Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Phenylpropanoid acid esters from Korean propolis and their antioxidant activities In-Kyoung Lee, Myung-Suk Han, Dae-Won Kim, Bong-Sik Yun ⇑ Division of Biotechnology and Advanced Institute of Environment and Bioscience, Chonbuk National University, 79 Gobong-ro, Iksan-si 570-752, Republic of Korea
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 20 March 2014 Revised 9 May 2014 Accepted 16 May 2014 Available online xxxx
Ten phenylpropanoic acid esters were isolated from an ethanolic extract of Korean propolis. Their structures were elucidated by spectroscopic methods including NMR and ESI-MS. Caffeic acid esters with catechol moiety exhibited significant ABTS and DPPH radical scavenging activity and protective effect against DNA damage by a Fenton reaction. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Propolis Phenylpropanoic acid esters Antioxidant activity
Propolis is a sticky resinous product built by honeybees by mixing their own waxes with resins collected from various plants, and it is used in the construction and maintenance of their hives. Propolis has been recently attracting the attention due to its various biological activities and therapeutic properties proven to be beneficial in allergies, asthma, diabetes, and hypertension.1,2 There is a lot of evidence that it is an abundant source of polyphenols, mainly flavonoids and phenolic acids, and they attribute to pharmacological
properties in propolis. Caffeic acid phenethyl ester (CAPE) has been identified as one of the major biologically active substance; it is best known as a phenylpropanoic acid ester in propolis and known for its antioxidant, anti-inflammatory, chemoprevention, and antitumor properties.3 The phenylpropanoic acid esters have been found to various extents in propolis, since the chemical composition of propolis depends on the regional vegetation, season, and honeybee species at the site of collection.
R1 5 9
3
R2
1
OR3
O
Table 1 Free radical scavenging activity of phenylpropanoic acid esters TEACa,b
Compounds
1: R1, R2 = OH, R3 = Phenethyl
c
2: R1, R2 = OH, R3 = Benzyl
1 2 3 4 5 6 7 8 9 10 BHA Trolox
3: R1, R2 = OH, R3 = Ethyl 4: R1 = OH, R2 = OCH3, R3 = Benzyl 5: R1 = OH, R2 = OCH3, R3 = 3,3-dimethylallyl 6: R1, R2 = OCH3, R3 = Cinnamyl 7: R1 = OH, R2 = H, R3 = Cinnamyl 8: R1 = OH, R2 = H, R3 = Benzyl 9: R1, R2 = H, R3 = Phenethyl 10: R1, R2 = H, R3 = Cinnamyl Figure 1. Structures of phenylpropanoic acid esters.
a b
⇑ Corresponding author. Tel.: +82 63 850 0839; fax: +82 63 850 0834. E-mail address: [email protected] (B.-S. Yun).
c d
ABTS radical
DPPHd radical
6.30 ± 0.43 3.42 ± 0.30 10.61 ± 0.39 24.82 ± 3.93 23.10 ± 4.79 >500 >500 >500 >500 >500 0.68 ± 0.06 1
0.46 ± 0.00 3.86 ± 0.79 6.30 ± 0.94 92.10 ± 7.86 105.00 ± 8.23 >500 >500 >500 >500 >500 0.84 ± 0.01 1
Expressed as IC50 of lM compounds/IC50 of lM trolox. Results presented as the mean (n = 3) ± SD. 2,20 -Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid). a,a-Diphenyl-b-picrylhydrazyl.
http://dx.doi.org/10.1016/j.bmcl.2014.05.065 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lee, I.-K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.065
2
I.-K. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Figure 2. Protective effects of compounds 1–10 against plasmid DNA breakage due to the Fenton reaction between ferrous and hydrogen peroxide. Butylated hydroxyanisole (BHA) and Trolox were used as controls. Supercoiled DNA from pBR322 was incubated for 30 min at 25 °C in 10.0 mM Tris–HCl buffer and mixed with 50.0 lM compounds, 0.25 mM H2O2, and 12.5 lM ferric chloride.
We previously reported on the isolation and characterization of flavonoids and their free radical scavenging activity from Korean propolis.4 In this study, we describe the isolation, structural elucidation of ten phenylpropanoic acid esters from Korean propolis, and their antioxidant properties, collected in the Yangpyeong region. Propolis (136.3 g) collected from the Yangpyeong region, Gyeonggi-do province in Korea was extracted with 95% ethanol for 24 h. The ethanolic extract was concentrated under reduced pressure and then subjected to silica gel column chromatography (70–230 mesh) eluted with a gradient with increasing amount (1–50%) of methanol in chloroform to afford two fractions, fractions A and B. Fraction A was chromatographed on a column of Sephadex LH-20 with chloroform: methanol (1:1, v/v) and then was purified with an ODS-Sepak cartridge using a gradient of increasing methanol (50–100%) in water to give three fractions, A-1, A-2, and A-3. Preparative reversed-phase HPLC was performed with 60–80% aqueous MeOH gradient for fraction A-1 to give compounds 9 (3.2 mg) and 10 (2.9 mg), 55% aqueous methanol for fraction A-2 to give compound 6 (2.1 mg), and 50% aqueous MeOH for fraction A-3 to give compounds 4 (4.6 mg) and 5 (1.8 mg). Fraction B was chromatographed on a Sephadex LH-20 column eluted with methanol to give two fractions, B-1 and B-2. Fraction B-1 was separated by an ODS-sepak cartridge using a gradient of increasing methanol (50–100%) in water, followed by preparative reversedphase HPLC with 60% aqueous methanol to afford compound 8 (9.6 mg). Compounds 1–3 (10.8, 31.4, and 3.9 mg, respectively) and compound 7 (4.1 mg) were isolated from fraction B-2 through preparative reversed-phase HPLC with 65% aqueous methanol. The structures of these compounds were established by spectroscopic method including NMR and ESI-MS, and determined by comparison with reference data.5,6 Their structures were identified as caffeic acid phenethyl ester (1), caffeic acid benzyl ester (2), caffeic acid ethyl ester (3), ferulic acid benzyl ester (4), ferulic acid 30 ,30 -dimethylallyl ester (5), 3,4-dimethoxycaffeic acid cinnamyl ester (6), coumaric acid cinnamyl ester (7), coumaric acid benzyl ester (8), cinnamic acid phenethyl ester (9), and cinnamic acid cinnamyl ester (10),7 as shown in Figure 1. Antioxidant effect of the 10 phenylpropanoid acid esters isolated was estimated using the method described in the literatures.8,9 The free radical scavenging activity was evaluated by measuring their scavenging effects against ABTS radical cation and DPPH radical. These assays provide information on the reactivity of test compounds with a stable free radical. The results were expressed as TEAC value (trolox equivalent antioxidant capacity, IC50 of lM compound/IC50 of lM trolox), as shown in Table 1. The active compounds showed radical scavenging activity in a dose-dependent manner. The most active compounds were 1, 2, and 3 (caffeic acid phenethyl ester, caffeic acid benzyl ester, caffeic acid ethyl ester), which contain catechol moiety known as an antioxidant pharmacophore.10 Compounds 4 and 5 (ferulic acid benzyl ester and ferulic acid 30 ,30 -dimethylallyl ester) exhibited moderate activities, and 6 (3,4-dimethoxycaffeic acid cinnamyl ester) showed no activity. In these compounds, the hydroxyl group of catechol moiety was masked by the methyl group, which causes a loss
of radical scavenging activity. Coumaric acid esters (7, 8) and cinnamic acid esters (9, 10) showed no activity up to 1.0 mM. The hydroxyl radical scavenging assay was evaluated by Fenton reaction-induced DNA single strand breakage method.8 Fenton chemistry produces strong oxidant hydroxyl radical with a reaction between iron and hydrogen peroxide, and causes breakage of the deoxyribose-phosphate backbone of DNA. This assay allows the quantification of single strand breakage DNA generated by a single nick in supercoiled form DNA. Compounds 1, 2, and 3 significantly inhibited the Fenton reaction in a concentration of 50 lM, resulting in protection against DNA damage, as shown in Figure 2. Compounds 4 and 5 exhibited moderate activity, but other compounds did not protection against DNA breakage. Phenolic compounds are known as powerful antioxidants because of their scavenging ability due to catechol moiety.10,11 It is well known that the semiquinone radical derived from H-atom donation of catechol can be stabilized by an intramolecular hydrogen bond and the electron-donating properties of the ortho-OH.11 In conclusion, this study provided evidence that caffeic acid benzyl ester and caffeic acid phenethyl ester are dominant constituents in Korean propolis, and caffeic acid esters with catechol moiety were considerably responsible for antioxidant activity of propolis. Acknowledgments This work was supported by a Grant from Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2012R1A1A3011559). References and notes 1. Aguero, M. B.; Gonzalez, M.; Lima, B.; Svetaz, L.; Sanchez, M.; Zacchino, S.; Feresin, G. E.; Palermo, J.; Wunderlin, D.; Tapia, A. J. Agric. Food Chem. 2010, 58, 194. 2. Cheng, P. C.; Wong, G. Bee World 1996, 77, 8. 3. Watanabe, M. A.; Amarante, M. K.; Conti, B. J.; Sforcin, J. M. J. Pharm. Pharmacol. 2011, 63, 1378. 4. Han, M. S.; Lee, I. K.; Kim, Y. S.; Kim, J. T.; Choe, K. R.; Yun, B. S. J. Korean Soc. Appl. Biol. Chem. 2010, 53, 512. 5. Hu, L. H.; Zou, H. B.; Gong, J. X.; Li, H. B.; Yang, L. X.; Cheng, W.; Zhou, C. X.; Bai, H.; Gueritte, F.; Zhao, Y. J. Nat. Prod. 2005, 68, 342. 6. Li, F.; Awale, S.; Tezuka, Y.; Esumi, H.; Kadota, S. J. Nat. Prod. 2010, 73, 623. 7. Caffeic acid phenethyl ester (1): ESI-MS m/z 285 [M+H]+, C17H16O4; 1H NMR (600 MHz, CD3OD) d: 7.50 (1H, d, J = 15.8 Hz, H-3), 7.20–7.30 (5H, m, H-40 –H80 ), 7.01 (1H, d, J = 2.0 Hz, H-9), 6.91 (1H, dd, J = 8.2, 2.0 Hz, H-5), 6.76 (1H, d, J = 8.2 Hz, H-6), 6.21 (1H, d, J = 15.8 Hz, H-2), 4.35 (2H, t, J = 6.9 Hz, H-10 ), 2.98 (2H, t, J = 6.9 Hz, H-20 ). Caffeic acid benzyl ester (2): ESI-MS m/z 271 [M+H]+, C16H14O4; 1H NMR (600 MHz, CD3OD) d: 7.57 (1H, d, J = 15.8 Hz, H-3), 7.30– 7.40 (5H, m, H-30 –H-70 ), 7.02 (1H, d, J = 2.0 Hz, H-9), 6.92 (1H, dd, J = 8.2, 2.0 Hz, H-5), 6.76 (1H, d, J = 8.2 Hz, H-6), 6.28 (1H, d, J = 15.8 Hz, H-2), 5.21 (2H, s, H10 ); 13C NMR (150 MHz, CD3OD) d: 167.7 (C-1), 148.3 (C-7), 145.9 (C-3), 145.5 (C-8), 136.5 (C-20 ), 126.4 (C-4), 128.2127.8 (C-30 –C-70 ), 121.7 (C-5), 115.2 (C6), 113.9 (C-9), 113.6 (C-2), 65.8 (C-10 ). Caffeic acid ethyl ester (3): ESI-MS m/z 209 [M+H]+, C11H12O4; 1H NMR (600 MHz, CD3OD) d: 7.53 (1H, d, J = 15.8 Hz, H3), 7.03 (1H, d, J = 2.0 Hz, H-9), 6.93 (1H, dd, J = 8.2, 2.0 Hz, H-5), 6.77 (1H, d, J = 8.2 Hz, H-6), 6.25 (1H, d, J = 15.8 Hz, H-2), 4.26 (2H, q, J = 6.9 Hz, H-10 ), 1.30 (3H, t, J = 6.9 Hz, H-20 ). Ferulic acid benzyl ester (4): ESI-MS m/z 285 [M+H]+, 307 [M+Na]+, C17H16O4; 1H NMR (600 MHz, CDCl3) d: 7.64 (1H, d, J = 15.8 Hz, H3), 7.30–7.40 (5H, m, H-30 –H-70 ), 7.01 (1H, d, J = 2.0 Hz, H-9), 7.06 (1H, dd, J = 8.2, 2.0 Hz, H-5), 6.89 (1H, d, J = 8.2 Hz, H-6), 6.32 (1H, d, J = 15.8 Hz, H-1),
Please cite this article in press as: Lee, I.-K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.065
I.-K. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx 5.23 (2H, s, H-10 ), 3.91 (3H, s, 8-OCH3); 13C NMR (150 MHz, CDCl3) d: 168.7 (C1), 150.2 (C-7), 149.2 (C-8), 146.8 (C-3), 137.6 (C-20 ), 128.9–129.3 (C-30 –C-70 ), 127.4 (C-4), 123.9 (C-5), 116.2 (C-6), 114.9 (C-2), 111.5 (C-9), 66.9 (C-10 ), 56.2 (C-8-OCH3). Ferulic acid 30 ,30 -dimethylallyl ester (5): ESI-MS m/z 285 [M+Na]+, C15H18O4; 1H NMR (600 MHz, CD3OD) d: 7.58 (1H, d, J = 15.8 Hz, H-3), 7.17 (1H, d, J = 2.0 Hz, H-9), 7.06 (1H, dd, J = 8.2, 2.0 Hz, H-5), 6.79 (1H, d, J = 8.2 Hz, H-6), 6.33 (1H, d, J = 15.8 Hz, H-2), 5.40 (1H, t, J = 7.4, H-20 ), 4.66 (2H, d, J = 7.4, H-10 ), 3.88 (3H, s, 8-OCH3), 1.77 (3H, s, H-40 ), 1.75 (3H, s, H-50 ); 13C NMR (150 MHz, CD3OD) d: 168.1 (C-1), 149.9 (C-7), 148.5 (C-8), 145.9 (C-3), 139.2 (C-30 ), 126.5 (C-4), 122.3 (C-5), 119.1 (C-20 ), 113.9 (C-2), 111.1 (C-9), 22.6 (C-40 ), 17.1 (C-50 ). 3,4-Dimethoxycaffeic acid cinnamyl ester (6): ESI-MS m/z 325 [M+H]+, C20H20O4; 1H NMR (600 MHz, CDCl3) d: 7.66 (1H, d, J = 15.8 Hz, H-3), 7.40 (2H, d, J = 7.6 Hz, H-50 , H-90 ), 7.33 (2H, t, J = 7.6 Hz, H-60 , H-80 ), 7.25 (1H, m, H70 ), 7.11 (1H, dd, J = 8.2, 2.0 Hz, H-5), 7.05 (1H, d, J = 2.0 Hz, H-9), 6.86 (1H, d, J = 8.2 Hz, H-6), 6.70 (1H, d, J = 15.8 Hz, H-30 ), 6.35 (1H, m, H-20 ), 6.34 (1H, d, J = 15.8 Hz, H-2), 4.86 (2H, d, J = 6.2 Hz, H-10 ) 3.91 (3H, s, 7-OCH3), 3.90 (3H, s, 8-OCH3). Coumaric acid cinnamyl ester (7): ESI-MS m/z 303 [M+Na]+ in positive mode, m/z 279 [MH] in negative mode, C18H16O3; 1H NMR (600 MHz, CDCl3) d: 7.67 (1H, d, J = 15.8 Hz, H-3), 7.44 (2H, d, J = 8.2 Hz, H-5, H-9), 7.41 (2H, d, J = 7.6 Hz, H-50 , H-90 ), 7.34 (2H, t, J = 7.6 Hz, H-60 , H-80 ), 7.25 (1H, m, H-70 ), 6.85 (2H, d, J = 8.2 Hz, H-6, H-8), 6.70 (1H, d, J = 15.8 Hz, H-30 ), 6.34 (1H, m, H-20 ), 6.33 (1H, d, J = 15.8 Hz, H-2), 4.86 (2H, d, J = 6.2 Hz, H-10 ); 13C NMR (150 MHz, CDCl3) d: 167.3 (C-1), 157.8 (C-7), 144.9 (C-3), 136.4 (C-40 ), 134.2 (C-30 ), 130.1 (C-5, C-9), 128.7 (C-60 , C-70 , C-80 ), 128.1 (C-4), 126.7 (C-50 , C-90 ), 123.5 (C-20 ), 116.0 (C-6, C-8), 115.5 (C-2), 65.1 (C-10 ). Coumaric acid benzyl ester (8): ESI-MS m/z 277 [M+Na]+, C16H14O3; 1H NMR (600 MHz, CDCl3) d: 7.67 (1H, d,
8. 9. 10. 11.
3
J = 15.8 Hz, H-3), 7.42 (2H, d, J = 8.2 Hz, H-5, H-9), 7.30–7.40 (5H, m, H-30 –H70 ), 6.83 (2H, d, J = 8.2 Hz, H-6, H-8), 6.34 (1H, d, J = 15.8 Hz, H-2), 5.25 (2H, s,H10 ); 13C NMR (150 MHz, CDCl3) d: 167.4 (C-1), 157.8 (C-7), 145.0 (C-3), 136.1 (C-20 ), 130.0 (C-5, C-9), 128.2–128.6 (C-30 C-70 ), 127.1 (C-4), 115.9 (C-6, C-8), 115.2 (C-2), 66.3 (C-10 ). Cinnamic acid phenethyl ester (9): ESI-MS m/z 275 [M+Na]+, C17H16O2; 1H NMR (600 MHz, CD3OD) d: 7.65 (1H, d, J = 15.8 Hz, H-3), 7.58 (2H, m, H-5, H-9), 7.39 (3H, m, H-6H-8), 7.20–7.34 (5H, m, H-4–H-8), 6.49 (1H, d, J = 15.8 Hz, H-2), 4.39 (2H, t, J = 6.8, H-10 ), 3.00 (2H, t, J = 6.8, H-20 ); 13 C NMR (150 MHz, CD3OD) d: 168.5 (C-1), 146.3 (C-3), 139.4 (C-30 ), 135.7 (C4), 131.5 (C-7), 130.0 (C-40 , C-80 ), 129.9 (C-50 , C-70 ), 129.5 (C-6, C-8), 129.2 (C-5, C-9), 127.6 (C-60 ), 118.8 (C-2), 65.4 (C-10 ), 35.4 (C-20 ). Cinnamic acid cinnamyl ester (10): ESI-MS m/z 287 [M+Na]+, C18H16O2; 1H NMR (600 MHz, CD3OD) d: 7.73 (1H, d, J = 15.8 Hz, H-3), 7.60 (2H, m, H-5, H-9), 7.42 (2H, d, J = 6.8 Hz, H-50 , H-90 ), 7.39 (3H, m, H-6–H-8), 7.30 (2H, m, H-60 , H-80 ), 7.25 (1H, m, H-70 ), 6.73 (1H, d, J = 15.8 Hz, H-30 ), 6.58 (1H, d, J = 15.8 Hz, H-2), 6.40 (1H, d, J = 15.8 Hz, H-20 ), 4.85 (2H, m, H-10 ); 13C NMR (150 MHz, CD3OD) d: 167.4 (C-1), 145.8 (C2), 136.8 (C-40 ), 135.0 (C-4), 134.5 (C-30 ), 130.8 (C-7), 129.3 (C-6, C-8), 128.9 (C60 , C-80 ), 128.5 (C-5, C-9), 128.3 (C-70 ), 126.9 (C-50 , C-90 ), 123.7 (C-20 ), 118.1 (C2), 65.5 (C-10 ). Lee, I. K.; Jung, J. Y.; Kim, Y. S.; Rhee, M. H.; Yun, B. S. Bioorg. Med. Chem. 2009, 17, 4674. Lee, I. K.; Yun, B. S. Bioorg. Med. Chem. 2007, 15, 3309. Chang, Y. C.; Lee, F. W.; Chen, C. S.; Huang, S. T.; Tsai, S. H.; Huang, S. H.; Lin, C. M. Free Radical Biol. Med. 2007, 43, 1541. Zhang, H. Y.; Sun, Y. M.; Wang, X. L. Chem. Eur. J. 2003, 9, 502.
Please cite this article in press as: Lee, I.-K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.065
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Phenylpropanoid acid esters from Korean propolis and their antioxidant activities In-Kyoung Lee, Myung-Suk Han, Dae-Won Kim, Bong-Sik Yun ⇑ Division of Biotechnology and Advanced Institute of Environment and Bioscience, Chonbuk National University, 79 Gobong-ro, Iksan-si 570-752, Republic of Korea
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 20 March 2014 Revised 9 May 2014 Accepted 16 May 2014 Available online xxxx
Ten phenylpropanoic acid esters were isolated from an ethanolic extract of Korean propolis. Their structures were elucidated by spectroscopic methods including NMR and ESI-MS. Caffeic acid esters with catechol moiety exhibited significant ABTS and DPPH radical scavenging activity and protective effect against DNA damage by a Fenton reaction. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Propolis Phenylpropanoic acid esters Antioxidant activity
Propolis is a sticky resinous product built by honeybees by mixing their own waxes with resins collected from various plants, and it is used in the construction and maintenance of their hives. Propolis has been recently attracting the attention due to its various biological activities and therapeutic properties proven to be beneficial in allergies, asthma, diabetes, and hypertension.1,2 There is a lot of evidence that it is an abundant source of polyphenols, mainly flavonoids and phenolic acids, and they attribute to pharmacological
properties in propolis. Caffeic acid phenethyl ester (CAPE) has been identified as one of the major biologically active substance; it is best known as a phenylpropanoic acid ester in propolis and known for its antioxidant, anti-inflammatory, chemoprevention, and antitumor properties.3 The phenylpropanoic acid esters have been found to various extents in propolis, since the chemical composition of propolis depends on the regional vegetation, season, and honeybee species at the site of collection.
R1 5 9
3
R2
1
OR3
O
Table 1 Free radical scavenging activity of phenylpropanoic acid esters TEACa,b
Compounds
1: R1, R2 = OH, R3 = Phenethyl
c
2: R1, R2 = OH, R3 = Benzyl
1 2 3 4 5 6 7 8 9 10 BHA Trolox
3: R1, R2 = OH, R3 = Ethyl 4: R1 = OH, R2 = OCH3, R3 = Benzyl 5: R1 = OH, R2 = OCH3, R3 = 3,3-dimethylallyl 6: R1, R2 = OCH3, R3 = Cinnamyl 7: R1 = OH, R2 = H, R3 = Cinnamyl 8: R1 = OH, R2 = H, R3 = Benzyl 9: R1, R2 = H, R3 = Phenethyl 10: R1, R2 = H, R3 = Cinnamyl Figure 1. Structures of phenylpropanoic acid esters.
a b
⇑ Corresponding author. Tel.: +82 63 850 0839; fax: +82 63 850 0834. E-mail address: [email protected] (B.-S. Yun).
c d
ABTS radical
DPPHd radical
6.30 ± 0.43 3.42 ± 0.30 10.61 ± 0.39 24.82 ± 3.93 23.10 ± 4.79 >500 >500 >500 >500 >500 0.68 ± 0.06 1
0.46 ± 0.00 3.86 ± 0.79 6.30 ± 0.94 92.10 ± 7.86 105.00 ± 8.23 >500 >500 >500 >500 >500 0.84 ± 0.01 1
Expressed as IC50 of lM compounds/IC50 of lM trolox. Results presented as the mean (n = 3) ± SD. 2,20 -Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid). a,a-Diphenyl-b-picrylhydrazyl.
http://dx.doi.org/10.1016/j.bmcl.2014.05.065 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lee, I.-K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.065
2
I.-K. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Figure 2. Protective effects of compounds 1–10 against plasmid DNA breakage due to the Fenton reaction between ferrous and hydrogen peroxide. Butylated hydroxyanisole (BHA) and Trolox were used as controls. Supercoiled DNA from pBR322 was incubated for 30 min at 25 °C in 10.0 mM Tris–HCl buffer and mixed with 50.0 lM compounds, 0.25 mM H2O2, and 12.5 lM ferric chloride.
We previously reported on the isolation and characterization of flavonoids and their free radical scavenging activity from Korean propolis.4 In this study, we describe the isolation, structural elucidation of ten phenylpropanoic acid esters from Korean propolis, and their antioxidant properties, collected in the Yangpyeong region. Propolis (136.3 g) collected from the Yangpyeong region, Gyeonggi-do province in Korea was extracted with 95% ethanol for 24 h. The ethanolic extract was concentrated under reduced pressure and then subjected to silica gel column chromatography (70–230 mesh) eluted with a gradient with increasing amount (1–50%) of methanol in chloroform to afford two fractions, fractions A and B. Fraction A was chromatographed on a column of Sephadex LH-20 with chloroform: methanol (1:1, v/v) and then was purified with an ODS-Sepak cartridge using a gradient of increasing methanol (50–100%) in water to give three fractions, A-1, A-2, and A-3. Preparative reversed-phase HPLC was performed with 60–80% aqueous MeOH gradient for fraction A-1 to give compounds 9 (3.2 mg) and 10 (2.9 mg), 55% aqueous methanol for fraction A-2 to give compound 6 (2.1 mg), and 50% aqueous MeOH for fraction A-3 to give compounds 4 (4.6 mg) and 5 (1.8 mg). Fraction B was chromatographed on a Sephadex LH-20 column eluted with methanol to give two fractions, B-1 and B-2. Fraction B-1 was separated by an ODS-sepak cartridge using a gradient of increasing methanol (50–100%) in water, followed by preparative reversedphase HPLC with 60% aqueous methanol to afford compound 8 (9.6 mg). Compounds 1–3 (10.8, 31.4, and 3.9 mg, respectively) and compound 7 (4.1 mg) were isolated from fraction B-2 through preparative reversed-phase HPLC with 65% aqueous methanol. The structures of these compounds were established by spectroscopic method including NMR and ESI-MS, and determined by comparison with reference data.5,6 Their structures were identified as caffeic acid phenethyl ester (1), caffeic acid benzyl ester (2), caffeic acid ethyl ester (3), ferulic acid benzyl ester (4), ferulic acid 30 ,30 -dimethylallyl ester (5), 3,4-dimethoxycaffeic acid cinnamyl ester (6), coumaric acid cinnamyl ester (7), coumaric acid benzyl ester (8), cinnamic acid phenethyl ester (9), and cinnamic acid cinnamyl ester (10),7 as shown in Figure 1. Antioxidant effect of the 10 phenylpropanoid acid esters isolated was estimated using the method described in the literatures.8,9 The free radical scavenging activity was evaluated by measuring their scavenging effects against ABTS radical cation and DPPH radical. These assays provide information on the reactivity of test compounds with a stable free radical. The results were expressed as TEAC value (trolox equivalent antioxidant capacity, IC50 of lM compound/IC50 of lM trolox), as shown in Table 1. The active compounds showed radical scavenging activity in a dose-dependent manner. The most active compounds were 1, 2, and 3 (caffeic acid phenethyl ester, caffeic acid benzyl ester, caffeic acid ethyl ester), which contain catechol moiety known as an antioxidant pharmacophore.10 Compounds 4 and 5 (ferulic acid benzyl ester and ferulic acid 30 ,30 -dimethylallyl ester) exhibited moderate activities, and 6 (3,4-dimethoxycaffeic acid cinnamyl ester) showed no activity. In these compounds, the hydroxyl group of catechol moiety was masked by the methyl group, which causes a loss
of radical scavenging activity. Coumaric acid esters (7, 8) and cinnamic acid esters (9, 10) showed no activity up to 1.0 mM. The hydroxyl radical scavenging assay was evaluated by Fenton reaction-induced DNA single strand breakage method.8 Fenton chemistry produces strong oxidant hydroxyl radical with a reaction between iron and hydrogen peroxide, and causes breakage of the deoxyribose-phosphate backbone of DNA. This assay allows the quantification of single strand breakage DNA generated by a single nick in supercoiled form DNA. Compounds 1, 2, and 3 significantly inhibited the Fenton reaction in a concentration of 50 lM, resulting in protection against DNA damage, as shown in Figure 2. Compounds 4 and 5 exhibited moderate activity, but other compounds did not protection against DNA breakage. Phenolic compounds are known as powerful antioxidants because of their scavenging ability due to catechol moiety.10,11 It is well known that the semiquinone radical derived from H-atom donation of catechol can be stabilized by an intramolecular hydrogen bond and the electron-donating properties of the ortho-OH.11 In conclusion, this study provided evidence that caffeic acid benzyl ester and caffeic acid phenethyl ester are dominant constituents in Korean propolis, and caffeic acid esters with catechol moiety were considerably responsible for antioxidant activity of propolis. Acknowledgments This work was supported by a Grant from Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2012R1A1A3011559). References and notes 1. Aguero, M. B.; Gonzalez, M.; Lima, B.; Svetaz, L.; Sanchez, M.; Zacchino, S.; Feresin, G. E.; Palermo, J.; Wunderlin, D.; Tapia, A. J. Agric. Food Chem. 2010, 58, 194. 2. Cheng, P. C.; Wong, G. Bee World 1996, 77, 8. 3. Watanabe, M. A.; Amarante, M. K.; Conti, B. J.; Sforcin, J. M. J. Pharm. Pharmacol. 2011, 63, 1378. 4. Han, M. S.; Lee, I. K.; Kim, Y. S.; Kim, J. T.; Choe, K. R.; Yun, B. S. J. Korean Soc. Appl. Biol. Chem. 2010, 53, 512. 5. Hu, L. H.; Zou, H. B.; Gong, J. X.; Li, H. B.; Yang, L. X.; Cheng, W.; Zhou, C. X.; Bai, H.; Gueritte, F.; Zhao, Y. J. Nat. Prod. 2005, 68, 342. 6. Li, F.; Awale, S.; Tezuka, Y.; Esumi, H.; Kadota, S. J. Nat. Prod. 2010, 73, 623. 7. Caffeic acid phenethyl ester (1): ESI-MS m/z 285 [M+H]+, C17H16O4; 1H NMR (600 MHz, CD3OD) d: 7.50 (1H, d, J = 15.8 Hz, H-3), 7.20–7.30 (5H, m, H-40 –H80 ), 7.01 (1H, d, J = 2.0 Hz, H-9), 6.91 (1H, dd, J = 8.2, 2.0 Hz, H-5), 6.76 (1H, d, J = 8.2 Hz, H-6), 6.21 (1H, d, J = 15.8 Hz, H-2), 4.35 (2H, t, J = 6.9 Hz, H-10 ), 2.98 (2H, t, J = 6.9 Hz, H-20 ). Caffeic acid benzyl ester (2): ESI-MS m/z 271 [M+H]+, C16H14O4; 1H NMR (600 MHz, CD3OD) d: 7.57 (1H, d, J = 15.8 Hz, H-3), 7.30– 7.40 (5H, m, H-30 –H-70 ), 7.02 (1H, d, J = 2.0 Hz, H-9), 6.92 (1H, dd, J = 8.2, 2.0 Hz, H-5), 6.76 (1H, d, J = 8.2 Hz, H-6), 6.28 (1H, d, J = 15.8 Hz, H-2), 5.21 (2H, s, H10 ); 13C NMR (150 MHz, CD3OD) d: 167.7 (C-1), 148.3 (C-7), 145.9 (C-3), 145.5 (C-8), 136.5 (C-20 ), 126.4 (C-4), 128.2127.8 (C-30 –C-70 ), 121.7 (C-5), 115.2 (C6), 113.9 (C-9), 113.6 (C-2), 65.8 (C-10 ). Caffeic acid ethyl ester (3): ESI-MS m/z 209 [M+H]+, C11H12O4; 1H NMR (600 MHz, CD3OD) d: 7.53 (1H, d, J = 15.8 Hz, H3), 7.03 (1H, d, J = 2.0 Hz, H-9), 6.93 (1H, dd, J = 8.2, 2.0 Hz, H-5), 6.77 (1H, d, J = 8.2 Hz, H-6), 6.25 (1H, d, J = 15.8 Hz, H-2), 4.26 (2H, q, J = 6.9 Hz, H-10 ), 1.30 (3H, t, J = 6.9 Hz, H-20 ). Ferulic acid benzyl ester (4): ESI-MS m/z 285 [M+H]+, 307 [M+Na]+, C17H16O4; 1H NMR (600 MHz, CDCl3) d: 7.64 (1H, d, J = 15.8 Hz, H3), 7.30–7.40 (5H, m, H-30 –H-70 ), 7.01 (1H, d, J = 2.0 Hz, H-9), 7.06 (1H, dd, J = 8.2, 2.0 Hz, H-5), 6.89 (1H, d, J = 8.2 Hz, H-6), 6.32 (1H, d, J = 15.8 Hz, H-1),
Please cite this article in press as: Lee, I.-K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.065
I.-K. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx 5.23 (2H, s, H-10 ), 3.91 (3H, s, 8-OCH3); 13C NMR (150 MHz, CDCl3) d: 168.7 (C1), 150.2 (C-7), 149.2 (C-8), 146.8 (C-3), 137.6 (C-20 ), 128.9–129.3 (C-30 –C-70 ), 127.4 (C-4), 123.9 (C-5), 116.2 (C-6), 114.9 (C-2), 111.5 (C-9), 66.9 (C-10 ), 56.2 (C-8-OCH3). Ferulic acid 30 ,30 -dimethylallyl ester (5): ESI-MS m/z 285 [M+Na]+, C15H18O4; 1H NMR (600 MHz, CD3OD) d: 7.58 (1H, d, J = 15.8 Hz, H-3), 7.17 (1H, d, J = 2.0 Hz, H-9), 7.06 (1H, dd, J = 8.2, 2.0 Hz, H-5), 6.79 (1H, d, J = 8.2 Hz, H-6), 6.33 (1H, d, J = 15.8 Hz, H-2), 5.40 (1H, t, J = 7.4, H-20 ), 4.66 (2H, d, J = 7.4, H-10 ), 3.88 (3H, s, 8-OCH3), 1.77 (3H, s, H-40 ), 1.75 (3H, s, H-50 ); 13C NMR (150 MHz, CD3OD) d: 168.1 (C-1), 149.9 (C-7), 148.5 (C-8), 145.9 (C-3), 139.2 (C-30 ), 126.5 (C-4), 122.3 (C-5), 119.1 (C-20 ), 113.9 (C-2), 111.1 (C-9), 22.6 (C-40 ), 17.1 (C-50 ). 3,4-Dimethoxycaffeic acid cinnamyl ester (6): ESI-MS m/z 325 [M+H]+, C20H20O4; 1H NMR (600 MHz, CDCl3) d: 7.66 (1H, d, J = 15.8 Hz, H-3), 7.40 (2H, d, J = 7.6 Hz, H-50 , H-90 ), 7.33 (2H, t, J = 7.6 Hz, H-60 , H-80 ), 7.25 (1H, m, H70 ), 7.11 (1H, dd, J = 8.2, 2.0 Hz, H-5), 7.05 (1H, d, J = 2.0 Hz, H-9), 6.86 (1H, d, J = 8.2 Hz, H-6), 6.70 (1H, d, J = 15.8 Hz, H-30 ), 6.35 (1H, m, H-20 ), 6.34 (1H, d, J = 15.8 Hz, H-2), 4.86 (2H, d, J = 6.2 Hz, H-10 ) 3.91 (3H, s, 7-OCH3), 3.90 (3H, s, 8-OCH3). Coumaric acid cinnamyl ester (7): ESI-MS m/z 303 [M+Na]+ in positive mode, m/z 279 [MH] in negative mode, C18H16O3; 1H NMR (600 MHz, CDCl3) d: 7.67 (1H, d, J = 15.8 Hz, H-3), 7.44 (2H, d, J = 8.2 Hz, H-5, H-9), 7.41 (2H, d, J = 7.6 Hz, H-50 , H-90 ), 7.34 (2H, t, J = 7.6 Hz, H-60 , H-80 ), 7.25 (1H, m, H-70 ), 6.85 (2H, d, J = 8.2 Hz, H-6, H-8), 6.70 (1H, d, J = 15.8 Hz, H-30 ), 6.34 (1H, m, H-20 ), 6.33 (1H, d, J = 15.8 Hz, H-2), 4.86 (2H, d, J = 6.2 Hz, H-10 ); 13C NMR (150 MHz, CDCl3) d: 167.3 (C-1), 157.8 (C-7), 144.9 (C-3), 136.4 (C-40 ), 134.2 (C-30 ), 130.1 (C-5, C-9), 128.7 (C-60 , C-70 , C-80 ), 128.1 (C-4), 126.7 (C-50 , C-90 ), 123.5 (C-20 ), 116.0 (C-6, C-8), 115.5 (C-2), 65.1 (C-10 ). Coumaric acid benzyl ester (8): ESI-MS m/z 277 [M+Na]+, C16H14O3; 1H NMR (600 MHz, CDCl3) d: 7.67 (1H, d,
8. 9. 10. 11.
3
J = 15.8 Hz, H-3), 7.42 (2H, d, J = 8.2 Hz, H-5, H-9), 7.30–7.40 (5H, m, H-30 –H70 ), 6.83 (2H, d, J = 8.2 Hz, H-6, H-8), 6.34 (1H, d, J = 15.8 Hz, H-2), 5.25 (2H, s,H10 ); 13C NMR (150 MHz, CDCl3) d: 167.4 (C-1), 157.8 (C-7), 145.0 (C-3), 136.1 (C-20 ), 130.0 (C-5, C-9), 128.2–128.6 (C-30 C-70 ), 127.1 (C-4), 115.9 (C-6, C-8), 115.2 (C-2), 66.3 (C-10 ). Cinnamic acid phenethyl ester (9): ESI-MS m/z 275 [M+Na]+, C17H16O2; 1H NMR (600 MHz, CD3OD) d: 7.65 (1H, d, J = 15.8 Hz, H-3), 7.58 (2H, m, H-5, H-9), 7.39 (3H, m, H-6H-8), 7.20–7.34 (5H, m, H-4–H-8), 6.49 (1H, d, J = 15.8 Hz, H-2), 4.39 (2H, t, J = 6.8, H-10 ), 3.00 (2H, t, J = 6.8, H-20 ); 13 C NMR (150 MHz, CD3OD) d: 168.5 (C-1), 146.3 (C-3), 139.4 (C-30 ), 135.7 (C4), 131.5 (C-7), 130.0 (C-40 , C-80 ), 129.9 (C-50 , C-70 ), 129.5 (C-6, C-8), 129.2 (C-5, C-9), 127.6 (C-60 ), 118.8 (C-2), 65.4 (C-10 ), 35.4 (C-20 ). Cinnamic acid cinnamyl ester (10): ESI-MS m/z 287 [M+Na]+, C18H16O2; 1H NMR (600 MHz, CD3OD) d: 7.73 (1H, d, J = 15.8 Hz, H-3), 7.60 (2H, m, H-5, H-9), 7.42 (2H, d, J = 6.8 Hz, H-50 , H-90 ), 7.39 (3H, m, H-6–H-8), 7.30 (2H, m, H-60 , H-80 ), 7.25 (1H, m, H-70 ), 6.73 (1H, d, J = 15.8 Hz, H-30 ), 6.58 (1H, d, J = 15.8 Hz, H-2), 6.40 (1H, d, J = 15.8 Hz, H-20 ), 4.85 (2H, m, H-10 ); 13C NMR (150 MHz, CD3OD) d: 167.4 (C-1), 145.8 (C2), 136.8 (C-40 ), 135.0 (C-4), 134.5 (C-30 ), 130.8 (C-7), 129.3 (C-6, C-8), 128.9 (C60 , C-80 ), 128.5 (C-5, C-9), 128.3 (C-70 ), 126.9 (C-50 , C-90 ), 123.7 (C-20 ), 118.1 (C2), 65.5 (C-10 ). Lee, I. K.; Jung, J. Y.; Kim, Y. S.; Rhee, M. H.; Yun, B. S. Bioorg. Med. Chem. 2009, 17, 4674. Lee, I. K.; Yun, B. S. Bioorg. Med. Chem. 2007, 15, 3309. Chang, Y. C.; Lee, F. W.; Chen, C. S.; Huang, S. T.; Tsai, S. H.; Huang, S. H.; Lin, C. M. Free Radical Biol. Med. 2007, 43, 1541. Zhang, H. Y.; Sun, Y. M.; Wang, X. L. Chem. Eur. J. 2003, 9, 502.
Please cite this article in press as: Lee, I.-K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.065
