한국식품영양과학회지 (Journal of the Korean Society of Food Science and Nutrition)

  • 제44권10호
  • /
  • Pages.1439-1449
  • /
  • 2015
  • /
  • 1226-3311(pISSN)
  • /
  • 2288-5978(eISSN)
한국식품영양과학회 (The Korean Society of Food Science and Nutrition)
권호 표지
DOI QR코드

DOI QR Code

복숭아꽃 에탄올 추출물과 분획물의 in vitro 항산화 효과 및 RAW 264.7 대식세포에서의 항염증 효과

In vitro Antioxidant and Anti-Inflammatory Activities of Ethanol Extract and Sequential Fractions of Flowers of Prunus persica in LPS-Stimulated RAW 264.7 Macrophages

  • 곽충실 (서울대학교 노화고령사회연구소) ;
  • 최혜인 (서울대학교 노화고령사회연구소)
  • 투고 : 2015.06.15
  • 심사 : 2015.08.12
  • 발행 : 2015.10.31
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

우리나라에서는 전통적으로 복숭아꽃을 차로 마셔왔으며 피부 부스럼 등의 치료에 이용하였다는 기록이 있어 복숭아꽃 추출물이 산화적 스트레스와 염증반응을 억제시키는 효과가 있는지를 본 연구에서 확인하고자 하였다. 복숭아꽃을 건조시킨 다음 에탄올 추출 농축액(EtOH)을 얻었고, 이로부터 다시 hexane(Hx), dichloromethane(DM), ethyl acetate(EA), n-butanol(BtOH) 및 water(DW) 순으로 순차적 용매 분획을 시행하여 획득한 각 농축액에서 총 페놀화합물 함량, 플라보노이드 함량, DPPH 라디칼과 ABTS 라디칼 소거능을 측정하였고, LPS를 처리한 RAW 264.7 대식세포에서 이들 추출물들이 NO, PGE2, IL-6, TNF-${\alpha}$ 생성에 미치는 영향을 측정하였다. 그 결과 총 페놀화합물 함량과 플라보노이드 함량은 EA 분획(394.6 mg TA/g, 253.7 mg RT/g)이 가장 높았고, 그다음이 BtOH 분획(128.3 mg TA/g, 93.1 mg RT/g), DM 분획(79.5 mg TA/g, 52.9 mg RT/g), EtOH 추출물(78.1 mg TA/g, 55.3 mg RT/g) 순이었다. DPPH 라디칼 소거능은 EA> BtOH${\geq}$ EtOH> DM=DW> Hx 순이었으며, ABTS 라디칼 소거능은 EA> BtOH> EtOH=DM> Hx=DW 분획 순으로 항산화 효과는 EA, BtOH 분획이 가장 우수하였다. 총 페놀화합물의 함량은 DPPH 라디칼 소거능($IC_{50}$)(r=-0.6081, P<0.01), ABTS 라디칼 소거능(r=0.9683, P<0.001)과 유의적인 상관관계를 나타내었으며, DPPH 라디칼 소거능($IC_{50}$)과 ABTS 라디칼 소거능은 서로 유의한 상관관계(r=-0.7172, P<0.001)를 나타내었다. LPS를 처리한 대식세포에 각 추출시료를 세포독성이 없는 농도로 처리한 결과 EtOH 추출물과 Hx, DM, EA 분획이 NO 생성을 유의하게 감소시켰으며(P<0.05), EtOH 추출물과 DM, EA 분획이 PGE2 생성을 유의하게 감소시켰다(P<0.05). 염증성 사이토카인인 IL-6와 TNF-${\alpha}$ 생성은 EtOH 추출물과 Hx, DM, EA, BtOH 분획 모두 유의하게 감소시켰다(P<0.05). 더 나아가 LPS를 처리한 대식세포에서 복숭아꽃 EtOH 추출물을 처리하였을 때 농도의존적으로 iNOS와 COX-2의 합성을 효과적으로 억제시킴으로써 주요한 염증 매개물질인 NO와 PGE2의 생성이 억제되는 기전을 제시하였다. 한편 대식세포에서 분비된 아질산염의 농도는 TNF-${\alpha}$ 농도(r=0.6477, P<0.05) 및 PGE2 농도(r=0.6377, P<0.05)와 유의한 상관관계를 나타내었으며, 아질산염, PGE2, IL-6 농도는 총 페놀화합물 함량이나 항산화 효과 지표들과 유의한 상관성을 보이지 않았다. 다만 TNF-${\alpha}$ 농도만 총 페놀화합물 함량(r=0.6524, P<0.05), 플라보노이드 함량(r=0.6914, P<0.05), DPPH 라디칼 소거능($IC_{50}$)(r=-0.6839, P<0.05), ABTS 라디칼 소거능(r=0.7921, P<0.01)과 각각 유의한 상관관계를 나타내었으며, 염증반응의 매개물질인 PGE2 및 IL-6 농도와는 유의한 상관성이 없었다. 본 실험 결과를 종합하면 복숭아꽃 에탄올 추출물은 항산화 효과와 항염증 효과가 우수하였으며, 그로부터 얻은 분획물 중에서 특히 EA와 BtOH 분획은 항산화 효과가 매우 우수하였고, DM과 EA 분획은 항염증 효과가 우수하였다. 따라서 복숭아꽃 에탄올 추출물을 비롯한 이들 분획물들은 산화적 스트레스 및 염증반응의 상승과 관련되어 있는 만성질환을 예방하는 건강기능성 식품의 개발을 위한 천연소재로 이용 가능할 것으로 기대된다.

Prunus persica Flos (PPF) were investigated for their antioxidant and anti-inflammatory activities to find a natural functional food resource preventing degenerative diseases associated with excessive oxidative stress and chronic inflammation. PPF was extracted using ethanol (EtOH) and then sequentially fractioned by hexane (Hx), dichloromethane (DM), ethyl acetate (EA), n-butanol (BtOH), and water (DW). Contents of total phenolics and flavonoids, as well as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities were measured. Anti-inflammatory effects in terms of nitric oxide (NO), prostaglandin (PG) E2, and pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-${\alpha}$ production were also measured using LPS-treated RAW 264.7 macrophages. EtOH extract showed relatively high antioxidant activity with high total phenolic (78.1 mg tannic acid/g) and flavonoid contents (55.3 mg rutin/g). EA fraction contained the highest total phenolic and flavonoid contents (394.6 mg tannic acid/g, 253.7 mg rutin/g), followed by BtOH (128.3 mg tannic acid/g, 93.1 mg rutin/g). EA and BtOH fractions and EtOH extract showed higher DPPH radical and ABTS radical scavenging activities than the others (P<0.05). In LPS-treated RAW 264.7 macrophages, EtOH extract ($200{\mu}g/mL$) showed significantly reduced (P<0.05) NO, PGE2, and TNF-${\alpha}$ production levels to 38.5%, 32.3%, and 48.9% of the control, respectively, as well as reduced iNOS and COX-2 protein expression. DM fraction ($50{\mu}g/mL$) showed significantly reduced (P<0.05) NO, PGE2, IL-6, and TNF-${\alpha}$ production levels to 43.5%, 13.3%, 38.7%, and 41.3% of the control, respectively, and EA fraction ($50{\mu}g/mL$) showed significantly reduced NO, PGE2, IL-6, and TNF-${\alpha}$ production levels to 44.8%, 22.4%, 45.7%, and 62.0% of the control, respectively. Taken together, EtOH extract of PPF showed potent antioxidant and anti-inflammatory activities, and EA and BtOH fractions showed comparatively stronger antioxidant activities while DM and EA fractions showed stronger anti-inflammatory activities. It can be concluded that EtOH extract of PPF and its fractions are good candidates as natural resources for the development of anti-oxidative and anti-inflammatory functional food products.

키워드

참고문헌

  1. Lodovici M, Guglielmi F, Meoni M, Dolara P. 2001. Effect of natural phenolic acids on DNA oxidation in vitro. Food Chem Toxicol 39: 1205-1210. https://doi.org/10.1016/S0278-6915(01)00067-9
  2. Kim SH, Choi JH, Oh HT, Chung MJ, Cui CB, Ham SS. 2008. Cytoprotective effect of antioxidant activity of Codonopsis lanceolata and Platycodon grandiflorum ethyl acetate fraction in human HepG2 cells. Korean J Food Sci Technol 40: 696-701.
  3. Li C, Wang MH. 2011. Antioxidant activity of peach blossom extracts. J Korean Soc Apple Biol Chem 54: 46-53.
  4. Bak MJ, Jeong JH, Kang HS, Jin KS, Ok S, Jeong WS. 2009. Cedrela sinensis leaves suppress oxidative stress and expressions of iNOS and COX-2 via MAPK signaling pathways in RAW 264.7 cells. J Food Sci Nutr 14: 269-276. https://doi.org/10.3746/jfn.2009.14.4.269
  5. Zhang R, Brennan ML, Shen Z, MacPherson JC, Schmitt D, Molenda CE, Hazen SL. 2002. Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation. J Biol Chem 277: 46116-46122. https://doi.org/10.1074/jbc.M209124200
  6. Coulibaly AY, Kiendrebeogo M, Kehoe PG, Sombie PA, Lamien CE, Millogo JF, Nacoulma OG. 2011. Antioxidant and anti-inflammatory effects of Scoparia dulcis L. J Med Food 14: 1576-1582. https://doi.org/10.1089/jmf.2010.0191
  7. Guo D, Xu L, Cao X, Guo Y, Ye Y, Chan CO, Mok DK, Yu Z, Chen S. 2011. Anti-inflammatory activities and mechanisms of action of the petroleum ether fraction of Rosa multiflora Thunb. hips. J Ethnopharmacol 138: 717-722. https://doi.org/10.1016/j.jep.2011.10.010
  8. Jung SH, Kim SJ, Jun BG, Lee KT, Hong SP, Oh MS, Jang DS, Choi JH. 2013. ${\alpha}-Cyperone, isolated from the rhizomes of Cyperus rotundus, inhibits LPS-induced COX-2 expression and PGE2 production through the negative regulation of NF${\kappa}B signalling in RAW 264.7 cells. J Ethnopharmacol 147: 208-214. https://doi.org/10.1016/j.jep.2013.02.034
  9. Koh YJ, Cha DS, Ko JS, Park HJ, Choi HD. 2010. Anti-inflammatory effect of Taraxacum officinale leaves on lipopolysaccharide-induced inflammatory responses in RAW 264.7 cells. J Med Food 13: 870-878. https://doi.org/10.1089/jmf.2009.1249
  10. Liang YC, Huang YT, Tsai SH, Lin-Shiau SY, Chen CF, Lin JK. 1999. Suppression of inducible cyclooxygenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse macrophages. Carcinogenesis 20: 1945-1952. https://doi.org/10.1093/carcin/20.10.1945
  11. Shan J, Fu J, Zhao Z, Kong X, Huang H, Luo L, Yin Z. 2009. Chlorogenic acid inhibits lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 cells through suppressing NF-kappaB and JNK/AP-1 activation. Int Immunopharmacol 9: 1042-1048. https://doi.org/10.1016/j.intimp.2009.04.011
  12. Lee EJ, Kim C, Kim JY, Kim SM, Nam D, Jang HJ, Kim SH, Shim BS, Ahn KS, Choi SH, Jung SH, Ahn KS. 2012. Inhibition of LPS-induced inflammatory biomarkers by ethylacetate fraction of Patrinia scabiosaefolia through suppression of NF-${\kappa}B$ activation in RAW 264.7 cells. Immunopharmacol Immunotoxicol 34: 282-291. https://doi.org/10.3109/08923973.2011.602412
  13. Lee AK, Sung SH, Kim YC, Kim SG. 2003. Inhibition of lipopolysaccharide-inducible nitric oxide synthase, TNF-${\alpha}$ and COX-2 expression by auchinone effects on I-${\kappa}B{\alpha}$ phosphorylation, C/EBP and AP-1 activation. Br J Pharmacol 139: 11-20. https://doi.org/10.1038/sj.bjp.0705231
  14. Lee JY, An BJ. 2010. Antioxidant and anti-inflammatory effects of Prunus persicae Flos. J Appl Biol Chem 53: 162-169. https://doi.org/10.3839/jabc.2010.029
  15. Han W, Xu JD, Wei FX, Zheng YD, Ma JZ, Xu XD, Wei ZG, Wang W, Zhang YC. 2015. Prokinetic activity of Prunus persica (L.) batsch flowers extract and its possible mechanism of action in rats. Biomed Res Int 2015: 569853.
  16. Heo MY, Kim SH, Yang HE, Lee SH, Jo BK, Kim HP. 2001. Protection against ultraviolet B- and C-induced DNA damage and skin carcinogenesis by the flowers of Prunus persica extract. Mutat Res 496: 47-59. https://doi.org/10.1016/S1383-5718(01)00218-2
  17. Lee JY, An BJ. 2012. Antioxidant and anti-inflammatory effects of fractions from Pruni persicae Flos. Kor J Herbology 27: 55-63.
  18. Singleton VL, Orthofer R, Lamuela-Raventos RM. 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol 299: 152-178. https://doi.org/10.1016/S0076-6879(99)99017-1
  19. Chae SK, Kang GS, Ma SJ, Bang KW, Oh MW, Oh SH. 2002. Standard food analysis. Jigu-moonwha Sa, Seoul, Korea. p 381-382.
  20. Senba Y, Nishishita T, Saito K, Yoshioka H, Yoshioka H. 1999. Stopped-flow and spectrophotometric study on radical scavenging by tea catechins and model compound. Chem Pharm Bull 47: 1369-1374. https://doi.org/10.1248/cpb.47.1369
  21. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26: 1231-1237. https://doi.org/10.1016/S0891-5849(98)00315-3
  22. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63. https://doi.org/10.1016/0022-1759(83)90303-4
  23. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. 1982. Analysis of nitrate, nitrite, and [$^{15}N$]nitrate in biological fluids. Anal Biochem 126: 131-138. https://doi.org/10.1016/0003-2697(82)90118-X
  24. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 240-254.
  25. Rice-Evans C, Miller N, Paganga G. 1997. Antioxidant properties of phenolic compounds. Trends Plant Sci 2: 152-159. https://doi.org/10.1016/S1360-1385(97)01018-2
  26. Nakayama T, Niimi T, Osawa T, Kawakishi S. 1992. The protective role of polyphenols in cytotoxicity of hydrogen peroxide. Mutat Res 281: 77-80. https://doi.org/10.1016/0165-7992(92)90039-K
  27. Toda M, Okubo S, Hiyoshi R, Shimamura T. 1989. The bactericidal activity of tea and coffee. Lett Appl Microbiol 8: 123-125. https://doi.org/10.1111/j.1472-765X.1989.tb00255.x
  28. Middletone E Jr, Kandaswami CC. 1994. Potential healthpromoting properties of citrus flavonoids. Food Technol 48: 115-119.
  29. Lee S, You Y, Kim K, Park J, Jeong C, Jhon DY, Jun W. 2012. Antioxidant activities of native Gwangyang Rubus coreanus Muq. J Korean Soc Food Sci Nutr 41: 327-332. https://doi.org/10.3746/jkfn.2012.41.3.327
  30. Epe B, Ballmaier D, Roussyn I, Briviba K, Sies H. 1996. DNA damage by peroxynitrite characterized with DNA repair enzymes. Nucleic Acids Res 24: 4105-4110. https://doi.org/10.1093/nar/24.21.4105
  31. Bogdan C. 2001. Nitric oxide and the immune response. Nat Immunol 2: 907-916. https://doi.org/10.1038/ni1001-907
  32. Guo JY, Huo HR, Yang YX, Li CH, Liu HB, Zhao BS, Li LF, Ma YY, Guo SY, Jiang TL. 2006. 2-Methoxycinnamaldehyde reduces IL-$1{\beta}$-induced prostaglandin production in rat cerebral endothelial cells. Biol Pharm Bull 29: 2214-2221. https://doi.org/10.1248/bpb.29.2214
  33. Chang ST, Wu JH, Wang SY, Kang PL, Yang NS, Shyur LF. 2001. Antioxidant activity of extracts from Acacia confusa bark and heartwood. J Agric Food Chem 49: 3420-3424. https://doi.org/10.1021/jf0100907
  34. Monzon ME, Casalino-Matsuda SM, Forteza RM. 2006. Identification of glycosaminoglycans in human airway secretions. Am J Respir Cell Mol Biol 34: 135-141. https://doi.org/10.1165/rcmb.2005-0256OC
  35. Guha M, Mackman N. 2001. LPS induction of gene expression in human monocytes. Cell Signal 13: 85-94. https://doi.org/10.1016/S0898-6568(00)00149-2
  36. Kim YS, Lee SJ, Hwang JW, Kim EH, Park PJ, Jeong JH. 2012. Anti-inflammatory effects of extracts from Ligustrum ovalifolium H. leaves on RAW264.7 macrophages. J Korean Soc Food Sci Nutr 41: 1205-1210. https://doi.org/10.3746/jkfn.2012.41.9.1205
  37. Botting RM. 2006. Inhibitors of cyclooxygenases: mechanisms, selectivity and uses. J Physiol Pharmacol S5: 113-124.
  38. Bekir J, Mars M, Souchard JP, Bouajila J. 2013. Assessment of antioxidant, anti-inflammatory, anti-cholinesterase and cytotoxic activities of pomegranate (Punica granatum) leaves. Food Chem Toxicol 55: 470-475. https://doi.org/10.1016/j.fct.2013.01.036
  39. Kwak CS, Lee KJ, Chang JH, Park JH, Cho JH, Park JH, Kim KM, Lee MS. 2013. In vitro antioxidant, anti-allergic and anti-inflammatory effects of ethanol extracts from Korean sweet potato leaves and stalks. J Korean Soc Food Sci Nutr 42: 369-377. https://doi.org/10.3746/jkfn.2013.42.3.369

피인용 문헌

  1. Prevention Effect of Prunus persica Flos Extract from Reactive Oxygen Species Generation and Matrix Metalloproteinases Production Induced by UVB Irradiation in Human Skin Cells vol.14, pp.2, 2016, https://doi.org/10.20402/ajbc.2016.0043
  2. Topical or oral treatment of peach flower extract attenuates UV-induced epidermal thickening, matrix metalloproteinase-13 expression and pro-inflammatory cytokine production in hairless mice skin vol.12, pp.1, 2018, https://doi.org/10.4162/nrp.2018.12.1.29
  3. 용매별 초석잠 추출물의 항산화 및 항당뇨 활성 vol.24, pp.5, 2015, https://doi.org/10.11002/kjfp.2017.24.5.615
  4. 조팝나무 뿌리 열수 추출물이 RAW264.7 세포에서 미치는 항산화 및 항염증 활성 vol.30, pp.4, 2015, https://doi.org/10.7732/kjpr.2017.30.4.335
  5. 섬애약쑥 용매별 추출물의 생리활성 vol.29, pp.11, 2019, https://doi.org/10.5352/jls.2019.29.11.1241
  6. 아스파라거스를 이용한 전통장류의 항산화 효과 vol.49, pp.1, 2021, https://doi.org/10.48022/mbl.2008.08006
  7. Biological Activities and Anti-wrinkle Effects of Pinus koraiensis Siebold et Zucc. Leaf Extract vol.29, pp.2, 2021, https://doi.org/10.7783/kjmcs.2021.29.2.117
  8. Antioxidant Activity, Skin Whitening, and Anti-Wrinkle Effects of Various Agastache rugosa Fractions vol.50, pp.7, 2015, https://doi.org/10.3746/jkfn.2021.50.7.679