176 Yoo-Hyun Kim and Ho Park. Anti-Inflammatory Activity and Mechanism of Medicinal Plants ORIGINAL ARTICLE Korean J Clin Lab Sci. 2016, 48(3):176-182 http://dx.doi.org/10.15324/kjcls.2016.48.3.176 pissn 1738-3544 eissn 2288-1662 Study on the Anti-Inflammatory Activity and Mechanism of Medicinal Plants Used in the Treatment of Arthritis Yoo-Hyun Kim, Ho Park Department of Clinical Laboratory Science, Wonkwang Health Science University, Iksan 54538, Korea 관절염치료에사용되는한약재들의항염증활성과기전에관한연구 김유현, 박호 원광보건대학교임상병리과 When inflammatory reaction is in progress, the macrophages release inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor- (TNF- ), and product inflammatory mediators, including inducible nitric oxide synthase (inos) and prostaglandin E2 (PGE2). We conducted this study to evaluate the anti-inflammatory efficacy on each water extract of Acanthopanacis cortex, Achyranthes radix, and Eucommiae cortex, and to investigate whether they inhibit the expression of pro-inflammatory cytokine. Acanthopanacis cortex, Achyranthes radix, and Eucommiae cortex were extracted with water and freeze-dried. Acanthoside D, 20-hydroxyecdysone, and pinoresinol diglucoside as an index material were analyzed by high-performance liquid chromatography (HPLC) to ensure that the components of each extracts were extracted well. RAW 264.7 cell line, stimulated with lipopolysaccharide (LPS) to cause an inflammatory response, was treated with each water extract at various concentrations to determine the anti-inflammatory efficacy. Then, the anti-inflammatory efficacy was confirmed by a nitric oxide (NO) assay, and the mrna expression levels of pro-inflammatory cytokines were measured by real time PCR. As a result, the indicator materials were detected from each extract, and Acanthopanacis cortex water extract (ACWE) and Achyranthes radix water extract (ARWE) were shown to have a high activity than Eucommiae cortex water extract (ECWE) in NO assay. In Korea, traditionally it prescribed a combination of medicinal herbs. This study confirmed the anti-inflammatory response of these medicinal plants in arthritis and its synergistic effect when used in combination with western medicine. Key words: Anti-inflammation, RAW 264.7, IL-6, inos, TNF- Corresponding author: Yoo-Hyun Kim Department of Clinical Laboratory Science, Wonkwang Health Science University, 514 Iksandae-ro, Iksan 54538, Korea Tel: 82-63-840-1218 Fax: 82-63-840-1219 E-mail: yhkim@wu.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright 2016 The Korean Society for Clinical Laboratory Science. All rights reserved. Received: July 11, 2016 Revised: July 29, 2016 Accepted: July 29, 2016 서론염증은생체조직이손상되었을때원래의상태로복구시키려는방어작용중하나이다. 외부자극에반응하여혈관내에서 serotonin, histamine, prostaglandin 등의물질이분비되어혈관투과성이증대되면염증반응이진행된다 [1]. 이때작동되는면역세포중대표 적인세포가대식세포이다 [2]. 염증반응이진행되면대식세포에서 interleukin-6 (IL-6), tumor necrosis factor- (TNF- ) 등의 cytokine들이발현되어 inducible nitric oxide synthase (inos), prostaglandin E2 (PGE2) 등의염증유발인자가생성된다 [3]. 이러한과정을 in vitro 실험에서재현할때, 주로사용되는물질이 lipopolysaccharide (LPS) 이며, LPS는그람음성세균의세포벽의
Korean J Clin Lab Sci. Vol. 48, No. 3, September 2016 177 구성성분중하나로대식세포를자극하여염증매개인자들을발현시키는대표적인물질이다 [4]. 임상적으로항염증작용에주로사용되는비스테로이드성진통소염제 (non-steroidal anti-inflammatory drugs, NSAIDs) 로는 asprin, magnesium salicylate, salsalate, diclofenac, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, meloxicam, nabumetone, naproxen, piroxicam, sulindac, tolmetin, zaltoprofen, celecoxib 등많은종류가있다. 그러나이들제제는위장관부작용, 간독성, 신장기능저하, 인지장애, 혈소판기능저하, 알레르기성피부반응등부작용이알려져있다 [5-7]. 특히위장관부작용이가장많이보고되고있으며이는소염제특성상위장관점막의손상과 prostaglandin 생성의억제가원인이다. 규칙적으로 NSAIDs 를처방받는환자의약 30% 가위나십이지장의궤양을보이고위출혈, 위천공, 위장관폐색등의부작용을보이기도한다 [8]. 최근의염증분야연구에서는신령버섯자실체의메탄올추출물이 LPS로자극한 RAW 264.7 세포주에서유의적인항염증효능이있다는보고와같이염증매개물질의생산과분비를억제하는천연적인소염물질을찾는경향을보인다 [9]. 이는기존합성제제들같은소염제가부작용을보이기때문이다 [10]. 우리나라의전통적인치료법은여러가지한약재를조합하여처방하여왔다. 이것은한약재를단독으로사용하는것보다조합을통하여시너지효과를보이거나질병치료에불필요한성분을중화시키는것을목적으로한것이다. 관절염치료에주로사용되어왔던오가피 (Acanthopanacis cortex), 우슬 (Achyranthes radix), 두충 (Eucommiae cortex) 등도이렇게조합에의해사용되어왔던대표적인한약재들이다. 그러나이들한약재들의치료제로서의과학적근거는매우미흡한실정이다. 본연구에서는마우스의대식세포주인 RAW 264.7 세포주에 LPS를사용하여염증을유발하고, 한약재인오가피, 우슬, 두충의항염증활성과그의기전을확인하여항염증에있어시너지효능에대한근거를마련하고자한다. 재료및방법 1. 오가피, 우슬, 두충의추출건조및분쇄한오가피, 우슬, 두충은각각 100 g씩을 1 L의증류수에넣은후 103 o C의 heating mentle로 3시간동안교반하며환류추출하고여과하였다. 잔류액을모두감압농축한후동결건조하고건조약재대비동결건조분말로하였다. 2. 오가피, 우슬, 두충추출물의 HPLC 분석오가피, 우슬, 두충을추출하여동결건조시킨분말을 1 mg/ml 의농도로각각에탄올에녹여초음파추출한후 pore size 0.2 m 에여과하여 high-performance liquid chromatography (HPLC; Agilent, Santa Clara, CA, USA) 로분석하여유효성분추출의정도를확인하였다. 각약재에대한지표물질로오가피는 acanthoside D, 우슬은 20-hydroxyecdysone, 두충은 pinoresinol diglucoside 를모두 Sigma Aldrich (St. Louis, MO, USA) 에서구입하였으며, 사용된유기용매는모두 HPLC grade (J.T Baker, USA) 를사용하였다. 각지표물질은에탄올에녹여 1 ml/min의농도로만들고, 2 배수희석을통해희석한후 pore size 0.2 m에여과하여 HPLC로분석하였다. Acanthoside D의분석은 C18 (4.6 250 mm, 5 m, Agilent) 컬럼을사용하고, 컬럼온도는 40 o C, 검출기는 UV 210 nm, 이동상으로는 10% acetonitrile 과 30% acetonitrile 을사용하여 gradient profile로실시하였으며유속은 1 ml/min으로하였다. Gradient 조건은 Table 1과같다 [11]. 20-hydroxyecdysone 의분석조건은 C18 (4.6 250 mm, 5 m, Agilent) 컬럼을사용하고, 칼럼온도는 35 o C, 검출기는 UV 254 nm, 이동상으로는 100% acetonitrile 과물을 gradient profile 로하였고, 그조건은 Table 2 와같으며유속은 1 ml/min으로실험하였다 [12]. 두충의지표물질인 pinoresinol diglucoside 는대한약전의두충정량법으로분석하였다. C18 (4.6 250 mm, 5 m, Agilent) 컬럼을사용하고, 컬럼온도는 35 o C, 검출기는 UV 230 nm, 이동상으로는 0.1% formic acid의물과 100% acetonitrile 을사용하여 gradient profile 로실시하였고, 유속은 1 ml/min으로하였다. Gradient 조건은 Table 3과같다. 오가피, 우슬, 두충추출물에대한유효성분 Table 1. Gradient condition of acanthoside D Time (min) Mobile phase 10% Acetonitrile 30% Acetonitrile 0 90 10 5 85 15 15 85 15 20 90 10 30 90 10 Table 2. Gradient condition of 20-hydroxyecdysone Time (min) Acetonitrile Mobile phase Water 0 15 85 8 15 85 15 30 70 30 30 70
178 Yoo-Hyun Kim and Ho Park. Anti-Inflammatory Activity and Mechanism of Medicinal Plants Table 3. Gradient condition of pinoresinol diglucoside Time (min) Acetonitrile Mobile phase 0.1% Formic acid 0 5 95 8 20 80 15 20 80 30 5 95 은각지표물질의농도별로얻어진값으로표준곡선을그리고, 기울기값에지표물질분석조건과같은방법으로얻어진각약재추출액의 peak 면적값을대입하여계산하였다. 3. 세포배양 마우스의대식세포인 RAW 264.7 세포주는한국세포주은행 (No. 40071; KCLB, Seoul, Korea) 에서구입하여사용하였다. 세포는 10% fetal bovine serum (FBS; Gibco, Rockville, MD, USA) 과 1% antibiotic-antimycotic이함유된 Dulbecco s minimum essential medium (DMEM; Gibco) 을사용하여 37 o C와 5% CO 2 조건하에서배양하였다. 4. 오가피, 우슬, 두충추출물의 NO assay RAW 264.7세포주가활성화되면 NO를생성하고생성된 NO는세포배양배지에서 NO 2 의형태로존재한다. 그양을 Griess 시약 [0.1% N-(1-naphthyl) ethylene diamide dihydrochloride, 1% sulfanilamide in 5% phosphoric acid; Sigma Aldrich] 과의반응을통해측정하였다. RAW 264.7 세포주를 96 well plate에 1 10 6 cell/ml의농도로분주하고 37 o C와 5% CO 2 incubator에서 24시간동안배양하였다. 1 g/ml LPS (Sigma Aldrich) 로자극시킨후각각의추출물을다양한농도 (200, 100, 50, 25 g/ml) 로처리한후 24시간동안배양하였다. 상층액을옮겨담고 Griess 시약을동량첨가한후 540 nm의 microplate reader (Tecan, Australia) 로흡광도를측정하여 NO 2 의농도는 sodium nitrite 의표준곡선에대입하여계산하였다. Griess 활성시험후, 각 well에 MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; Sigma Aldrich] 용액 ( 최종 0.5 g/ml) 을첨가하고 4시간동안 37 o C 에서배양하였다. 배지를제거하고 dimethyl sulfoxide (DMSO; Samchun Chemical, Korea) 를첨가하여생성된 formazan을용해시켰다. 570 nm에서흡광도를측정하였고 control group과비교하여세포생존율 (%) 을측정하였다. 5. 역전사중합반응 1 g/ml의 LPS로자극된 RAW 264.7 세포주에오가피, 우슬, 두충각각의추출물을농도별 (200, 100, 50, 25 g/ml) 로처리한후 TRI regent (Sigma Aldrich) 를이용하여총 cellular RNA를추출하였다. 1 g의 RNA를 oligo-(dt)15 primer와 avian myeloblastosis virus (AMV) reverse transcriptase (Promega, USA) 를이용하여역전사반응을진행하였다. PCR은 cdna, 0.2 mm dntp mixture (Promega, Madison, WI, USA), 10 pmol target gene-specific primers와 0.25 unit Taq DNA polymerase (Promega) 를섞은후 GeneAmp PCR system 2400 (Applied Biosystems, Foster, CA, USA) 을이용하여증폭하였다. 초기 denaturation을 94 o C에서 4분간진행하고 25 30 cycle로하였으며 denaturation 은 94 o C 에서 4분, annealing은 55 o C 에서 30 초, elongation 은 72 o C 에서 30초, extension은 72 o C 에서 5분간진행하였다. PCR산물을확인하기위하여 2% agarose gel로전기영동하였고 SYBR-Gold (Molecular Probes, Eugene, OR, USA) 와영상분석장치를이용하였다. 6. 통계모든실험은최소 3회반복하였으며데이터는평균 ± 표준편차로표시하였다. 통계처리는 analysis of variation (ANOVA) 방식으로 SPSS 버전 18.0 (IBM, Armonk, NY, USA) 을사용하였다. 결과 1. 오가피, 우슬, 두충의추출오가피, 우슬, 두충을열수로환류추출하고여과후동결건조하여분말의무게를측정하였다. 각각 19.3±1.27%, 38.2±1.49%, 14.2±0.76% 의수율을보였으며 HPLC 분석결과모든약재에서각각의지표성분이검출되었다. 우슬의 20-hydroxyecdysone 과오가피의 acantoside D는지표성분의검출이어렵지않았으나두충의 pinoresinol diglucoside 는분석이용이하진않았지만 HPLC 의 UV 검출기로가능하였다 (Fig. 1). 2. 오가피, 우슬, 두충추출물의 NO assay NO는 LPS의자극으로인하여활성화되는것으로 NO 합성효소에의하여생성된다. RAW 264.7 대식세포주에 LPS로자극시키면다량의 NO를생성하며이에의한세포독성은염증반응, 종양발생등에도관여한다 [13]. 본연구에서는오가피, 우슬, 두충각각의추출물에대하여 NO assay를진행하고항염증활성을농도별로비
Korean J Clin Lab Sci. Vol. 48, No. 3, September 2016 179 Fig. 1. Analysis of the indicator components for each of the medicines. (a) Acanthoside D and ACWE, (b) 20-hydroxyecdysone and ARWE, (c) Pinoresinol diglucoside and ECWE. The chromatogram of the standard material above is gray and chromatogram of the following medicines is black. Abbreviation: ACWE, Acanthopanacis cortex water extract; ARWE, Achyranthes radix water extract; ECWE, Eucommiae cortex water extract. 교하였다. 전체적으로는 샘플의 농도가 높아질수록 발생되는 NO 의 량이 감소하는 것으로 보아 농도 의존적으로 항 염증 효능이 있 다는 것을 확인할 수 있었다. 그러나 우슬, 오가피에 비하여 두충은 유의적으로 낮은 활성을 보였다(Fig. 2a). 실험에 사용된 오가피, 우 슬, 두충 추출물의 농도 별 세포독성을 확인한 MTT assay에서는 모 든 농도에서 85% 이상의 생존율을 보여 각 추출물은 RAW 264.7 세포주에서 독성을 나타내지 않음을 확인하였고 NO assay의 결과 가 세포사멸에 의한 것이 아님을 확인 할 수 있었다(Fig. 2b). 3. 역전사 중합반응 오가피, 우슬, 두충 추출물 각각의 항 염증 기전을 확인하기 위하 여 inos, TNF-, IL-6의 mrna 발현량을 real time PCR로 확인 하였다. inos와 IL-6의 억제율은 NO를 생성시키는 pathway에 속하는 pro-inflammatory cytokine이기에 NO와 비슷한 경향으 로 발현됨을 확인하였다(Fig. 3a,c). 그러나 TNF- 억제율에서는 NO assay에서 낮은 항 염증 활성을 보인 우슬 추출물이 가장 높은 활성을 보였다(Fig. 3b). 오가피, 우슬과 두충은 서로 다른 기전으로 항 염증 활성을 이끌었으므로 오가피와 두충, 또는 우슬과 두충의 조합은 in vivo 또는 임상실험에서 충분한 시너지를 보일 가능성이 높았으며 이들의 조합은 양질의 천연 소염제를 개발하는 데에 충분 한 가능성을 시사하였다. 고 찰 신체에 염증이 발생되면 면역조절의 중요한 인자인 cytokine들 은 감염에 대한 숙주반응으로서 발현된다[14]. NO를 형성하는 Fig. 2. Inhibitory effects of each of the medicine hot water extracts on LPS-induced NO production in macrophage cells. (a) RAW 264.7 cells were stimulated with 1 g/ml LPS in a presence or absence of ACWE, ARWE, ECWE. (b) The cell viability was measured by MTT method. After 20 h, cultured media were collected and analyzed nitrite concentration using Griess reaction. The values are means±sd. ***p.0001, **p 0.001, *p 0.05 compared to the control (LPS treated group). +p 0.05 compared to ECWE. #p 0.05 compared to ARWE. p 0.05 compared to ACWE. Abbreviation: ACWE, Acanthopanacis cortex water extract; ARWE, Achyranthes radix water extract; ECWE, Eucommiae cortex water extract. NO synthase (NOS)에는 endothelial NOS (enos), neuronal NOS (nnos) 그리고 iducible NOS (inos) 세가지가 있다. 이중 enos와 nnos는 상피세포와 신경에 존재하여 정상적인 생리기능
180 Yoo-Hyun Kim and Ho Park. Anti-Inflammatory Activity and Mechanism of Medicinal Plants Fig. 3. Effects of ACWE, ARWE, ECWE on LPS-induced pro-inflammatory cytokine mrna expression in macrophage cells. (a) The amount of inos was measured in proportion to the -actin and electrophoresis. (b) The amount of TNF- was measured in proportion to the -actin and electrophoresis. (c) The amount of IL-6 was measured in proportion to the -actin and electrophoresis. RAW 264.7 cells were treated with LPS (1 g/ml) and ACWE, ARWE, ECWE for 4 h. Total RNA was isolated and further analyzed by RT-PCR. Data were representative of three separated experiments. -actin was used as an internal standard. The values are means±sd. ***p<.0001, **p<0.001, *p<0.05 compared to the control (LPS treated group). + p<0.05 compared to ECWE. # p<0.05 compared to ARWE. p<0.05 compared to ACWE. Abbreviation: ACWE, Acanthopanacis cortex water extract; ARWE, Achyranthes radix water extract; ECWE, Eucommiae cortex water extract. 을조절하기위해지속적으로 NO를소량씩생성한다. 하지만 inos는대식세포나혈관세포에서 NO를과량생성하여면역질환및패혈증을야기시킨다 [15]. IL-6은항암효과와간세포의단백질합성을유도하고조직손상에대한발열반응을초래하며, IL-1과협동적으로작용하여 B세포에서의면역글로불린유리를촉진시키고흉선세포와 T세포의분화에관여한다 [16,17]. TNF- 는주로대식세포에서생성되어면역반응을증가시키고, 혈관성장자극, 에너지유출증가, 감염, 염증과창상치유의촉진과종양에항증식작용을한다 [18]. 본연구에서시험된염증성 cytokine 들은염증기전에서지금까지보고되어온 pathway중거의마지막단계이다. 본연구에서이러한 cytokine 의발현량이차이가있다는것은오가피, 우슬, 두충의추출물들이항염증효능을가짐에있어서염증을차단시키는경로가다르다는것을의미한다. 두충의경우에는 TNF- 를유의적으로차단시키는역할을하였고오가피, 우슬은주로 inos를차단시키는역할을했다. 위결과를유추해볼때, 오 가피와두충, 또는우슬과두충이조합된처방은각각의한약재의약효뿐아니라시너지효과를보일수있다는것을알수있다. 오가피는전통적으로중국과우리나라에서관절염치료에사용되어온식물로최근에는국내에서많이자생하고있는오가피에대한생리활성및가공적합성에대한다양한연구가진행되고있다. 오가피의성분은 stigma sterol, campesterol, -sitosterol 등의 sterol, sesamin과 savinin, chlorogenic acid, sesquiterpene 등으로이루어져있으며특히오가피의뿌리와줄기부분은대표적인생리활성성분으로알려져있는 eleutheroside E, eleutheroside B를다량함유하고있다고보고되었다 [19]. 우슬은아시아지역에광범위하게분포하는다년초식물뿌리로서보고된약리작용으로부종, 진통작용, 혈압강하, 이뇨작용, 류머티스등면역반응에대한민간요법으로널리사용하였다 [20,21]. 우슬에는항보체효과를갖는 oleanolic acid계 saponin 이함유되어있으며중추미각계의신호전달을억제하는 -aminobutyric acid 등이알려져있다 [22]. 우
Korean J Clin Lab Sci. Vol. 48, No. 3, September 2016 181 슬은그밖에도 steroid 계열인 -sitosterol, stigmasterol, stigmasterol glycoside, rubrosterone 등과 triterpenoid 계열의 inkosterone 과같은화합물들이보고되어있다 [23]. 두충은지금까지알려진오래된약초중의하나로 phytochemical 이많이들어있는데대표적인것이 polyphenolics pyrogallol, protocatechuic acid, coumaric acid, chlorogenic acid, triterpone, 그리고 quercetin, kaempherol, astragalin 을함유한 flavonoid 등이있다 [24]. 이와같이두충에는배당체, alkaloid, pectin, 지질, 유기산, 당류및 vitamin C 등과 phenol성화합물이함유되어있어혈압강하작용, 이뇨작용과항당뇨활성, cholesterol 저하및비만방지의효과가있는것으로알려져있다 [25]. 오가피, 우슬, 두충은한방에서는관절염치료에다빈도로사용되는약재이다. 본연구는이러한약재들의항염증활성과기전에대한연구를하였으며, 그결과각약재들이항염증활성이있음을확인하였고, 관여하는기전을이해함으로한약재의조합을통하여치료에시너지효과를연구하는데에기여할수있기를바란다. 요약대식세포에서염증반응이진행되면, interleukin-6 (IL-6), tumor necrosis factor- (TNF- ) 등의 cytokine들이발현되어 inducible nitric oxide synthase (inos), prostaglandin E2 (PGE2) 등의염증유발인자가생성된다. 오가피, 우슬, 두충각각의추출물이어느정도의항염증효능을보이며어떤 pro-inflammatory cytokine의발현을억제하는지에대한연구를진행하였다. 오가피, 우슬, 두충은물추출하고동결건조시켰다. 각각의추출물의구성성분들이잘추출되었는지확인하기위하여지표물질인 acanthoside D, 20-hydroxyecdysone, pinoresinol diglucoside를 HPLC로분석하였다. 항염증효능을확인하기위하여 lipopolysaccharide (LPS) 로 RAW 264.7 세포주를자극하여염증반응을일으킨상태에서각각의추출물을농도별로처리하고 NO assay를통해항염증효능을확인하였으며 real time PCR로 pro-inflammatory cytokine들의발현량을측정하였다. 결과적으로각각의추출물은지표성분들이검출되었으며오가피와우슬이두충보다 NO assay에서높은활성을보였다. Cytokine 발현량측정에서는오가피와우슬은 inos와 IL-6의발현을억제하였고, 우슬은 TNF- 의발현을억제하였다. 우리나라는전통적으로약재를조합하여처방하여왔다. 본연구는관절염에전통적으로사용해오던약재들이어떤기전에의하여항염증반응을보이는지확인하고이들을조합하여사용하였을때어떤근거에의하여시너지효능을보이는지확인하였다. Acknowledgements: 이논문은 2016년도원광보건대학교교내연구비지원으로이루어졌으며지원에감사드립니다. Funding: None Conflict of interest: None References 1. Song JH, Kim MJ, Kwon HD, Park IH. Antimicrobial activity of fractional extracts from Houttuynia cordata root. J Kor Soc Food Sci Nutr. 2003;32:1053-1058. 2. Higuchi M, Higashi N, Taki H, Osawa T. Cytolytic mechanism of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanism acts as synergistically as the major cytolytic mechanism of activated macrophages. J Immunol. 1990;144:1434-1441. 3. Stock M, Schilling M, Burkhardt K, Prestel R, Abendroth D, Hammer C. Production of pro-inflammatory cytokines and adhesion molecules in ex-vivo xenogeneic kidney perfusion. Transpl Int. 1994;7:647-649. 4. Kwon PS. Comparison of quantitative endotoxin against 5 species of Enterobacteriaceae. Korean J Clin Lab Sci. 2016;48: 124-129. 5. Bloom BS. Direct medical costs of disease and gastrointestinal side effects during treatment for arthritis. Am J Med. 1988; 84:20-24. 6. Bloom BS. Risk and cost of gastrointestinal side effects associated with nonsteroidal anti-inflammatory drugs. Arch Intern Med. 1989;149:1019-1022. 7. Fries JF. NSAIDS gastropathy: The second most deadly rheumatic disease epidemiology and risk appraisal. J Rheumatol. 1991;18:6-10. 8. Roujeau JC, Kelly JP, Naldi L, Rzany B, Stern RS, Anderson T, et al. Medication use and the risk of Steven-Johnson syndrome or toxic epidermal necrolysis. N Engl J Med. 1995;333:1600-1607. 9. Yoon KN, Jang HS, Jin GH. Antioxidant, anti-diabetic, anti-cholinesterase, and nitric oxide inhibitory activities of fruiting bodies of Agaricus brasiliensis. Korean J Clin Lab Sci. 2015;47:194-202. 10. Lee MH, Jeong JH, Jeong MS, Chang SH, Her E. Anti-inflammatory function of the Sophora japonica extract rutin: The inhibitory effect of rutin of Korean Sophora japonica on the productions of NO and TNF alpha from mouse peritoneal macrophages. Korean J Medicinal Crop Sci. 2010;18:105-112. 11. Hong SS, Hwang JS, Lee SA, Hwang BY, Ha KW, Ze KR, et al. Isolation and quantitative analysis of acanthoside D from Acanthopanacis cortex. Kor J Pharmacogn. 2001;32:316-321. 12. Son KH, Hwang JH, Lee SH, Park JH, Kang SJ, Chang SY, Lee KS. Isolation and quantitative determination of 20-hydroxyecdysone from Achyranthes radix. Kor J Pharmacogn. 1999;30:335-339. 13. Weisz A, Cicatiello L, Esumi H. Regulation of the mouse inducible-type nitric oxide synthase gene promoter by interferon-, bacterial lipopolysaccharide, and NG-monomethyl-Larginine. Biochem J. 1996;316:209-215. 14. Lee SJ, Kim JK. Inflammation and insufficient or disordered sleep. Korean J Clin Lab Sci. 2015;47:97-104.
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