Journal of Korean Society of Oral Health Science 2018, March. 6(1): 39~44 ISSN: 2288-6079 급성안면부통증모델에서한약재료 ( 용규, 포공영, 마치현, 토복령 ) 추출물의통증조절효과 김희진, 김윤경, 최자형, 이민경 동의대학교일반대학원보건의과학과 Anti-nociceptive effects of herb extract (Solanum nigrum L., Taraxacum platycarpum, Portulaca oleracea L., Smilax china) on acute orofacial pain in rats Hee-Jin Kim, Yun-Kyung Kim, Ja-Hyung Choi, Min-Kyung Lee Department of Biomedical Health Science, Dong-Eui University Correspondence to Lee M-K, Tel: +82-51-890-4238 Fax: +82-0505-182-6878 E-mail: lmk849@deu.ac.kr Received Feb 28, 2018 Revised Mar 5, 2018 Accepted Mar 16, 2018 ABSTRACT Solanum nigrum L., Taraxacum platycarpum, Portulacea oleracea L., and Smilax china are natural products which have an antibiotic effect. They are known for being anti-oxidant, anticancer, anti-nociceptive and detoxification in effect. In oriental medical science they are renowned for protecting kidney and urinary functions. However, the study of influence on relieving inflammatory orofacial pain is insufficient. So, this study deals with influence on orofacial pain control. Experimental 7 to 8-week-old male Sprague-Dawley rats weighing 240 280g were used. In order to induce acute orofacial pain, 50 μl formalin (5%) was injected under the vibrissapad of the rats. The rats were divided into 6 groups (n=3); formalin, formalin after distilled water (vehicle) or 4 herb extracts (T. platycarpum, S. nigrum L., P. oleracea L and S. china). The number of reactive behavioral responses including rubbing or scratching of the facial region was recorded for 9 successive 5-min intervals following formalin injection. Inflammatory orofacial pain was substantially reduced in the 4 herb extracts compared to the control group (vehicle). The pain was reduced in order of (T. platycarpum, S. nigrum L., P. oleracea L and S. china). According to the results, the 4 herb extracts can be considered as potential medicine for lessening acute inflammatory pain in orofacial area. Key words : Herb extracts, Orofacial pain, Formalin Ⅰ. 서론 통증제어는통증을완화시키는것으로, 의료에있어서필수적이다. 통증제어방법으로는약물, 신경차단, 전기자극등다양하다 [1]. 통증을치료하기위해염증반응의주요매개체를억제하는비스테로이드성약물 (non-steroidal antiinflammatory drugs) 이주로사용되고있으나, 이들약물은위 장관독성, 특히장기간복용시혈전증등의부작용을일으킬수있어서사용에주의가요구되고있다 [2]. 이에화학약품대신자연적이고건강을보하는한방제품과의약품을찾는소비자들이증가하고있으며 [3], 활발한연구가진행중이다. 천연물및한약재료로연구한논문들의화합물의범주는 flavonoid가가장많이연구되어져있고, triterpenoid, saponin, coumarin 등의화합물에대한연구들도시행되어지 Copyright@ 2018 by the Korean Society of Oral Health Science (KOHS) 39
Journal of Korean Society of Oral Health Science Vol. 6 No. 1 March 2018 고있다 [4]. 이중식물성분의풍부한종류중하나인 flavonoid 의생리활성에관한연구는 1936년 Szent-Gyorgyi 에의해 hesperidin, rutin, eriodictin 등의모세혈관투과율을저하시킨다고보고한것이처음이며, 그는이들에대해 vitamin P 라명명하였다. 그후 flavonoid의생리및약리활성에관한많은연구가진행되어대표적인약리작용 [5] 으로항암제, 항균제, 항바이러스제, 항염증제, 면역조절제및항혈전제등과같은다양한생물학적, 약리학적활성을나타내는것으로알려져있다 [6]. Kim 등 [7] 의연구에서는생약재고량강 (Alpinia officinarum Hance) 의 MeOH 추출물이 phenylephrine 전처리로유발한흰쥐적출혈관의수축을농도의존적으로이완시킴을확인하였으며, 고량강의 MeOH 추출물이보여준혈관평활근이완효과는이들 flavonoid 화합물에기인한것으로보고하였다 [7]. 또한 Kim 등 [8] 의연구에서는 streptozotocin (STZ) 을복강투여하여당뇨를유발시킨 rat에발효홍삼을경구투여하여확인한실험에서 STZ 투여에의해증가된혈청내 alanine aminotransferase와 aspartate aminotransferase의활성이발효홍삼추출물의투여로감소하였으며, 발효홍삼추출물의투여로항산화효소활성이유의적으로증가하였음을확인하였다 [8]. 이처럼천연으로부터항산화물질을찾고자하는연구가활발히진행되고있다. 합성항산화제는탁월한효과와경제성으로폭넓게사용되고있으나, 발암의가능성과인체에대한안전성에문제가제기되기때문에다양한약용식물들이각종의약품과식품의첨가제로개발되고있다 [9]. 축뇨제통탕은신장과방광기능을보하는육미지황탕에 20여가지의약재를가미한처방이며열을내리면서독을풀어내는효능이있는금은화, 소변기능을개선하는복분자와오미자, 천연항생제라할수있는용규 (Solanum nigrum L.), 포공영 (Taraxacum platycarpum), 마치현 (Portulaca oleracea L.), 토복령 (Smilax china) 등을가미해빈뇨를개선하고염증을사라지게하는목적의한약이다 [10]. 그중생체내에서항염증작용, 혈당저하, 혈액응고저하등의효능이있는용규 [11] 와한방에서해열, 발한, 강장해독, 임파선염, 급성기관지염, 위염, 담낭염, 부인병등의치료에사용되고있으며항산화및항암효과가우수한것으로보고되고있는포공영 [12] 그리고대장균, 장티푸스균, 이질간균, 포도상균등에서현저한억제작용으로새롭게보고되어급 만성장염의높은완치율로천연항생제라고평가되고있는마치현 [13] 이천연항생제의효능을보이고있으며, 토복령또한임상에서이뇨, 해독, 소 염효능을이용하여위암, 식도염, 만성피부질환등의치료에사용되고있다. 토복령의약리효과에관한연구를통해서항돌연변이원성작용, 항암작용, phospholipase A2 (PLA2) 저해작용등이확인되었고, 염증매개물질인 inos, COX-2의발현을억제하는효과가있는것으로밝혀졌다 [14]. 축뇨제통탕에포함된천연항생제라할수있는용규, 포공영, 마치현, 토복령에대한여러가지효능이다양한분야에서입증되고있다. 그러나구강안면부위통증에대한연구는미비한실정이다. 본연구에서는이와같은한약재료추출물들이구강안면부위에급성통증을유발한흰쥐에서통증조절효과가있는지를알아보고자한다. Ⅱ. 연구대상및방법 1. 실험동물 실험동물은 7-8주령의 Sprague-Dawley계수컷흰쥐 (240-280 g) 를효창사이언스 (Deagu, Korea) 에서공급받아사용하였다. 23 25 의일정한환경에서 12시간주 / 야순환주기하였고물과사료는자유로이공급하였다. 행동적인억압등에의한실험전스트레스를가능한최소화하였다. 본연구는의식이있는동물의실험에관한통증연구학회의윤리적규정을준수하고동의대학교동물실험윤리위원회의의승인 (A2017-022) 을받아연구를시행하였다. 2. 시약준비한약재료용규, 포공영, 마치현, 토복령은부산진전통약재시장 (Busan, Korea) 에서구입하였다. 4가지의건조된한약재료를각 20 g씩전기식지시저울 (DRETEC사, Japan) 로정확히달고각각 500 ml의증류수와혼합하여 1시간동안열수추출한뒤여과한후사용하였다. 4가지의한약재료의총추출량은각각 250 ml로측정되었으며, 4 냉장보관하여실험에이용하였다. 실험군은 formalin 주입군, distilled water (DW) 투여 30분경과후 5% formalin 주입군, 한약재료추출물 ( 용규, 포공영, 마치현, 토복령 ) 투여 30분경과후 5% formalin 주입군으로분류하였다. 40
Anti-nociceptive effects of herb extract (Solanum nigrum L., Taraxacum platycarpum, Portulaca oleracea L., Smilax china) on Acute Orofacial Pain in Rats 3. Formalin 으로유도한급성안면부통증 formalin으로유도한급성안면부통증반응은 Park[15] 등의연구에서사용된방법과동일하게수행하였다. 실험동물의스트레스를최소화하기위해플라스틱통에 10분이상적응시켰으며대조군 (n=3) 과실험군 (n=3) 은실험동물에각각 distilled water 1mL과한약재료추출물 1 ml를경구투여한뒤 30분경과시오른쪽안면부에 5% formalin (50 µl) 을인슐린주사기 (31 gauge) 를이용하여주입하였다. 주입직후부터 5분간격으로 45분동안통증행위반응을관찰하였으며약물이주입된안면부를문지르거나긁는행위를관찰하였다. 4. 통계분석실험결과는 IBM SPSS Statistic ver. 22.0 (IBM Co., Armonk, NY, USA) 을이용하여다중그룹에서일원배치분산분석을하였고, 사후분석은 LSD post-hoc test를이용하였다. Sigmaplot 2001 (SPSS Inc., Chicago, IL, USA) 을이용하여그래프를나타내었다. 모든결과는평균 ± 표준오차 (standard error of the mean) 로표시하였고통계적인비교를위하여유의수준은 0.05로설정하였다. Ⅲ. 결과 1. 행위반응에서용규, 포공영, 마치현, 토복령의안면부통증조절효과 실험동물의안면부피하에 formalin을주입하여유도한안면부통증모델에서한약재료추출물투여로인한통증행위반응의결과는 <Fig. 1> 과같다. 1차통증행위반응은 formalin 주입군, 대조군 (Veh+ F), 용규, 포공영, 마치현, 토복령추출물주입군간의유의한차이가없었으며, 2차통증행위반응에서는 formalin 주입군, 대조군에비해한약재료추출물주입군모두통증행위반응이유의하게감소됨을나타내었다. <Fig. 1> The effects of herb extracts on behavior responses following injection of formalin (5%, 50 µl) into vibrissa pad. herb extracts reduced behavioral responses during the second phase compared with 5% formalin-treated group. *p<0.05 vs. F, #p<0.05 vs. vehicle (veh)+f. S.N: Solanum nigrum L., T.P: Taraxacum platycarpum, P.O: Portulaca oleracea L., S.C: Smilax china 2. 시간흐름에서용규, 포공영, 마치현, 토복령의안면부통증조절효과 통증행위반응은시간의경과에따라변화를나타내었으며한약재료추출물투여로인한통증행위반응의조절효과는 <Fig. 2> 과같다. formalin의주입으로인한통증행위반응은 15분이후부터증가하여 20분에가장높게나타났으며 35분까지지속되다가 40분에감소되었다. 한약재료추출물주입군의경구투여에서는포공영이 20분에서 30분사이가장유의하게감소하였으며마치현, 용규, 토복령순으로모두통증행위반응이유의하게감소하였다. 41
Journal of Korean Society of Oral Health Science Vol. 6 No. 1 March 2018 <Fig. 2> Time response curve for number of scratches in formalin after adminstration of herb extracts. herb extracts significantly reduced formalin-induced nociceptive response at 15 40 min.(n=3 in each group). *p<0.05 vs. F, #p<0.05 vs. vehicle (veh)+f. Ⅳ. 고찰및결론 통증은많은질환에서나타나는중요한증상이다. 통증을발생시키는자극은조직손상, 염증발생등에의해통증성신경전달물질이유리되고신경전달물질이세포막의수용기에작용하여흥분성활동전위를야기한다. 말초의자극에의해일차구심성신경이활성화되어전기적임펄스를발생하고, 이신호가일련의신경전달과정을거치면서중추신경계에전달되어인지된다. 이러한통증은생체의이상을개체에게알리는경고신호로써생체를보호하기위한방어기전이다. 그러나통증이방어적인역할을다한뒤에도계속남아있게되면, 이통증자체가하나의질병이된다 [1,16]. 구강안면부통증 (orofacial pain) 은두부, 안면및모든구강내구조물의경조직및연조직과관련된통증을포함하는용어로서, 치아질환을비롯하여측두하악장애뿐아니라삼차신경통같은신경병성통증까지다양한질환들이유사한형태의동통으로발현되므로진단이어려우며, 두통, 근골격성통증, 신경성통증, 심인성통증, 암등의중요한질환으로부터발생하는통증을모두포함하고있다 [17]. 이러한통증을조절하기위해마약성진통제, 비마약성진통제, 향정신성약물그리고비스테로이드성소염진통제 (nonsteroidal anti-inflammatory drugs, NSAIDs) 등을사용하고있으며, 이중 NSAIDs의치료효과는주로 cyclooxygenase (COX)-2의억제로이루어지고 COX-1의억제는위장관의부작용을일으킨다고한다. NSAIDs는현재세계적으로가장많이처방되고사용되는약이나, 현재까지이러한부작용을피할수없는것으로되어있으며, NSAIDs는많은부작용을가지고있어간, 신장, 피부, 위장관등에영향을미치는데특히위장관에발생하는부작용은 COX-1 억제의결과로보고있으며, 장관점막의보호작용과항상성유지를담당하는 prostaglandin (PG) 이억제됨으로서위장관점막의손상을초래한다고알려져있다 [18]. 따라서 COX-1보다는 COX-2를선택적으로저해하는것이염증억제를위해중요하다. Jeong[19] 의연구에서는아로니아추출물의항산화제와항알레르기치료제로써의유효성을알아본결과 COX-1과 COX-2의저해비율비교를통해 COX-2를선택적으로저해하고있음을확인하였으며, 천연물인아로니아추출물은항산화및항알레르기효능을갖고있으며, 알레르기성비염이나아토피와같은알레르기관련질병치료에유효하게사용될것이라시사하여 [19], 천연물에대한효능을입증하였다. 본연구에시약으로사용된한약재료중용규와관련된실험을살펴보았을때 Chang[20] 은용규를메탄올에추출한추출물의투여로인간골수단핵구성백혈병세포에대한세포독성및 Apoptosis 에미치는영향과그기전을알아보았다. 그결과백혈병세포를자멸하게유발하는 cytochrome C 유리양이증가됨을확인하였으며 [20], 포공영에관한 Lee[21] 등의실험에서는포공영의에탄올추출물의중금속중독에의한손상억제효과를측정하였다. 현대사회의환경으로부터오염이우려되는납과수은을실험동물에노출시켰고그결과포공영을투여받은실험군은중금속투여로인하여발생하는체중감소의개선효과가나타났으며, 혈액세포의변화에있어서도중금속노출로감소되었던 RBC (red blood cell count) 의수를증가시킴과동시에증가된 WBC (white blood cell count) 및 platelet의수를정상수준으로회복시켜포공영에탄올추출물이체내노출된납과수은에대한독성을완화시켜주는것으로확인하였다 [21]. Joo[22] 등의연구에서마치현추출물이 mouse macrophage RAW 264.7 cells의세포생존율과지질다당체 (lipopolysaccharide; LPS) 로유발된 RAW 264.7 cells의 nitric oxide (NO), vascular endothelial, growth factor (VEGF), platelet derived growth factor (PDGF)-BB 등의염증촉발인자 42
Anti-nociceptive effects of herb extract (Solanum nigrum L., Taraxacum platycarpum, Portulaca oleracea L., Smilax china) on Acute Orofacial Pain in Rats (proinflammatory mediator) 와세포내칼슘의생성증가에미치는영향을측정하여, 대식세포에독성을유발하지않으면서 LPS로유발된대식세포의 NO, VEGF, PDGF-BB, 세포내 calcium의생성증가를억제하는등의항염효능을보고하였다 [22]. 축뇨제통탕에천연진통제및항생제로사용된용규, 포공영, 마치현, 토복령의효능을선행연구에서확인하였으며, 이러한한약재료의효능을기초로하여본연구는다양한통증이존재하는구강안면부위영역에한약재료추출물의통증조절에대한효과를확인하고자하였다. 한약재료추출물투여후안면부피하에 formalin을주입하여유발한통증모델에서포공영, 용규, 마치현, 토복령순으로안면부통증행위반응을유의하게감소시킴을확인하였으며, 이번연구에서는축뇨제통탕에천연항생제로포함된용규, 포공영, 마치현, 토복령각각의안면부통증행위반응에대해실험하였으며유의한결과를입증하였다. 후속연구에서는이들의병용효과에대한연구를통해서혼합시나타날수있는상승효과를연구할필요가있다고사료되며, 추가적인연구를통하여향후천연물진통제및소염제개발에있어서유용한후보물질이될수있을것으로생각된다. 참고문헌 1. Ko SH, Kang SK, Auh QS, Kim EC, Hong JP, Chun YH: Effect of Peripheral Opioid Receptor on Masticatory Muscle Pain Control. Korean Journal of Oral Medicine 38(2):164-174, 2013. 2. Kim YK. Lee MK: Analgesic Effects of Triptolide and N -nitro-l-arginine Methyl Ester in Rat s Temporomandibular Joint Pain Model. Journal of Dental Hygiene Science 15(6):800-806, 2015. 3. Kim JY: Political Economy of Traditional Knowledge: Bioeconomization of Korean Medicine and the Dispute on Botanical Drug. Discourse 201 17(1):77-111, 2014. 4. Park HR, Park EH: Development of Anti-Obesity functional foods in Herbal Medicine. Journal of the Korea Academia-Industrial cooperation Society 2:748-750, 2014. 5. Moon TC, Park JO, Chung KW, et al.: Anti-inflammatory Activity of the Flavonoid Components of Lonicera japonica. Yakhak Hoeji 43(1):117-123, 1999. 6. Kim HP, Son KH, Chang HW, Kang SS: Effects of Naturally Occurring Flavonoids on Inflammatory Responses and Their Action Mechanisms. Natural Product Sciences 6(4):170-178, 2000. 7. Kim HJ, Yoo MY, Kim KH, et al.: Vasorelaxation Effect of the Flavonoids from the Rhizome Extract of Alpinia officinarum on Isolated Rat Thoracic Aorta. Kor. J. Pharmacogn 37(1):56-59, 2006. 8. Kim HJ, Lee SG, Park SJ, et al.: Antioxidant Effects of Extracts from Fermented Red Ginseng Added with Medicinal Herbs in STZ-induced Diavetic Rats. Korean Journal of Food Science and Technology 44(3):367-372, 2012. 9. Ahn SK, Goo YM, Ko KH, et al.: Study on the Evaluation of Nutritional Values and Antioxidant Activities for Herbal Medicine By-products. Journal of Agriculture & Life Science 48(2):101-110, 2014. 10. Son GJ, Oh HJ, Lee JG, Nam SK, Kim CJ, Cho CS: Clinical Study of 25 Interstitial Cystitis Patients. The Korean Journal of Internal Medicine 33(2):222-230, 2012. 11. Cho SK, Kim MK, Lee JJ, Kwak YC, Lee SC, Lee Y: Effects of the Solanum nigrum L. and Glycyrrhiza uralensis Fisch for the Substitution of Antibiotics and the Performance on Broiler Chickens. Korean Journal of Poultry Science 36(3):215-222, 2009. 12. Lim AK, Kim JO, Jung MJ, Jung HK, Hong JH, Kim DI: Functional Biological Activity of Hot Water and Ethanol Extracts from Taraxaci herba. Journal of the Korean Society of Food Science and Nutrition 37(10):1231-1237, 2008. 13. Park SR, Han JW, Kang JY, Kil KJ, Yoo JH: Antioxidant Activities of Hot Water and Ethanol Extracts from Portulacae herba. The Korea Journal of Herbology 32(4):39-46, 2017. 14. Oh SW, Kim BW: Anti-inflammatory Effects of Smilacis 43
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