레이아웃 1

OriginalArticle 대한침구의학회지제 32 권제 4 호 (2015 년 12 월 ) : 77-89 The Acupuncture Vol. 32 No. 4 December 2015 : 77-89 pissn 1229-1137 eissn 2287-7797 http://dx.doi.org/10.13045/acupunct.2015063 SD(Sprague-Dawley) 랫드를이용한신바로약침의 4 주반복근육투여독성시험 이진호, 하인혁, 김미령, 김민정, 이재웅, 이인희, 정화진, 김은지 * 자생의료재단, 척추관절연구소 [Abstract] Multiple-doseIntramuscularToxicityStudyofShinbaroPharmacopuncture insprague-dawleyratsovera4-weekperiod JinHoLee,InHyukHa,MeRiongKim,MinJeongKim,JaeWoongLee,InHeeLee, HwaJinChungandEunJeeKim * JasengSpineandJointResearchInstitute,JasengMedicalFoundation Key words : Shinbaro;Pharmacopuncture;Multipledosetoxicitytest; Spraque-Dawleyrat Objectives : Toevaluatethesafetyofmultiple-doseintramuscularShinbaroPharmacopuncture inmaleandfemalesprague-dawley(sd)ratsoveraperiodof4weeks(12sessions). Methods : Inordertotestthesafetyofmultiple-doseintramuscularShinbaroPharmacopuncture weused40healthymaleandfemale6-weekoldsdrats(maleweight171.79~196.37g,female weight127.93~146.43g).shinbaropharmacopuncturewasadministeredintramuscularlyto maleandfemalesdratsatdosesof4.6(lowdosegroup,n=10),9.2(moderatedosegroup, n=10),and18.5mg/kg(highdosegroup,n=10),respectively.generalsymptoms,bodyweight changes,bloodtests,biochemicaltesting,necropsy,organweightandhistopathogicalfindings wereexaminedovera4-weekperiod. Results : 1.Nomortalitiesoradverseeffectswerecausedbytheinvestigationalsubstancewereobservedduringthestudyperiod. 2.Therewasnosignificantdifferenceinbodyweightcausedbythetheinvestigationalsubstanceacrossallgroups. 3.Nosignificantbetween-groupdifferencewasfoundtobecausedbytheinvestigational substanceinbloodtestsandbiochemicaltesting. 4.Noabnormalitiesweredetectedbyanecropsyexaminationwiththeunaidedeyeatthe macrolevelaftertreatmentwiththeinvestigationalsubstance. 5.Differenceinorganweightbetweengroupscausedbytheinvestigationalsubstancewas notfound. 6.Allgroupsdidnotexhibitpathologicalfindingscausedbytheinvestigationalsubstancein histopathogicalexamination. Conclusions : Accordingtotheseresults,ShinbaroPharmacopuncturehasnosystemicororgan toxicitywithmultiple-doseintramuscularadministrationsinmaleandfemalesdratsovera4- weekperiod(12sessions).theseresultsimplythatnoadverseeffectsareobservedatalevel (NOAEL)ofShinbaroPharmacopunctureof18.5mg/kg. Received : 2015. 11. 13. Revised : 2015. 12. 09. Accepted : 2015. 12. 11. On-line : 2015. 12. 18. Theauthorsaregratefultokoreapromotioninstitutefortraditionalmedicineindustry(KOT- MIN)fortheirsupport(No.20150602383). Correspondingauthor:JasengSpineandJointResearchInstitute,JasengMedicalFoundation, 5,Eonju-ro170-gil,Gangnam-gu,Seoul,RepublicofKorea Tel:+82-10-5342-7720 E-mail:letmelove57@hanmail.net ThisisanOpen-AccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/bync/3.0)whichpermitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. TheAcupuncture isthejournalofkoreanacupuncture&moxibustionmedicinesociety.(http://www.theacupuncture.org) Copyright 2014KAMMS.KoreanAcupuncture&MoxibustionMedicineSociety.Allrightsreserved. http://dx.doi.org/10.13045/acupunct.2015063 77

TheAcupunctureVol.32No.4December2015 Ⅰ. 서론 Ⅱ. 재료및방법 약침요법은기존의침법과한약을결합한것으로극소량의한약재를침놓는자리 ( 경혈 ) 에주입함으로써한약의작용과침의작용을극대화시키는신침요법이다. 현재까지약침연구는봉독약침에대한연구가다수보고되어있지만, 최근에는여러종류의약침에대한연구수가점차증가하고있다 1). 김 (2014) 1) 에의하면경추통증환자의치료에이용되고있는일반적인약침은봉독약침, 홍화약침, 지네약침, 어혈약침, 황련약침, 코퍼스약침, 소염약침, 황련해독탕약침, 신바로약침등이사용되고있다고보고되어있다. 이중신바로약침은연골보호 2), 항염증 3), 신경재생효과 4) 등의효능을지닌약침으로자생한방병원의골관절질환치료한약인 청파전 (GCSB-5) 을바탕으로개발된약침이다. 신바로약침을요추추간판탈출증환자의치료에사용한결과봉독약침과유의한수준의효과를내며부작용은없는것으로확인되었다 5). 또한, 요추추간판탈출증환자에게신바로약침처치군과비처치군으로무작위대조군실험을시행한결과, 요통의 S 가 2배이상감소되어신바로약침이허리통증을개선시키는데유효한효능이있는것으로보고되었다 6). 이처럼, 신바로약침의효능에대한연구에비하여표준화및안전성에대한연구가부족한시점에서, 최근신바로약침중대표적인구성약재인구척, 식방풍, 두충, 오가피, 우슬의표준화작업에대한연구를실시하여과학적인품질관리에대한방안을제시하였다 7). 또한 rats 와 Beagle dogs 을사용해단회독성에대한연구를진행하여급성독성이없음을확인하였다 8). 과학적인검증을통해신약의안전성을확보하기위한독성연구는독성시험의기간에따라단회투여독성시험 ( 급성독성실험 ) 및반복투여독성시험으로분류할수있다. 이에 CA- 약침 9), Sweetbee venom 10), 신바로3( 천수근 ) 약침 11) 등은 rats 나 Beagle dogs 을이용해단회독성시험과반복독성시험을통하여약침에대한안전성을확보하고있다. 따라서본연구는신바로약침의무독성량및표적장기를 Sprague-Dawley(SD) 계통암 수 rats 에 4주 (12 회 ) 반복근육투여를하여독성시험을실시하고유의한결과를얻어보고하는바이다. 1. 신바로 약침의 조제방법 신바로약침의약재인구척, 식방풍, 두충, 오가피, 우슬, 오공, 강활, 독활, 작약으로 9가지약재는그린명품제약 ( 경기도남양주 ) 에서구입하였으며총 150 g을넣고 70 % 주정을이용하여 3시간동안환류추출하였다. 이때추출에사용된물은 water purification system(model : ABB- OTA NEO) 을이용하여제조된 3차증류수를사용하였고, 추출액여과후여액을감압농축하였다. 농축된추출물을 80~90 % 주정을이용하여알코올수침을통해정제하였으며정제된추출물은여과후동결건조하여약침의원료로사용하였다. 정제된약침원료를정제수에녹이고 121 에서 15분멸균하여사용하였다. 2. 실험동물 1) 실험동물 및 사육환경 특정병원균이부재 (Specific Pathogene Free, SPF) 된 SD 계통의 rats 는오리엔트바이오에서구입하였으며, 5주령된암수각 55마리를 5일이상순화기간을두었다. 실험에사용한 rats 는순화기간중일반증상을관찰하여건강한 6주령 ( 수컷 171.79~196.37 g, 암컷 127.93~146.43 g) 의암수각 40마리를선택하여사용하였다. 동물실및사육환경은온도 22.40.9, 상대습도 45.24.4 % RH, 환기횟수 10~15 회 /hr을유지하였으며, 명암 cycle 은 12hr(08 : 00 점등 ~20 : 00 소등 ) 과조도 280 Lux 로조절하였다. 실험동물의사육밀도는검역, 순화, 투여및관찰기간중스테인레스제망사육상자 (250 W 350 L 180 H mm) 에 2마리이하로수용하였다. 사료 (Teklad Certified Irradiated Global 18 % Protein Rodent Diet, Harlan Co. Ltd., USA) 는급이기에넣고, 물은음용상수도수를정수시킨후폴리카보네이트제물병에넣어자유섭취시켰다. 해당시험은본시험기관동물실험윤리위원회의승인 ( 승인번호 : IA13-00074) 을거쳐수행되었다. 2) 시험군의 구성군분리는투여전일에체중을측정하고이를토대로암 수각각대조군 10마리와신바로시험군 ( 저, 중, 고용량 ) 10 마리씩분리하였다. 78 http://dx.doi.org/10.13045/acupunct.2015063

SD(Sprague-Dawley) 랫드를이용한신바로약침의 4 주반복근육투여독성시험 본시험군은임상예정용량인 1.11 mg/60 kg ( 약 18.5 μg/kg) 의약 1,000 배인 18.5 mg/kg 을고용량투여군으로정하고, 공비를 2로두어각각 9.2 및 4.6 mg/kg 을중용량과저용량으로설정하였으며, 주사용수를투여하는부형제대조군을두었다. 이를 4주반복투여용량결정시험을실시하였다 (Table 1). Table1.Groupcompositionforassessmentoffour weeksrepeated-dosetoxictestinsdrats Group Sex Animal No. 1 Dose (mg/kg/day) 2 Male G1-1~G1-10 G1 Female G1-51~G1-60 G2 G3 G4 Male Female Male Female Male Female G2-16~G2-25 G2-66~G2-75 G3-26~G3-35 G3-76~G3-85 G4-36~G4-45 G4-86~G4-95 1. No: Numbers, 2. G1: Control group(0 mg/kg), G2~G4 : Experimental group (4.6, 9.2, 18.5 mg/kg) 3) 시술 용량의 설정 및 시술방법 시술용량및방법은사람에대한임상예정경로로서근육투여시의독성을알아보기위하여선택하였다. 투여경로는동물을대퇴부내측이위로오도록배부에서요부의피부를보정하고알코올솜을이용하여투여부위를소독하였다. 대퇴부좌측근육내에주사침을찌르고피스톤을살짝후퇴해서혈액의유입이없으면시험물질을천천히주입하였다. 투여횟수및시각은 1회 / 일, 3일 / 주, 4주간 12 회투여하였고, 투여당일오전중에투여하였다. 본시험물질을이용한 2주반복투여용량결정시험과동일하게가장최근에측정한체중을기준으로 1 ml/kg 으로투여량을계산하였다. 이는식품의약품안전청고시제 2012-86 호 (2012 년 08월 24일 ) 의약품등의독성시험기준 및 OECD Guideline for the Testing of Chemicals No. 407 Repeated Dose 28-day Oral Toxicity Study in Rodents (Adopted 27th July, 1995) 에의거하였다. 0 4.6 9.2 18.5 3. 관찰 및 검사 1) 일반 증상 관찰 투여전기간에걸쳐 1일 1회투여직후의일반증상을관찰하였다. 일반증상의관찰은사망여부, 증상의종류, 발현일및증상의정도를개체별로기록하였다. 2) 체중측정모든동물에대하여입수시, 군분리시, 투여개시시, 투여후주 1회및부검일에측정하였다. 3) 혈액학적 검사혈액학적검사는부검동물을하룻밤절식한후 CO 2 가스에마취하에개복하여복부대동맥으로부터혈액을채취하였으며, 채취한혈액을항응고제인 EDTA-2K 를사용하였다. 혈액학적검사는보관된혈액을혈액분석기 (ADVIA 2120, SIEMENS, Germany ; MAI-105-01) 를이용하여검사를실시하였다. 검사항목은 White blood cell count(wbc), Red cell distribution width(rdw), Red blood cell count(rbc), Platelet(PLT), Hemoglobin conc.(hb), Mean Platelet Volume(MPV), Hematocrit(HCT), 백혈구감별계산, Mean corpuscular volume (MCV), Large unstained cells(luc), Mean corpuscular hemoglobin(mch), Reticulocyte(Reti), Meancorpuscular-hemoglobin conc.(mchc) 으로총 13가지항목에대한혈액학적검사를실시하였다. 4) 혈액생화학적 검사혈액생화학적검사는부검시복대동맥에서채혈한혈액을 3,000 rpm, 10분간원심분리하여얻은혈청을이용하였다. 이때사용한기기는혈액생화학검사기 (Hitachi7180, HITACHI, Japan ; MAI-059-01) 를이용하여실시하였다. 검사항목은 Aspartate aminotransferase(ast), Albumin(ALB), Alanine aminotransferase (ALT), Total bilirubin(t-bil), Alkaline phosphatase(alp), A/G ratio, Gamma(γ)-glutamyl transferase(ggt), Uric acid(ua), Lactate dehydrogenase(ldh), Triglyceride(TG), Blood urea nitrogen(bun), Calcium(Ca), Creatinine(CRE), Inorganic phosphorus(ip), Glucose (GLU), Chloride(Cl), Total cholesterol(cho), Magnesium(Mg), Total protein(tp), Sodium(Na), Creatine phosphokinase(cpk), Potassium(K) 으로총 22 가지항목에대한혈액생화학적검사를실시하였다. http://dx.doi.org/10.13045/acupunct.2015063 79

TheAcupunctureVol.32No.4December2015 5) 부검부검은투여종료후에부형제대조군및모든시험군의생존동물에대하여최종계획부검을실시하여모든장기에대하여상세한육안검사를시행하였다. 6) 장기중량측정장기중량측정은투여종류후부검하여, 고환, 전림선, 난소, 자궁, 비장, 간장, 흉선, 부신, 신장, 심장, 폐, 뇌, 뇌하수체의장기를적출하여전자저울을이용하여측정하였다. 7) 조직병리학적 검사조직병리학적검사는부형제대조군및고용량군의모든고정장기에대해조직표본을제작하여검경하였다. 4. 통계처리 1) 연속자료의 분석 ( 체중, 혈액학적 검사, 혈액생화학적 검사, 장기중량 ) 일반적인정규성을가정하고통계처리를실시하였다. 일원배치분산분석을통하여군간유의성을확인하고등분산을검정하였다. 일원배치분산분석에서유의성이인정되고등분산이인정되면 Duncan test 를, 등분산이인정되지않으면 Dunnett's T test 를사용하였다. Ⅲ. 결과 1. 일반 증상 및 사망률 실험기간동안암 수모든투여군에서사망동물및특이한이상증상은관찰되지않았다. 통계처리는암 수각각부형제대조군과시험군 ( 저, 중, 고용량 ) 간의비교를하였으며, 일반적으로모수적인다중비교 (parametric multiple comparison procedures) 혹은비모수적인다중비교 (non parametric multiple comparison procedures) 를사용하였다. 발생률의표기는일반적으로백분율로나타내었다. 통계방법은상용되는통계패키지인 SPSS 12.0K 프로그램을이용하였다. 2. 체중변화 주요군암컷 18.5 mg/kg 신바로약침투여군에서투여 4주차체중이대조군에비하여통계학적으로유의하게감소하였다 (p<0.05). 그외모든투여군에서통계학적으로유의한변화는관찰되지않았다 (Fig. 1). Fig.1.BodyweightchangesofmaleandfemaleSDratwithshinbaropharmacopunctureforfourweeks * : Significant difference between control and 18.5 mg/kg group value by Dunnett's T test, p<0.0 80 http://dx.doi.org/10.13045/acupunct.2015063

SD(Sprague-Dawley) 랫드를이용한신바로약침의 4 주반복근육투여독성시험 3. 혈액학적 검사 주요군수컷 4.6 mg/kg, 9.2 mg/kg 투여군의 neutrophil (NE) 수치, 주요군수컷 9.2 mg/kg 투여군의 percentage of neutropohil(nep) 수치가부형제대조군에비하여통 계학적으로유의하게증가 (p<0.05) 하였고, 주요군암컷 4.6 mg/kg, 9.2 mg/kg 투여군의 MCHC 농도가부형제대조군에비하여통계학적으로유의하게감소하였다 (p<0.05). 그외모든투여군에서통계학적으로유의한변화는관찰되지않았다 (Table 2). Table2.HematologicalvaluesofmaleandfemaleSDratwithshinbaropharmacopunctureforfourweeks sex: Male Hematological G1 24 (n=10) G2 (n=10) G3 (n=10) G4 (n=10) 0 mg/kg 4.6 mg/kg 9.2 mg/kg 18.5 mg/kg WBC 1 (K/μL) 8.992.42 11.042.82 10.162.72 9.243.26 NE 2 (K/μL) 0.890.24 1.370.47* 1.380.40* 1.090.23 LY 3 (K/μL) 7.682.15 9.272.42 8.412.35 7.842.95 MO 4 (K/μL) 0.180.05 0.220.08 0.180.08 0.160.08 EO 5 (K/μL) 0.050.01 0.050.02 0.050.02 0.050.03 BA 6 (K/μL) 0.000.00 0.010.00 0.010.01 0.010.01 LUC 7 (K/μL) 0.190.15 0.120.06 0.130.10 0.100.07 NEP 8 (%) 10.252.42 12.352.75 13.772.51* 12.362.28 LYP 9 (%) 85.142.94 84.042.78 82.492.91 84.322.41 MOP 10 (%) 1.950.37 1.920.48 1.840.71 1.680.44 EOP 11 (%) 0.630.21 0.490.31 0.530.23 0.510.23 BAP 12 (%) 0.050.05 0.080.04 0.070.08 0.110.12 LUP 13 (%) 1.971.23 1.120.70 1.261.22 0.990.41 RBC 14 (M/μL) 7.900.20 8.180.32 8.120.36 7.940.24 Hb 15 (g/dl) 17.220.47 17.860.67 17.580.78 17.350.62 HCT 16 (%) 47.301.49 48.552.02 48.171.57 47.721.48 MCV 17 (fl) 59.901.64 59.432.12 59.371.93 60.131.65 MCH 18 (pg) 21.800.55 21.820.60 21.690.85 21.880.66 MCHC 19 (g/dl) 36.380.46 36.760.74 36.510.67 36.350.97 RDW 20 (%) 11.390.30 11.130.30 11.370.36 11.170.40 PLT 21 (K/μL) 1077.9089.33 1154.80129.89 1115.10218.20 1117.5092.20 MPV 22 (fl) 6.760.36 6.480.24 6.910.39 6.690.54 Reti 23 (%) 2.610.48 2.460.37 2.470.48 2.650.23 1: White blood cell, 2: Neutrophils, 3: Lymphocyte, 4: Monocyte, 5: Eosinophil, 6: Basophil, 7: Large unstained cell, 8: Percent of neutrophils, 9: Percent of lymphocyte, 10: Percent of monocyte, 11: Percent of eosinophil, 12: Percent of basophil, 13: Percent of large unstained cel, 14: Red blood cell, 15: Hemoglobin, 16: Hematocrit, 17: Mean corpuscular volume, 18: Mean corpuscular hemoglobin, 19: Mean corpuscular hemoglobin concentration, 20: Red cell distribution width, 21: Platelet, 22: Mean platelet volume, 23: Reticulocyte, 24. G1: Control group(0 mg/kg), G2~G4 : Experimental group(4.6, 9.2, 18.5 mg/kg), * : Significant difference compared with control value by Dunnett's T test, p <0.05 http://dx.doi.org/10.13045/acupunct.2015063 81

TheAcupunctureVol.32No.4December2015 Table2.HematologicalvaluesofmaleandfemaleSDratwithshinbaropharmacopunctureforfourweeks(continued) sex: Female Hematological G1 24 (n=10) G2 (n=10) G3 (n=10) G4 (n=10) 0 mg/kg 4.6 mg/kg 9.2 mg/kg 18.5 mg/kg WBC 1 (K/μL) 7.020.88 7.721.43 6.411.06 7.112.40 NE 2 (K/μL) 0.760.31 1.592.36 0.690.21 0.510.21 LY 3 (K/μL) 5.910.68 5.661.88 5.480.94 6.352.36 MO 4 (K/μL) 0.150.05 0.270.36 0.120.03 0.110.03 EO 5 (K/μL) 0.070.04 0.050.03 0.050.01 0.050.02 BA 6 (K/μL) 0.010.01 0.010.01 0.010.00 0.010.00 LUC 7 (K/μL) 0.120.13 0.140.14 0.080.03 0.090.06 NEP 8 (%) 10.703.80 18.0920.42 10.742.51 8.054.43 LYP 9 (%) 84.464.90 75.9622.76 85.402.54 88.314.83 MOP 10 (%) 2.170.50 3.013.12 1.850.44 1.670.44 EOP 11 (%) 0.990.56 0.710.36 0.760.24 0.720.26 BAP 12 (%) 0.090.07 0.120.09 0.100.04 0.090.05 LUP 13 (%) 1.591.35 2.092.29 1.150.36 1.150.48 RBC 14 (M/μL) 7.840.34 7.760.36 7.810.39 7.830.37 Hb 15 (g/dl) 17.260.80 16.630.79 16.880.83 16.930.94 HCT 16 (%) 45.442.43 44.562.23 45.682.41 45.302.89 MCV 17 (fl) 57.921.10 57.482.01 58.521.70 57.861.56 MCH 18 (pg) 22.000.38 21.450.70 21.640.36 21.650.53 MCHC 19 (g/dl) 38.020.84 37.340.45* 37.000.72* 37.400.57 RDW 20 (%) 10.620.98 10.510.38 10.430.28 10.330.24 PLT 21 (K/μL) 1240.30147.30 1209.30129.69 1165.00169.52 1193.5088.04 MPV 22 (fl) 6.630.29 6.690.42 6.760.28 6.660.25 Reti 23 (%) 2.501.16 2.280.45 2.220.35 2.110.45 1: White blood cell, 2: Neutrophils, 3: Lymphocyte, 4: Monocyte, 5: Eosinophil, 6: Basophil, 7: Large unstained cell, 8: Percent of neutrophils, 9: Percent of lymphocyte, 10: Percent of monocyte, 11: Percent of eosinophil, 12: Percent of basophil, 13: Percent of large unstained cel, 14: Red blood cell, 15: Hemoglobin, 16: Hematocrit, 17: Mean corpuscular volume, 18: Mean corpuscular hemoglobin, 19: Mean corpuscular hemoglobin concentration, 20: Red cell distribution width, 21: Platelet, 22: Mean platelet volume, 23: Reticulocyte, 24. G1: Control group(0 mg/kg), G2~G4 : Experimental group(4.6, 9.2, 18.5 mg/kg), Group(n) / Dose(mg / kg) * : Significant difference compared with control value by Dunnett's T test, p<0.05 82 http://dx.doi.org/10.13045/acupunct.2015063

SD(Sprague-Dawley) 랫드를이용한신바로약침의 4 주반복근육투여독성시험 4. 혈액생화학적 검사 혈액생화학적검사결과, 암 수모든투여군에서통계학적으로유의한변화는관찰되지않았다 (Table 3). Table3.SerumbiochemicalvaluesofmaleandfemaleSDratwithshinbaropharmacopunctureforfourweeks sex: Male Serum G1 23 (n=10) G2 (n=10) G3 (n=10) G4 (n=10) 0 mg/kg 4.6 mg/kg 9.2 mg/kg 18.5 mg/kg AST 1 (IU/L) 133.2029.70 130.1035.62 121.6028.92 121.0032.86 ALT 2 (IU/L) 31.104.01 37.707.39 34.606.90 34.206.14 ALP 3 (IU/L) 707.8057.14 785.10137.19 681.5083.95 719.70174.69 GGT 4 (IU/L) 0.900.30 0.800.40 0.900.30 1.000.00 LDH 5 (IU/L) 1288.80709.57 1061.70915.57 847.20770.71 921.40766.96 BUN 6 (mg/dl) 17.692.34 19.092.06 17.941.33 17.502.34 CRE 7 (mg/dl) 0.520.05 0.550.05 0.550.05 0.510.02 GLU 8 (mg/dl) 213.8022.10 218.4027.10 203.6025.80 206.3041.68 CHO 9 (mg/dl) 68.4012.00 80.9010.25 72.0011.94 73.5011.67 TP 10 (g/dl) 6.680.27 6.880.26 6.790.31 6.770.21 CPK 11 (U/L) 843.30404.87 726.90519.65 584.60392.44 648.80447.59 ALB 12 (g/dl) 2.750.11 2.810.13 2.780.14 2.760.10 T-BIL 13 (mg/dl) 0.040.02 0.030.02 0.030.20 0.040.02 A/G ratio 14 0.700.04 0.690.04 0.690.03 0.690.03 TG 15 (mg/dl) 50.2022.17 51.3025.41 62.9016.92 64.0026.59 UA 16 (mg/dl) 2.630.61 3.240.74 3.060.59 2.880.49 Ca 17 (mg/dl) 13.130.61 13.180.41 13.050.47 13.350.67 IP 18 (mg/dl) 12.540.97 12.720.75 12.650.99 12.870.75 Cl 19 (mmol/l) 105.701.42 106.101.14 106.201.33 106.001.41 Mg 20 (mg/dl) 3.100.26 3.360.34 3.200.32 3.210.28 Na 21 (mmol/l) 144.801.17 144.901.45 145.400.92 146.101.64 K 22 (mmol/l) 5.410.62 5.720.28 6.060.91 5.880.67 1: Aspartate aminotransferase, 2: Alanine aminotransferase, 3: alkaline phosphatase, 4: Gamma(γ)-glutamyl transferase, 5: Lactate dehydrogenase, 6: Blood urea nitrogen, 7: Creatinine, 8: Glucose, 9: Total cholesterol, 10: Total protein, 11: Creatine phosphokinase, 12: Albumin, 13: Total bilirubin, 14: Albumin/Globulin ratio, 15: Triglyceride, 16: Uric acid, 17: Calcium, 18: Inorganic phosphorus, 19: Chloride, 20: Magnesium, 21: Sodium, 22: Potassium, 23. G1: Control group(0 mg/kg), G2~G4 : Experimental group(4.6, 9.2, 18.5 mg/kg), Group(n) / Dose(mg / kg) * : Significant difference compared with control value by Dunnett's T test, p<0.05 http://dx.doi.org/10.13045/acupunct.2015063 83

TheAcupunctureVol.32No.4December2015 Table3.SerumbiochemicalvaluesofmaleandfemaleSDratwithshinbaropharmacopunctureforfourweeks(continued) sex: Female Serum G1 23 (n=10) G2 (n=10) G3 (n=10) G4 (n=10) 0 mg/kg 4.6 mg/kg 9.2 mg/kg 18.5 mg/kg AST 1 (IU/L) 163.4057.47 129.6036.10 116.7018.32 139.7031.07 ALT 2 (IU/L) 37.1013.20 34.805.74 30.104.66 34.807.53 ALP 3 (IU/L) 427.60103.61 408.70116.02 460.8086.69 438.1085.90 GGT 4 (IU/L) 0.300.46 0.300.46 0.500.50 0.600.49 LDH 5 (IU/L) 1316.50704.54 1164.40941.13 835.20518.99 1346.80716.47 BUN 6 (mg/dl) 20.552.39 20.003.35 21.514.38 19.491.75 CRE 7 (mg/dl) 0.590.06 0.560.05 0.610.07 0.570.05 GLU 8 (mg/dl) 165.4040.76 167.1026.18 160.9028.77 170.3031.23 CHO 9 (mg/dl) 85.1013.58 96.409.33 99.0023.32 102.0017.98 TP 10 (g/dl) 7.350.37 7.110.41 7.180.51 7.170.38 CPK 11 (U/L) 807.50321.17 682.90472.68 488.60217.21 731.90297.78 ALB 12 (g/dl) 3.170.24 3.090.23 3.120.27 3.160.21 T-BIL 13 (mg/dl) 0.060.04 0.060.03 0.050.02 0.050.02 A/G ratio 14 0.760.10 0.770.05 0.770.05 0.790.06 TG 15 (mg/dl) 26.8011.14 29.006.32 33.4014.57 26.1011.16 UA 16 (mg/dl) 2.670.52 2.550.47 2.640.45 2.960.62 Ca 17 (mg/dl) 13.101.06 13.672.23 13.711.62 12.900.52 IP 18 (mg/dl) 11.461.27 12.090.70 12.090.87 11.590.83 Cl 19 (mmol/l) 106.201.99 105.301.55 106.201.83 106.904.18 Mg 20 (mg/dl) 3.370.17 3.380.21 3.460.39 3.400.47 Na 21 (mmol/l) 143.501.69 141.701.55 143.401.91 144.305.50 K 22 (mmol/l) 5.301.00 5.901.04 5.350.86 5.630.91 1: Aspartate aminotransferase, 2: Alanine aminotransferase, 3: alkaline phosphatase, 4: Gamma(γ)-glutamyl transferase, 5: Lactate dehydrogenase, 6: Blood urea nitrogen, 7: Creatinine, 8: Glucose, 9: Total cholesterol, 10: Total protein, 11: Creatine phosphokinase, 12: Albumin, 13: Total bilirubin, 14: Albumin/Globulin ratio, 15: Triglyceride, 16: Uric acid, 17: Calcium, 18: Inorganic phosphorus, 19: Chloride, 20: Magnesium, 21: Sodium, 22: Potassium, 23. G1: Control group(0 mg/kg), G2~G4 : Experimental group(4.6, 9.2, 18.5 mg/kg), Group (n) / Dose(mg / kg) * : Significant difference compared with control value by Dunnett's T test, p<0.05 84 http://dx.doi.org/10.13045/acupunct.2015063

SD(Sprague-Dawley) 랫드를이용한신바로약침의 4 주반복근육투여독성시험 5. 부검 부검은투여종료후에모든장기에대하여상세한육안검사를시행하였을때암 수모든투여군에서이상소견은발견되지않았다. 따라서암 수모든투여군에서통계학적으로유의한변화는관찰되지않았다 (Table 4). Table4.GrossfindingsofmaleandfemaleSDratwithshinbaropharmacopunctureforfourweeks Sex Organ Sign G1 1 (n=10, 0) G2 (n=10, 4.6) Group (mg/kg) G3 (n=10, 9.2) G4 (n=10, 18.5) Male All organs Normal 10 / 10 10 / 10 10 / 10 10 / 10 Female All organs Normal 10 / 10 10 / 10 10 / 10 10 / 10 * Number of animals with the sign / Number of animals examined 1. G1: Control group(0 mg/kg), G2~G4 : Experimental group(4.6, 9.2, 18.5 mg/kg) 6. 장기중량 장기중량측정결과, 암 수모든투여군에서통계학적으로유의한변화는관찰되지않았다 (Table 5). Table5.OrganweightsofmaleandfemaleSDratwithshinbaropharmacopunctureforfourweeks sex: Male (g) Organ G1 1 (n=10) G2 (n=10) G3 (n=10) G4 (n=10) 0 mg/kg 4.6 mg/kg 9.2 mg/kg 18.5 mg/kg Body weigh 362.4630.55 349.1632.27 347.9433.89 356.7137.30 Testis(Lt.) 1.610.08 1.690.15 1.640.13 1.710.11 Testis(Rt.) 1.640.09 1.680.13 1.640.12 1.690.12 Prostate 0.580.12 0.620.13 0.620.10 0.660.10 Spleen 0.770.09 0.760.06 0.690.07 0.750.12 Liver 10.531.05 10.151.15 10.421.35 10.751.54 Adrenal gland(lt.) 0.030.00 0.14.34 0.030.00 0.030.00 Adrenal gland(rt.) 0.030.01 0.030.01 0.030.00 0.030.00 Kidney(Lt.) 1.260.12 1.190.39 1.280.10 1.330.12 Kidney(Rt.) 1.300.11 1.310.09 1.330.11 1.360.12 Heart 1.330.11 1.230.10 1.280.14 1.340.14 Lung 1.470.14 1.520.14 1.450.19 1.530.20 Brain 2.020.09 2.060.10 2.030.08 2.040.06 Pituitary 0.010.00 0.010.00 0.010.00 0.010.00 Thymus 0.510.15 0.490.14 0.480.09 0.460.10 1. G1: Control group(0 mg/kg), G2~G4 : Experimental group(4.6, 9.2, 18.5 mg/kg), * : Significant difference compared with control value by Dunnett's T test, p<0.05, Group(n) / Dose(mg /kg) http://dx.doi.org/10.13045/acupunct.2015063 85

TheAcupunctureVol.32No.4December2015 Table5.OrganweightsofmaleandfemaleSDratwithshinbaropharmacopunctureforfourweeks(continue) sex: Female (g) Organ G1 1 (n=10) G2 (n=10) G3 (n=10) G4 (n=10) 0 mg/kg 4.6 mg/kg 9.2 mg/kg 18.5 mg/kg Body weigh 226.0111.21 219.978.85 223.469.87 220.4612.84 Testis(Lt.) 0.050.01 0.050.01 0.050.01 0.050.00 Testis(Rt.) 0.050.01 0.050.01 0.050.00 0.050.00 Prostate 0.380.07 0.530.24 0.490.10 0.510.15 Spleen 0.530.10 0.530.11 0.520.09 0.510.08 Liver 6.820.44 6.860.37 6.970.57 6.830.75 Adrenal gland(lt.) 0.030.00 0.030.00 0.030.00 0.040.00 Adrenal gland(rt.) 0.030.00 0.030.00 0.030.01 0.040.00 Kidney(Lt.) 0.780.09 0.790.06 0.830.07 0.830.09 Kidney(Rt.) 0.800.07 0.830.08 0.860.09 0.850.08 Heart 0.830.08 0.880.06 0.880.10 0.850.07 Lung 1.180.11 1.190.13 1.240.08 1.270.22 Brain 1.850.06 1.880.07 1.910.11 1.880.06 Pituitary 0.010.00 0.010.00 0.010.00 0.010.00 Thymus 0.450.06 0.450.06 0.460.06 0.420.07 1. G1: Control group(0 mg/kg), G2~G4 : Experimental group(4.6, 9.2, 18.5 mg/kg), * : Significant difference compared with control value by Dunnett's T test, p<0.05, Group(n) / Dose(mg /kg) 86 http://dx.doi.org/10.13045/acupunct.2015063

SD(Sprague-Dawley) 랫드를이용한신바로약침의 4 주반복근육투여독성시험 7. 조직병리학적 검사 암 수부형제대조군및 18.5 mg/kg 시험물질투여군간에서단핵구성세포침윤및국소성의괴사, 신장에서국소성의호염기성세뇨관및피질에낭, 갑상선의아가미소 체낭및이소성흉선, 폐에국소성의폐포대식세포축적, 혈관주위에부종, 골화생, 심장에국소성의단핵구성세포침윤, 뇌하수체원위부에낭, 전립선간질에단핵구성세포침윤등이관찰되었다 (Table 6). Table6.HistopathologicalfindingsofmaleandfemaleSDratwithshinbaropharmacopunctureforfourweeks Liver Thyroid Lung Heart Kidney Organ / findings (Number of animals) -Bile duct hyperplasia -Cell infiltration, mononuclear, perivascular -Cell infiltration, mononuclear, multifocal -Cell infiltration, mononuclear, focal G1 1 (n=10, 0 mg/kg) 2 (n=2) Male G4 (n=10, 18.5mg/kg) G1 (n=10, 0mg/kg) Female G4 (n=10, 18.5mg/kg) 3 (n=4) -Necrosis,focal -Ultimobranchial cyst -Ectopic thymus -Osseous metaplasia -Accumulation, alveolarmacrophage, focal -Edema,perivascular -Cellinfiltration, mononuclear, focal -Basophilic tubule, with inflammatory cell infiltration, focal, cortex (n=2) (n=3) (n=2) (n=3) -Cyst, with inflammatory cell infiltration, cortex Pituitary -Cyst, parsdistalis There were no remarkable changes in the other organ. 1. G1: Control group(0 mg/kg), G2~G4 : Experimental group (4.6, 9.2, 18.5 mg/kg), 2. Grade: minimal(), mild(+), 3. : No remarkable lesions http://dx.doi.org/10.13045/acupunct.2015063 87

TheAcupunctureVol.32No.4December2015 Ⅳ. 고찰및결론 한약제제는오랜기간동안임상적효능이있음을경험을통해입증되어왔으며, 여러질병에대하여효능이있음에도불구하고과학적인데이터가부족하여안전성과유효성에대한문제들이끊임없이제기되고있다. 이렇듯천연물을이용한약침의원료는자연에서생산되기때문에자연조건에따라성분량도달라지고제제공정중에서성분의분해와변화가일어날수있다. 최근이를극복하기위해임상적효능 (clinical studies) 뿐아니라분석연구 (analytic research), 단회및반복독성시험등과같이안전한약침을사용하기위해서여러방법으로천연물을이용한약침에대한연구가보고되고있다 1). 그중신바로약침은자생한방병원의청파전 (GCSB-5) 약재를기반으로만든약침으로동물실험을통해항염증효과 3) 와신경재생효과 4) 를보고한바있다. 따라서 SD rats 와 Beagle dogs 을이용한단회독성에대해신바로약침의독성 8) 은미약한것으로판단되었기때문에 4주 (12 회 ) 반복근육투여를하여신바로약침의독성에대한안전성을평가하여임상적용약의안전을확보하기위해실험을시행하였다. 본연구는 SD 계통암 수 rats 에시험물질인신바로약침을 4주 (12 회 ) 반복근육투여에의한독성시험을통하여무독성량및표적장기를관찰하였다. 시험물질은 4.6( 저용량군 ), 9.2( 중용량군 ) 및 18.5( 고용량군 ) mg/kg 용량으로설정하였으며, 실험기간동안일반증상, 체중변화, 혈액학적, 혈액생화학적검사, 부검결과, 장기중량측정및조직병리학적검사를실시하였다. 시험기간동안암 수모든투여군에서사망동물및특이일반증상은관찰되지않았다. 체중측정결과, 암컷 18.5 mg/kg 투여군에서투여 4주차체중이감소하였으나일시적인사료섭취량의감소로판단되며암 수상관성을보이지않거나생물학적변동범위에속하므로신바로약침투여에의한영향은아닌것으로판단되었다. 혈액학적검사결과, 적혈구및백혈구계의변동은암 수상관성또는용량의존성을보이지않거나생물학적변동범위에속하므로시험물질에의한영향은아닌것으로판단되었다. 혈액생화학적검사결과, 암 수모든투여군에서시험물질과관련된통계학적유의한변화는관찰되지않았다. 부검결과암 수모든투여군에서육안적이상소견은관찰되지않았다. 장기중량측정결과암 수모든투여군에서시험물질에 의한장기중량의차이는관찰되지않았다. 조직병리학적검사결과, 신바로약침에의한특이소견은관찰되지않았고, 단발성또는자연발생적병변으로보이는이상소견들이있었지만이는독성학적의미는없는것으로보고되고있다 12). 본연구결과로보아, 본시험조건에서시험물질인신바로약침을설치류인 SD rats 을이용한 4주간 (12 회 ) 반복근육투여를실시한결과시험물질에의한전신적인독성학적변화는관찰되지않았다. 따라서유해한영향이관찰되지않는화학물질의최대섭취량인 NOAEL(No observed adverse effect level) 은임상예정용량인 1.11 mg/60 kg ( 약 18.5 μg/kg) 의약 1,000 배인 18.5 mg/kg ( 고용량 ) 투여군의암 수 rats 에서독성과표적장기는관찰되지않았음을확인할수있었다. Ⅴ. References 1. Kim SH, Jung DJ, Choi YM, Kim JU, Yook TH. Trend of Pharmacopuncture Therapy for Treating Cervical Disease in Korea. J Pharmacopuncture. 2014 ; 17(4) : 7-14. 2. Kim JK, Park SW, Kang JW et al. Effect of GCSB-5, aherbalformulation, onmonosodium Iodoacetate-Induced Osteoarthritis in Rats. Evid. Based Complement. Alternat. Med. 2012 ; 2012 : 1-11. 3. Chung HJ, Lee HS, Shin JS et al. Modulation of acute and chronic inflammatory processes by a traditional medicine preparation GCSB-5 both invitroandinvivoanimalmodels. J. Ethnopharmacol. 2010 ; 130(3) : 450-9. 4. Kim TH, Yoon SJ, Lee WC et al. Protective effect of GCSB-5, an herbal preparation, against peripheral nerve injury in rats. J. Ethnopharmacol. 2011 ; 136(2) : 297-304. 5. Park OJ, Kim SG, Lee JJ, Lee SM, Kim SJ, Cho NG. The Effect of Shinbaro and Bee Venom Pharmacopuncture in Treating Lumbar Disc Herniations. The Acupuncture. 2013 ; 30(5) : 41-50. 6. Jun BC, Kim ES, Kim DS, Kim TH, Kim JY. Ef- 88 http://dx.doi.org/10.13045/acupunct.2015063

SD(Sprague-Dawley) 랫드를이용한신바로약침의 4 주반복근육투여독성시험 fectiveness of ShinBaro Pharmacopuncture on Lumbar Spinal Herniated Intervertebral Disc : A Randomized Controlled Trial. J. Korea CHUNA Med. Spine & Nerves. 2011 ; 6(2) : 109-19. 7. Lee JH, Kim MJ, Lee JW, Kim MR, Lee IH, Kim EJ. A Study on Standardization of Shinbaro Pharmacopuncture Using Herbal Medicines Identification Test and HPLC-DAD. The Acupuncture. 2015 ; 32(2) : 1-9. 8. Lee JH, Chung HJ, Lee IH, Lee JW, Kim EJ, Kim MJ. Study on Single-dose Intramuscular Toxicity of Shinbaro Pharmacopuncture in Sprague-Dawley (SD) Rats and Beagle Dogs. JKMR. 2015 ; 25(3) : 1-9. 9. Jung C, Lee MS. Single Intramuscular Dose Toxicity Study of CA-YAKCHIM in Sprague- Dawley Rats. J. Korea Immuno-yakchim society. 2014 ; 3(1) : 19-26. 10. Kwon HY, Kwon KR. Study of four weeks repeated-dose toxic test of Sweet Bee Venom in rats. J. Pharmacopuncture. 2011 ; 14(1) : 5-24 11. Lee JH, Lee IH, Lee JW, Kim EJ, Kim MJ. Single-dose Intramuscular Toxicity Studies of Shinbaro3 Pharmacopunture in Sprague- Dawley Rats and Beagle Dogs. JKMR. 2015 ; 25(2) : 73-80. 12. Park UJ, Na JB, Kang DH et el. 13 weeks oral repeated dose toxicity study and Toxico- kinetics study of stevioside in mice. The Annual Report of KNTP. 2005 ; 4(2) : 451-76. http://dx.doi.org/10.13045/acupunct.2015063 89