한방비만학회지제 18 권제 1 호, 2018 J Korean Med Obes Res 2018;18(1):36-49 https://doi.org/10.15429/jkomor.2018.18.1.36 pissn 1976-9334, eissn 2288-1522 JKOMOR Original Article 삼정환의랫드를이용한 90 일반복경구투여독성시험 김민지 이명종 김호준 동국대학교한의과대학한방재활의학교실 Repeated Dose 90-Day Oral Toxicity Study of Modified Samjung-Hwan in Sprague-Dawley Rats Min-Jee Kim, Myeong-Jong Lee, Hojun Kim Department of Rehabilitation Medicine of Korean Medicine, College of Korean Medicine, Dongguk University Received: May 13, 2018 Revised: June 5, 2018 Accepted: June 14, 2018 Correspondence to: Hojun Kim Department of Rehabilitation Medicine of Korean Medicine, Dongguk University Ilsan Oriental Hospital, 27 Dongguk-ro, Ilsandong-gu, Goyang 10326, Korea Tel: +82-31-961-9111 Fax: +82-31-961-9009 E-mail: kimklar@dongguk.ac.kr Copyright 2018 by The Society of Korean Medicine for Obesity Research Objectives: The study is aimed at evaluating the possible toxicity in 90-day repeated oral administration of modified Samjung-hwan (msjh) in Sprague-Dawley (SD) rats. This study was conducted to detect the no-observed adverse effect level (NOAEL). Methods: Modified SJH extract was administered orally in male and female SD rats at dose of 0, 1,000, 2,000, 4,000 mg/kg. Each group consisted of 10 rats of each gender. The modified SJH extract was given once a day for 90 days. We monitored the changes of mortalities, clinical signs, body weight changes, food consumption, ophthalmologic findings, urine analysis, hematology, serum biochemistry, necropsy findings, organ weights, histological markers of all animals treated with modified SJH extract during the study period. Results: There were no toxicologically significant changes in mortalities, clinical signs, body weight gains, food consumption, ophthalmologic findings, urine analysis, hematology, serum biochemistry, necropsy findings, organ weights, histological markers in any of rats tested. Conclusions: The NOAEL of the modified SJH extract in male rats and no observed effect level (NOEL) in female rats are considered 4,000 mg/kg. Key Words: No-observed-adverse-effect Level, Safety, Toxicity tests 서론 이상지질혈증 (dyslipidemia) 은총콜레스테롤과 low-density lipoprotein 콜레스테롤, 중성지방농도의증가와 highdensity lipoprotein 콜레스테롤의농도가감소되는비정상적인혈중지질상태이다 1). 이는남녀모두에게심혈관질환의위험요소로알려져있으며 2), 최근서구화된식습관과운동부족과같은생활습관에따라증가하는추세이다 3). 2014년질병관리본부통계에따르면, 만 30세이상성인의이상지질혈증중고콜레스테롤혈증의유병률은남성의경우 2005년 7.2% 에서 2013년 13.6% 로, 여성의경우 2005년 8.4% 에서 2013년 15.9% 로성별에관계없이약 2 배정도증가하였으며, 7명중 1명이고콜레스테롤혈증에 이환된것으로나타났다. 이상지질혈증의위험요인으로는연령과성별, 체질량지수 4), 주거지역 5), 고혈압, 당뇨병의만성질환, 흡연과음주, 경제상태, 교육수준 6) 이있다. 이상지질혈증의치료는식이요법, 운동, 금연등의생활습관개선이우선시되어야하며생활요법개선후에도조절이잘되지않으면약물요법을병행하고있다 3). 하지만간독성, 소화장애와같은부작용가능성이있어이를보완하기위해천연한약물에서의연구도활발히이루어지고있다 7). 최근삼정환의지질대사개선효과에관한연구에서고지방식이유도비만마우스에삼정환추출물을투여한결과세포내지질축적을유의하게감소시켰으며체중, 간중량및체지방량증가가감소되었다. 또한 adenosine monophosphate-activated protein kinase와 ace- 36 www.jkomor.org
김민지외 : 삼정환의랫드를이용한 90 일반복경구투여독성시험 tylcoenzyme A carboxylase의인산화를촉발시켰을뿐만아니라렙틴 (leptin) 의발현이증가되는것으로보고된바있다 8). 동의보감신형문 9) 에서삼정환 (Samjung-hwan) 은 오랫동안먹으면몸이가벼워지고오래살게되며, 얼굴이어린아이처럼된다 ( 久服輕身延年益壽面如童子 ). 고기록되어있다. 선행연구에서삼정환의항우울효과가밝혀졌는데, stress군에비해삼정환투여군에서혈청 corticosterone, adrenocorticotropic hormone 수치의감소가나타났다 10). 이외에도신경보호효과 11), 항산화효과 12), 면역개선효과 13) 가있다고알려졌다. 그리고삼정환의제조과정에숙성과정 ( 발효 ) 이포함되어있는발효삼정환에관한연구도있었는데, 체중감소와총콜레스테롤및혈당수치감소효과가밝혀졌다 14). 또한삼정환의구성약재인창출 (Atractylodes japonica) 의지질대사개선효과 15), 지골피 (Cortex lycii radicis) 의혈당강하, 혈압강압효과 16,17), 상심자 (Morus alba Linne) 의항산화효과 18,19) 등에관한연구도있다. 이에삼정환을비만, 지방간을비롯한대사질환과관련하여지질대사개선에적용할수있을것이라판단된다. 본시험은삼정환추출물의독성반응과그안전성을평가하고자식품의약품안전처에서고시한제2015-82호의약품등의독성시험기준 (2015) 과 OECD guideline (2008) 20) 에따라 90일간암수랫드 (Sprague-Dawley) 에반복경구투여시중장기간내에발현되는독성적특성을질적, 양적으로검사하였다. 재료및방법 1. 시험물질및투여 1) 시험물질 본시험에사용한삼정환추출물은 대평 (Daepyung, Busan, Korea) 에서갈색의분말형태로공급받아사용하였으며, 각약재모두지표물질기준함량을준수하였다 ( 창출의 Atractylodin 0.00034%, 상심자의 Rutin 0.02137%, 지골피의 Betaine 0.85764%). 상심자, 지골피, 창출을 3:1:1 비율로섞은후중량의 5배에달하는 30% 에탄올을투입하여 21) 75~ 80 에서 4시간가열추출하여 40 이하에서냉각후여과하였다. 그리고다시중량의 2.5배에달하는 30% 에탄올을투입하여 75~80 에서 2시간추출하 여 40 이하냉각후여과하였다. 이후 50±5 에서감압농축하여농축액건조물과덱스트린을 7:3 비율로혼합, 용해하여 Spray dryer로건조하였다 ( 수율 21.9%). 조제후 1~2시간내에투여하는것을원칙으로하며, 90일간 ( 투여전기간 ) 매일조제하였다. 고용량군은멸균증류수 (KAI5038; JW Pharmaceutical Corporation, Seoul, Korea) 를이용하여 4,000 mg/10 ml (40%) 로조제하였고, 중, 저용량군은동일한멸균증류수로단계희석하여각각 2,000 mg /10 ml (20%), 1,000 mg/10 ml (10%) 로조제하였다. 투여시침전물이없도록 magnetic stirrer로충분히섞어사용하였다. 2) 투여절차시험물질의임상예정경로로서경구투여를선택하였으며, 경구투여용존데를장착한주사관을이용하여매일 90일간 1일 1회 08:00~12:00에위내에직접투여하였다. 투여액량은최근체중측정일의체중을기준으로 10 ml/kg 으로산출하였다. 3) 군분리및투여용량설정순화후평균체중에가장가까운암수각 40마리를선택하였다. 선택한동물들은각군평균체중이균등하도록무작위로암수각 4군, 군당 10마리로군분리하였다. 투여용량설정은본시험물질을이용한 28일간의반복경구투여용량설정시험의결과인암수랫드의 no observed adverse effect level (NOAEL, 최대무독성용량 ) 4,000 mg/ kg/day를참고하여결정되었다. 본시험에서는 4,000 mg/ kg/day를고용량으로설정하고공비 2를적용한 2,000 및 1,000 mg/kg/day를중용량및저용량으로설정하였다. 대조군에서는부형제인멸균증류수를투여하였다. 2. 시험동물및사육환경 1) 시험동물시험동물의이용은대구가톨릭대학교동물실험윤리위원회의승인하에이루어졌다 ( 제IACUC-2016-005호 ). 시험동물은모두특정병원균부재 (specific pathogen free, [SPF]) Sprague-Dawley (SD) 계랫드 (Daehanbiolink, Eumseong Gun, Korea) 를사용하였다. SPF Sprague Dawley (SD) 계랫드는일반독성시험에널리사용되고있어시험기초자료가풍부하여, 시험결과의해석및평가에이러한자료를활용 www.jkomor.org 37
한방비만학회지제 18 권제 1 호, 2018 할수있어선택하였다 21). 입수시에는 5주령수컷 48마리, 134.0~160.8 g, 5주령암컷 48마리, 115.8~140.3 g이었으며투여개시에는 6주령수컷 40마리, 193.5~220.0 g, 6주령암컷 40마리, 148.5~183.7 g이었다. 동물입수시검수및검역을실시하였으며, 동물의순화는입수후수컷 7일, 암컷 8일간해당동물실내에서실시하였다. 순화기간중매일 1회일반증상을관찰하고, 투여전일및투여일에체중을측정하여동물에이상이없음을확인하였다. 2) 사육환경사육환경은시험의전기간동안온도 22±3, 상대습도 30~70%, 환기횟수시간당 10~20회, 조명 12시간명암주기 ( 점등 8:00, 소등 20:00), 조도 150~300 Lux, 소음 60 db이하, 암모니아농도 20 ppm 이하로설정되었다. 순화검역, 투여및관찰기간에는 Polysulfone 사육상자 (280 [width, W] 420 [length, L] 200 [height, H] mm) 에, 절식및요검사기간에는대사케이지 (310 W 450 L 210 H mm) 에서수용하였다. 검역, 순화기간에는 1~3마리, 관찰기간에는 2마리사육하였다. 사육상자는주 1회, 급수병은주 2회교환하였으며, 모든사육기자재는멸균하여사용하였다. 실험동물용고형사료는감마선멸균사료 (Harlan Laboratories, Inc., Itingen, Switzerland) 를자유섭취시켰으며음수는자외선유수살균기로여과후자유섭취시켰다. 3. 관찰및검사 1) 일반증상관찰모든동물에대해서 1일 1회일반증상의종류, 발현및증상의정도를관찰하였으며, 1일 2회사망및빈사동물을관찰하였다. 관찰은모든동물에대하여투여일로부터 90일간실시하였다. 2) 체중측정모든동물의체중을투여개시일에측정하고, 이후에는주 1회, 부검전일및부검일에측정하였다. 부검일체중은절식을실시하였으므로, 체중평가에서제외하고부검전일체중을평가하였다. 3) 사료섭취량산정투여개시전의사료섭취량은군분리일부터투여개시일까지 1일간의섭취량을측정하였다. 투여기간에는 7일 간의섭취량을측정하여 1일의평균섭취량을산출하였다. 투여 13주째에는 6일간의섭취량을측정하여 1일의평균섭취량을산출하였다. 4) 안과학적검사동물도입시모든동물에대하여육안으로눈의외관및전안부를관찰하였다. 투여마지막주에는모든동물을육안으로눈의외관을관찰한후, 각군당 5마리에대해서는안구에산동제 ( 이솝토아트로필, Alcon, Seoul, Korea; Lot No.: 14K19AF) 를점적하여안저사진기 (Genesis, Kowa Co. Ltd., Aichi, Japan) 로전안부, 중간투광체및안저를관찰하였다. 5) 요검사각군당 5마리에대하여투여 13주에요검사를실시하였다. 동물을대사케이지에수용하여 3~4시간동안채뇨한신선뇨중약 1 ml를취하여요검사용시험지 (Multistix 10SG, SIEMENS, New Orleans, LA, USA), 요자동분석기 (CliniTek Advantus TM, SIEMENS, New Orleans, LA, USA) 및요침사검사로분석하였다. 또한 24시간동안계속채집한요로요총량을측정하였다. 요분석항목은요당 (glucose [GLU]), 빌리루빈 (total bilirubin [BIL]), 케톤체 (ketone body, KET), 백혈구 (leukocyte [LEU]), 잠혈 (occult blood [OB]), ph, 단백질 (protein [PRO]), 유로빌리노겐 (urobilinogen [URO]), 아질산염 (nitrite [NIT]) 및요비중 (specific gravity [SG]) 등을검사하였다. 요침사검사는적혈구 (red blood cell [RBC]), 백혈구 (white blood cell [WBC]), 상피세포및원주 (casts) 를관찰하였다. 6) 혈액학적검사부검시까지생존한모든동물에대하여혈액학적검사를실시하였다. 혈액학적검사는랫드를하룻밤절식시킨후채혈한혈액을항응고제인 ethylene diamine tetraacetic acid-2k가들어있는 vacutainer tube (Vacutainer 3 ml, Franklin Lakes, NJ, USA) 에주입한후자동혈액분석기 (ADVIA 2120, SIEMENS, New Orleans, LA, USA) 를통해이루어졌다. 분석항목은백혈구 (WBC) 의수, 적혈구 (RBC) 의수, 혈색소량 (hemoglobin [HGB]), 적혈구백분율 (hematocrit [HCT]), 평균적혈구용적 (mean corpuscular volume [MCV]), 평균적혈구혈색소량 (mean corpuscular hemoglobin [MCH]), 38 www.jkomor.org
김민지외 : 삼정환의랫드를이용한 90 일반복경구투여독성시험 평균적혈구혈색소농도 (mean corpuscular hemoglobin concentration [MCHC]), 적혈구분포폭 (red cell distribution [RDW]), 혈액소분포폭 (hemoglobin distribution width [HDW]), 혈소판수 (platelet [PLT]), 평균혈소판용적 (mean platelet volume [MPV]), 망상적혈구 (reticulocyte [RET]), 백혈구백분비 neutrophil (NEU), lymphocyte (LYM), monocyte (MONO), eosinophil (EOS), basophil (BASO), large unstained cells (LUC) 등이었다. 7) 혈액생화학적검사혈액생화학적검사를위해혈액일부를 clot activator가들어있는 vacutainer tube (Vacutainer 3.5 ml, Franklin Lakes, NJ, USA) 에주입하고 10~15분간상온에방치하여응고시켰다. 이후 3,000 rpm으로 10분동안원심분리 (MF80, Hanil, Gimpo, Korea) 하여얻은혈청으로혈액생화학분석기 (KONELAB 20XT, Thermo, Waltham, MA, USA) 를사용하여측정하였다. 측정항목은 aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-gtp (γ-glutamyl transpeptidase), blood urea nitrogen (BUN), creatinine (CRE), glucose (GLU), total cholesterol (CHO), total protein (PRO), creatine phosphokinase (CPK), albumin (ALB), total bilirubin (BIL), triglyceride (TG), inorganic phosphorus (IP), albumin/globulin ratio (A/G), Ca2+ 였다. 또한 Na+, K+, Cl- 함량은전해질분석기 (744 Na+/K+/Cl-Analyzer, SIEMENS, New Orleans, LA, USA) 로측정하였다. 8) 혈액응고시간검사부검시채혈한혈액중 1.8 ml를 3.2% sodium citrate 0.2 ml가들어있는 vacutainer tube (Vacutainer 1.8 ml, Franklin Lakes, NJ, USA) 에주입한후, 3,000 rpm으로 10 분동안원심분리 (MF80, Hanil, Gimpo, Korea) 하여얻은혈청으로 protrombin time (PT), activated partial thromboplastin time (APTT) 를측정하였다. 혈액응고시간검사기 (ACL 7000 Instrumentation Laboratory, Bedford, MA, USA) 를사용하여 Nephelometric Analysis 방법으로초 (sec) 단위로측정하였다. 9) 부검및채혈부검전날 17시간이상절식한랫드를부검당일에 Isoflurane으로흡입마취하여복대동맥에서혈액학적검사및혈액생화학적검사를위한채혈을실시하였다. 이후복대동맥및복대정맥을절단하여방혈치사시킨다음, 모든장기에대해부검소견을관찰하였다. 10) 장기중량측정부검시장기를적출한후정밀저울을이용하여중량을측정하였다. 양측성장기는양측장기를각각측정하였다. 측정장기는난소 (ovary), 부신 (adrenal gland), 뇌하수체 (pituitary), 가슴샘 (thymus), 전립샘 (prostate), 고환 (testis), 부고환 (epididymis), 비장 (spleen), 신장 (kidney), 심장 (heart), 폐 (lung), 뇌 (brain) 및간 (liver) 등이었다. 11) 조직병리학적검사부검을실시한모든동물에대하여장기를적출하여 10% 중성완충포르말린용액에고정하였으며또한안구는 Davidson 용액에, 고환과부고환은 Boulin 용액에고정하였다. 고정장기는고환, 부고환, 정낭, 전립샘, 난소, 자궁, 질, 방광, 비장, 위, 췌장, 십이지장, 공장, 회장, 맹장, 결장, 직장, 장간막림프절, 부신, 신장, 간, 대퇴골, 턱밑림프절, 침샘, 흉골, 가슴샘, 심장, 폐, 대동맥, 흉척수, 혀, 기관, 식도, 갑상샘, 안구, 하더샘, 뇌, 뇌하수체, 피부 ( 젖샘 ) 이었다. 고정장기 조직은삭정, 탈수및파라핀포매등의일반적인조직처리과정을거쳐조직절편을제작하였다. 이후박절하여 Hematoxylin & Eosin (H&E) 염색을실시하였다. 대조군, 고용량군 (4,000 mg/kg/day) 의모든고정장기 조직에대하여검경하였고, 이상이있는장기에대해서저용량군 (1,000 mg/kg/day) 및중용량군 (2,000 mg/kg/day) 을추가검경하였다. 12) 통계학적방법대조군과투여군간의평균비교에는모수적인다중비교 (parametric multiple comparison procedures) 혹은비모수적인다중비교 (non parametric multiple comparison procedures) 를사용하였으며, 통계학적분석은상용으로널리사용되고있는통계패키지인 SPSS ver 19.0 (IBM Corp., Armonk, NY, USA) 을이용하였다. 체중, 사료섭취량, 혈액학적검사, 혈액생화학적검사, 장기중량등과같은연속적인자료는 one-way analysis of variance (ANOVA) test 로평균치에대한유의성을검정하였다. 유의성이있으면 www.jkomor.org 39
한방비만학회지제 18 권제 1 호, 2018 Levene test로등분산성을검정하여등분산일경우에는 Duncan multiple range test, 이분산일경우에는 Dunnett s T-test를이용하였다. 요검사결과와같이불연속인자료의분석척도변환을통해중증도 (severity) 로나타내어통계분석이이루어졌다. Kruskal-Wallis H-test를실시한후, 유의성이있으면 Mann-Whitney U-test를적용하여대조군과의유의성을확인하였다. 요색조는 Fisher s exact test 를적용하였다. 13) 독성판정기준삼정환추출물의반복투여독성시험을통해얻은변화또는독성은독성의정도와양상에따라 Weight-based classification ( 결과- 경중에따른분류 ) 로이루어졌다. Weightbased classification은이전연구 22-24) 를참고하여시험물질- 유래중요한변화 (Important compound-related changes), 시험물질-유래경미한변화 (Minor compound-related changes) 및비시험물질-유래변화 (Noncompound-related changes) 의 3단계로분류된다. Weight-based classification의 3단계는본시험의목적인 no observed effect level (NOEL, 최대무영향용량 ), no observed adverse effect level (NOAEL, 최대무독성용량 ) 및 lowest observed adverse effect level (LOAEL, 최소독성용량 ) 등의독성지표를얻기위해이들각각의정의에따라시험물질-유래중요한변화는시험물 질에의한 adverse effect로, 시험물질-유래경미한변화는시험물질에의한 non-adverse effect로, 비시험물질-유래변화는시험물질에의한 no effect로분류되었다. 일반적으로 NOEL은시험물질에의한독성학적및약리학적변화를유발하지않는최대무영향용량이다. NOAEL은시험물질에의한 adverse effect를유발하지않거나명확한질환과연계되지않는 non-adverse effect를나타내는최대무독성용량이며 LOAEL은 adverse effect를유발하는최소독성용량을말한다 24). 이러한독성지표의정의에따라 weight-based classification에의한독성지표를 Table 1과같이도출하였다. 결과 1. 일반증상및사망동물 삼정환추출물투여에의해대조군을포함한모든시험군에서전시험기간동안사망동물은없었다. 투여기간중일반증상관찰결과, 이상증상은관찰되지않았다. 2. 체중변화삼정환추출물투여에의한체중변화 (weight changes) 및증체량 (weight gains) 측정결과, 대조군과비교하여모든시험군에서이상소견은관찰되지않았다 (Fig. 1, 2). Table 1. Definitions of Lowest Observed Adverse Effect Level (LOAEL), No Observed Adverse Effect Level (NOAEL) and No Observed Effect Level (NOEL) by Three Steps of Weight-based Classification in Toxicity Test Weight-based classification Definitions of criteria Criteria of toxicity Evaluation endpoints Important compound-related changes Statistically and biologically significant * histopathological changes that are dose-dependent at all doses. Though not dose-dependent, histopathological changes that are statistically significant and that do not occur in the control group. Minor compound-related changes Clinic pathological and histopathological changes that are dose-dependent in certain doses or are not dose-dependent at a certain administered dose. Reversibility is anticipated or there is a histopathological change in the control group. Dose-dependent or non-dose-dependent change that is statistically and biologically significant and is thought to be due to the pharmacological effects of the test substance. Noncompound-related changes Statistically significant change that is obtained through comparison with the control group results that are beyond historical data. Changes due to mistakes in the test process or spurious changes. Adverse effect Non-adverse effect No effect LOAEL NOAEL * Biological significance: statistical significance of the difference between outcomes in a test and historical data; Historical data: mean±standard deviation (SD) of controls from other Repeated Dose 90 days Oral Toxicity Study. NOEL 40 www.jkomor.org
김민지외 : 삼정환의랫드를이용한 90 일반복경구투여독성시험 Fig. 1. modified Samjung-hwan (msjh) was administered orally for 90days. There were no significant differences between any of the groups. Body changes in male Sprague-Dawley rat after once oral administration of modified Samjung-hwan (SJH). Values are presented as mean±standard deviation (n=10). Fig. 3. Kruskal-Wallis' H-test for urine protein in male Sprague-Dawley rat after once oral administration of modified Samjung-hwan (SJH). Values are presented as severity (n=20). *Represents a significant difference at P<0.05 level compared with the vehicle control. PRO: protein, : sterile water administration group, : modified Samjung-hwan (msjh) extract 1,000 mg/kg/day administration group, : msjh extract 2,000 mg/kg/day administration group, : msjh extract 4,000 mg/kg/day administration group. Fig. 2. modified Samjung-hwan (msjh) was administered orally for 90days. There were no significant differences between any of the groups. Body changes in female Sprague-Dawley rat after once oral administration of modified Samjung-hwan (SJH). Values are presented as mean±standard deviation (n=10). 3. 사료섭취량암수평균사료섭취량에서대조군과비교하여모든시험군에서이상소견은관찰되지않았다. 4. 안과학적검사 군분리시외안검사와투여마지막주에수행된안저검사결과에서이상소견은관찰되지않았다. 5. 요검사 요검사결과, 수컷 KET 및 PRO 수치가고용량군 (4,000 mg/kg/day) 에서대조군과비교하여통계적으로유의하게증가하여, 시험물질- 유래경미한변화로판단하였다 (P<0.05) Fig. 4. Kruskal-Wallis' H-test for urine ketone in male Sprague-Dawley rat after once oral administration of modified Samjung-hwan (SJH). Values are presented as severity (n=20). *Represents a significant difference at P<0.05 level compared with the vehicle control. KET: ketone body, : sterile water administration group, : modified Samjung-hwan (msjh) extract 1,000 mg/kg/day administration group, : msjh extract 2,000 mg/kg/day administration group, : msjh extract 4,000 mg/kg/day administration group. (Table 2, Fig. 3, 4). 6. 혈액학적검사 암수모두에서대조군과비교하여이상소견이관찰되지않았다 (Table 3, 4). 7. 혈액생화학적검사 수컷에서 CHO 수치가대조군과비교하여고용량군 (4,000 mg/kg/day) 에서통계적으로유의하게감소하여, 시험물질-유래경미한변화로판단하였다 (P<0.05) (Table 5). www.jkomor.org 41
한방비만학회지제 18 권제 1 호, 2018 Table 2. Urinalysis of Male and Female Standard Deviation Rats Treated Orally with Modified Samjung-Hwan for 90 Days Items Values Severity Male Groups (mg/kg/day) Female GLU - 0 5 5 5 5 5 5 5 5 BIL - 0 5 5 5 5 5 5 5 5 KET - 0 4 3 3 5 5 5 5 +/- 1 1 2 1 1 1+ 2 1 4 SG 1.010 1 4 3 3 5 4 4 3 1.015 2 2 1 2 1 1 2 1.020 3 1 1 1.025 4 1 2 ph 6.5 0 7.0 1 7.5 2 2 1 8.0 3 1 3 5 1 2 3 8.5 4 3 4 2 3 5 3 2 9.0 5 PRO - 0 5 5 3 2 +/- 1 3 1 1 1 3 1+ 2 2 4 1 1 2+ 3 2 3 1 3+ 4 1 1 URO 0.2 0 5 5 5 4 5 5 5 5 1 1 1 NIT - 0 5 5 5 5 5 4 5 5 + 1 1 OB - 0 1 2 3 4 5 5 5 5 +/- 1 4 3 2 1+ 2 2+ 3 3+ 4 1 LEU - 0 1 5 5 2 2 +/- 1 3 2 2 1 1 3 1+ 2 1 1 1 2 1 2+ 3 1 1 2 1 1 3+ 4 1 RBC - 0 5 5 5 5 5 5 5 5 WBC - 0 5 5 5 5 5 5 5 5 Epithelial cell - 0 4 5 4 5 4 5 4 5 +/- 1 1 1 1 1 Casts - 0 4 5 5 4 5 4 5 5 +/- 1 1 1 1 : sterile water administration group, : modified Samjung-hwan (msjh) extract 1,000 mg/kg/day administration group, : msjh extract 2,000 mg/kg/day administration group, : msjh extract 4,000 mg/kg/day administration group. GLU: glucose, BIL: bilirubin, KET: ketone body, SG: specific gravity, PRO: protein, URO: urobilinogen, NIT: nitrite, OB: occult blood, LEU: leukocyte, RBC: red blood cell, WBC: white blood cell, -: negative, +: positive. * The unit of Urobilinogen is Ehrlich unit/dl; Urine sediments. 42 www.jkomor.org
김민지외 : 삼정환의랫드를이용한 90 일반복경구투여독성시험 Table 3. Hematological Values in Male and Female Standard Deviation Rats Treated Orally with Modified Samjung-Hwan for 90 Days Tests * Male Groups (mg/kg/day) Female RBC (10 6 /µl) 8.73±0.345 8.71±0.341 8.45±0.380 8.58±0.470 8.00±0.353 8.10±0.429 7.75±0.781 8.03±0.215 HGB (g/dl) 14.79±0.428 14.53±0.907 14.25±0.406 14.37±0.738 14.29±0.498 14.46±0.448 13.91±1.294 14.55±0.538 HCT (%) 44.51±1.165 44.73±1.360 43.22±1.149 43.93±2.247 42.42±1.702 42.90±1.532 41.16±4.002 43.34±1.470 MCV (fl) 51.03±1.634 51.37±1.296 51.20±2.012 51.30±2.298 53.04±0.763 53.05±1.631 53.15±1.333 53.99±1.683 MCH (pg) 16.93±0.572 16.70±0.932 16.91±0.895 16.79±0.998 17.87±0.416 17.89±0.547 17.95±0.613 18.13±0.668 MCHC (g/dl) 33.21±0.433 32.51±2.028 33.00±0.583 32.72±0.627 33.70±0.447 33.72±0.413 33.77±0.469 33.61±0.517 RDW (%) 12.98±0.731 12.56±0.470 13.21±0.788 13.13±0.869 11.41±0.814 11.34±0.420 11.92±1.923 11.44±0.474 HDW (g/dl) 2.71±0.136 2.68±0.139 2.76±0.289 2.81±0.359 2.37±0.127 2.42±0.108 2.46±0.246 2.44±0.146 PLT (10 3 /µl) 928.50±176.477 986.30±153.770 986.40±85.004 955.60±119.821 1044.90±99.430 995.40±93.385 991.20±156.818 1026.80±109.274 MPV (fl) 7.35±0.513 7.21±0.285 7.33±0.267 7.32±0.257 7.24±0.267 7.24±0.255 7.32±0.439 7.24±0.201 RET (%) 1.46±0.192 1.40±0.285 1.70±0.414 1.81±0.505 1.46±0.329 1.52±0.543 1.44±0.335 1.64±0.538 : sterile water administration group, : modified Samjung-hwan (msjh) extract 1,000 mg/kg/day administration group, : msjh extract 2,000 mg/kg/day administration group, : msjh extract 4,000 mg/kg/day administration group, RBC: red blood cell, HGB: hemoglobin, HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, RDW: red cell distribution, HDW: hemoglobin distribution width, PLT: platelet, MPV: mean platelet volume, RET: reticulocyte. * Represents a significant difference at P<0.05 level compared with the vehicle control. Table 4. Changes of White Blood Cell Number in Male and Female Standard Deviation Rats Treated Orally with Modified Samjung-Hwan for 90 Days Tests Unit Male Groups (mg/kg/day) Female WBC 10 3 /µl 3.65±1.647 3.44±1.573 3.62±0.931 3.53±0.869 1.52±0.517 1.68±0.592 2.15±1.623 1.88±0.534 NEU % 24.62±11.811 24.80±18.972 22.78±7.356 22.82±3.878 16.69±4.956 20.12±8.055 16.80±6.584 17.16±5.787 10 3 /µl 0.82±0.329 0.87±0.896 0.82±0.330 0.82±0.275 0.25±0.118 0.32±0.128 0.29±0.138 0.32±0.151 LYM % 68.95±13.738 69.34±22.744 72.42±7.612 72.43±4.718 77.55±6.078 74.11±7.938 78.03±7.986 76.93±5.981 10 3 /µl 2.60±1.413 2.37±1.506 2.62±0.764 2.55±0.628 1.18±0.433 1.26±0.518 1.76±1.613 1.45±0.447 MONO % 2.59±1.145 3.53±4.809 2.42±0.700 2.30±0.724 2.42±0.977 2.66±0.715 2.24±1.239 2.19±0.479 EOS % 3.03±2.093 1.84±0.836 1.55±0.334 1.81±0.872 2.59±1.478 2.44±1.270 2.13±1.067 2.95±2.530 BASO % 0.19±0.074 0.17±0.116 0.17±0.095 0.17±0.095 0.20±0.141 0.15±0.118 0.21±0.120 0.19±0.099 LUC % 0.63±0.510 0.32±0.253 0.64±0.327 0.49±0.238 0.55±0.284 0.55±0.317 0.59±0.296 0.55±0.314 : sterile water administration group, : modified Samjung-hwan (msjh) extract 1,000 mg/kg/day administration group, : msjh extract 2,000 mg/kg/day administration group, : msjh extract 4,000 mg/kg/day administration group, WBC: white blood cell, NEU: neutrophils, LYM: lymphocytes, MONO: monocytes, EOS: eosinphils, BASO: bosophils, LUC: large unstained cells. 8. 혈액응고시간검사암수모두에서대조군과비교하여이상소견이관찰되지않았다 (Table 6). 9. 부검소견암컷중용량군 (2,000 mg/kg/day) 의 1예에서자궁 (uterus) 내맑은액체저류 (retention of clear fluid) 가관찰되었지만, 다른개체에서는관찰되지않아비시험물질-유래변화로판단하였다. 10. 장기중량장기중량결과는절대중량 (absolute organ weights) 과부검전체중대비상대중량 (relative organ weights) 을바탕으로판정하였다. 수컷의왼쪽고환 (testis) 의절대중량에 www.jkomor.org 43
한방비만학회지제 18 권제 1 호, 2018 Table 5. Blood Chemistry Values in Male and Female Standard Deviation Rats Treated Orally with Modified Samjung-Hwan for 90 Days Tests * Male Groups (mg/kg/day) Female ALB (g/dl) 3.1±0.09 3.1±0.03 3.1±0.09 3.2±0.10 3.4±0.15 3.4±0.13 3.4±0.21 3.4±0.14 ALP (U/L) 204.1±29.56 205.5±50.69 206.9±18.86 476.7±809.51 132.3±26.64 127.2±21.49 141.6±31.04 141.4±34.77 ALT (U/L) 26.1±6.17 27.0±5.79 25.3±3.98 29.2±7.35 20.8±4.07 18.8±3.49 21.8±5.04 21.9±3.03 AST (U/L) 88.0±12.98 97.5±21.98 83.0±19.18 94.1±24.27 82.3±19.13 75.7±14.28 83.2±21.11 80.7±13.22 BIL (mg/dl) 0.1±0.02 0.1±0.04 0.1±0.02 0.1±0.02 0.2±0.02 0.1±0.03 0.2±0.08 0.5±0.06 BUN (mg/dl) 15.2±2.52 15.6±1.61 15.8±2.39 16.3±3.06 19.38±5.45 19.3±4.21 15.2±3.28 14.8±2.35 CHO (mg/dl) 106.7±20.23 91.2±12.76 99.0±17.05 86.85±15.30 92.9±21.17 80.2±22.22 82.8±18.46 87.2±14.66 CPK (U/L) 195.2±168.57 290.8±190.00 203.4±146.03 256.6±214.43 181.1±141.75 162.4±66.03 202.5±192.81 185.3±133.51 CRE (mg/dl) 0.6±0.06 0.6±0.04 0.6±0.04 0.6±0.04 0.8±0.28 0.7±0.18 0.6±0.09 0.7±0.06 GGT (U/L) 3.7±0.65 3.2±0.48 3.2±0.73 3.3±0.74 3.6±0.58 3.7±0.74 3.6±0.41 3.8±0.50 GLU (mg/dl) 199.9±23.14 188.3±16.92 187.2±11.48 191.6±18.04 142.0±26.34 135.7±21.83 155.1±20.32 129.0±15.93 IP (mg/dl) 6.0±0.44 6.0±0.87 6.1±0.31 6.1±0.55 5.2±0.62 5.4±0.66 5.0±0.72 5.1±0.64 PRO (g/dl) 5.6±0.19 5.7±0.22 5.8±0.19 5.8±0.31 5.8±0.26 5.7±0.31 5.8±0.43 5.8±0.27 TG (mg/dl) 39.6±10.99 36.9±12.06 44.4±15.09 44.5±15.47 31.2±4.11 30.9±10.14 34.2±6.12 35.1±7.34 A/G ratio 1.2±0.06 1.2±0.10 1.1±0.07 1.2±0.08 1.5±0.11 1.5±0.09 1.5±0.09 1.4±0.07 Ca 2+ (mg/dl) 9.7±0.26 9.5±0.22 9.7±0.27 9.8±0.24 10.2±0.14 10.1±0.32 10.1±0.27 10.3±0.26 Na + (mg/dl) 141.2±0.67 141.5±0.64 142.1±0.60 141.7±1.11 141.2±1.66 140.7±1.25 140.4±1.16 140.5±1.11 K + (mmol/l) 4.7±0.37 4.8±0.49 4.7±0.30 4.8±0.28 4.3±0.28 4.3±0.30 4.1±0.31 4.2±0.21 Cl - (mmol/l) 105.1±0.72 105.3±1.42 106.2±1.35 105.7±1.39 106.8±1.714 106.9±1.30 106.6±1.69 106.6±1.40 : sterile water administration group, : modified Samjung-hwan (msjh) extract 1,000 mg/kg/day administration group, : msjh extract 2,000 mg/kg/day administration group, : msjh extract 4,000 mg/kg/day administration group, ALB: albumin, ALP: alkaline phosphatase, ALT: alanine aminotransferase, AST: aspartate aminotransferase, BIL: total bilirubin, BUN: blood urea nitrogen, CHO: total cholesterol, CPK: creatine phosphokinase, CRE: creatinine, GGT: gamma-glutamyltransferase, GLU: glucose, IP: inorganic phosphorus, PRO: total protein, TG: triglyceride, A/G ratio: albumin/globulin ratio, Ca 2+ : calcium ion, Na+: sodium ion, K+: potassium ion, Cl-: chloride ion. * Represents a significant difference at P<0.05 level compared with the vehicle control. Table 6. Plasma Coagulation Values in Male and Female Standard Deviation Rats Treated Orally with Modified Samjung-Hwan for 90 Days Tests Male Groups (mg/kg/day) Female PT (sec) 9.47±0.501 9.66±0.454 9.40±0.473 9.43±0.449 9.36±0.531 9.55±0.751 9.45±0.561 9.26±0.494 APTT (sec) 20.3±1.03 1.92±2.93 18.8±1.48 19.1±1.77 20.0±1.40 19.2±1.78 19.2±1.37 18.5±2.54 : 0 mg/kg/day, :1,000 mg/kg/day, : 2,000 mg/kg/day, : 4,000 mg/kg/day, PT: protrombin time, APTT: activated partial thromboplastin time. 서대조군과비교하여저용량군 (1,000 mg/kg/day) 및중용량군 (2,000 mg/kg/day) 에서통계적으로유의하게감소하였으나용량의존성및좌측고환에영향이없는정상범위 (historical data) 내의변화로비시험물질-유래변화로판단하였다 (P<0.05) (Table 7). 반면암컷에서는장기중량의유의한변화는관찰되지않았다 (Table 8). 11. 조직병리학적검사조직병리학적검사에서나타나는결과는중등도 (severity) 에따라 minimal ( 아주경증 ), slight ( 경증 ), moderate ( 중증 ), severe ( 상당한중증 ) 의 4단계로구분하여나타냈다. 암컷피부 (skin) 에서낭포 (cyst) 가고용량군 (4,000 mg/kg/day) 에서 minimal 로 1예가나타났으나발생빈도가낮아비시험물질-유래변화로판단하였다. 44 www.jkomor.org
김민지외 : 삼정환의랫드를이용한 90 일반복경구투여독성시험 Table 7. Absolute (g) and Relative (%) Organ Weights in Male Standard Deviation Rats (n=40) Treated Orally with Modified Samjung-Hwan for 90 Days Groups (mg/kg/day) Organs Adrenal (L) (g) 0.0277±0.00433 0.0277±0.00506 0.0264±0.00399 0.0281±0.00603 Percentage (%) 0.0055±0.00079 0.0056±0.00118 0.0053±0.00061 0.0056±0.00114 Adrenal (R) (g) 0.273±0.00601 0.0299±0.00592 0.0282±0.00314 0.0281±0.00558 Percentage (%) 0.0054±0.00101 0.00859±0.00069 0.0056±0.00064 0.0056±0.00101 Pituitary (g) 0.0146±0.00152 0.0151±0.00213 0.0144±0.0083 0.0140±0.00155 Percentage (%) 0.0029±0.00033 0.0031±0.00052 0.0029±0.00029 0.0028±0.00026 Testis (L) (g) 2.2532±0.19915 2.0690±0.17673 * 2.0751±0.16925 * 2.1223±0.13738 Percentage (%) 0.4488±0.03414 0.4164±0.04098 0.4158±0.03249 0.4236±0.03511 Testis (R) (g) 2.1901±0.22137 2.0382±0.19586 2.0953±0.17746 2.0809±0.13745 Percentage (%) 0.4362±0.03766 0.4098±0.03944 0.4197±0.03113 0.4153±0.03466 Epididymis (L) (g) 0.7345±0.06676 0.7440±0.06412 0.7229±0.07862 0.6977±0.05935 Percentage (%) 0.1464±0.01336 0.01501±0.01750 0.1447±0.01406 0.1391±0.01256 Epididymis (R) (g) 0.7197±0.06118 0.7445±0.06761 0.7055±0.07411 0.7232±0.08201 Percentage (%) 0.1435±0.01196 0.1500±0.01662 0.1410±0.01081 0.1445±0.01963 Thymus (g) 0.4385±0.12049 0.4968±0.16093 0.4729±0.11082 0.4837±0.09800 Percentage (%) 0.0873±0.02307 0.0979±0.02287 0.0947±0.02323 0.0961±0.01710 Prostate (g) 0.6929±0.16213 0.6640±0.15732 0.5854±0.09933 0.6941±0.15308 Percentage (%) 0.1395±0.03990 0.1345±0.03475 0.1168±0.01602 0.14383±0.02982 Spleen (g) 0.9145±0.13154 0.9041±0.19825 0.9694±0.11095 0.9272±0.14224 Percentage (%) 0.1813±0.01705 0.1796±0.02346 0.1935±0.01351 0.1844±0.02460 Kidney (L) (g) 1.5566±0.13740 1.5130±0.18088 1.5819±0.16563 1.5507±0.12337 Percentage (%) 0.3099±0.02272 0.3027±0.01690 0.3160±0.02160 0.3085±0.01382 Kidney (R) (g) 1.5331±0.10402 1.5358±0.17972 1.5851±0.15875 1.5689±0.14416 Percentage (%) 0.3055±0.01939 0.3077±0.02299 0.3166±0.01762 0.3122±0.02127 Heart (g) 1.4444±0.13072 1.4335±0.20866 1.4578±0.14844 1.4587±0.07729 Percentage (%) 0.2872±0.01643 0.2860±0.01574 0.2912±0.01520 0.2911±0.02095 Lung (g) 1.8800±0.12812 1.8491±0.14987 1.8970±0.16929 1.8573±0.09562 Percentage (%) 0.3742±0.01463 0.3718±0.03303 0.3797±0.02777 0.3768±0.01776 Brain (g) 2.1926±0.05457 2.1889±0.09843 2.1985±0.10363 2.2194±0.08003 Percentage (%) 0.4378±0.02955 0.4416±0.04197 0..4425±0.04748 0.4432±0.03201 Liver (g) 13.4832±0.99265 13.0017±1.67276 13.8935±1.97693 14.3159±1.32183 Percentage (%) 2.6855±0.15655 2.5989±0.12716 2.7658±0.17128 2.8483±0.19245 : sterile water administration group, : modified Samjung-hwan (msjh) extract 1,000 mg/kg/day administration group, : msjh extract 2,000 mg/kg/day administration group, : msjh extract 4,000 mg/kg/day administration group, L: left, R: right. * Represents a significant difference at P<0.05 level compared with the vehicle control. 수컷뇌하수체 (pituitary) 에서낭포 (cyst) 가고용량군에서 minimal 로 1예가나타났으나대조군에서도 minimal 로 1예가나타나비시험물질-유래변화로판단하였다. 암컷뇌하수체에서도낭포가고용량군에서 minimal 로 1예가나타났으나대조군에서도 minimal 로 2예가나타나비시험물질-유래변화로판단하였다. 수컷전립샘 (prostate) 에서염증세포침윤 (infiltration of inflammatory cells) 이고용량군에서 minimal 로 1예가나타났지만대조군에서도 minimal 로 1예가나타나비시험물질-유래변화로판단하였다. 수컷신장 (kidney) 에서세뇨관 (renal tubule) 재생 (regeneration) 이고용량군에서 minimal 로 1예, 염증세포침윤이고용량군에서 minimal 로 1예가나타났으나대조군에서도염증세포침윤이 minimal 로 2예가나타나비시험물질-유래 www.jkomor.org 45
한방비만학회지제 18 권제 1 호, 2018 Table 8. Absolute (g) and Relative (%) Organ Weights in Female Standard Deviation Rats (n=40) Treated Orally with Modified Samjung-Hwan for 90 Days Groups (mg/kg/day) Organs Adrenal (L) (g) 0.0341±0.00482 0.0334±0.00344 0.0327±0.00222 0.0323±0.00429 Percentage (%) 0.0126±0.00178 0.0122±0.00133 0.0118±0.00067 0.0117±0.00171 Adrenal (R) (g) 0.0332±0.00360 0.0346±0.0047 0.0318±0.00145 0.0337±0.00417 Percentage (%) 0.0123±0.00175 0.0126±0.00131 0.0115±0.00103 0.0122±0.00162 Pituitary (g) 0.0167±0.00200 0.0153±0.00211 0.0155±0.00235 0.0157±0.00166 Percentage (%) 0.0062±0.00073 0.0056±0.00093 0.0056±0.00092 0.0057±0.00058 Ovary (L) (g) 0.0676±0.00858 0.0682±0.00836 0.0619±0.01038 0.0654±0.00834 Percentage (%) 0.0251±0.00437 0.0249±0.00324 0.0223±0.00346 0.0236±0.00310 Ovary (R) (g) 0.0679±0.00782 0.0726±0.01004 0.0631±0.00980 0.0689±0.00868 Percentage (%) 0.0251±0.00374 0.30265±0.00311 0.0227±0.00329 0.0248±0.00247 Thymus (g) 0.3255±0.08114 0.3288±0.06143 0.3318±0.08951 0.3141±0.03945 Percentage (%) 0.1190±0.03015 0.1198±0.02206 0.1194±0.03138 0.1142±0.02016 Spleen (g) 0.5778±0.06290 0.5815±0.07877 0.6360±0.19500 0.5620±0.05106 Percentage (%) 0.2125±0.01788 0.2120±0.02698 0.2308±0.07951 0.2029±0.01648 Kidney (L) (g) 0.8849±0.08610 0.8686±0.05604 0.8803±0.07593 0.8669±0.07386 Percentage (%) 0.3260±0.02907 0.3173±0.02393 0.3179±0.03144 0.3130±0.02300 Kidney (R) (g) 0.8791±0.08162 0.8863±0.0554 0.8971±0.08354 0.8792±0.06333 Percentage (%) 0.3237±0.02558 0.3240±0.02563 0.3240±0.03473 0.3175±0.01831 Heart (g) 0.8953±0.06342 0.9222±0.07450 0.9120±0.12305 0.8943±0.04516 Percentage (%) 0.3300±0.002265 0.3364±0.02231 0.3286±0.04188 0.3238±0.02637 Lung (g) 1.3573±0.08748 1.4178±0.09770 1.3853±0.10071 1.3612±0.10099 Percentage (%) 0.5002±0.02873 0.5178±0.03793 0.4997±0.03603 0.4931±0.05025 Brain (g) 1.9584±0.08726 2.0014±0.06540 2.0037±0.07336 1.9997±0.04320 Percentage (%) 0.7236±0.06209 0.7323±0.05821 0.7252±0.06681 0.7248±0.06168 Liver (g) 6.9063±0.84855 7.0742±1.07608 7.1424±0.56172 6.8777±0.84140 Percentage (%) 2.5402±0.24859 2.5860±0.42754 2.5747±0.17181 2.4743±0.17757 Represents a significant difference at P<0.05 level compared with the vehicle control. : sterile water administration group, : modified Samjung-hwan (msjh) extract 1,000 mg/kg/day administration group, : msjh extract 2,000 mg/kg/day administration group, : msjh extract 4,000 mg/kg/day administration group, L: left, R: right. 변화로판단하였다. 암컷장막간림프절 (lymph node) 에서장간막림프절의색소 (pigment) 와임파동적혈구증다증 (sinus erythrocytosis) 이고용량군에서 minimal 로 1예가나타났으나발생빈도가낮아비시험물질-유래변화로판단하였다. 수컷폐 (lung) 에서무기질침착 (mineralization) 이고용량군에서 minimal 로 1예가나타났으나발생빈도가낮아비시험물질-유래변화로판단하였다. 암컷폐에서는무기질침착이대조군 (0 mg/kg/day) 에서만 minimal 로 2예가나타나비시험물질-유래변화로판단하였다. 수컷심장 (heart) 에서염증세포침윤이고용량군에서 minimal 로 2 예가나타났지만대조군에서도 minimal 로 2예가나타나비시험물질-유래변화로판단하였다. 고찰 본시험은시험물질인삼정환추출물의반복투여에따른독성을평가하기위하여 6주령 Sprague-Dawley계랫드에시험물질을 1,000, 2,000 및 4,000 mg/kg의용량으로군당암수각 10마리에 90일간경구투여하였다. 독성지표를위해일반증상관찰, 체중측정, 사료섭취량측정, 안검사, 요검사, 전해질, 혈액학적및혈액생화학적검사, 부검 46 www.jkomor.org
김민지외 : 삼정환의랫드를이용한 90 일반복경구투여독성시험 시장기의중량측정, 육안적검사및조직병리학적검사등이수행되었다. 이때본독성시험으로부터정량적지표의정확한설정을위해반복투여독성시험을위한독성평가기준이응용되었다. 관찰기간동안암수시험물질투여에의한사망예는관찰되지않았다. 시험의결과, 독성판정기준에따라삼정환추출물은수컷의 NOAEL, 암컷의 NOEL이모두 4,000 mg/kg/day로판단되었다. 수컷에서 NOAEL의판단기준이되는시험물질-유래경미한변화가관찰되었는데, 요검사에서 KET 및 PRO 수치증가와생화학검사에서 CHO 수치감소이다. 우선 KET 및 PRO 수치가고용량군에서증가하였는데, KET 이상의원인으로는당뇨병또는식이섭취저하로인한기아등이있으며, 요검사및생화학결과에서 Glucose 수치가정상으로나타났고, 체중및사료섭취량에서도이상이관찰되지않았다. PRO 수치이상의원인은신장및요로의이상시나타날수있는데, 이러한증상을뒷받침해줄혈액학및생화학검사의이상과조직병리검사의결과에서신장및요로의이상이관찰되지않았다. 따라서요검사의 KET 및 PRO 수치증가는시험물질의약리적변화에의한 Non-adverse effect로시험물질-유래경미한변화로판단하였다. 또한 CHO 수치가대조군과비교하여고용량군에서 18.7% 감소하였다. 이러한변화는간의이상과관련있는데, 생화학분석결과및조직병리소견에서이상이관찰되지않아독성으로판단하기는힘들며, 시험물질의약리적변화에의한 non-adverse effect로시험물질- 유래경미한변화로판단하였다. 암컷에서 NOEL의판단기준이되는비시험물질-유래변화가관찰되었는데, 부검소견에서중용량군의 1예에서자궁내액체저류의관찰이다. 이는나이든암컷의성주기와관련된증상으로비시험물질-유래변화로판단하였다. 조직병리검사에서피부에서낭포가고용량군에서 1예가나타났으나발생빈도수가적어시험물질영향이아니라고판단하였다. 뇌하수체낭포가고용량군에서 1예가나타났지만, 뇌하수체낭포는나이가들어감에따라나타나는자연발생증상으로 25) 고용량군에서나타난낭포는시험물질에의한영향은아니라고판단되었다. 장간막림프절에서장간막림프절의색소와임파동적혈구증다증이고용량군에서 1예가나타났으나발생빈도수가적어시험물질영향이아니라고판단하였다. 폐에서무기질침착이대조군에서만 2예가나타나시험물질에의한영향은아 니라고판단되었다. 수컷에서도비시험물질-유래변화가관찰되었는데, 장기중량에서왼쪽고환의절대중량에서대조군과비교하여저, 중용량군에서통계적으로유의하게각각 7.2%, 7.5% 감소하였으나용량의존성및좌측고환에영향이없는정상범위내의변화로시험물질영향이아닌것으로판단하였다. 뇌하수체낭포가고용량군에서 1예가나타났지만뇌하수체낭포는나이가들어감에따라나타나는자연발생증상으로 25) 고용량군에서나타난낭포는시험물질에의한영향은아니라고판단되었다. 전립샘에서염증세포침윤이고용량군에서 1예가나타났으나대조군에서도 1 예가나타났으며, 전립선의미약한염증은나이가들어감에따라나타나는자연발생증상으로 25) 고농도군에서나타난국소적인염증은시험물질에의한영향은아니라고판단되었다. 신장에서세뇨관재생과염증세포침윤이고용량군에서각각 1예가나타났지만, 세뇨관재생과염증세포침윤은자연발생적으로나타나는증상으로 25) 대조군에서도나타나고빈도가낮아시험물질에의한영향은아닌것으로판단되었다. 폐에서무기질침착이고용량군에서 1예가있으나발생빈도가낮고, 암컷대조군에서도나타나시험물질영향이아닌것으로판단하였다. 심장에서염증세포침윤이고용량군에서 2예가나타났지만대조군에서도 2예가나타나시험물질영향이아닌것으로판단하였다. 즉시험물질을투여한시험군의피부, 뇌하수체, 전립선, 신장, 림프관, 폐, 심장등에서병변이나타났으나대조군에서도나타나는개체특이적현상이거나나이가많아짐에따라나타나는자연발생적병변으로판단되어시험물질의영향이아닌것으로판단하였으며, 본시험에서시험물질에의한뚜렷한표적장기는없었다. 물론이전연구와는달리암컷, 수컷모두삼정환의체중감소및혈당수치감소에대한효과는본시험에서유의하게관찰되지않았으며 CHO 수치는수컷에서만유의하게감소하였다. 선행연구에서는고지방식이를한점, 삼정환을상온에서혹은유산균과함께발효하였다는점이본연구와차이가있다. 따라서고지방식이를한군에서발효하지않은삼정환을복용하였을때나타나는지질대사개선효과에대해추가적인연구가필요할것으로사료된다. www.jkomor.org 47
한방비만학회지제 18 권제 1 호, 2018 결론 삼정환추출물의 90일반복독성시험결과를바탕으로시험의독성판정기준에따라판정할때, 수컷은 NOAEL, 암컷은 NOEL이 4,000 mg/kg/day로판단된다. 그러나식약처고시제2015-82호 (2015.11.11.) 의약품등의독성시험기준 에따라판정할경우암수모두 NOAEL이 4,000 mg/kg/day로추정할수있다. 감사의글 본연구는한국보건사업진흥원을통해보건복지부 한의약선도기술개발사업 의재정지원을받아수행된연구임 ( 과제고유번호 : HI14C0556). References 1. Ballantyne CM, O' Keefe JH, Gotto AM. Dyslipidemia & Atherosclerosis Essentials 2009. 4th ed. Burlington, Massachusetts : Jones & Bartlett Publishers. 2009 : 5. 2. Jee SH, Jang Y, Oh DJ, Oh BH, Lee SH, Park SW, et al. A coronary heart disease prediction model: the Korean Heart Study. BMJ Open. 2014 ; 4(5) : e005025. 3. Committee for Guidelines for Management of Dyslipidemia. 2015 Korean guidelines for management of dyslipidemia. J Lipid Atheroscler. 2015 ; 4(1) : 61-92. 4. Khader YS, Batieha A, El-Khateeb M, Al Omari M, Ajlouni K. Prevalence of dyslipidemia and its associated factors among Jordanian adults. J Clin Lipidol. 2010 ; 4(1) : 53-8. 5. Wang S, Xu L, Jonas JB, You QS, Wang YX, Yang H. Prevalence and associated factors of dyslipidemia in the adult Chinese population. PLoS One. 2011 ; 6(3) : e17326. 6. Sun GZ, Li Z, Guo L, Zhou Y, Yang HM, Sun YX. High prevalence of dyslipidemia and associated risk factors among rural Chinese adults. Lipids Health Dis. 2014 ; 13 : 189. 7. Jang WS, Kim YS, Seol IC. Antioxidant and lipid-lowering effects of artemisia capillaris on a rat model of hyperlipidemia. The Journal of Korean Oriental Medicine. 2012 ; 33(2) : 11-24. 8. Lim DW, Bose S, Wang JH, Choi HS. Kim YM, Chin YW, et al. Modified SJH alleviates FFAs-induced hepatic steatosis through leptin signaling pathways. Sci Rep. 2017 ; 7 : 45425. 9. Huh J. Dongyibogam. 2nd ed. Seoul : Bubin Press. 2012 : 221. 10. Lee DJ, Jeong JC. Peroxynitrite scavenging activity of Samjunghwan. Korean J Orient Intern Med. 2006 ; 27(1) : 178-87. 11. Kim HG, Ju MS, Park H, Seo Y, Jang YP, Hong J, et al. Evaluation of Samjunghwan, a traditional medicine, for neuroprotection against damage by amyloid-beta in rat cortical neurons. J Ethnopharmacol. 2010 ; 130(3) : 625-30. 12. Han KS, Wang J, Lim D, Chin YW, Choi YH, Choi HS, et al. Anti-oxidative and anti-obesity effect of combined extract and individual extract of samjunghwan. J Korean Med Obes Res. 2014 ; 14(2) : 47-54. 13. Kim SY, Jeong JC. Effects of samjunghwan on the IL-1 gene expression in the macrophage. Korean J Orient Intern Med. 2006 ; 27(1) : 228-36. 14. Song MY, Bose S, Kim HJ. Anti-Obesity Effects of Fermented Samjung-hwan in Hign Fat Diet Rats. J Korean Med Obes Res. 2013 ; 13(1) : 17-23. 15. Kim AJ, Park SJ, Rho JO. Mulberry fruit extract consumption is inversely associated with hyperlipidemia in middle-aged men. Korean J Food Nutr. 2008 ; 21(2) : 121-6. 16. Han Y, Park YK. The comparisons of lycii radicis cortex and corni fructus water extract effects on streptoxotocin-induced diabetes in rats. Korea J Herbol. 2013 ; 28(6) : 71-7. 17. Jung YS, Park CH, Shin HC. Antioxidative effects of Lycium chinense Miller on cisplatin-induced nephrotoxicity in rats. Korean J Orient Int Med. 2014 ; 35(1) : 92-105. 18. Lee EJ, Bae JH. Study on the alleviation of an alcohol induced hangover and the antioxidant activity by mul- 48 www.jkomor.org
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