23(2)-12(김성연).fm

J. Fd Hyg. Safety Vol. 23, No. 2, pp. 169~176 (2008) Journal of Food Hygiene and Safety Available online at http://www.foodhygiene.or.kr 랫트에서 WK-38 에대한 13 주반복경구투여독성에관한연구 장보윤 1 김윤철 1 강대길 2 이호섭 2 김성연 1 * 1 원광대학교약학대학, 2 원광대학교한의학전문대학원 Thirteen-week Repeated Oral Dose Toxicity Study of WK-38 in Rats Bo Yoon Chang 1, Yoon Chul Kim 1, Dae Gill Kang 2, Ho Sub Lee 2, and Sung Yeon Kim 1 * 1 College of Pharmcy, Wonkwang University, Shinyoung-dong 344-2, Iksan Chungbuk 570-749, Korea 2 Korea Professional Graduate School of Oriental Medicine, Research Institute (MeRRI), Wonkwang University, Iksan, Jeonbuk 570-749, Korea (Received February 6, 2008/Accepted May 6, 2008) ABSTRACT The subcronic toxicity of WK-38, a herbal preparation for the atherosclerosis, was examined in male and female Sprague-Dawley rats. WK-38 is composed of Rhei Rhizoma, Magonoliae Cortx, Moutan Cortex Radicis. Rats were treated with the test substance at a dose of 5 mg/kg, 50 mg/kg and 500 mg/kg intragastrically for 13 weeks. Control rats were treated with vehicle only. No death and abnormal clinical signs were observed throughout the administration period. Also there was no difference in net body weight gain, food and water consumption, organ weight, gross pathological findings, and urine analysis among the groups of rats treated with different doses of the WK-38. Hematological findings and biochemical examination revealed no evidence of specific toxicity related to WK-38. The results suggested that under the condition employed in this study no observation effect level (NOEL) of WK-38 would be 500 mg/kg/day. Key words: WK-38, repeated oral toxicity, Rats, Atherosclerosis 서 최근통계청에서발표한자료에의하면우리나라주요사망원인중뇌혈관질환및심장질환과같은순환기계질환에의한사망이전체만성질환에의한사망률중 40% 이상을차지하고있다 1). 순환기계질환의심각성에대한인식이점차확대되어가고있는가운데, 이러한순환기계질환의주원인중하나로혈관내벽에지방이나콜레스테롤등의침착으로나타나는혈액순환장애현상인죽상경화증 (atherosclerosis) 이제시되고있다. 효능뿐만아니라안전성이확보된자생식물을이용한죽상경화증에효과적인기능성식품및약품의개발이절실히요구되고있다. 죽상경화증등장기간에걸쳐약물투여를해야하 *Correspondence to: Sung Yeon Kim, College of Pharmcy, Wonkwang University, Shinyoung-dong 344-2, Iksan Chungbuk 570-749, Korea Fax: 063-850-7309 E-mail: sungykim@wonkwang.ac.kr 론 는만성질환에는천연의약품이부작용면에서매우유리할수있다. 그러나천연물들의약효는고전적인대중요법과경험에근거한것이므로, 현대약물학적관점으로는해석이어려운경우가많다. 특히, 일부한약재들은잠재적독성을가지고있어장기간복용하는경우건강에치명적인영향을초래할수있음이보고되고있다 2-4). WK-38는대황 ( 大黃, Rhei Rhizoma), 후박 ( 厚朴, Magonoliae Cortx), 목단피 ( 牧丹皮, Moutan Cortex Radicis) 의복합물로서죽상경화증의예방및치료를목적으로구성된처방이다. WK-38의원료물질인대황, 후박, 목단피는한방처방에많이사용되는한약재나그의안전성과독성에관한심도있는연구가이루어지지않았다. 안전성연구의일환으로 WK-38 단회경구투여실험을실시한결과 5) 반수치사량이 2,000 mg/kg을상회하는것으로평가되었다. WK- 38본연구는최근발생이증가하고있는순환기질환의대표적원인인죽상경화증예방과치료를목적으로조성된새로운한방처방인 WK-38의장기복용시안전성을평가하고자웅성과자성랫트에 13주간반복투여실험을수행하였다. 169

170 Bo Yoon Chang, Yoon Chul Kim, Dae Gill Kang, Ho Sub Lee, and Sung Yeon Kim 실험재료및방법 실험동물의선정 7주령 Sprague-Dawley 계통의특정병원균부재 (Specific pathogen free, SPF) 랫트를 ( 주 ) 샘타코 BIO KOREA에서구입후 1주간의적응기를둔후체중이유사한랫트를한그룹으로실험에투입하였다. 이동물들은인수시및적응기간동안일반증상의이상이관찰되지않았다. 온도 22±2 o C, 상대습도 55±10%, 조명시간 12시간 ( 오전 6시 ~ 오후 6시 ) 및조도 150~300 Lux로설정뒤수행하고, 방사선조사로멸균된실험동물용고형사료 (( 주 ) 오리엔트 ) 와정수시스템을이용한물 (tap water) 을자유섭취하도록하였다. 실험물질의제조및투여 WK-38은한방임상에서가장널리사용되는전탕추출법을을이용하여제조하였다. 대황 ( 大黃 ), 후박 ( 厚朴 ), 목단피 ( 牧丹皮 ) 각 100 g을증류수 2,500 ml가들어있는삼각플라스크에넣고 150분간가열하였다. 추출액을 3000 rpm 으로 20분간원심분리하여얻은상등액을감압농축후동결건조하였다. 수율은 47 g(15.6%) 이었다. 랫트의시험물질 WK-38의단회경구투여독성시험에서 2,000 mg/kg에서약물에의한사망개체및 14일간의관찰기간중이상증상이없었다. 이에근거하여반복투여독성시험에서는 500 mg/kg을최고용량으로정하고, 정제증류수로희석하여 50 mg/kg, 5 mg/kg의시험물질을제조하였다. 투여경로는한방임상에서가장널리이용되고있는경구투여를선택하였다. 금속제동물용위관 (Sondae) 을이용하여위내에직접주입하였다. 1일 1회이며액량은 5 ml/ kg 으로계산하여 13주간투여하였다. 관찰및검사항목 일정시간후잔량을측정하여마리당평균섭취량 (g/rat/ day) 및섭수량 (ml/rat/day) 을산출하였다. 안과학적검사 적응기간중시험동물에대하여눈의외관을관찰하였다. 최종시험물질투여후모든동물의눈의외관을육안으로관찰하였다. 뇨검사 뇨검사는대조군및 WK-38(5, 50, 500 mg/kg) 군에서마지막시험물질투여후 24시간동안채뇨하여얻은검체에대해수행되었다. 유리스켄 10 에스지엘시험지 (URISCAN, 영동제약 ) 를이용하여잠혈 (occult blood), 빌리루빈 (bilirubin), 우로빌리노겐 (urobilinogen), 케톤체 (ketones), 단백질 (protein), 아질산염 (nitrite), 포도당 (glucose), ph, 비중 (specific gravity), 백혈구 (white blood cell) 를측정하였다. 혈액학적검사 실험종료후 ethyl ether로마취하여복대동맥으로부터채혈하고 Coulter counting을위하여혈액응고방지제 EDTA-2K가들어있는 CBC채혈병 (BD Vacutainer) 에혈액 4 ml 취한후, 5분이상혼합하여백혈구수 (WBC), 적혈구수 (RBC), 헤마토크리트치 (HCT), 혈색소량 (HGB), 평균적혈구용적 (MCV), 평균적혈구혈색소량 (MCH), 평균적혈구혈색소농도 (MCHC) 및혈소판수 (PLT) 그리고백혈구백분율로호중구 (neutrophil), 호산구 (eosinophil), 호염기구 (basophil), 림프구 (lymphocyte), 단핵구 (monocyte) 검사를수행하였다. 혈액응고시간에있어서 prothrombin time(pt) 와 activated partial thromboplastin time(aptt) 을측정하기위해일부의혈액을 3.8% Sodium citrate가처리된채혈병 (BD Vacutainer) 에취한후혼합하여원심분리 (3000 rpm, 10 min) 하여얻은혈장을이용하였다. 위의지표들은 ( 주 ) 켐온에의뢰하여측정하였다. 일반증상및사망동물의관찰 식품의약품안전청의의약품등의독성시험기준 6) 을참고하여, 전동물에대하여전시험기간동안매일일회씩의일반증상관찰과사망의유무를관찰하였다. 체중변화, 섭이량및섭수량의측정 모든동물에대하여시험기간중매주 1회랫트의체중을측정하였고, 주 1회사료및물섭취량을측정하였다. 측정방법은체중측정일에사료및물정량을급여한후 혈액생화학적검사 실험종료후복대동맥으로부터채혈하여얻어진혈액을실온에서 30분간방치하여응고시킨후원심분리 (3,000 rpm, 15 min) 해서얻은혈청에대하여혈액생화학적검사를다음의항목 alanine transaminase(alt), aspartate transaminase(ast), alkaline phosphatase(alp), cholesterol, glucose, total protein(t-protein), blood urea nitrogen (BUN), creatinine, total bilirubin(t-bil) 치를자동분석장치 (SPOTCHEM EZ, AP-4430, ARKRAY) 를이용하여측정

Thirteen-week Repeated Oral Dose Toxicity Study of WK-38 in Rats 171 하였다. 전해질자동분석장치 (NOVA5, Waltham, MA, USA) 를이용하여 sodium(na + ), potassium(k + ), chloride(cl ) 를측정하였다. 전해질을제외한혈액생화학적지표들은 ( 주 ) 켐온에의뢰하여측정하였다. 부검및장기중량측정 실험종료후모든랫트에대하여에테르마취하에치사시킨후장기를적출하고, 육안적병변관찰및심장, 간, 신장 ( 좌, 우 ), 비장, 고환 ( 좌, 우 ), 난소 ( 좌, 우 ), 부신 ( 좌, 우 ), 폐, 뇌, 갑상선및위의중량을측정하였다. 통계동물의사망이관찰되지않아치사량을구하는통계는실시하지않았다. 대조군과시험물질투여군체중의변화의차이는 Student's t-test 로비교하여유의성을검정하였다. 뇨검사의결과들은 Kurskal-Wallis' H test 로통계처리하였다. 결 과 일반증상및사망률식품의약품안전청의독성시험기준을참고하여전동물에대하여전시험기간동안매일일회씩의일반증상관찰과사망의유무를관찰한결과모든시험군에서전시험 Fig. 2. Food consumption changes in A) male and B) female rats treated orally with WK-38 for 13 weeks. Fig. 1. Body weight increases in A) male and B) female rats treated orally with WK-38 for 13 weeks. Fig. 3. Water consumption changes in A) male and B) female rats treated orally with WK-38 for 13 weeks.

172 Bo Yoon Chang, Yoon Chul Kim, Dae Gill Kang, Ho Sub Lee, and Sung Yeon Kim Table 1. Urinalysis of male and female rats orally administered with WK-38 for 13 weeks Parameter Occult blood Bilirubin Urobilinogen (mg/100 ml) Ketone (mg/dl) Protein (mg/100 ml) Nitrite Glucose (mg/100 ml) ph Specific Gravity Neucocyte (WBC/µL) Sex Male Female Dose (mg/kg) Control 5 50 500 Control 5 50 500-5 0 3 4 0 5 5 5 ± 0 5 0 0 1 0 0 0 + 10 0 0 1 0 1 0 0 0 ++ 50 0 0 0 0 3 0 0 0 +++ 250 0 0 1 1 0 0 0 0-5 5 5 5 5 5 5 5 ± 0 0 0 0 0 0 0 0 + 0.5 0 0 0 0 0 0 0 0 ++ 1.0 0 0 0 0 0 0 0 0 +++ 3.0 0 0 0 0 0 0 0 0-0.1 5 5 4 4 0 0 0 0 ±1 0 0 0 1 3 1 1 0 + 4 0 0 0 0 1 1 4 3 ++8 0 0 1 0 1 3 0 2 +++12 0 0 0 0 0 0 0 0-0 0 0 0 0 5 1 5 ±5 2 4 3 2 4 0 4 0 + 10 1 1 1 2 1 0 0 0 ++ 50 1 0 1 1 0 0 0 0 +++ 100 1 0 0 0 0 0 0 0-4 0 0 0 0 5 0 1 ±10 1 0 0 3 1 0 0 0 + 30 0 2 0 2 0 0 3 3 ++ 100 0 2 4 0 3 0 1 1 +++ 300 0 1 1 0 1 0 1 0 ++++ 1000 0 0 0 0 0 0 0 0-5 5 5 5 5 4 2 5 + 0 0 0 0 0 1 3 0-5 5 5 5 5 5 5 5 ±100 0 0 0 0 0 0 0 0 + 250 0 0 0 0 0 0 0 0 ++ 500 0 0 0 0 0 0 0 0 +++ 1000 0 0 0 0 0 0 0 0 ++++ 2000 0 0 0 0 0 0 0 0-5.0 0 0 0 0 0 1 1 1 ±6.0 3 1 1 0 1 0 1 2 + 7.0 2 1 4 2 1 4 3 1 ++8.0 0 3 0 0 0 0 0 1 +++9.0 0 0 0 3 3 0 0 0-1.000 1 0 1 1 0 0 1 0 ±1.005 0 0 1 2 0 0 1 1 + 1.010 2 2 0 0 5 3 1 2 ++ 1.020 2 3 2 2 0 2 2 1 +++ 1.025 0 0 1 0 0 0 0 1 ++++ 1.030 0 0 0 0 0 0 0 0-2 0 0 0 0 0 1 0 + 25 0 2 5 4 3 2 1 0 ++ 75 1 1 0 1 2 3 3 1 +++ 500 2 2 0 0 0 0 0 4

Thirteen-week Repeated Oral Dose Toxicity Study of WK-38 in Rats 173 기간동안사망동물은관찰되지않았다. 단회투여에서일시적으로관찰되었던혈관확장효과는 5) 본실험조건에서는나타나지않았다. WK-38의반복투여는관찰된모든일반증상에서대조군과차이를유발하지않았다. 체중의변화섭이량및섭수량의변화시험물질에의한체중변화관찰결과 WK-38 의반복투여는자성웅성랫트모두에서체중에유의적인영향을 주지않았다 (Fig. 1). 전시험기간을통해동물당일일사료섭취량은대조군및시험물질투여군에서유의적인차이를나타내지않았다 (Fig. 2). 시험기간중웅성랫트의대조군, 저용량군, 중용량군, 고용량군의동물당평균일일섭이량은 23.9±5.3, 24.2±5.1, 24.3±5.4, 24.4±5.8(g) 이었다. 자성의랫트에있어서도시험기간중대조군과시험물질의투여군모두에서섭이량의변화는인정되지않았으며대 Table 2. Hematological values of rats treated orally with WK-38 for 13 weeks sex Male Female Item Dose (mg/kg) 0 5 50 500 0 5 50 500 WBC (K/mL) 5.3±1.63 6.05±2.14 5.40±2.08 7.07±2.62 2.8±0.39 3.20±2.22 2.94±1.60 3.9±0.66 RBC (M/mL) 8.1±1.06 7.43±1.42 8.62±0.61 8.41±0.41 7.2±0.21 6.64±2.07 7.49±0.42 7.39±0.20 HGB (g/dl) 14.4±1.91 12.97±2.67 14.76±0.67 14.46±0.51 13.3±0.4 13.18±1.47 13.58±0.52 13.36±0.53 HCT (%) 39.4±5.12 35.5±7.2 39.92±2.15 39.52±47.06 36.4±0.8 33.22±10.49 37.22±1.28 36.38±1.51 MCV (fl) 48.6±0.52 47.86±2.63 46.42±3.37 47.06±1.13 50.6±1.12 49.98±1.29 49.82±1.64 49.24±1.90 MCH (pg) 17.8±0.11 17.46±0.81 17.18±1.2 17.20±0.37 18.5±0.62 21.84±7.98 18.18±0.41 18.10±0.68 MCHC (g/dl) 36.6±0.22 36.5±0.50 37.02±0.47 36.58±0.26 36.5±0.69 43.86±16.52 36.50±0.71 36.74±0.26 PLT (K/mL) 611.5±432.5 665.0±386.7 911.2±165.8 899.6±87.3 938.0±72.6 612.5±400.0 903.0±86.2 983.0±47.5 NEU (K/mL) 0.71±0.32 0.62±0.25 0.87±0.30 1.06±0.25 0.44±0.12 0.38±0.24 0.39±0.12 0.52±0.10 LYM (K/mL) 4.09±1.12 4.80±1.83 3.92±1.90 5.45±2.32 2.09±0.46 2.46±1.97 2.29±1.36 3.06±0.54* MONO(K/uL) 0.28±0.16 0.44±0.22 0.44±0.22 0.31±0.04 0.13±0.05 0.17±0.17 0.13±0.09 0.17±0.05 EOS (K/mL) 0.10±0.04 0.05±0.02 0.08±0.02 0.09±0.05 0.08±0.03 0.13±0.09 0.08±0.05 0.10±0.04 PT (sec) 16.3±1.5 15.9±1.05 16.8±0.96 16.3±0.76 17.3±0.7 16.5±1.41 15.8±0.61 16.7±1.16 APTT (sec) 18.4±4.61 15.0±2.56 17.3±1.62 16.9±.26 16.8±1.79 18.3±6.76 16.2±2.12 18.5±4.93 WBC: white blood cell count, RBC: red blood cell count, HGB: hemoglobin, HCT: hematocrit, MCV: mean corpuscular volume, MCH; mean corpuscular hemoglobin, MCHC; mean corpuscular hemoglobin concentration, PLT: Platelet, NEU; neutrophil, LYM; lymphocyte, MONO; monocyte, EOS; eosinophile, PT; prothrombin time, APTT; activated partial thromboplastin time. Values are presented as mean ± standard deviation (SD). Statistically significant from control (* p<0.05) Table 3. Biochemical serum values of rats treated orally with WK-38 for 13 weeks Item sex Male Female Dose 0 5 50 500 0 5 50 500 (mg/kg) T-protein (g/dl) 6.6±1.1 6.5±0.8 7.2±0.8 7.0±0.8 6.9±0.8 7.2±0.8 7.5±±0.6 7.3±0.7 T-bilirubin (mg/dl) 0.7±0.2 0.5±0.1 0.7±0.2 0.6±0.1 0.6±0.4 1.1±1.2 0.8±0.7 0.6±0.5 Glucose (mg/dl) 134.1±21.4 164.0±11.6** 139.1±21.7 165.6±33.1 161.7±16.3 177.9±14.3 174.4±29.2 176.4±19.8 AST (IU/L) 217.9±61.9 189.0±41.5 177.6±32.1 183.7±24.0 157.4±35.7 173.7±58.5 171.9±43.8 174.3±48.2 ALT (IU/L) 48.6±8.4 42.0±8.5 47.7±14.7 37.3±12.2 40.1±12.5 65.1±68.1 53.7±23.8 43.4±16.5 ALP (IU/L) 459.±130.6 437.1±52.2 393.4±108.3 351.7±78.4 502.9±181.4 508.1±148.2 494.0±186.0 494.7±189.9 cholesterol (mg/dl) 82.9±9.4 87.9±9.7 76.9±28.6 91.1±13.1 84.0±15.9 100.7±34.3 95.1±20.0 103.9±14.9 BUN (mg/dl) 19.4±3.3 19.0±3.9 17.4±1.9 19.6±2.1 17.4±2.6 19.4±3.3 17.7±1.5 17.6±3.4 Creatinine (mg/dl) 0.7±0.2 0.6±0.2 0.7±0.1 0.8±0.2 1.0±0.1 0.9±0.01 0.9±0.1 0.7±0.01 Na (meq/l) 158.6±7.6 160.0±3.3 159.6±7.8 157.6±13.5 158.7±7.9 157.6±4.2 163.6±8.6 155.9±6.6 K (meq/l) 5.0±2.4 3.2±0.3 4.0±1.6 3.8±0.7 3.2±0.4 3.2±0.6 3.1±0.5 3.3±0.6 Cl (meq/l) 95.4±10.8 88.4±1.4 91.3±9.7 92.9±8.3 95.1±9.1 89.9±3.1 88.9±3.0 91.0±7.3 ALT: alanine transaminase, AST: aspartate transaminase, ALP : alkaline phosphatase, T-protein: total protein, BUN: blood urea nitrogen. Values are presented as mean ± standard deviation (SD). Statistically significant from control (**p<0.01).

174 Bo Yoon Chang, Yoon Chul Kim, Dae Gill Kang, Ho Sub Lee, and Sung Yeon Kim 조군, 저용량군, 중용량군, 고용량군의동물당평균일일섭이량은각각 16.9±4.4, 17.0±5.5, 16.9±3.5, 16.7±2.9(g) 이었다. 섭수량에있어서도 13주투여기간동안본시험물질투여와관련된유의적변화는관찰되지않았다 (Fig. 3). 뇨검사 Table 1에나타낸것과같이, 측정된잠혈 (occult blood), 빌리루빈 (bilirubin), 우로빌리노겐 (urobilinogen), 케톤체 (ketones), 단백질 (protein), 아질산염 (nitrite), 포도당 (glucose), ph, 비중 (specific gravity), 백혈구 (white blood cell) 의지 표에서대조군과비교하여 WK-38 반복투여에의한유의적인변화가관찰되지않았다. 안과학적검사소견 WK-38 의반복투여와관련된특기할만한변화는관찰되지않았다. 육안적부검자웅성랫트의전용량에서시험물질의투여에의하여발생되었다고인정되는조직및장기의변화는육안적으로관찰되지않았다. Table 4. Absolute organ weights (g) of male and female rats orally administered with WK-38 for 13 weeks Organs (g) sex Male Female Dose (mg/kg) 0 5 50 500 0 5 50 500 Body Weight 501.5±33.3 500.2±21.3 488.4±40.6 486.0±33.9 301.0±18.4 287.0±19.8 272.0±33.0 290.0±23.8 Liver 14.1±0.9 14.9±0.9 14.5±1.3 15.2±1.3 8.5±1.1 9.0±1.6 8.2±0.6 8.4±0.6 Spleen 0.87±0.07 0.81±0.05 0.90±0.13 0.80±0.16 0.57±0.07 0.53±0.08 0.55±0.13 0.54±0.08 Heart 1.6±0.2 1.5±0.1 1.4±0.1 1.4±0.1 0.99±0.13 0.85±0.17 0.97±0.13 0.93±0.06 Stomach 1.9±0.4 2.0±0.4 2.0±0.3 1.9±0.3 1.2±0.2 1.2±0.3 1.2±0.2 1.2±0.3 Brain 2.2±0.1 2.2±0.1 2.2±0.3 1.8±0.6 1.9±0.2 1.9±0.1 1.9±0.1 1.9±0.1 Thyroid gland 0.40±0.16 0.50±0.18 0.50±0.18 0.60±0.20 0.20±0.04 0.20±0.08 0.30±0.10 0.30±0.07 Lung 2.1±0.4 2.1±0.4 2.1±0.2 1.9±0.2 1.4±0.2 1.5±0.3 1.4±0.2 1.6±0.2 Testis R 1.8±0.2 1.9±0.2 1.9±0.2 1.8±0.2 0.07±0.01 0.07±0.02 0.07±0.02 0.07±0.02 L 1.7±0.2 1.9±0.3 1.9±0.2 1.8±0.2 0.09±0.02 0.07±0.02 0.07±0.02 0.07±0.01 Adrenal gland R 0.04±0.01 0.04±0.02 0.03±0.02 0.03±0.01 0.04±0.01 0.06±0.03 0.05±0.02 0.03±0.01 L 0.03±0.01 0.03±0.01 0.04±0.01 0.03±0.02 0.05±0.01 0.06±0.02 0.05±0.03 0.04±0.01 Kidney R 1.6±0.1 1.6±0.1 1.5±0.1 1.6±0.2 0.9±0.1 0.9±0.1 0.8±0.2 0.9±0.1 L 1.6±0.2 1.6±0.1 1.5±0.1 1.6±0.1 1.1±0.3 0.8±0.2 0.9±0.1 0.9±0.1 Values are presented as mean ± standard deviation (SD). Table 5. Relative organ weights (%) of male and female rats orally administered with WK-38 for 13 weeks Organs (%) sex Male Female Dose (mg/kg) 0 5 50 500 0 5 50 500 Body Weight (g) 501.5±33.3 500.2±21.3 488.4±40.6 486.0±29.7 301.0±18.4 287.0±19.8 272.0±33.0 290.0±23.8 Liver 2.83±0.23 2.98±0.22 2.98±0.36 3.12±0.23 2.82±0.36 3.15±0.50 3.07±0.55 2.93±0.29 Spleen 0.17±0.04 0.16±0.01 0.18±0.04 0.18±0.06 0.19±0.03 0.19±0.03 0.21±0.07 0.19±0.03 Heart 0.32±0.05 0.30±0.03 0.30±0.03 0.28±0.02 0.33±0.03 0.29±0.06 0.36±0.08 0.32±0.03 Stomach 0.38±0.10 0.41±0.08 0.40±0.05 0.40±0.08 0.41±0.06 0.44±0.12 0.46±0.15 0.43±0.11 Brain 0.45±0.02 0.44±0.03 0.44±0.07 0.37±0.13 0.64±0.09 0.66±0.05 0.71±0.08 0.66±0.04 Thyroid gland 0.09±0.04 0.11±0.04 0.11±0.04 0.12±0.04 0.06±0.01 0.08±0.03 0.11±0.06 0.10±0.02 Lung 0.43±0.09 0.42±0.09 0.43±0.06 0.39±0.05 0.46±0.06 0.53±0.11 0.54±0.08 0.55±0.08 Testis R 0.36±0.04 0.38±0.06 0.39±0.06 0.38±0.05 0.02±0.004 0.02±0.005 0.02±0.013 0.02±0.006 L 0.35±0.05 0.37±0.05 0.39±0.06 0.37±0.04 0.03±0.009 0.03±0.007 0.03±0.011 0.03±0.005 Adrenal gland R 0.009±0.001 0.008±0.002 0.008±0.002 0.007±0.004 0.008±0.002 0.017±0.011 0.018±0.010 0.017±0.004 L 0.009±0.004 0.008±0.004 0.008±0.004 0.007±0.001 0.008±0.004 0.017±0.008 0.018±0.010 0.017±0.005 Kidney R 0.32±0.04 0.32±0.03 0.32±0.02 0.33±0.03 0.32±0.02 0.32±0.03 0.32±0.10 0.32±0.02 L 0.32±0.06 0.32±0.03 0.32±0.03 0.34±0.04 0.36±0.12 0.30±0.06 0.35±0.07 0.32±0.03 Values are presented as mean ± standard deviation (SD).

Thirteen-week Repeated Oral Dose Toxicity Study of WK-38 in Rats 175 혈액학적검사 WK-38 투여에따른혈액학적검사결과자성랫트고용량투여군에서림프구의유의적증가가나타났으나, 용량의존적이지않았으며웅성랫트에서는전체용량투여군과대조군사이에유의적인차이가나타나지않았다. 이외의지표에서는물질투여와관련된유의적인변화가관찰되지않았다 (Table 2). 혈액생화학적검사혈액생화학적소견에서총단백질량및총 bilirubin의함량은대조군과비교할때유의성있는차이를나타내지는않았다. 혈액생화학적지표중웅성랫트저용량투여군에서혈당 (glucose) 의증가가관찰되었다. 이외에측정된모든혈액생화학적인지표에서는 WK-38 투여에의한유의한변화는관찰되지않았다 (Table 3). 절대장기중량및상대장기중량투여물질에의한절대장기중량의변화는웅성과자성모든랫트에서나타나지않았다 (Table 4). 상대적장기무게도시험물질의 13주간반복투여에의한변화는관찰되지않았다 (Table 5). 고 WK-38은죽상경화증의예방및치료를목적으로조성된시험물질로, 13주반복경구투여에의한독성을식품의약품안전청의독성시험기준 6) 에근거하여연구하였다. 시험물질을 13주간반복경구투여하고사망률, 일반증상, 체중변화, 뇨검사, 부검소견및장기무게및혈액학적, 혈액생화학적지표등을관찰하였다. 시험에사용된 WK-38는대황 ( 大黃 ), 후박 ( 厚朴 ), 목단피 ( 牧丹皮 ) 의복합물로서순환기계질환발생의주원인중하나인죽상경화증의예방및치료를목적으로본연구자들에의해자체구성된처방이다. 황량 ( 黃良 ), 화삼 ( 火蔘 ) 이라고불리는대황 ( 大黃 ) 은성미는차고쓰다. 독성은없는것으로알려져있으며, 그성분중 d-catechin에항응혈작용이있음이알려져있다 7). 후박 ( 厚朴 ) 은복부의팽만감을주증으로하는소화기질환과불안, 신경증등을포함한광의의정신신경질환에사용되며 8), 최근후박의주성분인 magnolol은혈관내의산화형의 LDL(low density lipoprotein) 에의한활성산소종 (ROS; reactive oxygen species) 의생성을억제하여죽상경화성혈관질환 (atherosclerotic vascular disease) 에보호효과가있음이보고되었다 9). 진경 ( 鎭痙 ), 진통 ( 鎭痛 ), 소염 ( 消炎 ), 지혈 ( 止血 ), 항어혈 ( 抗瘀血 ) 작용을하는것으로알려진목단피 ( 牧丹皮 ) 는 10) 혈관내피로부터 nitric oxide 분비를증가시켜혈관확장작용을나타냄이밝혀졌다 11). 찰 WK-38을 Sprague-Dawley 계통의랫트에 13주간반복경구투여결과, 모든시험군에서시험기간시험물질로인한일반증상및사망동물은관찰되지않았다. 시험기간동안체중의지속적인증가가관찰되었으며통계학적으로유의적인차이는나타나지않았다안검사및뇨검사에서모든투여군에서대조군과비교하여시험물질투여에기인한유의성있는변화는관찰되지않았다. WK-38 투여는혈액학적검사및혈액생화학적검사중자성랫트고용량투여군에서림프구의증가와중웅성랫트저용량투여군에서서혈당 (glucose) 의증가가관찰되었지만이는용량상관성이결여되었으며정상범위이내의변화로본시험물질에의한독성학적변화로는판단되지않았다. 이상의결과로보아본시험조건하에서 WK-38의랫트에대한 13주반복경구투여시험에서독성학적이상변화는관찰되지않았다. 따라서무독성량은 500 mg/kg을상회하는것으로판단되었다. 요 죽상경화증 (arteriosclerosis) 의예방과치료를목적으로조성된새로운한방처방인 WK-38을웅성과자성랫트에 13주간반복투여하여독성을평가하였다. WK-38은대황 ( 大黃, Rhei Rhizoma), 후박 ( 厚朴, Magonoliae Cortx), 목단피 ( 牧丹皮, Moutan Cortex Radicis) 의복합물로구성되었다. 실험동물에게 5 mg/kg, 50 mg/kg 또는 500 mg/kg 을경구로투여하였다. 투여기간동안사망, 일반증상, 섭이량, 섭수량, 및체중증가등을관찰하였다. 투여된 WK-38 모든용량에서사망하는개체는없었다. 시험기간동안체중의지속적인증가가관찰되었으며통계학적으로유의적인차이는나타나지않았다. 안검사및뇨검사에서모든투여군에서대조군과비교하여시험물질투여에기인한유의성있는변화는관찰되지않았다. WK-38 투여는혈액학적검사및혈액생화학적검사결과시험물질에의한독성학적변화로는판단되는지표는없었다. 이상의결과에근거하여본시험조건하의 WK-38의랫트에대한 13주반복경구투여시험에서는독성학적변화가관찰되지않았다. 따라서무독성량은 500 mg/kg을상회하는것으로판단된다. 약 감사의말씀 이연구는자생식물이용기술사업단의과제지원 (PF03201-01-00) 을받아수행하였음 참고문헌 1. 통계청 : 2005 년사망원인통계결과 (2006).

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