약학박사학위논문 급 만성위염치료제로유용한목별자 추출물및유효성분 Momordicae cochichinensis Seed Extract as a Therapeutic Agent for Acute & Chronic Gastritis and Its Constituents 20

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2 약학박사학위논문 급 만성위염치료제로유용한목별자 추출물및유효성분 Momordicae cochichinensis Seed Extract as a Therapeutic Agent for Acute & Chronic Gastritis and Its Constituents 2012 년 8 월 서울대학교대학원 약학과생약학전공 정기원 i

3 국문초록 위점막은위산, 알코올, 헬리코박터균 (Helicobacter pylori), 각종약물 ( 대표적으로 NSAIDs; non-steroidal anti-inflammatory drugs) 등의공격인자또는스트레스로인한위점막혈액순환저하에의해쉽게손상을받는다. 공격인자에의해위점막이손상되면발적, 출혈, 부종, 미란을동반하는위염이발생하게되고, 그증상이심한경우에는점막하 ( 粘膜下 ) 조직과근육층까지손상이미치는위궤양이발생하게된다. 현재, 위장관병변에대한표준치료제로위산의분비를억제하는프로톤펌프억제제 (PPIs; proton pump inhibitors) 또는 H 2 수용체길항제 (H 2 -receptor antagonists) 가주로사용되며임상에서우수한치료효과를보이고있지만, 약물복용을중단한경우위장관병변이쉽게재발하거나일부만성및난치성위염및위궤양에대해서는치료의한계를보이는경우가있다. 이러한단점을극복하기위해위점막보호제를위산분비억제제와병용하는사례가급격히증가하고있으나, 약효와작용기전이뚜렷하지않거나복용량이많은단점이있어서우수한약물을개발할필요성이여전히있다. 본연구에서는각종한의서와문헌검색등을통하여위점막보호 ( 급성및만성위염치료 ) 효능을보일가능성이있는생약후보들을선별하였으며, 선별된생약에대해동물모델스크리닝을실시하였다. 스크리닝을위해에탄올유발위염모델과 NASID 중하나인 i

4 디클로페낙 (diclofenac) 유발위염모델실험을수행하였으며, 그결과목별자추출물이가장우수한활성을나타냄을알수있었고, 이러한결과로목별자를최종후보생약으로선정하였다. 목별자는중국남부지방과베트남에널리분포하는다년생의박과 (Cucurbitaceae) 식물인목별 ( 木鱉, Momordica cochichinensis Springer) 의종자로서소염작용이강하고우수한상처치료효과와함께류머티즘성동통및근경련등에효과가있는것으로알려져있다. 선정된목별자에대해에탄올유발위염모델에서분획활성연구를수행하였으며, 이를통해활성을보인 n-buoh 층에대해물질분리를시도하였다. 그결과 3종의사포닌을분리하였고, 각종분석광학적데이터를활용하여구조분석을실시한결과, 화합물 1은 gypsogenin-3-oβ-d-galactopyranosyl(1 2)-[α-L-rhamnopyranosyl(1 3)]-β-Dglucuronopyranoside, 화합물 2는 quillaic acid-3-o-β-d-galactopyranosyl(1 2)- [α-l-rhamnopyranosyl(1 3)]-β-D-glucuronopyranoside, 그리고화합물 3은 gysogenin-3-o-β-d-galactopyranosyl(1 2)-[α-L-rhamnopyranosyl(1 3)]-β-D- glucuronopyranosido-28-o-β-d-xylopyranosyl(1 3)-β-D-glucopyranosyl(1 3)- [β-d-xylopyranosyl(1 4)-α-L-rhamnopyranosyl(1 2)-β-D-fucopyranoside로구조를규명하였다. 화합물 1는천연물에서처음분리된물질이며, 화합물 2는 Momordica 속에서처음분리된물질이고, 화합물 3은목별자에서이미분리, 보고된 Momordica saponin I으로동정하였다. 3종화합물에대한에탄올유도위염모델에서약리활성을측정한 ii

5 결과, 통계적유의성은없었으나모두항위염활성을나타내었고, 화합물 3이가장높은효능을나타내는경향을보였다. 이러한결과로볼때, 위점막보호활성이어느정도사포닌에기인하는것으로사료된다. 분리된화합물의경우수율이낮고분리가어려우며, 활성도상대적으로추출물보다높지않다고판단되고, 합성자체도어려운문제점이있기때문에단일화합물이아닌목별자에탄올추출물 (MSE; Momordicae Semen Extract) 로연구를수행하였다. MSE에대한위점막보호활성을검증하기위해각종동물모델및기전관련된실험을수행하였다. 그결과, 에탄올및디클로페낙 (diclofenac) 으로유발된급성위염모델, 그리고 H. pyroli로유발된만성위염모델모두에서우수한보호활성을보였으며, 이러한우수한약효는위점액 (gastric mucus) 분비촉진효과, 상처치료 (wound-healing) 효과, 항염증및진통효과에기인함을알수있었다. 또한, 이러한약리작용은기전연구를통해위점막보호활성을지닌 neuropeptide인 calcitonin gene-related peptide (CGRP) 와가스트린 (gastrin) 분비를억제하는인자로보고된 somatostatin이주로역할을담당할것으로추정하였다. 약효와더불어 MSE에대한랫드독성시험을실시하였는데, 그결과반복경구투여최소치사용량 (MLD) 와 4주반복독성시험에서무해용량 (NOAEL) 이 2,000 mg/kg 임을알수있었다. 목별자원생약자원의안정적인확보를위해 LC-MS similarity analysis를수행하였으며, 분석결과중국운남성산지의목별자가기준에 iii

6 적합함을알수있었다. 이와더불어목별자원생약의수급과정중에동속식물과의혼돈가능성에대해문헌적연구를실시하였는데, 그결과동속식물과혼재될가능성이희박하고, 만약혼재되어있다고하더라도다른동속식물과육안으로쉽게구분가능함을알수있었다. 또한, MSE의함량분석법에대한신뢰성을확보하기위해밸리데이션연구를수행하였으며, 이를통해분석법에대한신뢰성을확보하였다. 이러한결과들은목별자의 GCAP (Good Collecting & Agricultural Practice) 를위한기초데이터로활용할수있을것으로사료되었다. 이상의연구결과들을종합해볼때, 목별자추출물은다양한급성및만성위염모델에서우수한효능을보였고, 다양한 in vivo 및 in vitro 기전모델에서도우수한효과를나타내었기에향후임상에적용했을경우에서도뛰어난위점막보호효과를보일가능성이높다고생각되며, 단회및반복경구투여독성시험을통해우수한안전성을확인할수있었다. 더욱이원생약에대한문헌적연구, similarity analysis, 분석법밸리데이션연구를통해 GCAP를위한다양한기초데이터를확보함에따라, 위점막보호활성을갖는신규천연물의약품개발의가능성이높을것으로판단된다. 주요어 : Momordica cochichinensis Sprenger, Gastroprotective, Calcitonin generelated peptide, Somatostatin, Momordica saponin 학번 : iv

7 목차 국문초록 i 목차 v List of Schemes ix List of Tables x List of Figures xiii List of Abbreviations xvi I. 서론 1 1. 위염의원인및치료 1 2. 목별자 (Seed of Momordica cochichinensis Springer) 의특징 5 3. 목별자의식물화학적성분 6 4. 급성및만성위염동물모델및기전 위염동물모델 위염관련기전 12 II. 실험재료및방법 실험재료 식물재료 기기및시약 위염치료효과를지닌생약의선별 목별자로부터급 만성위염활성분획물제조, 활성성분의분리및구조분석 목별자의추출및분획물제조 20 v

8 3.2. 목별자의활성성분의분리 화합물 1의분리및구조분석 화합물 2의분리및구조분석 화합물 3의분리및구조분석 화합물 1과 2의당분석 위염관련동물모델및기전연구 에탄올유발위염모델 디클로페낙유발위염모델 H. pylori 유발만성위염모델 Alcian Blue-Staining of Gastric Mucosal Croton oil 유발 Ear Edema 모델 진통모델 (Formalin test) 상처치료모델 (Wound healing model) CGRP 수용체특이적조직 Assay 위장조직내 CGRP 분비측정 Somatostatin 수용체특이적조직 Assay 단회및반복경구투여독성시험연구 Liquid chromatography mass spectrometry (LC MS) 를이용한 Similarity Analysis 연구 목별자동속식물에대한연구 목별자추출물의분석법밸리데이션연구 46 III. 결과및고찰 후보생약의선정 49 vi

9 2. 동물위염모델에서의후보생약의선별 목별자로부터활성성분의분리및이의구조규명 목별자분획활성연구 화합물 1의구조 화합물 2의구조 화합물 3의구조 화합물 1, 2 및 3의활성연구 목별자추출물의급 만성위염치료효능 목별자추출물의에탄올유발위염모델에서의효능 목별자추출물의디클로페낙유발위염모델에서의효능 목별자추출물의 H. pylori 유발위염모델에서의효능 목별자추출물의위점액분비촉진모델에서의효능 목별자추출물의상처치료모델에서의효능 목별자추출물의 Croton oil로유도된귀부종실험모델에서의효능 목별자추출물의진통모델 (Formalin test) 에서의효능 목별자추출물의 CGRP 수용체특이적조직 Assay에서의활성 목별자추출물의위조직에서 CGRP 분비에미치는영향 목별자추출물의 Somatostatin 수용체특이적조직 Assay에서의활성 목별자추출물의독성시험연구 랫드단회경구투여급성독성시험 83 vii

10 5.2. 랫드 4주반복경구투여독성시험 LS-MS를이용한목별자산지별 Similarity Analysis 연구 목별자의동속식물에대한연구 목별자의동속식물에대한문헌적연구 여주 (M. charantia) 내 Momordica saponin I의존재유무에대한연구 목별자추출물의분석법밸리데이션연구 114 IV. 결론 126 V. 참고문헌 131 Abstract 142 viii

11 List of Schemes Scheme 1. Extraction and fractionation scheme of M. cochinchinensis 20 Scheme 2. Isolation of compounds 1, 2, and 3 from n-buoh soluble fraction of M. cochichinensis 21 Scheme 3. Maximum likelihood tree for Momordica obtained from combined plastid, nuclear and mitochondrial sequence data 91 Scheme 4. The historical biogeography of Momordica inferred on the preferred maximum likelihood tree under maximum likelihood optimization 92 Scheme 5. Momordica species from Asia geographic origin 94 ix

12 List of Tables Table 1. Classification for the anti-gastritis and anti-gastric ulcer agents 4 Table 2. Constituents of M. cochinchinensis in its various parts 10 Table 3. Table 4. Table 5. Table C- and 1 H-NMR spectroscopic data of aglycon for compound 1 (in Pyridine-d 5 ) C- and 1 H-NMR spectroscopic data of sugar moieties for compound 1 (in Pyridine-d 5 ) C- and 1 H-NMR spectroscopic data of aglycon for compound 2 (in Pyridine-d 5 ) C- and 1 H-NMR spectroscopic data of sugar moieties for compound 2 (in Pyridine-d 5 ) 31 Table 7. Instruments and condition for analytical method validation 47 Table 8. List of candidates for anti-gastritis agent 49 Table 9. Optimized mobile phase condition for LC-MS similarity analysis 85 Table 10. Correlation analysis chart among M. cochichinensis samples from Vietnam and China 87 Table 11. Classification and naming of M. cochinchinensis 89 Table 12. Momordica species originated from Asia 93 Table 13. Extraction and summary: Momordica species from Asia geographic origin 95 Table 14. Extraction and summary: Momordica species of Cucurbitaceae inhabiting in China 96 x

13 Table 15. Extraction and summary: Momordica species as an economic plants in Asia 98 Table 16. Extraction and summary: Momordica species having an apparent habitat (national) in Asia 99 Table 17. Classification of Momordica species by habitats 101 Table 18. The excluded Momordica spices from the research subject and the reasons 102 Table 19. The final Momordica species for research subject 103 Table 20. Information about M. cochinchinensis 104 Table 21. Information about M. subangulata Blume 105 Table 22. Information about M. dioica 106 Table 23. Morphology of Group I species 107 Table 24. The gross output of Gac fruit in Hai Duong region, Vietnam 109 Table 25. The validation items and specifications for the analytical method 114 Table 26. The result of standard solution in specificity test 115 Table 27. The result of testing solution in specificity test 115 Table 28. The result of purity test by PDA 116 Table 29. Summary of the test results in specificity test 116 Table 30. The result of standard solution in linearity test 117 Table 31. Summary of the results in linearity test 118 Table 32. The result of standard solution in accuracy test 119 xi

14 Table 33. The result of testing solution in accuracy test spiking 120 Table 34. The result of testing solution in accuracy test addition 120 Table 35. Summary of results in accuracy test 121 Table 36. The result of injection repeatability - 100% standard solution (14.0 μg/ml) (ten times injection) 121 Table 37. The result of method repeatability - 100% standard solution (800 μg/ml) (After making 6 samples, five times injection) 122 Table 38. The result of injection reproducibility - 100% standard solution (14.0 μg/ml) (Ten times injection) 123 Table 39. The result of method reproducibility. 100% standard solution (800 μg/ml) (After making 6 samples, five times injection) 123 Table 40. Cumulative Results 124 Table 41. Summary of the results in precision test 124 Table 42. Summary of results in validation for analytical method 125 xii

15 List of Figures Figure 1. Results of chronic gastric H. pylori infection 2 Figure 2. Structures of terpenoids in M.cochinchinensis 7 Figure 3. Structures of saponins in M.cochinchinensis 9 Figure 4. Relation between the activity of vagal nerves and enteric cholinergic and GRP-releasing neurons and gastrin released from the G-cells (A) in intact and H. pylori infected mucosa. 14 Figure 5. Morphology of M. cochinchinensis seeds 15 Figure 6. UPLC chromatogram of 50% ethanol extract of M. cochinchinensis 23 Figure 7. Pictures of the raw materials of candidates 50 Figure 8. Gastroprotective effects of candidates (MP09~MP15) in rat model of NSAID (diclofenac)-induced acute gastritis 51 Figure 9. Gastroprotective effects of candidates (MP09~MP15) in rat model of NSAID (diclofenac)-induced acute gastritis erosion 52 Figure 10. Gastroprotective effects of candidates (MP09, 12, 15) in rat model of ethanol-induced acute gastritis 53 Figure 11. Gastroprotective effects of MP09, MP09 EtOAc fraction, and MP09 n- BuOH fraction in rat model of ethanol-induced acute gastritis 54 Figure 12. The molecular structure of compound 1 57 Figure H-NMR spectrum of compound 1 (600 MHz, Pyridine-d 5 ) 58 Figure C-NMR spectrum of compound 1 (150 MHz, Pyridine-d 5 ) 59 Figure 15. HMBC spectrum of compound 1 60 xiii

16 Figure 16. The molecular structure of compound 2 62 Figure H-NMR Spectrum of compound 2 (600 MHz, Pyridine-d 5 ) 63 Figure C-NMR Spectrum of compound 2 (150 MHz, Pyridine-d 5 ) 64 Figure 19. HMBC spectrum of compound 2 65 Figure 20. The molecular structure of compound 3 67 Figure H-NMR spectrum of compound 3 (900 MHz, Pyridine-d 5 -D 2 O (1:1)) 68 Figure C-NMR spectrum of compound 3 (225 MHz, Pyridine-d 5 -D 2 O (1:1)) 69 Figure 23. Gastroprotective effects of MP09 n-buoh fraction, compounds 1, 2, and 3 in rat model of ethanol-induced acute gastritis 70 Figure 24. Figure 25 Figure 26. Comparisons of the gastric damage indices in rat models of ethanolinduced gastritis 72 Comparisons of the gastric damage indices in rat models of diclofenac-induced gastritis 73 Comparisons of ulceration scores in mouse model of H. pyloriinduced gastritis 74 Figure 27. Histological comparison of depth of the gastric mucus between MSEtreated and water-treated rats after ethanol challenge 75 Figure 28. Time course of wound healing in mouse cutaneous injury model 77 Figure 29. Anti-inflammatory effect of MSE in croton oil-induced ear edema test 78 Figure 30. Analgesic effect of MSE in formalin test 79 xiv

17 Figure 31. Dose response curve of MSE in CGRP-specific tissue inotropy assay 80 Figure 32. CGRP secretion effect from gastric tissue of MSE 81 Figure 33. Dose response curve of MSE in somatostatin-specific tissue neurogenic twitch assay 82 Figure 34. LC-MS chromatogram of 50% ethanol extract of M. cochinchinensis 86 Figure 35. LC-MS Chromatograms of 50% ethanol extract of M. cochinchinensis (MCREF), MCC2, and MCV6 88 Figure 36. Momordica species in genus of Cucurbitaceae 96 Figure 37. Native distribution of Momordica species in Asia 97 Figure 38. Momordica species in Herbaria around the world 99 Figure 39. Synonyms of Momordica species 100 Figure 40. Morphology of M. charantia 107 Figure 41. Morphology of M. balsamina 107 Figure 42. Pattern chromatograms of MSE, M. charantia, and Spiking 111 Figure 43. Overlay chromatogram of MSE, M. charantia & Spiking 112 Figure 44. LC-MS SIM of MSE and M. charantia 113 Figure 45. UPLC Chromatogram of MSE 116 Figure 46. Linearity graph of standard (Momordica saponin I) 118 xv

18 List of Abbreviations AcCN ANOVA Ave. n-buoh CaCl 2 CD CFU CGRP CMC CO 2 Conc. COSY CT 50 EC 50 ESI EtOAc EtOH FID Gal GC GCAP : Acetonitrile : Analysis of variance : Average : Normal buthanol : Calcium chloride : Circular dichroism : Colony-forming unit : Calcitonin gene-related peptide : Carboxymethyl cellulose : Carbon dioxide : Concentration : Correlation spectroscopy : Half closure time : 50% effective concentration : Electrospray ionization : Ethyl acetate : Ethanol : Flame ionization detector : Galactose : Gas chromatography : Good collecting and agricultural practice xvi

19 GC-MS GlcA GRIN GRP HEPES HMBC H 2 O HPLC HR-FAB-MS HSQC ICR ID IL-1β IL-8 IR IV KCl Lab LC-MS MC MCC MCV MeOH : Gas chromatography-mass spectrometry : Glucuronic acid : Germplasm resourced information network : Gastrin-releasing peptide : 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid : Heteronuclear multiple bond correlation : Water : High performance liquid chromatography : High resolution-fast atom bombardment-mass spectroscopy : Heteronuclear single-quantum correlation spectroscopy : Imprinting control region : Internal diameter : Interleukin-1 beta : Interleukin-8 : Infrared absorption : Intra venous : Potassium chloride : Laboratory : Liquid chromatography-mass chromatography : Methyl cellulose : Momordica cochichinensis from China : Momordica cochichinensis from Vietnam : Methanol xvii

20 MgCl 2 MLD mp MP MPLC MS MS I MSE NaCl NaHCO 3 NMR NO NOAEL NSAID PBS PDA PG PPI RF Rha ROESY RP RSD : Magnesium chloride : Minimum lethal dose : Melting point : Mucosa protective : Medium pressure liquid chromatography : Mass spectrometry : Momordica saponin I : Momordicae cochichinensis seed extract : Sodium chloride : Sodium bicarbonate : Nuclear magnetic resonance : Nitric oxide : No observable adverse effect level : Non-steroidal anti-inflammatory drug : Phosphate buffered saline : Photodiode array : Prostaglandin : Proton pump inhibitors : Response factor : Rhamnose : Rotating frame nuclear overhauser effect spectroscopy : Reversed-phase : Relative standard deviation xviii

21 RT SEM SD SIM S/N SPF SST : Retention time : Standard error of mean : Sprague-Dawley : Single ion monitoring : Signal-to-noise ratio : Specific-pathogen-free : Somatostatin SSTR2 : Somatostatin receptor 2 TMS TNF-α TOCSY TOP UPLC UV VEGF : Tetramethylsilane : Tumor necrosis factor-alpha : Total correlation spectroscopy : Topically : Ultra performance liquid chromatography : Ultraviolet absorption : Vascular endothelial growth factor xix

22 I. 서론 1. 위염의원인및치료 위를보호하는위점막층은여러종류의인자로부터공격을받아손상받기쉽다. 대표적인공격인자로서위산, 알코올, 각종약물 [nonsteroidal anti-inflammatory drugs (NSAID), 골다공증치료제 ( 알렌드로네이트 ) 등 ], H. pylori, 스트레스에의해유도된위점막혈액순환 (microcirculation) 장애와혈압등이있다 (Amaral et al., 2010; Frezza et al., 2001; Mizuno, 1987; Suzuki et al., 1993). 이러한요인으로인해점막층이손상되었을때미란, 발적, 출혈, 부종을동반한염증이발생하게되며, 손상이심하여점막하조직 (submucosa) 까지손상되었을때이를위궤양이라한다. 또한, 위와직접연결되어있는십이지장도비슷한요인에의해염증과십이지장궤양이발생할수있다. 참고로위에언급된공격인자에의해만성표재성위염이발생하며, 그정도가심해짐에따라위축성위염, 장상피화생, 위점막이형성증과같은임상적증상을보이게되고, 마지막단계인위암이발생하게된다. 위축성위염은위염이만성화되어위점막이얇아지고주름이생기는것을말한다. 장상피화생 (intestinal metaplasia) 은위점막의상피세포가장점막의상피세포로변화하는것을말하며, 위점막에서소화액을분비하지못하는상황이지속될때생긴다 (Stemmermann, 1994). 장상피화생환자중 0.25% 가위암으로진전되는데, 이는정상인보다 10배이상위암이발생할가능성이있는것이다. 위암의전단계로 - 1 -

23 알려진위점막이형성증 (gastric epithelial dysplasia) 은위암으로진행되는 위점막상피에나타나는종양성증식을말한다 (Lauwers, 1999). Figure 1. Results of chronic gastric H. pylori infection. This infection results either in antral predominant gastritis with hypergastrinemia and hyperchlohydria leading to duodenal or gastric peptic ulceration, while corpus predominant H. pyloriinduced atrophic gastritis leads to hypochlohydria and increased gastrin release, resulting in the cancerogenesis. Most of infected patients (~80%) show only mild mixed gastritis and normal gastric acid secretion without significant gastric diseases (Konturek et al., 2006). 위에제시된공격인자로인해유발된위및십이지장의염증 / 궤양을치료하기위해서는위산분비억제, H. pylori의증식억제, 점액분비촉진, 상피세포재생촉진, 항염증등의효능을가진약물이필요하다. 현재가장대표적인위염및위궤양치료제로는위산분비억제의 - 2 -

24 효능을지닌 H 2 -receptor antagonist와 proton pump inhibitor (PPI) 류의약물이며임상에서우수한효과가입증되었다 (Bell et al, 1992; Feldman et al, 1990; Schunack et al., 1989; Mark et al., 1990; Mills et al., 1989). PPI의대표적인성분은 omeprazole, esomeprazole, lansoprazole, pantoprazole, rabeprazole 등이있으며, H 2 -receptor antagonist의대표적인성분은 cimetidine, ranitidine 등이있다. 이들약물은임상적으로우수한치료효과를보이고있지만약물복용을중단한경우위장관병변이쉽게재발하거나일부만성 난치성위염및위궤양에대해서는치료의한계를보이는경우가있다 (Berardi et al., 1987; Fullarton et al., 1989; Szabo and Bynum, 1988). 위장관병변에대한치료효율을높이는동시에치료후재발률을낮추기위해서는위점막손상유발인자인위산을지속적으로억제하는것도중요하지만한편으로는점액의분비를촉진하고상피세포의재생을촉진하여점막을강화시키고항염증과항산화작용을통하여위점막을보호하는효능을지닌위점막보호제 (gastroprotectant) 를함께사용하는것이매우중요하다. 또한노년층의증가와더불어퇴행성질환을치료하기위해위장관의손상을유발하기쉬운 NSAID류약물또는알렌드로네이트등의장기간복용이급속도로증가함에따라이들약물에의한위장손상을예방하기위하여위산억제제보다는위점막보호제를위손상유발약물과병행처방하는하는것이바람직하기에위점막보호제의수요는급격히증가하고있다. 현재위점막보호제로서애엽 95% 에탄올연조엑스 ( 스티렌정 ), 레바미피드 (rebamipide, 뮤코스타정 ), 에카베트나트륨 (Ecabet Sodium, - 3 -

25 가스트렉스과립 ), 테프레논 (teprenone), 수크랄페이트 (sucralfate), 폴라프레징크 (Polaprezinc, 프로멕과립 ), 쏘팔콘 (sofalcone, 쏘론정 ) 등의약물이개발되어있으나 (Yamasaki et al., 1989; Takei, 2012; Terano et al., 2007; Higuchi et al., 2010), 일반적으로속효성이적고작용기전이뚜렷하지않거나복용량이많은단점이있어서개선된약물의개발이필요하다. Table 1. Classification for the anti-gastritis and anti-gastric ulcer agents (except H. pylori infection medicines) 위점막보호제 PPI H 2 receptor antagonist 제산제 위점막피복, 위산분비 위산분비기전의 알칼리성물질로 개요 점액합성및 분비촉진등을 통한위점막 최종단계인 proton pump 차단 하나인 histamine 수용체차단 위산을중화시킴 보호 위궤양 가장강력한 위궤양및 즉각적인속쓰림 재발방지효과 효과를가지나 위염에모두 개선효과가 있으며, 고가임. 약물 적용가능. Half 있으나, 장기 공격인자 복용중단후 dose 발매로 복용시위장내 장 단점 차단제 (PPI, H 2 -RA) 와병용함. 쉽게재발가능성존재 제산제대체추세. 위산분비 환경이알칼리화되어소화장해 처방 base 약물 억제효과가 유발. 변비또는 PPI 에비해 설사유발 약함 주요제품 스티렌, 무코스타, 가스트렉스 란스톤, 판토록, 파리에트, 레바넥스 잔탁, 큐란 겔포스엠 - 4 -

26 2. 목별자 (Seed of Momordica cochichinensis Springer) 의특징 목별자는박과 (Cucurbitaceae) 에속하는다년생덩굴식물로써목별 (Momordica cochinchinensis Sprenger) 의건조된성숙한종자이다. 중국에서몰루카스제도까지분포하며동아시아와동남아시아에서전통생약으로사용되어왔다. 목별은다년생초질등본식물로팽대한덩이모양의뿌리가있다. 줄기에는세로의모서리가있다. 덩굴손은굵고튼튼하며잎과대생하여한개이고분지하지않았다. 잎은어긋나고원형내지넓은달걀모양이며길이는 7~14 cm이고보통 3개로얕게갈라지거나깊게갈라진다. 산비탈, 숲, 토층이비교적두터운지역에서자라며재배하기도한다 ( 중약대사전, 2006; Pharmacopoeia of the People s Republic of China, 1997). 목별자는편평한원판 ~ 거북잔등모양이며지름 2~3 cm, 두께약 5 mm이다. 바깥면은회갈색 ~ 회흑색이며양쪽에거친톱니상의돌기가 10여개씩있다. 외종피는굳으며내종피는회록색의얇은막으로되어있고그안에 2장의황백색의떡잎이들어있다. 이약은특이한기름냄새가있고맛은쓰다. 양품의경우알이크고고르며무겁고깨어진것이없고기름기가많은것이어야한다 ( 대한약전외한약 ( 생약 ) 규격집, 2007). 목별자는상용하는중약의하나이나대개는외용으로사용하고아주 - 5 -

27 드물게복용으로사용한다. 목별자는맛이쓰고, 떫고, 성질이차며, 열증의억제, 해열, 해독의효과가있어간, 비장이상, 치질, 상처, 타박상, 종기, 서혜선종 (buboes), 볼거리 (mumps), 하지부종등에사용되어왔으며, 화농성피부감염증에효과를지닌것으로알려져있다 (Cheung et al., 1985; Gao et al., 2005; Perry et al., 1980). 최근문헌에의하면목별자추출물이랫드모델에서위궤양치료와혈관신생성효과를증진시키는것으로보고되었다 (Kang et al., 2009; Kang et al., 2010) 3. 목별자의식물화학적성분 목별자에함유된지방산에는 decanoic acid, palmitic acid, stearic acid, peanut acid이있으며, 그중함량이제일높은것은 stearic acid로 51.68% 를차지한다. 불포화지방산은 hexanoic acid, 2-heptadecenoic acid, oleic acid, (Z,Z)-9,12-octadecadienoic acid, linoleic acid, 10-nonadecenoic acid과 11-eicosenoic acid가존재하며, 함량이가장높은것은 oleic acid이며지방산총량의 21.62% 를차지한다. karounidiol, isokarounidiol, 5- dehydrokarounidiol, 7-oxodihydrokarounidiol과같은 triterpenoid와 β-sitosterol, stigmast-en-3β-alcohol, stigmast-7,22-diene-3β-alcohol과같은 sterol 성분이분리되었으며 (De Shan et al., 2001; Kan et al., 2006), 목별자에만존재하는화학성분인 momordica saponin I과 momordica saponin II (Iwamoto et al., 1985), 그리고 momordic acid, oleanolic acid, momorcochin 등이보고되었다. 면역과항암등의활성을지닌단백질류와펩타이드류도보고되었다 (Chan et al., 2009; Chuethong et al., 2007; Tsoi et al., 2004; Wong et al., 2004)

28 참고로 oleanolic 배당체의경우에탄올또는인도메타신으로유도된위염모델에서항위염효능을지니는것으로보고되었다 (Matsuda et al., 1998). 목별자에서분리된 terpenoid는구조는아래와같다 (Iwamoto et al., 1985; Kubota et al., 1971; Shan et al., 2001). Columbin Vergatic acid Chondrillasterol Nardol Figure 2. The molecular structures of terpenoids in M. cochinchinensis - 7 -

29 Momordicine III Momordin I Momordin IC Momordin ID Momordin IE Hemsloside MA-1-8 -

30 Momordin IIA Momordin IIB Momordin IIC Momordin IID Momordin IIE Momordin III Figure 3. The molecular structures of saponins in M.cochinchinensis - 9 -

31 Table 2. Constituents of M. cochinchinensis in its various parts Compound Name 성분계열 Part Chondrillasterol Stigmasterol found in tuber; TradiMed* Columbin Diterpenoid found in root; Waterman et al., 1985 Momorcochin Protein found in fruit; TradiMed Momordica saponin I Gypsogenin saponin found in seed; Iwamoto et al., 1985 Momordica saponin II Quillaic acid saponin found in seed; Iwamoto et al., 1985 Momordicine III Cucurbitacine saponin found in root; TradiMed Momordin I Oleanolic acid saponin found in root; Iwamoto et al., 1985(2) 6'-Methyl momordin I Oleanolic acid saponin found in root; Nawamura et al., 1988 Momordin Ia Oleanolic acid saponin found in root; Nawamura et al., 1988 Momordin Ib Oleanolic acid saponin found in root; Nawamura et al., 1988 Momordin Ic Oleanolic acid saponin found in root; Nawamura et al., 1988 Momordin Id Oleanolic acid saponin found in root; Nawamura et al., 1988 Momordin Ie Oleanolic acid saponin found in root; Nawamura et al., 1988 Momordin II Oleanolic acid saponin found in root; Iwamoto et al., 1985 (2) Momordin IIa Oleanolic acid saponin found in root; Nawamura et al., 1988 Momordin IIb Oleanolic acid saponin found in root; Nawamura et al., 1988 Momordin IIc Oleanolic acid saponin found in root; Nawamura et al., 1988 Momordin IId Oleanolic acid saponin found in root; Nawamura et al., 1988 Momordin IIe Oleanolic acid saponin found in root; Nawamura et al., 1988 Momordin III Epoxy-oleanolide found in root; Iwamoto et al., 1985 (2) Nardol Sesquiterpenoid TradMed Vergatic acid Triterpenoid TradMed * TradiMed : 전통동양약물데이터베이스 (

32 4. 급성및만성위염동물모델및기전 위점막은생리적인보호막으로서 1차적으로점막상피를보호하는점액층을분비한다. 그러나, 끊임없이많은공격인자에노출될수있으며, 여러요소들이위점막보호기전에기여하는것으로알려져있다 위염동물모델 위염동물모델의경우, 위염을일으킬수있는다양한공격인자를제거한여러동물모델이만들어져왔다. 주로사용되는동물모델유발인자로는에탄올, 디클로페낙, 인도메타신, 아세트산, 암모니아, 스트레스 (water immersion restraint stress), H. pylori가있다. 그리고, NSAID에의해발생하는위염또는위궤양치료의효능을검증하기위해주로디클로페낙또는인도메타신으로유발된모델을사용한다 (Hsu et al., 2008; Yamasaki et al., 1999; Urushidani et al., 1977). 참고로본연구에서는에탄올, 디클로페낙, H. pylori로유발된위염모델을사용하였다. 각모델의경우 alcian blue staining을이용하여위점액분비여부를측정할수있다 (Kitagawa et al., 1986). 이와더불어위염의효능을간접적으로증명할수있는모델로함염모델 (croton oil or arachidonic acid-induced ear edema model) 과진통모델 (formalin test, tail flick model, writhing test), 상처치료모델 (wound-healing model) 을들수있다 (Hunskaar et al., 1985; Inoue et al., 1991; D Amour et al., 1941; Montesinos et al., 1997)

33 4.2. 위염관련기전 점막액을형성하는 Paracrine과신경조절기전은 neuropeptide의한종류인 calcitonin gene-related peptide (CGRP), nitric oxide (NO) 그리고 prostaglandin (PG) 로보고되었으며 (Akiba et al., 2000; Akiba et al., 2001; Ichikawa et al., 2000), CGRP의경우랫드모델에서 NO 의존적으로 mucin 생성을자극하였고 (Ichikawa et al., 2000), 고농도의에탄올을처리한랫드의위조직에서 CGRP의농도가감소됨이관찰되었다 (Evangelista et al., 1993). 단기적으로 capsaicin을경구투여로 afferent fiber를자극하는경우위점막의혈류량이뚜렷하게증가되는것으로나타났으며, 이러한활성의주요매개체는 CGRP라는것이밝혀졌다 (Holzer, 1998; Holzer et al., 1991; Holzer, 2007). 또한, CGRP는혈관평활근에직접작용하기도하며, NO 형성을증가시킴으로써부분적으로혈관이완을유도하는것으로보고되었다 (Lambrecht et al., 1993). 즉, 위점막보호에관련하는구조로 capsaicin-sensitive afferent fiber를경유하여 CGRP 분비가촉진되는것으로추측된다 (Hayashi, 2001). 이러한결과점막미세혈류가증가되고점막세포사이에침투한위산의청소가원활하게되며점액생성이증가될것으로추측되고있다. 최근위점액증가에관한기전으로 PG와 NO 의존성경로및 heat shock protein이관여한다는연구결과들이보고되고있는데위점막보호제로쓰이고있는 geranylgeranlyaceton의경우 PG와는관계없이 NO- 의존성경로및 heat shock protein이관여하리라추측되고있다 (Brzozowski et al., 2005)

34 Somatostatin의경우위장호르몬인가스트린분비를억제하여위점막보호효능을지닌것으로알려져있는데 (Avgerinos et al., 2005; Konturek et al., 2006), UCB에서 Somatostatin-ucb TM 라는이름의위궤양치료제로개발되었다. UCB에서출시된또다른위궤양치료제인 Stogar (lafutidine, H 2 -receptor antagonist) 의경우 H. pylori 감염된환자에게투여시, CGRP와 somatostatin의혈장농도를유의성있게낮추었는데이는 capsaicin-sensitive neuron을경유하여작용하는것으로추측된다 (Shimatani et al. 2006). 한편, CGRP는 somatostatin의방출을증가시키며, 이와동시에가스트린방출을억제하는것으로보고되었다 (Manela et al., 1995)

35 Figure 4. Relation between the activity of vagal nerves and enteric cholinergic and GRP-releasing neurons and gastrin released from the G-cells (A) in intact and H. pylori infected mucosa. Meal and IV injection of gastrin-releasing peptide (GRP) induces gastric acid secretion via releasing gastrin and subsequent stimulation of the parietal cells to secrete acid that in turn inhibits further release of antral hormone by stimulating the D-cells and releasing somatostatin (SST). The latter peptide inhibits gastrin release via SST receptors (SSTR2) and thus attenuates gastric acid secretion (Konturek et al., 2006)

36 II. 실험재료및방법 1. 실험재료 1.1. 식물재료 실험에사용한목별자 (20 kg) 는 2008 년 6 월중국운남성에서 구입하여김진웅교수로부터감정을받은후본실험에사용하였다. 표본은서울대학교약학대학약초원 (KJ 0024C) 에보관되어있다. Figure 5. Morphology of seeds of M. cochinchinensis

37 1.2. 기기및시약 Alcian blue (Sigma-Aldrich, USA) Carboxymethyl cellulose (Sigma-Aldrich, USA) CGRP linked immunoassay kit (SPI-Bio, Australia) α-cgrp (8-37) (Bachem, Switzerland) CGS (Tocris, USA) Clarithromycin (Abbott, USA) Columns and cartridge ACQUITY UPLC BEH C18, 1.7 μm, mm (Waters, Milford, USA) Ascentis Express C18, 150 mm 4.6 mm, 5 μm (Sigma-Aldrich, USA) Biotage SNAP Cartridge (Biotage, Charlotte, NC, USA) BPX-50, 0.25 mm 30 m (SGE, Melbourne, Australia) J sphere ODS H-80, S-4 μm, mm i.d., 80A (YMC, Tokyo, Japan) YMC*GEL ODS-A, S-150 μm, 450 mm 20 mm i.d. (YMC, Tokyo, Japan) Croton oil (Sigma-Aldrich, USA) Diclofenac (Sigma-Aldrich, USA) Ether (SkySoltech, SK Chemicals, Korea) First grade reagent for analysis (Deajung Chemical & Metals Co. Ltd., Korea) Flash purification system (Isolera Flash Purification System; Biotage, Sweden) Formalin (Formaldehyde, Aqueous solution of 37%; Sigma-Aldrich, USA) FT-IR (JASCO FT/IR-300E spectrophotometer;, Jasco, Tokyo, Japan) FT-NMR (Bruker AVANCE 900 / Bruker AVANCE 600 spectrometer; Bruker,

38 Rheinstetten, Germany) GC-MS system (Perkin-Elmer Clarus 600 gas chromatograph and Clarus 600T mass spectrometer; Perkin-Elmer, Norwalk, CT, USA) Graph-Prism (Graph Software, USA) Helicobacter pylori selective supplements ( colony-forming unit/0.4 ml/mouse; Clinical isolates, National Taiwan University Hospital Laboratory) Hexobarbital (Sigma-Aldrich, USA) HPLC Solvents (Fisher Scientific, Korea) HPLC system Agilent 1100 series (Agilent Technologies, Palo Alto, CA, USA) ACQUITY UPLC (Waters Corporation, Milford, USA) HR-FAB-MS (JEOL JMS 700 MStation; Akishima, Tokyo, Japan) Image-ProPlus Version (Media Cybernetics, CA,USA) Krebs-Henseleit buffer (In-house) LC-ESI-MS (Finnigan LCQ ion-trap mass (ThermoQuest; San Jose, CA, USA) Male CD-1 (Crl.) (under Charles River Laboratories) Mass spectrometer (JEOL JMS-700; Akishima, Tokyo, Japan) MS System (LCQ Deca XP; Thermo Finnigan, CA, USA) Melting Point Tester (Buchi B-540; Buchi, Flawil, Switzerland) Microbalance (Mettler AE 50; Zürich, Switzerland) Omeprazole (Astra, Sweden) Phosphate Buffered Saline ( ph 7.4; Sigma-Aldrich, USA) Pipette (Pipetman P20; Gilson, France)

39 Polarimeter (JASCO DIP-1000; Jasco, Tokyo, Japan) Pyrogen free saline (Sintong, Republic of China) Rebamipide (Sigma-Aldrich, USA) Rotary evaporator (N-1100; EYELA, Japan) Sharp punch, ID 12 mm (Sinter, Republic of China). SigmaStat version 3.5 (Systat Software, Chicago, IL USA) Sodium chloride (Wako, Japan) Somatostatin 28 (Sigma-Aldrich, USA) Sucrose (Sigma-Aldrich, USA) Sterile distilled water (In-house) Tyrode buffer (In-house) UPLC system (ACQUITY UPLC ; Waters Corp, Milford, USA) UV spectrometer (Beckman DU 650 spectrophotometer; Fullerton, USA)

40 2. 위염치료효과를지닌생약의선별 위점막보호 ( 급성및만성위염치료 ) 효능을지닌후보생약을선정하기위하여, 각종정보 [KFDA 생약종합정보시스템, 전통동양약물데이터베이스 (TradiMed; Pubmed, 중양대사전, 향약대사전, 동의보감, 한의사자문 ] 를활용하였다. 이를통해 1차로생약을선별하였으며, 선별된생약에대해신규성과학술적가치를재검토하여 2차후보생약을선정하였고, 이에대해동물모델스크리닝을실시하였다. 선정된생약의 1차스크리닝을위해 in vivo 모델인디클로페낙유발위염모델을수행하였으며, 비교약물로레바미피드를사용하였다. 그리고 1차스크리닝을통해선별된생약에대해 2차스크리닝을실시하였는데, 이를위해 in vivo 모델인에탄올유발위염모델을수행하여 ( 비교약물로레바미피드사용 ) 최종후보모델을도출하였다. 참고로 in vivo 모델에대한실험방법은하기 4.1. In vivo 위염모델 에서술되어있다

41 3. 목별자로부터급 만성위염활성분획물제조, 활성성분분리및 구조분석 3.1. 목별자의추출및분획물제조 목별자의껍질을제거한 kernel 1 kg (dry weight) 을 5배 (v/w) 의 50% EtOH를가하여 80 C에서 4시간동안 2회추출하였다. 추출액을여과지로여과한후여액을감압농축하고동결건조하여목별자 50% EtOH 추출물 55 g을수득하였다 ( 수율 5.5%). 수득한 50% EtOH 추출물을증류수에현탁한후극성지향적분리기법에따라 n-hexane, EtOAc 및 n- BuOH로분획하여각각 3.5 g ( 수율 0.35%), 5.7 g ( 수율 0.57%) 및 10.3 g ( 수율 1.03%) 의분획물을얻었다 (Scheme 1). 이중 EtOAc과 n-buoh 분획은에탄올유발위염모델에서보호활성을측정하였다. Scheme 1. Extraction and fractionation scheme of M. cochinchinensis

42 3.2. 목별자의활성성분의분리 목별자추추물의 n-buoh 분획으로부터화합물 1, 2 및 3을분리하기위하여 flash purification system 및 RP-HPLC를이용하였다. 즉, n-buoh 분획 10.3 g을정제수에현탁시킨후, Biotage SNAP cartridge가장착된 flash purification System (Isolera One with fixed 210 nm wavelength detector and single fraction collector bed) 에 loading하였으며, 0.3% formic acid과 MeOH를 gradient elution (9:1 at 0 min to 100% methanol at 30 min) 한이동상조건으로분리하였다. 이를통해 5개의분획 (F01~F05) 을얻었으며, 이중 F05 분획에대해 RP-HPLC (YMC J Sphere ODS-H80, 150 mm 4.6 mm i.d., YMC Co. Ltd.; flow rate of 2 ml/min; AcCN-H 2 O (29:71, 0.1% trifluoroacetic acid)) 에적용하여화합물 1 (145 mg), 2 (130 mg) 및 3 (250 mg) 을얻었다 (Scheme 2). Scheme 2. Isolation of compounds 1, 2, and 3 from n-buoh soluble fraction of M. cochichinensis

43 참고로위의방법의경우우선 n-buoh로분획을해야하고, 고순도의화합물을얻기가어려운단점이있어, 에탄올수용액추출물을아세톤으로침전시킨후, 침전물을 MPLC와 HPLC를사용하여분리하는또다른방법을고안하였다. 목별자의껍질을제거하여얻어진 kernel 1 kg (dry weight) 을분쇄하여 50% 에탄올 5 L을넣은후 80 o C에서 3시간동안추출을하였으며, 이과정을 2회반복하였다. 추출액을여과지로여과하였으며여액을 60 o C에서감압농축을실시한후, 감압건조기로잔류용매를제거하였다. 이를통해얻어진추출물중 10 g을취하여 100 ml의정제수에넣은후현탁시켰으며, 현탁액에아세톤을넣어주어최종 50% (v/v) 아세톤수용액이되게하였다. 1시간동안방치한후에침전된침전물을여과지에여과하여따로분리하였으며, 여액인아세톤수용액에추가로아세톤 400 ml를첨가하여재침전시켰다. 이때생성된침전물도여과하여이전침전물과합친후, 감압농축및감압건조하여 750 mg의침전물을얻었다. 침전물에서성분을분리하기위해 column chromatography를실시하였다. 역상컬럼 (YMC*GEL ODS-A 12 nm, S-150 μm, 450 mm 20 mm i.d., YMC Co. Ltd.) 을사용하였으며, 용매조건으로 MeOH과 H 2 O 혼합용액을선택하여 1:9 비율에서 7:3 비율로 gradient elution을시켰다. 이를통해 7개의분획 (F01~F07) 분하였으며, 이중 F07 분획에대해 column chromatography를실시하였다 (YMC J Sphere ODS-H80, 150 mm 4.6 mm i.d., YMC Co. Ltd.; flow rate of 2 ml/min; AcCN-H 2 O (29:71, 0.1% trifluoroacetic acid)). 그결과 98% 이상의순도를갖는화합물 1, 2 및 3을분리할수있었다 (data not shown)

44 Figure 6. UPLC chromatogram of 50% ethanol extract of M. cochinchinensis

45 3.3. 화합물 1 의분리및구조분석 목별자추추물의 n-buoh 분획을 flash purification system과 RP-HPLC (YMC J Sphere ODS-H80, 150 mm 4.6 mm i.d., YMC Co. Ltd.; flow rate of 2 ml/min; AcCN-H 2 O (29:71, 0.1% trifluoroacetic acid)) 에적용하여화합물 1을분리및정제하였으며, 아래와같이구조분석을위한데이터를확보하였다. Compound 1 C 48 H 74 O 19, An amorphous powder [α] 24 D: (c 0.21, MeOH) mp C IR: ν max 3400, 2935, 1733, 1691, 1089, 1045 cm -1 HR-FAB-MS (negative ion mode): m/z [M-H] - 1 H-NMR (Pyridine-d 5, 600 MHz): δ 9.90 (s, H-23), 6.06 (br s, Gal-H-1), 5.44 (s, H-12), 5.17 (d, J = 7.62 Hz, Gal H-1), 4.97 (br s, GalH-2), 4.92 (m, Gal H-5), 4.82 (d, J = 7.20 Hz, GlcA H-1), 4.55 (dd, J = 9.33, 0.33 Hz, Gal H-3), 4.52 (m, J = Hz, GlcA H-4), 4.52 (m, Gal H-4), 4.46 (m, GlcA H-5), 4.46 (m, Gal H-6a), 4.43 (m, Gal H-2), 4.42 (m, Gal H-6b), 4.36 (t, J = 9.33 Hz, Gal H-4), 4.27 (t, J = 7.92 Hz, GlcA H-2), 4.22 (t, J = 8.73 Hz, GlcA H-3), 4.00 (dd, J = 9.51, 3.27 Hz, Gal-H-3), 3.95 (dd, J = 11.82, 4.44 Hz, H-3), 3.90 (t, J = 6.24 Hz, Gal H-5), 3.27 (dd, J =

46 13.65, 3.70 Hz, H-18), 2.12 (m, H-2a), 2.08 (m, H-15a), 2.08 (m, H-16a), 2.03 (m, H-7a), 1.94 (m, H-16b), 1.84 (m, H-11), 1.82 (m, H-7b), 1.81 (m, H-2b), 1.78 (m, H-19a), 1.66 (m, H-9), 1.66 (d, J = 6.12 Hz, Gal-H-6), 1.44 (m, H-21a), 1.41 (m, H-22a), 1.40 (m, H-6a), 1.37 (s, H-24), 1.36 (m, H-5), 1.35 (m, H-1a), 1.27 (m, H- 19b), 1.25 (s, H-27), 1.21 (m, H-21b), 1.13 (m, H-22b), 1.12 (m, H-15b), 1.01 (s, H-30), 0.96 (s, H-29), 0.96 (m, H-6b), 0.93 (s, H-26), 0.82 (m, H-1b), 0.76 (s, H-25) 13 C-NMR (Pyridine-d 5, 150 MHz): δ (C-23), (C-28), (GlcA C - 6), (C-13), (C-12), (Gal C-1), (GlcA C-1), (Rha C- 1), 85.4 (C-3), 85.4 (GlcA C-3), 79.1 (GlcA C-2), 78.1 (GlcA C-4'), 77.3 (Gal C-5), 75.7 (Gal C-3), 74.4 (Rha C-4), 73.6 (Gal C-2), 73.3 (Rha C-3), 72.9 (Rha C-2), 71.9 (GlcA C -5), 70.9 (Rha C-5), 70.5 (Gal C-4), 62.3 (Gal C-6), 55.6 (C-4), 49.3 (C-5), 48.3 (C-9), 47.2 (C-17), 46.9 (C-19), 42.6 (C-14), 42.5 (C-18), 40.5 (C-8), 38.5 (C-1), 36.7 (C-10), 34.7 (C-21), 33.8 (C-29), 33.7 (C-7), 33.0 (C-22), 31.5 (C- 20), 28.7 (C-15), 26.6 (C-27), 25.9 (C-2), 24.3 (C-30), 24.2 (C-11), 24.1 (C-16), 21.0 (C-6), 19.0 (Gal C-6), 17.8 (C-26), 16.1 (C-25), 11.7 (C-24)

47 Table C- and 1 H-NMR spectroscopic data of aglycon for compound 1 (in Pyridine-d 5 ) a) Position 13 C (δ) b) 1 H (δ) c) / / / / / / / / / a) The assignments were based on 1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC, TOCSY and NOESY, b) 150 MHz for 13 C-NMR, c) 600 MHz for 1 H-NMR

48 Table C- and 1 H-NMR spectroscopic data of sugar moieties for compound 1 (in Pyridine-d 5 ) a) Position 13 C (δ) b) 1 H (δ) c) glucuronic acid (d, 7.2) (t, 7.9) (t, 8.7) (m) (m) galactose (d, 7.6) (m) (dd, 9.5, 3.3) (m) (t, 6.2) (m) 4.53 (m) rhamnose (br s) (br s) (m) (t, 9.3) (m) a) The assignments were based on 1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC, TOCSY and NOESY, b) 150 MHz for 13 C-NMR, c) 600 MHz for 1 H-NMR

49 3.4. 화합물 2 의분리및구조분석 목별자추출물의 n-buoh 분획을 flash purification system과 RP-HPLC (YMC J Sphere ODS-H80, 150 mm 4.6 mm i.d., YMC Co. Ltd.; flow rate of 2 ml/min; AcCN-H 2 O (29:71, 0.1% trifluoroacetic acid)) 에적용하여화합물 2를분리및정제하였으며, 아래와같이구조분석을위한데이터를확보하였다. Compound 2 C 48 H 74 O 20, White amorphous powder [α] 24 D: (c 0.3, MeOH); mp IR: ν max 3417, 2943, 1718, 1078, 1044 cm -1 High Resolution (HR)-FAB-MS (negative ion mode): m/z [M-H] - 1 H-NMR (Pyridine-d 5, 600 MHz): δ 9.87 (s, H-23), 6.06 (br s, Rha-H-1), 5.60 (br s, H-12), 5.23 (br s, H-16), 5.17 (d, J = 7.62 Hz, Gal-H-1), 4.97 (br s, Rha-H-2), 4.92 (m, Rha-H-5), 4.83 (d, J = 7.20 Hz, GlcA-H-1), 4.55 (dd, J = 9.33, 3.33 Hz, Rha-H-3), 4.53 (m, GlcA-H-4), 4.51 (m, Gal-H-4), 4.46 (m, H- GlcA-5), 4.46 (m, Gal-H-6), 4.43 (m, Gal-H-2), 4.42 (m, Gal-H-6b), 4.35 (t, J = 9.33 Hz, Rha-H-4), 4.27 (t, J = 7.92 Hz, GlcA-H-2), 4.22 (t, J = 8.73 Hz, GlcA-H-3), 4.00 (dd, J = 9.63, 3.33 Hz, Gal-H-3), 3.97 (d, J = 4.98 Hz, H-3), 3.90 (t, J = 6.24 Hz, Gal-H-5), 3.60 (dd, J = 14.25, 4.05 Hz, H-18), 2.83 (m, H-19), 2.51 (m, H-21), 2.43 (m, H-22),

50 2.31 (m, H-15), 2.25 (m, H-22b), 2.15 (m, H-2), 1.93 (m, H-11), 1.83 (m, H-9), 1.83 (m, H-2b), 1.80 (s, H-27), 1.68 (m, H-15b), 1.67 (d, J = 6.36 Hz, Rha-H-6), 1.54 (m, H-7), 1.47 (s, H-24), 1.40 (m, H-1), 1.38 (m, H-5), 1.38 (m, H-6b), 1.38 (m, H-19b), 1.38 (m, H-21b), 1.19 (m, H-7b), 1.18 (s, H-29), 1.07 (s, H-30), 0.96 (s, H-26), 0.94 (m, H-6), 0.89 (m, H-1b), 0.79 (s, H-25) 13 C-NMR (Pyridine-d 5, 150 MHz): δ (C-23), (C-28), (C- GlcA-6), (C-13), (C-12), (C- Gal-1), (GlcA-C-1), (Rha-C-1), 85.3 (C-3), 85.3 (C- GlcA-3), 79.1 (C-GlcA-2), 78.1 (C- GlcA-4), 77.3 (Gal-C-5), 75.7 (Gal-C-3), 75.2 (C-16), 74.4 (Rha-C-4), 73.6 (Gal-C-2), 73.3 (Rha- C-3), 72.9 (Rha-C-2), 71.9 (C- GlcA-5), 70.9 (Rha-C-5), 70.5 (Gal-C-4), 62.3 (Gal-C-6), 55.6 (C-4), 49.3 (C-17), 49.3 (C-5), 47.8 (C-19), 47.5 (C-9), 42.6 (C-14), 41.9 (C-18), 40.6 (C-8), 38.6 (C-1), 36.7 (C-10), 36.7 (C-21), 36.6 (C-15), 33.8 (C- 29), 33.4 (C-22), 33.3 (C-7), 31.6 (C-20), 27.7 (C-27), 25.9 (C-2), 25.2 (C-30), 24.2 (C-11), 21.0 (C-6), 19.0 (Rha-C-6), 17.9 (C-26), 16.2 (C-25), 11.7 (C-24)

51 Table C- and 1 H-NMR spectroscopic data of aglycon for compound 2 (in Pyridine-d 5 ) a) Position 13 C (δ) b) 1 H (δ) c) / / / / / / / / a) The assignments were based on 1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC, TOCSY and NOESY, b) 150 MHz for 13 C-NMR, c) 600 MHz for 1 H-NMR

52 Table C- and 1 H-NMR spectroscopic data of sugar moieties for compound 2 (in Pyridine-d 5 ) a) a) Position 13 C (δ) b) 1 H (δ) c) glucuronic acid (d, 7.2) (t, 7.9) (t, 8.7) (m) (m) galactose (d, 7.6) (m) (dd, 9.6, 3.3) (m) (t, 6.2) (m) 4.53 (m) rhamnose (br s) (br s) (m) (t, 9.3) (m) The assignments were based on 1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC, TOCSY and NOESY, b) 150 MHz for 13 C-NMR, c) 600 MHz for 1 H-NMR

53 3.5. 화합물 3 의분리및구조분석 목별자추추물의 n-buoh 분획을 flash purification system과 RP-HPLC (YMC J Sphere ODS-H80, 150 mm 4.6 mm i.d., YMC Co. Ltd.; flow rate of 2 ml/min; AcCN-H 2 O (29:71, 0.1% trifluoroacetic acid)) 에적용하여화합물 3을분리및정제하였으며, 아래와같이구조분석을위한데이터를확보하였다. Compound 3 (Momordica saponin I) C 48 H 74 O 20, White amorphous powder [α] (c = 0.2, MeOH-H D 2O (1:2)) mp IR: ν max (OH), (CH 3, CH 2 ), (-COOR), (-COO-), (C-O-) cm -1 High Resolution (HR)-FAB-MS (negative ion mode): m/z [M-H] - 1 H-NMR (Pyridine-d 5 -D 2 O (1:1), 900 MHz): δ 9.79 (s, H-23), 5.86 (d, J = 8.1 Hz, anomeric), 5.82 (br s, anomeric), 5.75 (br s, anomeric), 5.29 (d, J = 8.1 Hz, anomeric), 5.26 (d, J = 8.1 Hz, anomeric), 5.13 (d, J = 8.1 Hz, anomeric), 5.02 (d, J = 7.2 Hz, anomeric), 4.88 (d, J = 6.3 Hz, GlcA-H-1), 1.73 (d, J = 6.3 Hz, CH 3, sugar), 1.64 (d, J = 5.4 Hz, CH 3, sugar), 1.55 (d, J = 6.3 Hz, CH 3, sugar), 1.50 (s, CH 3 ), 1.28 (s, CH 3 ), 1.03 (s, CH 3 ), 0.99 (s, CH 3 ), 0.94 (s, CH 3 ), 0.92 (s, CH 3 )

54 13 C-NMR (Pyridine-d 5 -D 2 O (1:1), 225 MHz): δ (C-23), (C-28), (GlcA-H-6), (C-13), (C-12), (anomeric), (anomeric), (anomeric), (anomeric), (anomeric), (anomeric), (anomeric), 96.6 (Fuc-H-1), 88.7, 87.0 (GlcA-C-1), 86.4, 83.8, 80.2, 79. 6, 79.5, 78.6, 78.7, 78.4, 77.2, 76.9, 76.4, 75.7, 75.7, 75.0, 74.2, 74.1, 73.5, 73.3, 72.3, 72.3, 71.9, 72.0, 70.8, 70.6, 68.0, 63.9, 63.7, 63.4, 57.5, 49.9, 49.5, 49.0, 48.1, 44.0, 43.6, 41.8, 39.9, 37.7, 36.4, 35.6, 34.9 (CH 3 ), 34.2, 34.0, 32.4, 32.4, 29.9, 27.7 (CH 3 ), 26.6, 25.4 (CH 3 ), 25.1, 22.4, 21.7, 20.9, 20.4 (CH 3, sugar), 19.5 (CH 3, sugar), 19.1 (CH 3 ), 18.3 (CH 3, sugar), 17.7, 17.5 (CH 3 ), 15.7, 12.4 (C-24) 3.6. 화합물 1 과 2 의당분석 화합물 1 과 2 의당분석을위해기존의문헌의방법과같은방법으로 GC-MS 에적용하여분석하였다 (Yoon et al., 2007). [Capillary column: BPX-50 (0.25 mm 3 m; SGE, Melbourne, Australia), detector: flame ionization detector (FID), column temperature: 210 C, injector temperature: 270 C, detection temperature: 300 C, carrier gas: He (2.0 kg/cm)] 화합물 1 과 2 의산가수물은 trimethylsilyated 된표준물질 (Dgalactose, D-glucuronic acid 및 L-rhamnose) 의 GC-MS 상의 retention time 과비교하여검증하였다

55 4. 위염관련동물모델및기전연구 4.1. 에탄올유발위염모델 에탄올유발위염모델은에탄올에의해발생하는급성위점막손상여부를측정하는모델로아래와같은시험방법으로효능을평가하였다. 랫드를 18시간동안절식시킨후경구투여용랫드존데를이용하여 control, 시험물질, 대조물질의각각의용량을경구로투여하였다. 경구투여 1 시간후에위점막손상유발인자로서 100% ethanol 5 ml/kg를경구투여하고그로부터 6시간후에 ether 마취하에치사시킨후, 개복하여위를적출하였다. 적출한위를수술용가위를이용하여바깥쪽둘레를따라반쯤절개한후안의내용물을 saline으로세척하면서깨끗이제거하였다. 적당한크기의코르크판에위의점막부분이위를향하게하고팽팽하게펼치면서핀으로고정시켰다. 위점막손상정도는숙련된전문가를통해아래와같은 gross ulcer index 식에따라평가하였다 (Nam et al., 2005; Park et al., 2004). (number of type I lesions) + (number of type II lesions) 2 + (number of type II lesions) 3 - Type I: 부종, 충혈또는한군데점막하의점모양의출혈존재 - Type II: 작은미란을수반하는점막하의출혈병변존재 - Type III: 미란과침습성병변을수반하는깊은궤양존재

56 4.2. 디클로페낙유발위염모델 디클로페낙유발위염모델은 NSAID로인한급성위점막의손상을효능을평가하는모델로아래와같은시험방법으로효능을평가하였다. 랫드를 18시간동안절식시킨후경구투여용랫드존데를이용하여 control, 시험물질, 대조물질의각각의용량을경구로투여하였다. 경구투여 1 시간후에디클로페낙을 40 mg/kg로경구투여하고 6 시간후에 ether 마취하에치사시킨후개복하여위를적출하였다. 적출한위를수술용가위를이용하여바깥쪽둘레를따라반쯤절개한후안의내용물을 saline으로 washing하면서깨끗이제거하였다. 적당한크기의코르크판에위의점막부분이위를향하게하고팽팽하게펼치면서핀으로고정시켰다. 위점막손상정도는숙련된전문가를통해아래와같은 gross ulcer index 식에따라평가하였다 (Nam et al., 2005; Urushidani et al., 1977). (number of type I lesions) + (number of type II lesions) 2 + (number of type II lesions) 3 - Type I: 부종, 충혈또는한군데점막하의점모양의출혈존재 - Type II: 작은미란을수반하는점막하의출혈병변존재 - Type III: 미란과침습성병변을수반하는깊은궤양존재

57 4.3. H. pylori 유발만성위염모델 실제임상적으로위점막손상의가장중요한원인인세균인 H. pylori에의해유발되는만성위점막손상을측정하는모델로아래와같은시험방법으로효능을평가하였다. 마우스를 18시간동안절식시킨후 H. pylori (suspension at CFU/0.4 ml/mouse) 를경구투여하여접종하였다. 시험물질과 vehicle (2% Tween 80) 을 H. pylori를접종한후 1시간시점에서경구투여하며, 이를 7일간매일경구투여하였다. 대조물질 (omeprazole 1 mg/kg + clarithromycin 10 mg/kg in combination) 도시험물질도하루에한번씩일주일간경구투여한다. 감염후 8일이되는시점 ( 마우스를 overnight 절식 ) 에마우스를치사시킨후개복하여위를적출하였다. 적출한위를수술용가위를이용하여바깥쪽둘레를따라반쯤절개한후안의내용물을 saline으로 washing하면서깨끗이제거하였다. 적당한크기의코르크판에위의점막부분이위를향하게하고팽팽하게펼치면서핀으로고정시켰다. Ulceration score는점막의손상도에따라 4단계로평가하였다 (0 = normal appearance, 1 = mild red spots, 2 = moderate red spots and/or hemorrhage spots, 3 = marked hemorrhage spots) (Marchetti et al., 1995) Alcian Blue-Staining of Gastric Mucosal 랫드에시험약물을경구투여한후 30 분에에탄올 1.5 ml 을경구 투여하였다. 1 시간후랫드를치사시킨다음위를적출하여위점막의

58 일부를취한후 ice-cold M Sucrose로씻어내었다. 씻어낸위점막부위를세절하여, 0.16 M of sucrose buffered with 0.05 M of sodium acetate(ph 5.8) 에녹여있는 0.1% alcian blue에 2시간동안담가염색시켰다. 결합되지않은염색액은 0.25 M sucrose로 15분내지 45분동안깨끗이씻어내었다. 염색된위점막을현미경으로관찰하여정성적으로평가하였다 (Kitagawa et al., 1986) Croton oil 유발 Ear Edema 모델 시험물질의항염효과를평가하기위하여아래와같은방법으로시험하였다. ICR 마우스를미리실험실로옮겨 30분이상순화시킨후, 시험약물과대조약물을경구투여하고빈 cage에옮겨 1시간동안안정화시켰다. 경구투여 1시간후마우스를왼손으로보정한뒤아세톤에용해시킨 2.5 % croton oil을오른쪽귀의안쪽과바깥쪽에각각 pipette (PIPETMAN P20) 을이용하여 12.5 μl씩고르게도포한후입으로불어 acetone이휘발되도록하였다. 1군당분주된 1개의발염제 tube를사용하였다. 발염 4시간후 ICR mouse를 ether chamber에넣고과마치사시켰다. 과마치사시킨 ICR mouse를 thickness gauge로속도변화법을이용해왼쪽, 오른쪽의순서로양쪽귀의두께를측정하였다. 오른쪽귀의두께에서왼쪽귀의두께를뺀값을왼쪽귀의두께로나누고이값에 100을곱하여발염율을계산하였다 (Gabor, 2000; Park et al., 2004). 발염율 = [( 오른쪽귀의두께-왼쪽귀의두께 ) 왼쪽귀의두께 ]ⅹ

59 4.6. 진통모델 (Formalin test) 시험물질의진통효과를평가하기위하여아래와같은방법으로시험하였다. ICR 마우스를미리실험실로옮겨 30분이상순화시킨후, 시험약물과대조약물 morphine을경구투여하고거울이달린빈 cage에옮겨 1시간동안안정화시켰다. 시험약물과대조약약물을경구투여하고, 1시간후 29.5 gauge의 insulin syringe를사용하여미리준비한 formalin을마우스의왼쪽발바닥한가운데에 20 μl씩피하로주사하고관찰 cage로옮겨 5분동안 1차측정 (early phase) 하였다. 10분후같은방법으로 2차측정 (late phase) 을실시하였다. 측정은 2마리씩진행하며, 발바닥을핥는시간 (licking time) 을 timer로기록하였다. Formalin 피하주사시발바닥에피가나거나 formalin이바깥으로샌경우, 개체이상으로인하여관찰 cage 안에서심하게뛰거나 cage 밖으로뛰어나가는경우, 그리고측정결과발바닥을핥는시간이 10초이하로 formalin에의한통증유발이제대로되지않은경우에는그개체의데이터를최종결과에서제외하였다 (Tjølsen et al., 1992; Yang et al., 2002) 상처치료모델 (Wound healing model) 시험물질의상처치료효과를평가하기위하여아래와같은방법으로 시험하였다. 준비된마우스를 hexobarbital (90 mg/kg, i.p.) 로마취시킨후, 어깨와

60 등쪽부위의털을깎았다. 직경 12 mm의날카로운펀지로근육층 (Panniculus carnosus) 과이에부착된피부조직을제거하며, 그즉시시험물질 (MSE 50 μg/mouse) 또는대조물질 (CGS μg/mouse) 을국소적으로발라주었으며, 이후 10일동안하루에한번지속적으로국소적으로발라주었다. 상처부위는 Image-ProPlus 기기를사용하여 1, 3, 5, 7, 9, 11일되는날에측정하였다. 상처치료율 (%) 을측정하였으며, wound half-closure time (CT 50 ) 은 Graph-Prism 소프트웨어를사용하여선형회귀방법으로분석하였다 (Montesinos et al., 1997) CGRP 수용체특이적조직 Assay MSE가 CGRP 수용체에대한자극을촉진시키는지여부를증명하기위해 organ bath 상에서기닉피그좌심방조직을사용하여 CGRP 수용체에특이적인조직수축시험을수행하였다 (Ohno et al., 2008). 다양한농도의시험물질을기닉피그좌심방조직이놓여있는 organ bath의반응완충액에혼합하였다. 수축정도는 human α-cgrp (8-37) (0.03 μm) (Bachem, Switzerland) 과비교하여측정하였다 위장조직내 CGRP 분비측정 시험물질의 CGRP 분비효과를평가하기위하여아래와같은 방법으로시험을실시하였다. 7 주령랫드를 18 시간동안절식후, 마취하에서위를적출하였다

61 적출된위를절개하여점막부위가밖으로나오게하였다. 점막조직을깨끗하게씻은후, 혼합산소포화 modified Krebs-Henseleit 용액 (in mm: NaCl 110, KCl 4.7, CaCl 2 1.0, MgCl , NaHCO 3 25, glucose 5.6 and HEPES 10, ph 7.4) 에서 1시간동안안정화시켰다. 95% O 2 와 5% CO 2 로포화시킨 modified Krebs-Henseleit 용액 2 ml이포함된시험관에시험물질을넣은후조직을배양하였다. 30분후배양액을 4 C에서 3,000 rpm으로 5분간원심분리를하였다 (Nishihara et al., 2002; Inaba et al., 1996). 상등액의 CGRP 농도를 CGRP linked immunoassay (SPI-Bio, Massy Cedex, France) 로측정하였다 (Frobert et al., 1999) Somatostatin 수용체특이적조직 Assay MSE가 somatostatin 수용체에대한자극을촉진시키는지여부를증명하기위해 organ bath 상에서기닉피그회장조직을사용하여 somatostatin 수용체에특이적인조직수축시험을수행하였다 (Feniuk et al., 1993). 다양한농도의시험물질을기닉피그좌심방조직이놓여있는 organ bath의반응완충액에혼합하였다. 수축정도는 somatostain 28 (0.03 μm) (Sigma-Aldrich, USA) 과비교하여측정하였다

62 5. 단회및반복경구투여독성시험연구 시험물질의반복경구투여독성을평가하기위해 Sprague-Dawley (SD) 본실험은식품의약품안정청 (KFDA) 에서제공하는의약품독성시험가이드라인에따라수행하였다. MSE 500, 1,000 및 2,000 mg/kg 또는 vehicle (0.5% MC 용액 ) 을 12.5 ml/kg 용량으로군당 10마리 ( 암수각 5마리 ) 랫드에반복경구투여한후사망률, 일반증상, 체중및부검소견을 14일간관찰하여대조군과비교하였다. 14일째되는날랫드를이산화탄소로희생시킨후주요장기들의변화를평가하였다. 또한, 4주반복투여독성연구를위해 MSE를 500, 1,000 및 2,000 mg/kg/day의용량으로군당암수각각 10마리 ( 회복군은 15마리 ) 에투여한후, 4주동안사망률, 체중변화, 사료및물섭취량, 안검사및임상적징후를관찰하였다. 4주후랫드를희생시켜혈액학및혈액생화학적검사, 요검사, 장기중량, 부검소견및조직병리학적인소견을관찰하였다

63 6. Liquid chromatography mass spectrometry (LC MS) 를이용한 Similarity Analysis 연구 일반적으로생약은많은성분들이포함되어있으며, 다수의성분이상호 Synergy를일으키며 multi-target 효과를나타내는것으로알려져있다 (Xie et al., 2008). 생약의이러한특징때문에많은성분중에활성이있는특정성분을가려내기가쉽지않으며, 결국천연물의약품을표준화하고품질을관리하는것이어렵다는것을반증한다고할수있다. 이를극복하기위한노력으로특정지표성분이나활성성분들에대해함량을측정하는방법으로품질을관리하고있으나 (marker approach) (Mok and Chau, 2006), 이러한전략은천연물의약품에대한품질관리방법으로는부족한것으로여겨진다 (Li et al., 2010). 이러한문제점을극복하기위해많은노력들이있었으며, 최근에는 HPLC, GC, NMR 등에서얻어지는다양한화학적 profile을이용하여품질관리를시도하고있다. 본연구에서는 LC-MS를이용하여 pattern chromatogram에대한유사성분석을시도하였으며, 유사성분석을위해베트남과중국에서확보된목별자생약을사용하였다

64 유사성분석을위한분석조건은아래와같다. - LC-ESI-MS: Finnigan LCQ ion-trap mass - Column: Ascentis Express C 18 (150 mm 4.6 mm) - Flow rate: 0.3 ml/min - Mobile phase: A, formic acid 0.1% in water; B, acetonitrile A (%) B (%) 시료는목별자 50% 에탄올추출물을사용하였으며, 유사성분석을위해 LC-MS chromatogram 상에의미있는 peak 들을선정하고, 선정된피크에대해상관분석법을사용하여분석을시도하였다. 상관계수에따른유사성에대해자체기준 0.9 이상으로설정하였으며, 0.9 이상의수치가나올경우유사하다고판단하기로하였다

65 7. 목별자동속식물에대한연구 목별자의경우다수의동속식물이존재하여원생약수급시혼돈의문제가발생할가능성이존재하기에이를방지하기위한기초적인연구를수행하였다. 우선목별자동속식물에대한문헌적연구를실시하여목별자가혼돈될가능성이있는지에대한여부를알아보았다. 이를위해목별자의계통도및명명에관한기초조사를실시하였고, Momordica 속식물계통도, 원산지분류 (Historical biogeography), 아시아지역에분포하는 species 에대한근거자료취합, Synonyms DB (TROPICOS database) 를통해 Momordica 속식물을조사하여혼돈가능성여부를확인하였다. 이와더불어실제적인연구를위해목별자의지표성분인 Momordica saponin I이여주 (M. charantia) 추출물에함유되었는지에대한정성적비교, 분석을실시하였으며, 이를통해동속식물과혼돈가능성을배제하고자하였다. 참고로 Momordica saponin I은 TradiMed ( 및문헌조사에의하면, 목별자의동속식물및그외다른종에서는발견되지않는것으로알려졌다. 시험을위해 UPLC를이용한 spiking 법으로분석하여여주내 MS I의존재유무를확인하였고, 다시 LS-MS를이용한 selected ion monitoring (SIM) 법으로분석하여재확인시험을진행하였다. 분석조건은아래와같다

66 [UPLC 분석조건 ] - 분석기기 : Waters UPLC - Solvent: A: 0.1% phosphoric acid, B: acetonitrile - 칼럼 : ACQUITY UPLC BEH C18, 1.7 μm, mm - 온도 : Sample temperature 25, column oven 30 - Detector: UV 210 nm [LC-MS 분석조건 ] - HPLC System: Agilent Solvent: A: 0.1% formic acid, B: acetonitrile - 칼럼 : YMC Hydrosphere C18, S-5 μm, 12 nm, mm - 온도 : Sample temperature 25, column oven 30 - Detector: UV 210 nm - MS System: LCQ Deca XP (Thermo Finnigan) - Ionization: ESI / positive mode - Full ms: ~ SIM ms: ~

67 8. 목별자추출물의분석법밸리데이션연구 목별자의지표성분을정량하고자할때이에대한시험법을통해얻어진데이터가신뢰할수있음을증명하고자분석법밸리데이션연구를실시하였다. 밸리데이션을위해사용되는용어의정의는아래와같다. 1) 정확성 (accuracy/trueness) : 시험방법으로얻어진시험결과 ( 실측치 ) 와진실의수치 ( 참값 ) 와일치하는정도를나타내는것이다. 정확성은종종기지량을첨가한검체의분석에의한회수율로나타내며시험법의정확도를재는척도이다. 2) 정밀성 (precision) : 균일한재료로부터다수의검체를시험했을때시험결과간에일치 ( 재현성 ) 하는정도를나타내는것을말한다. 일반적으로시험법의정도는표준편차또는상대적표준편차 ( 변동계수 ) 로나타낸다. 3) 특이성 (specificity) : 특이성이란공존이예측되는불순물, 분해물, 배합성분등의존재하에서이성분들의영향을받지않고분석대상물을정확하고특이적으로측정할수있는능력을말한다. 4) 직선성 (linearity) : 시험법에있어서의직선성이란검체중에함유되어있는분석대상물의양또는농도에대하여직선적인비례관계를나타내는능력을말한다. 분석법밸리데이션을위한분석기기및조건은아래 Table 7 와같다

68 Table 7. Instruments and condition for analytical method validation Instrument UPLC sample manager Sample loop Wash Solvent Model Acquity sample manager Partial loop with needle overfill Strong solvent: acetonitrile Temperature Column: 30 ± 1 Weak solvent: H 2 O-Acetonitrile, 90:10 (v/v) Sample manager: 25 ± 3 Detector Tunable UV detector (210 nm) PDA UV detector Manager S/W Analytical column Mobile phase Injection volume Flow rate Empower ACQUITY UPLC BEH C18, 1.7 μm 0.2% phosphoric acid-acetonitrile 60:40 (v/v) 2.0 μl 0.5 ml/min 분석법밸리데이션시험항목별시험방법은아래와같다. 1) 특이성 (specificity), 분리능 (resolution), 대칭성 (symmetry) : 표준액및검액을 HPLC로분석하였을때다른모든피크와주성분의피크가겹치지않음을확인한다. 2) 정밀성 (precision) - Repeatability : 동일검체를최소 6회이상반복조제하여조제간의상대표준편차를구하고자한다. - Reproducibility : 다른날, 다른실험실, 다른기기에서동일검체를최소 6회이상반복조제하여조제간의상대준편차를구한다

69 3) 직선성과범위 (linearity and range): 함량분석농도의 300% 이상의표준액을이동상으로희석제조하여 UPLC 분석하여직선성을확인한다. 4) 정확성 (accuracy) - 표준액정확성 : 약 60%, 100%, 140% 양에해당하는표준품을취하여희석액에녹여샘플을제조하여분석후, 첨가한표준품과계산된표준품의회수량을비교하여회수율을평가한다. - 검액정확성 : 약 60%, 100%, 140% 양에해당하는검체를취하여희석액에녹여샘플을제조한후, 제조된샘플을계산된표준액에 spiking 하거나, 샘플에계산된표준품을추가한후에각각첨가한표준품과계산된표준품의회수량을비교하여회수율을평가한다

70 III. 결과및고찰 1. 후보생약의선정 각종정보 [KFDA 생약종합정보시스템, 전통동양약물데이터베이스 (TradiMed; Pubmed, 중양대사전, 향약대사전, 동의보감, 한의사자문 ] 를바탕으로후보생약에대한 1차및 2차선별을한결과, 15종후보생약을선별하였으며선정된리스트는아래와같다 (Table 8). 생약코드에서 MP는 mucosa protective를의미한다. Table 8. List of candidates for anti-gastritis agent 생약코드 생약명 생약코드 생약명 생약코드 생약명 MP01 포공영 MP06 진교 MP11 진피 ( 귤 ) MP02 곽향 MP07 골쇄보 MP12 백작약 MP03 인동 MP08 황금 MP13 화피 MP04 진피 MP09 목별자 MP14 파고지 MP05 백출 MP10 황련 MP15 만형자

71 Figure 7. Pictures of the raw materials of candidates

72 2. 동물위염모델에서의후보생약의선별 선정된 15종생약에대해디클로페낙유발위염모델에서효능을측정한결과 MP09 ( 목별자 ), MP1 2( 백작약 ), MP15 ( 만형자 ) 50% 추출물이상대적으로우수한효능을보였으며 (Figure 8 & 9), 특히, 목별자추출물의경우 3.6점으로 vehicle 대비 (10.2점) 가장우수한효능을나타냄을알수있었다. 참고로스크리닝단계이기때문에통계적유의성이있는데이터를확보하는목적보다는상대적우의성을가리기위한실험을진행하였다. 결과는제시하지는않았으나 MP01~08의경우효능을보이지않았다. 이로써 MP09, MP12, MP15를후보생약으로선별하였고, 3종후보에대해추가로에탄올유발위염모델을수행하였다. Figure 8. Gastroprotective effects of candidates (MP09~MP15) in rat model of NSAID (diclofenac)-induced acute gastritis. Results are mean ± SEM from six animals per group

73 Figure 9. Gastroprotective effects of candidates (MP09~MP15) in rat model of NSAID (diclofenac)-induced acute gastritis erosion. Results are mean ± SEM from six animals per group. 디클로페낙유발모델로선정된 3종생약에대해추가적으로에탄올유발위염모델을수행한결과 MP09 ( 목별자 ) 추출물의 gastric damage score가 3.0점으로가장우수하였으며, vehicle 대비 (9.2점) 통계적유의성을나타내었다 (P=0.031) (Figure 10). 이러한결과를토대로목별자를향후연구를위한최종생약후보로선정하였다

74 Figure 10. Gastroprotective effects of candidates (MP09, MP12, and MP15) in rat model of ethanol-induced acute gastritis. Results are mean ± SEM from six animals per group. *P < 0.05 compared with vehicle group

75 3. 목별자로부터활성분리및이의구조규명 3.1. 목별자분획활성연구 선정된목별자생약을 n-hexane, EtOAc, n-buoh 층으로분획하였으며, 이중지방성분이많이포함되는것으로판단되는 n-hexane 층을제외한 EtOAc와 n-buoh 층에대해에탄올유발위염모델을실시하였다. 본실험에서는우의성에대한변별력을높이기위해 100 mg/kg의농도에서비교하였다. 시험결과통계적유의성을보이지않지만 n-buoh층이활성이더좋은것으로판단하여 n-buoh 층에대해성분연구를실시하였으며, 이를통해 3종의화합물을분리하게되었다. Figure 11. Gastroprotective effects of MP09, MP09 EtOAc fraction, and MP09 n- BuOH fraction in rat model of ethanol-induced acute gastritis. Results are mean ± SEM from six animals per group

76 3.2. 화합물 1 의구조 화합물 1은흰색무정형의가루형태의물질로서선광도는 [α] D (C=0.21, MeOH) 이었으며, high-resolution (HR)-FAB-MS (negative ion mode) 에서 m/z [M-H] - 의분자이온피크를보여이화합물의분자식을 C 48 H 74 O 19 으로추정하였다. 13 C-NMR Spectrum (Figure 13) 을통해 48개의 carbon signal을확인 하였으며, 그중 30개는 aglycon, 18개는 sugar moiety에서유래된것으로확인하였다. 1 H-NMR spectrum (Figure 14) 을통해 δ 0.76, 0.93, 0.96, 1.01, 1.25, 1.37에서 6개의 tertiary methyl proton singlet을확인하였으며, δ 5.44 (br s) 에서 olefinic proton 및 δ 9.90 (s) 에서 aldehyde proton을확인하였다. 또한, 13 C-NMR spectrum을통해 δ 180.6에서 carboxyl carbon signal을확인하였다. 이러한데이터를기존의문헌과주의깊게비교한결과화합물 1의 aglycon이 gypsogenin임을확인할수있었다 (Bouguet-Bonnet et al., 2002; Zheng et al., 2007). 1 H-NMR에서 anomeric proton signal을 δ 4.82 (d, J = 7.2 Hz, GlcA-H-1), 5.17 (d, J = 7.6 Hz, Gal-H-1), 6.06 (br s, Rha-H-1) 에서확인하였으며, 13 C-NMR spectrum에서 anomeric carbon signal을 δ (Rha C-1), (Gal C-1), (GlcA C-1) 에서확인하였다. NOESY spectrum을확인한결과, δ H 9.90 (H-23) 피크와 3.95 (H-3) 피크그리고 1.25 (H-27) 피크와 0.96 (H-29) 피크사이에강한 correlation을보임에따라, aldehyde group이 α-orientation을이루고있음을알수있었다. 화합물 1을가수분해한후 trimethylsilyated 유도체화하여 GC-MS 분석을한결과, D- galactose, L-rhamnose 및 D-glucuronic acid가존재함을알수있었고,

77 glucuronic acid 와 galactose anomeric proton 의 J value 가각각 7.2 및 7.6 Hz 를보임에따라 glucuronopyranosyl 과 galactopyranosyl moiety 가 β- configuration 으로되어있음을확인할수있었다. Rhamnopyranosyl moiety 의 configuration 의경우 anomeric signal 이 1 H-NMR 과 13 C-NMR spectrum에서각각 δ 6.06 (br s) 및 δ 103.9에서관찰됨에따라 α- orientation으로정하였다 (Jia et al., 1998). 당과 gypsogenin과의결합위치는 HMBC spectrum을통해 C-3위치임을확인하였다 (Figure 15). 또한, 5.17 (Gal-H-1) 에서의 proton signal은 δ C 79.1 (GlcA-C-2) 과 long range correlation이확인되어 galactose가 glucuronic acid C-2 위치와결합되어있음을알수있었고, δ H 6.06 (Rha-H-1) 과 85.4 (GlcA-C-3) signal 사이에 cross 피크를보임에따라 rhamnose가 glucuronic acid C-3 위치에연결되어있음을확인하였다. 이상의결과를근거로하여화합물 1의구조를 gypsogenin-3-o-β-dgalactopyranosyl(1 2)-[α-L-rhamnopyranosyl(1 3)]-β-D-glucuronopyranoside 로동정하였으며, 이화합물은천연물에서처음분리, 보고되는물질이다 (Figure 12)

78 Figure 12. The molecular structure of compound 1 (gypsogenin-3-o-β-dgalactopyranosyl(1 2)-[α-L-rhamnopyranosyl(1 3)]-β-Dglucuronopyranoside)

79 Figure H-NMR spectrum of compound 1 (600 MHz, Pyridine-d 5 )

80 Figure C-NMR spectrum of compound 1 (150 MHz, Pyridine-d 5 )

81 Figure 15. HMBC spectrum of compound

82 3.3. 화합물 2 의구조 화합물 2는흰색무정형의가루형태의물질로써선광도는 [α] 24 D (C=0.3, MeOH) 이었으며, high-resolution (HR)-FAB-MS (negative ion mode) 에서 m/z [M-H] - 의분자이온피크를보여이화합물의분자식을 C 48 H 74 O 20 으로추정하였다. 13 C-NMR spectrum (Figure 17) 을통해 48개의 carbon signal을 확인하였으며, 그중 30개는 aglycon, 18개는 sugar moiety에서유래된것으로확인하였다. 1 H-NMR spectrum (Figure 18) 을통해 δ 0.79, 0.96, 1.07, 1.18, 1.47, 1.80에서 6개의 tertiary methyl proton singlet을 δ 5.60 (br s) 에서 doublet olefinic proton 및 δ 9.87 (s) 에서 aldehyde proton을확인하였으며, δ 5.23 (br s) 에서 hydroxymethylene proton signal (H-16β) 을확인하였다. 또한, 13 C-NMR spectrum을통해 δ 180.5에서 carboxyl carbon signal을확인함에 따라, 화합물 2의 aglycon이 quillaic acid임을확인하였다 (Bouguet-Bonnet et al., 2002; Luo et al., 2006; Zheung et al., 2007). 1 H-NMR spectrum을통해 δ 4.83 (d, J = 7.20 Hz, GlcA-H-1), 5.17 (d, J = 7.62 Hz, Gal-H-1), 6.06 (br s, Rha-H- 1) 에서 3개의 anomeric proton을확인하였으며, 이에해당하는 13 C-NMR Spectrum에서의 anomeric carbon signal은 δ 104.4, 105.0, 103.9에서확인하였다. 당에의해생성되는모든 proton peak는 HSQC, TOCSY, ROESY, HMBC correlation을활용하여주의깊게 Assign하였다. 화합물 2을가수분해한후 trimethylsilyated 유도체화하여 GC-MS 분석을한결과, D-galactose, L-rhamnose 및 D-glucuronic acid가존재함을알수있었다. Glucuronopyranosyl과 galactopyranosyl moiety의 β anomeric configuration은

83 3 J H1, H2 coupling constant 수치 ( Hz) 로확인할수있었다. Aglycon 과 당과의연결, 그리고당사이의결합은 Figure 19 에서와같이 HMBC correlation을통해확인하였다. [δ H 5.17 (Gal-H-1) with δ C 79.1 (GlcA-C-2), δ H 6.06 (Rha-H-1) with 85.3 (GlcA-C-3), δ H 4.83 (GlcA-H-1) with 85.3 (C-3)] 이상의결과를근거로하여화합물 2을 quillaic acid-3-o-β-dgalactopyranosyl(1 2)-[α-L-rhamnopyranosyl(1 3)]-β-D-glucuronopyranoside 로동정하였다 (Guo et al., 1998). 이화합물은 Momordica 속에서처음분리, 보고되는물질이다. Figure 16. The molecular structure of compound 2 (quillaic acid-3-o-β-d- galactopyranosyl(1 2)-[α-L-rhamnopyranosyl(1 3)]-β-D- glucuronopyranoside)

84 Figure H-NMR spectrum of compound 2 (600 MHz, Pyridine-d 5 )

85 Figure C-NMR spectrum of compound 2 (150 MHz, Pyridine-d 5 )

86 Figure 19. HMBC spectrum of compound

87 3.4. 화합물 3 의구조 화합물 3 은흰색무정형의화합물로써선광도는 [α] 24 D (C=0.2, MeOH-H 2 O(1:2)) 이었으며, high-resolution (HR)-FAB-MS (negative ion mode) 에서 m/z [M-H] - 의분자이온피크를보여이화합물의분자식을 C 76 H 120 O 40 으로추정하였다. 융해점 mp는 이었으며, IR ν max 값은 (-OH), (CH 3, CH 2 ), (-COOR), (-COO-), (C-O-) cm -1 이었다. 13 C-NMR Spectrum을통해 76개의 carbon signal을확인하였으며, 그중 30개는 Algycon, 46개는 sugar moiety에서유래된것으로확인하였다. 1 H- NMR spectrum을통해 δ 0.92, 0.94, 0.99, 1.03, 1.28, 1.50에서 6개의 Tertiary methyl proton singlet을 δ 5.44 (br s) 에서 doublet olefinic proton 및 δ 9.79 (s) 에서 aldehyde proton을확인하였다. 또한, 13 C-NMR spectrum을통해 δ 에서 carboxyl carbon signal을확인함에따라, 화합물 2의 aglycon이 gypsogenin임을확인하였다 (Bouguet-Bonnet et al., 2002). 1 H-NMR spectrum을통해 4.88 (d, J = 6.3 Hz, GlcA-H-1), 5.02 (d, J = 7.2 Hz, anomeric), 5.13 (d, J = 8.1 Hz, anomeric), 5.26 (d, J = 8.1 Hz, anomeric), 5.29 (d, J = 8.1 Hz, anomeric), 5.75 (br s, Rha-H-1), 5.82 (br s, Rha-H-1), 5.86 (d, J = 8.1 Hz, Fuc-H-1) 에서 8개의 anomeric proton을확인하였으며, 이에해당하는 13 C-NMR spectrum에서의 anomeric carbon signal은의 δ , , , , , , , 96.57에서확인하였다. Aglycon C-3 및 C-28과 sugar unit과의 linkage는 HMBC correlation을통해확인하였다 [δ H 4.88 (GlcA-H-1) with δ C (C-3), δ H 5.86 (Fuc-H-1) with

88 (C-28)]. 이상의결과를바탕으로기존문헌에제시된데이터와의비교및, 결과는제시하지않았지만, standard와의 UPLC 정성분석을통해화합물 3을 gysogenin-3-o-β-d-galactopyranosyl(1 2)-[α-L-rhamnopyranosyl(1 3)]-β- D-glucuronopyranosido-28-O-β-D-xylopyranosyl(1 3)-β-D-glucopyranosyl(1 3) -[β-d-xylopyranosyl(1 4)-α-L-rhamnopyranosyl(1 2)-β-D-fucopyranoside로동정하였으며, 이는목별자에서이미분리, 동정된 Momordica saponin I (MS I) 으로판명되었다 (Iwamoto et al., 1985). Figure 20. The molecular structure of compound 3 (Momordica saponin I)

89 Figure H-NMR spectrum of compound 3 (Pyridine-d 5 -D 2 O (1:1))

90 Figure C-NMR spectrum of compound 3 (Pyridine-d 5 -D 2 O (1:1))

91 3.5. 화합물 1, 2 및 3 의활성연구 분리된화합물 1, 2 및 3에대해에탄올유발위염모델에서활성연구를실시하였다. n-buoh 층 (100 mg/kg) 과레바미피드 (100 mg/kg) 를대조약물로하였으며, 화합물 1, 2 및 3은 20 mg/kg의농도로투여하였다. 실험결과, 통계적유의성을보이지않지만, 세가지화합물모두활성이나타나는경향을보였으며통계적유의성은없지만이중화합물 3이상대적으로가장좋은효능을보였다. Figure 23. Gastroprotective effects of MP09 n-buoh fraction, compounds 1, 2, and 3 in rat model of ethanol-induced acute gastritis. Rats were orally administrated with MP09 n-buoh fraction (100 mg/kg), compounds 1, 2, and 3 (20 mg/kg) or rebamipide (100 mg/kg), and 30 minutes later, ethanol (1.5 ml) was administrated using gastric sonde. Results are mean ± SEM from six animals per group

92 하지만화합물 1, 2 및 3의경우 20 mg/kg에서활성을나타내는경향을보이긴하였으나, n-buoh 분획대비뚜렷한약효를보이지않았을뿐만아니라, 함량이낮고 ( 화합물 3의경우수율이원생약대비약 0.025%) 분리가용이하지않아약효시험을수행하기가어려운단점이있다. 이러한단점에의해화합물 1, 2 및 3을위염치료제로개발하는것이어렵다고판단되었다. 그리하여단일화합물대신추출물로활성연구수행하기로하였다. 10% 에탄올을추출용매로하여향후실험을위한추출물을얻었으며, 이추출물을 MSE (Momordicae cochinchinensis extract) 로명명하였다. 데이터를제시하지는않았지만에탄올유도위염모델에서예비실험을실행한결과 MSE는 n-buoh 분획물과유사한위염활성을보였다. 화합물 3 (MS I) 의경우 MSE를대표하는화합물로사용하기에적합하다고판단하여지표물질로선정하였다

93 4. 목별자추출물의급 만성위염치료효능 4.1. 목별자추출물의에탄올유발위염모델에서의효능 MSE의에탄올에의해유발되는급성위점막손상에대한예방효과를확인하기위해랫드를이용한에탄올유발급성위점막손상모델시험을수행한결과, vehicle 군대비 gastric damage index 수치를현저히낮추었으며농도의존성도보였다 (Figure 24). 대조군인레바미피드가 200 mg/kg에서유의적인효과를보인것과비교할때, 에탄올유발위염모델에서는 MSE가레바미피드보다더강한위염예방효과를나타내는것으로사료된다. 12 Gastric Damage Index ** ** ** ** Vehicle MSE (50 mg/kg) MSE (100 mg/kg) MSE (200 mg/kg) Rebamipide (100 mg/kg) Rebamipide (200 mg/kg) Figure 24. Comparisons of the gastric damage indices in rat models of ethanolinduced gastritis. The data shown are the means of gastric damage index induced by ethanol. Rats were given MSE at 50, 100, and 200 mg/kg or rebamipide at 100 mg/kg followed by the administration of ethanol (1.5 ml). Results are mean ± SEM from six animals per group. *P < 0.05 and **P < 0.01 compared with vehicle group

94 4.2. 목별자추출물의디클로페낙유발위염모델에서의효능 Non-Steroidal Anti-Inflammatory drug (NSAID) 은 PG의생성을억제하여강력한항염증작용을발휘하는동시에위장관에서는 PG의위장관보호기능이감소되어위점막의손상을유발하는부작용이있다. NSAID 약물로유발된위점막손상에대한 MSE의보호효능을알아보기위해 40 mg/kg 디클로페낙을이용한급성위점막손상모델시험을수행하였다. 시험결과, 디클로페낙으로유발된위점막손상은 MSE의전처리에의해현저히감소되었으며위점막손상억제율은 200 mg/kg에서 vehicle 군대비각각 16%, 42%, 62% 를나타내었다. 반면대조군인레바미피드의경우두농도모두효능을나타내지않았다 (Figure 25). 10 Gastric Damage Index * ** Vehicle MSE (50 mg/kg) MSE (100 mg/kg) MSE (200 mg/kg) Rebamipide (100 mg/kg) Rebamipide (200 mg/kg) Figure 25. Comparisons of the gastric damage indices in rat models of diclofenacinduced gastritis. The data shown are the means of gastric damage index induced by diclofenac. Rats were given MSE at 50, 100, and 200 mg/kg or rebamipide at 100 mg/kg followed by the administration of diclofenac (40 mg/kg). Results are mean ± SEM from six animals per group. *P < 0.05 and **P < 0.01 compared with vehicle group

95 4.3. 목별자추출물의 H. pylori 유발위염모델에서의효능 이전연구에서 MSE가에탄올또는디클로페낙에의해유도되는위점막손상을효과적으로억제하는효과를확인하는것과더불어실제임상적으로위점막손상의가장중요한원인인 H. pylori에의해유발되는만성위점막손상을억제또는치유하는데있어서 MSE의효과를확인하였다. 실험결과 MSE를 50, 100, 200 mg/kg의용량으로투여한경우 vehicle 군대비위점막손상을각각 45, 64, 82% 억제하였으며, 200 mg/kg에서통계적유의성을보였다. 반면 Omeprazole + clarithromycin 투여군은 vehicle 군대비위점막손상이 55% 억제되었다 (Figure 26). 3 Ulceration score 2 1 * Vehicle MSE (50 mg/kg) MSE (100 mg/kg) MSE (200 mg/kg) Omeprozole 0.1 mg/kg + Clarithromycin 10 mg/kg 0 Figure 26. Comparisons of ulceration scores in mouse model of H. pylori-induced gastritis. Gastric damage was scored at four levels according to the degree of hemorrhage and severity of erosive lesions: 0 = normal appearance, 1 = mild red spots, 2 = moderate red spots and/or hemorrhage spots, and 3 = marked hemorrhage spots. Results are mean ± SEM from six animals per group. *P < 0.05 compared with vehicle group

96 4.4. 목별자추출물의위점액분비촉진모델에서의효능 위점막은위산으로부터위를보호하는가장중요한장벽이다. MSE를처리한군과처리하지않은군에대해위벽에존재하는점막두께를현미경으로분석하였는데, 위조직에대한조직학적슬라이드를비교한결과, Figure 27에서볼수있듯이 MSE를경구투여한군에서의점막두께가더두꺼웠다 (Figure 27). 이는 MSE가위점액분비를통해위점막보호에중요한역할을할수있음을보여주는근거로사료된다. Figure 27. Histological comparison of depth of the gastric mucus between MSEtreated and water-treated rats after ethanol challenge. Rats were treated with MSE (100, 200 mg/kg) or water (control group) 30 minutes before oral administration of pure ethanol (1.5 ml). One hour after the ethanol challenge, rat s stomach was taken, sliced and stained with alcian blue dye, which acid mucopolysaccharides and glycosaminoglycans blue to bluish-green

97 4.5. 목별자추출물의상처치료모델에서의효능 상피세포의증식과이동촉진 (proliferation & migration of epithelial cells) 은손상된위점막의회복을촉진하므로위염위궤양의치료제에있어서중요한요소이면서동시에일반적인상처치료의촉진에있어서도매우중요하다. 이에따라피부의상처치료에있어서 MSE의영향을비교하였다. 직경 12 mm 펀치로마우스의등에상처를낸후 MSE를 vehicle에녹여 10일간매일바르면서상처의면적을분석한결과 50, 100, 200 μg /mouse의용량으로 MSE를상처에바른경우 wound half-closure time (CT 50 ) 이각각 5.3 ± 0.1, 5.7 ± 0.1, 5.6 ± 0.2 day이었으나, vehicle 군의경우 CT 50 은 6.9 ± 0.3 day 로서통계적으로유의하게 MSE를처리한군의상처치유속도가빨라진것을알수있었다. 상처치료과정을촉진시키는것으로알려진양성대조군 CGS (Adenosine A 2A subtype receptor agonist; Montesinos et al., 1997) 을처리한실험군의 CT 50 은 5.2 ± 0.13 day로서 MSE 처리군과유사하였다 (Figure 28). 이실험을통해서 MSE가우수한상처치료효능을가지고있음을확인할수있었다

98 Vehicle (0.5% CMC/PBS), 20 ml/mouse 10, TOP MSE, 200 mg/mouse 10, TOP CGS-21680, 10 mg/mouse 10, TOP Figure 28. Time course of wound healing in mouse cutaneous injury model. Test substance was administered topically (TOP) once daily for 10 consecutive days. MSE at 50 μg/mouse or CGS at 10 μg/mouse or Vehicle (0.5% CMC/PBS) administered topically once daily for 10 consecutive days (n=5 per group). Significant differences are considered compared to vehicle at *P <

99 4.6. 목별자추출물의 Croton oil 로유도된귀부종실험모델에서의효능 본연구에서는 MSE의항염증효과를검증하기위해 croton oil 유도귀부종실험을수행하였다. 그결과 MSE가용량의존적으로부종을감소시키는것이확인되었으며그효과는비교약물로사용한디클로페낙, 레바미피드의효능과동등또는그이상이었다 (Figure 29). MSE의우수한항염증효과는 H. pylori나여러위점막인자공격인자에의해유발되는염증반응을억제함으로서위점막손상이확대되는것을막고신속하게회복시키는데매우중요한역할을할것으로생각된다. 참고로 목별자추출물의 somatostatin 수용체특이적조직 assay 결과에서 MSE가 somatostatin와의연관가능성이있음을보여주었는데, somatostatin은항염증과관련된다는여러보고가있다 (Markovics et al., 2011; Dror et al., 2008; Helyes et al., 2006; Pintér et al., 2006). Figure 29. Anti-inflammatory effect of MSE in croton oil-induced ear edema test. Results are mean ± SEM. **P < 0.01 and ***P < compared with vehicle group

100 4.7. 목별자추출물의진통모델 (Formalin Test) 에서의효능 본실험에서는일반적으로많이사용되는 formalin test를실시하여 MSE의진통효과를확인하였다. 시험결과 formalin test의 phase 1시험에서는양성대조약물로사용한 morphine (30 mg/kg) 이 67% 의진통효과를보였으며 MSE는 50, 100, 200 mg/kg용량에서 18%, 33% 72% 의용량의존적인진통효과를보였다. 특히, MSE가 200 mg/kg에서보인진통효과는 morphine의활성에대등한것이어서주목할만하다. 그러나 formalin test의 phase 2 시험에서는 morphine (30 mg/kg) 이 87% 의진통효과를보인반면 MSE는모든용량에서유의한진통효과를보이지못했다. 일반적으로 formalin test의 phase 2에서나타나는통증은염증과관련된만성통증이며, phase 1에서유발되는통증은 pain receptor가직접관여하는 nociceptive pain이다. 이러한결과는위염과위궤양으로인해위점막에서발생하는통증을 MSE가경감시킬수있는가능성을보여준다고할수있다. Figure 30. Analgesic effect of MSE (50, 100, and 200 mg/kg) in formalin test. Results are mean ± SEM. *P < 0.05 and **P < 0.01 compared with vehicle group

101 4.8. 목별자추출물의 CGRP 수용체특이적조직 Assay 에서의활성 CGRP는강력한혈관이완성 neuropeptide로써위점막의혈류량을증가시키며점액과 bicarbonate 분비를촉진하여위점막보호활성을나타낸다. 또한, PGI 2 (prostacycline I 2 ), PGE 2 (prostacycline E 2 ), NO의발현을증가시키며, 염증성 cytokine (TNF-α, IL-1β, IL-8) 분비를억제시키고, acetylcholine의유리를유도한다. 따라서 CGRP tissue assay를통해 MSE가 CGRP의 agonist 역할을하는지알아보았다. 실험결과 MSE는 100 μg/ml 농도에서 30 nm hcgrpα 에의해유발되는수축대비 85% 의수축 (agonist-like positive inotropy of 85%) 을보였으며, MSE의 EC 50 은 62.4 μg / ml인것으로나타났다. CGRP의경우 specific antagonist인 α-cgrp (8-37) 을처리할경우 MSE가보인양성반응이억제되는지를확인하였으며 (Lawson et al., 1994), 그결과 94% 억제되었다. 이와같은결과를통해 MSE가 CGRP 수용체의 agonist로서작용할가능성이있음을확인할수있었다. 100 % Inotropy Relative to hcgrpα (0.03 M) MSE (μ g/ml) Figure 31. Dose response curves of MSE in CGRP-specific tissue inotropy assay (EC 50 = 62.4 μg/ml)

102 4.9. 목별자추출물의위조직에서 CGRP 분비에미치는영향 위조직에서 MSE가 CGRP 분비에미치는영향을확인하기위해위조직을적출한후에 buffer에서안정화시킨다음 MSE의처리에따른 CGRP release의변화를관찰하였다. 실험결과, MSE를 100 μg/ml 이상의농도로처리한경우 CGRP의분비가증가하였으며, 1,000 μg/ml의농도에서는통계적유의성을보였다. 이러한결과를통해이전에실시한 CGRP receptor tissue assay에서의양성반응이 CGRP의발현을증가시켜서유도되었음을추측할수있다. 하지만, 적출한조직을이용한실험결과를 in vivo 실험을통해서평가할필요가있다고사료된다. Figure 32. CGRP secretion effect from gastric tissue of MSE. Results are mean ± SEM. *P < 0.05 compared with vehicle group

103 4.10. 목별자추출물의 Somatostatin 수용체특이적조직 Assay 에서의활성 Somatostatin은강하게위산을억제하여가스트린분비촉진과함께위산도를조절하는 neuropeptide이며, 점액과 bicarbonate의분비를촉진하여위점막보호효과가있는것으로알려져있다. 따라서 MSE의점막보호활성의작용기전을탐색하기위해 somatostatin tissue assay를수행하였다 (Dennis, 1989; Feniuk et al., 1993). 실험결과 12.5, , 100 μg / ml의용량에서 MSE가용량의존적으로유의하게양성반응을나타내었으며, MSE의 EC 50 은 25.1μg / ml인것으로나타났다. 이로써 MSE가 somatostatin 수용체에자극하는역할을할가능성이있음을확인할수있다. % Reduction of neurogenic twitch Relative to SST (0.03 M) MSE (μ g/ml) Figure 33. Dose response curves of MSE in somatostatin-specific tissue neurogenic twitch assay (EC 50 = 25.1 μg/ml)

104 5. 목별자추출물의독성시험연구 5.1. 단회경구투여급성독성시험 MSE에대하여 Sprague-Dawley (SD) 계통암수랫드에단회경구투여하여급성독성시험을실시하였다. 시험물질을 500, 1,000 및 2,000 mg/kg의용량으로, 군당 10 마리 ( 암수각 5 마리 ) 에반복경구투여한후사망률, 일반증상, 체중및부검소견을 14일간관찰하여부형제대조군과비교하였으며그결과, 1) 실험기간중사망동물은관찰되지않았다. 2) 일반증상관찰결과, 투여당일암수동물에서설사와항문주위오염이용량상관성있게관찰되었다. 3) 체중변화관찰결과, 유의한변화는관찰되지않았다. 4) 부검소견관찰결과, 육안적이상소견은관찰되지않았다. 이상의결과로보아, MSE을 Sprague-Dawley (SD) 계통의암수랫드에단회경구투여하였을때, 본시험물질의최소치사량 (MLD: Minimum Lethal Dose) 은암수모두 2,000 mg/kg을상회하는것으로판단된다 랫드 4 주반복경구투여독성시험 본시험은 MSE를 SD 계통의랫드에 4 주간반복경구투여하였을때나타나는독성을조사하며, 2 주간의회복기간을두어회복성을알아보고자수행하였다. 시험물질을 500, 1,000 및 2,000 mg/kg/day의용량으로군당암수각각 10 마리 ( 회복군은 15 마리 ) 에투여한후

105 사망률, 일반증상, 체중변화, 사료및물섭취량, 안검사, 요검사, 혈액학및혈액생화학적검사, 장기중량, 부검소견및조직병리학적인소견을관찰한결과, 1) 실험기간동안시험물질의투여와관련된일반증상의이상이나사망동물은없었다. 2) 체중, 사료및물섭취량측정결과, 독성학적으로시험물질의투여와관련된특이한변화는확인되지않았다. 3) 요검사결과, 시험물질의투여와관련된특이한소견은없었다. 4) 혈액학및혈액생화학적검사결과, 독성학적으로시험물질의투여와관련된특이한변화는없었다. 5) 장기중량측정결과, 시험물질의투여와관련된독성학적인변화는없었다. 6) 육안적인부검소견및조직병리학적검사결과, 시험물질의투여와관련된이상소견은없었다. 이상의결과를종합하여볼때, MSE를 SD 계통의암수랫드에 4 주간반복경구투여하였을때, 본시험조건하에서본시험물질의무독성량 (NOAEL; no observed adverse effect level) 은암수모두 2,000 mg/kg/day 이상으로판단하였으며, 표적장기는관찰되지않았다

106 6. LS-MS 를이용한목별자산지별 Similarity Analysis 연구 목별자 50% 에탄올추출물에대한유사성분석을위해분리조건을최적화시키는작업을하였다. 이를위해 Ascentis Express C 18 (150 mm 4.6 mm, 5 μm) 컬럼을사용하였으며, 이동상은 formic acid 0.1% 와 AcCN을 gradient 조건으로사용하였다. 최적의이동상조건은 Table 9과같으며, 이조건에서얻어진대표 LC- MS chromatogram은 Figure 34과같다. 참고로추출및농축의용의성을위해 50% 에탄올을추출용매로사용하였다. Table 9. Optimized mobile phase condition for LC-MS similarity analysis Formic acid 0.1% Acetonitrile 0 95% 5% 45 30% 70% 46 0% 100% 55 0% 100% 56 95% 5% 70 95% 5%

107 Figure 34. LC-MS chromatogram of 50% ethanol extract of reference M. cochinchinensis (MCREF) Figure 34에서검출된피크중전체를대표할수있는 peak 11종을선택하여 similarity analysis를수행하였다. 구체적으로 RT 4.39, 5.91, 13.56, 13.87, 19.78, 23.60, 26.96, 28.36, 30.00, 31.41, 35.35분에존재하는피크를선택하였으며, 선택된피크에대해 Table 10에서와같이상관분석을실시하였다

108 Table 10. Correlation analysis chart among M. cochichinensis samples from Vietnam and China. MCREF MCV1 MCV2 MCV3 MCV4 MCV5 MCV6 MCV7 MCV8 MCC1 MCC2 MCREF 1 MCV MCV MCV MCV MCV MCV MCV MCV MCC MCC 상관분석결과 Reference 목별자의 50% 에탄올추출물 (MCREF) 은중국운남성 (MCC2) 목별자와가장유사함을보였으며, 상관계수를보았을때자체기준 (> 0.9) 에적합한수치를보였다 (> 0.906). 다른지역의샘플들과의상관계수도 사이에값을가지고있어어느정도유사성을보였으나, 베트남목별자 (MCV6) 의경우는완전히다른 pattern을보여 (-0.028) 적합한목별자를수급하기위해서는유사성분석이필요하다고판단하였다

109 Figure 35. LC-MS Chromatograms of 50% ethanol extract of reference M. cochinchinensis (MCREF), MCC2, and MCV6-88 -

110 7. 목별자의동속식물에대한연구 7.1. 목별자의동속식물에대한문헌적연구 본연구에서는목별자의동속식물에대한문헌적연구를통해 목별자수급을위한 GCAP 에대한기초자료를얻고자하였다 목별자의계통도 목별자의계통도몇명명은 Table 11 와같다. Table 11. Classification and naming of M. cochinchinensis Classification Naming Kingdom Plantae Haeckel, plants Subkingdom Viridaeplantae Cavalier-Smith, green plants Phylum Tracheophyta Sinnott, 1935 ex Cavalier-Smith, vascular plants Subphylum Euphyllophytina Infraphylum Radiatopses, Kenrick & Crane, 1997 Class Magnoliopsida, Brongniart, 1843 Subclass Dilleniidae, Takhtajan, 1967 Superorder Violanae, R. Dahlgren ex Reveal, 1992 Order Cucurbitales, Dumortier, 1829 Family Cucurbitaceae, Durande, cucumber family Subfamily Cucurbitoideae Tribe Joliffieae Genus Momordica C. Linnaeus,

111 [ 참고 ] Lour.: João de Loureiro A Portuguese Jesuit missionary, paleontologist, physician and botanist ( ). In 1742 he travelled to Cochinchina, remaining there for 30 years. He became a specialist in Asian flora and on his return published Flora Cochinchinensis (1790). Spreng. : Curt Polycarp Joachim Sprengel A German botanist and physician. (August 3, March 15, 1833) He also made many improvements in the details of both the Linnaean and the natural systems of classification. The standard botanical author abbreviation Spreng. is applied to species he described Momordica 속식물조사 1) Momordica 속식물계통도 Scheme 3 은 Momordica 속식물의염기서열로부터상호유연관계 확인하고, 이를 maximum likelihood 통계법으로처리한결과수치를 나타낸 dendrogram 이다

112 Scheme 3. Maximum likelihood tree for Momordica obtained from combined plastid, nuclear and mitochondrial sequence data (Schaefer and Renner, 2010) 2) 원산지분류 (Historical biogeography) Scheme 4는 Momordica 속식물원산지를나타내는 dendrogram으로이중동아시아및동남아시아로에서기원한 species는 9종임확인할수있다 (Table 12)

113 Scheme 4. The historical biogeography of Momordica inferred on the preferred maximum likelihood tree under maximum likelihood optimization (Schaefer and Renner, 2010)

114 Table 12. Momordica species originated from Asia Genus Distribution Species Momordica East & Southeast Asia M. clarkeana M. cochinchinensis M. sphaeroidea M. macrophylla M. denticulata M. suringarii M. laotica M. subangulata West Asia M. denudata M. renigera M. dioica Asia 지역에분포하는 species 에대한근거자료취합 1) 근거자료 I Momordica 속중아시아지역에분포하고있는 species 만을붉은사각형으로표시하였으며, 이를추출하여자료로사용하였다 (Schaefer and Renner, 2010). 그외지역은대부분이아프리카지역이고극히일부북미 / 호주지역이확인된다 (Scheme 5 & Table 13)

115 Scheme 5. Momordica species from Asia geographic origin

116 Table 13. Extraction and summary: Momordica species from Asia geographic origin Nation Korea China India Indonesia Malaysia Philippines Papua New Guinea Sri Lanka Thailand Species M. charantia M. cochinchinensis M. macrophylla M. subangulata M. renigera M. trofolia (Ambon) M. denticulata (Borneo) M. clarkeana (Borneo) M. subangulata (Sumatra) M. denticulata M. sphaeroidea M. sphaeroidea M. denudata M. dioica M. charantia M. laoitca M. suringarii 2) 근거자료 II TROPICOS 데이터베이스에서중국에서식하는식물들의자료를추출하였는데, 좌측창은 Momordica의 family인 Cucurbitaceae를선택하였고우측창에서 Momordica 속식물들을확인한후이를자료로추출하였다 (Figure 36 & Table 14). * TROPICOS was originally created for internal research but has since been made available to the world s scientific community. All of the nomenclatural, bibliographic, and specimen data accumulated in MBG s electronic databases during the past 25 years are publicly available here. This system has over one million scientific names and 3.5 million specimen records

117 Figure 36. Momordica species in genus of Cucurbitaceae (TROPICOS database) Table 14. Extraction and summary: Momordica species of Cucurbitaceae inhabiting in China (TROPICOS database) Family Genus Species Cucurbitaceae M. charantia L. M. cochinchinensis (Lour.) Spreng M. dioica Roxb. Ex Willd. M. subangulata Blume 3) 근거자료 III Germplasm Resources Information Network (GRIN) 데이터베이스는미국농무부산하 National Genetic Resources Program의자료로전세계 46,000 여종의자료가있으며, economic plants에대한자료위주로되어있다. 이자료로부터 Momordica 속식물중아시아지역에분포되어있는작물을추출하여자료로사용하였다 (Figure 37 & Table 15)

118 Figure 37. Native distribution of Momordica species in Asia (Temperate & Tropical) (GRIN Taxonomy)

119 Table 15. Extraction and summary: Momordica species as an economic plants in Asia Distribution Species Asia - Temperate M. balsamina L. M. charantia L. M. charantia subsp. charantia M. cochinchinensis (Lour.) Spreng. Asia - Tropical M. balsamina L. M. charantia L. M. charantia subsp. Charantia M. cochinchinensis (Lour.) Spreng. M. cymbalaria Hook. f. M. dioica Roxb. ex Willd 4) 근거자료 IV India National Bureau of Plant Genetic Resources에서발행한자료로 Momordica 속에대한자세한설명과 Momordica species의 India 내 geographical distribution 및 ecological 자료등이있는 400여페이지에걸쳐조사된자료이다 (Joseph, 2004). 그중산지가아시아로확인된 species 만을추출하여자료로사용하였다 (Figure 38 & Table 16)

120 Figure 38. Momordica species in Herbaria around the world Table 16. Extraction and summary: Momordica species having an apparent habitat (national) in Asia Nation Species Burma or Myanma Indonesia (Borneo) Malaysia M. rengera M. acuminata M. clarkeana M. subangulata Philippine Sri Lanka M. cochinchinensis M. denudata

121 Synonyms 지금까지추출하여얻은종들중에서이명 ( 異名 ) 이있는종들의확인이필요하였다. 이를위해신뢰도가높은 TRIPICOS 데이터베이스를통해 Momordica species의 synonyms 를추출하여 M. cochinchinensis 와 M. subangulata 가이명이있음을확인하였다 (Figure 39). M. cochinchinensis M. subangulata Figure 39. Synonyms of Momordica species

122 Momordica cochinchinensis 동속식물조사범위결정 1) 아시아지역에서확인되는종들과그서식지 ( 서아시아지역제외 ) 지금까지수집한자료및조건에적합한 Momordica 속식물을추출한후, 식물의학명과그서식지역을정리하였다 (Table 17). 자료는 species 의 alphabet 순으로정리하였다. 단, 목별자 (M. cochinchinensis) 는가장상단에위치시켰다. Table 17. Classification of Momordica species by habitats Species Country M. cochinchinensis (= M. macrophylla) China, Asia, Philippines M. acuminata Indonesia M. balsamina China, India M. charantia Korea, China, Asia M. clarkeana Indonesia, Malaysia M. denticulata Malaysia, Indonesia M. denudata Sri Lanka M. dioica Sri Lanka, China M. renigera India, Myanmar M. sphaeroidea Philippines, Papua New Guinea M. subangulata (= M. laotica) China, Malaysia, Indonesia Thailand M. suringarii Thailand

123 2) 조사제외 species Table 17에있는자료중서식지역이중국이나 Cochin china가아닌종은우선제외하였다 (Table 18. Group I). 본연구에사용된목별자는중국과베트남에한정하여채집하였고, 이들은주로인도네시아, 태국, 미얀마등지에서서식하여추후원생약의구입이나 GCP에서혼용의여지가낮다고판단하였다. 참고로본연구에사용된목별자는중국과베트남에서구입하였으므로조사지역을중국과베트남지역으로한정하고자하였다. 다음으로 M. charantia, M. balsamina의경우는서식지역은추출조건에적합하나, dendrogram 계통도상에서서로상이한 species로이두종은그형태학적인자료만조사하기로하였다 (Table 18. Group II). Table 18. The excluded Momordica spices from the research subject and the reasons Group 제외이유 Species I China 및 Cochin china 이외의지역에서서식함 M. acuminata (Indonesia) M. clarkeana (Indonesia, Malaysia) M. denticulata (Malaysia, Indonesia) M. denudata (Sri Lanka) M. renigera (India, Myanma) M. sphaeroidea (Philippines, Papua New Guinea) M. suringarii (Thailand) II Likelihood가상이함 M. charantia M. balsamina

124 3) 조사종들선택 Table 17와 Table 18으로부터최종정리된자료로모든추출조건에적합한 M. cochinchinensis, M. subangulata, M. dioica 에대해서는자세한조사를하였고, M. charantia, M. balsamina (Group II) 에대해서는앞에서밝힌바와같이열매와 seed 의형태학적인간단한조사만을하였다 (Table 19). Table 19. The final Momordica species for research subject 조사방법 자세한조사 (Group II) 형태조사 (Group II) M. cochinchinensis M. subangulata M. dioica M. charantia M. balsamina Species

125 M. cochinchinensis 및유사동속식물 5 종조사 1) 상세조사 1-1) M. cochinchinensis ( 목별자 ) Table 20. Information about M. cochinchinensis Name Momordica cochinchinensis 목별자 gấc (in Vietnamese) 木鳖果, mùbiēguǒ (in Chinese) Baby Jackfruit, Spiny Bitter Gourd, Sweet Gourd, or Cochinchin Gourd (common name) Distribution Grown widely in Southeast Asia, as well as China and Japan Use The powder is indicated to promote the subsidence of nodulation and swelling, combat poisons and cure sores and is orally consumed for the treatment of sores and inflammatory swellings, mastitis, scrofula, haemorrhoids, anal fistula, chronic eczema, neurodermatitis and scalds (Pharmacopoeia of the People s Republic of China, 1997). Substances Chondrillasterol, Columbin, Hemsloside MA-1, 6'-Methylmomordin I, Momorcochin Momordica saponin I, Momordica saponin II, Momordicine III, Momordin I, Momordin IC, Momordin ID, Momordin IE, Momordin IIA, Momordin IIB, Momordin IIC, Momordin IID, Momordin IIE, Momordin III, Nardol, Vergatic acid (Tradimed) Figure

126 1-2) M. subangulata Blume Table 21. Information about M. subangulata Blume Name Ao e mu bie (in Chinese) Momordica subangulata Blume peria katak, kambas Bitter gourd (common name) Distribution Use Guangdong; Guangxi; Guizhou; Yunnan Being eaten as vegetable and is believed can help to lower the blood pressure and cure diabetic. Figure

127 1-3) M. dioica Table 22. Information about M. dioica Name Yun nan mu bie (in Chinese) Momordica dioica Teasle Gourd, Spine gourd Kakrol, Kankro, Kartoli, Kantoli, Kantola or Kantroli Distribution Substances A native plant of the Indian sub-continent, Yunnan α-spinasterol octadecanonate, α-spinasterol-3-o-β-d-glucopyranoside, 3-O-β-D--glucuronopyranosyl gypsogenin, 3-O-β-D-glucopyranosyl gypsogenin, 3-O-β-D--glucopyranosyl hederagenin Figure

128 1-4) Seed Length & Width 자료 Table 23. Morphology of Group I species Species Mean length (mm) Mean width (mm) M. cochinchinensis 20 ~ ~ 30 M. subangulata 9~10 7 M. dioica 6~7 7 2) 형태조사 (Group II) 1) M. charantia ( 여주 ) Figure 40. Morphology of M. charantia 2) M. balsamina Figure 41. Morphology of M. balsamina

129 Momordica cochinchinensis 및유사동속식물조사결론 Momordica L. 의 Likelihood 계통도자료 (Schaefer and Renner, 2010) 를기준으로하여 TRIPICOS, GRIN, Pubmed, Tradimed 와기타논문및인터넷자료등을검색한결과 China 및 Cochinchina에주로서식하는 Momordica 속식물은 5 종 (M. balsamina, M. charantia, M. cochinchinensis, M. dioica 및 M. subangulata) 임을확인하였다. 그중목별자 (M. cochinchinensis) 를포함한유사도가높은 M. subangulata 및 M. dioica에대한생약명, 분포지역, 사용예, 구성성분, Pubmed 확인논문, 과육혹은종자의모양등을조사한결과, 본문에제시된각생약의사진에서도알수있듯이과육의형태와색상을통해서로쉽게구별할수있었으며, 원료생약인종자는크기 (Length) 도구별이용이하였다. 또한, 중국약전및관련논문자료에서도 M. dioica, M. subangulata 의크기는 M. cochinchinensis 크기의약 30 ~ 50 % 정도인것으로기록되어있어, 목별자를구매하거나 GCAP 를하는데있어서유사종과혼용되는일은없을거라예상된다. Group II에속한 2 종 (M. balsamina, M. charantia) 의경우는형태로만조사를실시하였으며, 형태로보았을때목별자와는열매나 seed의모양이상이함을알수있었기때문에이종도목별자와혼용되는일은없을것으로판단된다. 결론적으로목별자와유사종으로판단되는 4가지생약은목별자와혼용되거나혼돈되는경우가없을것으로판단되나, 보다과학적인증명을위해추후유사생약의종자를직접확보하여실제적인연구를

130 통해증명하는작업이필요하리라생각된다. [ 참고 ] 목별자산지조사상기목별자동속식물연구더불어목별자의주산지에대해조사를하였다. 목별자는중국남부광서성 / 운남성및베트남북부이다. 베트남에서는목별자의열매를 Gac이라부르는데, Gac 의과육에존재하는 β-carotene은그함량이당근보다높다. 특히 Gac 이외에는 β- carotene 섭취가어려운베트남인들을위해 Gac의생산은정부주도차원에서대규모로이루어지고있다. 특히, 북부 Hai Duong 지역은그생산량이여타국가나지역과는비교가되지않을정도로많은데, 그생산량은다음과같다. Table 24. The gross output of Gac fruit in Hai Duong region, Vietnam (KOTRA report) Year The weights of Gac fruit 110,000 tons 250,000 tons An average gac fruit weighing 1 kg yields approximately 190g of fruit pulp and 130 g of seeds 따라서대규모생약의확보를위해서는베트남 Hai Duong 산목별자를수급하는것이유리하다고판단된다. 참고로데이터로제시되지는않았지만, 활성및독성은산지 ( 중국광서, 운남, 호북, 장시, 베트남등 ) 에크게영향을받지않는것으로판단된다

131 7.2. 여주 (M. charantia) 내 Momordica saponin I 존재유무에대한연구 목별자의학명은 Momordica cochichinensis Spr. (Cucurbitaceae) 로그구성성분인 momordica saponin I (MS I) 은 Iwamoto 등이분리, 구조규명을하였으며, 이성분은 Tradimed DB ( 및문헌조사에의하면목별자의동속식물및그외다른종에서는아직발견되지않는것으로알려져있다. 여주의학명은 Momordica charantia (Cucurbitaceae) 로목별자의동속식물이다. 본연구는 MS I을통해목별자와여주를구별하고, 지표성분으로써의가능성을확인하는데있다. 실험결과는 Figure 42와 43과같다

132 a. MSE b. M. charantia c. spiking Figure 42. Pattern chromatograms of MSE, M. charantia, and Spiking

133 Figure 43. Overlay chromatogram of MSE, M. charantia & Spiking (Red: M. charantia, Blue: MSE, Purple: spiking) UPLC pattern 및 spiking 분석결과여주에 MS I 가존재하지않는 것으로확인되었으나, 보다확실한결론을얻기위해 LC-MS 분석을 진행하였으며, 결과는아래와같다

134 a. MSE b. M. charantia Figure 44. LC-MS SIM of MSE and M. charantia MS I의 accurate molecular weight는 이다. MSE의 HPLC pattern 및 SIM 결과로부터 MS I peak는 25.8 분경에 [M+Na] + 의형태로검출되는것을확인하였다. 이를토대로여주추출물을측정한결과 25.8±1.5 분경에 SIM에서확인할수있는 peak는검출되지않았다. 따라서여주추출물에는 MS I이존재하지않음을재확인하였다. 이상의 HPLC-UV와 LC-MS 실험결과로여주추출물에는 MS I이존재하지않음을확인할수있었으며, MS I이지표물질로사용하는것이적합함을알수있었다

135 8. 목별자추출물의분석법밸리데이션연구 원료의약품내지표성분 Momordica saponin I의함량을측정하기위한분석기기는 UPLC (Waters, USA) 로결정하였다. UPLC 분석조건의확립을위해이동상의 ph, buffer의종류, buffer 내용매조성을결정하는실험을수행하였다. 그결과 0.2% 인산수용액과 acetonitrile 을 6:4 (v/v) 의비율로혼합했을때지표성분피크의 S/N, 대칭성및분리능이우수함을확인하였다. 이조건에서검액의최적농도를찾고자, 검액내지표성분피크의 S/N 이 100 이상농도를기준으로설정하였고이농도는 800 μg/ml 임을확인하였다. MSE의분석법에대한밸리데이션은아래 Table 25의분석항목과그에대한기준에의거하여수행하였다. Table 25. The validation items and specifications for the analytical method 시험항목 Validation 정량법 / 함량시험법 기준 특이성 - 확인 + 적합 - 분리능 이상 - 대칭성 이하 직선성 이상 범위 + 적합 정확성 (RSD) + 2.0% 이하 정밀성 (RSD) - Repeatability % 이하 - int. precision + * 2.0 % 이하 - Reproducibility (+) 2.0 % 이하 정량한계 - 최소 검출한계 - 최소 * Reproducibility 실험을했을경우불필요

136 MSE 의분석법에대한밸리데이션결과는아래와같다. 8.1 특이성 (Specificity) 아래의시험결과를통해검액, 표준액및희석용액사이에간섭이 없음을확인하였다. 따라서이분석법은특이성이확보되었다고 판단되었다. Table 26. The result of standard solution in specificity test Peaks Inj. 1 Inj. 2 Inj. 3 Inj. 4 Inj. 5 Mean %RSD RT 면적 이론단수 대칭성 간섭여부 없음 없음 없음 없음 없음 없음 없음 Table 27. The result of testing solution in specificity test Peaks Inj. 1 Inj. 2 Inj. 3 Inj. 4 Inj. 5 Mean %RSD RT 면적 이론단수 대칭성 분리능 간섭여부 없음 없음 없음 없음 없음 없음 없음

137 Figure 45. UPLC Chromatogram of MSE Table 28. The result of purity test by PDA Purity Angle 0.90 Purity Threshold 1.08 Table 29. Summary of the results in specificity test No. Parameter Acceptance criteria Observation 1 검액내표준품피크의 RT 2.9 ~ 검액내표준품피크의이론단수 2000 이상 검액내표준품피크의대칭성 1.5 이하 검액내표준품피크의간섭여부 간섭없음 간섭없음 5 검액내표준품피크의분리능 2.0 이상 검액내표준품피크의 Purity Angle < Threshold 0.90 <

138 8.2 직선성 (Linearity) 아래의시험결과를통해표준액의직선성상관계수는 이상으로나타나기준에적합하였으며, 분석가능한범위의피크면적 %RSD가 2.0 이하인 4.0 ~ 50.0 μg/ml 이었다. 따라서이분석법은특이성이확보되었다고판단된다. Table 30. The result of standard solution in linearity test 검액내함량 0.50% 0.75% 1.00% 1.50% 1.75% 2.00% 2.50% 4.17% 6.25% 표준품농도 (μg/ml) Inj Inj Inj Inj Inj Mean %RSD RF Range 설정 4.0 ~ 50.0 Ave. (RF) %RSD (RF) R 직선식 Area = 1086 * Conc. (μg/ml)

139 Figure 46. Linearity graph of standard (Momordica saponin I) Table 31. Summary of the results in linearity test No. Parameter Acceptance criteria Observation 1 표준액직선상관계수 이상 표준액직선범위 ~ 50.0 (μg/ml) 3 범위내피크면적 RSD 2.0 이하 <

140 8.3. 정확성 (Accuracy) 시험결과, 각회수율의 RSD는 2.0% 이하이며, 각농도에서의회수율은모두기준에적합하였다. 검액내표준액의농도에관계없이정확성이있음을두종류의정확성실험방법에서확인하였다. 그러므로이분석법은정확성이있는결과를확보할수있는분석법으로판단된다. Table 32. The result of standard solution in accuracy test Weight (μg/ml) Inj Inj Inj Inj Inj Mean Calculated (μg/ml) %Recovery Ave. (%Recovery) 99.7 %RSD (%Recovery)

141 Table 33. The result of testing solution in accuracy test - spiking ( 검액 : 표준액 = 1:1 (v/v)) Sample 저농도검액 μg/ml 중농도검액 μg/ml 고농도검액 μg/ml 검액내함량 (μg/ml) # Calculated (μg/ml) Inj Inj Inj Mean Calculated (μg/ml) %Recovery Ave. (%Recovery) %RSD (%Recovery) 0.53 Table 34. The result of testing solution in accuracy test addition Sample 저농도검액 중농도검액 고농도검액 Added Conc. (μg/ml) Inj Inj Inj Mean Calculated (μg/ml) %Recovery Ave. (%Recovery) %RSD (%Recovery)

142 Table 35. Summary of results in accuracy test No. Parameter Acceptance criteria Observation 1 표준액회수율 %RSD 2.0 이하 검액내회수율 %RSD (Spiking) 2.0 이하 검액내회수율 %RSD (Addition) 2.0 이하 정밀성 (Precision) 1) Repeatability 시험결과, 각정밀성의 %RSD 는 2.0% 이하이므로모두기준에 적합하다. 그러므로이분석법은정밀성이있는결과를확보할수있는 분석법으로판단된다. Table 36. The result of injection repeatability - 100% standard solution (14.0 μg/ml) (Ten times injection) Inj. # 면적 이론단수 대칭성 Calculated (μg/ml) %Recovery Ave. (%Recovery) %RSD (%Recovery) Ave. ( 이론단수 ) Ave. ( 대칭성 ) Ave. (%Recovery) 99.9 %RSD (%Recovery)

143 Table 37. The result of method repeatability - 100% standard solution (800 μg/ml) (After making 6 samples, five times inject.) 날짜 실험실 Lab 1 기기 컬럼 분석자 UPLC LS1 ACQUITY UPLC BEH C μm KWS Vial # Conc. (μg/ml) Inj Inj Inj Inj Inj Mean % Assay 이론단수 (inj. 3) 대칭성 (inj. 3) 분리능 (inj. 3) Ave. (%Assay) 1.82 %RSD (%Assay) 0.47 Ave. ( 이론단수 ) Ave. ( 대칭성 ) 1.04 Ave. ( 분리능 )

144 2) Reproducibility Table 38. The result of injection reproducibility - 100% standard solution (14.0 μg/ml) (Ten times injection) Inj. # 면적 이론단수 대칭성 Ave. ( 면적 ) %RSD ( 면적 ) 1.22 Ave. ( 이론단수 ) Ave. ( 대칭성 ) 0.99 Table 39. The result of method reproducibility. 100% standard solution (800 μg/ml) (After making 6 samples, five times injection) 날짜 실험실 Lab 2 기기 UPLC NP1 컬럼 ACQUITY UPLC BEH C μm 분석자 JKW Vial # Conc. (μg/ml) Inj Inj Inj Inj Inj Mean % Assay 이론단수 (inj. 3) 대칭성 (inj. 3) 분리능 (inj. 3) Ave. (%Assay) 1.88 %RSD (%Assay) 0.64 Ave. ( 이론단수 ) Ave. ( 대칭성 ) 0.98 Ave. ( 분리능 )

145 Table 40. Cumulative Results %Assay #1 #2 #3 #4 #5 #6 Lab Lab Cumulative Ave. (%Assay) 1.85 Cumulative %RSD (%Assay) 1.91 Table 41. Summary of the results in precision test No. 1 Parameter Acceptance criteria Observation Repeatability 중 Injection의 %RSD 2.0 이하 0.27 Repeatability 중 %Assay 의 %RSD 2.0 이하 Reproducibility 중 %Injection 의 %RSD 2.0 이하 1.22 Reproducibility 중 %Assay 의 %RSD 2.0 이하 각 %Assay 의 cumulative %RSD 2.0 이하

146 8.5. 결과종합 MSE 함량시험법의시험농도인 800 μg/ml에서위의시험법밸리데이션결과를보아목별자추출물의함량분석법의특이성, 정밀성, 정확성, 직선성등을확인할수있었다 (Table 42). 그러므로본시험법이목별자추출물의함량시험에사용하기에적합하다고판단된다. Table 42. Summary of results in validation for analytical method 특이성 정확성 정밀성 Analytical Content Acceptance criteria Results 확인 MSE 확인 확인 분리능 > 대칭성 < 직선성 R 2 > 범위 (μg/ml) Range of Standard 4.0 ~ 50.0 Spiking %RSD of % Recovery 0.53% Addition < 2.0% 1.53% Repeatability Injection 0.27% Method 0.47% Reproducibility Injection %RSD < 2.0% 1.22% Method 0.64% Cumulative 1.91%

147 IV. 결론 본연구의목적은급성및만성위염치료효능을지닌물질또는추출물을찾기위함이며, 이를위해 1차적으로각종한의서와문헌검색등을통하여위점막보호 ( 급성및만성위염치료 ) 효능을나타낼가능성이있는생약후보를검색하였다. 그결과 50종의생약을선별하였으며, 선별된 50종생약에대해신규성이나학문적가치의기준으로추가검토하여 15 종의스크리닝후보생약을최종선정하였다. 1차스크리닝을위해디클로페낙유발위염모델을수행하였으며, 실험결과에따라 3종의후보생약 ( 목별자, 백작약, 만형자 ) 을선별하였다. 이후 2차스크리닝을실시하였으며, 이를위해사용된모델은에탄올유발위염모델이었다. 실험결과목별자 ( 木鱉子, seed of Momordica cochinchinensis) 추출물이가장우수한활성을나타내었고, 목별자를본연구를위한최종후보생약으로선정하였다. 선정된목별자에대한분획연구를실시하였으며, 분획물에대해에탄올유발위염모델을수행하여약효우의성을검정하였다. 실험결과유의성을보이지는않았지만 n-buoh 층이활성이높았으며, MPLC 및 HPLC를이용하여 n-buoh 층에대한성분분리를시도하였다. 그결과 3종의사포닌을분리하였고 ( 화합물 1, 2 및 3), 각종분석광학적데이터를활용하여구조분석을실시한결과, 화합물 1은 gypsogenin-3-oβ-d-galactopyranosyl(1 2)-[α-L-rhamnopyranosyl(1 3)]-β-Dglucuronopyranoside, 화합물 2는 quillaic acid-3-o-β-d-galactopyranosyl(1 2)- [α-l-rhamnopyranosyl(1 3)]-β-D-glucuronopyranoside, 그리고화합물 3은

148 gysogenin-3-o-β-d-galactopyranosyl(1 2)-[α-L-rhamnopyranosyl(1 3)]-β-D- glucuronopyranosido-28-o-β-d-xylopyranosyl(1 3)-β-D-glucopyranosyl(1 3)- [β-d-xylopyranosyl(1 4)-α-L-rhamnopyranosyl(1 2)-β-D-fucopyranoside로구조를결정하였다. 화합물 1은천연물에서처음분리된물질이었으며, 화합물 2는 Momordica 속에서처음분리된물질이었고, 화합물 3은목별자에서이미분리, 보고된 Momordica saponin I이었다. 이후 3종화합물에대해에탄올유도위염모델에서활성을측정하였으며, 실험결과유의성은없었으나화합물 1, 2 및 3 모두활성이있었고, 화합물 3 (Momordica saponin I) 이상대적으로가장좋은효능을보였다. 이러한결과로볼때, 위점막보호활성이사포닌에기인할가능성이높음을알수있었다. 하지만, 분리된사포닌경우수율이낮고 (0.025% 이하 ), 활성이추출물대비뛰어나지않으며, 합성자체도거의불가능하기때문에단일성분이아닌목별자에탄올추출물 (MSE; Momordicae Semen Extract) 을이용하여이후연구를수행하였다. 단, Momordica saponin I의경우목별자추출물의지표성분으로서의가치가있다고판단되었다. 최종적으로선택된목별자추출물 (MSE) 에대한위점막보호활성을검증하기위해각종동물모델및기전과관련된실험을수행하였다. 에탄올및디클로페낙으로유발된급성위염모델, 그리고 H. pyroli로유발된만성위염모델을수행하였으며, 실험결과급 만성위염모델모두에서우수한활성을보였다. 이러한결과들은 MSE가위점막손상을억제할수있다는명백한증거를보여주었다. 이러한우수한급 만성위염모델에서의약효를뒷받침하기위하여

149 위점액분비촉진모델, 상처치료 (wound-healing) 모델, croton oil 유발 ear edema 모델, formalin test 모델에서 MSE에대한효능을실험하였으며, 그결과우수한위점액 (Gastric mucus) 분비촉진효과, 상처치료효과, 항염증및진통효과를확인할수있었다. 참고로궤양부근에서의혈관신생성이위궤양치료를촉진시키는데있어중요한역할을하며 (Szabo et al., 1995), 홍삼으로부터분리된사포닌류가 VEGF 생성촉진을통해혈관신생성을일으켜상처치료에효과를나타내는것이보고되었는데 (Kimura et al., 2006), 이와같은문헌적근거를종합해볼때, MSE에존재하는사포닌성분들이상처치료효과를보일가능성이높으며, 이러한상처치료효능이결과적으로 MSE의위점막보호활성에기여할수있음을보여준다고할수있겠다. 또한, 이러한약리작용의기전을밝히기위해, 위점막보호활성을지닌 neuropeptide 중하나인 CGRP와가스트린분비를억제하는인자로알려진 somatostatin와관련된기전실험을수행하였다. CGRP가위궤양치료를촉진시킬뿐만아니라에탄올로유발된위점막손상을예방할수있음이기존의문헌에서보고되었는데 (Hayashi et al., 2001; Ohno et al., 2008), 본연구결과 MSE가 CGRP 수용체특이적조직 assay에서 positive inotropic 효능을나타내었고, 이러한효능이 CGRP 수용체의 antagonist인 α-cgrp (8-37) 에의해억제되는것으로보았을때, CGRP가 MSE의위염치료효과에있어중요한매개체역할을할가능성을보여준다고할수있다. Somatostatin의경우 SSR2를경유하여가스트린방출을억제하고위점막의혈류속도를저하시켜위궤양출혈을막는중요한역할을하는것으로알려졌으며 (Li et al., 1996), 최근에위점막

150 보호활성이 somatostatin과연관되어있음이보고되었고 (De Almeida et al., 2012), 합성 somatostatin analogue인 octreotide가만성스트레스로유발된랫드의위병변을억제하는것으로보고되었다 (Nassar et al., 2011). 본연구에서 MSE를 somatostatin 수용체특이적조직 assay에적용한결과, 12.5~100 μg/ml 범위에서농도의존적으로기닉픽회장의수축을증가시켰는데, 기존문헌의내용으로유추해보았을때 MSE가 somatostatin 수용체를경유하여위점막보호활성을나타낼가능성이높다고할수있다. MSE의안전성을증명하기위해급성및반복경구투여독성시험을실시하였다. 단회경구투여독성시험결과, MSE의최소치사량 (MLD) 은 2,000 mg/kg/day였으며, 4주반복경구투여독성시험결과무해용량 (NOAEL) 이 2,000 mg/kg이었다. 이러한결과는 MSE가임상적으로도매우안전한약물일가능성을보여준다. 목별자원생약의안정적인수급을위하여 LC-MS Similarity Analysis를수행하였으며, 분석결과중국운남성산지의목별자가기준에적합함을알수있었다. 이와더불어목별자원생약을수급하는과정중에동속식물과의혼용가능성에대해문헌적연구를실시하였는데, 그결과동속식물과혼재될가능성이희박하고, 만약혼재되어있다고하더라도다른동속식물과육안으로쉽게구분이가능함을알수있었다. 또한, MSE의함량분석법에대한신뢰성을확보하기위해밸리데이션연구를수행하였고, 이를통해현분석법에대한신뢰성을확보할수있었다. 이러한결과들은목별자의 GCAP (Good Collecting & Agricultural Practice) 를위한기초자료로활용할수있을것으로사료된다

151 결론적으로, MSE는다양한급성및만성위염모델에서현재표준치료제로사용하고있는레바미피드와비교했을때우수한효능을보였고, 다양한 in vivo 및 in vitro 기전모델에서도뛰어난효과를나타냈으며, 단회및반복경구투여독성시험에서우수한안전성을보였다. 또한, 원생약에대한문헌적연구, similarity analysis, 분석법밸리데이션연구를통해 MSE를개발하는데있어 GCAP를위한다양한기초데이터를확보할수있었다. 본연구를통해얻어진이러한결과들을종합해볼때, MSE를위점막보호활성을갖는신규천연물의약품으로개발할경우성공가능성이충분하다고판단된다

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163 Abstract Momordicae cochichinensis Seed Extract as a Therapeutic Agent for Acute & Chronic Gastritis and Its Constituents Jung, Kiwon College of Pharmacy (Pharmacognosy) The Graduate School Seoul National University The gastric mucosa is the mucous membrane layer that protects the stomach against harsh acidic conditions. A mounting body of research has shown that this protective layer can be damaged by various factors, including excessive alcohol consumption, use of non-steroidal anti-inflammatory drugs (NSAIDs), exposure to Helicobacter pylori, disturbance of microcirculation of gastric mucosa, and stressinduced hypotension. The damaged mucosal layer becomes susceptible to inflammation, such as flare, hemorrhage, and edema, which ultimately results in ulceration in the stomach. Over the past decades, H 2 receptor antagonists and proton pump inhibitors (PPIs), which prevent the secretion of gastric acid, have been used for the treatment of gastric ulcer. However, these drugs have a severe drawback of frequent relapse upon the cessation of administration. Recently, a novel class of drugs, known as mucosal protective agents, such as rebamipide, polaprezinc, and sofalcone, has been introduced to clinical practices. Unlike the H 2 receptor antagonists and PPIs, the mucosal protective drugs can promote the regeneration of the mucosal tissue to ensure protection against the recurrence of gastritis. However, the mucosal

164 protective drugs clinical value is limited due to the requirement of high dose and slow onset. Many attempts have been made to obtain a novel class drug with improved therapeutic efficacy and safety. In this study, to develop a new herbal gastroprotective agent, we referred a variety of references, for example traditional Chinese medicine archives, journal papers, commercial DBs, and consultants opinions so that we chose 15 candidates and performed in vivo models as tools for the screening. After two steps of in vivo screening, we have achieved a final herbal candidate, Momordicae cochinchinensis seed, Momordica cochichinensis Springer (Cucurbitaceae), which has long been known in traditional Chinese medicine for its various therapeutic benefits including effective relief of boils, rheumatic pain, muscular spasm, hemorrhoids, and hemangiomas. We conducted bioactivities-guided fractionation and revealed that n-buoh fraction showed a most potent anti-gastritis activity. The n-buoh fraction was separated by column chromatography to yield three saponins, compounds 1, 2, and 3 which showed gastroprotective effects in rat model of ethanol-induced gastritis. On the basis of spectroscopic evidences, compound 1 was assigned as gypsogenin- 3-O-β-D-galactopyranosyl(1 2)-[α-L-rhamnopyranosyl(1 3)]-β-Dglucuronopyranoside, compound 2 as quillaic acid-3-o-β-d-galactopyranosyl (1 2)-[α-L-rhamnopyranosyl(1 3)]-β-D-glucuronopyranoside, and compound 3 as a known ingredient, Momordica saponin I. Compound 1 was isolated for the first time from natural resources. To the best of our knowledge, compound 2 was firstly determined from Genus Momordica. Although the compound 3 (momordica saponin I) have shown the relative gastroprotective effect in gastritis model, due to the very low separating yield and

165 difficulties in isolating or synthesizing, we decided to investigate ethanol extract of Momordicae cochinchinensis seed (MSE) instead of pure compound in the remaining part of our study. Nevertheless, even though further explorations are needed, momordica saponin I might be used as an active marker for MSE. One of the most prevalent methods to assess the efficacy of gastritis treatment using the animal model is to induce damage on the gastric mucosa, which uses NSAID, ethanol or H. pylori. In the animal models that use ethanol, diclofenac, and H. pylori, the gastric damage indexes of MSE-treated group were found to be lower than those values of the positive control, rebamipide which has been commercially used for mucosal protection and treatment of gastritis. Therefore, it is evident that MSE is able to protect the damage of the gastric mucosa effectively. In addition, MSE not only increased secretion of gastric mucus, but it showed wound-healing effect, anti-inflammatory effect, and analgesic effect, which presumably are responsible for increasing the gastroprotective effect s of MSE. To investigate the mechanism of gastroprotective effects induced by MSE, we conducted calcitonin gene-related peptide (CGRP) and somatostatin-specific tissue assays. CGRP, a 37-amino acid neuropeptide, plays strong gastroprotective role by either promoting secretion of gastric mucus and bicarbonate, or stimulating gastric mucosal blood circulation. On the other hand, somatostatin has been known to inhibit gastrin release via somatostatin receptor 2 and thus, attenuate gastric acid secretion. From the results of our studies, CGRP and somatostatin most likely plays a role in the ulcer healing by MSE in gastritis models. To assure the safety of MSE, single and repeated oral dose toxicity studies were conducted. The minimal lethal dose of MSE was determined to be 2,000 mg/kg/day in a sing oral dose toxicity study. The NOAEL was set to be 2,

166 mg/kg/day from the repeated oral dose toxicity study. These results clearly demonstrate that MSE might have a high therapeutic index. For the purpose of setting up basic data for Good Collection and Agricultural Practice (GCAP), we performed LC-MS Similarity Analysis of sourced herbal materials from various regions, literature studies of Momordica species, and validation of analytical method. The results demonstrated i) Momordicae cochichinensis seed of Yunnan Sheng in China was suitable for the specification, ii) there may be little chances of adulteration in sourcing the raw material, and iii) the quantitative method of MSE has been well validated. In conclusion, we demonstrated that MSE has reduced progressing acute and chronic gastrointestinal disorders, such as gastritis and gastric ulcer, when compared to that of the current standard therapy in various in vivo and in vitro models. In addition, MSE showed good safety profiles in the oral dose toxicity studies. These results warrant that MSE could be a promising herbal medicine for the treatment of gastric disorders. Keywords : Momordica cochichinensis Sprenger, Gastroprotective, Calcitonin gene-related peptide, Somatostatin, Momordica saponin Student Number :

167 감사의글 8년이란긴박사학위과정을마치고감사의글을적고있는지금, 정신없이보낸세월을핑계로고마운분들에대한감사의마음을잊고살았다는느낌이듭니다. 어릴때는모든일이저로인해이루어지는줄만알고어줍잖은자만에빠졌었으나, 이제는제주위에계신모든분들의손길덕에제인생이더욱값지게된다는생각에하루하루를감사히살고있습니다. 이러한교훈이본논문으로얻게되는학위보다더욱값진것이라생각됩니다. 언제나저를믿어주시고, 올바른길로인도하여주신김진웅교수님께가장먼저감사의말씀을드리고자합니다. 지금와서생각해보니박사학위기간동안교수님께많은어려움을드렸던것같습니다. 부디철들지못했던지난날의저의행동과생각들을용서하여주시기바랍니다. 이에대한보답의길은더욱열심히, 그리고의미있게사는것이라고생각합니다. 교수님, 진심으로감사드립니다. 그리고한결같은인자하심으로부족한저에게신뢰와용기를주신김영중교수님께진심으로감사드리며, 학위기간동안많은조언과도움을아끼지않으셨던성상현교수님께도깊은감사를드립니다. 비록설익은논문이지만좋은논문이될수있도록많은조언을아끼지않으신박정일교수님과김선여교수님, 강건욱교수님께도깊은감사를드립니다. 이번박사학위를위해많은도움을주신선후배님들과동기분들이있습니다. 청춘이꽃필무렵부터같이생활하였고, 이제는정신적인멘토가되어주신진영원교수님과못난동기때문에귀한시간을희생해주었고걱정을같이한박규환교수, 윤기동교수, 김철영교수, 정윤희교수, 허태회교수에게지면으로나마감사를말을전합니다. 그리고선배를위해고생해준후배종혜와동영이에게감사드리며,

168 바쁘신와중에도내일처럼도움을주신채희성박사님께도감사 드립니다. 일일이이름을나열하진못했으나저의학위논문을위해 도움을주신모든학교선후배, 동기여러분께도깊은감사를드립니다. 또한, 제마음의고향을만들어준나의영원한잡사친구, 효일이, 재학이, 순재, 태식이, 현이, 희상이, 일균이, 홍득이, 석민이, 창림이, 명우그리고지금은하늘에있는소중한친구성태에게도깊은감사를드립니다. 모두들힘든시기를겪고있지만앞으로도우리들의영원한우정을지켰으면합니다. 그리고우리아이들을위해항상즐겁게봉사하시는조아모회원, 양진환아버님, 김태균아버님, 이창래아버님, 김형태아버님, 오제민아버님, 남동훈아버님께도감사의말씀을드립니다. 박사학위준비하느라신경을많이못쓴것같아죄송합니다. 벌써 SK케미칼에근무한지 15년이되었습니다. 회사업무를하면서박사학위를준비한다는것이쉬운일이아니었으나많은분께서도와주셔서여기까지오게되었습니다. 김훈택실장님, 민동선팀장님, 류근호팀장님, 김재선팀장님, 오준교팀장님, 박양혜차장님, 서정민과장님, 신호철차장, 유헌승과장덕분으로무사히박사학위를마칠수있게되었습니다. 특히, 김재선팀장님께서많은도움을주셨으며어려운시기를잘극복할수있도록커다란의지가되어주셨습니다. 진심으로감사합니다. 그리고저의상사셨던한국해양연구원신희재박사님께도감사드립니다. 박사님의따뜻한마음과열정이제인생에있어커다란길잡이가되고있습니다. 비록언급되지않았으나여러모로도움을주신회사분들께도진심으로감사의말씀을드립니다. 무엇보다도어려운환경속에서도바른사람이되도록가르쳐주시고한결믿음으로저를길러주신아버지, 어머니께깊은감사를드립니다. 마냥철부지였던막내가드디어박사가되었습니다! 그리고먼발치에서도항상든든한후원자가되시는누나와매형, 형과형수님께도감사드립니다. 또한, 첫째, 둘째, 셋째형님께도감사의

169 말씀을드립니다. 바쁘다는핑계로자주연락드리지못해죄송한 마음뿐입니다. 지금은비록하늘나라에계시지만진심으로존경하는장모님과장인어른께이부족한논문을바치고자합니다. 지금이라도 정서방, 축하하네 ~ 라고말씀하실것같아눈시울이뜨거워지지만, 지난날의기억들이되새기며따뜻한마음으로살고자합니다. 항상하늘에서인자하신모습으로저를지켜봐주실거라믿고열심히그리고성실하게살겠습니다. 이제는더이상저희걱정은마시고편안히쉬시기바랍니다. 마지막으로지금이순간에도곁에있는, 현재까지그랬고앞으로도믿음과사랑으로같이할사랑스러운저의아내혜경이와하나뿐인우리딸수현이에게진심으로고맙다는말을전하고싶습니다. 긴박사학위과정중에힘든일이많았을텐데잘견뎌줘서고맙고, 솔직히미안한마음이큽니다. 앞으로는행복하고좋은일들만가득할것이며, 오늘보다더행복한우리가족이될수있도록최선을다하겠습니다. 더욱사랑하겠습니다. 다시한번저의박사학위를위해도움을주신모든분들께진심으로 감사를말씀을드리며, 부족하지만이논문을바칩니다. 항상행복하시길 기원! 하겠습니다