[논문]왕산방풍의 뿌리로부터 제조한 유기용매 분획물에서의 항산화 활성 및 RAW264.7 세포주에서의 항염증 효능

김진익; 최용원; 최근준; 강지안; 이인영; 나란투야 난딘셋세그; 오명석; 조식제; 문자영

doi:10.5352/jls.2021.31.1.17

왕산방풍의 뿌리로부터 제조한 유기용매 분획물에서의 항산화 활성 및 RAW264.7 세포주에서의 항염증 효능
Antioxidant Activities of Peucedanum insolens Kitagawa Root Extracts and Their Anti-inflammatory Effects on LPS-treated RAW264.7 Cells 원문보기

생명과학회지 = Journal of life science, v.31 no.1, 2021년, pp.17 - 27

김진익 (창원대학교 생명보건학부) , 최용원 (창원대학교 생명보건학부) , 최근준 (창원대학교 생명보건학부) , 강지안 (창원대학교 생명보건학부) , 이인영 (창원대학교 생명보건학부) , 나란투야 난딘셋세그 (창원대학교 생명보건학부) , 오명석 (창원대학교 생명보건학부) , 조식제 (새우리 국제특허법률사무소) , 문자영 (창원대학교 생명보건학부)

초록
AI-Helper

본 연구는 Peucedanum insolens Kitagawa 뿌리의 여러 유기용매 분획물에 대한 항산화 활성과 LPS 처리된 RAW264.7 세포에서 항염증 효과를 조사하기 위해 수행되었다. 이 연구를 위해 P. insolens Kitagawa 뿌리를 먼저 실온에서 1 주일 동안 건조하여 잘게 썰고 70% 에탄올로 추출한 다음 hexane, chloroform, ethyl acetate 및 물로 연속적으로 분획하였다. DPPH (1,1-diphenyl-2-picrylhydrazyl) 라디칼 소거능 분석과 total polyphenol 및 total flavonoid 함량을 측정하여 유기용매 분획물의 항산화 활성을 평가했다. RAW264.7 세포에서 iNOS, COX-2, IL-1β 및 IL-6과 같은 염증 매개 유전자의 발현 억제 수준을 측정하여 분획의 항 염증 효능을 평가했다. 본 연구 결과는 P. insolens Kitagawa 뿌리의 ethyl acetate 분획물이 상대적으로 높은 함량의 total flavonoid (34.08±1.68 ㎍ QE/mg)와 total polyphenol (154.1±3.2 ㎍의 GE/mg)을 함유하고 있음을 분명히 보여주었다. DPPH 분석 결과 P. insolens Kitagawa는 ethyl acetate 분획에서 강력한 자유 라디칼 소거 활성을 가지고 있음을 보여주었다. Ethyl acetate 분획 및 hexane 분획은 모두 RAW264.7 세포에서 24시간 동안 LPS (1 ㎍/ml) 처리에 의해 유도된 산화질소 생성에 대해 강력한 억제력을 나타냈다. 본 연구결과는 또한 P. insolens Kitagawa의 hexane 분획 및 ethyl acetate 분획 모두 RAW264.7 세포에서 24시간 동안 LPS 처리에 의해 과발현된 iNOS, IL-1β 및 IL-6의 mRNA 수준을 강력하게 억제하는 것으로 나타났다. 이러한 결과는 P. insolens Kitagawa가 항 염증 활성에 강한 효능을 가진 화합물을 포함할 수 있음을 시사한다. 염증 조절에 대한 P. insolens Kitagawa 분획물의 더 자세한 효과를 이해하기 위하여 이 분획물이 cytokine 신호 전달 경로 및 염증 신호 경로에 대한 조절작용과 같은 추가적인 작용기전 연구가 필요하다.

Abstract ▼ AI-Helper

This study was performed to investigate the antioxidant activities of subfractions of Peucedanum insolens Kitagawa root in various organic solvents and their anti-inflammatory effects on LPS-treated RAW264.7 cells. First, P. insolens Kitagawa roots were dried at room temperature for one week, chopped, and extracted with 70% ethanol. The resulting extracts were successively sub-fractionated with hexane, chloroform, ethyl acetate, and water. The antioxidant potential of the fractions was evaluated using a DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay and by measuring total polyphenol and flavonoid contents. The anti-inflammatory potency of the fractions was evaluated by measuring the inhibition levels of the expressions of inflammatory-mediated genes and proteins (e.g., iNOS, COX-2, IL-1β, and IL-6) in RAW264.7 cells. The results clearly showed that the ethyl acetate fraction of the P. insolens Kitagawa root contained relatively high total flavonoid (34.08±1.68 ㎍ of quercetin equivalents per mg) and total polyphenol (154.1±3.2 ㎍ of gallic acid equivalents per mg) contents. The DPPH assay results showed that the P. insolens Kitagawa root possessed strong free radical scavenging activity in the ethyl acetate fraction. Both the ethyl acetate and hexane fractions showed strong inhibitory potencies to nitric oxide production induced by lipopolysaccharide (1 ㎍/ml) treatment for 24 hr in RAW264.7 cells. The results also showed that both the hexane and ethyl acetate fractions of the P. insolens Kitagawa root strongly inhibited mRNA levels of iNOS, IL-1β, and IL-6, which were overexpressed by LPS treatment for 24 hr in the RAW264.7 cells. These results suggest that P. insolens Kitagawa root may contain compounds that possess strong potency for anti-inflammatory activity. Further studies are needed to discover more detailed modes of action of P. insolens Kitagawa root fractions against inflammation modulation, such as the regulation of cytokine signaling and inflammatory signaling pathways.

주제어

표/그림 (8)

$Fig. 1. Scheme for preparation of organic sol-vent-fractions of Peucednum insolens Kitagawa root.$ 그림 Fig. 1. Scheme for preparation of organic sol-vent-fractions of Peucednum insolens Kitagawa root.
표 Table 1. Primer sequences and lengths used for qRT-PCR
표 Table. 2. Total polyphenols and total flavonoids in the organic solvent extracts of P. insolens Kitagawa root
$Fig. 2. DPPH radical-scavenging activities of organic solvent-fractions of P. insolens Kitagawa root. RH, RC and RE represent the fractions of hexane, chloroform, and ethyl acetate of P. insolens Kitagawa root, respectively.$ 그림 Fig. 2. DPPH radical-scavenging activities of organic solvent-fractions of P. insolens Kitagawa root. RH, RC and RE represent the fractions of hexane, chloroform, and ethyl acetate of P. insolens Kitagawa root, respectively.
$Fig. 3. Cytotoxicity of organic solvent-fractions of P. insolens Kitagawa root in RAW264.7 cells. Cells were treated with various concentrations of hexane (RH), chloroform (RC) or ethyl acetate (RE)-fraction of P. insolens Kitagawa root extracts. After treatment for 24 hr, cell viability was measured with the MTT assay. Values are the mean ± SD of experiments in triplicate (n=3).$ 그림 Fig. 3. Cytotoxicity of organic solvent-fractions of P. insolens Kitagawa root in RAW264.7 cells. Cells were treated with various concentrations of hexane (RH), chloroform (RC) or ethyl acetate (RE)-fraction of P. insolens Kitagawa root extracts. After treatment for 24 hr, cell viability was measured with the MTT assay. Values are the mean ± SD of experiments in triplicate (n=3).
$Fig. 4. Protective effects of organic solvent-fractions of P. insolens Kitagawa root against cytotoxicity induced by LPS treat-ment in RAW264.7 cells. RAW264.7 cell were treated with both LPS (1 μg/ml) and either hexane (RH), chloro-form (RC) or ethyl acetate (RE)-fraction of P. insolens Kitagawa root extracts simultaneously for 24 hr. All ex-periments were performed in triplicate and results are expressed as mean ± SD. *p<0.05, **p<0.01 (untreated group vs each treated group); n.s., not significant.$ 그림 Fig. 4. Protective effects of organic solvent-fractions of P. insolens Kitagawa root against cytotoxicity induced by LPS treat-ment in RAW264.7 cells. RAW264.7 cell were treated with both LPS (1 μg/ml) and either hexane (RH), chloro-form (RC) or ethyl acetate (RE)-fraction of P. insolens Kitagawa root extracts simultaneously for 24 hr. All ex-periments were performed in triplicate and results are expressed as mean ± SD. *p<0.05, **p<0.01 (untreated group vs each treated group); n.s., not significant.
$Fig. 5. Nitrite-scavenging activities of organic solvent-fractions of P. insolens Kitagawa root in LPS-stimulated RAW 264.7 cells. RAW264.7 cells were treated with either hexane (RH), chloroform (RC) or ethyl acetate (RE)-fractions at indicated concentrations in the absence or presence of LPS (1 μg/ml) for 24 hr. After the incubation period, cell culture supernatants were harvested and nitrite production was determined by Griess reagent. Values are expressed as described in the Materials and Methods section. All experiments were performed in triplicate and results are expressed as mean ± SD. ***p<0.001 (untreated group vs each treated group).$ 그림 Fig. 5. Nitrite-scavenging activities of organic solvent-fractions of P. insolens Kitagawa root in LPS-stimulated RAW 264.7 cells. RAW264.7 cells were treated with either hexane (RH), chloroform (RC) or ethyl acetate (RE)-fractions at indicated concentrations in the absence or presence of LPS (1 μg/ml) for 24 hr. After the incubation period, cell culture supernatants were harvested and nitrite production was determined by Griess reagent. Values are expressed as described in the Materials and Methods section. All experiments were performed in triplicate and results are expressed as mean ± SD. ***p<0.001 (untreated group vs each treated group).
$Fig. 6. Real time inhibitory effects of organic solvent-fractions of P. insolens Kitagawa root on the expressions of iNOS (A), IL-1β (B) and IL-6 (C) genes in LPS-stimulated RAW 264.7 cells. RAW264.7 cells were treated with 1 μg/ml LPS in the absence or presence of either hexane (RH), chloroform (RC) or ethyl acetate (RE) fractions of P. in-solens Kitagawa roots at indicated concentrations (50 or 100 μg/ml) for 24 hr. After the incubation period, total RNAs were extracted from the treated cells and cDNAs were reverse transcribed using first strand cDNA syn-thesis kit as described in Materials and Methods. Expression levels of iNOS, IL-1β and IL6 mRNA were amplified by real-time PCR analysis. All experiments were conducted in triplicate and results are expressed as mean ± SD. ***p<0.001 (untreated group vs each treat-ed group); n.s., not significant.$ 그림 Fig. 6. Real time inhibitory effects of organic solvent-fractions of P. insolens Kitagawa root on the expressions of iNOS (A), IL-1β (B) and IL-6 (C) genes in LPS-stimulated RAW 264.7 cells. RAW264.7 cells were treated with 1 μg/ml LPS in the absence or presence of either hexane (RH), chloroform (RC) or ethyl acetate (RE) fractions of P. in-solens Kitagawa roots at indicated concentrations (50 or 100 μg/ml) for 24 hr. After the incubation period, total RNAs were extracted from the treated cells and cDNAs were reverse transcribed using first strand cDNA syn-thesis kit as described in Materials and Methods. Expression levels of iNOS, IL-1β and IL6 mRNA were amplified by real-time PCR analysis. All experiments were conducted in triplicate and results are expressed as mean ± SD. ***p<0.001 (untreated group vs each treat-ed group); n.s., not significant.

AI 본문요약
AI-Helper

데이터처리

Student’s t-test and one-way ANOVA were used to determine the statistical significance of the difference between values for the various experimental and control groups

참고문헌 (46)

Agrawal, S., Kulkarni, G. T. and Sharma, V. N. 2011. A comparative study on the antioxidant activity of methanolic extracts of Terminalia paniculata and Madhuca longifolia. Free Radic. Antiox. 1, 62-68.
Bendary, E., Francis, R. R., Ali, H. M. G., Sarwat, M. I. and El Hady, S. 2013. Antioxidant and structure-activity relationships (SARs) of some phenolic and anilines compounds. Ann. Agric. Sci. 58, 173-181.

상세보기
Bertolini, A., Ottani, A. and Sandrini, M. 2002. Selective COX-2 inhibitors and dual acting anti-inflammatory drugs: critical remarks. Curr. Med. Chem. 9, 1033-1043.

상세보기
Brand-Williams, W., Cuvelier, M. E. and Berset, C. 1995. Use of a free radical method to evaluate antioxidant activity. LWT - Food Sci. Technol. 28, 25-30.

상세보기
Brune, B., Dehne, N., Grossmann, N., Jung, M., Namgaladze, D., Schmid, T., Von Knethen, A. and Weigert, A. 2013. Redox control of inflammation in macrophages. Antioxid. Redox Signal 19, 595-637.

상세보기
Chi, H. J. and Han, D. S. 1976. New or Noteworthy Medicinal Plants from Korea (II). Kor. J. Pharmacog. 7, 69-71.
Chi, H. J. and Kim, H. S. 1981. Coumarins from the Root of Peucedanum insolens Kitagawa. Kor. J. Pharmacog. 12, 79-81.
Chi, H. J. and Kim, H. S. 1982. Pharmacognostical Studies on the Root of Peucedanum insolens Kitagawa (II). Kor. J. Pharmacog. 13, 48-48
Choi, Y. H., Kim, G. Y. and Lee, H. H. 2014. Anti-inflammatory effects of cordycepin in lipopolysaccharide-stimulated RAW264.7 macrophages through toll-like receptor 4-mediated suppression of mitogen-activated protein kinases and NF-κB signaling pathways. Drug Des. Devel. Ther. 8, 1941-1953.

상세보기
Cote, J., Caillet, S., Doyon, G., Sylvain, J. F. and Lacroix, M. 2010. Bioactive compounds in cranberries and their biological properties. Crit. Rev. Food Sci. Nutr. 50, 666-679.

상세보기
Davis, D. W. 1947. Determination of flavonones in citrus juice. Anal. Chem. 19, 46-48.

상세보기
Du, Z., Liu, H., Zhang, Z. and Li, P. 2013 Antioxidant and anti-inflammatory activities of Radix Isatidis polysaccharide in murine alveolar macrophages. Int. J. Biol. Macromol. 58, 329-335.

상세보기
Goraca, A., Huk-Kolega, H., Kleniewska, P., PiechotaPolanczyk, A. and Skibska, B. 2013. Effects of lipoic acid on spleen oxidative stress after LPS administration. Pharmacol. Rep. 65, 179-186.

상세보기
Han, J. M., Lee, E. K., Gong, S. Y., Sohng, J. K., Kang, Y. J. and Jung, H. J. 2019. Sparassis crispa exerts antiinflammatory activity via suppression of TLR-mediated NFκ B and MAPK signaling pathways in LPS-induced RAW264.7 macrophage cells. J. Ethnopharmacol. 231, 1018.
Han, S. Y., Yi, Y. S., Jeong, S. G., Hong, Y. H., Choi, K. J., Hossain, M. A., Hwang, H., Rho, H. S., Lee, J., Kim, J. H. and Cho, J. Y. 2018. Ethanol extract of Lilium bulbs plays an antiinflammatory role by targeting the IKK-mediated NF-κB pathway in macrophages. Am. J. Chin. Med. 46, 12811296.
Heo, S. J., Jang, J., Ye, B. R., Kim, M. S., Yoon, W. J., Oh, C., Kang, D. H., Lee, J. H., Kang, M. C. and Jeon, Y. J. 2014. Chromene suppresses the activation of inflammatory mediators in lipopolysaccharide-stimulated RAW264.7 cells. Food Chem. Toxicol. 67, 169-175.

상세보기
Hewett, J. A. and Roth, R. A. 1993. Hepatic and extrahepatic pahtobiology of bacterial lipopolysaccharides. Pharmacol. Rev. 45, 382-411.
Hwang, Y. J., Lee, E. J., Kim, H. R. and Hwang, K. A. 2013. In vitro antioxidant and anticancer effects of solvent fractions from Prunella vulgaris var lilacina. BMC Complement. Altern. Med. 13, 310.

상세보기
Khan, S., Choi, R. J., Shehzad, O., Kim, H. P., Islam, M. N., Choi, J. S. and Kim, Y. S. 2013. Molecular mechanism of capillarisin-mediated inhibition of MyD88/TIRAP inflammatory signaling in in vitro and in vivo experimental models. J. Ethnopharmacol. 145, 626-637.

상세보기
Khan, S., Shin, E. M., Choi, R. J., Jung, Y. H., Kim, J. W., Tosun, A. and Kim, Y. S. 2011. Suppression of LPS-induced inflammatory and NF-κB responses by anomalin in RAW 264.7 macrophages. J. Cell Biochem. 112, 2179-2188.

상세보기
Kim, Y. H., Lee, S. H., Lee, J. Y., Choi, S. W., Park, J. W. and Kwon, T. K. 2004. Triptolide inhibits murine-inducible nitric oxide synthase expression by down-regulating lipopolysaccharide-induced activity of nuclear factor-κB and c-Jun NH 2-terminal kinase. Eur. J. Pharmacol. 494, 1-9.

상세보기
Kroncke, K. D., Fehsel, K. and Kolb-Bachofen, V. 1998. Inducible nitric oxide synthase in human diseases. Clin. Exp. Immunol. 113, 147-156.

상세보기
Losada-Barreiro, S. and Bravo-Diaz, C. 2017. Free radicals and polyphenols: The redox chemistry of neurodegenerative diseases. Eur. J. Med. Chem. 133, 379-402.

상세보기
Madamanchi, N. R., Vendrov, A. and Runge, M. S. 2005. Oxidative stress and vascular disease. Arter. Thromb. Vasc. Biol. 25, 29-38.

상세보기
Maione, F., Russo, R., Khan, H. and Mascolo, N. 2016. Medicinal plants with anti-inflammatory activities. Nat. Prod. Res. 30, 13431352.
Mercurio, F. and Manning, A. M. 1999. Multiple signals converging on NF-κB. Curr. Opin. Cell Biol. 11, 226-232.

상세보기
Mills, E. L. and O'Neill, L. A. 2016. Reprogramming mitochondrial metabolism in macrophages as an anti-inflammatory signal. Eur. J. Immunol. 46, 13-21.

상세보기
Mordan, L. J., Burnett, T. S., Zhang, L. X., Tom, J. and Cooney, R. V. 1993. Inhibitors of endogenous nitrogen oxide formation block the promotion of neoplastic transformation in C3H 10 T1/2 fibroblasts. Carcinogenesis 14, 1555-1559.

상세보기
Nam, J. Y. and Ryu, K. S. 1975. Pharmacognostical Studies on Korean 'Bang Poong'. Kor. J. Pharmacog. 6, 151-159
Niu, X., Xing, W., Li, W., Fan, T., Hu, H. and Li, Y. 2012. Isofraxidin exhibited anti-inflammatory effects in vivo and inhibited TNF-α production in LPS-induced mouse peritoneal macrophages in vitro via the MAPK pathway. Int. Immunopharmacol. 14, 164-171.

상세보기
Oberoi, H. S. and Sandhu, S. K. 2015. Therapeutic and nutraceutical potential of bioactive compounds extracted from fruit residues AU-Babbar, Neha. Crit. Rev. Food Sci. Nutr. 55, 319-337.

상세보기
Ohshima, H. and Bartsch, H. 1994. Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat. Res. 305, 253-264.

상세보기
Pan, S. Y., Zhou, S. F., Gao, S. H., Yu, Z. L., Zhang, S. F., Tang, M. K., Sun, J. N., Ma, D. L., Han, Y. F., Fong, W. F. and Ko, K. M. 2013. New perspectives on how to discover drugs from herbal medicines: CAM's outstanding contribution to modern therapeutics. Evid. Based Complement. Alternat. Med. 2013, 627375-627401.

상세보기
Perkins, N. D. 2000. The REL/NF-κB family: Friend and foe. Trends Biochem. Sci. 25, 434-440.

상세보기
Rahman, K. Studies on free radicals, antioxidants, and cofactors. 2007. Clin. Interv. Aging 2, 219-236.
Rajapakse, N., Kim, M. M., Mendis, E. and Kim, S. K. 2008. Inhibition of inducible nitric oxide synthase and cyclooxy-genase-2 in lipopolysaccharide-stimulated RAW264. 7 cells by carboxybutyrylated glucosamine takes place via downregulation of mitogen-activated protein kinase-mediated nuclear factor-κB signaling. Immunology 123, 348-357.

상세보기
Singleton, V. L. and Rossi, J. A. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents, Am. J. Enol. Vitic. 16, 144-158.
Soobrattee, M. A., Neergheen, V. S., Luximon-Ramma, A., Aruoma, O. I. and Bahorun, T. 2005. Phenolics as potential antioxidant therapeutic agents: Mechanism and actions. Mutat. Res. 579, 200-213.

상세보기
Takamata, A. 1992. Effect of vagotomy on cardiovascular adjustment to hyperthermia in rats. Jpn. J. Physiol. 42, 641-652.

상세보기
Tapiero, H., Tew, K.D., Ba, N. and Mathe, G. 2002. Polyphenols: do they play a role in the prevention of human pathologies? Biomed. Pharmacother. 56, 200-207.

상세보기
Tungmunnithum, D., Thongboonyou, A., Pholboon, A. and Yangsabai, A. 2018. Flavonoids and other phenolic com pounds from medicinal plants for pharmaceutical and med ical aspects: An overview. Medicines 5, 93.

상세보기
Wadsworth, T. L. and Koop, D. R. 1999. Effects of wine polyphenolics quercetin and resveratrol on pro-inflammatory cytokine expression in RAW 264.7 macrophages. Biochem. Pharmacol. 57, 941-949.

상세보기
Wang, X. and Lau, H. 2006. Prostaglandin E2 potentiates the immunologically stimulated histamine release from human peripheral blood-derived mast cells through EP1/EP3 receptors. Allergy 61, 503-506.

상세보기
Yang, C. S., Landau, J. M., Huang, M. T. and Newmark, H. L. 2001. Inhibition of carcinogenesis by dietary polyphenolic compounds. Annu. Rev. Nutr. 21, 381-406.

상세보기
Yeom, M., Park, J., Lim, C., Sur, B., Lee, B., Han, J. J., Choi, H. D., Lee, H. and Hahm, D. H. 2015. Glucosylceramide attenuates the inflammatory mediator expression in lipopolysaccharide-stimulated RAW264. 7 cells. Nutr. Res. 35, 241-250.

상세보기
Yoshino, S., Sasatomi, E. and Ohsawa, M. 2000. Bacterial lipopolysaccharide acts as an adjuvant to induce autoimmune arthritis in mice. Immunobiology 99, 607-614.

저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증