ORAC Assay 에 의한 인삼의 항산화 활성 연구

김성환,김영목,Kim, Sung-Hwan,Kim, Young-Mok 동아시아식생활학회 2007 동아시아식생활학회지 Vol.17 No.3

인삼의 여러 생리 활성 가운데 항산화 정도를 알아보기 위하여 백삼(6 년근), 백삼(5 년근), 피부백삼(5 년근), 피부백삼 (4 년근) 의 80% 에탄올 엑기스, 에틸아세테이트 분획, 수포화 부탄올 분획, 물 분획을 얻은 후 LC/Mass 를 사용하여 사포닌 함량을 조사하고 기존의 여러 가지 항산화 작용 측정 방법들의 오류를 없애고 더욱 정확한 결과를 낼 수 있는 대처 방안으로 선정된 ORAC Assay에 의해 항산화 활성을 검토하였다. 인삼 중 사포닌은 ginsenoside Rg 1 과 Rb1 이 주요 성분으로 다량 함유하고 있었으며, Rc, Rb2, Re 등이 뒤를 이었고, 그밖에도 Rd, Rg3, Rh1가 널리 분포하고 있었다. 피부백삼 5 년근의 경우 에탄올 엑기스와 수포화 부탄올 분획에서 다른 인삼 분획에 비해 높은 함유량을 보였으나 실험의 한계상 인삼 재배기간과 인삼 종류별 각각의 분획에 대한 사포닌 함량 비교는 어려웠다. 인삼의 각 분획별 항산화 활성은 80% 에탄올 엑기스, 에틸아세테이트 분획, 수포화 부탄올 분획, 물 분획 모두에서 나타났고 비교적 전체 유기 용매 분획의 값이 비슷하였으며, 수층 분획이 다소 낮은 활성을 보였다. 검체 인삼들의 각 용매추출 분획 상호간의 유의성 비교에서는 모든 인삼 검체의 에틸아세테이트 분획에서만 유의성을 나타내었다(p>0.05). 인삼 중 항산화 활성은 에틸아세테이트 층으로 이행되는 폴리페놀 계통 성분이나 일부 비극성의 사포닌에 의한 것으로 추측되고 있으나 모든 분획에서 나타난 것으로 보아 이들 외에 산성 다당체, 당 단백질, 수용성 다당류 등 다른 생리활성 물질에 대한 연구가 요구된다. This study was performed to investigate the antioxidant activity of Korean ginseng using an ORAC(Oxygen Radical Absorbance Capacity) assay. Four fractions each (80% ethanol, ethyl acetate, water saturated 1-butanol, and water) were obtained from different ginseng samples (White Ginseng: ; 6 yrs-., 5 yrs-., ; Cork Ginseng: ; 5 yrs-., 4 yrs-.). The saponin content of each fraction was quantified by LC/MS, and the antioxidant capacity of the ginseng was measured by the ORAC assay. The ORAC method, which was recently validated using automatic liquid handling systems, has been adapted for manual handling with the use of a conventional fluorescence microplate reader. Furthermore, the ORAC assay provides a direct measure of hydrophilic chain-breaking antioxidant capacity against peroxy radical, which is the exiting and emission of 2,2'-Azobis (2-methylpropionamidine)-dihychloride (AAPH). As a result of our experiments, ginsenosides Rg1 and Rb1 were the two major saponins found in the ginseng samples, and Rc, Rb2, Re, Rd, Rg3, and Rh1 were detected in a small quantities. For the antioxidant capacities of the fractions (80% ethanol, ethyl acetate, butanol, and water), we found that the organic solvent fraction had similar antioxidant capacities, and were higher than the capacity of the water fraction. When determining the similarities in each fraction, only the ethyl acetate fraction showed similarity compared to other fractions (p>0.05). The antioxidant capacity of ginseng may come from phenolic compounds and some nonpolar saponins. However, based on the results of this study, we hypothesize that some acidic polysaccharides and other biological components may contribute to its antioxidant capacity. Additional research is required to determine other possible biological response modifiers that contribute to the antioxidant capacity of ginseng.

Effect of Enzyme Treatment with β-Glucosidase on Antioxidant Capacity of Mulberry (Morus alba L.) Leaf Extract

Gyo-Nam Kim,Hae-Dong Jang 한국식품과학회 2010 Food Science and Biotechnology Vol.19 No.5

This study was carried out to investigate the effect of enzyme treatment with β-glucosidase on antioxidant capacity of the mulberry leaf extract (MLE) using oxygen radical absorbance capacity (ORAC) and cellular antioxidant capacity (CAC) assay. The MLE was prepared by autoclaving at 121℃ for 15 min and treated with β-glucosidase for 9 hr. High pressure liquid chromatography (HPLC) analysis showed that only qercetin-3-β-D-glucose (QT-G) among quercetin (QT) glycosides of MLE, including QT-G, quercetin-3-O-glucose-6"-acetate (QT-GA), and rutin (RT), was converted into QT by 3 hr treatment with β-glucosidase. The in vitro peroxyl radical- and hydroxyl radical scavenging capacity significantly increased after the enzyme treatment using β-glucosidase for 6 and 9 hr,respectively. The metal chelating activity increased after the enzyme treatment using β-glucosidase for 3 hr. The intracellular antioxidant capacity of MLE to protect AAPH- and Cu2+-induced oxidative stress in HepG2 cells significantly increased after the enzyme treatment using β-glucosidase for 3 and 6 hr, respectively, indicating that QT may be released from QT-G by β-glucosidase and penetrate into cell membrane so that it can contribute to the intracellular antioxidant capacity of MLE.

Microplate-Based Oxygen Radical Absorbance Capacity(ORAC) Assay of Hydrophilic and Lipophilic Compartments in Plasma

Ho-Kyung Kwak,Jeffrey B. Blumberg,Chung-Yen Chen,Paul E. Milbury 한국영양학회 2006 Nutritional Sciences Vol.9 No.1

Methods have been developed to evaluate the total antioxidant capacity of foods and plasma but limitations are associated with their ability to determine precisely the contribution of lipophilic antioxidants in a lipid milieu as well as interactions among them Thus, we modified the Oxygen Radical Absorbance Capacity (ORAC) assay to determine the peroxyradical scavenging ability of both hydrophilic and lipophilic compartments in plasma. The hydrophilic ORAC assay was performed in a phosphate buffer system utilizing 2,2'-azobis (2-amidinopropane) dihydrochloride as a peroxyradical generator and fluorescein as the target. The lipophilic ORAC assay was carried out in a dimethylsulfoxide: butyronitrile (DMSO/BN, 9:1 v/v) system using2,2'-azobis (2,4-dimethyl valeronitrile) as a peroxyradical generator and BODIPY C11 581/591 as the target. Analyses were conducted in bovine serum supplemented with water-and lipid-soluhle antioxidants and in human plasma. Albumin(0.5~5g/㎗) and uric acid(0.1~0.5 m㏖/ℓ) increased hydrophilic ORAC values in a dose-dependent fashion(R²=0.97 and 0.98, respectively) but had no impact on lipophilic ORAC values. α-Tocopherol (15~200 μ㏖/ℓ) increased lipophilic ORAC values in a dose-dependent fashion (R²=0.94); neither α-tocopherol nor β-carotene had an impact on hydrophilic ORAC values. However, addition of β-carotene at physiological concentrations (0.23~1.86 μ㏖/ℓ), either alone or in combination with other carotenoids, had no significant impact on lipophilic ORAC values. Thus, while assays of "total antioxidant capacity" in biological matrices would be a useful research and clinical tool, existing methods are limited by the lack of complete responsiveness to the full range of dietary antioxidants.

Microplate-Based Oxygen Radical Absorbance Capacity (ORAC) Assay of Hydrophilic and Lipophilic Compartments in Plasma

Kwak Ho Kyung,Blumberg Jeffrey B.,Chen Chung Yen,Milbury Paul E. The Korean Nutrition Society 2006 Nutritional Sciences Vol.9 No.1

Methods have been developed to evaluate the total antioxidant capacity of foods and plasma but limitations are associated with their ability to determine precisely the contribution of lipophilic antioxidants in a lipid milieu as well as interactions among them Thus, we modified the Oxygen Radical Absorbance Capacity (ORAC) assay to determine the peroxyradical scavenging ability of both hydrophilic and lipophilic compartments in plasma The hydrophilic ORAC assay was performed in a phosphate buffer system utilizing 2,2'-azobis (2-amidinopropane) dihydrochloride as a peroxyradical generator and fluorescein as the target The lipophilic ORAC assay was carried out in a dimethylsulfoxide :butyronitrile (DMSO/BN, 9:1 v/v) system using 2,2'-azobis (2,4-dimethyl valeronitrile) as a peroxyradical generator and BODIPY C11 581/591 as the target Analyses were conducted in bovine serum supplemented with water - and lipid - soluble antioxidants and in human plasma. Albumin (0.5$\sim$5 g/dL) and uric acid (0.1$\sim$0.5 $\mu$mol/L) increased hydrophilic ORAC values in a dose-dependent fashion ($R^{2}$=0.97 and 0.98, respectively) but had no impact on lipophilic ORAC values. $\alpha$-Tocopherol (15$\sim$200 $\mu$mol/L) increased lipophilic ORAC values in a dose-dependent fashion ($R^{2}$=0.94); neither $\alpha$-tocopherol nor $\beta$-carotene had an impact on hydrophilic ORAC values. However, addition of $\beta$-carotene at physiological concentration (0.23$\sim$1.86 $\mu$mol/L), either alone or in combination with other carotenoids, had no significant impact on lipophilic ORAC values. Thus, while assays of 'total antioxidant capacity' in biological matrices would be a useful research and clinical tool, existing methods are limited by the lack of complete responsiveness to the full range of dietary antioxidants.

Peroxyl Radical Scavenging Capacity of Methanolic Extracts of Vietnamese Medicinal Plants

Bui Huu Tai,Phan Van Kiem,Hae Dong Jang,Young Ho Kim 충남대학교 약학대학 의약품개발연구소 2014 藥學論文集 Vol.29 No.-

Eighty eight Vietnamese medicinal plants were screened for their effect on peroxyl radical scavenging capacity using modified Oxygen Radical Absorbance Capacity (ORAC) assay. The results showed that the methanol extracts from leaves and twigs of Alchornea rugosa, Alniphyllum fortunei, Ampelopsis cantoniensis, Cayratia geniculata, Elaeocarpus silvestris, Enkianthus quinqueflorus, Ficus subpyriformis, Glochidion sphaerogynum, Phoebe attenuata, Psychotria poilanei, Rhododendron simsii, Syzygium sterropyllum showed significant inhibition on the active of peroxyl radical. The ORAC values was determined ranging from 10.52 to 14.80 fold-up net protection provided by 1 μM of Trolox. Our preliminary data may prompted further pharmacological study as well as antioxidant properties of those plants which have not been reported previously.

Evaluation of the Antioxidant Activities of Natural Components of Artemisia iwayomogi

YANXITAO,Yan Ding,이상현,이위,선아난,양서영,장해동,김영호 한국생약학회 2014 Natural Product Sciences Vol.20 No.3

The antioxidant activities of 29 components isolated from the aerial parts of Artemisia iwayomogi were evaluated in vitro and in cell culture. Among the tested compounds, 2, 6, 8, 10, 13, and 14 exhibited the greatest peroxyl radical-scavenging activities in the oxygen radical absorbance capacity (ORAC) assay, and 2, 10, and 14 also showed significant reducing capacities. However, all compounds showed weak metal chelating activities. Their cellular antioxidant activities were evaluated in HepG2 cells. At 10 μM, compounds 6, 8, and 14 exhibited stronger protection against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress than compounds 2, 10, and 13. Moreover, Compounds 2 and 8 were more effective in protecting against Cu2+-induced oxidative stress than compounds 6, 10, 13, and 14 at 10 μM. These results suggest that the phenolic compounds in A. iwayomogi have the potential to be developed as natural antioxidants for the treatment of oxidative stress-related diseases.

Evaluation of the Anti-osteoporosis and Antioxidant Activities of Phenolic Compounds from Euphorbia maculata

Bui Thi Thuy Luyen,BUIHUU TAI,Nguyen Phuong Thao,이상현,장해동,이영미,김영호 한국응용생명화학회 2014 Applied Biological Chemistry (Appl Biol Chem) Vol.57 No.5

Antioxidant and anti-osteoporosis activities of extractsand chemical constituents from the whole plant of Euphorbiamaculata were investigated. The MeOH extract, as well as EtOAcand H2O fractions (10.0 μg/mL), exhibited potent antioxidantactivities. Their oxygen radical absorbance capacity and cupricion reducing antioxidant capacity values were 27.07±0.31 to28.47±0.36 and 43.86±0.26 to 46.67±0.34 fold higher than thoseof 1.0 μM Trolox, respectively. The MeOH extract and EtOAcfraction (at 10.0 μg/mL) also significantly suppressed excessivebone resorption by osteoclasts with tartrate-resistant acid phosphatase(TRAP) activity values of 154.90±4.25 and 163.95±9.77%,respectively. Bioassay guided isolation of the EtOAc and H2Ofractions afforded 19 known compounds (1−19). Of these,compounds 18, and 13−15 showed good antioxidant activitybased on peroxyl radical-scavenging and reducing capacity assays,whereas compounds 1, 4, 7, and 14 showed the most significantinhibitory effect with TRAP activity values ranging from 121.31±1.41 to 110.00±3.74% relative to the control.

In vitro and Cellular Antioxidant Activity of Arginyl-fructose and Arginyl-fructosyl-glucose

Jung-Sook Lee,Gyo-Nam Kim,Sang-Hyun Lee,Eui-Su Kim,Kyoung-Soo Ha,Young-In Kwon,Heon-Sang Jeong,Hae-Dong Jang 한국식품과학회 2009 Food Science and Biotechnology Vol.18 No.6

Arginyl-fructose (AF) and arginyl-fructosyl-glucose (AFG) were chemically synthesized and purified. Their in vitro and cellular antioxidant activity was investigated using oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity assay, respectively. The peroxyl radical scavenging activity of AF was much higher than that of AFG, which was in good agreement with their reduction capacity to donate electrons or hydrogen atoms. On the other hand, the hydroxyl radical scavenging activity of AF was weaker than that of AFG, which was consistent with their metal chelating activity, suggesting that AFG-Cu²? complex may be less redox-active than AF-Cu²? complex due to 1 glucose molecule attached. The cellular antioxidant activity of AF and AFG appeared to depend on both their permeability into cell membrane and the scavenging activity on peroxyl or hydroxyl radicals. These results indicate that AF and AFG, Maillard reaction products, may have a high potential as a material for the development of nutraceutical food with antioxidant activity.

Nicotine Detoxification of Rutin, Quercitrin, and Chlorogenic Acid Isolated from Houttuynia cordata by Reducing Reactive Oxygen Species and Inducing Conversion from Nicotine to Cotinine

Kim, Kyeong Mu,Shim, Soon-Mi The Korean Society for Applied Biological Chemistr 2014 Applied Biological Chemistry (Appl Biol Chem) Vol.57 No.4

The hypothesis of the present study is that methanol extract of Houttuynia cordata Thunb (MEH) and its targeted bioactive components including rutin, quercitrin, and chlorogenic acid can be effective in reducing reactive oxygen species (ROS) caused by nicotine and promoting nicotine to cotinine in HepG2 cell. Oxygen radical absorbance capacity (ORAC) of bioactive components and MEH was measured to assess free radical scavenging capacity. ROS inhibition ability of bioactive components and MEH was measured by 2',7'-dichlorodihydrofluorescein diacetate assay. The conversion rate of nicotine to cotinine by bioactive components and MEH was determined by the direct barbiturate assay method. ORAC value confirmed that MEH and its bioactive components provided an antioxidant capacity ranging from 126 to $138{\mu}M$ trolox equivalents/100 g. Compared to nicotine only, pretreatment of MEH, rutin, and quercitrin was revealed to effectively inhibit ROS production in HepG2 cell by up to 9, 7.4, and 14%, respectively. Nicotine conversion to cotinine after 120 min incubation was 1.7 and 1.4 times higher in rutin and chlorogenic acid pretreatment than the control, respectively. H. cordata and its targeted bioactive components could be a valuable natural ingredient for inhibiting ROS formation by nicotine as well as enhancing the rate of nicotine to cotinine turnover.

추출 용매에 따른 인삼과 압출 성형 인삼의 사포닌 함량 및 항산화 활성 연구

김성환 ( Sung Hwan Kim ) 한국식품영양학회 2011 韓國食品營養學會誌 Vol.24 No.4

This study was conducted to investigate the changes in saponin content and antioxidant activity of crude ginseng and extruded ginseng by using different solvent extraction methods. Each of the fractions was first extracted by 80% ethanol followed by ether treatment to remove the lipid components. Water soluble components were separated by ethylacetate and water saturated butanol. Four fraction, including 80% ethanol, ethylacetate, butanol and water were obtained from crude and extruded ginsengs to analyze saponin content and antioxidant activity. Saponin content and antioxidant capacity of each of the four fractions were measured by LC/MS analysis and ORAC(Oxygen Radical Absorbance Capacity) assay, respectively. It was found that a major portion of saponin was present in ethyl acetate and water saturated butanol fractions. When extracted by 80% ethanol, ginsenoside Rb1 and Rg1 were mostly found in crude ginseng, while ginsenoside Re and Rb1 were detected in extruded ginseng. Even though Rh1 and Rg3 were found in a very small quantity in crude ginseng, there was a significant quantity of both in extruded ginseng when extracted by 80% ethanol. Similar tendency was also observed in extruded ginseng fraction when extracted with ethyl acetate and butanol. In crude ginseng, the level of Rg1 was the highest among other ginsenosides upon extraction by ethyl acetate, while Rh1 and Rg3 were predominantly found by employing similar solvent extraction in the extruded ginseng. Also, Rg1, Re and Rb1 were also found in the extruded ginseng with small quantity. Rg1, Re and Rb1 were found in crude ginseng by butanol extraction, while Rb1 and Re were extracted from the extruded ginseng. Overall, there was no difference in the saponin content between crude ginseng and extruded ginseng when extracted by butanol and water, but twice as much of saponin was obtained by 80% ethanol extraction and 6 times more saponin were obtained in ethyl acetate fraction in the extruded ginseng. Antioxidant capacity of crude ginseng as determined by ORAC assay was higher in 80% ethanol(high in many different kinds of biological compounds) and water saturated butanol(high in polar saponin) fractions than the ethyl acetate and water fractions. No difference in antioxidant capacity was observed between crude and extruded ginseng. However, antioxidant capacity of ethyl acetate and water fractions in extruded ginseng was significantly higher than crude ginseng(P>0.05). All the fractions in both, crude and extruded ginseng possessed antioxidant capacity and even water fractions that contained almost no saponin had some antioxidant capacity. While determining correlation coefficient between fractions in extruded ginseng by Pearson correlation, it was observed that 80% ethanol fraction was in correlation with ethyl acetate(P>0.01) and ethanol(P>0.001) and in the case of ethylacetate, correlation was observed only with butanol fraction(P>0.05).