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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
A new tetrahydrofuran-type lignan with anti-inflammatory activity from Asarum heterotropoides Fr. Schmidt var. mandshuricum a
a
a
b
Jian Huang , Hui-Qing Wang , Cui Zhang , Guo-Yu Li , Rui-Chao c
Lin & Jin-Hui Wang
ab
a
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
Click for updates
b
School of Pharmacy, Shihezi University, Shihezi 832002, China
c
National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China Published online: 05 Aug 2013.
To cite this article: Jian Huang, Hui-Qing Wang, Cui Zhang, Guo-Yu Li, Rui-Chao Lin & Jin-Hui Wang (2014) A new tetrahydrofuran-type lignan with anti-inflammatory activity from Asarum heterotropoides Fr. Schmidt var. mandshuricum, Journal of Asian Natural Products Research, 16:4, 387-392, DOI: 10.1080/10286020.2013.820713 To link to this article: http://dx.doi.org/10.1080/10286020.2013.820713
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2014 Vol. 16, No. 4, 387–392, http://dx.doi.org/10.1080/10286020.2013.820713
A new tetrahydrofuran-type lignan with anti-inflammatory activity from Asarum heterotropoides Fr. Schmidt var. mandshuricum Jian Huanga, Hui-Qing Wanga, Cui Zhanga, Guo-Yu Lib*, Rui-Chao Linc and Jin-Hui Wanga,b* a
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School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China; bSchool of Pharmacy, Shihezi University, Shihezi 832002, China; cNational Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China (Received 30 March 2013; final version received 26 June 2013) A new tetrahydrofuran-type lignan, episesaminone (1), was isolated from Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. Its structure was established by spectroscopic techniques (HR-MS, 1D NMR, 2D NMR, and circular dichroism). The anti-inflammatory activity in RAW 264.7 macrophages was carried out on 1 and other eight known compounds, the epimer of 1 (2) and seven known furofurans-type lignan (3 – 9) obtained from A. heterotropoides Fr. Schmidt var. mandshuricum. Compounds 1, 2, 3, 4, 5, 7, and 9 showed significant anti-inflammatory activity, particularly 50 mM compound 3 inhibited 69.2% NO production compared with the lipopolysaccharide group. Keywords: Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag.; episesaminone; NMR; CD; anti-inflammatory activity
1.
Introduction
Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag., a traditional Chinese medicine known as ‘Liaoxixin’, is widely distributed in the north-east of China. The herb has been used for the treatment of cold, analgesic, antitussive, or anti-allergic remedy [1]. Some phytochemical studies on the herb revealed the presence of volatile oil [2], flavonoids [3], aristolochic acids [4], and furofurans-type lignan [5]. Takasaki et al. [6] reported that furofurans-type lignan showed potent anti-tumor-promoting activities. Additionally, Macrae and Towers [7] claimed that furofurans-type lignan also played an important biological role in antimitosis, antimicrobe, and antioxidation. Interested in finding more biologically active substances from this folk medicine, we further undertook the investigation on its phytochemical com-
position. As a result, a new tetrahydrofuran-type lignan, named episesaminone (1), together with eight known furan-type lignans, sesaminone (2) [8], 4-((1S,3aR, 4R,6aR)-4-(benzo[d][1,3]dioxol-5-yl)hexahydrofuro[3,4-c]furan-1-yl)benzene-1, 2-diol (3) [9], epipinoresinol (4) [10], (1R,2S,5R,6R)-50 -O-methylpluviatilol (5) [11], piperitol (6) [12], pluviatilol (7) [13], (2)-sesamin (8) [12], and asarinin (9) [12], was isolated from the dried roots of A. heterotropoides Fr. Schmidt var. mandshuricum (Figure 1). This paper deals with their structural elucidation and anti-inflammatory activity in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages.
2. Results and discussion Compound 1 was obtained as a colorless solid (MeOH) with the molecular formula C20H18O7 as determined by the HR-ESI-
*Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis
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J. Huang et al. O 4' O 3'
5' 6' 1'
2' HO
7' O 9
8' 9'
8
6
7
5 4
1
O
O 2
3 O
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Figure 1. Structure of compound 1.
MS at m/z 371.1122 [M þ H]þ, indicating twelve degrees of unsaturation. The 1H NMR spectrum of compound 1 showed six aromatic proton signals at dH 7.80 (1H, dd, J ¼ 8.1, 1.5 Hz, H-6), 7.57 (1H, d, J ¼ 1.5 Hz, H-2), 6.93(1H, d, J ¼ 8.1 Hz, H-5), 6.82 (1H, br s, H-20 ), and 6.77 (2H, br s, H-50 , 60 ), which were assigned to two 1,3,4-trisubstituted benzene rings. The presence of each of 2H singlets at dH 6.06 and dH 5.92, assigned to methylene carbons (dC 103.5 and 102.3), suggested
that both benzene rings have 3,4-methylenedioxyl groups. The fact that the carbonyl group is connected to a part of the phenyl group was indicated by a carbonyl carbon signal at dC 199.9 and the conjugated property absorption peak shown in the IR spectrum at 1669 cm21. The 1H NMR spectrum also exhibited resonances of a 2,3,4-trisubstituted tetrahydrofuran [dH 4.98 (1H, d, J ¼ 6.3 Hz, H-70 ), 4.42 (1H, t, J ¼ 8.1 Hz, H-9a), 4.30 (1H, m, H-8), 4.06 (1H, dd, J ¼ 8.4, 5.4 Hz, H-9b), and 2.86 (1H, qd, J ¼ 7.5, 6.3 Hz, H-80 )] (Table 1). And this was further confirmed by the signals [dC 83.1 (C-70 ), 70.5 (C-9), 51.2 (C-80 ), and 50.4 (C-8)] in the 13C NMR spectrum (75 MHz, MeOD) (Table 1). Careful analysis of the correlations in the HMQC and HMBC spectra led to the establishment of the planar structure of compound 1 (Figure 1). The hydroxymethyl was linked to C-8 0 , which was deduced from the long-range
Table 1. 1H and 13C NMR spectral data of compound 1 (MeOD) (1H: 300 MHz, 13C: 75 MHz, d: ppm, J: Hz). 1
H NMR
No 1 2 3 4 5 6 7 8 9a 9b 10 20 30 40 50 60 70 80 90 a 90 b OCH2O OCH2O
1
13
Sesaminone
7.57, d, J ¼ 1.5
7.46
6.93, d, J ¼ 8.1 7.80, dd, J ¼ 8.1, 1.5
6.84 7.57
4.30, m 4.42, t, J ¼ 8.1 4.06, dd, J ¼ 8.1, 5.4
4.12 4.12 4.25
6.82, br s
6.96
6.77, br s 6.77, br s 4.98, d, J ¼ 6.3 2.86, qd, J ¼ 7.5, 6.3 3.18, d, J ¼ 7.5 3.18, d, J ¼ 7.5 6.06, s 5.92, s
6.76 6.84 4.65 2.84 3.64 3.74 6.06 5.95
C NMR 1 132.4 109.2 149.8 153.7 108.9 126.6 199.9 50.4 70.5 133.6 107.6 148.2 149.1 108.8 120.3 83.1 51.2 62.1 103.5 102.3
NOESY 1
HMBC 1
8
C-3, 4, 6, 7
8
C-1, 3, 4 C-4, 5, 7
2, 6, 90 a, 90 b 90 a 70 , 80
C-80 , 90 , 9 C-70 , 80 , 8, 7
70 , 90 a, 90 b
C-10 , 30 , 40 , 60
70 , 90 a, 90 b 9b, 20 , 60 9b 9a, 20 , 60 , 8 8, 20 , 60
C-70 C-50 , 40 , C-10 , 20 , 60 C-70 , 80 , 8 C-3, 4 C-30 , 40
Journal of Asian Natural Products Research
O O
O HO
O
O O
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Figure 2. Key HMBC correlations of compound 1.
correlations in the HMBC spectrum (Figure 2) from the protons at dH 3.18 (2H, br d, J ¼ 7.5 Hz) to C-8 (dC 50.4), C80 (dC 51.2), and C-70 (dC 83.1). The correlations from H-70 at dH 4.98 to C-20 at dC 107.6, C-60 at dC 120.3, and C-100 at dC 133.6 suggested that C-70 of tetrahydrofuran was connected with C-10 of the benzene ring. The correlations from H-9b at dH 4.06, H-9a at dH 4.42 to C-7 at dC 199.9 indicated that the carbonyl carbon linked to C-8 of the tetrahydrofuran ring. The relative stereostructure was established by the NOESY correlations. The NOESY cross-peaks between H-9b and H70 , H-9b and H-80 , H-8 and H-90 a, H-8 and H-90 b suggested the relative configuration of H-70 and H-80 to be cis and H-80 and H-8 to be trans (Figure 3). Furthermore, this relative configuration of 1 was also supported by the fact that H-9a0 and H9b0 showed a upfield shift of 0.46 and 0.56 ppm, respectively, by comparison of
Figure 3.
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the 1H NMR data with those of the known compound, sesaminone [8], which was due to the change of shielding effect of the benzene rings linked to C-70 on the hydroxymethyl. The absolute configuration of 1 was elucidated by the circular dichroism (CD) exciton chirality method [14]. A negative Cotton effect at 269.0 nm (2 0.20) (Figure 4) caused by the exciton coupling of the benzoyl and benzene chromphores suggested that the absolute configuration of 8, 70 , and 80 was S, R, and R, respectively. On the basis of this evidence, the structure of 1 is shown in Figure 1, named as episesaminone. The anti-inflammatory effects of the nine compounds isolated from A. heterotropoides Fr. Schmidt var. mandshuricum were investigated in LPS-triggered RAW264.7 macrophages. The results showed that nine compounds exhibited the different degree of anti-inflammatory effect, among which compounds 1, 2, 4, 5, 7, and 9 at 50 mM, respectively, inhibited 29.7, 29.4, 31.5, 29.0, 26.9, and 26.0% NO production, while compound 3 decreased 69.2% NO level compared with the LPS group (Table 2).
3.
Experimental
3.1 General experimental procedures Optical rotations were determined on a Perkin-Elmer 241 MC polarimeter
Key NOESY correlations of compound 1.
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Figure 4. The negative exciton chirality of compound 1.
(Perkin-Elmer, Norwalk, CT, USA). UV spectra were obtained on a Shimadzu UV1700 spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were recorded on a Bruker IFS 55 spectrophotometer (Bruker, Karlsruhe, Germany). CD spectra were gotten on Biologic MOS-450 CD spectrometer (Bio-Logic, Grenoble, France). The NMR data were recorded on a Bruker AV-300 spectrometer (Bruker, Bremen, Germany). The HR-ESI-MS data were obtained on a Waters LCT Premier XE time-of-flying mass spectrometer (Waters, Milford, CT, USA). Column chromatography (CC) was performed on silica gel (200 – 300 mesh; Qingdao
Marine Chemical Group, Co., Qingdao, China), octadecylsilane (ODS) (30 – 50 mm; Tianjin Mical Reagent Co., Tianjin, China), and preparative HPLC [Hitachi-L-7110 pump, Hitachi L-7420 UV spectrophotometric detector at 220 nm (Hitachi, Tokyo, Japan), YMC C 18 reversed-phase column (5 mm, 10 mm £ 250 mm; flow rate: 2.0 ml/min). Fetal calf serum was from the Beijing Yuanhengshengma Biology Technology Research Institute (Beijing, China). Dulbecco’s Modified Eagle’s Medium (DMEM) was from Gibco/BRL (Gaithersburg, MD, USA). 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Sigma (St Louis, MO, USA).
3.2
Plant material
The roots of A. heterotropoides Fr. Schmidt var. mandshuricum were collected in Benxi, Liaoning of China in September 2010, and were identified by Prof. Jincai Lu (School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University). A voucher specimen (No. 20100912) was deposited in the Research Department of Natural Medicine, Shenyang Pharmaceutical University.
Table 2. The anti-inflammatory activity of the isolated nine compounds in RAW264.7 macrophages triggered with 1 mg/ml LPS. No LPS 1 2 3 4 5 6 7 8 9 Indomethacin
Concentration of drug (mM)
Cell viability (%)
Concentration of NO (mM)
Inhibitory rate of NO (%)
— 50 50 50 50 50 50 50 50 50 50
93.8 ^ 3.1 91.2 ^ 1.8 98.5 ^ 1.5 89.7 ^ 2.8 94.2 ^ 5.7 99.8 ^ 1.2 98.4 ^ 1.5 94.8 ^ 5.1 94.2 ^ 5.7 100.6 ^ 4.6 95.57 ^ 4.1
131.8 ^ 3.8 92.7 ^ 1.7 93.0 ^ 2.1 40.5 ^ 2.3 90.2 ^ 2.9 93.5 ^ 2.3 116.3 ^ 2.5 96.3 ^ 1.5 113.0 ^ 1.6 97.5 ^ 2.1 41.3 ^ 2.5
— 29.7 29.4 69.2 31.5 29.0 11.7 26.9 14.2 26.0 68.6
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Journal of Asian Natural Products Research 3.3 Exaction and isolation Dried roots of A. heterotropoides Fr. Schmidt var. mandshuricum (7.5 kg) were extracted three times with 60 liters 95% EtOH under reflux conditions for 3 h to give a crude extract, which was suspended in H2O and extracted with petroleum ether (PE), CHCl3, and AcOEt, successively, to yield a PE-soluble fraction (10.0 g), a CHCl3-soluble fraction (32.0 g), and an AcOEt-soluble fraction (18 g). The three parts were combined (50 g) and subjected to CC (silica gel, gradient of PE:AcOEt, 100:1 – 0:100) to afford 61 fractions (1 – 61). Fractions 7 and 25 were recrystallized to obtain compounds 9 (99.6 mg) and 7 (48.9 mg), respectively. Fraction 9 was resubjected to CC (silica gel, gradient of PE:CHCl3:AcOEt, 100:0:0 –0:100:100) to give a Fraction 9.6. Fraction 9.6 was recrystallized to get compound 8 (16.9 mg). Fraction 27 was fractionated by CC (silica gel, gradient of PE:CHCl3: AcOEt, 100:0:0 –0:100:100) to give Fraction 27.5. Fraction 27.5 was applied to preparative reversed-phase HPLC (MeOH:H2O, 60:40, flow rate 2.0 ml/min, wavelength 220 nm) and afforded compound 6 (19.0 mg, Rt 39.6 min). Fraction 35 was resubjected to CC (silica gel, gradient of PE:CHCl3:AcOEt, 100:0:00:100:100) to give a Fraction 35.5. Fraction 35.5 was separated by CC (ODS, MeOH:H2O, 60:40) to yield Fraction 35.5.5. Fraction 35.5.5 was further subjected to HPLC (MeOH:H2O, 65:35, flow rate 2.0 ml/min, wavelength 220 nm) to obtain 1 (15.3 mg, Rt 25.4 min). Fraction 40 was resubjected to CC (silica gel, gradient of PE:CHCl 3:AcOEt, 100:0:0 – 0:100:100) to give Fraction 40.2. Fraction 40.2 was applied to HPLC (MeOH:H2O, 60:40, flow rate 2.0 ml/min, wavelength 220 nm) to get compound 5 (18.4 mg, Rt 28.1 min). Fraction 45 was separated by CC (silica gel, gradient of PE: CHCl3:AcOEt, 100:0:0 – 0:100:100) to give Fraction 45.6. Fraction 45.6 was
391
also purified by HPLC (MeOH:H2O, 60:40, flow rate 2.0 ml/min, wavelength 220 nm) to get compounds 4 (7.4 mg, Rt 17.6 min), 3 (25.7 mg, Rt 21.5 min), and 2 (24.1 mg, Rt 31.8 min). 3.3.1
Episesaminone (1)
Colorless solid (MeOH); ½a21 D 2 23:1 (c ¼ 0.55, MeOH). UV (MeOH) lmax: 208.6, 230.0, and 280.4 nm; IR (KBr) vmax: 3420, 2926, 1720, 1628, 1384, 1113, 1029, and 670 cm21. CD (MeOH) D1238.0 nm þ0.21, D1269.0 nm –0.20, D1339.0 nm þ 0.14; 1 H and 13C NMR spectral data are shown in Table 1; HR-ESI-MS: m/z 371.1122 [M þ H]þ (calcd for C20H19O7, 371.1131). 3.4
Anti-inflammation activity assay
3.4.1 Measurement of NO releasing RAW264.7 cells were seeded in 96-well plates (2 £ 105/ml) and incubated at 378C in a humidified atmosphere containing 5% CO2 overnight. After 12 h, the cells were treated with 50 mM compounds for 1 h, then added LPS (1 mg/ml) for 24 h. NO levels were determined by measuring nitrite levels using the supernatant (100 ml) mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride, and 2.5% phosphoric acid) and the absorbance was measured at 570 nm [15]. The nitrite concentration was determined by using a standard curve of sodium nitrite made up in DMEM free of phenol red. C: concentration. NO inhibitory rate ð%Þ ¼
C NO ðLPSÞ 2 C NO ðcompoundÞ £ 100 CNO ðLPSÞ
3.4.2 Measurement of RAW264.7 cell viability RAW264.7 cells viability was measured by MTT assay. The cells were seeded in 96well plates (2 £ 105/ml) and incubated at
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378C in a humidified atmosphere containing 5% CO2 overnight. After 12 h, the cells were treated with 50 mM compounds for 1 h, then added LPS (1 mg/ml) for 24 h. MTT (5 mg/ ml) was subsequently added and incubated for 4 h. The crystals were dissolved in DMSO. The optical densities at 490 nm were measured with a microplate reader (TECAN SPECTRA, Wetzlar, Germany). cell viability rate ð%Þ A490 ðcompoundÞ 2 A490 ðblankÞ ¼ £ 100 A490 ðcontrolÞ 2 A490 ðblankÞ
Acknowledgments This work was financially supported by Key Projects of the National Science and Technology Pillar Program (2012BAI30B02) and National Science and Technology Major Project (2010ZX09401-304).
References [1] P.G. Xiao, Mod. Chin. Mater. Med. 3, 219 (2002). [2] D.F. Yang, R.N. Yan, and C.S. Yang, China J. Chin. Mater. Med. 22, 426 (1997). [3] T. Iwashina, J. Kitajima, Y. Shiuchi, and Y. Itou, Biochem. Syst. Ecol. 33, 571 (2005).
[4] X. Lu, D. Wu, Z.H. Wu, H.Y. Gao, B.H. Sun, and L.J. Wu, J. Shenyang Pharm. Univ. 27, 707 (2010). [5] X. Lu, L. Xu, J. Huang, H.Y. Gao, and L.J. Wu, J. Shenyang Pharm. Univ. 25, 702 (2008). [6] M. Takasaki, T. Konoshima, I. Yasuda, T. Hamano, and H. Tokuda, Biol. Pharm. Bull. 20, 776 (1997). [7] W.D. Macrae and G.H.N. Towers, Phytochemistry 23, 1207 (1984). [8] Y.M. Chung, H. Hayashi, H. Matsumoto, T. Otani, K.I. Yoshida, M.Y. Huang, R. X. Chen, J.R. Liu, and M. Nakayama, J. Antibiot. 47, 487 (1994). [9] T. Namino, N. Masaaki, O. Yoshiko, K. Yoshinori, K. Yoshinobu, and S. Hiroshi, Biol. Pharm. Bull. 35, 709 (2012). [10] M. Schottner, J. Reiner, and F.S.K. Tayman, Phytochemistry 46, 1107 (1997). [11] T.H. Quang, N.T.T. Ngan, C.V. Minh, P. V. Kiem, B.H. Tai, N.P. Thao, S.B. Song, and Y.H. Kim, Bioorg. Med. Chem. Lett. 22, 2527 (2012). [12] F. Abe, S. Yahara, K. Kubo, H. Okabe, and I. Nishioka, Chem. Pharm. Bull. 22, 2650 (1974). [13] S.Z. Choi, M.C. Yang, S.U. Choi, and K.R. Lee, Arch. Pharm. Res. 29, 203 (2006). [14] N. Harada and K. Nakanishi, J. Am. Chem. Soc. 91, 3989 (1969). [15] M.A. Titheradge, Methods Mol. Biol. 100, 83 (1998).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
A new tetrahydrofuran-type lignan with anti-inflammatory activity from Asarum heterotropoides Fr. Schmidt var. mandshuricum a
a
a
b
Jian Huang , Hui-Qing Wang , Cui Zhang , Guo-Yu Li , Rui-Chao c
Lin & Jin-Hui Wang
ab
a
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
Click for updates
b
School of Pharmacy, Shihezi University, Shihezi 832002, China
c
National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China Published online: 05 Aug 2013.
To cite this article: Jian Huang, Hui-Qing Wang, Cui Zhang, Guo-Yu Li, Rui-Chao Lin & Jin-Hui Wang (2014) A new tetrahydrofuran-type lignan with anti-inflammatory activity from Asarum heterotropoides Fr. Schmidt var. mandshuricum, Journal of Asian Natural Products Research, 16:4, 387-392, DOI: 10.1080/10286020.2013.820713 To link to this article: http://dx.doi.org/10.1080/10286020.2013.820713
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [UNAM Ciudad Universitaria] at 15:42 19 December 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2014 Vol. 16, No. 4, 387–392, http://dx.doi.org/10.1080/10286020.2013.820713
A new tetrahydrofuran-type lignan with anti-inflammatory activity from Asarum heterotropoides Fr. Schmidt var. mandshuricum Jian Huanga, Hui-Qing Wanga, Cui Zhanga, Guo-Yu Lib*, Rui-Chao Linc and Jin-Hui Wanga,b* a
Downloaded by [UNAM Ciudad Universitaria] at 15:42 19 December 2014
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China; bSchool of Pharmacy, Shihezi University, Shihezi 832002, China; cNational Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China (Received 30 March 2013; final version received 26 June 2013) A new tetrahydrofuran-type lignan, episesaminone (1), was isolated from Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. Its structure was established by spectroscopic techniques (HR-MS, 1D NMR, 2D NMR, and circular dichroism). The anti-inflammatory activity in RAW 264.7 macrophages was carried out on 1 and other eight known compounds, the epimer of 1 (2) and seven known furofurans-type lignan (3 – 9) obtained from A. heterotropoides Fr. Schmidt var. mandshuricum. Compounds 1, 2, 3, 4, 5, 7, and 9 showed significant anti-inflammatory activity, particularly 50 mM compound 3 inhibited 69.2% NO production compared with the lipopolysaccharide group. Keywords: Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag.; episesaminone; NMR; CD; anti-inflammatory activity
1.
Introduction
Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag., a traditional Chinese medicine known as ‘Liaoxixin’, is widely distributed in the north-east of China. The herb has been used for the treatment of cold, analgesic, antitussive, or anti-allergic remedy [1]. Some phytochemical studies on the herb revealed the presence of volatile oil [2], flavonoids [3], aristolochic acids [4], and furofurans-type lignan [5]. Takasaki et al. [6] reported that furofurans-type lignan showed potent anti-tumor-promoting activities. Additionally, Macrae and Towers [7] claimed that furofurans-type lignan also played an important biological role in antimitosis, antimicrobe, and antioxidation. Interested in finding more biologically active substances from this folk medicine, we further undertook the investigation on its phytochemical com-
position. As a result, a new tetrahydrofuran-type lignan, named episesaminone (1), together with eight known furan-type lignans, sesaminone (2) [8], 4-((1S,3aR, 4R,6aR)-4-(benzo[d][1,3]dioxol-5-yl)hexahydrofuro[3,4-c]furan-1-yl)benzene-1, 2-diol (3) [9], epipinoresinol (4) [10], (1R,2S,5R,6R)-50 -O-methylpluviatilol (5) [11], piperitol (6) [12], pluviatilol (7) [13], (2)-sesamin (8) [12], and asarinin (9) [12], was isolated from the dried roots of A. heterotropoides Fr. Schmidt var. mandshuricum (Figure 1). This paper deals with their structural elucidation and anti-inflammatory activity in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages.
2. Results and discussion Compound 1 was obtained as a colorless solid (MeOH) with the molecular formula C20H18O7 as determined by the HR-ESI-
*Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis
388
J. Huang et al. O 4' O 3'
5' 6' 1'
2' HO
7' O 9
8' 9'
8
6
7
5 4
1
O
O 2
3 O
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Figure 1. Structure of compound 1.
MS at m/z 371.1122 [M þ H]þ, indicating twelve degrees of unsaturation. The 1H NMR spectrum of compound 1 showed six aromatic proton signals at dH 7.80 (1H, dd, J ¼ 8.1, 1.5 Hz, H-6), 7.57 (1H, d, J ¼ 1.5 Hz, H-2), 6.93(1H, d, J ¼ 8.1 Hz, H-5), 6.82 (1H, br s, H-20 ), and 6.77 (2H, br s, H-50 , 60 ), which were assigned to two 1,3,4-trisubstituted benzene rings. The presence of each of 2H singlets at dH 6.06 and dH 5.92, assigned to methylene carbons (dC 103.5 and 102.3), suggested
that both benzene rings have 3,4-methylenedioxyl groups. The fact that the carbonyl group is connected to a part of the phenyl group was indicated by a carbonyl carbon signal at dC 199.9 and the conjugated property absorption peak shown in the IR spectrum at 1669 cm21. The 1H NMR spectrum also exhibited resonances of a 2,3,4-trisubstituted tetrahydrofuran [dH 4.98 (1H, d, J ¼ 6.3 Hz, H-70 ), 4.42 (1H, t, J ¼ 8.1 Hz, H-9a), 4.30 (1H, m, H-8), 4.06 (1H, dd, J ¼ 8.4, 5.4 Hz, H-9b), and 2.86 (1H, qd, J ¼ 7.5, 6.3 Hz, H-80 )] (Table 1). And this was further confirmed by the signals [dC 83.1 (C-70 ), 70.5 (C-9), 51.2 (C-80 ), and 50.4 (C-8)] in the 13C NMR spectrum (75 MHz, MeOD) (Table 1). Careful analysis of the correlations in the HMQC and HMBC spectra led to the establishment of the planar structure of compound 1 (Figure 1). The hydroxymethyl was linked to C-8 0 , which was deduced from the long-range
Table 1. 1H and 13C NMR spectral data of compound 1 (MeOD) (1H: 300 MHz, 13C: 75 MHz, d: ppm, J: Hz). 1
H NMR
No 1 2 3 4 5 6 7 8 9a 9b 10 20 30 40 50 60 70 80 90 a 90 b OCH2O OCH2O
1
13
Sesaminone
7.57, d, J ¼ 1.5
7.46
6.93, d, J ¼ 8.1 7.80, dd, J ¼ 8.1, 1.5
6.84 7.57
4.30, m 4.42, t, J ¼ 8.1 4.06, dd, J ¼ 8.1, 5.4
4.12 4.12 4.25
6.82, br s
6.96
6.77, br s 6.77, br s 4.98, d, J ¼ 6.3 2.86, qd, J ¼ 7.5, 6.3 3.18, d, J ¼ 7.5 3.18, d, J ¼ 7.5 6.06, s 5.92, s
6.76 6.84 4.65 2.84 3.64 3.74 6.06 5.95
C NMR 1 132.4 109.2 149.8 153.7 108.9 126.6 199.9 50.4 70.5 133.6 107.6 148.2 149.1 108.8 120.3 83.1 51.2 62.1 103.5 102.3
NOESY 1
HMBC 1
8
C-3, 4, 6, 7
8
C-1, 3, 4 C-4, 5, 7
2, 6, 90 a, 90 b 90 a 70 , 80
C-80 , 90 , 9 C-70 , 80 , 8, 7
70 , 90 a, 90 b
C-10 , 30 , 40 , 60
70 , 90 a, 90 b 9b, 20 , 60 9b 9a, 20 , 60 , 8 8, 20 , 60
C-70 C-50 , 40 , C-10 , 20 , 60 C-70 , 80 , 8 C-3, 4 C-30 , 40
Journal of Asian Natural Products Research
O O
O HO
O
O O
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Figure 2. Key HMBC correlations of compound 1.
correlations in the HMBC spectrum (Figure 2) from the protons at dH 3.18 (2H, br d, J ¼ 7.5 Hz) to C-8 (dC 50.4), C80 (dC 51.2), and C-70 (dC 83.1). The correlations from H-70 at dH 4.98 to C-20 at dC 107.6, C-60 at dC 120.3, and C-100 at dC 133.6 suggested that C-70 of tetrahydrofuran was connected with C-10 of the benzene ring. The correlations from H-9b at dH 4.06, H-9a at dH 4.42 to C-7 at dC 199.9 indicated that the carbonyl carbon linked to C-8 of the tetrahydrofuran ring. The relative stereostructure was established by the NOESY correlations. The NOESY cross-peaks between H-9b and H70 , H-9b and H-80 , H-8 and H-90 a, H-8 and H-90 b suggested the relative configuration of H-70 and H-80 to be cis and H-80 and H-8 to be trans (Figure 3). Furthermore, this relative configuration of 1 was also supported by the fact that H-9a0 and H9b0 showed a upfield shift of 0.46 and 0.56 ppm, respectively, by comparison of
Figure 3.
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the 1H NMR data with those of the known compound, sesaminone [8], which was due to the change of shielding effect of the benzene rings linked to C-70 on the hydroxymethyl. The absolute configuration of 1 was elucidated by the circular dichroism (CD) exciton chirality method [14]. A negative Cotton effect at 269.0 nm (2 0.20) (Figure 4) caused by the exciton coupling of the benzoyl and benzene chromphores suggested that the absolute configuration of 8, 70 , and 80 was S, R, and R, respectively. On the basis of this evidence, the structure of 1 is shown in Figure 1, named as episesaminone. The anti-inflammatory effects of the nine compounds isolated from A. heterotropoides Fr. Schmidt var. mandshuricum were investigated in LPS-triggered RAW264.7 macrophages. The results showed that nine compounds exhibited the different degree of anti-inflammatory effect, among which compounds 1, 2, 4, 5, 7, and 9 at 50 mM, respectively, inhibited 29.7, 29.4, 31.5, 29.0, 26.9, and 26.0% NO production, while compound 3 decreased 69.2% NO level compared with the LPS group (Table 2).
3.
Experimental
3.1 General experimental procedures Optical rotations were determined on a Perkin-Elmer 241 MC polarimeter
Key NOESY correlations of compound 1.
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Figure 4. The negative exciton chirality of compound 1.
(Perkin-Elmer, Norwalk, CT, USA). UV spectra were obtained on a Shimadzu UV1700 spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were recorded on a Bruker IFS 55 spectrophotometer (Bruker, Karlsruhe, Germany). CD spectra were gotten on Biologic MOS-450 CD spectrometer (Bio-Logic, Grenoble, France). The NMR data were recorded on a Bruker AV-300 spectrometer (Bruker, Bremen, Germany). The HR-ESI-MS data were obtained on a Waters LCT Premier XE time-of-flying mass spectrometer (Waters, Milford, CT, USA). Column chromatography (CC) was performed on silica gel (200 – 300 mesh; Qingdao
Marine Chemical Group, Co., Qingdao, China), octadecylsilane (ODS) (30 – 50 mm; Tianjin Mical Reagent Co., Tianjin, China), and preparative HPLC [Hitachi-L-7110 pump, Hitachi L-7420 UV spectrophotometric detector at 220 nm (Hitachi, Tokyo, Japan), YMC C 18 reversed-phase column (5 mm, 10 mm £ 250 mm; flow rate: 2.0 ml/min). Fetal calf serum was from the Beijing Yuanhengshengma Biology Technology Research Institute (Beijing, China). Dulbecco’s Modified Eagle’s Medium (DMEM) was from Gibco/BRL (Gaithersburg, MD, USA). 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Sigma (St Louis, MO, USA).
3.2
Plant material
The roots of A. heterotropoides Fr. Schmidt var. mandshuricum were collected in Benxi, Liaoning of China in September 2010, and were identified by Prof. Jincai Lu (School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University). A voucher specimen (No. 20100912) was deposited in the Research Department of Natural Medicine, Shenyang Pharmaceutical University.
Table 2. The anti-inflammatory activity of the isolated nine compounds in RAW264.7 macrophages triggered with 1 mg/ml LPS. No LPS 1 2 3 4 5 6 7 8 9 Indomethacin
Concentration of drug (mM)
Cell viability (%)
Concentration of NO (mM)
Inhibitory rate of NO (%)
— 50 50 50 50 50 50 50 50 50 50
93.8 ^ 3.1 91.2 ^ 1.8 98.5 ^ 1.5 89.7 ^ 2.8 94.2 ^ 5.7 99.8 ^ 1.2 98.4 ^ 1.5 94.8 ^ 5.1 94.2 ^ 5.7 100.6 ^ 4.6 95.57 ^ 4.1
131.8 ^ 3.8 92.7 ^ 1.7 93.0 ^ 2.1 40.5 ^ 2.3 90.2 ^ 2.9 93.5 ^ 2.3 116.3 ^ 2.5 96.3 ^ 1.5 113.0 ^ 1.6 97.5 ^ 2.1 41.3 ^ 2.5
— 29.7 29.4 69.2 31.5 29.0 11.7 26.9 14.2 26.0 68.6
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Journal of Asian Natural Products Research 3.3 Exaction and isolation Dried roots of A. heterotropoides Fr. Schmidt var. mandshuricum (7.5 kg) were extracted three times with 60 liters 95% EtOH under reflux conditions for 3 h to give a crude extract, which was suspended in H2O and extracted with petroleum ether (PE), CHCl3, and AcOEt, successively, to yield a PE-soluble fraction (10.0 g), a CHCl3-soluble fraction (32.0 g), and an AcOEt-soluble fraction (18 g). The three parts were combined (50 g) and subjected to CC (silica gel, gradient of PE:AcOEt, 100:1 – 0:100) to afford 61 fractions (1 – 61). Fractions 7 and 25 were recrystallized to obtain compounds 9 (99.6 mg) and 7 (48.9 mg), respectively. Fraction 9 was resubjected to CC (silica gel, gradient of PE:CHCl3:AcOEt, 100:0:0 –0:100:100) to give a Fraction 9.6. Fraction 9.6 was recrystallized to get compound 8 (16.9 mg). Fraction 27 was fractionated by CC (silica gel, gradient of PE:CHCl3: AcOEt, 100:0:0 –0:100:100) to give Fraction 27.5. Fraction 27.5 was applied to preparative reversed-phase HPLC (MeOH:H2O, 60:40, flow rate 2.0 ml/min, wavelength 220 nm) and afforded compound 6 (19.0 mg, Rt 39.6 min). Fraction 35 was resubjected to CC (silica gel, gradient of PE:CHCl3:AcOEt, 100:0:00:100:100) to give a Fraction 35.5. Fraction 35.5 was separated by CC (ODS, MeOH:H2O, 60:40) to yield Fraction 35.5.5. Fraction 35.5.5 was further subjected to HPLC (MeOH:H2O, 65:35, flow rate 2.0 ml/min, wavelength 220 nm) to obtain 1 (15.3 mg, Rt 25.4 min). Fraction 40 was resubjected to CC (silica gel, gradient of PE:CHCl 3:AcOEt, 100:0:0 – 0:100:100) to give Fraction 40.2. Fraction 40.2 was applied to HPLC (MeOH:H2O, 60:40, flow rate 2.0 ml/min, wavelength 220 nm) to get compound 5 (18.4 mg, Rt 28.1 min). Fraction 45 was separated by CC (silica gel, gradient of PE: CHCl3:AcOEt, 100:0:0 – 0:100:100) to give Fraction 45.6. Fraction 45.6 was
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also purified by HPLC (MeOH:H2O, 60:40, flow rate 2.0 ml/min, wavelength 220 nm) to get compounds 4 (7.4 mg, Rt 17.6 min), 3 (25.7 mg, Rt 21.5 min), and 2 (24.1 mg, Rt 31.8 min). 3.3.1
Episesaminone (1)
Colorless solid (MeOH); ½a21 D 2 23:1 (c ¼ 0.55, MeOH). UV (MeOH) lmax: 208.6, 230.0, and 280.4 nm; IR (KBr) vmax: 3420, 2926, 1720, 1628, 1384, 1113, 1029, and 670 cm21. CD (MeOH) D1238.0 nm þ0.21, D1269.0 nm –0.20, D1339.0 nm þ 0.14; 1 H and 13C NMR spectral data are shown in Table 1; HR-ESI-MS: m/z 371.1122 [M þ H]þ (calcd for C20H19O7, 371.1131). 3.4
Anti-inflammation activity assay
3.4.1 Measurement of NO releasing RAW264.7 cells were seeded in 96-well plates (2 £ 105/ml) and incubated at 378C in a humidified atmosphere containing 5% CO2 overnight. After 12 h, the cells were treated with 50 mM compounds for 1 h, then added LPS (1 mg/ml) for 24 h. NO levels were determined by measuring nitrite levels using the supernatant (100 ml) mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride, and 2.5% phosphoric acid) and the absorbance was measured at 570 nm [15]. The nitrite concentration was determined by using a standard curve of sodium nitrite made up in DMEM free of phenol red. C: concentration. NO inhibitory rate ð%Þ ¼
C NO ðLPSÞ 2 C NO ðcompoundÞ £ 100 CNO ðLPSÞ
3.4.2 Measurement of RAW264.7 cell viability RAW264.7 cells viability was measured by MTT assay. The cells were seeded in 96well plates (2 £ 105/ml) and incubated at
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378C in a humidified atmosphere containing 5% CO2 overnight. After 12 h, the cells were treated with 50 mM compounds for 1 h, then added LPS (1 mg/ml) for 24 h. MTT (5 mg/ ml) was subsequently added and incubated for 4 h. The crystals were dissolved in DMSO. The optical densities at 490 nm were measured with a microplate reader (TECAN SPECTRA, Wetzlar, Germany). cell viability rate ð%Þ A490 ðcompoundÞ 2 A490 ðblankÞ ¼ £ 100 A490 ðcontrolÞ 2 A490 ðblankÞ
Acknowledgments This work was financially supported by Key Projects of the National Science and Technology Pillar Program (2012BAI30B02) and National Science and Technology Major Project (2010ZX09401-304).
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