농약과학회지 (Korean J. Pestic. Sci.) Vol. 20, No. 3, pp. 197-202 (2016) Open Access http://dx.doi.org/10.7585/kjps.2016.20.3.197 Online ISSN 2287-2051 Print ISSN 1226-6183 ORIGINAL ARTICLES / SYNTHESIS / FORMULATION / CONTROL 계피또는데리스추출물을주원료로하는유기농업자재의약효성분안정성 최근형 진초롱 박병준 임성진 노진호 문병철 공승헌 1 김진효 1, * 농촌진흥청국립농업과학원화학물질안전과 1 경상대학교농업생명과학연구원응용생명과학부 (BK21 plus) The Shelf-life of Agricultural Organic Materials Containing Cinnamon or Derris Extract: Thermal Stability of Cinnamyl Derivatives and Rotenoids Geun-Hyoung Choi, Cho-Long Jin, Byung-Jun Park, Sung-Jin Lim, Jin-Ho Rho, Byung-Cheol Moon, Seung-Heon Kong 1 and Jin Hyo Kim 1, * Chemical Safety Division, National Institute of Agricultural Sciences, RDA, Wanju, Korea 1 Division of Applied Life Science (BK21 plus), Institute of Agriculture and Life Science (IALS), Gyeongsang National University, Jinju, Korea (Received on September 13, 2016. Revised on September 26, 2016. Accepted on September 28, 2016) Abstract The stabilities of bioactive compounds in cinnamon or derris extract were investigated in commercial agricultural organic materials (biopesticide) during storage on different temperature conditions (0 o C, 23 o C, 35 o C, 45 o C, and 54 o C). The selected bioactive compounds were cinnamaldehyde, and cinnamyl alchol in cinnamon extract and deguelin, and rotenone in derris extract. Half-lives of the total cinnamyl derivatives in biopesticide (A, B, C, and D) ranged from 15.1 to 46.2 days on the different temperature and cinnamaldehyde was more stable than cinnamyl alcohol in the biopesticide. The half-lives of total rotenoid ranged from 1.7 to 173 days on the different temperature in the tested biopesticide (E, F, and G) containing derris extract. The stabilities of deguelin, and rotenon in the biopesticide showed similar values in the same condition. Key words Biopesticide stability, cinnamaldehyde, cinnamon, derris, rotenoid. 서 론 잔류화학물질에대한우려와지속가능농업에대한소비자의관심이높아짐에따라친환경농산물에대한수요와이에따른관련인증이꾸준히증가하고있다 (RDA 2011). 하지만, 각종병해충으로인해상품성이높은친환경농산물의단위면적당생산량은관행농업과큰차이를보이고있고, 이와같은문제해결을위해병해충관리용유기농업자재의수요는지속적으로증가하고있다 (RDA 2011). 현재국내에유통되는유기농업자재는목록공시제와품질인증제 *Corresponding author E-mail: jhkim75@gnu.ac.kr 를통해제품에대한품질관리와평가를받고있지만, 농자재의약효까지보장하는품질인증제등록제품은전체등록제품 1406개중 1.4% 에지나지않는다 (RDA 2016). 이와같은품질인증등록제품의절대적부족원인으로고비용이소요되는유기농업자재품질관리기술개발과여기에맞춘효능평가시스템구축을들수있다. 따라서, 국내에서주로사용되는병해충관리용유기농업자재의원료와이를주성분으로하여제조한제품에대한안정성연구를지속적으로수행하여병해충관리용유기농업자재품질관리에관한기술개발을할필요가있다. 이에따라, 작물병해충관리용유기농업자재에주로사용되는식물추출물인님, 고삼, 계피, 데리스추출물등에대한지표물질설정연구가최근보고된바있고 (Lee et al. 2013, Lim et al. 2014a, Lim et al. 197
198 최근형 진초롱 박병준 임성진 노진호 문병철 공승헌 김진효 2014b, Lim et al. 2015), 이와함께, 님과고삼추출물은주요살충성분인 azadirachtins와 matrines를지표물질로하여유기농업자재제품중안정성이보고된바있다 (Kim et al. 2015). 본연구에서는님, 고삼과함께유기농업자재원료로많이사용되는계피와데리스추출물을주원료로사용한제품에대해지표물질을중심으로제품안정성을연구하였다. 계피의지표물질은 Lim 등 (2014a) 이보고한것을바탕으로효능성분으로다량함유되어있는성분인 cinnamaldehyde 와 cinnamyl alcohol을계피의지표물질로선정하였고, 데리스추출물에서는 deguelin과 rotenone 을품질평가지표물질로선정하였다 (Lim et al. 2015). Cinnamaldehyde와 cinnamyl alcohol은계피유에다량함유되어있는성분으로 L-phenylalanine으로부터생합성되는것으로알려져있으며, 살균과살충기능을갖는물질이다 (Liu et al. 2014, Shreaz et al. 2016). 이들물질의안정성은여러문헌에서확인할수있고, 제품의안정성과관련이깊은수중분해반감기는 15 days, 대기중광분해반감기는 cinnamaldehyde 0.31 days과 cinnamyl alcohol 0.1 days로알려져있다 (ECHA, 2016a, ECHA 2016b). 또한, 데리스의주요살충성분으로알려진 deguelin과 rotenone은빛과산소에민감한성분으로분해가매우빨라광분해반감기 <30 min, 수중분해반감기가 0.5 days로알려져있다 (Cabizza et al. 2004, Dawson et al. 2011). 따라서, 본연구에서는병해충관리용유기농업자재원료로많이사용되는계피와데리스추출물의효능성분에대한유통제품중의안정성을비교하여유기농업자재에대한유통기한설정연구에도움을주고자하였다. 재료및방법 시료계피또는데리스추출물을주원료로하는 목록공시 등록제품중국내에서제조된제품을대상으로, 계피제품의경우고상제품 2종 (Biopesticide A, B) 과액상제품 2종 (Biopesticide C, D) 을무작위선정하였고, 데리스제품은액상제품 3종 (Biopesticide E, F, G) 을시중에서구매하여안정성평가시험에사용하였다. 시험에사용된제품내포함된보조제는 Table 1에나타내었다. 표준품및시약 Cinnamaldehyde, cinnamyl alcohol, deguelin, rotenone 등표준품은 Sigma-Aldrich Co. (MO, USA) 에서구매하였다. Acetonitrile, methanol, dichloromethane, sodium sulfate anhydrous 는 Merck 사 (Darmstadt, German) 제품을사용하였으며, 시료전처리에사용되는 Hydrophilic-Liphophilic Balanced (HLB) solid-phase extraction (SPE) cartridge (60 mg, 3 ml) 는 Waters Co. (MA, USA) 제품을사용하였다. 열안정성평가식물추출물및이를주원료로하는제품을질소기류하에서용기개봉후즉시암갈색밀폐용기에나눠옮겨담고, 4 o C, 23 o C, 35 o C, 45 o C, 54 o C로설정된항온기에빛을차단하여각각보관한후일정간격으로시료를취하여유효성분함량을분석하였다. 계피추출물유효성분분석계피추출물의유효성분인 cinnamaldehyde와 cinnamyl alcohol은 Lim 등 (2014a) 이보고한방법에따라액상시료는증류수로 100배희석후시험에사용하였고, 희석된시료 1 ml를 HLB cartridge에 loading 한뒤증류수 2mL로세척후 acetone (3 ml 2) 으로 elution한뒤감압농축후 GC- FID (Agilent 7890, Agilent Co., Santa Clara, USA) 를사용하여정량분석하였다. 고상제품시료는 acetone으로추출한뒤감압농축후시험에사용하였다. 기기분석조건은아래의 Table 2에자세히나타내었다. 분석검량선은 cinnamaldehyde와 cinnamyl alcohol 모두 0.5-20 mg/l (r 2 0.999) 에서작성하였고, 전처리완료시료는 acetone으로희석후분석에사용하였다. Table 1. Ingredient list of the tested commercial biopesticides Biopesticide A Cinnamon oil, paraffin oil Biopesticide B Cinnamon oil, plant extracts Biopesticide C Cinnamon extract, plant extract Biopesticide D Cinnamon oil, plant oil, paraffin oil Biopesticide E Derris extract, detergent, emulsifier, diluents Biopesticide F Derris extract, citronella oil, others (not specified) Biopesticide G Derris extract, plant extract, ethanol Table 2. Instrumental condition for the analysis of cinnamaldehyde and cinnmayl alcohol Instrument Agilent 6890 with FID detector Column RTX-5 (30 m 0.25 mm, 0.25 µm, Restek, PA, USA) Gas (Flow rate) He (6 ml min 1 ) Temperature Injector : 230 o C, Detector 300 o C Oven Temp. Initial 70 o C (2 min holding), Ramping 3 o C min 1 (93 o C), 30 o C min 1 (130 o C), 3.5 o C min 1 (140 o C), 40 o C min 1 (300 o C).
계피또는데리스추출물을주원료로하는유기농업자재의약효성분안정성 199 데리스추출물유효성분분석. 데리스추출물을함유한유기농자재의주요지표성분인 deguelin과 rotenone의분석은 Lim 등 (2015) 의방법에따라액액분배법과 HLB cartridge를사용한정제를수행하였다. 먼저, 증류수 (distilled water, DW) 로 10배희석한유기농자재를 dichloromethane을사용하여분액정제하고, 감압농축한다음 2mL의 5% methanol 수용액으로재용해하여정제용시료로사용하였다. HLB cartridge (60 mg, 3 ml) 에정제용시료 2mL를 loading 한뒤 2mL의 5% methanol 수용액을흘려주어세정하고, methanol 5 ml로지표성분을용출하였다. 용출액은감압농축후 methanol로재용해한뒤 UPLC를이용하여분석하였다. 기기분석에는 Acquity BEH-C18 (1.7 µm, 2.1 100 mm, Waters Co., Boston, USA) 칼럼을사용하여 0.05% formic acid 와 acetonitrile을이동상으로사용하여 Lim 등 (2015) 의조건에따라 295 nm 에서정량분석을실시하였다. 분석검량선은 deguelin과 rotenone 모두 0.1-20 mg/l (r 2 0.999) 에서작성하였으며, 전처리완료시료는 methanol로희석후분석에사용하였다. 결과및고찰 계피추출물을주성분으로한유기농업자재계피추출물을주성분으로하는유기농업자재의유효성분중품질평가지표성분은 cinnamaldehyde와 cinnamyl alcohol로정하고, 이에대한제품중열안정성을평가하였다. 시험에사용된계피추출물을사용한유기농업자재 4종 (Biopesticide A, B, C, D) 중고상제품 2종 (Biopesticide A, B) 과액상제품 2종 (Biopesticide C, D) 에대한분석결과, 제품내 cinnamyl 유도체총함량은 3.4-16.4% 로확인되었으며 (Table 3), 이를기준으로농약에적용되는유통기한설정에적합한유효성분허용범위는 90-110% 로확인되었다 (Kim et al. 2015). 최대 30일까지진행된시험에서시험기간중층분리, 침전물발생과같은물성변화는관찰되지않았다. 반면, cinnamyl 유도체의총함량은 54 o C에서 14일 보관후 >10% 지표성분감소가관찰되었으며, 45 o C 이하의시험조건에서도농약에설정된유통기한설정 (1년) 규정을충족시키지는못하였다. 보관온도에따른 cinnamyl derivatives의제품별분해속도 k는 single exponential equation을사용하여산출하였으며, 상온보관을기준으로총 cinnamyl derivatives의 k 0.018-0.097로 (Fig. 1), 이에따른반감기는 7.1-38.5 days로나타났다 (Table 4). Cinnamaldehyde와 cinnamyl alcohol에대한각성분별제품내분해속도는 cinnamyl alcohol이 cinnamaldehyde보다최대 5배 (Biopesticide D) 빨랐으며, 모든시험대상제품에서 cinnamyl alcohol의안정성이 cinnamaldehyde보다낮은것으로나타났다 (Table 5). 이는 cinnamyl alcohol의이화학적안정성에관한여러문헌과일치하는결과이다 (ECHA 2016b). 처리온도별분해반감기는 Table 4에나타낸바와같이 15.1-43.3 days (Biopesticide A), 21.7-46.2 days (Biopesticide B), 10.0-13.1 days (Biopesticide C), 4.7-9.4 days (Biopesticide D) 로보관온도가낮을수록지표물질의안정성이높아지는것으로확인되었다. 특히, 액상제품의분해반감기는물질이 Fig. 1. An example for stability of total cinnamyl derivatives (cinnamaldehyde and cinnamyl alcohol) in commercial biopesticides during storage at 23 o C. Table 3. Total contents of the selected bioactive compounds in the biopesticides Bioactive compound contents (%) Cinnamaldehyde Cinnamyl alcohol Deguelin Rotenone Biopesticide A 16.4 0.03 ND a) ND Biopesticide B 3.4 ND ND ND Biopesticide C 10.8 0.27 ND ND Biopesticide D 6.02 0.19 ND ND Biopesticide E ND ND 0.13 0.19 Biopesticide F ND ND 1.56 2.05 Biopesticide G ND ND 1.52 1.21 a) Not detected.
200 최근형 진초롱 박병준 임성진 노진호 문병철 공승헌 김진효 Table 4. Half-life of total cinnamyl derivatives or rotenoids in commercial biopesticide under different temperature Half-life (days) a) 4 o C 23 o C 35 o C 45 o C 54 o C Biopesticide A b) 43.3 30.1 24.8 13.9 15.1 Biopesticide B b) 46.2 38.5 34.7 21.7 21.7 Biopesticide C b) 13.1 11.7 11.6 10.5 10.0 Biopesticide D b) 9.4 7.1 7.1 5.1 4.7 Biopesticide E c) 25.0 6.7 2.9 2.4 1.5 Biopesticide F c) 173 138 30.9 3.7 3.5 Biopesticide G c) 92.4 77.0 58.9 8.8 6.6 a) The half-life was mean value of triplicate replication (R 2 >0.60). b) The values were calculated with the total cinnamyl derivatives. c) The halflife was calculated with the total rotenoids Table 5. Half-life of cinnamaldehyde, cinnamyl alcohol, deguelin and rotenone in commercial biopesticide at 23 o C Half-life (days) at 23 o C Cinnamaldehyde Cinnamyl alcohol Deguelin Rotenone Biopesticide A 30.1 21.7 NT a) NT Biopesticide B 38.5 NT NT NT Biopesticide C 11.9 52.1 NT NT Biopesticide D 57.2 51.4 NT NT Biopesticide E NT NT 5.9 7.1 Biopesticide F NT NT 138 138 Biopesticide G NT NT 77.0 86.6 a) Not tested. 갖는수중분해반감기 (15 days) 와유사하였고, 이는제품내포함된물이물질의분해과정에직접관여하여나타난결과로추정되었다. 이와달리, 고상제품은물과의접촉이제한되어상대적으로긴반감기를나타낸것으로나타났다. 따라서, 계피와같이안정성이낮은효능물질을포함한제품의유통기한연장을위해서는저온유통혹은질소충진포장, 천연안정제사용과같은기술개발이보완되어야할것으로판단된다. 데리스추출물및이를주성분으로한유기농업자재데리스추출물을주성분으로하는유기농업자재 3종 (Biopesticide E, F, G) 에대해서는 rotenoid계효능성분인 deguelin과 rotenone을지표물질로설정하여제품내지표성분의안정성을평가하였다. 안정성평가는설정온도에서최대 180일간보관하면서관능적변화와지표성분변화를관찰하였다. 시험에사용된유기농업자재에포함된총 rotenoid 함량은 0.32-3.61% 로확인되었다 (Table 3). 상온보관시험결과, 총시험기간동안데리스추출물을주성분으로한유기농업자재의관능적품질변화는관찰되지않았다. 또한, 54 o C에서 14일보관시평가대상제품 3종모두 rotenoid 함량이기준농도이상감소하여유통기한 1년기준을충족시 Fig. 2. An example for stability of total rotenoids (deguelin and rotenone) in commercial biopesticides during storage at 23 o C. 키지못하는것으로확인되었다. 상온에서총 rotenoids 를기준으로한제품별분해속도 k 는 0.005-0.103으로확인되었으며 (Fig. 2), 이에따른반감기는 6.7-138 days로나타났다 (Table 4). 제품내포함된 deguelin 과 rotenone에대한각성분별분해속도 k를비교하면, deguelin은 0.005-0.117이었고, rotenone은 0.005-0.097로확인되었다. 이에따른반감기는 deguelin이 5.9-138 days,
계피또는데리스추출물을주원료로하는유기농업자재의약효성분안정성 201 rotenone이 7.1-138 days로제품에따라각주요성분의반감기가큰차이를나타내었다 (Table 5). 하지만, 동일제품내 degeulin과 rotenone의안정성은유사한것으로확인되었다. 보관온도별분해반감기는 Table 4에나타낸바와같이총 rotenoid 함량을기준으로볼때, 1.5-25.0 days (Biopesticide E), 3.5-173 days (Biopesticide F), 6.6-92.4 days (Biopesticide G) 로낮은온도에서지표성분의안정성이높은경향을보였다. 이와같은시험결과는물질이갖는광분해반감기 (<0.5 h) 및수중분해반감기 (0.5 days) 와큰차이를보이고있으며, 이는 rotenoid계물질의분해반응에물이외의인자가더중요한역할을하는것으로판단된다. 또한, 제품별반감기차이가크게나타나는것은추출물과함께제공된여러보조제의영향으로고려되며, 제형에관한추가연구가요구된다. 이와더불어, 본연구에서선정한지표성분이전체약효를대표할수있는지에대한보완연구가필요할것이다. 감사의글 본연구는농촌진흥청공동연구사업 ( 과제번호 : PJ010896) 의지원에의해이루어진것입니다. Literature cited Cabizza, M., A. Angioni, M. Melis, M. Cabras, C. V. Tuberoso and P. Cabras (2004) Rotenone and rotenoids in Cubè resins, formulations and residues on olives. J. Agric. Food Chem. 52:288-293. Dawson, V. K., W. H. Gingerich, R. A. Davis and P. A. Gilderhus (2011) Rotenone persistence in freshwater ponds: Effects of temperature and sediment adsorption. N. Am. J. Fish Manage. 11:226-231. ECHA (2016a) Cinnamaldehyde - Biodegradation in water and sediment: simulation tests. https://echa.europa.eu/registrationdossier/-/registered-dossier/14462/5/3/3. ECHA (2016b) Cinnamyl alcohol - Biodegradation in water and sediment: simulation tests. https://echa.europa.eu/ registration-dossier/-/registered-dossier/12023/5/3/3. Kim, J. H., G. H. Choi, J. E. Kang and B. J. Park (2015) Stability of representative active compounds on commercial biopesticides based on neem or Sophora flavescens extract under controlled temperature. Korean J. Pestic. Sci. 19:88-92. Lee, J. W., C. L. Jin, K. C. Jang, G. H. Choi, H. D. Lee and J. H. Kim (2013) Investigation on the insecticidal limonoid content of commercial biopesticides and neem extract using solid phase extraction. J. Agric. Chem. Environ. 2:81-85. Lim, S. J., D. Y. Jeong, G. H. Choi, B. J. Park and J. H. Kim (2014a) Quantitative analysis of matrine and oxymatrine in Sophora flavescens extract and its biopesticides by UPLC. J. Agric. Chem. Environ. 3:64-73. Lim, S. J., J. H. Lee, J. H. Kim, G. H. Choi, N. J. Cho and B. J. Park (2014b) Quantitative analysis of cinnamaldehyde, cinnamylalcohol and salicylaldehyde in commercial biopesticides containing cinnamon extract using gas chromatography-flame ionization detector. Korean J. Environ. Agric. 33:213-219. Lim, S. J., J. H. Kim, G. H. Choi and B. J. Park (2015) Quantitative analysis of rotenone and deguelin in biopesticides containing derris extract by ultra performance liquid chromatography. Korean J. Environ. Agric. 34:27-31. Liu, X. C., J. Cheng, N. N. Zhao and Z. L. Liu (2014) Insecticidal activity of essential oil of Cinnamomum cassia and its main constituent, trans-cinnamaldehyde, against the Booklice, Liposcelis bostrychophila. Trop. J. Pharm. Res. 13:1697-1702. RDA (2011) Organic is life. In RDA Interrobang. Eds. B. S.Lee, J. S. Shin, W. S. Cho, C. Y. Min, Rural Development of Administration, Suwon, Korea. pp 1-20. RDA (2016) List of organic farming material, Rural Development Administration. http://nongsaro.go.kr/portal/ ps/psa/psab/psabl/openapipurchaseinfolst.ps?menuid= PS02479 Shreaz, S., W. A. Wani, J. M. Behbehani, V. Raja, M. Irshad, M. Karched, I. Ali, W. A. Siddiqi and L. T. Hun (2016) Cinnamaldehyde and its derivatives, a novel class of antifungal agents. Fitoterapia 112:116-131.
202 최근형 진초롱 박병준 임성진 노진호 문병철 공승헌 김진효 계피또는데리스추출물을주원료로하는유기농업자재의약효성분안정성 최근형 진초롱 박병준 임성진 노진호 문병철 공승헌 1 김진효 1, * 농촌진흥청국립농업과학원화학물질안전과 1 경상대학교농업생명과학연구원응용생명과학부 (BK21 plus) 요약계피추출물의지표성분으로알려진 cinnamaldehyde 와 cinnamyl alcohol, 데리스추출물의지표성분인 deguelin 과 rotenone 을대상으로유기농업자재제품중안정성을평가하였다. 시험은 4 o C, 23 o C, 35 o C, 45 o C, 54 o C 에서각각진행하였으며, 계피추출물을주성분으로하는유기농업자재중고상제품 (Biopesticide A, B) 의유효성분반감기가 15.1-46.2 days 로액상제품 (Biopesticide C, D) 에비해보관중안정성이높게나타났다. 또한, 동일제품내에서는 cinnamaldehyde 의안정성이 cinnamyl alcohol 보다높은것으로나타났다. 데리스추출물을사용한제품 (Biopesticide E, F, G) 의 rotenoid 계유효성분반감기는 1.5-173 days 로제품별보관온도에따른지표성분안정성이큰차이를나타내었으며, 동일제품에서 deguelin 과 rotenone 의안정성은큰차이를나타내지않았다. 색인어 계피, 데리스, 안정성, 로테논, 신남알데히드, 생물농약