동의생리병리학회지제 22 권 2 호 Korean J. Oriental Physiology & Pathology 22(2):340 345, 2008 발효에의한오가피의항당뇨활성촉진 함성호 임병락 1 유가화 2 가선오 2 박병현 2 * 한서제약, 1 : 에이치앤비티코리아, 2 : 전북대학교의과학연구소 의학전문대학원생화학교실 Fermentation Increases Antidiabetic Effects of Acanthopanax Senticosus Seongho Ham, Byunglak Lim 1, Jiahua Yu 2, Sun-O Ka 2, Byung-Hyun Park 2 * Han Seo Pharm. Co., Ltd., 1:H&BT Co, Ltd., 2:Department of Biochemistry, Medical School and Institute for Medical Sciences, Chonbuk National University Extract of Acanthopanax senticosus has recently been demonstrated to possess significant antidiabetic potential, in accordance with the traditional use of this plant as an antidiabetic natural health product. The present study evaluated the effects of fermented extract (FE) of this plant on glucose-stimulated insulin secretion, glucose uptake, and streptozotocin-induced type 1 diabetes model. A 3 h pretreatment with FE prevented IL-1β and IFN-γ toxicity in isolated rat islets. However, it did not affect insulin-stimulated glucose uptake in C2C12 myotubes. In addition, pretreatment of mice with FE blocked the destruction of streptozotocin-induced islets and the development of type 1 diabetes. FE reduced blood glucose level, increased insulin secretion, and improved glucose tolerance in streptozotocin-treated mice, whereas nonfermented extract (NFE) had moderate effects. Immunohistochemical staining for insulin clearly showed that pretreatment with FE blocked the STZ-induced islets destruction and restored the number of islet cells that secreted insulin to the level of the control. Although the active principles and their mechanisms of action remain to be identified, FE may nevertheless represent a novel complementary therapy and a source of novel therapeutic agents against type 1 diabetes mellitus. Key words : Acanthopanax senticosus, fermentation, β cells, cytokine, streptozotocin 서론 인삼 (Panax ginseng) 이가진 saponin 배당체는장내미생물에의하여탈당화되고, 일부탈당화된 monoglycoside 또는완전탈당화된비당부 (aglycon) 가흡수되어인삼 saponin의활성을나타내는것으로알려졌다 1,2). 황금 (Scutellariae Radix) 의 baicalin, 치자 (Gardenia Furctus) 의 geniposide, 감초 (Glycyrrhiza uralensis Fisch) 의 glycyrrhizin은인삼사포닌과는 aglycon의구조는다르나모두배당체 (glycoside) 로존재하고있으며, 장내미생물의영향을받아 baicalin은 baicalein으로 3), geniposide는 monoglycoside로 4), glycyrrhizin은 glycyrrhetinic acid 5) 로대사되어흡수되는것으로알려져있다. 따라서장내세균에의한천연물질의대사과정을이해하는것은천연물질의약 * 교신저자 : 박병현, 전주시덕진구금암동 2-20 전북대학교의학전문대학원 E-mail : bhpark@chonbuk.ac.kr, Tel : 063-270-3139 접수 : 2008/01/24 채택 : 2008/02/12 리활성을이해하는데매우중요하다. 오가피 (Acanthopanax senticosus) 는오갈피라고도불리우며, 인삼, 산삼과함께오가과에속한다. 인삼과산삼이음지식물인데반해오가피는양지식물이다. 오가피에의해면역증강 6), 혈당강하 7), 항바이러스작용 8), 항암작용 9), 노화방지 10), 스트레스저항 11), 및지구력향상기능이보고되었다 12). 최근에본연구진은오가피가지질대사에중요한 lipoprotein lipase의효소활성을증가시켜중성지방의대사를촉진시킬가능성을보고한바있다 13). 배당체는그구조에따라비배당부와배당부로나뉜다. 즉비배당부에당이결합된형태를배당체라한다. 오가피에들어있는 lupane 배당체는사람의장내유산균에의해비배당부로생물전환된다는보고가있으며 14,15), 이러한비배당부가흡수되어약리효과를나타낸다는것은잘알려져있다. 대표적인예로인삼의 saponin은다마란사포닌에당이결합된사포닌으로서경구투여시쉽게흡수되지않고대신장내미생물에의하여 monoglycoside 또는 aglycon 형태로흡수되어약리효과를나타 - 340 -
발효에의한오가피의항당뇨활성촉진 낸다 1,2,16). 오가피중 (Acanthopanax senticosus araliaceae) 에함유한활성성분으로는 lignan, terpenoid, saponin, polysaccharide 등이알려져있으며, 이중에서도 oleanolate 계열의 saponin 배당체들이항당뇨활성을가진것으로보고되었다 17). 우리몸속의장내세균은다양한방법을통해인간의건강에영향을미친다. 외부에서침입한세균에의해설사, 식중독등의부작용이나타나기도하지만, 정상적인장내유산균은영양물질의흡수, 점막보호, 장내혈관생성의조절, 이물질의대사, 면역증강등을일으킨다 18-20). 이외에도장내유산균은천연물질의배당체를비배당부로전환시키는역할을수행하여천연물질의약리활성을조절한다 2,21,22). 오가피를비롯한천연물질의약리적효과는개인에따라그차이가크다. 이는아마도개인마다장내유산균의종류및숫자의차이에의해천연물질의장내대사가다르기때문일것으로추정하고있다. 이에본연구에서는이러한장내미생물의역할을근거하여유산균으로오가피추출물을발효시키고이를쥐에복용시킨후항당뇨효과에미치는영향을관찰하였다. 재료및방법 1. 세포배양, 균주및시약 C2C12 세포는 American Type Culture Collection (ATCC) 에서구입하여 DMEM 배지에서 10% fetal bovine serum, 2 mm glutamine, 100 units/ml of penicillin, 100 μg/ml of streptomycin, 2.5 μg/ml of amphotericin B를첨가한후 CO 2 incubator에서배양하였다. IL-1β와 IFN-γ는 R&D에서구입하였고, 기타다른시약은 Sigma에서구입하였다. Lactobacillus casei (ATCC 80382) 는한국미생물보존센터에서공급받았으며, MRS (Difco) 배지에증식시킨다음 colony를멸균된식염수에현탁하여 3000 rpm으로 5 분간원심분리한다음침전물을얻어다시멸균식염수로현탁하고이를발효용균주액으로사용하였다. 2. 오가피추출물 (NFE) 의조제오가피는시중에서구입하였으며, 오가피 1 kg에정제수 10 리터를넣고 80~90 를유지하면서 3 시간동안추출하고여과하였다. 다시동일하게 1 회더반복추출한다음여액을모아 60 이하를유지하면서총량이 1 kg이되도록감압농축하여만들었다. 3. 유산균에의한오가피발효물 (FE) 의조제오가피추출물 (NFE) 1 리터에포도당 10 g을가해녹인다음고온고압멸균하고실온으로냉각하고, 유산균액 10 ml를가하고 30~32 를유지하면서 30 일간배양하여배양액을만들었다. 이액을다시고온고압멸균한다음 60 이하를유지하면서진공건조하여건조물약 85 g을얻어이를오가피발효물 (FE) 로사용하였다. 4. 췌장 islet의분리췌장 islets은수컷 Sprague-Dawley rats로부터 collagenase digestion 방법에의해분리하였다 6). 분리한후 islets은 2 mm L-glutamine, 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin이첨가된 RPMI-1640 medium에서하루동안안정화시킨후실험에사용하였다. 5. 인슐린분비능분석 (glucose-stimulated insulin secretion assay) Islets에싸이토카인을 24 시간동안처리한후 3 mm D-glucose가포함된Krebs-Ringer bicarbonate buffer (25 mm Hepes, 115 mm NaCl, 24 mm NaHCO 3, 5 mm KCl, 1 mm MgCl 2, 2.5 mm CaCl 2, 0.1% bovine serum albumin) 로 3번 washing 한후 5.5 mm 또는 20 mm D-glucose에 30 분간노출시킨후분비된인슐린양을 radioimmunoassay kit (Linco Research) 를이용하여측정하였다. 6. Glucose uptake study C2C12 세포를 Krebs-Ringer phophate buffer (KRB: 25 mm HEPES, ph 7.4, 118 mm NaCl, 4.8 mm KCl, 1.3 mm CaCl 2, 1.2 mm KH 2PO 4, 1.3 mm MgSO 4, 5 mm NaHCO 3, 0.07% BSA, and 5.5 mm glucose) 로세척한후 serum-free media로 3 시간안정화시켰다. 오가피를처리한후에세포를 0.5 Ci of 2-deoxy-D-[ 3 H] glucose가포함된 KRB로 20 분간반응시켰다. 차가운 PBS를처리함으로써반응을종결시켰다. 0.1% SDS를처리하여세포를깬후 radioactivity를 liquid scintillation counter 를사용하여측정하였다. 7. 제 1 형당뇨병유발 5-6 주령의 ICR mice를사용하였다. 당뇨병을유발하기위해하루에한번씩, 5 일간 stpreptzotocin (50 mg/kg body weight; 0.2 ml) 을 sodium citrate buffer (ph 4.0) 에녹여복강내로주사하였다. 오가피의효과를관찰하기위해마우스에게 streptozotocin을주사하기전에오가피를 3 일간 5 g/kg/day씩복강내로주사하였다. 마우스는다음과같이 4 군으로나누어실험을했다. 1) 아무것도처리하지않은대조군, 2) streptozotocin 처리군, 3) 오가피 (NFE) + streptozotocin 처리군, 4) 발효된오가피 (FE) + streptozotocin 처리군 (n=7, 각군 ). 처음 streptozotocin을처리한날을 day 1으로정의하였다. 대조군은 citrate buffer만투여하였다. 5 일째마우스를희생시키고, 혈액을채취하여 1 mg/ml EDTA가들어있는 tube에보관하여 insulin과 glucose 농도를측정하였다. Glucose 농도는 glucose oxidase 방법 (Sigma) 을통해측정하였고, insulin 농도는 radioimmunoassay kit (Linco Research) 를통해측정하였다. 동물실험에관한모든사항은전북대학교실험동물위원회 (Institutional Animal Care and Use Committee at Chonbuk National University) 의승인을얻은후시행하였다. 8. Glucose tolerance test 마우스를 16 시간동안금식을시키고복강내로 glucose를 1.5 g/kg of body weight 농도로주사한후꼬리의끝을잘라 glucometer를이용하여 30 분, 1 시간, 2 시간, 3 시간에혈중 - 341 -
함성호 임병락 유가화 가선오 박병현 glucose 농도를측정하였다. 슐린에의한 glucose uptake 에아무런영향을미치지않았다. 9. 췌장조직의면역염색면역염색을위해, DAKO Envision system (DAKO) 을이용하였다. 췌장을떼어서즉시고정액 (10% formalin solution in 0.1 M PBS) 에넣은후 4 시간후에 0.1 M PBS로세척하였다. Paraffin으로 block을만든후 4 µm 두께로잘라 section을만들었다. Deparaffinization 과정을거친후에 Protein Block Serum-Free (DAKO) 로실온에서 10 분간반응시켜비특이적반응을제거하였다. 2 시간동안실온에서인슐린에대한항체 (Santa Cruz Biochemicals) 반응시켜면역염색을실시하였다. 10. 통계분석통계분석은 Student's t-test와 ANOVA를 이용하였고, p<0.05를통계적으로유의한것으로간주하였다. 결과 1. 췌장 islet에서오가피에의한인슐린분비능유지췌장 islet을분리한후 3 시간동안 500 g/ ml의오가피를전처리한후싸이토카인인 IL-1β (2 ng/ml) 와 IFN-γ (100 U/ml) 를병합하여 24 시간동안처리하였다. 대조군 islets의경우 20.1 ± 2.2 ng/ml 농도의인슐린을분비하였으나, 싸이토카인처리군은 10.4 ± 3.2 ng/ml 농도의인슐린을분비하였다 (p<0.01). 발효된오가피 (FE) 를처리한경우발효되지않은오가피 (NFE) 에비해인슐린분비능유지가뚜렷하게증가함을관찰하였다 (Fig. 1). Fig. 2. The effect of Acanthopanax senticosus extract on glucose uptake in C2C12 myotubes. C2C12 cells were pretreated with or without Acanthopanax senticosus extractas as indicated and then treated with 100 nm insulin for 2 h. Glucose uptake was measured as described in Materials and Methods. The results represent the mean ±SEM for two independent assays in triplicate. **p<0.01 vs. untreated control. 3. 당뇨병유발마우스에서오가피가혈중 glucose, insulin 및조직에미치는영향 In vitro 실험을통해확인한오가피의항당뇨효과를 in vivo 실험에서확인하고자 streptozotocin 유발당뇨쥐모델을사용하였다. ICR mice에오가피 (5 g/kg/day) 을 3 일간복강내로투여하였다. 그런다음 streptozotocin을 50 mg/kg 농도로 5일간복강내로주사하였다. 먼저혈중 glucose 농도가증가함에따라췌장 islets으로부터인슐린분비능력을측정하기위해 glucose tolerance test를시행하였다. 마우스를 16 시간굶긴후 glucose (1.5 g/kg body weight) 를당뇨유발쥐의복강내로주사했을때 60 분후에최고치에도달하였다 (Fig. 3). Fig. 1. Acanthopanax senticosus extract inhibits IL-1β and IFN-γ -induced reduction of glucose-stimulated insulin secretion in rat islets Rat islets (10 islets/500 μl) were treated with IL-1β and IFN-γ with or without a 3 h pretreatment with Acanthopanax senticosus extract. Following 24 h of incubation, glucose-stimulated insulin secretion was quantified. The results of triplicate samples are expressed as the mean ± the SEM. **p<0.01 vs. untreated control; #p<0.05 vs. IL-1β + IFN-γ. 2. C2C12 myotubes에서오가피에의한 glucose uptake에미치는영향 다음으로오가피가제 2형당뇨병에이용이가능한지를알아보기위해 C2C12 myotubes를이용하여 glucose uptake를관찰하였다. Fig. 2에서보는바와같이 C2C12 myotubes에 100 nm 농도의인슐린을처리하면 glucose uptake가약 1.8 배정도증가하였다. 오가피를전처리한경우발효여부와관계없이인 Fig. 3. Oral glucose tolerance test of diabetic mice and diabetic mice treated with Acanthopanax senticosus extract ICR mice were injected with Acanthopanax senticosus extract via i.p. in a daily dose of 5 g/kg/day, and then injected with STZ (50 mg/kg) via i.p. for 5 days. After 16 h fasting, tail blood samples (5 μl) were collected at 0, 30 min, 1h, 2h, and 3h after the intraperitoneal glucose challenge (1.5 g/kg) and measured plasma glucose. The results of triplicate samples are expressed as the mean ± the SEM. **p<0.01 vs. STZ; #p<0.05 vs. NFE of Acanthopanax senticosus. 오가피를미리먹인쥐의경우혈중 glucose 농도의증가가유의하게감소하였으며, 발효시킨오가피 (FE) 의경우그렇지않은오가피 (NFE) 에비해혈중 glucose 농도증가가더유의하게감소하였다. 또한오가피를처리한마우스에있어공복시혈중 glucose 농도와인슐린농도는 streptozotocin 유발당뇨쥐와비교했을때 glucose 농도는더낮고, 인슐린농도는더높았다 - 342 -
발효에의한오가피의항당뇨활성촉진 (Fig. 4). 마찬가지로발효오가피를사용한경우혈중혈중 glucose 농도및인슐린농도는더욱더정상쥐에가까운수치를보였다. 다음으로췌장조직을떼어 islets의파괴정도를 H&E 염색 (Fig. 5a-5d) 및인슐린에대한 immunohistochemistry (Fig. 5e-5h) 을통해관찰하였다. 당뇨병유발마우스의경우 lymphocyte의침윤과 islets의퇴행성변화를관찰할수있었다. 또한 immunohistochemistry 결과의경우에도 insulin-immunoreactive β cells이현저하게감소하였다. 하지만오가피를전처리한경우, 특히발효된오가피 (FE) 를전처리한경우 streptozotocin 유발 islets의파괴를막을수있었고, insulin에반응하는 β cells가유의하게증가하였다 (Fig. 5d & 5h). 고 찰 본연구에서오가피를발효시키면싸이토카인및 streptozotocin에대한저항성이증가하여혈당강하효과가증가됨을관찰하였다. 전통적으로오가피를비롯하여많은천연물질이임상에적용되어왔다. 하지만이런천연물질은개인마다차이를보이고, 이를한의학에서는체질에따른차이로여겨왔다 24). 또한최근의연구결과에의하면장내에기생하는세균의종 Fig. 4. The effect of Acanthopanax senticosus extract on blood glucose and insulin levels All experimental conditions for inducing diabetes were same as described in Fig. 3. Levels of fasting glucose and insulin were determined. The results of samples are expressed as the mean ± the SEM. **p<0.01 vs. untreated control; #p<0.05, ##p<0.01 vs. STZ. Fig. 5. Acanthopanax senticosus extract protects islets from STZ-induced destruction Pancreases were obtained from normal control (a, e), STZ-injected (b, f), NFE of Acanthopanax senticosus and STZ-injected (c, g), and FE of Acanthopanax senticosus and STZ-injected mice (d, h). The cellular morphologies of these same islets and of the adjoining exocrine regions were then counterstained with H-E (a-d). Islets were labeled for insulin antibody and peroxidase-labeled anti-rabit IgG, and then examined by microscopy (e-h). 류혹은그양에의해천연물질을복용한후생물학적효능에차이가나타남이보고되어왔다 2,21,22). 즉천연물질이우리몸으로흡수되기전에장내에서활성화된상태로전환되는과정이필요하다. 이에최근에는약리활성을지니는비배당부만을선택적으로얻기위한노력이진행되어왔다. 이를위해초기에는화학적으로황산또는수산화나트륨과같이산이나알카리를처리하여배당체를가수분해하는방법이시도되었다. 이경우비선택적으로가수분해가일어나고, 최종산물을추가적으로중화시키는과정이필요하다. 이런번거로움을극복한방법이장내에기생하는유산균을이용하여발효를시키는방법이다. 예를들면 Canadian blueberry juice의경우발효과정을통해 C2C12 myotubes에서 deoxyglucose의 uptake를 2 배증가시키고 25), 인삼의경우장내세균에의해 deglycosylation 된후 fatty acid esterification 과정을통해활성화된다 2). 발효된오가피의항당뇨효과를나타내는기전은일차적으로 islets의인슐린분비능을유지함에의해나타났다. 싸이토카인을 islets에처리하였을때 islets이파괴되어인슐린분비능이감소하였다. 하지만오가피를전처리하면싸이토카인에의한 islets의손상이억제되었다. 싸이토카인에의한 islets의손상기전은 NF-κB 활성과밀접한관계가있다 23). 비록본연구에서세포수준에서오가피가싸이토카인에의한 NF-κB 활성을억제함을보여주지는못했지만, 오가피특히발효된오가피를전처리하면 islets의인슐린분비능이거의정상 islets과비슷한정도로회복함을관찰할수있었다. 또한 multiple low-dose streptozotocin 을투여하는제 1형당뇨병동물모델을만들었을때형태학적으로췌장 islets에서인간의제 1형당뇨병과비슷한 immune cell infiltration, β-cell degeneration, 인슐린에대한 immunoreactivitiy 등의소견이나타났지만, 오가피를전처리했을때이런소견이사라지고, 기능적으로혈중 glucose 및인슐린농도가정상군과비슷한농도로회복됨을관찰할수있었다. 따라서오가피는제 1형당뇨병의진행예방및 islets transplantation 후생존율을높이는데좋은보조제로사용이가 - 343 -
함성호 임병락 유가화 가선오 박병현 능함을의미해주고있다. 한편오가피는제 2형당뇨병모델에서는아무런효과를보여주지못했다. C2C12 myotubes를이용하여인슐린에의한 glucose uptake를배가시키는지여부를관찰하였지만아무런효과가없었다. 최근 Liu 등의보고에의하면 26) 제 2형당뇨병모델에서오가피를처리하면혈당이감소함을관찰하였다. In vivo 조건에서인슐린의효과를증가시키는기전은다양하게일어난다. 예를들면직접인슐린신호전달체계를증가시킬수도있고, 인슐린에영향을받는지방세포나근육세포의분화및성장에영향을미쳐인슐린의효과를배가시킬수도있다 27). 본실험에서는 C2C12 myotubes를사용하여 glucose uptake를관찰하는 in vitro 실험만을수행하였기때문에제 2형당뇨병동물모델에서오가피의항당뇨효과를보일수있는가능성을배제할수없다. Liu 등의보고와다른결과를보인까닭은아마도본연구에사용한오가피의종류, 재배지역의차이및추출방법의차이등에기인할것으로추측한다. 20 세기항생제의발견은전염병으로부터인간이자유로워질수있어근대의학의가장위대한업적중의하나로평가되고있다. 하지만항생제의무분별한남용은항생제내성, 원내감염등의부작용을일으킬뿐만아니라정상적인인간장내세균의조성및수량에변화를초래하여천연물질의흡수및생물학적활성의변화를초래하였다. 천연물질은오랫동안경험을통해그안정성및효능이이입증되었고, 특정한유산균을통한천연물질의선택적인활성화는그천연물질의효능을배가시킬뿐만아니라복용량의감소로인해예상되는부작용을최소화할수있는장점이있다. 이에본연구는발효과정을통한천연물질의효능증가에대한좋은예가될수있을뿐만아니라제 1형당뇨병의예방및치료에오가피를사용할수있는이론적토대를제공할것으로생각한다. 감사의글 이논문은전북과학연구단지육성사업의지원에의하여연구되었음. 참고문헌 1. Bae, E.A., Han, M.J., Kim, E.J., Kim, D.H. Transformation of ginseng saponins to ginsenoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants. Arch Pharm Res 27: 61-67, 2004. 2. Hasegawa, H. Proof of the mysterious efficacy of ginseng: basic and clinical trials: metabolic activation of ginsenoside: deglycosylation by intestinal bacteria and esterification with fatty acid. J Pharmacol Sci 95: 153-157, 2004. 3. Akao, T., Kawabata, K., Yanagisawa, E., Ishihara, K., Mizuhara, Y., Wakui, Y., Sakashita, Y., Kobashi, K. Baicalin, the predominant flavone glucuronide of scutellariae radix, is absorbed from the rat gastrointestinal tract as the aglycone and restored to its original form. J Pharm Pharmacol 52: 1563-1568, 2000. 4. Akao, T., Kobashi, K., Aburada, M. Enzymic studies on the animal and intestinal bacterial metabolism of geniposide. Biol Pharm Bull 17: 1573-1576, 1994. 5. Akao, T., Kobashi, K. Glycyrrhizin β-d-glucuronidase of Eubacterium sp. from human intestinal flora. Chem Pharm Bull (Tokyo) 35: 705-710, 1987. 6. Steinmann, G.G., Esperester, A., Joller, P. Immunopharmacological in vitro effects of Eleutherococcus senticosus extracts. Arzneimittelforschung 51: 76-83, 2001. 7. Sui, D.Y., Lu, Z.Z., Li, S.H., Cai, Y. Hypoglycemic effect of saponin isolated from leaves of Acanthopanax senticosus (Rupr. et Maxin.) Harms. Zhongguo Zhong Yao Za Zhi 19: 683-685, 703, 1994. 8. Glatthaar-Saalmuller, B., Sacher, F., Esperester, A. Antiviral activity of an extract derived from roots of Eleutherococcus senticosus. Antiviral Res 50: 223-228, 2001. 9. Hibasami, H., Fujikawa, T., Takeda, H., Nishibe, S., Satoh, T., Fujisawa, T., Nakashima, K. Induction of apoptosis by Acanthopanax senticosus HARMS and its component, sesamin in human stomach cancer KATO III cells. Oncol Rep 7: 1213-1216, 2000. 10. Lin, C.C., Huang, P.C. Antioxidant and hepatoprotective effects of Acathopanax senticosus. Phytother Res 14: 489-494, 2000. 11. Gaffney, B.T., Hugel, H.M., Rich, P.A. The effects of Eleutherococcus senticosus and Panax ginseng on steroidal hormone indices of stress and lymphocyte subset numbers in endurance athletes. Life Sci 70: 431-442, 2001. 12. Szolomicki, J., Samochowiec, L., Wojcicki, J., Drozdzik, M. The influence of active components of Eleutherococcus senticosus on cellular defence and physical fitness in man. Phytother Res 14: 30-35, 2000. 13. Yang, J.Y., Lee, K.S., Kim, M.K., Moon, S.K., Kang, M.K., Park, B.H., Kim, J.S., Park, J.W. Effect of Acanthopanax senticosus on lipoprotein lipase in 3T3-L1 adipocytes. Phytother Res 18: 160-163, 2004. 14. Park, S.Y., Chang, S.Y., Yook, C.S., Nohara, T. New 3,4-seco-lupane-type triterpene glycosides from Acanthopanax senticosus forma inermis. J Nat Prod 63: 1630-1633, 2000. 15. Yoshizumi, K., Hirano, K., Ando, H., Hirai, Y., Ida, Y., Tsuji, T., Tanaka, T., Satouchi, K., Terao, J. Lupane-type saponins from leaves of Acanthopanax sessiliflorus and their inhibitory activity on pancreatic lipase. J Agric Food Chem 54: 335-341, 2006. - 344 -
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