQC experiment. The signal were observed in the HMBC experiment.
(1H, d, J = 3.6 Hz, H-3), 3.16, (1H, dd, J = 6.0, 2.4 Hz, H-5), 6.72, (1H, d, J = 6.0 Hz, H-6), 3.46, (1H, dd, J = 7.2, 2.4 Hz, H-7), 3.51, (1H, dd, J = 7.2, 1.8 Hz, H-8). In the 1H–1H COSY spectrum (Fig. 2), the correlations from H-1 to H2 and H-8, H-8 to H-7, H-7 to H-5, and H-5 to H-6 together with the HMBC correlations (Fig. 2) between H-2/6 and C-1, H-3/5 and C-4 displayed the existence of an unusual bicycle-[2.2.2]-oct-5-en-4-one ring moiety which was established by eight carbons from C-1 to C-8. Moreover, the HMBC correlations from H-7 to C-1″ and C-2″/6″, H-8 to C-12, and H-9 to C-1 indicated that the ABX-spin aromatic ring was located at C-7, the carbethoxy was attached at C-8, and the α,βunsaturated carbonyl moiety was connected with C-1. Finally, the propanoid acid moiety was linked to C-11 on the basis of the correlations of H-8′ with C-11. The relative stereochemistry of 1 was determined by the NOE experiment. Irradiation of δH 3.51 (H-8) caused a NOE enhancement of the signal at δH 3.16 (H-5), indicating that the C-12 was in an exo position. The peak at δH 3.78 (H-2) showed NOE correlations with the peaks at δH 3.71 (H-3) and at δH 3.46 (H-7), which established the hydroxyl group and the phenyl group in an endo position, respectively. Irradiation of δH 7.34 (H-9) did not increase the intensity of δH 6.14 (H-10), which indicated that the double bond geometry between C-9 and C-10 was in the E configuration. The absolute configuration of 1 was determined by application of CD method. Compound 1 showed a split CD curve with a negative Cotton effect at 270 nm followed by positive Cotton effect at
64
G.-X. Ma et al. / Fitoterapia 105 (2015) 61–65
Fig. 2. Key 1H–1H COSY (bond) and HMBC (arrows) correlations for compound 1.
318 nm, which is in good agreement with the λ max of the 3,4dihydroxyphenyl (270 nm) and β,γ-unsaturated ketone (318 nm) (Fig. 3). Due to the existing of carbonyl-carbon-π system as an inherent dissymmetric chromophore in 1, the absolute configuration was successfully determined as (2S, 3S, 5S, 7S, 8R) by applying the detailed octant rule [19]. Thus, the structure of 1 was determined, and named as cleroden A. Compound 1 contains an unusual bicycle [2.2.2] octane moiety representing a new class of phenolic acid. Compound 2 was obtained as a yellowish amorphous powder with an [α]25 D + 145.7 (c 0.12, MeOH) and assigned as C37H33O15 on the basis of its negative HRESIMS result (m/z 717.1799 [M–H]−). The NMR (Tables 1 and 2) and IR spectroscopic data for this compound was analogous to those of 1, except for the appearance of a 3-(3,4dihydroxyphenyl) lactic acid moiety at δH 6.73 (2H, d, J = 8.4 Hz), 7.07 (2H, d, J = 8.4 Hz), 3.06 (1H, dd, J = 14.4, 7.8 Hz), 3.12 (1H, dd, J = 14.4, 4.8 Hz), 5.20 (1H, dd, J = 7.8, 4.8 Hz), 3.69 (3H, s); δC 128.2, 131.7 (overlapped), 116.5 (overlapped), 157.7, 38.1, 75.1, and 172.2.
In the HMBC spectrum, the long-range correlation between δH 5.20 (1H, dd, J = 7.8, 4.8 Hz, H-8′) and C-11 (δC 168.1) indicated that the additional lactic acid moiety was located at C-11. Similarly, the propanoid acid moiety was attached to C-12 on the basis of correlation of H-8‴ with C-12. The relative configuration and absolute configuration of 2 were consistent to 1 by comparison with their NOESY and CD spectra data. Therefore, compound 2 was determined as cleroden B. Compound 3 exhibited the molecular formula C37H33O15 according to HRESIMS (m/z 717.1795 [M–H]−). The 1H NMR and 13C APT data were closely related to those of 2 (Tables 1 and 2). The differences were in the position of the methoxy group which was placed at C-9′ in 2, but at C-9‴ in 3. The HMBC correlations from δH 3.54 (3H, s) to C-9‴ (δC 171.6) confirmed the locations of methoxy at C-9‴. Taking together with the NOESY and CD spectra data, compound 3 was established as shown and named as cleroden C. Compound 4 exhibited the molecular formula C36H31O15 according to the negative HRESIMS (m/z 703.1638 [M–H]−). The 1H and 13C APT spectra (Tables 1 and 2) of 4 were comparable with those of 2. The differences were shown that the methoxy group in 2 disappeared in 4 which was fully supported by the molecular formula C36H31O15 above. The absolute configuration of 4 was determined by NOESY spectrum and CD method. Accordingly, the structure of compound 4 was established as cleroden D. Compounds 1–4 represent novel metabolites of the shikimate pathway. The bicycle [2.2.2] octane moiety of clerodens A–D strongly suggested a biogenesis as shown in Scheme 1. After the loss of a proton and electron, the caffeic acid molecule was oxidated and formed an intermediate A with a diene carrying three electron-attracting groups. The intermediate combined with the dienophile bearing an electrondonating 3,4-dihydroxyphenyl group lead to the acceleration of the Diels–Alder reaction. Then, the final esterification reaction generated compounds 1–4. Considering this medicinal herb as therapeutical agent of nephritis and cystitis, the isolated compounds (1–4) were studied for their antiinflammatory activities on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7. The results showed that compounds 1–3 showed moderate inhibitory activities with IC50 values ranging from 12.4 to 18.9 μM, in particular, compound 4 displayed significant inhibitory activities with IC50 value of 6.8 μM (Table 3). From the
Fig. 3. CD spectrum of compound 1, recorded in MeOH.
G.-X. Ma et al. / Fitoterapia 105 (2015) 61–65
65
Scheme 1. The proposed biogenetic pathways for the new compounds 1–4.
Table 3 Inhibitory activity of compounds 1–4 on LPS-induced NO production in RAW 264.7 macrophages. a
Compounds
IC50 (μM)
1 2 3 4 Aminoguanidineb
18.9 ± 1.2 14.7 ± 0.48 12.4 ± 1.5 6.8 ± 0.92 1.53 ± 0.23
a b
Value present mean ± SD of triplicate experiments. Positive control substance.
biological results, it can be inferred that the number of phenolic hydroxyl groups in the structure may enhance the anti-inflammatory activity.
Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.
Acknowledgments This work was financially supported by the technological large platform for comprehensive research and development of new drugs in the Twelfth Five-Year “Significant New Drugs Created” Science and Technology Major Projects (No. 2012ZX09301-002-001-032), National Natural Science Foundation of China (No. 30973626), the Science and Technology Grant of Guangxi Province (No. 0639039) and special purpose of basic scientific research operation grant for Commonweal Academy and Institute of Central Authorities (No. YZ-1-24), and innovation capacity-building in Guangxi Science and Technology Agency (0443002-2).
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