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J. Fd Hyg. Safety Vol. 26, No. 4, pp. 424~434 (2011) Journal of Food Hygiene and Safety Available online at http://www.foodhygiene.or.kr 한약재중아플라톡신분석에관한연구 이성득 * 김연선 1 김남훈 정희정 정삼주 김화순 김경식 한기영 서울시보건환경연구원, 1 한양여자대학 A study on Aflatoxins Analysis in The Herb Medicines Sung-deuk Lee*, Yeon-sun Kim 1, Nam-hoon Kim, Hee-jung Jung, Sam-joo Jung, Hwa-soon Kim, Kyung-sik Kim, and Ki-young Han Seoul Metropolitan Government Research Institute of Public Health and Environment 1 Hanyang Women's College (Received August 31, 2011/Revised October 16, 2011/Accepted December 7, 2011) ABSTRACT - The increase in the consumption of herb medicines have made their use a public health problem due to the potential fungal contamination and the risk of the presence of mycotoxins. 360 samples of herb medicines were evaluated for the aflatoxin contamination. The natural occurrence of aflatoxins in these samples were determined using immunoaffinity column clean up and high performance liquid chromatography (HPLC) with post-column derivatization. For samples analyzed, mean levels (incidence) of AFB1, AFB2, AFG1 and AFG2 in positive samples were 1.4 µg/kg (46.4%), 0.4 µg/kg (25.4%), 1.1 µg/kg (37.8%) and 0.9 µg/kg (24.3%), respectively. Recoveries of the full analytical procedure were 71.7~99.7% for AFB1, 88.1~99.2% for AFB2, 82.8~95.5% for AFG1 and 77.9~90.0% for AFG2. The excess cancer risk estimated using the cancer potency of aflatoxin B1 (7 (mg/kg/day) 1 for HBsAg and 230 (mg/kg/day) 1 for HBsAg + ) were 1.30 10 5 ~ 1.22 10 7 for hepatits B surface antigen negative (HBsAg ) and 3.31 10 4 ~3.12 10 6 for hepatits B surface antigen positive (HBsAg + ) respectively. In conclusion, although the contamination levels of samples used in the study were low, further actions are also required to undertake a program of herbal surveys in order to access mycotoxin contamination overall so that the safety of public will be protected. Key words: herb medicines, aflatoxins, HPLC, recoveries, excess cancer risk 약용식물은오래전부터질병의예방및치료의목적으로사용되어왔으며, 최근세계적으로식품재료와대체또는보완약재로서수요가급격히증가함과동시에약용식물의자체의독성과부작용에관한안전문제가대두되고있다 1). 또한선진국의약용식물소비증가로국제무역규모는매해 7% 씩증가되고있어 2), 한약재의국제적유통으로곰팡이오염에의한아플라톡신의우려는세계적문제가되고있다 3). 우리나라에서한약재란약사법과한약재품질및유통관리규정에따라한약또는한약제제를제조하기위하여원료로사용되는생약과원료약재로규정하고있고, 약제의기원에따라약용식물, 약용동물, 약용광물로구분할수있는데, 그중식물성약제가한약재의약 87% 에해당된 *Correspondence to: Sung-deuk Lee, Seoul Metropolitan Government Research Institute of Public Health and Environment, 5, Yangnyeongjungang-ro, Dongdaemun-gu, Seoul 130-864, Korea Tel: 82-2-968-5098, Fax: 82-2964-8175 E-mail: lesudu@seoul.go.kr 다 4). 대부분식물성한약재는재배된약용식물을단순히건조, 절단하여유통소비되어, 재배와수확후보관기간동안외부의오염원인미생물의오염과번식을방지하여농산물자원이안전성을확보하는것이필요하다. 곰팡이에오염된약용식물은상품성이저하되어경제적손실을초래하고, 원료의화학적조성의변화는약리성분을감소시키고 3), 또한 2차대사산물인아플라톡신, 제랄레논, T-2 등의새로운독성물질이생성되어 5), 기대하였던약리효과뿐아니라독성물질을섭취함으로써부작용을초래할수있다. 특히아플라톡신은곰팡이독소중가장강력한독성물질로서장기간고농도의아플라톡신을섭취할경우면역체계저하시키고 6), 간경변과간암을유발시켜국제암연구소에서는 1군발암물질로분류하고있다 7). 현재아플라톡신은 20여종이알려져있으나 8), Aspergillus flavus와 Aspergillus parasiticus 등에의하여생성되는아플라톡신 B1, B2, G1 및 G2 (Fig. 1) 9) 가흔히발견되며, 그중아플라톡신 B1의독성이가장큰것으로보고되어있다 10). 일단생성된아플라톡신독소는열에안정하여일 424

A study on Aflatoxins Analysis in The Herb Medicines 425 360건을구입하여분쇄후분말로균질화하여시험재료로사용하였다. Fig. 1. Chemical structure of major aflatoxins. 반적인조리법으로는감소되거나분해되지않기때문에 11) 오염된것을섭취하지않는것이최선의방법으로고려되고있다. 우리나라에서소비되는한약재의 70~80% 가수입한약재이며 12), 그지역의토양, 기후및매개곤충등의재배환경과수확과정및보관방법이열악한경우곰팡이의오염을피할수없다 13). 특히아플라톡신의생성은기후가비교적높은열대또는아열대지방에서발생이용이하기때문에, 그지역에서수입된한약재의경우에는아플라톡신오염에관한주의가더욱필요하다. 이러한약용식물의독소대처방안으로세계보건기구등에서는약용식물의안전한사용을위한허용기준, 지침, 평가기준을마련하였고 14-16). 대부분의국가에서는식품과사료에서곰팡이독소의허용기준 ( 아플라톡신 B1 5 µg/kg; 총아플라톡신 20 µg/kg) 2) 을설정하여관리하고있으나, 현재까지한약재에서기준을설정하고있는나라는이탈리아 ( 아플라톡신 B1 5 µk/kg; 총아플라톡신 10 µg/kg) 등일부국가에한정되어있고 17), 우리나라에서는최근한약재에서곰팡이독소중아플라톡신 B1의허용기준을감초등 9품목에한하여 10 µg/kg 이하로설정하였고 18), 다시 2009년에감초등 19품목으로확대하여관리하고있다 19). 최근국내외로곰팡이의오염과관련하여식품및사료에관한아플라톡신오염의연구는다양하게진행되고있으나, 국민건강이관련된한약재를대상으로는소수로진행되고있다. 따라서본연구에서는서울시내에서유통중인아플라톡신허용기준설정한약재를구입하여아플라톡신의함량을분석하고위해성을평가하여한약재의안전성을확보하기위한기초자료로제시하고자한다. 재료및방법 실험재료서울시내에서 2010년 1월 ~12월동안한약규격품으로포장되어유통중인감초 (54건), 결명자 (22건), 괄루인 (20 건 ), 귀판 (2건), 도인 (36건), 목과 (44건), 반하 (9건), 백자인 (8건), 백편두 (14건), 빈랑자 (23건), 산조인 (22건), 연자육 (41건), 울금 (12건), 원지 (17건), 육두구 (2건), 지구자 (3건), 파두 (1건), 행인 (17건), 홍화 (14건) 19품목 분석시약아플라톡신표준품은 Mix kit-m (Supelco, USA) 을사용하였으며, 시료의추출과액체크로마토그래프의이동상의조제에는 methanol (Fisher, USA), acetonitirle (Merck, Germany) 를사용하였으며, 시료의여과와정제에는유리섬유여과지 (Whatman 1.6 µm, England) 와아플라톡신및오크라톡신용면역친화성칼럼 (Aflaochra, Vicam, USA) 을각각사용하였다. 분석기기시료를분말로균질화하고추출하기위하여믹서 ( 대성아트론 DA338, 한국 ) 와교반기 (JEIO TECH SK-600, 한국 ) 를사용하였으며, 아플라톡신분석은액체크로마토그래프 (Waters e2695, USA) 와칼럼유도화장치 (Aura PHRED, USA) 를사용하였다. 표준용액조제표준용액은표준품으로구입한 Mix kit-m (B1 = 1 µg/ml, B2 = 0.3 µg/ml, G1 = 1 µg/ml and G2 = 0.3 µg/ml) 을희석하여아플라톡신 B1과 G1을각각 5, 10, 20 ng/ml, B2 와 G2 를각각 1.5, 3, 6 ng/ml으로 3단계로희석하여조제하였다. 시험용액제조및분석 시료추출 시료약 500 g를잘분쇄한후가루로하여약 5.0 g를정밀하게달아물 메탄올혼합액 (3:7) 100 ml을넣어 1 시간동안추출하였다. 추출액을유리섬유여과지를이용하여여과후다시 100 ml로맞춘후다시이액 10 ml를취하여 80 ml 되게희석하여추출용액으로하였다. 추출용액의정제 추출용액 40 ml을면역친화성칼럼을통과시킨후물 10 ml을 3mL/ 분의유속으로 2회통과시켜세척한후면역친화성칼럼에주사기로 10초간공기를통과시켜건조하였다. 건조된면역친화성칼럼에메탄올 1.0 ml를통과시켜용출액을시험용액으로하였고, 용출액이투명하지않을경우 0.45 µm 필터로여과하여시험용액으로하였다. 액체크로마토그래피분석 표준용액과시험용액을형광검출기와칼럼유도화장치 ( 광화학반응장치 ) 가부착된액체크로마토그래프에주입하여표준용액과시험용액의머무름시간과면적을비교하여분석하였으며기기의분석조건은 Table 1과같다.

426 Sung-deuk Lee et al. Table 1. The analytical conditions of HPLC for aflatoxins Instrument HPLC (Watere2695, USA) Column Symmetry (R) C18 5 µm, 4.6 250 mm Injection volumn 10 µl Flow rate 1.2 ml/min Florescene detector Excitation 365 nm, Emission 435 nm Mobile phase Acetonitrile : Methanol : water = 15 : 25 : 60 PHRED (Photochemical reactor for Postcolumn derivatives enhanced detection) 회수율검정 아플라톡신 B1, B2, G1 및 G2의회수율은생약중곰팡이독소기준이설정된 19품목중예비분석결과아플라톡신이검출되지않은 5개품목 ( 감초, 괄루인, 목과, 빈랑자, 연자육 ) 을선정한후, 각품목별로표준용액을첨가하여회수율시험용액의최종농도가아플라톡신 B1, B2, B1 및 G2가각각 1.5 ng/ml, 5 ng/ml, 1.5 ng/ml 및 5ng/ml이되도록하여, 3회반복시험하여회수율을구했다. 위해성평가 한약재의복용으로인한아플라톡신 B1 위해성평가는 FAO/WHO 20) 의만성노출평가방법에따라아플라톡신 B1 의오염도와일일섭취량을체중으로나누어일일인체노출량을구한후아플라톡신 B1의발암력을곱하여간암발생에대한초과발암위해도를계산하였다. 일일인체노출량 한약재의일일평균섭취량은이 12) 가실시한소매상중한의원을대상으로실시한일평균섭취량II 중곰팡이독소기준이설정된반하, 백편두, 빈랑, 산조인의일평균섭취량을평균한 8.557 g와, 평균체중은 2005년산업자원부기술표준원 21) 에서제시하고있는남자 69.6 kg, 여자 56.4 kg 의평균값인 63.0 kg을이용하였다. Dietary Exposure = n i = 1 CAFB1i IRi -------------------------------- BW CAFB1i : Concentration of aflatoxin B1 i : Number of herb medicine IRi : Ingestion rate BW : Body weight 초과발암위해도 아플라톡신 B1의발암력은 Yeh 등 22) 이제시한 B형간염비보균자의경우 9 (mg/kg/day) 1 와 B형간염보균자의경우 230 (mg/kg/day) 1 을일일인체노출량에곱하여구하였다. Excess Cancer Risk = Dietary Exposure(mg/kg/day) Cancer Potency(mg/kg/day) 1 결과및고찰 회수율검정액체크로마토그래피상에서 Fig. 2와같이아플라톡신 B1, B2, G1 및 G2는명확하게 peak가분리되었으며, Fig. 3과같이각아플라톡신표준용액의검량선은직선성을나타냈다. 전체독소에대한시료의회수율은 Table 2와같이감초는 71.7~88.1%, 괄루인은 90.0~96.7%, 목과는 85.2~95.1%, 빈랑자는 82.1~99.1% 그리고연자육은 84.4~99.7% 로감초를제외하고다른품목들은비교적우수하게나타났다. 또한아플라톡신독소별회수율은 B1, B2, 및 G1은 90% 이상으로높게나타났으나, 아플라톡신 G2의회수율은다른독소에비하여낮게나타났다. 이것은시료의구성성분과희석액의영향을받는면역친화성칼럼의친화력이낮기때문이며 2), 아플라톡신 G2의회수율을높이기위해서는시료성분의농도가낮을때가능하며 19), 면역친화성칼럼의회수율이낮은경우는항체의양이부족하거나, 곰팡이독소에대한낮은친화성에기인한다고보고된바있다 2). Table 3과같이약용식물에대하여회수율을조사한결과 Romagnoli 등 17) 은 76.1~78.1%, Rief 등 23) 은 57.8~99.4%, Ventura 등 24) 은 77.6~110.4% 그리고 Han 등 25) 은 89.5~109.0% 이었는데, 이러한회수율차이는시료의종류및정제방 Fig. 2. Chromatogram of aflatoxins.

A study on Aflatoxins Analysis in The Herb Medicines 427 Fig. 3. Calibration curve of aflatoxin B1, B2, G1 and G2. Table 2. Recoveries(average) of added aflatoxins in herb medicines (%) * AFB1 ** AFB2 *** AFG1 **** AFG2 Glycyrrhizae Radix et Rhizoma 71.7 88.1 82.8 77.9 Trichosanthis Semen 96.7 96.4 90.1 90.0 Chaenomelis Fructus 95.1 94.6 92.3 85.2 Arecae Semen 99.1 98.0 90.8 82.1 Nelumbinis Semen 99.7 99.2 95.5 84.4 * AFB1; aflatoxinb1, ** AFB2; aflatoxinb2, *** AFG1; aflatoxing1, **** AFG2; aflatoxing2. 법과유도화장치의종류등시험방법에따라차이가있는것으로추측되며, 특히시험용액의 ph는회수율에영향을미치는데, 분석과정중시료를 70% 메탄올로추출하고다시물로희석할때시료의 ph가낮아지는경우회수율이낮아질수있고, 특히아플라톡신 G2의경우는 ph가 4 이하이거나 8이상인경우에는회수율이감소하기때문에희석용액으로 PBS (phosphate buffer saline) (ph 7.4) 또는 0.1M PB (phosphate buffer) (ph 8.0) 를사용하여적정 ph 로조정해야하는데 26), 도인을제외하고본회수율시험에사용된품목들의 ph는 4~5사이로측정되어 ph에의한영향은없는것으로추측되었다. 또한아플라톡신 B1 및 G1은자연적으로적은형광성을띠기때문에 27) 정밀한분석을위하여분석칼럼전후에유도화장치가필요하여 28), 본조사에서는유도화장치로 PBPB (pyridinium bromide perbromide), Kobra cell( 브롬전기발생장치 ) 등의방법중에서 PHRED( 광화학반응장치 ) 를부착하여분석하였다. 한약재중아플라톡신오염실태서울시내에서 2010년 1월에서 12월까지유통중인한약규격품 19품목 360건을구입하여아플라톡신의오염실태를조사하였다. 시료의제조시기는 2007년에서 2010년사이에제조되어포장된제품으로외관상으로는대부분시료에서곰팡이의오염을알수없었다. 분석결과아플라톡신의평균검출량은 Fig. 4와같다. 전체시료에서아플라톡신 B1, B2, G1 및 G2는 1.4 ± 1.8 µg/ kg, 0.4 ± 1.1 µg/kg, 1.1 ± 5.0 µg/kg 및 0.9 ± 3.4 µg/kg의범위로각각검출되었으며, 아플라톡신 G1은시료별로검출범위가가장넓게나타났다. 또한각품목별아플라톡신의검출범위는 Table 4와같다. 한약재별곰팡이독소허용기준인아플라톡신 B1 10 µg/ kg 이하를초과한품목은백자인과빈랑자 2품목이었고, 다 Table 3. Recoveries of added aflatoxins in herb medicines (%) * AFB1 ** AFB2 *** AFG1 **** AFG2 Ref. Romagnoli. B 77.0 78.1 76.1 78.1 17 Reif. K 78.6~99.4 70.9~93.5 78.2~97.1 57.8~78.7 23 Ventura. M 110.4 89.9 81.0 77.6 24 Han. Z 98.5~101.3 93.2~105.7 89.5~109.0 97.4~106.8 25 * AFB1; aflatoxinb1, ** AFB2; aflatoxinb2, *** AFG1; aflatoxing1, **** AFG2; aflatoxing2.

428 Sung-deuk Lee et al. Fig. 4. The range of aflatoxins in herb medicines. Table 4. The amounts of aflatoxins in herb medicines (µk/kg) Type of medicines No. of samples * AFB1 ** AFB2 *** AFG1 **** AFG2 Glycyrrhizae Radix et Rhizoma 54 0.8 ± 1.6 1) 0.3 ± 0.6 8.2 ± 25.9 1.9 ± 6.8 (0.1~7.4) 2) (0.1~3.5) (0.1~138.3) (0.1~40.9) Cassiae Semen 22 0.5 ± 1.0 0.1 ± 0.4 11.1 ± 14.9 1.8 ± 5.3 (0.1~4.5) (0.2~1.4) (0.1~63.3) (0.2~25.4) Trichosanthis Semen 20 0.1 ± 0.2 0.3 ± 1.0 0.8 ± 1.4 5.9 ± 25.0 (0.1~0.9) (0.1~4.7) (0.1~5.2) (0.5~115.0) Testudinis Plastrum 2 0.5 ± 0.3 0.1 ± 0.1 (0.2~0.8) 0.3 Persicae Semen 36 1.1 ± 2.1 0.5 ± 1.4 1.3 ± 7.0 0.4 ± 1.6 (0.1~9.0) (0.1~6.9) (0.1~42.5) (0.2~9.2) Chaenomelis Fructus 44 0.1 ± 0.1 0.1 ± 0.2 1.6 ± 7.9 2.3 ± 12.7 (0.1~0.6) (0.1~1.0) (0.1~52.8) (0.1~85.1) Pinelliae Tuber 8 0.5 ± 1.2 0.3 ± 0.6 0.1 (0.1~3.8) (0.2~1.9) 0.1 Thujae Semen 8 10.6 ± 14.5 1.3 ± 1.7 0.1 ± 0.2 0.2 (0.3~36.2) (0.2~4.1) (0.7~0.7) 0.2 Dolichoris Semen 14 0.1 0.1 ± 0.3 0.1 ± 0.2 (0.1~0.2) (0.1~1.2) (0.1~0.8) Arecae Semen 23 3.0 ± 3.8 0.1 ± 0.3 3.6 ± 10.4 0.1 ± 0.2 (0.2~11.6) (0.1~1.0) (0.1~50.5) (0.1~1.2) Zizyphi Semen 22 1.0 ± 2.0 0.1 ± 0.3 2.7 ± 11.4 2.8 ± 8.4 (0.1~7.5) (0.1~0.9) (0.2~54.9) (0.1~31.7) Nelumbinis Semen 41 0.7 ± 1.6 0.4 ± 1.6 0.9 ± 3.2 0.6 ± 1.9 (0.1~8.3) (0.1~10.0) (0.1~16.3) (0.2~11.3) Curcumae Radix 12 1.5 ± 2.8 0.1 ± 0.4 0.9 ± 1.6 0.3 ± 0.7 (0.1~8.3) (1.4~1.4) (0.4~5.5) (0.5~2.6) Polygalae Radix 17 0.8 ± 1.4 2.1 ± 8.0 0.8 ± 1.7 0.1 ± 0.2 (0.3~6.1) (0.1~34.0) (0.1~7.0) (0.1~0.6) Myristicae Semen 2 3) Hoveniae Semen Cum Fructus 3 0.1 ± 0.1 0.1 ± 0.1 (0.1~0.3) (0.1~0.3) Crotonis Semen 1 4.0 0.1 Armeniacae Semen 17 0.4 ± 1.2 1.2 ± 3.9 0.1 ± 0.5 0.3 ± 0.8 (0.1~5.1) (0.1~16.7) (0.1~2.1) (0.1~3.6) Carthami Flos 14 0.4 ± 0.8 0.1 ± 0.4 2.6 ± 7.2 0.1 (0.1~2.8) (0.1~1.5) (0.2~27.0) 0.1 * AFB1; aflatoxinb1, ** AFB2; aflatoxinb2, *** AFG1; aflatoxing1, **** AFG2; aflatoxing2. 1) Mean ± standard deviation 2) Detection range 3) ; Not Detected.

A study on Aflatoxins Analysis in The Herb Medicines 429 Table 5. Aflatoxin content in herb medicines from each country (µk/kg) Year Region * AFB1 ** AFB2 *** AFG1 **** AFG2 Ref. Tassaneeyakul, W 2004 Thailand 2.2~11.3 0.5~1.0 0.6~2.0 0.4 31 Ali, N 2004 Malaysia Indonesia 0.3 0.1 0.1 detected 2 Romagnoli, B 2005 Itlay 1) 17 Han, Z 2010 China 1.4 1.3 0.5 0.9 25 * AFB1; aflatoxinb1, ** AFB2; aflatoxinb2, *** AFG1; aflatoxing1, **** AFG2; aflatoxing2 1) ; Not Detected. 른품목들은모두기준에적합하였으며, 육두구는전혀검출되지않았다. 백자인과빈랑자를제외한모든품목에서아플라톡신 B1, B2, G1 및 G2는 0.1~9.0 µg/kg, 0.1~34.0 µg/ kg, 0.1~138.3 µg/kg 및 0.1~115.0 µg/kg의범위로각각검출되었으며, 품목별로아플라톡신 G1과 G2는아플라톡신 B1 및 B2에비하여동일품목중시료간의검출량의차이가많았다. 모든품목에서아플라톡신 B1과 B2는검출량의차이가적었으나감초, 결명자, 빈랑자, 산조인은아플라톡신 G1의검출량차이가크게나타났고, 괄루인과목과는아플라톡신 G2의검출량차이가크게나타났다. 시료별로백자인에서아플라톡신 B1, 원지에서는아플라톡신 B2, 감초에서는아플라톡신 G1, 괄루인은아플라톡신 G2가가장높게검출되었다. 본조사에서아플라톡신 B1은미량검출되었지만, 대부분의약용식물이한가지이상의곰팡이에오염되어있어, 여러종류의곰팡이독소에교차오염될경우상승효과를나타내어, 동시에여러종류의곰팡이독소를섭취하였을경우건강의위해요소는더욱커지게된다 5). 최근약용식물중아플라톡신에관한연구결과에의하면 Rizzo 등 29) 은 152건에서의아플라톡신 B1 및 B2를 10~ 2000 µg/kg, Roy 등 30) 은아플라톡신 B1을 170~670 µg/kg을검출하여본조사와는차이가있었지만 Tassaneeyakul 등 31), Ali 등 2), Romagnoli 등 17) 및 Han 등 25) 의연구결과에서는미량검출되어본조사의결과와유사하였다 (Table 5). 각조사별차이는약용식물의종류, 재배지역과추출용매의 양과추출시간등분석방법에따른차이로추측된다 32). Fig. 5는네종류의아플라톡신 (B1 + B2 + G1 + G2) 의전체검출량을나타내고있는데, 일부품목을제외하고시료들의아플라톡신독소의검출량은차이가많았으며, 백자인과빈랑자의평균검출량은다른품목에비하여높게나타났다. 감초와괄루인의평균검출량은 10 µg/kg 이하로나타났지만, 다른품목에비하여동일품목중시료들의검출량의차이가크게나타났다. Fig. 6은우리나라에서한약재중허용기준이설정되어있는품목의아플라톡신 B1의검출량을나타내고있는데백자인과빈랑자를제외하고모든시료가기준에적합하였고, 대부분의품목들은시료별로검출량의차이가적게나타났다. Fig. 7은전체시료 360건중아플라톡신이검출된건수를나타내고있는데, 아플라톡신 B1은 168건 (46.4%), 아플라톡신 B2는 92건 (25.4%), 아플라톡신 G1은 137건 (37.8%), 아플라톡신 G2는 88건 (24.3%) 검출되어, 아플라톡신 B1의발생률이가장높았다. Table 6은품목별로아플라톡신의검출건수를나타내고있는데, 감초, 귀판, 백자인, 빈랑자, 파두, 울금, 원지및지구자는아플라톡신 B1, 백자인및파두는아플라톡신 B2, 감초, 결명자및지구자는아플라톡신 G1, 결명자및귀판은아플라톡신 G2가 50% 이상검출되었다. 또한전체시료중유일하게감초는아플라톡신 B1 및 G1의발생건수가 50% 를초과하여구입에주의를필요로한다. Fig. 5. The amounts of total aflatoxins (B1, B2, G1. G2) in herb medicines.

430 Sung-deuk Lee et al. Fig. 6. The amounts of aflatoxin B1 in herb medicines. Fig. 7. Incidence of aflatoxins all the medicines (case). 감초, 산조인, 연자육, 결명자, 목과, 백편두, 지구자, 귀판, 울금은식약공용한약재로서식품재료로사용될경우한약규격품과는달리아플라톡신오염에대한검증절차없이유통소비되어건강의위해요소로작용할가능성이 있어, 같은한약재를식품용도로사용할경우에도아플라톡신의허용기준을적용해야될것으로생각된다. 또한한약재를탕제로복용할경우한약재에서탕제로의이행률을감안하여오염된아플라톡신양보다적게섭취할수있으나, 환제로제조하여섭취하는경우는오염된전량을섭취하는결과를초래하여건강상심각한장애를유발시킬수있다. 아플라톡신의반수치사량 LD 50 (lethal dose) 은 5mg/kg로추측되어, 급성중독은예외적인현상이며, 일반적으로아플라톡신에의한중독은면역억제작용과간경변같은만성적인독성이문제화되기때문에 13), 일상생활에서적은양 Table 6. Incidence of aflatoxins in herb medicines (case) Type of medicines No. of samples * ** *** **** AFB1 AFB2 AFG1 AFG2 case % case % case % case % Glycyrrhizae Radix et Rhizoma 54 31 57.4 23 42.6 35 64.8 20 37.0 Cassiae Semen 22 10 45.5 3 13.6 17 77.3 11 50.0 Trichosanthis Semen 20 6 30.0 7 35.0 9 45.0 3 15.0 Testudinis Plastrum 2 2 100.0 0 0.0 0 0.0 1 50.0 Persicae Semen 36 16 44.4 11 30.6 8 22.2 5 13.9 Chaenomelis Fructus 44 12 27.3 6 13.6 14 31.8 5 11.4 Pinelliae Tuber 8 3 37.5 0 0.0 2 25.0 1 12.5 Thujae Semen 8 7 87.5 6 75.0 1 12.5 1 12.5 Dolichoris Semen 14 3 21.4 0 0.0 2 14.3 4 28.6 Arecae Semen 23 19 82.6 7 30.4 11 47.8 4 17.4 Zizyphi Semen 22 10 45.5 7 31.8 5 22.7 6 27.3 Nelumbinis Semen 41 20 48.8 10 24.4 12 29.3 13 31.7 Curcumae Radix 12 7 58.3 1 8.3 5 41.7 2 16.7 Polygalae Radix 17 10 58.8 3 17.6 7 41.2 8 47.1 Myristicae Semen 2 0 0.0 0 0.0 0 0.0 0 0.0 Hoveniae Semen Cum Fructus 3 2 66.7 0 0.0 2 66.7 0 0.0 Crotonis Semen 1 1 100.0 1 100.0 0 0.0 0 0.0 Armeniacae Semen 17 4 23.5 5 29.4 4 23.5 3 17.6 Carthami Flos 14 5 35.7 2 14.3 3 21.4 1 7.1 Total 360 168 46.4 92 25.4 137 37.8 88 24.3 * AFB1; aflatoxinb1, ** AFB2; aflatoxinb2, *** AFG1; aflatoxing1, **** AFG2; aflatoxing2.

A study on Aflatoxins Analysis in The Herb Medicines 431 Table 7. The cases of herb medicines from country (case) Type of medicines Total Imported domestic China South Africa Vetnam Indonesia others Korea Glycyrrhizae Radix et Rhizoma 54 52 1 1 Cassiae Semen 22 9 1 12 Trichosanthis Semen 20 19 1 Testudinis Plastrum 2 2 Persicae Semen 36 5 31 Chaenomelis Fructus 44 1 1 42 Pinelliae Tuber 8 8 Thujae Semen 8 8 Dolichoris Semen 14 14 Arecae Semen 23 2 18 2 1 Zizyphi Semen 22 13 8 1 Nelumbinis Semen 41 9 1 31 Curcumae Radix 12 9 1 2 Polygalae Radix 17 17 Myristicae Semen 2 1 1 Hoveniae Semen Cum Fructus 3 1 2 Crotonis Semen 1 1 Armeniacae Semen 17 12 2 2 1 Carthami Flos 14 14 Total 360 194 36 31 22 16 61 의아플라톡신이라도섭취를억제해야한다. 약용식물에서식하는곰팡이는주로 Aspergillus와 Penicillum 및 Rhizopus 이나 33), 곰팡이중아플라톡신을생성하는균주는일부로한정되어있지만, 특히열대나아열대지방에서는 Aspergillus flavus와 Aspergillus parasiticus가널리분포하고있어, 이곰팡이들은세계각지에서소비되는주요식량과밀접한관계가있다 13). 식물체는재배되는동안다른곰팡이들에의하여동시에오염되기때문에, 이들곰팡이들은한가지이상의독소를생성하고 34), 곰팡이의오염은식물체원료의구성성분을변화시켜약용식물의약리작용을저하시키며 3). Santos 등 5) 은약용식물 84건중 73건이동시에 4가지이상의곰팡이독소 (T-2, zealenon, aflatoxin, ochratoxin, deoxynivalenol) 에오염되어있음을보고하였다. 또한 Romagnoli 등 17) 이조사한 27종의약용식물에서는아플라톡신이검출되지않았으며. Rizzo 등 29) 은약용식물 152건을조사한결과 52% 가 Aspergillus에오염되어있었고, 그중곰팡이가분리된 40건중 20건 (50.0%) 에서아플라톡신을검출하여, 연구자별로차이가있었다. Table 7은품목별로수입된국가를나타내고있으며, 전체시료 360건의시료중국내산한약재가 61건 (16.9%) 이었고, 수입된한약재가 299건 (83.1%) 이었고, 수입산한약재중중국산한약재가 194건 (64.9%) 으로과반수이상을차지하였다. 시료중국내산한약재는결명자와목과가대부분이었고, 도인은남아프리카, 연자육은베트남, 빈랑자는인도 네시아에서수입된것을제외하고는대부분의품목은중국에서수입되었다. 곰팡이의독소의생성과농도는원산지의환경적, 지형학적인요인과, 식물의구성성분에따라달라지기때문에 5,30), 약용식물이재배된원산지가중요하다. 곰팡이독소중아플라톡신의생성은대부분수확후보관중부적절한수분과온도에서저장할때발생하지만, 식물체가성장중가뭄과같은가혹조건에서는곰팡이는식물체의조직에상당한양의아플라톡신을생성하여경제적손실을초래할수있다 13). 향신및약용식물은곰팡이독소생성을억제하는필수오일을함유하고있어다른곡류에서보다아플라톡신의생성이억제되지만 35), 저장조건과저장기간에따라독소생성량은증가하며, 특히습도가높을수록, 저장기간이길수록생성량은증가하기때문에, 혐기성상태에서는 Aspergillus의성장을저해하여, 건조한상태에서프로필렌용기에저장하여방지할수있다 36). 다른저장방법으로곰팡이의성장억제를위하여화학합성물질이이용되었지만부작용을수반하여최근백리향등의식물성추출물을이용하여저장중곰팡이의성장의억제하려는연구가진행되고있고 37), 아플라톡신에오염된농산물은암모니아수나과산화수소로처리하여활성을억제하는방법을사용하고있다 36). 본조사에서는아플라톡신독소의발생비율 46.7% 에비하여상대적으로아플라톡신의오염량은아플라톡신 B1이 0.1~9.0 µg/kg의범위로검출되어, 현재의기준으로는안전

432 Sung-deuk Lee et al. Table 8. Excess cancer risk for primary liver cancer due to dietary exposure by aflatoxin B1 Type of medicines *AFB1 Mean (µk/kg) Dietary Exposure (mg/kg/day) Excess Cancer Risk HBsAg- HBsAg+ Glycyrrhizae Radix et Rhizoma 0.8 1.09 10 7 9.78 10 7 2.50 10 5 Cassiae Semen 0.5 6.79 10 8 6.11 10 7 1.56 10 5 Trichosanthis Semen 0.1 1.36 10 8 1.22 10 7 3.12 10 6 Testudinis Plastrum 0.5 6.79 10 8 6.11 10 7 1.56 10 5 Persicae Semen 1.1 1.49 10 7 1.34 10 6 3.44 10 5 Chaenomelis Fructus 0.1 1.36 10 8 1.22 10 7 3.12 10 6 Pinelliae Tuber 0.5 6.79 10 8 6.11 10 7 1.56 10 5 Thujae Semen 10.6 1.44 10 6 1.30 10 5 3.31 10 4 Dolichoris Semen 1) - - - Arecae Semen 3.0 4.07 10 7 3.67 10 6 9.37 10 5 Zizyphi Semen 1.0 1.36 10 7 1.22 10 6 3.12 10 5 Nelumbinis Semen 0.7 9.51 10 8 8.56 10 7 2.19 10 5 Curcumae Radix 1.5 2.04 10 7 1.83 10 6 4.69 10 5 Polygalae Radix 0.8 1.09 10 7 9.78 10 7 2.50 10 5 Myristicae Semen - - - Hoveniae Semen Cum Fructus 0.1 1.36 10 8 1.22 10 7 3.12 10 6 Crotonis Semen 4.0 5.43 10 7 4.89 10 6 1.25 10 4 Armeniacae Semen 0.4 5.43 10 8 4.89 10 7 1.25 10 5 Carthami Flos 0.4 5.43 10 8 4.89 10 7 1.25 10 5 * AFB1 Mean; aflatoxinb1 Mean ; Not Detected. 한수준으로판단되지만, 약용식물의경우한가지이상의곰팡이독소에오염될가능성이많기때문에여러독소에의한상승효과를억제하기위하여, 약용식물에다른곰팡이독소의허용기준을추가로설정하여관리해야될것으로사료된다. 또한소량의곰팡이독소라도여러경로를통하여장기간섭취하면만성적인중독이가능하므로, 소량이라도섭취하지않는것이중요하다. 위해성평가한약재의복용으로인한아플라톡신 B1의만성일일섭취량및초과발암위해도는 Table 8과같다. 초과발암위해도는일반적으로발암성물질에의한위해도를말하며, 개인이유해오염물질에장기간인체노출되었을때암이발생할수있는확률로써 10의 ( 1) 지수값으로나타내고, 유해물질에대한초과발암위해도는 10 5 ~10 6 의범위에서결정된다 38). U.S.EPA 39) 에서는초과발암위해도는 10 6 을초과하지않으면무시할수있는위험기준에해당하고, 초과발암위해도가 10 4 ~10 6 의범위이면일반적으로수용할수있는범위로제안하고있다. 아플라톡신 B1에오염된한약재의장기복용으로인하여간암이발생할초과발암위해도는시료중백자인이 B형간염비보균자의경우 1.30 10 5 이었고 B형간염보균자의경우는 3.31 10 4 로가장높았고, 전체시료의초과발암위해도는 B형간염비보균자의경우는 1.30 10 5 ~1.22 10 7 이었으며, B형간염보균자의경우는 3.31 10 4 ~3.12 10 6 의범위이었다. 이는아플라톡신 B1에오염된한약재를평생복용하였을경우각각만명당 1명에서천만명당 1명및천명당 3명에서백만명당 3명의비율로간암이발생되는것을의미한다. 본결과에서는 U.S.EPA 39) 에서제안된기준을적용하면 B형간염비보균자의경우는비교적안전한것으로나타났으나, B형간염보균자의경우는아플라톡신 B1이가장높게검출된시료를섭취할경우천명당 3명의비율로위험한수준으로판단된다. 일상생활에서아플라톡신B1이오염된한약재및식품을섭취함으로써초과발암위해도는증가할것으로예상할수있어, 곰팡이에오염되지않은한약재와식품을섭취하지않는것이최선의방법으로고려된다. 아플라톡신의오염을방지하기위해서는약용식물의재배에서수확후보관, 유통, 소비에이르기까지전과정에서곰팡이의오염을방지해야하며, 약용식물의최대수출국인중국과인도등의국가들이약용식물에대한품질검증절차를강화해야될것이고, 유통소비되는한약재의많은양이수입되므로정부로서도철저한정밀검사를통하여수입절차를강화해야할것으로생각된다, 요약 서울시내에서 2010년중유통된한약규격품 19종 (360 건 ) 을구입하여면역친화성칼럼과유도화장치가부착된액체크로마토그래프를이용하여아플라톡신의함량을분석

A study on Aflatoxins Analysis in The Herb Medicines 433 하였다. 그결과전체시료 360건중아플라톡신 B1은 168건 (46.4%), 아플라톡신 B2는 92건 (25.4%), 아플라톡신 G1은 137건 (37.8%) 그리고아플라톡신 G2는 88건 (24.3%) 검출되었고, 검출량은아플라톡신 B1 1.4 ± 1.8 µg/kg, 아플라톡신 B2는 0.4 ± 1.1 µg/kg, 아플라톡신 G1은 1.1 ± 5.0 µg/ kg 그리고아플라톡신 G2는 0.9 ± 3.4 µg/kg이었으며, 백자인과빈랑자를제외한모든시료에서아플라톡신 B1 허용기준 (10 µg/kg 이하 ) 에적합하였다. 위해성평가결과전체시료의초과발암위해도는 B형간염비보균자의경우는 1.30 10 5 ~1.22 10 7 이었고, B형간염보균자의경우는 3.31 10 4 ~3.12 10 6 의범위이었다. 본연구의대부분시료에서아플라톡신의오염량은적은것으로평가되었으나, 다른종류의곰팡이독소와다른경로에의한아플라톡신의섭취를감안하여, 향후아플라톡신을비롯한포괄적인곰팡이독소에관한연구가진행되어야될것으로생각된다. 참고문헌 1. Joshi, B.S. and Kaul, P.N.: Alternative medicine: Herbal drugs and their critical appraisal part 1. Prog Drug Res, 56, 1-76 (2001). 2. Ali, N., Hashim, N.H., Saad, B., Safan, K., Nakajima, M. and Yoshizawa, T.: Evaluation of a method to determine the natural occurrence of aflatoxins in commercial traditional herbal medicines from Malaysia and Indonesia. Food Chem. Toxicol, 43, 1763-1772 (2005). 3. Dubey, N.K., Kumar, A., Singh, P. and Shukla, R.: Microbial contamination of raw materials: A major reason for the decline of India's share in the global herbal market. Curr. Sci, 95, 717-718 (2008). 4. 한국보건산업진흥원. 다빈도한약재소비형태및가격구조실태조사연구. 보건복지부연구보고서, A0063-65610- 57-0109 (2001). 5. Santos, L., Marin, S., Sanchis, V. and Ramos, A.J.: Screening of mycotoxin multicontamination in medicinal and aromatic herbs sampled in Spain. J. Sci. Food. Agric, 89, 1802-1807 (2009). 6. Conner, D.E.: Naturally occuring compounds. In: Davinson, P.L., Branen, A.L. (Eds.), Antimicrobials in Foods. Marcel Dekker, New York, pp. 441-486 (1993). 7. International Agency for Research on Cancer. Some traditional medicines, Some mycotoxins, naphthalene and stylene. IARC Mongr Eval Carcinog Risks Hum, Suppl 7, 1-440 (1987). 8. Reiter, E., Zentek, J. and Razzazi, E.: Review on sample preparation strategies and methods used for the analysis of aflatoxins in food and feed. J. Mol. Nutri. Food Res, 53, 508-524 (2009). 9. Cole, R.J. and Cox, R.H.: Handbook of toxic fungal metabolites, Academic Press, New York (1981). 10. Eaton, D.L. and Groopman J.D.: The toxicology of aflatoxins; Human health, Veterinary and Agricultural Significance, 1st edn, Academic Press, San Diago, CA (1994). 11. Mido, A.F., Campos, R.R. and Sabino, M.: Occurrence of aflatoxins B1, B2, G1 and G2 in cooked food components of whole meals marketed in fast food outlets of the city of Sao Paulo, SP, Brazil. Food Addit. Contam, 18, 445-448 (2001). 12. 이종태 : 한국인의한약재복용실태조사연구. 식품의약품안전청연구보고서 (2006). 13. Moss, M.O.: Risk assessment for aflatoxins in foodstuffs. Int. Biodeterior Biodegrad, 50, 137-142 (2002). 14. The Europen Parliament and the council of the European union. directive 2004/24/EC of the European Parliament and the council of 31 March. Official Journal of the European Union L 136, 85-90 (2004). 15. International Agency for Research on Cancer. Some traditional medicines, Some mycotoxins, naphthalene and stylene. IARC Mongr Eval Carcinog Risks Hum, Lyon, 160-336 (2002). 16. World health organization. General guidelines for methodologies on research and evaluation of traditional medicines. WHO/EDM/TRM, WHO, Genova (2000). 17. Romagnoli, B., Menna, V., Gruppioni. N. and Bergamini, C.: Aflatoxins in spices, aromatic herbs, herb-teas and medicinal plants marketed in Italy. Food Control, 18, 697-701 (2007). 18. 식품의약품안전청. 생약등의잔류오염물질기준및시험방법. 식품의약품안전청고시 2009-104 (2009). 19. Gobel, R. and Lusky, K.: Simultaneous determination of aflatoxins, ochratoxin A and zearalenone in grain by new immunoaffinity column/liquid chromatography. J. AOAC Int, 87, 411-416 (2004). 20. Food and agriculture organization/world health Organization. Dietary exposure assessment of chemicals in food. Report of a joint FAO/WHO consultation, Annapolis, Maryland, USA, 2-6, May (2005). 21. 산업자원부기술표준원. 한국인의인체치수조사사업의학술용역연구결과보고서 (2004). 22. Yeh, F.S., Yum M.C., Mo, C.C., Luo, S.L, Tong, M.J., and Henderson, B.E.: Hepatitis B virus, aflatoxins and hepatocellular carcinomain southern Guangxi, China, Cancer Res, 49, 2506-2509 (1989). 23. Rief, K. and Metzer, W.: Determination of aflatoxins in medicinal herbs and plant extracts. J. Chromatogr. A, 692, 131-136 (1995). 24. Ventura, M., Gomez, A., Anaya, I., Diaz, J., Broto, F., Agut, M. and Comellas L.: Determination of aflatoxins B1, G1, B2 and G2 in medicinal herbs by liquid chromtography-tantem mass spectrometry. J. Chromtogr. A, 1048, 25-29 (2004). 25. Han, Z., Zheng, Y., Luan, L., Cai, Z., Ren, Y. and Wu, Y.: An ultra-high- performance liquid chromatogarphy-tandem mass spectrometry method for simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in traditional Chinese medicines. Anal Chim Acta, 664, 165-171 (2010). 26. Ip, S.P. and Che, C.T.: Determination of aflatoxins in chinese medicinal herbs by high-performance liquid chromatography using immunoaffinity column cleanup improvement of recov-

434 Sung-deuk Lee et al. ery. J. Chromatogr. A, 1135, 241-244 (2006). 27. Reiter E, Zentek J and Razzazi E.: Review on sample preparation strategies and methods used for the analysis of aflatoxins in food and feed. Mol. Nutr. Food Res. 53, 508-524 (2009). 28. Traag, W.A., Van Trijp, J.M., Tuinstra, L.G.M.T. and Kok, W.T.: Sample clean-up and post-column derivatization for the determination of aflatoxin B1 in feedstuff by liquid chromatography. J. chromatogr. A, 396, 389-394 (1987). 29. Rizzo, I., Vedoya, G., Maurutto, S., Haidukowski, M. and Varsavsky, E.: Assesment of toxigenic fungi on Argentinean medicinal herbs. Microbiol Res. 159, 113-120 (2004). 30. Roy, A.K. and Chourasia, H.K.: Mycoflora, mycotoxin producibility and mycotoxins in traditional herbal drugs from India. J. Applied Microbiol, 36, 295-302 (1990). 31. Tassaneeyaakul, W., Razzaai-Fazeli, E., Porasuphatana, S. and Bohm, J.: Contamination of aflatoxins in herbal medicinal products in Thailand. Mycopathologia, 158, 239-244 (2004). 32. Gomez-Catalan, J., Pique, E., Falco, G., Borrego, N, Rodamilans, M. and Liobet, J.M.: Determination of aflatoxins in medicinal herbs by HPLC. An efficient method for routine analysis. Phytochem Anal, 16, 196-204 (2005). 33. Kumar, V., Basu, M.S. and Rajendran, T.P.: Mycotoxin research and mycoflora in some commercially important agricultural commodities. Crop Protection, 27, 891-905 (2008). 34. Monbaliu, S., Wu, A., Zhang, D., Peteghem, C.V. and Saeger, D.S.: Multi- mycotoxin UPLC-MS/MS for tea, herbal infusions and the derived drinkable products. J. Agri. Food Chem, 58, 12664-12671 (2010). 35. Patkar, K.L., Usha, C.M., Shetty, H.S., Paster, N. and Lacey, J.: Effect of spice essential oils on growth and aflatoxin B1 production by Aspergillus flavus. Lett Appl Microbiol, 17, 49-51 (1993). 36. Amjad Iqbal, S., Khalil, I.A. and Shah, H.: Aflatoxin contents of stored and artificially inoculated cereals and nuts. Food Chem, 98, 699-703 (2006). 37. Bluma, R., Amaiden, M.R. and Etcheverry, M.: Screening of Argentine plant extracts: Impact on growth parameters and aflatoxin B1 accumulation by Aspergillus section Flavi. Int. J. Food Microbiol, 122, 114-125 (2008). 38. 손종렬, 이철민 : 화학물질의위해성평가. 대한환경공학회지, 29, 477-488 (2007). 39. http://www.epa.gov/region8/r8risk/hh_risk.html#cancer.