Korean J. Plant Res. 27(1):011-021(2014) http://dx.doi.org/10.7732/kjpr.2014.27.1.011 Print ISSN 1226-3591 Online ISSN 2287-8203 Original Research Article 최혜경 1, 노항식 1, 정자영 1, 하헌용 2 * 1 식품의약품안전평가원독성연구과, 2 세명대학교자연약재과학과 Acute Oral Toxicity of Atractylodes macrocepala KOIDZ. Hye-Kyung Choi 1, Hang-Sik Roh 1, Ja-Young Jeong 1 and Hun-Yong Ha 2 * 1 Toxicological Research Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong 363-700, Korea 2 Department of National Medicine Resources, Semyung University, Jecheon 390-711, Korea Abstract - Atractylodes macrocepala KOIDZ. (AmK) is a herbal medicine and resources of functional food which has been used for the treatment of indigestion, anorexia, diarrhea and digestive dysfunction. Recently AmK is frequently used as resources of functional food and whitening cosmetics. In this study was carried out to evaluate the acute oral toxicity of Amk in Sprague-Dawley(SD) rats. male and female rats were administered orally with Amk extract of 1,000 mg / kg (low dosage group), 2,000 mg / kg (middle dosage group) and 4,000 mg / kg (high dosage group). We daily observed number of deaths, clinical signs and gross findings for 7 days. No dead SD rats and no clinical signs were found during the experiment period. Also other specific changes were not found between control and treated groups in hematology and serum biochemistry. But we found out feeble histopathological changes in liver fat tissues. In addition no significant changes of gross bady and individual organs weight. These results suggest that water soluble extract of AmK has not acute oral toxicity and oral LD 50 value was over 4,000 / mgkg in SD rats. Key words - Acute, Atractylodes macrocepala KOIDZ., Herbal medicine, LD 50, SD rat, Toxicity 서언 최근건강과웰빙에대한각계의관심이증가되는추세에있으며, 이에따른한약및생약에대한일반인의관심과수요의증가로인하여다양한한약과생약을원료로하는기능성식품과천연물의약품등의무분별한사용이늘고있다 (Oh et al., 2010; Kim et al., 2012). 또한천연물유래의생약과한약은오랜기간사용되어왔기에안전할것이라는인식으로그독성과부작용에대한과학적인근거가확보되지않아국민건강을위협하는요소로작용하고있다 (Park et al., 2008). 따라서천연물생약에대한과학적검증을통해안전성을확보할필요가있으며체계적인관리시스템을도입해야할필요가있다 (Hwang et al., 2004). * 교신저자 (E-mail) : siberiahusky@hanmail.net 특히백출은한약재가운데보기 ( 補氣 ) 의기능을가진약물로써일찍이고대한의처방에서다양하게응용되어왔다. 또한식품원료로도제한적으로사용되고있어그활용빈도가점차로증가하고있는추세이다 (Zhao et al., 2006). 뿐만아니라지방세포의지방합성을저해하는것으로알려져있으며 (Choi et al., 2010), 피부의멜라민색소의합성을저해하는것으로도알려져있어비만과미용분야에서다양하게활용될수있다 (Park et al., 1999; Kim et al., 2005). 국내에서는전통적으로 Atractylodes macrocepala KOIDZ. 이외에 Atractylodes japonica KOIDZ. ( 삽주 ) 의 1~2 년생근경을백출로혼용하여사용하고있다 (Kang et al., 2000). 최근에는백출을삽주로대용하는경우가감소하고있으며, 수입되는한약재또한 Atractylodes macrocepala KOIDZ. 가주류를이루고있다. 백출은국화과 (Compositae) 에속한다년생식물인백출 c 본학회지의저작권은 ( 사 ) 한국자원식물학회지에있으며, 이의무단전재나복제를금합니다. This is an Open-Access article c 본 distributed 학회지의 under 저작권은 the terms ( of 사 the ) 한국자원식물학회지에 Creative Commons -11- Attribution 있으며 Non-Commercial, 이의무단전재나 License (http://creativecommons.org/licenses/by-nc/3.0) 복제를금합니다. which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
韓資植誌 Korean J. Plant Res. 27(1) : 011~021(2014) (Atractylodes macrocepala KOIDZ.) 의근경으로, 山薊, 天薊, 山薑, 山精, 山連, 冬白朮등의이명으로불린다 (Ha, 2007; Hu et al., 1998). 백출의성질은온 ( 溫 ) 하고, 맛은감고 ( 甘苦 ) 하며, 비장과위장에주로작용하여보비익위 ( 補脾益胃 ), 조습화중 ( 燥濕和中 ) 하는작용이있어식욕부진, 권태무력, 비위허약및설사등의소화기기능저하를치료하는약물로알려져있다 (Zhao et al., 2006). 주요약리활성성분으로는 atractylone, eudesmol, palmitic acid, hinesol 등을함유하고있으며, 장관운동촉진, 간보호및이담작용, 항산화, 혈당강하, 위궤양예방등의효능이알려져있다 (Hu et al., 1998). 이에본연구에서는한약및제한적식품원료로사용되는백출의안전성에대한과학적근거를확보하기위해식품의약품안전청 비임상시험관리기준 ( 식약청고시제2012-61) 에따라단회투여독성시험을수행하였다. 재료및방법실험용약재선정본연구에사용된백출 (Atractylodes macrocepala KOIDZ.) 은한약재로사용이가능한기본적수치를거친것으로 ( 주 ) 퓨어마인드 (Yongchoen, Korea) 에서구입하였으며, 대한약전및생약규격집을근거로중앙약사심의위원회한약분과감별위원의 감별을받아사용하였다. 실험용추출물제조본연구에서는백출의일반적인경구투여에서가장빈번하게나타나는열수추출물에대한실험을진행하였으며, 이를위하여백출 500 g을압력추출기에넣고 HPLC 용 D.W. 4L 를넣은후, 약110 로 4시간동안가열 추출하고나온추출액을병에담았다. 열이적당히식으면여과지로여과하여추출액을감압농축기로농축을실시한뒤, 농축된시료를동결건조기로약 3일동안동결건조수분을제거하고냉동보관후실험에사용하였다. 실험동물동물은생후 7주된 150~180 g의 SD (Sprague-Dawley) Rat 를실험동물로사용하였다. 사료 ( 실험동물용고형사료 ) 는 ( 주 ) 퓨리나사료 (CAT NO. 38057) 을사용하였으며, 물은필터와유수살균기를이용하여여과살균된정제수를고온 고압에서멸균한뒤, 유리로된음수병을통하여자유롭게먹도록유지하였다. 사육장의온도는 21~24, 상대습도는 40~80% 로유지하고, 12시간마다낮과밤이반복되도록사육장내빛을조절하여사육하였으며, 안정기 7일동안체중변화를관찰하였다 (Table 1-1, 1-2). Table 1-1. Body weight of male rats in stable period day 0 1 2 3 4 5 6 7 Select group Except group 117 124 133 143 158 166 176 185 116 124 132 142 156 164 173 184 117 125 135 145 158 166 176 185 118 126 137 146 158 168 178 186 118 125 137 148 158 168 177 187 130 140 150 162 174 186 196 207 118 126 137 147 157 169 179 187 128 138 147 159 170 181 193 205 130 139 149 159 172 184 196 208 137 146 156 164 175 186 198 209 119 127 138 148 157 169 179 188 127 136 147 162 171 183 192 202 138 146 156 164 176 187 199 210 113 122 134 145 153 162 171 180 114 122 134 147 154 162 172 180 139 146 156 166 176 187 198 211-12-
Table 1-2. Body weight of female rats in stable period day 0 1 2 3 4 5 6 7 Select group Except group 119 125 131 138 146 153 161 169 105 108 114 118 122 128 136 145 108 111 116 123 130 137 144 151 114 118 124 130 138 146 153 159 111 114 118 125 132 138 145 153 114 117 121 129 137 145 152 159 112 115 120 128 136 143 150 157 113 116 120 127 136 144 151 157 105 108 113 117 122 128 135 142 106 109 113 118 124 130 137 145 114 119 123 131 140 147 153 160 105 107 113 118 122 129 136 143 103 105 108 113 118 123 128 135 120 127 135 142 149 156 162 170 119 126 132 139 146 154 162 170 104 106 109 114 119 124 127 135 시험군구성및투여량설정시험군의구성시험군은대조군, 저용량, 중용량, 고용량의 4개군으로나누었으며, 각군별암 수 3개체로구성하였다 (Table 2). 투여량설정본시험은 비임상시험관리기준 ( 식약청고시제2012-61) 을바탕으로실험을수행하였다. 이에따르면강제경구투여시기술적으로투여하는용량은 1,000~2,000 mg / kg이나, 예외적인경우에는 5,000 mg / kg까지투약이가능하다. 생약재의경우양약과다르게다량으로오랫동안섭취하기때문에최고용량을 2,000 mg / kg의배수인 4,000 mg / kg으로설정하였고, 용량결정시험에서는 4,000 mg / kg을포함하여, 저용량 1,000 mg / kg과중용량 2,000 mg / kg으로설정하여, 총 3단계의용량을처리하기로하였다. 군분리및잔여동물처리순화기간중건강한것으로판정된동물들의체중을측정하고, 평균체중에가까운동물들을암수각각 48 마리씩선택하였다. 선택한동물들은순위화한체중에따라 시험군의구성 표에명시된동물수가되도록하였다. 군분리후잔여동물은안락사시켰다. Table 2. Configuration of the test group Group Sex Number of animals Volume (ml/ kg ) Dose ( mg / kg ) Control M / F 3 / 3 10 0 Low dose M / F 3 / 3 10 1,000 Middle dose M / F 3 / 3 10 2,000 High dose M / F 3 / 3 10 4,000 시험방법 1) 본시험물질은임상적용시경구를통하여섭취하고있으므로, 시험물질을존데를이용하여경구로투여하였다. 2) 시험물질은투여전체중을측정하여투여량 (10 ml/ kg ) 을환산하여, 1일 1회, 단회투여후일주일동안관찰하였다. 3) 시험물질을투여한모든동물에대하여 1일 2회 ( 오전 10:00 이전, 오후 14:00 이후 ) 빈사동물및사망여부를확인하였고, 외관적이상및임상증상, 이상징후의발생여부와그정도를관찰하였다. 4) 부검계획도살동물에대하여혈액학적검사를실시하였다. 동물을하룻밤절식시킨후동물전용마취제 (Zoletil 50, ( 주 ) 버박코 -13-
韓資植誌 Korean J. Plant Res. 27(1) : 011~021(2014) 리아 ) 로마취후개복하여, 복대동맥을방혈및치사시켜장기조직을육안으로검사하였다. 검사항목혈청생화학적검사혈액학적검사를실시한동물에대하여혈청생화학적검사를실시하였다. 복대동맥으로부터채혈된혈액을 3,000 rpm, 15분간원심분리하여얻은혈청을이용하여다음과같은항목에대해측정하였다 (Table 3). 혈액학적검사계획도살동물에대하여혈액학적검사를실시하였다. 동물을하룻밤절식시킨후동물전용마취제 (Zoletil 50, ( 주 ) 버박코리아 ) 로마취후개복하여, 복대동맥으로부터채혈한혈액을이 용하여다음의항목을측정하였다 (Table 4). 장기중량측정부검후다음의장기를적출하여전자저울을이용하여측정하였으며, 실측치 ( 절대중량 ) 를측정하고, 체중에대한비 ( 상대중량 ) 도계산하였다. 또한, 양측정장기는모두측정하였다 (Table 5). 통계처리및결과판정본시험에서얻은측정치들은 ANOVA 분석을실시한후, 유의성이인정될경우 Dunnett s t-test 를실행하여대조군과각용량군간의통계학적유의성을검정하였다 (p < 0.05). 결과및고찰 Table 3. Serum biochemical value test items Items Aspartate aminotransferase, AST Alanine aminotransferase, ALT Lactate dyhydrogenase, LDH Alkaline phosphatase, ALP Blood urea nitrogen, BUN Creatinine, CREA Unit IU/l IU/l IU/l IU/l mg /dl mg /dl 사망률및임상증상관찰백출물추출물을이용한단회투여독성시험에서, 단회투여후일주일동안관찰한결과시험물질로인한폐사및특이적인임상증상이관찰되지않았다. 음수율및사료섭취량시험전기간동안대조군과비교하여시험물질투여로인한유의성있는음수율및사료섭취량변화는관찰되지않았다. Table 4. Hematological value test items Items Unit White blood cell count, WBC 10 3 / mm 3 Red blood cell count, RBC 10 6 / mm 3 Hemoglobin concentration, HGB g/dl Hematocrit, HCT % Mean corpuscular volume, MCV fl Mean corpuscular hemoglobin, MCH pg Mean corpuscular hemoglobin concentration, MCHC g/dl Cellular hemoglobin concentration mean, CHCM g/dl Cellular hemoglobin, CH pg Red cell distribution width, RDW % Hemoglobin distribution width, HDW g/dl Platelet, PLT 10 3 / mm 3 Mean plasma volume, MPV fl Table 5. Organ weights test items Items Brain Pituitary gland Thyroid Seminal vesicle Lung Prostate Male Thymus Adrenal gland (L), Right Heart Kidney (L), Right Spleen Testis (L), Right Liver Epididymis (L), Right Brain Pituitary gland Thyroid Liver Female Lung Uterus Thymus Adrenal gland (L), Right Heart Kidney (L), Right Spleen Ovary (L), Right -14-
Table 6-1. Body weight and absolute organ weights of male rats treated with Atracylodes macrocephala Koid (g) Control 1,000 mg / kg 2,000 mg / kg 4,000 mg / kg Body Weight 230.47 ± 17.78 z 230.54 ± 8.87 220.80 ± 14.23 235.61 ± 7.36 Pituitary gland 0.009 ± 0.002 0.008 ± 0.001 0.009 ± 0.001 0.008 ± 0.001 Brain 1.942 ± 0.163 1.923 ± 0.060 1.838 ± 0.137 1.893 ± 0.047 Thyroid 0.014 ± 0.005 0.016 ± 0.006 0.021 ± 0.004 0.019 ± 0.002 Adrenal gland(l) 0.021 ± 0.001 0.019 ± 0.002 0.022 ± 0.002 0.023 ± 0.001 Adrenal gland(r) 0.030 ± 0.020 0.021 ± 0.002 0.023 ± 0.001 0.021 ± 0.001 Kidney(L) 1.079 ± 0.147 1.085 ± 0.033 1.084 ± 0.143 1.063 ± 0.116 Kidney(R) 1.080 ± 0.195 1.096 ± 0.036 1.062 ± 0.099 1.063 ± 0.121 Lung 1.025 ± 0.022 1.061 ± 0.072 1.051 ± 0.045 1.013 ± 0.079 Thymus 0.728 ± 0.021 0.605 ± 0.081 0.663 ± 0.188 0.570 ± 0.095 Heart 0.934 ± 0.067 0.984 ± 0.070 0.884 ± 0.126 0.980 ± 0.108 Spleen 0.658 ± 0.006 0.594 ± 0.124 0.604 ± 0.105 0.598 ± 0.078 Liver 7.988 ± 0.589 7.530 ± 0.614 7.615 ± 1.166 8.345 ± 0.198 Testis(L) 1.148 ± 0.094 1.166 ± 0.047 1.188 ± 0.015 1.157 ± 0.030 Testis(R) 1.133 ± 0.091 1.178 ± 0.061 1.157 ± 0.050 1.177 ± 0.018 Epididymis(L) 0.173 ± 0.006 0.186 ± 0.004 0.164 ± 0.032 0.185 ± 0.019 Epididymis(R) 0.170 ± 0.028 0.186 ± 0.018 0.172 ± 0.017 0.188 ± 0.012 Seminal vesicle 0.329 ± 0.046 0.319 ± 0.077 0.296 ± 0.081 0.332 ± 0.164 Ventral Prostate 0.203 ± 0.003 0.186 ± 0.011 0.166 ± 0.026 0.180 ± 0.020 Table 6-2. Body weight and relative organ weights of male rats treated with Atracylodes macrocephala Koid (g%) Control 1,000 mg / kg 2,000 mg / kg 4,000 mg / kg Pituitary gland 0.004 ± 0.001 z 0.004 ± 0.001 0.004 ± 0.001 0.003 ± 0.001 Brain 0.783 ± 0.136 0.836 ± 0.054 0.832 ± 0.023 0.804 ± 0.038 Thyroid 0.006 ± 0.003 0.007 ± 0.002 0.010 ± 0.002 0.008 ± 0.001 Adrenal gland(l) 0.008 ± 0.002 0.008 ± 0.000 0.010 ± 0.001 0.010 ± 0.001 Adrenal gland(r) 0.013 ± 0.010 0.009 ± 0.001 0.010 ± 0.000 0.009 ± 0.001 Kidney(L) 0.430 ± 0.031 0.471 ± 0.019 0.490 ± 0.033 0.450 ± 0.036 Kidney(R) 0.428 ± 0.041 0.475 ± 0.004 0.481 ± 0.014 0.451 ± 0.039 Lung 0.413 ± 0.064 0.461 ± 0.036 0.476 ± 0.012 0.430 ± 0.022 Thymus 0.294 ± 0.049 0.263 ± 0.041 0.298 ± 0.065 0.242 ± 0.040 Heart 0.374 ± 0.031 0.428 ± 0.045 0.399 ± 0.038 0.416 ± 0.039 Spleen 0.265 ± 0.041 0.257 ± 0.049 0.272 ± 0.030 0.253 ± 0.027 Liver 3.197 ± 0.254 3.263 ± 0.149 3.438 ± 0.362 3.543 ± 0.093 Testis(L) 0.465 ± 0.092 0.507 ± 0.040 0.539 ± 0.027 0.491 ± 0.026 Testis(R) 0.459 ± 0.095 0.512 ± 0.046 0.524 ± 0.011 0.500 ± 0.024 Epididymis(L) 0.070 ± 0.012 0.081 ± 0.005 0.074 ± 0.013 0.079 ± 0.006 Epididymis(R) 0.069 ± 0.018 0.081 ± 0.008 0.078 ± 0.005 0.080 ± 0.003 Seminal vesicle 0.132 ± 0.025 0.139 ± 0.036 0.133 ± 0.030 0.140 ± 0.066 Ventral Prostate 0.081 ± 0.003 0.081 ± 0.006 0.075 ± 0.007 0.076 ± 0.006 z Values are presented as the means ± S.D. Relative weight; Ratio to body weight 100 (%). -15-
韓資植誌 Korean J. Plant Res. 27(1) : 011~021(2014) 체중및장기중량측정순화기간후 Rat 를군분리하여 0, 1,000, 2,000, 4,000 mg / kg의백출물추출물을각각단회경구투여한후, 일주일뒤부검을실시하였다. 수컷 Rat 의부검시체중은대조군은 230.47 ± 17.78 g, 1,000 mg / kg군은 230.54 ± 8.87 g, 2,000 mg / kg군은 220.80 ± 14.23 g, 4,000 mg / kg군은 235.61 ± 7.36 g이며, 시험물질투여로 2,000 mg / kg군에서체중이소폭감소하는경향을보였다. 부검시장기중량및상대중량을측정한결과, 대조군과시험물질투여군간에통계학적인유의성을관찰할수없었다 (Table 6-1, 6-2). 암컷 Rat 의부검시체중은대조군은 168.90 ± 20.51 g, 1,000 mg / kg군은 165.56 ± 7.95 g, 2,000 mg / kg군은 159.00 ± 4.24 g, 4,000 mg / kg군은 168.00 ± 6.30 g 이며, 시험물질투여로 1,000 mg / kg와 2,000 mg / kg에서유의성있는체중감소가관찰되었다. 부검시장기중량및상대중량을측정한결과, 대조군과시험물질투여군간에통계학적인유의성을관찰할수없었다 (Table 7-1, 7-2). 혈액학적검사수컷 Rat 에 0, 1,000, 2,000, 4,000 mg / kg의백출물추출물을 각각단회경구투여한후, 일주일뒤부검을실시하였다. 부검후복대동맥으로부터채혈한혈액을이용하여백혈구수 (WBC), 적혈구수 (RBC), 혈색소량 (HGB) 등을측정하였다. 그결과대조군과비교하여 1,000-2,000 mg / kg에서백혈구수가유의적으로증가하였으며, 적혈구수는모든실험군에서유의성있게증가하였다 (Table 8). 암컷 Rat 에 0, 1,000, 2,000, 4,000 mg / kg의백출물추출물을각각단회경구투여한후, 일주일뒤부검을실시하였다. 부검후복대동맥으로부터채혈한혈액을이용하여백혈구수 (WBC), 적혈구수 (RBC), 혈색소량 (HGB) 등을측정하였다. 그결과대조군과비교하여 1,000-2,000 mg / kg에서백혈구수가유의적으로증가하였으며, 적혈구수는모든실험군에서유의성있게증가하였다 (Table 9). 혈액생화학적검사수컷 Rat 에 0, 1,000, 2,000, 4,000 mg / kg의백출물추출물을각각단회경구투여한후, 일주일뒤부검을실시하였다. 부검후복대동맥으로부터채혈한혈액을원심분리하여얻은혈청을이용하여아스파테이트아미노기전이효소 (AST), 알라닌아미노기전이효소 (ALT), 젖산탈수효소 (LDH) 등을측정하였다. 그 Table 7-1. Body weight and absolute organ weights of female rats treated with Atracylodes macrocephala Koid (g) Control 1,000 mg / kg 2,000 mg / kg 4,000 mg / kg Body Weight 168.90 ± 20.51 z 165.56 ± 7.95 159.00 ± 4.24 168.00 ± 6.30 Pituitary gland 0.0356 ± 0.0472 0.0091 ± 0.0037 0.0099 ± 0.0025 0.0081 ± 0.0021 Brain 1.7645 ± 0.0982 1.8513 ± 0.2256 1.7925 ± 0.0284 1.7151 ± 0.0522 Thyroid 0.0232 ± 0.0018 0.0147 ± 0.0051 0.0413 ± 0.0441 0.0136 ± 0.0040 Adrenal gland(l) 0.0250 ± 0.0035 0.0259 ± 0.0016 0.0226 ± 0.0015 0.0207 ± 0.0032 Adrenal gland(r) 0.0276 ± 0.0005 0.0249 ± 0.0012 0.0240 ± 0.0028 0.0266 ± 0.0058 Kidney(L) 0.7396 ± 0.0362 0.7602 ± 0.0587 0.7027 ± 0.0168 0.7890 ± 0.0676 Kidney(R) 0.7422 ± 0.0705 0.7066 ± 0.0487 0.7121 ± 0.0305 0.7322 ± 0.0560 Lung 0.8355 ± 0.0660 0.8604 ± 0.0982 0.8799 ± 0.0582 0.8863 ± 0.0794 Thymus 0.5019 ± 0.1762 0.5627 ± 0.0790 0.5420 ± 0.0861 0.5356 ± 0.1483 Heart 0.6849 ± 0.1105 0.7763 ± 0.0270 0.6746 ± 0.0484 0.6984 ± 0.0527 Spleen 0.3724 ± 0.0409 0.4183 ± 0.0093 0.3609 ± 0.0217 0.4179 ± 0.0200 Liver 5.7180 ± 0.6455 5.8617 ± 0.3045 5.2984 ± 0.0880 5.4202 ± 0.1323 Uterus 0.3390 ± 0.1357 0.4478 ± 0.0447 0.2708 ± 0.0487 0.2495 ± 0.0321 Ovary(L) 0.0423 ± 0.0088 0.0297 ± 0.0085 0.0282 ± 0.0062 0.0326 ± 0.0054 Ovary(R) 0.0395 ± 0.0059 0.0374 ± 0.0051 0.0256 ± 0.0044 0.0305 ± 0.0070-16-
Table 7-2. Body weight and relative organ weights of female rats treated with Atracylodes macrocephala Koid (g%) Control 1,000 mg / kg 2,000 mg / kg 4,000 mg / kg Pituitary gland 0.022 ± 0.031 z 0.005 ± 0.002 0.006 ± 0.001 0.005 ± 0.001 Brain 1.009 ± 0.063 1.117 ± 0.112 1.137 ± 0.034 1.021 ± 0.023 Thyroid 0.009 ± 0.008 0.006 ± 0.006 0.027 ± 0.029 0.008 ± 0.002 Adrenal gland(l) 0.014 ± 0.003 0.016 ± 0.002 0.014 ± 0.001 0.012 ± 0.002 Adrenal gland(r) 0.016 ± 0.002 0.015 ± 0.001 0.015 ± 0.002 0.016 ± 0.004 Kidney(L) 0.425 ± 0.058 0.460 ± 0.042 0.446 ± 0.013 0.469 ± 0.024 Kidney(R) 0.426 ± 0.064 0.427 ± 0.022 0.452 ± 0.019 0.435 ± 0.019 Lung 0.478 ± 0.038 0.519 ± 0.034 0.558 ± 0.041 0.527 ± 0.029 Thymus 0.285 ± 0.090 0.339 ± 0.032 0.345 ± 0.061 0.321 ± 0.095 Heart 0.391 ± 0.057 0.470 ± 0.025 0.428 ± 0.038 0.416 ± 0.026 Spleen 0.214 ± 0.037 0.253 ± 0.015 0.229 ± 0.016 0.249 ± 0.012 Liver 3.265 ± 0.312 3.541 ± 0.067 3.362 ± 0.127 3.227 ± 0.045 Uterus 0.192 ± 0.069 0.270 ± 0.014 0.171 ± 0.026 0.149 ± 0.020 Ovary(L) 0.024 ± 0.003 0.018 ± 0.005 0.018 ± 0.004 0.019 ± 0.003 Ovary(R) 0.022 ± 0.002 0.023 ± 0.003 0.016 ± 0.002 0.018 ± 0.004 z Values are presented as the means ± S.D. Relative weight; Ratio to body weight 100 (%). Table 8. Hematological values of male rats treated with Atracylodes macrocephala Koid Control 1,000 mg / kg 2,000 mg / kg 4,000 mg / kg WBC( 10 3 mm 3 ) 7.67 ± 1.13 z 8.89 ± 2.82 8.75 ± 0.02 7.15 ± 1.24 RBC( 10 6 / mm 3 ) 6.66 ± 0.33 7.24 ± 2.50 6.94 ± 0.15 6.95 ± 0.23 HGB(g/dl) 13.60 ± 0.26 14.13 ± 5.27 14.10 ± 0.46 13.50 ± 0.62 HCT(%) 43.53 ± 1.27 46.10 ± 16.64 46.13 ± 0.90 45.33 ± 2.85 MCV(fl) 65.43 ± 1.48 63.67 ± 24.65 66.50 ± 0.89 65.17 ± 2.84 MCH(pg) 20.47 ± 0.59 19.53 ± 7.45 20.33 ± 0.29 19.47 ± 0.80 MCHC(g/dl) 31.27 ± 0.46 30.73 ± 11.43 30.57 ± 0.38 29.83 ± 0.64 CHCM(g/dl) 34.47 ± 0.21 34.37 ± 12.85 34.30 ± 0.26 33.90 ± 0.61 CH(pg) 22.40 ± 0.40 21.77 ± 8.30 22.67 ± 0.45 21.97 ± 0.85 RDW(%) 13.77 ± 0.70 13.67 ± 5.05 13.53 ± 0.84 13.80 ± 0.46 HDW(g/dl) 2.02 ± 0.12 1.98 ± 0.73 1.99 ± 0.01 2.06 ± 0.04 PLT( 10 3 / mm 3 ) 1423.33 ± 88.52 1345.67 ± 457.20 1280.33 ± 29.16 1495.33 ± 131.20 MPV(fl) 7.30 ± 0.44 9.33 ± 3.01 9.93 ± 0.15 10.13 ± 0.23 WBC; White blood cell count, RBC; Red blood cell count, HGB; hmoglobin concentration, HCT; Hematocrit, MCV; Mean corpuscular hemoglobin, MCH; Mean corpuscular hemoglobin concentration, CHCM; Cellular hemoglobin concentration mean, CH; Cellular hemoglobin, RDW; Red cell distribution width, HDW; Hemoglobin distribution width, PLT; Platelet, MPV; Mean plasma volume. -17-
韓資植誌 Korean J. Plant Res. 27(1) : 011~021(2014) Table 9. Hematological values of female rats treated with Atracylodes macrocephala Koid Control 1,000 mg / kg 2,000 mg / kg 4,000 mg / kg WBC( 10 3 / mm 3 ) 4.21 ± 1.93 z 4.89 ± 1.15 6.03 ± 0.26 6.38 ± 0.48 RBC( 10 6 / mm 3 ) 6.12 ± 1.55 7.00 ± 0.54 7.04 ± 0.35 7.01 ± 0.20 HGB(g/dl) 14.47 ± 0.47 14.17 ± 0.35 14.20 ± 1.04 14.33 ± 0.40 HCT(%) 38.83 ± 9.56 44.57 ± 1.08 44.77 ± 2.48 43.53 ± 0.83 MCV(fl) 63.63 ± 0.76 63.90 ± 3.42 63.57 ± 0.32 62.13 ± 2.73 MCH(pg) 24.80 ± 6.76 20.27 ± 1.01 20.17 ± 0.76 20.43 ± 0.83 MCHC(g/dl) 38.93 ± 10.21 31.73 ± 0.61 31.73 ± 1.12 32.90 ± 0.75 CHCM(g/dl) 35.20 ± 0.44 34.93 ± 0.75 34.83 ± 0.12 34.13 ± 0.32 CH(pg) 22.33 ± 0.32 22.20 ± 0.72 22.07 ± 0.23 21.13 ± 0.67 RDW(%) 12.93 ± 0.58 12.77 ± 0.40 13.37 ± 0.32 12.73 ± 0.67 HDW(g/dl) 1.92 ± 0.10 1.89 ± 0.10 1.85 ± 0.15 1.81 ± 0.05 PLT( 10 3 / mm 3 ) 1054.33 ± 173.64 1379.33 ± 92.46 1102.67 ± 562.86 1099.33 ± 247.11 MPV(fl) 7.63 ± 0.15 8.77 ± 0.51 10.03 ± 1.38 9.17 ± 0.55 WBC; White blood cell count, RBC; Red blood cell count, HGB; hmoglobin concentration, HCT; Hematocrit, MCV; Mean corpuscular hemoglobin, MCH; Mean corpuscular hemoglobin concentration, CHCM; Cellular hemoglobin concentration mean, CH; Cellular hemoglobin, RDW; Red cell distribution width, HDW; Hemoglobin distribution width, PLT; Platelet, MPV; Mean plasma volume. Table 10. Serum biochemical values of male rats treated with Atracylodes macrocephala Koid Control 1,000 mg / kg 2,000 mg / kg 4,000 mg / kg AST(IU/l) 128.00 ± 8.49 z 201.50 ± 17.68 148.50 ± 2.12 133.00 ± 15.56 ALT(IU/l) 27.50 ± 3.54 28.00 ± 5.66 26.50 ± 13.44 28.00 ± 7.55 LDH(IU/l) 1946.50 ± 120.92 1955.50 ± 132.23 1958.50 ± 55.86 1913.67 ± 174.27 ALP(IU/l) 349.00 ± 28.28 283.50 ± 27.58 316.50 ± 96.87 291.00 ± 26.06 BUN( mg /dl) 15.60 ± 2.26 14.20 ± 1.84 13.25 ± 2.05 14.43 ± 1.96 CREA( mg /dl) 0.68 ± 0.06 0.96 ± 0.29 0.81 ± 0.23 0.83 ± 0.17 AST; Aspartate aminotransferase, ALT; Alanine aminotransferase, LDH; Lactate dehydrogenase, ALP; Alkaline phosphatase, BUN; Blood urea nitrogen, CREA; Creatinine. Table 11. Serum biochemical values of female rats treated with Atracylodes macrocephala Koid Control 1,000 mg / kg 2,000 mg / kg 4,000 mg / kg AST(IU/l) 117.33 ± 25.72 z 264.00 ± 50.69 189.00 ± 53.25 167.67 ± 18.93 ALT(IU/l) 31.67 ± 2.52 18.33 ± 6.03 26.67 ± 12.50 21.67 ± 2.62 LDH(IU/l) 1589.33 ± 495.02 1652.67 ± 276.58 1911.00 ± 105.67 2012.67 ± 14.82 ALP(IU/l) 240.33 ± 27.30 184.00 ± 48.14 210.00 ± 10.15 231.33 ± 40.37 BUN( mg /dl) 18.47 ± 1.56 14.37 ± 1.42 18.73 ± 1.51 19.30 ± 0.86 CREA( mg /dl) 0.80 ± 0.01 0.86 ± 0.39 0.86 ± 0.15 0.96 ± 0.20 AST; Aspartate aminotransferase, ALT; Alanine aminotransferase, LDH; Lactate dehydrogenase, ALP; Alkaline phosphatase, BUN; Blood urea nitrogen, CREA; Creatinine. -18-
Fig. 1. Histopathological change of female and male rats treated with Atracylodes macrocephala Koid. (A, B: control; C, D: female 4,000 g/ kg ; E, F: male 4 g/ kg ) A,C, E(50X), B, D, F(200X) 결과시험물질투여군에서 AST 가증가하였으며, 저용량에서증가하였다가고용량으로갈수록소폭감소하는경향을보였다 (Table 10). 암컷 Rat 에 0, 1,000, 2,000, 4,000 mg / kg의백출물추출물을각각단회경구투여한후, 일주일뒤부검을실시하였다. 부검후복대동맥으로부터채혈한혈액을원심분리하여얻은혈청을이용하여아스파테이트아미노기전이효소 (AST), 알라닌아미노기전이효소 (ALT), 젖산탈수효소 (LDH) 등을측정하였다. 그결과시험물질투여군에서 AST 가증가하였으며, 고용량보다는저용량에서의증가가뚜렷하였다 (Table 11). 병리학적독성평가백출물추출물을단회투여한후, 부검을실시하였다. 부검후적출한모든장기를조직병리로판독한결과, 암수컷모두 3 마리중 1마리의간조직에서약한지방변성 (fatty change) 이관찰되었다 (Fig. 1). 현재관찰된지방변성정도는매우약한정도 이나, 일반적인 Rat 의간에서는잘나타나지않는증상이므로추가연구가필요한것으로사료된다. 한약및생약은약용자원으로써뿐만아니라주요한식품자원으로써그범위와빈도가점차증가하고있다. 특히국내에서의약품으로관리되는 514 종한약재중한약과식품겸용으로관리되는품목은 117종에달하며 (Kim, 1999; Kweon, 2012), 특별한규제없이일반에서구매와사용이가능한실정이다. 그러나지금까지경험적인사용법과용량에의존하여왔을뿐, 이들생약에대하여과학적인근거를제시하지못하고있다. 따라서천연물유래생약및한약의안전성에대한지속적이고체계적인연구가이루어져야할것으로사료된다 (Kang et al., 2013). 백출의경우국내에서오랜기간동안삽주 (Atractylodes japonica KOIDZ.) 를백출 (Atractylodes macrocepala KOIDZ.) 의대용으로사용해왔기때문에식물기원에대한혼란을야기할수있고, 그로인한오남용또한우려되는상황에있다 (Lee et al., 2002). 다만최근에는백출과삽주를구분하여한약을 -19-
韓資植誌 Korean J. Plant Res. 27(1) : 011~021(2014) 유통하는추세에있으며, 이로인한백출의안전성에대한과학적검증이반드시필요한상황이다. 본연구에서는단회투여독성평가에따른사망률, 임상증상, 음수율, 식이율, 체중및장기중량변화, 혈액학적검사및혈액생화학적검사, 해부병리학적조직검사등을암수별시험군으로나누어수행함으로써연구결과에대한근거를명확하게제시하고자하였다. 결과에서알수있듯이시험기간중사망한개체가없어 LD 50 의산출은불가하였으며, 기본적인임상증상이나이상소견은관찰되지않았다. 또한체중의변화나개별장기의중량변화는대조군과비교하여유의성이관찰되지않았으며, 혈액검사영역에서도유의성있는변화가관찰되지는않았으나, AST 가저용량에서증가하였다가용량이증가할수록감소되는경향을보였다. 이러한결과는백출을사용할때투여량에따라효능에차이를보일수있음을시사하며, 경구투여에대한용량기준의설정이필요할것으로사료된다. 또한 Rat 장기조직에대한병리학적검사에서일부미세한간조직의지방변성이관찰되었다. 이러한결과는특히한약및생약의섭취에있어서간과간세포에대한영향과관련이있을것으로사료되며, 추가적인연구가뒷받침되어야할것으로보인다. 사사본연구는식품의약품안전처 독성물질연구및지원시스템구축연구 (12181MFDS607) 의지원을받아수행되었습니다. 적요본연구는 Atractylodes macrocepala KOIDZ. 의급성경구독성을평가하기위하여 SD계 Rat 를이용하여농도별열수추출물을투여하고체중변화, 이상반응, 장기무게변화, 혈액학적이상반응, 조직병리학적이상반응등을측정하였다. Atractylodes macrocepala KOIDZ. 를투여한실험군에서비교적낮은농도에서체중의감소가관찰되었으나, 이상반응이나장기무게에있어유의성있는변화는관찰되지않았다. 혈액학적지표에있어서도비교적낮은농도에서 WBC 및 AST 의증가가관찰되었다. 조직병리학적소견에서일부간조직의지방변성이관찰되어간독성과간세포에미치는영향에대한연구가필요할것으로판단된다. 이와같은연구의결과를종합해볼때백출이경구투여에있어비교적안전한물질인것으로판단할수있다. 그 러나단회경구투여급성독성시험만으로천연물생약에대한독성유무를판단하기에는일정부분제약이있으므로, 추가적으로 2주 ( 또는 4주 ) 반복경구투여독성시험및 13주반복경구투여독성시험그리고유전독성에대한연구들이순차적으로수행되어야하며, 이를통하여백출에대한체계적인독성정보를구축함으로써보다정확하고과학적근거에입각한안전성자료가확보될수있을것으로기대한다. References Choi, Y.S., K.D. Choi, S.D. Kim, P. Owens and C.S. Chung. 2010. Extract of Korean medicinal plant extracts alter lipogenesis of pig adipose tissue and differentiation of pig preadipocytes in vitro. Journal of Animal Science and Tech. 52(5):383-388 (in Korean). Ha, H.Y. 2007. Bonchoyimyung. Passanpass, Seoul, Korea. pp. 461-462 (in Korean). Hu, X.M., W.K. Zhang, S.L. Yu and M.Z. Wang. 1998. Zhonghwabencao Jingxuanben. Shanghaikejichubanshe. Shanghai, China. pp. 1893-1902 (in Chinese). Hwang, S.Y., W. Kwon, H.Y. Chai, Y.M. Cho, N.J. Lee, J.M. Ryu, J.S. Sin, T.M. Kim, J.H. Cho, E.J. Kim, J.H. Park, J.K. Kang and Y.B. Kim. 2004. Four-week repeated-dose toxicity study on Mori radicis cortex. The Korean Journal of Laboratory Animal Sci. 20(3):283-290 (in Korean). Kang, B.S., Y.C. Ko, Y.B. Guk, G.Y. Kim, S.H. Kim, Y.L. Kim, H.C. Kim, S.H. Roh, Y.K. Park, B.I. Seo, Y.B. Seo, H.J. Song, M.G. Shin, D.G. Ahn, S.Y. Lee, Y.J. Lee, S.I. Cho, Y.S. Joo and H.Y. Choi. 2000. Bonchohak. Younglimsa, Seoul, Korea. pp. 536-537 (in Korean). Kang, S.Y., Y.C. Park and Y.K. Park. 2013. Thirteen-week repeated-dose oral toxicity study of KOB03, a polyherbal medicine for allergic rhinitis, in rats. Korean Journal of Herbol. 28(1):15-21 (in Korean). Kim, C.T., M.H. Jung, C.S. Moon, Y.H. Lim, S.J. Kang and W.G. Cho. 2005. Inhibitors of melanogenesis from Atractylodes rhizomes. Natural Product Sci. 36(1):60-63 (in Korean). Kim, K.S. 1999. Present status and perspectives of medicinal plant resources in Korea. Research of National Resour. 2:25-41 (in Korean). Kim, T.H., S. Jang, A.R. Lee, A.Y. Lee, G. Choi and H.K. Kim. 2012. The analysis of residual pesticides and sulfur dioxide in commercial medicinal plants. Korean Journal of Herbol. 27(6):43-48 (in Korean). -20-
Kweon, K.T. 2012. A reasearch on management system of herbal medicine in common use for food and medicine. Korean Journal of Herbol. 27(2):25-29 (in Korean). Lee. J.H., Y.K. Kim, S.P. Hong and C.S. Kim. 2002. Studies of taxonomic origins of Atractylodis rhizoma Alba and Atractylodis rhizoma. Korean Journal of Oriental Medic. 8(1):55-63 (in Korean). Oh. T.W., H.S. Bae, C.H. Yoon and Y.K. Park. 2010. Thirteen-week repeated-dose oral toxicity study of the modified Wenpitang-Hab-Wulingsan (WHWR) in spraguedawley rats. Korean Journal of Herbol. 25(3):43-51 (in Korean). Park, H.M., H.T. Shin and S.D. Lee. 2008. Herbal toxicological effects on rats fetus-focusing on ojeoksan-. Korean J. Oriental Preventive Medical Soc. 12(2):27-35 (in Korean). Park, J.S., Y.S. Kim, J.Y. Lee, L.G. Park, B.H. Jeon, W.H. Woo and W.Y. Jeong. 1999. Effect of rhizoma Atractylodis macrocephalae on the melanogenesis. Korean J. Oriental Medical Pathol. 13(2):91-98 (in Korean). Zhao, A.S., Y.L. Sun and L.S. Zhang. 2006. Safety assessment of baizhu. Chin. J. Public Heal. 22(1):43-45 (in Chinese). (Received 21 August 2013 ; Revised 14 October 2013 ; Accepted 29 October 2013) -21-