J. Exp. Biomed. Sci. 11 (2005) 237 242 Effects of the Hovenia dulcis THUMBER var. Koreana Nakai Extracts on Hepatoprotective Activities and Improvement of Hepatofunction Hyun-Ik Hwang, In-Soon Lee 1, Hee-Jun Chae and Hae-Yeon Moon Division of Food, Biological and Chemical Engineering, 1 Phytochemical Co., Ltd. Business Incubating Center, Daegu University, Gyeongbuk 712-714, Korea The Hovenia extracts showed a significant hepato-protective activity against alcohol and CCl 4 -induced hepatotoxicity. Injection of CCl 4 and alcohol induced liver injury and increased serum GOT (glutamic oxaloacetic transaminase) and GPT (glutamic pyuvic transaminase) levels in injured rats. The ADH (alcohol dehydrogenase)-like and ALDH (aldehyde dehydrogenase)-like activities in the Hovenia extracts were studied. Their activities in the extracts of Hovenia wood were higher than the extracts of other parts of Hovenia. And then Hovenia wood extracts have high effects on the alcohol and acetealdehyde degradation. The Hovenia extracts were decreased concentration of fusel oil. The ADH-like and ALDH-like activities in the Hovenia wood extract were 1.14 unit/µl, 0.33 unit/µl respectively. Key Words: Hoveina dulcis, ADH, ALDH, GOT, GPT, Alcohol 서 간 ( 肝 ) 은우리몸에서가장큰장기로우상복부에존재하며, 우리몸에필요로하는각종영양분의대사는물론뇌에필요한에너지를공급하고, 독성물질들을결합하고또해독시키는종합적인화학공장이다. 한편담즙산을분비하여지방산의분해및흡수를돕고, 여분의탄수화물, 비타민들을저장하고, 혈액의단백질을합성하고, 생체막구성에필요한콜레스테롤을합성한다. 이러한다양한기능을갖고있는간에이상이있으면곧황달, 빈혈을수반한여러가지부작용이나타난다. 간의이상원인으로는술이가장큰부분을차지하며주성분인 ethyl alcohol 이외에몸에해로운미량의 methyl alcohol, aldehyde 및 fusel oil류등의성분이포함되어있으며, 이들미량성분들이주로중추신경의마비, 두통, 속쓰림등숙취현상을유발하는요인으로밝혀지고있다 (Mcdonald 1973). 우리나라의간경변증환자의 20% 는사망시간암이합병되어있고간암환자의 80% 는간경변이같이발병되어나타난다. 간경변의원인은간암의환경적요인과같이바이러스, 영양장애, 간독성물질, 음주등모두가환경과관련이있는것들로알려져있으나정확한상 론 * 논문접수 : 2005년 5월 11일수정재접수 : 2005년 6월 7일 문혜연, ( 우 ) 712-714 경상북도경산시진량읍대구대학교공과대학식품 생명 화학공학부 Tel: 053-850-6552, Fax: 053-850-6559 e-mail: moonhy@daegu.ac.kr 관관계는아직밝혀진것이없다 (Kim et al., 2001; Choung et al., 2002; Roh et al., 2002). 기존에사용되는인공합성항암제의경우부작용으로인하여많은문제가야기되고있어이를대체할물질로서천연의생리활성물질로서간기능개선작용을갖는기능성물질을찾아내어치료제로개발하고자하는연구가활발하게이루어지고있다 (Kennedy et al., 1990; Kwak et al., 1999,). 따라서, 본연구는천연물중숙취해소와간기능개선, 간의독성물질제거효능이있는것으로알려진헛개나무 (Hovenia dulcis THUNBER var. koreana Nakai) 를이용하여간기능개선및간보호효과를검정하여생약으로써의개발가능성을타진하였다. 헛개나무는중국과일본, 우리나라에주로분포하고있는갈매나무과의낙엽교목으로서우리나라는주로강원도및황해도이남, 중부지방에분포하고있는수종으로매우드물게군락을형성하고있다. 헛개나무의약리적인기능에대한기록들을살펴보면간장의기능을좋게하고간에쌓인독을풀어주는효과가있어음주후에나타나는두통, 어지러움, 속불편함, 구취, 소갈등의다양한숙취증상을해소하는기능과위장병, 대장염등음주로인하여생긴병을고치는데효능이있는것으로알려지고있다 (Kim, 1996). 이러한헛개나무의효능을검정하기위해각부위별로추출물을얻은다음 alcohol과 acetaldehyde, fusel oil류의분해에미치는생화학적효과에대하여연구하였으며, 또한간기능에대한효능을알아보기위해간조직에미치는생리생화학적효과에대해서도확인하였다. - 237 -
재료및방법 1. 실험재료본실험에사용한헛개나무 (Hovenia dulcis THUNBER var. koreana Nakai) 는 5년생이상을재배농가로부터구입하여잎과가지, 수피, 목부부분으로나눈다음각각을잘게썰어건조후조직 100 g 당추출용매 1:10 (w/v) 의비율로하여추출하였다. 실험동물은 SD (Sprague-Dawley) 계웅성 6주령의랫트를한국실험동물협회로부터구입하여사용하였다. 2. Alcohol 분해효과검정 150 mm semicarbazide 50 µl와 50 mm sodium pyrophosphat (ph 9.6) 750 µl에 30 mm NAD + 50 µl, 3 M ethanol 50 µl, 증류수 1.5 ml의 reaction mixture에헛개나무각조직별추출물을 50 mg (dry weight) 첨가하여 37 에서 30 min 동안반응한다음생성된 NADH를 340 nm에서흡광도를측정하여산출하였다. 대조구는 Sigma사에서구입한 ADH 300 unit가생성하는 NADH의값으로하였다. 3. 숙취해소효과검정 Aldehyde dehydrogenase (ALDH) activity 측정 : 0.8 mm EDTA 100 µl와 0.8 mm mercaptoethanol 30 µl, 180 mm sodium pyrophosphate (ph 9.0) 750 µl에 30 mm NAD 50 µl, 15 mm pyrazole 50 µl, 30 mm acetaldehyde 50 µl가혼합된 reaction mixture에헛개나무각조직별추출물을 50 mg 첨가하여 37 에서 30 min 동안반응한다음생성되는 NADH를 340 nm에서흡광도를측정하여산출하였다. 대조구는 Sigma사에서구입한 ALDH 10 unit가생성하는 NADH의값으로하였다 (Lee et al., 1993). Fusel oil류에대한분해효과 : 150 mm semicarbazide 50 µl와 50 mm sodium pyrophosphat (ph 9.6) 750 µl에 30 mm NAD 50 µl, 3 M fusel oil류 50 µl, 증류수 1.5 ml과헛개나무각조직별추출물을 50 mg 첨가하여 37 에서 30 min 동안반응하여생성된 NADH를 340 nm에서흡광도를측정하여산출하였다. 대조구로는 ethanol을분해하는 ADH 300 unit가가지는효율을백분율로산출하여환산하였다. 4. CCl 4 를이용한간손상유도와치료 CCl 4 와 olive oil을 1:1로혼합한후랫트중량 100 g당 0.166 ml의 CCl 4 혼합물을 1일 1회 2일간경구투여하여간조직손상을유도하였다. 손상된간조직의치료를위해랫트중량 100 g당 0.2 ml의헛개나무각조직별추출물을 1일 2회 3일간경구투여한후해부를통하여간조직의손상및치료유무를육안으로확인하고간동맥으로부터채혈한혈액으로부터혈장만회수하여간기능지수를분석하였다. 실험동물중대조구는 CCl 4 혼합물대신동일양의증류수를투여하였다 5. Alcohol 을이용한간손상유도와치료 60 kg의성인이소주한병을섭취하는것을기준으로하여랫트중량 100 g당 1.2 ml의 alcohol을경구투여뒤 1시간경과후치료를위하여랫트중량 100 g당헛개나무각조직별추출물을 0.2 ml을경구투여하여 3일간치료를유도하였다. 이과정에서혈중 alcohol 농도의변화를알아보기위해 1시간간격으로 48시간동안렛트의꼬리동맥으로부터혈액을채취하여혈중 alcohol 농도를측정하였으며, 간기능손상과회복유무는해부를통하여육안으로관찰하였고간기능지수와혈중 alcohol 농도를측정하였다. 실험동물중대조구는 alcohol 대신동일량의증류수를경구투여하였다. 6. 간기능지수의측정간기능개선효과를진단하기위해 GOT (glutamic oxa- Table 1. The effect of the Hovenia extracts on the alcohol degradation in vitro. The control was calculated to effect of alcohol analyzed by 300 unit/ml ADH Control 1) Leaf extract Bark extract Branch extract Wood extract 23% Alcohol 300 192±6.2 a 345±23.4 462±12.1 564±24.5 a 15% Alcohol 300 201±12.5 b 360±25.3 474±8.6 b 570±25.0 1) alcohol dehydrogenase 300 unit/ml, a, b, ab Values followed by different letters are significantly different (P<0.05) Table 2. The effect of the Hovenia extracts on the acetaldehyde degradation in vitro. The control was calculated to effect of acetaldehyde degradation analyzed 10 unit ALDH per ml Control 1) Leaf extract Bark extract Branch extract Wood extract Acetaldehyde 30 mm 10 5.82±1.2 b 16.17±2.1 17.42±1.6 b 18.14±2.5 1) Aldehyde dehydrogenase (ALDH), a, b, ab Values followed by different letters are significantly different (P<0.05) - 238 -
loacetic transaminase), GPT (glutamic pyuvic transaminase) 와 total cholesterol, triglycerides 수치를측정하여비교하였다. 진단용 kit는아산제약에서생산된것을사용하였다 (Chung et al., 1982; Bickel et al., 1991). 7. 혈중 alcohol 농도측정 150 mm semicarbazide 50 µl와 50 mm sodium pyrophosphat (ph 9.6) 750 µl에 30 mm NAD 50 µl, 증류수 1.5 ml과 ADH (alcohol dehydrogenase) 10 unit가포함된 reaction mixture에채혈로얻은 serum 0.1 ml을첨가하여 37 에서 30 min 동안반응한다음생성된 NADH를 340 nm에서흡광도를측정하여분석하였다. 표준 alcohol 농도는위의 reaction mixture 에헛개나무각조직별추출물 50, 100, 300, 500, 800, 1,000 mg를각각첨가하여반응을유도한다음 340 nm에서측정된흡광도를표준 alcohol 농도로환산하여이용하였다. 높은것을확인되었지만, 잎추출물의경우다소미약한것으로나타났다 (Table 1). 이는 alcohol 분해효소인 ADH 300 unit의활성과비교한실험에서도잎추출물은 97 unit로다른조직에비해현저하게효소적활성이떨어지는것으로보아헛개나무추출물중 alcohol 분해효능이있는물질이헛개나무수령이늘어남에따라잎보다는가지나목부쪽으로약리성성분이축적되는것으로판단된다. 결과및고찰 헛개나무추출물의 alcohol 분해효과를검정하기위해시판중인주정중소주와청주를기준으로하여 23% 와 15% 로적용하였으며각각의 alcohol 농도에대한분해효과를검정하였다. Alcohol 분해에대한효과는 alcohol dehydrogenase 의생리활성에준하여 NAD + 가 NADH로변화되는농도를측정하여산출하였다. 헛개나무조직별 alcohol 분해효과를비교했을때목부추출물의 alcohol 분해효과가가장탁월한것으로나타났으며, 가지와수피또한 alcohol 분해효과가 Fig. 1. The effect of the Hovenia branch extracts on the fusel oils degradation. The control was calculated to effect of alcohol analyzed by 300 unit/ml ADH. : 23% alcohol : 15% alcohol Fig. 2. The effect of the Hovenia extracts on the GOT and GPT level in the CCl 4 -treated rats. The GOT and GPT level of CCl 4 -treated rats decreased significanly (P<0.05). GOT: Glutamate oxaloacetate transaminase, GPT : Glutamate pyruvate transaminase - 239 -
Acetaldehyde 분해효소인 ALDH 10 unit와헛개나무조직별추출물을비교한결과, 목부추출물의경우 ALDH 활성보다약 2배정도의높은분해효과를확인할수있었으며 (Table 2), 잎추출물의경우 ADH의결과와동일하게낮은활성값으로나타났다. 이는 alcohol 분해효과와유사한결과로헛개나무추출물에포함된성분이 alcohol 분해효능뿐만아니라 acetaldehyde 분해활성도가지고있으며 alcohol 섭취로인해발생하는생리적현상인숙취를해소하는약리적성분을포함하고있음을시사하는것이다. Fusel oil은 alcohol 발효시생성되는부산물로서체내흡수시두통, 메스꺼움을유발하는숙취의원인물질로알려져있다. 이들 fusel oil에대한헛개나무조직별추출물의분해효과를검정하기위해 ADH 300 unit와비교한결과가지추출물이가장분해효과가우수한것으로나타났으며, isobutanol에대한분해효율이가장높은것으로판명되었다 (Fig. 1). 이는헛개나무추출물이숙취현상을해소하는데탁월한효능이있음을보여주는것이며옛의서의기록을과학적으로뒷받침해주는중요한결과로판단된다. CCl 4 의간독성기작은간세포의과산화지질반응을일으켜간독성을나타내는것으로알려져있다. CCl 4 에의하여간손상이유도된랫트에헛개나무조직별추출물을경구 Fig. 3. The effect of the Hovenia extracts on the concentration of blood alcohol. 투여하여치료를유도한경우와헛개나무추출물로치료하지않은랫트의간을간기능지수인 GOT, GPT 수치를통하여비교하였다. GOT 수치의경우 CCl 4 에의하여손상된간에헛개나무목부추출물을투여한결과 48시간경과시정상치로회복되는것을확인하였으며, GPT 또한 48시간경과후정상수치로기능이회복되었다 (Fig. 2). 간의지방대사효과를확인할수있는척도인총콜레스테롤함량과중성지질의수치를측정하여간기능의정상유무를확인한결과목부추출물을투여한실험구에서는회복율이빠른것으로나타났다 (Table 3). 이러한결과로서헛개나무추출물은간기능개선및간세포재생에효과가있는것으로확인되었으며헛개나무조직중목부 > 가지 > 수피 > 잎의순서로간기능개선효능이우수한것으로생각된다. 헛개나무추출물의알코올분해효과를확인하기위하여실험동물인랫트에주정인소주를경구투여한다음헛개나무각조직별추출물로처리한결과 1시간경과후부터혈중알코올농도가현저하게감소하는것을관찰할수있었다 (Fig. 3). 헛개나무추출물로치료하지않은대조구와비교시혈중알코올농도가약 50% 정도빠르게감소하는것을관찰하였다. 이는헛개나무추출물이알코올을신속하게분해할수있는 ADH 활성을가지고있음을시사하고있으며, 간기능지수를나타내는 GOT, GPT 수치는 48시간경과후부터모두정상으로회복되는것을확인하였다 (Fig. 4). 총콜레스테롤과중성지질의수치회복율은목부추출물에서가장좋은결과로나타났다 (Table 4). 이결과는 CCl 4 에의한실험결과와동일한것으로헛개나무추출물중목부조직의추출물이수율면에서는낮은반면약리적인활성은가장우수한것으로확인되었다. 이들결과를종합해보면헛개나무추출물은간기능개선및생리현상회복에우수한효능이있는것으로판단되며헛개나무조직중목부, 가지, 수피, 잎의순서로간기능개선효과가높은것으로확인하였다. 이는다른천연물을이용한간기능개선연구결과와비교시약 30% 정도더높은회복율을나타내는것이다 (Jeong, 2000). 이러한결과를토대로헛개나무목부조직추출물에다른유기용매등을이용하여분획화하여추출한다음, 각추출액으로부터간암유발독성물질제거효과를조사하고더나아가간암세포에 Table 3. The effect of the Hovenia extracts to concentraton of cholesterol in blood plasma of rats 1) treated with CCl 4 Control 2) Leaf extract Bark extract Branch extract Wood extract TG 92.5±16.1 a 62.1±28.3 58.9±13.9 60.8±10.6 51.5±13.8 a TC 166.4±3.5 ab 124.2±2.4 b 119.4±2.4 121.3±1.6 b 118.0±1.0 1) Mean ± S.D (n=8), 2) No treatmented Hovenia extract a, ab Values followed by different letters are significantly different (P<0.05) TG: Triglycerides (normal value: male 50~155 mg/dl female 40~115 mg/dl) TC: Total cholesterol (normal value: 130~250 mg/dl) - 240 -
Fig. 4. The effect of the Hovenia extracts on the GOT and GPT level in the alcohol-treated rats. The GOT and GPT level of alcoholtreated rats decreased significantly (P<0.01). Table 4. The effect of the Hovenia extracts to concentration of cholesterol in blood plasma of rats 1) treated with alcohol control Leaf extract Bark extract Branch extract Wood extract TG 86.3±12.1 86.1±22.1 60.9±10.3 60.7±19.4 54.1±14.0 TC 171.0±2.9 122.7±4.9 120.1±1.7 122.0±6.1 119.1±2.4 1) Mean ± S.D (n=8), 2) No treatmented Hovenia extract a, ab Values followed by different letters are significantly different (P<0.05) TG: Triglycerides (normal value: male 50~155 mg/dl female 40~115 mg/dl) TC: Total cholesterol (normal value: 130~250 mg/dl) 대한세포분열억제효과등의항암작용에관해서연구할필요가있다. 감사의말씀이논문은 2003 학년도대구대학교학술연구비지원에의해수행된논문으로, 이에감사드립니다. REFERENCES An SY, Kim YK, Kim MH, Lee BI. Comparison of hepatic detoxification activity and reducing serum alcohol concentration of hovenia dulsis and alnus japonica. Korean J Med Crop Sci. 1999. 7: 263-268. Bae SK, Kim JH, Lee WP. The clinical significance of SGOT/ SGPT ratio in patients with primary liver cancer. Korean J Gastroenterol. 1982. 14. 167-170. Baek HS, Park SK, Yim CY. SGOT/SGPT ratios in various histologic types of hepatoma confirmed by needle biopsy. Korean J Gastroenterol. 1981. 13. 77-82. Behrens MJ, Hoerner M, Lasker JM, Liber CS. Formation of acetaldehyde adducts with ethanol-inducible P-450IIE1 in vitro. Biochem Biophys Res Commun. 1988. 154. 584-591. Bickel M, Bader E, Brocks DG, Engelbart K, Gunzler V, Schmidts HL, Vagel GH. Benefical effects of prolyl 4-hydrozylase in CCl4-induced fibroses of the liver in rats. J Hepatol. 1991. 13. 526-534. Blomstrand R, Kager L, Lantto O. The combustion of triolein-1-14 C and its inhibition by alcohol in man. Life Sci. 1973. 13. 1131-1139. Brodie NN, Maickel RP. Role of the sympathetic nervous system in drug-induced fatty liver. Ann New York Acad Sci. 1963. 104. 1049-1053. - 241 -
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