Advanced
Corilagin with Inhibitory Activity against NO Production from Euphorbia supina
Corilagin with Inhibitory Activity against NO Production from Euphorbia supina
Natural Product Sciences. 2014. Jun, 20(2): 126-129
Copyright © 2014, The Korean Society of Pharmacognosy
  • Received : January 29, 2014
  • Accepted : April 29, 2014
  • Published : June 30, 2014
Download
PDF
e-PUB
PubReader
PPT
Export by style
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Hee-Sung Chae
College of Pharmacy and RFIND-BKplus Team, Dongguk University-Seoul, 32 Dongguk-lo, Ilsandong-gu, Goyang, Gyeonggi-do 410-820, Republic of Korea
Young-Mi Kim
College of Pharmacy and RFIND-BKplus Team, Dongguk University-Seoul, 32 Dongguk-lo, Ilsandong-gu, Goyang, Gyeonggi-do 410-820, Republic of Korea
Eun Joo Lee
College of Pharmacy and Wonkwang Oriental Medicines Research Institute, Wonkwang University, Iksan, Jeonbuk, Republic of Korea
Hyuk Hwan Song
Immune Modulator Research Center, Bio-Therapeutics Research Institute, Korea Research Institute of Bioscience & Biotechnology; 685–1 Yangcheong-ri, Ochang-eup, Cheongwon-gun, ChungBuK 363–883, Republic of Korea
Sei-Ryang Oh
Immune Modulator Research Center, Bio-Therapeutics Research Institute, Korea Research Institute of Bioscience & Biotechnology; 685–1 Yangcheong-ri, Ochang-eup, Cheongwon-gun, ChungBuK 363–883, Republic of Korea
Young Hee Choi
College of Pharmacy and RFIND-BKplus Team, Dongguk University-Seoul, 32 Dongguk-lo, Ilsandong-gu, Goyang, Gyeonggi-do 410-820, Republic of Korea
Young-Won Chin
College of Pharmacy and RFIND-BKplus Team, Dongguk University-Seoul, 32 Dongguk-lo, Ilsandong-gu, Goyang, Gyeonggi-do 410-820, Republic of Korea
f2744@dongguk.edu

Abstract
Bioactivity-guided investigation on whole plant of Euphorbia supina , using LPS-induced Raw264.7 cells, led to the identificaiton of a tannin which was not reported from this plant along with four known constituents (quercetin, astragalin, juglanin and methyl gallate). The structure of the tannin was determined as corilagin by the interpretation of NMR (1D and 2D) and MS spectroscopic data. All the isolates were tested for the inhibitory activity against NO production in LPS-induced Raw264.7 cells. Among the tested isolates, corilagin was found to be the most active compound.
Keywords
Introduction
Euphorbia supina Raf. belongs to the family Euphorbiaceae and its whole plant has been used traditionally for the treatment of inflammatory and allergic diseases in Korea. Previous studies on this plant reported the presence of flavonoids as constituents of this plant, as well as antioxidative, anti-viral and peroxynitrite-scavenging activity of flavonoids. 1 As part of our ongoing search for biologically active constituents from medicinal plants, E. supina was selected for the follow-up isolation work since its MeOH extract showed NO inhibitory activity (IC 50 15.1 μg/ml) in LPS-induced Raw264.7 cells. Bioactivityguided separation on the MeOH extract of E. supina led to the isolation of a gallic acid derivative (methyl gallate), three flavonoids (quercetin, astragalin and juglanin) and a new constituent in this plant, corilagin. All the isolates were tested in LPS-induced Raw264.7 cells.
Experimental
General experimental procedures – NMR spectra were recorded on a Brucker DMX-800 MHz FT-NMR spectrometer with the tetramethylsilane as an internal standard. HRESIMS and LC-MS/MS were performed with on a Waters Q-Tof Premier spectrometer (Micromass UK Ltd., Manchester, UK) and API4000 triple quadrupole mass spectrometer (AB Sciex, Foster city, CA), respectively. Sephadex LH-20 (25 - 100 μm, Sigma-Aldrich, Steinheim, Germany), silica gel (230 - 400 mesh, SiliCycle Inc., Quebec, Canada) were used for column chromatography. TLC was performed on precoated Kiesel-gel 60 F 254 (0.25 mm, Merck, Darmstadt, Germany) and Kiesel-gel 60 RP-18F 254s (0.25 mm, Merck, Steinheim, Germany).
Plant materials – Whole plants of E. supina (4.3 kg) were collected in Chungbuk province, Korea in May 2011. These plant samples were identified by one of authors. A voucher specimen (DUCYW-0009) has been deposited at College of Pharmacy, Dongguk University, Seoul, Republic of Korea.
Extraction and isolation – Dried E. supina (4.3 kg) were pulverized and extracted with methanol at the room temperature three times (each 8 L) to obtain 520 g of solid extract. The methanol extract was suspended in H 2 O and then partitioned with n -hexane, chloroform, ethyl acetate, n -butanol (BuOH). All the fractions, n -hexane, chloroform, ethyl acetate and BuOH-soluble fractions, were tested in LPS induced RAW 264.7 cells and it was found the BuOH-soluble fraction potently inhibited NO production with IC 50 value 7.53 μg/ml. The BuOH-soluble extract (ESB) (4.5 g) was subjected to a column chromatography using a silica gel (500 g) eluting with gradient mixtures of chloroform: methanol (20 : 1 to 1 : 1) to give 13 fractions (ESB1 - ESB13). Fraction ESB2 (74.5 mg) was subjected to Sephadex LH-20, eluted with MeOH (100%), and afforded nine fractions (ESB2S1 - ESB2S9). Quercetin ( 1 ) (1 mg) was precipitated from ESB2S2 fraction. Fraction ESB3 (68.1 mg) was chromatographed on a medium pressure liquid chromatography (MPLC) column of RP C-18 (3.5 × 40 cm, 40C18-PREP), eluted with 50% MeOH and afforded five fractions (ESB3R1 - ESB3R5). Fraction ESB3R3 (19mg) was subjected to Sephadex LH- 20 eluted with MeOH (100%) and afforded five fractions (ESB3R3S1 - ESB3R3S5). Astragalin ( 2 ) (2 mg) was precipitated from ESB3R3S2 fraction. Fraction ESB5 (194mg) was chromatographed on medium pressure liquid chromatography (MPLC) column of RP C-18 (3.5 × 40 cm, 40C 18-PREP), eluted with 50% - 100% MeOH and afforded seven fractions (ESB5R1 - ESB5R7). Fraction ESB5R6 (52 mg) was subjected to Sephadex LH-20 eluted with MeOH (100%) and afforded five fractions (ESB5R6S1 - ESB5R6S5). Juglanin ( 3 ) (4.4 mg) was precipitated from ESB5R6S3 fraction. Fraction ESB4 (52.7 mg) was subjected to Sephadex LH-20 eluted with MeOH (100%) and afforded nine fractions (ESB4S1 - ESB4S9). Methyl gallate ( 4 ) (7 mg) was precipitated from ESB4S2 fraction. Corilagin ( 5 ) (18 mg) was recipitated from ESB9S2 fraction.
Quercetin (1) 1 H NMR (DMSO- d 6 , 400 MHz) δ 7.66 (1H, d, J = 1.2 Hz, H-2'), 7.53 (1H, dd, J = 8.6, 1.2 Hz, H-6'), 6.88 (1H, d, J = 8.6 Hz, H-5'), 6.38 (1H, d, J = 2.0 Hz, H-8), 6.17 (1H, d, J = 2.0 Hz, H-6); 13 C NMR (DMSO- d 6 , 100 MHz) δ 175.8 (C-4), 163.9 (C-7), 160.7 (C-5), 156.1 (C-9), 147.7 (C-4'), 146.8 (C-2), 145.0 (C-3'), 135.7 (C-3), 121.9 (C-1'), 120.1 (C-6'), 115.6 (C-5'), 115.2 (C-2'), 103.0 (C-10), 98.2 (C-6), 93.3 (C-8); ESI-MS: m/z 301 [M − H]
Astragalin (2) 1 H NMR (DMSO- d 6 , 400 MHz) δ 7.99 (2H, d, J = 8.8 Hz, H-2', 6'), 6.85 (2H, d, J = 8.8 Hz, H-3', 5') 6.01 (1H, brs, H-8), 5.91 (1H, brs, H-6) 5.35 (1H, d, J = 7.2 Hz, H-1"), 3.57 - 3.08 (glucose protons); 13 C NMR (DMSO- d 6 , 100 MHz) δ 176.1 (C-4), 160.9 (C-5), 159.9 (C-4'), 156.9 (C-9), 156.6 (C-2), 132.8 (C-3), 130.6 (C-2', 6'), 121.0 (C-1'), 115.1 (C-3', 5'), 103.8 (C-10), 101.4 (C-1"), 98.9 (C-6), 93.8 (C-8), 77.4 (C-5"), 76.5 (C-3"), 74.2 (C-2"), 69.8 (C-4") 62.8 (C-6"); ESIMS: m/z 447 [M − H]
Juglanin (3) 1 H NMR (DMSO- d 6 , 400 MHz) δ 8.02 (2H, d, J = 8.8 Hz, H-2', 6'), 6.89 (2H, d, J = 8.8 Hz, H-3', 5'), 6.44 (1H, d, J = 1.6 Hz, H-8), 6.20 (1H, d, J = 1.6 Hz, H-6), 5.62 (1H, d, J =0.8 Hz, H-1"), 4.15 (1H, d, J = 2.4 Hz, H-5"), 3.75 – 3.16 (arabinose protons); 13 C NMR (DMSO- d 6 , 100 MHz) δ 177.6 (C-4), 164.5 (C-7), 161.2 (C-5), 160.0 (C-4'), 156.7 (C-9), 156.4 (C-2), 133.4 (C-3), 130.9 (C-2'), 130.7 (C-6'), 120.7(C-1'), 115.4 (C-3', 5'), 108.0 (C-1"), 103.9 (C-10), 98.7 (C-6), 93.7 (C-8), 86.3 (C-4"), 82.1 (C-2"), 77.1 (C-3"), 60.4 (C-5"); ESI-MS: m/z 417 [M − H]
Methyl gallate (4) 1 H NMR (acetone- d 6 , 400 MHz) δ 7.11 (2H, s, H-2', 6'), 3.78 (-OCH3, s, H-1); ESI-MS: m/z 183 [M − H]
Corilagin (5)
PPT Slide
Lager Image
−37.9 (c 0.1, DMSO), 1 H NMR (DMSO- d 6 , 800 MHz) δ 6.98 (2H, s, H-2', H-6'), 6.53 (1H, s, H-6"'), 6.43 (1H, s, H-6"), 6.17 (1H, d, J = 7.5 Hz, H-1), 4.58 (1H, brs, H-3), 4.36 (1H, t, J = 8.1 Hz, H-5), 4.27 (1H, d, J = 3.0 Hz, H-4), 4.21 (1H, dd, J = 10.8, 7.7 Hz, H-6), 3.99 (1H, dd, J = 10.8, 10.2 Hz, H-6), 3.88 (1H, d, J = 7.5 Hz, H-2) ppm; 13 CNMR (DMSO- d 6 , 200 MHz) δ 167.1 (C-7"), 166.9 (C-7"'), 164.8 (C-7'), 145.6 (C-3'), 145.6 (C-5'), 143.7 (C-3"), 143.9 (C-3"'), 143.9 (C-5"'), 143.7 (C-5"), 139.0 (C-4'), 135.8 (C-4"), 135.4 (C-4"'), 123.9 (C-2"), 123.1 (C-2"'), 118.7 (C-1'), 116.1 (C-1"), 115.9 (C-1"'), 109.0 (C-2'), 109.0 (C-6'), 106.9 (C-6"), 105.9 (C-6"'), 92.2 (C-1), 77.5 (C-3), 76.4 (C-5), 71.7 (C-2), 63.9 (C-6), 62.2 (C-4); HRESIMS: m/z 633.0728 [M − H] (calcd for C 20 H 25 O 23 , 633.0787). CD (c 1.97 × 10 −4 M, MeOH) λ max (Δε) 239 (−1.83), 264 (0), 284 (−1.08) nm
LC-MS/MS analysis − Test sample was analyzed using an API4000 triple quadrupole mass spectrometer (AB Sciex, Foster city, CA) in the multiple reaction monitoring (MRM) mode with electrospray ionization interface used to negative ions ([M − H] ). The turbo ion-spay interface was operated in negative ion mode at −4500 V and 400℃. The mass transition used for test sample was m/z 633.1 → 301.0 (collision energy, −50 eV), respectively. Quadrupoles Q1 and Q3 were set to unit resolution. The analytical data were processed using Analyst software (Version 1.5.1; Applied Biosystems).
Measurement of nitric oxide (NO) production − NO production was assayed by measuring nitrite in supernatants of cultured RAW 264.7 cells. 2 Cells (1 × 10 6 /mL) were seeded in 96 well culture plates. After pre-incubation of RAW 264.7 cells for 18 hours, cells were pretreated with test samples (5 μg/ml) for 30min and then stimulated LPS (500 ng/mL) for 24 hours. The supernatant was mixed with an equal volume of Griess reagent and incubated at room temperature for 5min. The concentration of nitrite was measured at 570 nm.
Result and Discussion
Compounds 1 - 5 were identified as quercetin ( 1 ), astragalin ( 2 ), juglanin ( 3 ), and methyl gallate ( 4 ), 3 the known constituents from this plant, by comparison with the published values. 1 H NMR spectra displayed two singlet peaks at δ H 6.53 (1H, s) and 6.43 (1H, s) assignable to a hexahydroxydiphenoyl (HHDP) group, one singlet peak at δ H 6.98 (2H, s) from a galloyl group, and seven peaks in the range δ H 6.17-3.84 corresponding to a glucose unit. MS fragments at mz 301 ([M − H] -332, loss of a galloyl glucose) and mz 463 ([M − H] -170, loss of a galloyl group) supported the presence of a HHDP and a galloyl group in MS spectrum. Anomeric proton at δ H 6.17 with a coupling constant of 7.5 Hz indicated this glucose was β-confomer. The location of the HHDP and the galloyl group was established by the HMBC correlations ( Fig. 2 ). Long range couplings were observed between H-6 (δ H 4.21 and 3.99) and δ C 167.1, as well as H-3 (δ H 4.58) and δ C 166.9, suggesting that the HHDP was linked to C-3 and C-6. The remaining galloyl group was connected to C-1 via an ester linkage by the observed HMBC correlation of H-1 (δ H 6.17) to δ C 164.8. Based on all the data and the published values, 4 this compound was elucidated as corilagin. Furthermore, the stereochemistry of corilagin was confirmed as shown in Fig. 1 by CD measurement and comparison with the literature. 5 Corilagin ( 5 ) was isolated for the first time from this plant.
PPT Slide
Lager Image
Structures of compounds 1 - 5.
PPT Slide
Lager Image
Key HMBC correlations and 1H-1H COSY of corilagin (A); CD spectrum of corilagin (B).
Effects of compounds on production of NO in LPSinduced RAW 264.7 cells. The cells (1 × 105cells/ml) were pretreated with compounds (5 μg/ml) 30 min prior to stimulation with LPS (500 ng/ml) 24 hours after stimulation, the NO level of the supernatants was measured by Griess reagent. Statistical signicance: p < 0.05, as compared to the LPS treated group. Signicanct differences between treated groups were determined using the Dunnett’s t-test. Values shown are the mean S.E. of duplicate determinations from three separate experiments. 1: quercetin, 2: astragalin, 3: juglanin, 4: methyl gallate, 5: corilagin
PPT Slide
Lager Image
Effects of compounds on production of NO in LPSinduced RAW 264.7 cells. The cells (1 × 105 cells/ml) were pretreated with compounds (5 μg/ml) 30 min prior to stimulation with LPS (500 ng/ml) 24 hours after stimulation, the NO level of the supernatants was measured by Griess reagent. Statistical signicance: p < 0.05, as compared to the LPS treated group. Signicanct differences between treated groups were determined using the Dunnett’s t-test. Values shown are the mean S.E. of duplicate determinations from three separate experiments. 1: quercetin, 2: astragalin, 3: juglanin, 4: methyl gallate, 5: corilagin
The isolated compounds 1 - 5 were tested in LPSinduced NO production assay using Raw 264.7 cells and two compounds were found to inhibit NO production, significantly, in this assay system ( Table 1 ). The known structure of this plant, methyl gallate, was known to possess the inhibitory activity against NO production 6 - 8 and in the present study, similarly, the level of NO as decreased by the treatment of this compound. Of the isolates, corilagin demonstrated to inhibit potently NO production in LPS-induced Raw 264.7 cells, which was consistent with the previous studies. 9
In the current investigation, it was found that corilagin with inhibitory activity against NO production in LPSinduced Raw 264.7 cells, in part, may be responsible for anti-inflammatory activity of this plant.
Acknowledgements
This work was supported from a Grant of the NRF (2011-0014225, Y.-W. Chin) funded by the government of Korea (MEST).
References
Hong H. K. , Kwak J. H. , Kang S. C. , Lee J. W. , Park J. H. , Ahn J. W. , Kang H. S. , Choung E. S. , Zee O. P. 2008 Kor. J. Pharmacogn. 39 260 - 264
Chae H. S. , Kang O. H. , Choi J. G. , Oh Y. C. , Lee Y. S. , Jang H. J. , Kim J. H. , Park H. , Jung K. Y. , Sohn D. H. , Kwon D. Y. 2009 Biol. Pharm. Bull. 32 553 - 557    DOI : 10.1248/bpb.32.553
An R. B. , Kwon J. W. , Kwon T. O. , Chung W. T. , Lee H. S. , Kim Y. C. 2007 Kor. J. Pharmacogn. 38 291 - 295
Yamada H. , Nagao K. , Dokei K. , Kasai Y. , Michihata N. 2008 J. Am. Chem. Soc. 130 7566 - 7567    DOI : 10.1021/ja803111z
Okuda T. , Yoshida T. , Hatano T. , Koga T. , Toh N. , Kuriyama K. 1982 Tetrahedron Lett. 23 3937 - 3940    DOI : 10.1016/S0040-4039(00)87748-5
Chae H. S. , Kang O. H. , Choi J. G. , Oh Y. C. , Lee Y. S. , Brice O. O. , Chong M. S. , Lee K. N. , Shin D. W. , Kwon D. Y. 2010 Am. J. Chin. Med. 38 973 - 983    DOI : 10.1142/S0192415X10008391
Shin Y. J. , Jung D. Y. , Ha H. K. , Park S. W. 2004 Korean J. Food Sci. Technol. 36 968 - 973
Woo E. R. , Lee J. Y. , Cho I. J. , Kim S. G. , Kang K. W. 2005 Pharmacol. Res. 51 539 - 546    DOI : 10.1016/j.phrs.2005.02.002
Zhao L. , Zhang S. L. , Tao J. Y. , Pang R. , Jin F. , Guo Y. J. , Dong J. H. , Ye P. , Zhao H. Y. , Zheng G. H. 2008 Int. Immunopharmacol. 8 1059 - 1064    DOI : 10.1016/j.intimp.2008.03.003