Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Constituents from Apium graveolens and their anti-inflammatory effects Li-Han Zhu, Tian-Hui Bao, Yue Deng, Hua Li & Li-Xia Chen To cite this article: Li-Han Zhu, Tian-Hui Bao, Yue Deng, Hua Li & Li-Xia Chen (2017): Constituents from Apium graveolens and their anti-inflammatory effects, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2017.1381687 To link to this article: http://dx.doi.org/10.1080/10286020.2017.1381687
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Published online: 03 Oct 2017.
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Date: 04 October 2017, At: 21:59
Journal of Asian Natural Products Research, 2017 https://doi.org/10.1080/10286020.2017.1381687
Constituents from Apium graveolens and their anti-inflammatory effects Li-Han Zhua, Tian-Hui Baoa, Yue Denga, Hua Lia,b and Li-Xia Chena a
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Key Laboratory of Structure-Based Drug Design & Discovery, School of Traditional Chinese Materia Medica, Wuya College of Innovation, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China; bSchool of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
ABSTRACT
A phthalide glycoside, (3R, 4R)-4-O-β-D-glucopyranosyl-senkyunolide (1), and a megastigmane glycoside, (6S, 7R)-3-oxo-megastigma-4, 8-dien-7-O-β-D-glucoside (2), along with two known aglycones (3–4), were isolated from the 70% EtOH extract of fresh whole grass of Apium graveolens L. Their structures were elucidated by extensive spectroscopic analysis. All of these compounds were tested for their inhibitory effects on nitric oxide (NO) production in the RAW 264.7 macrophages. Among them, compounds 3 and 4 showed potent inhibitory activity against LPS-induced nitric oxide production in RAW 264.7 macrophages, with IC50 values of 24.0 ± 2.1 μM and 28.6 ± 2.4 μM, respectively.
ARTICLE HISTORY
Received 29 June 2017 Accepted 14 September 2017 KEYWORDS
Apium graveolens; phthalide glycoside; megastigmane glycoside; nitric oxide
O OH
O
OH O
HO HO
O
HO HO
OH 1
O OH 2
O O
1. Introduction The genus (Apium) with 20 species is distributed widely in temperate regions around the world, and two species were found in China. Apium graveolens has been used as a wellknown vegetable, and most of the people like it, which contains flavonoids [1,2], coumarins [3–6], phthalides [7–9], sesquiterpenes [10], volatile oil [11,12], and so on. A lot of studies showed that the crude extract of A. graveolens possessed prominent anti-inflammatory, anti-hypertensive, hypolipidemic, antioxidant, anti-cancer, and other effects [13–17]. CONTACT Li-Xia Chen [email protected]; Hua Li [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/10286020.2017.1381687. © 2017 Informa UK Limited, trading as Taylor & Francis Group
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L.-H. ZHU ET AL.
In order to find the anti-inflammatory material basis of A. graveolens, the EtOH extract of the whole grass of this plant was investigated, and two new compounds, along with two known compounds, were isolated. Their structures were elucidated by comprehensive spectroscopic analysis. All of these compounds were tested for their inhibitory effects on nitric oxide (NO) production in RAW 264.7 macrophages.
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2. Results and discussion The fresh whole grass of A. graveolens was cut into pieces and extracted with 70% EtOH. The extract was suspended in water and then partitioned with petroleum ether (PE) and ethyl acetate (EtOAc) successively. Then, the EtOAc layer was separated by silica gel, Sephadex LH-20, ODS open column chromatography (CC), and HPLC to produce two new compounds, a phthalide glycoside, (3R, 4R)-4-O-β-D-glucopyranosyl-senkyunolide (1), and a megastigmane glycoside, (6S, 7R)-3-oxo-megastigma-5,8-dien-7-O-β-D-glucoside(2), along with two known aglycones, sedanolide (3) [18] and (3S)-3-hydroxy-megastigma5,8-dien-7- one (4) [19] (Figure 1). Compound 1 was isolated as white powder (MeOH). Molish reaction was positive, and glucose was detected after acid hydrolysis. The molecular formula C18H26O8 was deduced by HR-ESI-MS data at m/z 393.1527 [M+Na]+, with six degrees of unsaturation. The anomeric proton signal at δH 4.42 (1H, d, J = 7.8 Hz) showed the glucose with β-anomeric configuration. The signals at δH 1.64 (1H, dd, J = 15.2, 6.7 Hz), 2.10–2.16 (1H, m), 1.37–1.42 (2H, m), 1.37–1.42 (2H, m), and 0.95 (3H, t, J = 7.1 Hz) in the 1H-NMR (Table 1) spectrum, in combination with the 1H-1H COSY correlations (Figure 2) of H-3 with H-1′, H-1′ with H-2′, H-2′ with H-3′, and H-3′ with H-4′ indicated that the presence of a n-butyl and two olefinic
Figure 1. Structures of compounds 1–4.
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Table 1. 1H- and 13C-NMR spectroscopic data for compounds 1 and 2 in CD3OD (at 300 and 75 MHz, respectively) 1
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Position 1 2
2
δC 173.5
δH (J in Hz)
3 4 5
83.9 69.1 31.9
6 7 7a 3a 8 9 10 11 12 13 1′
116.2 130.8 127.0 159.9
5.43, dd (7.5, 3.1) 4.80–4.87, m 2.76, br dd (15.6, 8.8) 2.88, dt (15.6, 4.5) 6.14, dt (9.5, 4.0) 6.23, br d (9.5)
2′ 3′ 4′ 5′ 6′
28.1 23.7 14.4
1″ 2″ 3″ 4″ 5″ 6″
103.5 74.9 78.2 71.5 78.0 62.7
32.7
1.64, dd (15.2, 6.7) 2.10–2.16, m 1.37–1.42, m 1.37–1.42, m 0.95, t (7.1)
4.42, d (7.8) 3.13, t (7.8) 3.33–3.36, m 3.28–3.31, m 3.32–3.39, m 3.71, dd (11.6, 6.9) 3.89, br d (11.6)
δH (J in Hz)
δC 37.2 48.4
2.00, d (16.8) 2.40, d (16.8)
202.1 126.2 166.0
5.84, br s
56.8 77.1
2.64, d (8.9) 4.30–4.38, m
138.3 128.9 21.2 23.9 28.2 27.7 102.5
5.73, dd (15.6, 6.5) 5.56–5.64, m 1.25, d (6.3) 1.90, s 0.99, s 0.97, s 4.31, d (7.8)
75.3 78.2 71.6 78.0 62.8
3.11–3.20, m 3.11–3.20, m 3.23–3.34, m 3.23–3.34, m 3.79, dd (11.8, 2.0) 3.64, dd (11.8, 5.1)
Figure 2. Key correlations of 1H-1H COSY, HMBC, and NOESY of compounds 1 and 2.
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L.-H. ZHU ET AL.
protons [δH 6.14 (1H, dt, J = 9.5, 4.0 Hz), 6.23 (1H, br d, J = 9.5 Hz)]. The 13C-NMR (Table 1) spectrum exhibited four characteristic carbon resonances at δC 173.5, 159.9, 127.0, and 83.9, typical signals of a five-membered α, β-unsaturated lactone. The HMBC correlations (Figure 2) from H-6 to C-7a/C-3a, H-7 to C-7a/C-3a indicated the presence of one pair of conjugated double bonds. The correlations of H-7 with C-7a, C-3a and H-5 with C-7a, C-3a, and C-4 revealed that the connection of the α, β-unsaturated lactone ring and the six-membered ring was through C-3a and C-7a [20]. The HMBC correlation of H-1′ with C-3 suggested that the butyl moiety was located at C-3, and the correlation of H-1″ with C-4 showed that the glucose moiety was attached to C-4. Thus, the structure of compound 1 was determined as 4-O-β-D-glucopyranosyl-senkyunolide. The relative configuration of compound 1 was elucidated based on the analysis of NOESY cross-peak. The NOESY correlation (Figure 2) of H-3 with H-4 suggested that H-3 and H-4 were on the same plane. The absolute configuration of compound 1 was determined by the application of CD octant rule and sector rule of carboxylic ester. The CD spectrum showed positive Cotton effect at 210 nm and negative Cotton effect at 240 nm, revealing that the configurations of C-3 and C-4 were R and R, respectively [20]. Thus, the structure of compound 1 was determined as (3R, 4R)-4-O-β-D-glucopyranosyl-senkyunolide. Compound 2 was isolated as colorless oil (MeOH). Molish reaction was positive, and the glucose was obtained after acid hydrolysis, indicating that compound 2 is a glucoside. The molecular formula C19H30O7 was determined by HR-ESI-MS data at m/z 393.1889 [M+Na]+, with five degrees of unsaturation. The signal at δH 4.31 (1H, d, J = 7.8 Hz) indicated that the glucose has β-anomer. The 1H-NMR (Table 1) spectrum revealed the appearance of four methyl groups [δH 1.25 (3H, d, J = 6.3 Hz), 1.90 (3H, s), 0.99 (3H, s), and 0.97 (3H, s)] and three olefinic protons [δH 5.84 (1H, br s), 5.73 (1H, dd, J = 15.6, 6.5 Hz), and 5.56–5.64 (1H, m)]. The 13C-NMR (Table 1) spectrum displayed nine typical signals, corresponding to four methyl carbons (δC 28.2, 27.7, 23.9, and 21.2), one carbonyl carbon (δC 202.1) and four olefinic carbons (δC 126.2, 128.9, 138.3, and 166.0). Among them, the signals at δC 202.1, 126.2, and 166.0 are characteristic signals of an α, β-unsaturated ketone group. In addition, a ring existed in the structure according to the five degrees of unsaturation. In the HMBC spectrum, the long-range correlations (Figure 2) of H-12, H-13 with C-1/C6/C-2, and H-11 with C-5 suggested the presence of a 1,1,5-trimethylcyclohex-4-enone ring. The proton signals (Table 1) at δH 5.73 (1H, dd, J = 15.6, 6.5 Hz), 5.56–5.64 (1H, m), and 1.25 (3H, d, J = 6.3 Hz), as well as the long-range correlations (Figure 2) from H-7 to C-1/C-6/C-8/C-9/C-10 in HMBC spectrum showed that a (E)-butenyl group was linked to C-6 of 1,1,5-trimethylcyclohex-4-enone ring. The HMBC correlations (Figure 2) of H-7 with C-1′ and H-1′ with C-7 suggested the glucose attached to C-7 [21]. The absolute configuration of C-6 in compound 2 was determined by CD spectrum with negative Cotton effect at 324 nm and positive Cotton effect at 250 nm, indicating that the configuration of C-6 was S [22]. The absolute configuration of C-7 in compound 2 was determined by the glucosidation shifts according to the reference [23]. When secondary allylic alcohols and β-D-glucoses connected to form glycosides, the chemical shifts of β-carbon decreased in comparison with those of the aglycone. In the structure with R or S configuration of the α-carbon, glucosidation shift of β-carbon with sp2 hybridization was −4.4 ± 0.5 ppm or −3.2 ± 0.5 ppm, respectively. The chemical shift of C-8 (β-C) of compound 2 was δC 135.0 in pyridine-d5, and that of the aglycone was δC 137.8, this difference value of chemical shift
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was −2.8 ppm, so the configuration of C-7 was R. Therefore, the structure of compound 2 was elucidated as (6S, 7R)-3-oxo-megastigma-4, 8-dien-7-O-β-D-glucoside. Inflammation is a defensive response to endogenous or exogenous stimuli in the living tissue of vascular system. Nitric oxide (NO) is an indispensable regulator of the immune system and can cause tissue damage due to its excessive production. Therefore, NO is not only an inflammatory factor, but also an immunoregulatory effector, and the amount of NO production plays a vital role in maintaining human health [24]. In our study, all of these isolated compounds were tested for their inhibitory activities against NO production in LPS-induced macrophages (Table 2). Compounds 1–4 showed no cytotoxicity (0–100 μM) on RAW 264.7 macrophage. Compounds 3 and 4 showed potent inhibitory activity against LPS-induced nitric oxide production in RAW 264.7 macrophages with IC50 values of 24.0 ± 2.1 μM and 28.6 ± 2.4 μM, respectively, comparable to the positive control of hydrocortisone (IC50 of 37.6 ± 2.9 μM). Certainly, the anti-inflammatory mechanisms of these compounds are required to further study.
3. Experimental 3.1. General experimental procedures Optical rotations were determined on a PerkinElmer 241 polarimeter (Perkin-Elmer, Waltham, MA, U.S.A.). HRESIMS were received on an Agilent 6210 TOF mass spectrometer (Palo Alto, U.S.A.). IR (4000−400 cm−1) spectra (KBr disks) were recorded on a Bruker IFS 55 spectrometer (Bruker Optics, Ettlingen, Germany), and UV spectra on a Shimadzu UV 1700UV-VIS recording spectrophotometer (Shimadzu Corporation, Kyoto, Japan). NMR spectra were performed on Bruker ARX-300 spectrometers (Bruck Biospin, Fallanden, Switzerland). Chemical shifts were based on the TMS, and the value was expressed in δ values (ppm), with the coupling constants were described with Hz. CD spectra were measured on a Bio-Logic Science MOS-450 spectrometer (Bio-Logic, Claix, France). Silica gel GF254 prepared for TLC and silica gel (200–300 mesh) for column chromatography (CC) were obtained from Qingdao Marine Chemical Factory (Qingdao, China). Octadecyl silica gel was purchased from Merck Chemical Company Ltd (Darmstadt, Germany). Sephadex LH-20 was a product of Pharmacia (Amersharm, Sweden). RP-HPLC separations were conducted using an LC-6AD liquid chromatography with a YMC Pack ODS-A column (250 × 20 mm, 5 μm, 120 Å) and SPD-10AVP UV/vis detector (Shimadzu, Kyoto, Japan). GC analysis was performed on GL-2010 (Shimadzu, Japan). All HPLC or analytical grade reagents were purchased from Tianjin Damao Chemical Company (Tianjin, China). Traces of samples on silica gel plates were detected under UV light and heating after spraying with anisaldehyde-H2SO4 reagent. Table 2. Inhibitory effects of compounds 1–4 on NO production induced by LPS in macrophages. Compound 1 2 3 4 Hydrocortisonea a
Positive control.
IC50 ± SD (μM) 50.7 ± 3.1 >100 24.0 ± 2.1 28.6 ± 2.4 37.6 ± 2.9
CC50 (μM) >100 >100 >100 >100 >100
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3.2. Plant materials The fresh whole plants of Apium graveolens were collected from Shenyang, Liaoning Province, China, and were indentified by Prof Qishi Sun, Department of Pharmacognosy, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. The specimen (AP-2007-716) was deposited in the Herbarium of the Department of Natural Products Chemistry, Shenyang Pharmaceutical University.
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3.3. Extraction and isolation The fresh whole grass of Apium graveolens was cut into pieces (100 kg) and extracted with 70% EtOH (3 × 150 L × 2 h). The crude extract was suspended in water and partitioned successively with petroleum ether (PE) and ethyl acetate (EtOAc) in the same volume for three times. The solvent of the EtOAc layer was evaporated under vacuum to yield the residue (75 g), which was divided on silica gel CC and eluted by CH2Cl2/MeOH (100:1 to 0:100) into fifteen fractions (E1-E15) according to TLC analysis. Fraction E13 (5.2 g) was subjected to Sephadex LH-20 CC (MeOH), which generated two fractions (E131-E132). E132 was separated by open ODS CC and eluted by MeOH/H2O (1:9 to 1:0), producing five fractions (E1321-E1325). E1325 was purified by preparative HPLC (55% MeOH/H2O, flow rate 6.0 ml/min) to afford 1 (36.5 mg, tR = 60 min). E1323 was separated via preparative HPLC (40% MeOH/H2O, flow rate 6.0 ml/min) to yield five subfractions (E13231-E13235), E13233 was further subjected to preparative TLC [CH2Cl2/MeOH (5:1)] to give 2 (9.5 mg). Fraction E7 (10 g) was subjected to Sephadex LH-20 CC and eluted with CH2Cl2/MeOH (1:1) to yield E71, E72, and E73. E72 was separated on ODS CC and eluted with MeOH/ H2O (1:9 to 1:0) to obtain five subfractions (E721-E725), and E721 was separated by preparative HPLC (30% MeOH/H2O, flow rate 6.0 ml/min) to afford 3 (8.8 mg, tR = 15 min). The solvent of the PE layer was evaporated to dryness under vacuum to yield the residue (48 g), which was separated on silica gel CC and eluted by CH2Cl2/acetone (100:1 to 0:100) to afford eleven fractions (P1-P11) according to TLC analysis. Fraction P5 was subject to preparative TLC [PE/EtOAc (4:1)] to give 4 (7.1 mg). 3.3.1. Compound 1 White power (MeOH); [𝛼]20 D −140.0 (c 0.40, MeOH); UV (MeOH) 𝜆max (log ε) 276 (3.33) nm; IR (KBr) vmax 3424, 2905, 1765, 1727, 1384, 1080 cm−1; 1H (300 MHz, CD3OD) and 13 C-NMR (75 MHz, CD3OD) spectral data, see Table 1; HR-ESI-MS: m/z 393.1527 [M+Na]+ (calcd for C18H26O8Na, 393.1525). 3.3.2. Compound 2 Colorless oil (MeOH); [𝛼]20 D −100.0 (c 0.30, MeOH); UV (MeOH) 𝜆max (log ε) 234 (3.92) nm; IR (KBr) vmax 3921, 2923, 1648, 1384 cm−1; 1H (300 MHz, CD3OD) and 13C-NMR (75 MHz, CD3OD) spectral data, see Table 1; HR-ESI-MS: m/z 393.1889 [M+Na]+ (calcd for C19H30O7Na, 393.1885). 3.4. Acid hydrolysis and GC analysis of compounds 1 and 2 The method for detecting D-sugar that we took was to hydrolyze compounds 1 and 2, and then compared the retention time with the standard D-glucose through GC analysis.
JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
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Compounds 1 and 2 were hydrolyzed with 15% HCl (2 ml) for 2 h at 90 °C, respectively. Then, the mixture was cooled to room temperature and extracted twice with an equal volume of ethyl acetate, the extracts were combined to give the aglycone fraction, and the aqueous layer was concentrated in vacuo to afford the residues. Hydroxylamine hydrochloride (2 mg) and pyridine (1 ml) were added to every residues and heated at 90 °C for 1 h to form sugar alcohol, and then acetic anhydride was added and heated to 90 °C for 1 h to form a solution of glycoside acetate derivative. The derivative was analyzed by GC, using the DM-5 column (30 × 0. 25 mm, 0.25 μm) and FID detector. The GC spectrum of compounds 1 and 2 showed the peaks at 19.3 min, and the standard D-glucose also presented the peak at 19.3 min.
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3.5. NO production bioassay The nitrite concentration in the medium was measured as an indicator of NO production according to the Griess reaction. Briefly, RAW 264.7 cells were seeded into 96-well tissue culture plates at a density of 1 × 105 cells/well and stimulated with 1 μg/ml of LPS with or without of test compounds. After incubation at 37 °C for 24 h, 100 μl of cell-free supernatant was mixed with 100 μl of Griess reagent [mixture of equal volumes of reagent A and reagent B, A : 1% (w/v) sulfanilamide in 5% (w/v) phosphoric acid, B : 0.1% (w/v) N-(1-naphthyl) ethylenediamine]. Absorbance (540 nm) was measured in a microplate reader. The NO concentrations and inhibitors rates were calculated by a calibration curve.
Disclosure statement No potential conflict of interest was reported by the authors.
References [1] S.K. Garg, S.R. Gupta, and N.D. Sharama, Planta Med. 38, 363 (1980). [2] J.L. Lv, R. Islam, A.H. Aisa, and L.X. Liao, Chin. Tradit. Pat. Med. 29, 406 (2007). [3] S.K. Garg, S.R. Gupta, and N.D. Sharma, Phytochemistry 17, 2135 (1978). [4] S.K. Garg, S.R. Gupta, and N.D. Sharma, Phytochemistry 18, 1580 (1979). [5] A.K. Jain, N.D. Sharma, S.R. Gupta, and D.R. Boyd, Planta Med. 52, 246 (1986). [6] S.K. Garg, S.R. Gupta, and N.D. Sharma, Phytochemistry 18, 1764 (1979). [7] R.A. Momin and M.G. Nair, J. Agric. Food. Chem. 49, 142 (2001). [8] D. Hefr, Pharmazie 34, 658 (1979). [9] R.A. Momin, R.S. Ramsewak, and M.G. Nair, J. Agric. Food. Chem. 48, 3785 (2000). [10] J. Kitajima, T. Ishikawa, and M. Satoh, Phytochemistry 64, 1003 (2003). [11] G. Macleod and J.M. Ames, Phytochemistry 28, 1817 (1989). [12] J. Tang, Y.G. Zhang, T.G. Hartman, and R.T. Rosen, J. Agric. Food. Chem. 38, 1937 (1990). [13] D. Tsi, N.P. Das, and B.K. Tan, Planta Med. 61, 18 (1995). [14] J. Yan, L. Yu, S. Xu, W.H. Gu, and W.M. Zhu, Sci. Hortic. Amsterdam 165, 218 (2014). [15] K.L. Zhou, F. Zhao, Z.H. Liu, Y.L. Zhuang, L.X. Chen, and F. Qiu, J. Nat. Prod. 72, 1563 (2009). [16] M.S. Kamdem, M.L. Sameza, P.M.J. Dongmo, and F.F. Boyom, J. Life. Sci. 16, 51 (2015). [17] H.Z. Marzouni, N. Daraei, N. Sharafi-Ahvazi, N. Kalani, and W. Kooti, J. Pharm. Pharm. Sci. 5, 1710 (2016). [18] R.A. Momin and M.G. Nair, J. Agric. Food. Chem. 49, 142 (2001). [19] R.A. Lloyd, C.W. Miller, D.L. Roberts, and J.A. Giles, Tobacco. Sci. 20, 125 (1976). [20] Q. Wei, J.B. Yang, J. Ren, A.G. Wang, T.F. Ji, and Y.L. Su, Fitoterapia 93, 226 (2014). [21] Y. Tao, W. Jiang, Y.Y. Cheng, and Y.F. Zhang, J. Asian Nat. Prod. Res. 14, 826 (2012).
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[22] C. Lee, S. Lee, and S.Y. Park, Nat. Prod. Sci. 19, 355 (2013). [23] I. Horibe, S. Seo, Y. Yoshimura, and K. Tori, Org. Magn. Reson. 22, 428 (1984). [24] A. Nagahisa, Y. Kanai, O. Suga, and K. Taniguchi, Eur. J. Pharmacol. 217, 191 (1992).
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Constituents from Apium graveolens and their anti-inflammatory effects Li-Han Zhu, Tian-Hui Bao, Yue Deng, Hua Li & Li-Xia Chen To cite this article: Li-Han Zhu, Tian-Hui Bao, Yue Deng, Hua Li & Li-Xia Chen (2017): Constituents from Apium graveolens and their anti-inflammatory effects, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2017.1381687 To link to this article: http://dx.doi.org/10.1080/10286020.2017.1381687
View supplementary material
Published online: 03 Oct 2017.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ganp20 Download by: [Tufts University]
Date: 04 October 2017, At: 21:59
Journal of Asian Natural Products Research, 2017 https://doi.org/10.1080/10286020.2017.1381687
Constituents from Apium graveolens and their anti-inflammatory effects Li-Han Zhua, Tian-Hui Baoa, Yue Denga, Hua Lia,b and Li-Xia Chena a
Downloaded by [Tufts University] at 21:59 04 October 2017
Key Laboratory of Structure-Based Drug Design & Discovery, School of Traditional Chinese Materia Medica, Wuya College of Innovation, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China; bSchool of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
ABSTRACT
A phthalide glycoside, (3R, 4R)-4-O-β-D-glucopyranosyl-senkyunolide (1), and a megastigmane glycoside, (6S, 7R)-3-oxo-megastigma-4, 8-dien-7-O-β-D-glucoside (2), along with two known aglycones (3–4), were isolated from the 70% EtOH extract of fresh whole grass of Apium graveolens L. Their structures were elucidated by extensive spectroscopic analysis. All of these compounds were tested for their inhibitory effects on nitric oxide (NO) production in the RAW 264.7 macrophages. Among them, compounds 3 and 4 showed potent inhibitory activity against LPS-induced nitric oxide production in RAW 264.7 macrophages, with IC50 values of 24.0 ± 2.1 μM and 28.6 ± 2.4 μM, respectively.
ARTICLE HISTORY
Received 29 June 2017 Accepted 14 September 2017 KEYWORDS
Apium graveolens; phthalide glycoside; megastigmane glycoside; nitric oxide
O OH
O
OH O
HO HO
O
HO HO
OH 1
O OH 2
O O
1. Introduction The genus (Apium) with 20 species is distributed widely in temperate regions around the world, and two species were found in China. Apium graveolens has been used as a wellknown vegetable, and most of the people like it, which contains flavonoids [1,2], coumarins [3–6], phthalides [7–9], sesquiterpenes [10], volatile oil [11,12], and so on. A lot of studies showed that the crude extract of A. graveolens possessed prominent anti-inflammatory, anti-hypertensive, hypolipidemic, antioxidant, anti-cancer, and other effects [13–17]. CONTACT Li-Xia Chen [email protected]; Hua Li [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/10286020.2017.1381687. © 2017 Informa UK Limited, trading as Taylor & Francis Group
2
L.-H. ZHU ET AL.
In order to find the anti-inflammatory material basis of A. graveolens, the EtOH extract of the whole grass of this plant was investigated, and two new compounds, along with two known compounds, were isolated. Their structures were elucidated by comprehensive spectroscopic analysis. All of these compounds were tested for their inhibitory effects on nitric oxide (NO) production in RAW 264.7 macrophages.
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2. Results and discussion The fresh whole grass of A. graveolens was cut into pieces and extracted with 70% EtOH. The extract was suspended in water and then partitioned with petroleum ether (PE) and ethyl acetate (EtOAc) successively. Then, the EtOAc layer was separated by silica gel, Sephadex LH-20, ODS open column chromatography (CC), and HPLC to produce two new compounds, a phthalide glycoside, (3R, 4R)-4-O-β-D-glucopyranosyl-senkyunolide (1), and a megastigmane glycoside, (6S, 7R)-3-oxo-megastigma-5,8-dien-7-O-β-D-glucoside(2), along with two known aglycones, sedanolide (3) [18] and (3S)-3-hydroxy-megastigma5,8-dien-7- one (4) [19] (Figure 1). Compound 1 was isolated as white powder (MeOH). Molish reaction was positive, and glucose was detected after acid hydrolysis. The molecular formula C18H26O8 was deduced by HR-ESI-MS data at m/z 393.1527 [M+Na]+, with six degrees of unsaturation. The anomeric proton signal at δH 4.42 (1H, d, J = 7.8 Hz) showed the glucose with β-anomeric configuration. The signals at δH 1.64 (1H, dd, J = 15.2, 6.7 Hz), 2.10–2.16 (1H, m), 1.37–1.42 (2H, m), 1.37–1.42 (2H, m), and 0.95 (3H, t, J = 7.1 Hz) in the 1H-NMR (Table 1) spectrum, in combination with the 1H-1H COSY correlations (Figure 2) of H-3 with H-1′, H-1′ with H-2′, H-2′ with H-3′, and H-3′ with H-4′ indicated that the presence of a n-butyl and two olefinic
Figure 1. Structures of compounds 1–4.
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Table 1. 1H- and 13C-NMR spectroscopic data for compounds 1 and 2 in CD3OD (at 300 and 75 MHz, respectively) 1
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Position 1 2
2
δC 173.5
δH (J in Hz)
3 4 5
83.9 69.1 31.9
6 7 7a 3a 8 9 10 11 12 13 1′
116.2 130.8 127.0 159.9
5.43, dd (7.5, 3.1) 4.80–4.87, m 2.76, br dd (15.6, 8.8) 2.88, dt (15.6, 4.5) 6.14, dt (9.5, 4.0) 6.23, br d (9.5)
2′ 3′ 4′ 5′ 6′
28.1 23.7 14.4
1″ 2″ 3″ 4″ 5″ 6″
103.5 74.9 78.2 71.5 78.0 62.7
32.7
1.64, dd (15.2, 6.7) 2.10–2.16, m 1.37–1.42, m 1.37–1.42, m 0.95, t (7.1)
4.42, d (7.8) 3.13, t (7.8) 3.33–3.36, m 3.28–3.31, m 3.32–3.39, m 3.71, dd (11.6, 6.9) 3.89, br d (11.6)
δH (J in Hz)
δC 37.2 48.4
2.00, d (16.8) 2.40, d (16.8)
202.1 126.2 166.0
5.84, br s
56.8 77.1
2.64, d (8.9) 4.30–4.38, m
138.3 128.9 21.2 23.9 28.2 27.7 102.5
5.73, dd (15.6, 6.5) 5.56–5.64, m 1.25, d (6.3) 1.90, s 0.99, s 0.97, s 4.31, d (7.8)
75.3 78.2 71.6 78.0 62.8
3.11–3.20, m 3.11–3.20, m 3.23–3.34, m 3.23–3.34, m 3.79, dd (11.8, 2.0) 3.64, dd (11.8, 5.1)
Figure 2. Key correlations of 1H-1H COSY, HMBC, and NOESY of compounds 1 and 2.
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protons [δH 6.14 (1H, dt, J = 9.5, 4.0 Hz), 6.23 (1H, br d, J = 9.5 Hz)]. The 13C-NMR (Table 1) spectrum exhibited four characteristic carbon resonances at δC 173.5, 159.9, 127.0, and 83.9, typical signals of a five-membered α, β-unsaturated lactone. The HMBC correlations (Figure 2) from H-6 to C-7a/C-3a, H-7 to C-7a/C-3a indicated the presence of one pair of conjugated double bonds. The correlations of H-7 with C-7a, C-3a and H-5 with C-7a, C-3a, and C-4 revealed that the connection of the α, β-unsaturated lactone ring and the six-membered ring was through C-3a and C-7a [20]. The HMBC correlation of H-1′ with C-3 suggested that the butyl moiety was located at C-3, and the correlation of H-1″ with C-4 showed that the glucose moiety was attached to C-4. Thus, the structure of compound 1 was determined as 4-O-β-D-glucopyranosyl-senkyunolide. The relative configuration of compound 1 was elucidated based on the analysis of NOESY cross-peak. The NOESY correlation (Figure 2) of H-3 with H-4 suggested that H-3 and H-4 were on the same plane. The absolute configuration of compound 1 was determined by the application of CD octant rule and sector rule of carboxylic ester. The CD spectrum showed positive Cotton effect at 210 nm and negative Cotton effect at 240 nm, revealing that the configurations of C-3 and C-4 were R and R, respectively [20]. Thus, the structure of compound 1 was determined as (3R, 4R)-4-O-β-D-glucopyranosyl-senkyunolide. Compound 2 was isolated as colorless oil (MeOH). Molish reaction was positive, and the glucose was obtained after acid hydrolysis, indicating that compound 2 is a glucoside. The molecular formula C19H30O7 was determined by HR-ESI-MS data at m/z 393.1889 [M+Na]+, with five degrees of unsaturation. The signal at δH 4.31 (1H, d, J = 7.8 Hz) indicated that the glucose has β-anomer. The 1H-NMR (Table 1) spectrum revealed the appearance of four methyl groups [δH 1.25 (3H, d, J = 6.3 Hz), 1.90 (3H, s), 0.99 (3H, s), and 0.97 (3H, s)] and three olefinic protons [δH 5.84 (1H, br s), 5.73 (1H, dd, J = 15.6, 6.5 Hz), and 5.56–5.64 (1H, m)]. The 13C-NMR (Table 1) spectrum displayed nine typical signals, corresponding to four methyl carbons (δC 28.2, 27.7, 23.9, and 21.2), one carbonyl carbon (δC 202.1) and four olefinic carbons (δC 126.2, 128.9, 138.3, and 166.0). Among them, the signals at δC 202.1, 126.2, and 166.0 are characteristic signals of an α, β-unsaturated ketone group. In addition, a ring existed in the structure according to the five degrees of unsaturation. In the HMBC spectrum, the long-range correlations (Figure 2) of H-12, H-13 with C-1/C6/C-2, and H-11 with C-5 suggested the presence of a 1,1,5-trimethylcyclohex-4-enone ring. The proton signals (Table 1) at δH 5.73 (1H, dd, J = 15.6, 6.5 Hz), 5.56–5.64 (1H, m), and 1.25 (3H, d, J = 6.3 Hz), as well as the long-range correlations (Figure 2) from H-7 to C-1/C-6/C-8/C-9/C-10 in HMBC spectrum showed that a (E)-butenyl group was linked to C-6 of 1,1,5-trimethylcyclohex-4-enone ring. The HMBC correlations (Figure 2) of H-7 with C-1′ and H-1′ with C-7 suggested the glucose attached to C-7 [21]. The absolute configuration of C-6 in compound 2 was determined by CD spectrum with negative Cotton effect at 324 nm and positive Cotton effect at 250 nm, indicating that the configuration of C-6 was S [22]. The absolute configuration of C-7 in compound 2 was determined by the glucosidation shifts according to the reference [23]. When secondary allylic alcohols and β-D-glucoses connected to form glycosides, the chemical shifts of β-carbon decreased in comparison with those of the aglycone. In the structure with R or S configuration of the α-carbon, glucosidation shift of β-carbon with sp2 hybridization was −4.4 ± 0.5 ppm or −3.2 ± 0.5 ppm, respectively. The chemical shift of C-8 (β-C) of compound 2 was δC 135.0 in pyridine-d5, and that of the aglycone was δC 137.8, this difference value of chemical shift
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was −2.8 ppm, so the configuration of C-7 was R. Therefore, the structure of compound 2 was elucidated as (6S, 7R)-3-oxo-megastigma-4, 8-dien-7-O-β-D-glucoside. Inflammation is a defensive response to endogenous or exogenous stimuli in the living tissue of vascular system. Nitric oxide (NO) is an indispensable regulator of the immune system and can cause tissue damage due to its excessive production. Therefore, NO is not only an inflammatory factor, but also an immunoregulatory effector, and the amount of NO production plays a vital role in maintaining human health [24]. In our study, all of these isolated compounds were tested for their inhibitory activities against NO production in LPS-induced macrophages (Table 2). Compounds 1–4 showed no cytotoxicity (0–100 μM) on RAW 264.7 macrophage. Compounds 3 and 4 showed potent inhibitory activity against LPS-induced nitric oxide production in RAW 264.7 macrophages with IC50 values of 24.0 ± 2.1 μM and 28.6 ± 2.4 μM, respectively, comparable to the positive control of hydrocortisone (IC50 of 37.6 ± 2.9 μM). Certainly, the anti-inflammatory mechanisms of these compounds are required to further study.
3. Experimental 3.1. General experimental procedures Optical rotations were determined on a PerkinElmer 241 polarimeter (Perkin-Elmer, Waltham, MA, U.S.A.). HRESIMS were received on an Agilent 6210 TOF mass spectrometer (Palo Alto, U.S.A.). IR (4000−400 cm−1) spectra (KBr disks) were recorded on a Bruker IFS 55 spectrometer (Bruker Optics, Ettlingen, Germany), and UV spectra on a Shimadzu UV 1700UV-VIS recording spectrophotometer (Shimadzu Corporation, Kyoto, Japan). NMR spectra were performed on Bruker ARX-300 spectrometers (Bruck Biospin, Fallanden, Switzerland). Chemical shifts were based on the TMS, and the value was expressed in δ values (ppm), with the coupling constants were described with Hz. CD spectra were measured on a Bio-Logic Science MOS-450 spectrometer (Bio-Logic, Claix, France). Silica gel GF254 prepared for TLC and silica gel (200–300 mesh) for column chromatography (CC) were obtained from Qingdao Marine Chemical Factory (Qingdao, China). Octadecyl silica gel was purchased from Merck Chemical Company Ltd (Darmstadt, Germany). Sephadex LH-20 was a product of Pharmacia (Amersharm, Sweden). RP-HPLC separations were conducted using an LC-6AD liquid chromatography with a YMC Pack ODS-A column (250 × 20 mm, 5 μm, 120 Å) and SPD-10AVP UV/vis detector (Shimadzu, Kyoto, Japan). GC analysis was performed on GL-2010 (Shimadzu, Japan). All HPLC or analytical grade reagents were purchased from Tianjin Damao Chemical Company (Tianjin, China). Traces of samples on silica gel plates were detected under UV light and heating after spraying with anisaldehyde-H2SO4 reagent. Table 2. Inhibitory effects of compounds 1–4 on NO production induced by LPS in macrophages. Compound 1 2 3 4 Hydrocortisonea a
Positive control.
IC50 ± SD (μM) 50.7 ± 3.1 >100 24.0 ± 2.1 28.6 ± 2.4 37.6 ± 2.9
CC50 (μM) >100 >100 >100 >100 >100
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3.2. Plant materials The fresh whole plants of Apium graveolens were collected from Shenyang, Liaoning Province, China, and were indentified by Prof Qishi Sun, Department of Pharmacognosy, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. The specimen (AP-2007-716) was deposited in the Herbarium of the Department of Natural Products Chemistry, Shenyang Pharmaceutical University.
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3.3. Extraction and isolation The fresh whole grass of Apium graveolens was cut into pieces (100 kg) and extracted with 70% EtOH (3 × 150 L × 2 h). The crude extract was suspended in water and partitioned successively with petroleum ether (PE) and ethyl acetate (EtOAc) in the same volume for three times. The solvent of the EtOAc layer was evaporated under vacuum to yield the residue (75 g), which was divided on silica gel CC and eluted by CH2Cl2/MeOH (100:1 to 0:100) into fifteen fractions (E1-E15) according to TLC analysis. Fraction E13 (5.2 g) was subjected to Sephadex LH-20 CC (MeOH), which generated two fractions (E131-E132). E132 was separated by open ODS CC and eluted by MeOH/H2O (1:9 to 1:0), producing five fractions (E1321-E1325). E1325 was purified by preparative HPLC (55% MeOH/H2O, flow rate 6.0 ml/min) to afford 1 (36.5 mg, tR = 60 min). E1323 was separated via preparative HPLC (40% MeOH/H2O, flow rate 6.0 ml/min) to yield five subfractions (E13231-E13235), E13233 was further subjected to preparative TLC [CH2Cl2/MeOH (5:1)] to give 2 (9.5 mg). Fraction E7 (10 g) was subjected to Sephadex LH-20 CC and eluted with CH2Cl2/MeOH (1:1) to yield E71, E72, and E73. E72 was separated on ODS CC and eluted with MeOH/ H2O (1:9 to 1:0) to obtain five subfractions (E721-E725), and E721 was separated by preparative HPLC (30% MeOH/H2O, flow rate 6.0 ml/min) to afford 3 (8.8 mg, tR = 15 min). The solvent of the PE layer was evaporated to dryness under vacuum to yield the residue (48 g), which was separated on silica gel CC and eluted by CH2Cl2/acetone (100:1 to 0:100) to afford eleven fractions (P1-P11) according to TLC analysis. Fraction P5 was subject to preparative TLC [PE/EtOAc (4:1)] to give 4 (7.1 mg). 3.3.1. Compound 1 White power (MeOH); [𝛼]20 D −140.0 (c 0.40, MeOH); UV (MeOH) 𝜆max (log ε) 276 (3.33) nm; IR (KBr) vmax 3424, 2905, 1765, 1727, 1384, 1080 cm−1; 1H (300 MHz, CD3OD) and 13 C-NMR (75 MHz, CD3OD) spectral data, see Table 1; HR-ESI-MS: m/z 393.1527 [M+Na]+ (calcd for C18H26O8Na, 393.1525). 3.3.2. Compound 2 Colorless oil (MeOH); [𝛼]20 D −100.0 (c 0.30, MeOH); UV (MeOH) 𝜆max (log ε) 234 (3.92) nm; IR (KBr) vmax 3921, 2923, 1648, 1384 cm−1; 1H (300 MHz, CD3OD) and 13C-NMR (75 MHz, CD3OD) spectral data, see Table 1; HR-ESI-MS: m/z 393.1889 [M+Na]+ (calcd for C19H30O7Na, 393.1885). 3.4. Acid hydrolysis and GC analysis of compounds 1 and 2 The method for detecting D-sugar that we took was to hydrolyze compounds 1 and 2, and then compared the retention time with the standard D-glucose through GC analysis.
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Compounds 1 and 2 were hydrolyzed with 15% HCl (2 ml) for 2 h at 90 °C, respectively. Then, the mixture was cooled to room temperature and extracted twice with an equal volume of ethyl acetate, the extracts were combined to give the aglycone fraction, and the aqueous layer was concentrated in vacuo to afford the residues. Hydroxylamine hydrochloride (2 mg) and pyridine (1 ml) were added to every residues and heated at 90 °C for 1 h to form sugar alcohol, and then acetic anhydride was added and heated to 90 °C for 1 h to form a solution of glycoside acetate derivative. The derivative was analyzed by GC, using the DM-5 column (30 × 0. 25 mm, 0.25 μm) and FID detector. The GC spectrum of compounds 1 and 2 showed the peaks at 19.3 min, and the standard D-glucose also presented the peak at 19.3 min.
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3.5. NO production bioassay The nitrite concentration in the medium was measured as an indicator of NO production according to the Griess reaction. Briefly, RAW 264.7 cells were seeded into 96-well tissue culture plates at a density of 1 × 105 cells/well and stimulated with 1 μg/ml of LPS with or without of test compounds. After incubation at 37 °C for 24 h, 100 μl of cell-free supernatant was mixed with 100 μl of Griess reagent [mixture of equal volumes of reagent A and reagent B, A : 1% (w/v) sulfanilamide in 5% (w/v) phosphoric acid, B : 0.1% (w/v) N-(1-naphthyl) ethylenediamine]. Absorbance (540 nm) was measured in a microplate reader. The NO concentrations and inhibitors rates were calculated by a calibration curve.
Disclosure statement No potential conflict of interest was reported by the authors.
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