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1 헛개나무열매로부터분리한 (+)-Catechin 의간해독및 Alcohol 분해활성에관한연구 강원대학교대학원식품생명공학과 이동일

2 Introduction

3 연구의필요성및배경 사회의급변화, 다양화로야기되는과도한긴장및스트레스해결의방법으로과다한음주 2000 년국민 1 인당주류소비량이 63.1 L 로계속적인증가추세 희석식소주나위스키등고함량 alcohol 주류의소비증대 간경변, 만성감염등 alcohol 섭취로인한만성간질환발생률의증가 만성간질환에효과가있는여러가지합성물질이알려져있으나부작용이알려지고있어최근에식물자원유래의천연생리활성물질에대한연구가많이이뤄짐

4 헛개나무의특성 갈매나무과교목으로지구자나무, 호깨나무, 허리깨나무, 拐棗, 白石木, 木密, 玄圃梨등으로불림 높이 10~20 m, 직경 40~80 cm 耐寒性, 耐陰性, 耐潮性및耐公害性 수피는흑회색, 열매는갈색이며닭의발톱모양이고은은한향기와단맛을지님 본초강목에따르면술을썩히는작용과생즙은술독을풀고구역질을멎게한다고함 한방에서종자는酒精中毒, 小便不利, 과경은, 滋養補血에이용

5 연구의개요 1. 강원도양양산헛개나무수피, 목부및열매로부터조추출물 (CE) 과 diethyether 분획물 (PE) 을조제하여간해독에관여하는 GST 효소활성을측정 2. 헛개나무의 alcohol 대사개선효과를살펴보기위하여식품공전에서식용으로가능한열매의 CE 와 PE 를쥐에급여후 ADH 와 ALDH 효소활성에미치는효과를측정 3. 헛개나무열매의 CE 와 PE 를쥐와사람에게급여후 alcohol 을투여하여각추출물의 alcohol 분해능을측정 4. 헛개나무열매추출물의유효한활성성분을얻기위하여대량추출및분획을실시하였고각종기기분석을통하여단일물질 (HD 1 ) 을분리하고그구조를확인 5. 분리된활성물질의 alcohol 대사효능검증을위하여쥐를대상으로 ADH, ALDH 효소활성을측정하였고, 활성물질과 alcohol 을사람과쥐에게투여후 alcohol 분해능을측정하였으며, 또한간보호예방효과를알아보기위하여 CCl 4 투여후 GOT, GPT 효소활성측정및간조직의병리소견을확인

6 Materials and Methods

7 실험재료및분석기기 1. 헛개나무재료 : 강원도양양군농업기술센터에서 2001 년에채취한것을사용 2. 시약 : GST, ALDH, ADH, NaOH, HCl 등분석시약은 Sigma 제품을사용하였고그외 ethanol, chloroform, butanol, diethylether 등의추출용매및분획용매는특급시약사용 3. 실험동물 : Sprague-dawley 계웅성 3 주령 rat 를한림대학교와 ICR mouse 를대한실험동물센터로부터구입하여사용 4. 실험기기 : UV/VIS spectrophotometer (U-3210, spectrophotometer, Hitachi) preparation HPLC (Delta prep. 4000, Waters), Alcohol 측정기는 (Alcon AL-200, Korea), 적외선 (IR) 스펙트럼은 (FT/IR-5300, JASCO), 질량 (MS) 스펙트럼은 JEOL JMS-600W, NMR 은 Varian UI 500 (Varian, USA) 를이용

8 GST 측정을위한추출물및분획물조제 수피, 목부, 열매부 세절및분쇄 10 배증류수, 70% ethanol 수피부, 열매부 70% ethanol로용해 chloroform, 물, diethylether 1: 9: 에서 6 시간 2 회반복추출 감압여과, 농축및동결건조 Diethylether 분획물 수층분획물 Chloroform 분획물

$2 Evaporate in vacuo 70 % ethanol extract H 2 O H 2 O-sat. BuOH H 2 O H 2 O fraction BuOH BuOH fraction Dry over Na 2 SO 4 anhyd. Filtrate Conc.$

9 활성성분분리를위한추출물의분획 Hovenia dulcis T HUNB Fruit Extract with total 5 L of 70 % ethanol under ultrasonication Filtrate through Toyo No. 2 Evaporate in vacuo 70 % ethanol extract H 2 O H 2 O-sat. BuOH H 2 O H 2 O fraction BuOH BuOH fraction Dry over Na 2 SO 4 anhyd. Filtrate Conc. To dryness EtOAc EtOAc fraction Dry over Na 2 SO 4 anhyd. Filtrate Conc. To dryness

간기능생리활성측정 (GST) 조제된반응시약농도별추출물첨가 1-chloro-2,4-dinitrobenzene TCA를가해반응종결 340 nm에서흡광도측정 37 에서 5 분반응 37 에서 2 분반응 원심분리 Ingredient Concentration Volume( ml ) Reduced glutathione 0.04 M 0.

10 간기능생리활성측정 (GST) 조제된반응시약농도별추출물첨가 1-chloro-2,4-dinitrobenzene TCA를가해반응종결 340 nm에서흡광도측정 37 에서 5 분반응 37 에서 2 분반응 원심분리 Ingredient Concentration Volume( ml ) Reduced glutathione 0.04 M GST solution 20 units 0.1 Phosphate buffer 0.1 M 0.5 Sample Each concentration 0.05 Total activity (units) = A340/9.6 희석배수 3 ml /0.1 crude extract ( ml ) Specific activity ( units/ mg protein ) = total activity / total protein 활성율 (%) = specific activity test / specific activity control 100

alcohol 농도측정 적출한간으로부터 cytosol 을분리 ADH 활성측정은 cytosol 을 ethanol, semicarbazide, NAD 혼합액에가한후 37 에서반응후 340 nm 에서흡광도측정 ALDH 활성은 cytosol 에 sodium pyrohposphate buffer,

11 추출물과분획물의 Alcohol 농도분해능및 ADH 와 ALDH 효소활성실험 동물을이용한 alcohol 분해능실험 ADH, ALDH 효소활성측정 사람을대상으로한 alcohol 분해능실험 웅성 S.D rat(300±12g) 30 마리를 3 군 ( 대조군, CE 군, PE 군 ) 으로분류 CE 추출물, PE 분획물 (50g/ 100ml) 을조제 투여량 (1g/kg body weight) 30 분후 alcohol (4g/kg body weight) 을투여 안와정맥으로부터혈액을채취하여혈중 alcohol 농도측정 적출한간으로부터 cytosol 을분리 ADH 활성측정은 cytosol 을 ethanol, semicarbazide, NAD 혼합액에가한후 37 에서반응후 340 nm 에서흡광도측정 ALDH 활성은 cytosol 에 sodium pyrohposphate buffer, pyrazol, acetaldehyde 혼합액을가한후 340 nm 에서흡광도측정 성인남성 (19 세 ~30 세 )30 명을 3 군 ( 대조군, CE 군, PE 군 ) 으로분류 CE 추출물, PE 분획물 (50g/ 100ml) 을조제 음주 1 시간전에투여량 (1.5ml/kg body weight) 투여 음용술은시판양주 (43, 3ml/kg) 를투여 호흡측정기를이용하여혈중 alcohol 농도측정

12 활성물질의분리및정제 Silicagel chromatography HPLC, prep-hplc 에의한정제 분리물질 (HD1) 구조및분자량확인 GST 활성이높은 BuOH 분획물을 CHCl 3 에용해 1 차 Silica gel column(5 35 cm) 에 loading 한후 CHCl 3 : MeOH (10:0 0:10) 으로 elution 하고 200 ml /fraction 분취하여 GST 활성측정 CHCl 3 를가해녹인후 2 차 silica gel column (3 50 cm) 에 loading 하고 CHCl 3 : MeOH (10:0 0:10) 으로 elution 하여각분획당 50 ml씩분취하여 GST 활성측정 TLC(CHCl 3 : MeOH: H 2 O=65:35:10) 254nm band 확인 HPLC 분석의 Solid phase 는 Nucleosil- ODS column ( mm) 를, Mobile phase 는 MeOH-H 2 O/1 % acetic acid 를사용 Programmed gradient 조건은 20 % MeOH (0 min) 80 % MeOH (25 min) 80 % MeOH (35 min) 20 % MeOH (45 min) in 1 % acetic acid 으로하여 detection 은 UV 254nm 에서함 Prep-HPLC 분석은 Solid phase 로 TSK-Gel ODS-80 TM column ( mm) 을, mobile phase 는 65 % MeOH/1 % acetic acid (isocratic elution) 을사용하여 UV 254 nm에서검출 (HD 1 ) UV 분석은 U-3210, spectrophotometer, Hitachi) IR 분석은 KBr 법을이용하여 (FT/IR-5300, JASCO) 로측정 질량 (EI-MS, EI-HR-MS) 스펙트럼은 JEOL JMS-600W(Japan), FAB-MS 는 negative ion FAB- MS (VG Quattro, Great Britain) 로측정하였고 glycerol 을표준물질함 핵자기공명 ( 1 H, 13 C-NMR COSY, NOESY, HMQC, HMBC) 은 Varian UI 500(Varian, USA) 을이용하였고 TMS 를표준물질로사용하여구조확인

13 분리물질 (HD 1 ) 의효능검증 동물을이용한 alcohol 분해능실험 사람을대상으로한 alcohol 분해능실험 CCl 4 투여에의한간보호예방효과실험 ICR mouse(30±3.2g) 5 마리를대조군과 HD 1 을투여한군으로분류 분리물질투여 (1g/kg body weight) 30 분후 alcohol (4g/kg body weight) 을투여 안와정맥으로부터혈액을채취하여혈중 alcohol 농도측정 성인남성 (20 세 ~27 세 ) 각 5 명을대조군과 HD 1 을투여한군으로분류 분리물질을 2g/ 1000ml 로조제 음주 1 시간전에조제시료 (1.5ml/kg body weight) 투여 음용술은시판양주 (43, 3ml/kg) 를투여 호흡측정기를이용하여혈중 alcohol 농도측정 ICR mouse( 체중 30 g ± 1.5 g) 5 마리를 1 군으로하여양성대조군과 CCl 4 를단독처리군, CCl 4 와분리물질을동시에투여한군으로분류 Dose-schedule (Table. 1) 에따라진행하여 48 시간경과후도살하여혈액을채취해 Reitman-Frankel method (Reitman & Frankel, 1957) 를사용하여 GOT, GPT 활성측정 간조직검사 (H & E)

14 Table 1. Dose schedule of experiment Groups Times Before 2 hours 0 24 hours 48 hours Normal 0.9 % saline 0.9 % saline 0.9 % saline CCl % saline CCl % saline (+)-Catechin (+)-Catechin CCl 4 (+)-catechin * Each group consist of 5 mice (ICR, 웅성 30 ± 1.5 g) ** CCl 4 (0.3ml/kg D.W) and (+)-catechin(1 mg) were oral administrated

15 Results

16 Table 2. The extraction and fraction yields of Hovenia dulcis T HUNB according to solvents. Samples Solvents Yields (%) Bark Water % ethanol 7.37 Diethyl ether Fr Chloroform Fr Water Fr Vessel area Water % ethanol 3.69 Diethyl ether Fr Chloroform Fr Water Fr Fruit Water % ethanol Diethyl ether Fr Chloroform Fr Water Fr

17 Specific relative activity(%) Bark H 2 O extract (0.5 mg / ml ) Bark 70 % EtOH extract (0.5 mg / ml ) Vessel H 2 O extract (0.5 mg / ml ) Vessel 70 % EtOH extract (0.5 mg / ml ) Fruit H 2 O extract (0.5 mg / ml ) Fruit 70 % EtOH extract (1.0 mg / ml ) Bark H 2 O extract (1.0 mg / ml ) Bark 70 % EtOH extract (1.0 mg / ml ) Vessel H 2 O extract (1.0 mg / ml ) Vessel 70 % EtOH extract (1.0 mg / ml ) Fruit H 2 O extract (1.0 mg / ml ) Fruit 70 % EtOH extract (1.0 mg / ml ) Fig. 1. Relative specific activities of GST in the extracts from Hovenia dulcis T HUNB.

18 Specific relative activity(%) Diethyl ether Frac.(bark, 0.5 mg / ml ) Chloroform Frac.( bark, 0.5 mg / ml ) Diethyl ether Frac.(fruit, 0.5 mg / ml ) Chloroform Frac.(fruit, 0.5 mg / ml ) Diethyl ether Frac.(bark, 1.0 mg / ml ) Chloroform Frac.(bark,1.0 mg / ml ) Diethyl ether Frac.(fruit, 1.0 mg / ml ) Chloroform Frac.(fruit 1.0 mg / ml ) Fig. 2. Relative specific activity of GST in the fractions from Hovenia dulcis T HUNB 70 % ethanol extracts.

19 Table 3. Comparison of residual alcohol concentrations in peripheral blood of Rats fed with or without Hovenia dulcis T HUNB extracts. Group Time(hr) Control Ethanol only 1 CE 2 PE * ± a ± a ± a ± ± b ± a ± b ± ± a ± a ± b ± ± c ± a ± b ± Mean ± S.E (n=10). + Alcohol concentration in the blood : % (v/w). a,b Significantly different from the control : p< % EtOH extracts 2 Diethylether fractons

20 Alcohol concentration (%) * * * * * * Time(hr) Fig. 3. Comparison of residual alcohol concentrations by uptaking alcohol with or without two kinds of Hovenia dulcis T HUNB extracts according to drinking time. Mean ± S.E (n=10) * Significantly different from the control : Control, : PE, : CE

21 Table 4. Comparison of the enhancement of ADH and ALDH activities at one hour after ethanol administration with or without feeding the extracts in rats. Activity Group Ethanol only 1 CE 2 PE ADH + activity(%) * 28.8 a ± b ± b ±0.04 ALDH + activity(%) 29.8 a ± b ± b ±0.08 Mean ± S.E (n=10) + Relative ADH and ALDH activities : (A 340 (control) -A 340 (sample) ) / A 340 (control) 100 a,b Significantly different from the Control : p< % EtOH extracts 2 Diethylether fractions

22 Table 5. Relative specific activities of GST in the fractions from Hovenia dulcis T HUNB fruit. (%) Fraction Activities H 2 O 115 BuOH 171 EtOAc 123

23 Relative specific activity (%) Fig. 4. Fraction of BuOH phase of Hovenia dulcis THUNB fruits extract by first silica gel column chromatography - O.D Relative specific activity

24 Relative specific activity (%) Fig. 5. Fraction of BuOH phase of Hovenia dulcis THUNB fruits extract by second silica gel column chromatography - O.D Relative specific activity

25 2.038 Fig. 6. HPLC elution profile of the active fractions separated from silica gel column chromatography.

26 Fig. 7. UV absorption pattern of HD1

27 Fig. 8. IR spectrum of HD1

28 Fig. 9. EI-MS spectrum of HD1

29 OH OH HO O m/z 290 OH OH RDA cleavage HO O OH OH OH m/z CH 2 HOHC CH m/z 152 OH H + 2 O m/z 123 OH Fig. 10. RDA fragmentation of HD 1

30 Fig. 11. FAB-MS spectrum of HD1

31 Fig. 12. H-NMR spectrum of HD 1 (CD 3 OD)

32 Fig C-NMR spectrum of HD 1 (CD 3 OD)

33 Fig. 14. DEPT spectrum (90 o ) of HD 1 (CD 3 OD)

34 Fig. 15. DEPT spectrum (135 o ) of HD 1 (CD 3 OD)

35 Fig H- 1 H COSY spectrum of HD 1 (CD 3 OD)

36 Fig. 17. NOESY spectrum of HD 1 (CD 3 OD)

37 Fig. 18. HMQC spectrum of HD 1 (CD 3 OD)

38 Fig. 19. HMBC spectrum of HD 1 (CD 3 OD)

39 Fig. 20. Determination of chemical structure of HD 1 (+)-catechin (C 15 H 14 O 6 )

40 Table 6. Comparison of the enhancement of ADH and ALDH activities at one hour after ethanol administration with or without (+)-catechin in mice. Control (+)-catechin ADH activity (%) 28.3±1.5 a 42.6±2.4 b ALDH activity (%) 29.1±1.3 a 59.8±1.9 b Mean ± S.E (n=5) a,b Significantly different from the control : p<0.05

41 Alcohol concentration (%) Hour Fig. 21. Comparison of residual alcohol concentrations in peripheral blood of mice with or without (+)-catechin. Mean ± S.E (n=5) *Significantly different from the control : p<0.05 : (+)-catechin : Control

42 Alcohol concentration (%) * * * * * * * Hour Fig. 22. Comparison of residual alcohol concentrations by uptaking alcohol in blood with or without (+)-catechin according to drinking time in human. Mean ± S.E (n=5) *Significantly different from the control : p<0.05 : (+)-catechin : Control

43 GOT(IU/L) ) b b 400 b b a a DAY Fig. 23. Changes in serum GOT activity after administration of (+)-catechin in normal and CCl 4 treated mice. Mean ± S.E (n=5) ab Significantly different from the control : p<0.05 : Normal, : CCl 4, : CCl 4 + (+)-catechin

44 GPT(IU/L ) b b 80 b b 60 a a DAY Fig. 24. Changes in serum GPT activity after administration of (+)-catechin in normal and CCl 4 treated mice. Mean ± S.E (n=5) ab Significantly different from the control : p<0.05 : Normal, : CCl 4, : CCl 4 + (+)-catechin

45 Fig. 25. Light microscopic photograph of the mice liver fed (+)-catechin in carbon tetra chloride treated mice A : Normal B : CCl 4 C : CCl 4 + (+)-catechin (1 day) D : CCl 4 + (+)-catechin (2 day)

46 Conclusion

47 헛개나무열매추출물의 GST 활성측정결과, 70 % ethanol 추출물 ( 조추출물 ) 이물추출물에비해높은활성을보였고특히 diethylether 분획물은조추출물에비해 GST 활성이약 11 % 정도증가 사람과동물을대상으로실시한 alcohol 분해능측정결과, 헛개나무열매조추출물의급여군은대조군에비해혈중 alcohol 분해능이높았고 diethylether 분획물급여군은조추출물급여군에비해 alcohol 분해능이더욱우수하였음 동물을대상으로한 ADH, ALDH 활성측정결과, 헛개나무열매조추출물과분획물군의활성이대조군보다높았고특히분획물의경우 ADH 와 ALDH 활성이각각 2.4 배와 1.8 배증가함 헛개나무열매로부터 silica gel chromatography, TLC, HPLC 를이용해활성물질 HD 1 을분리하였고, IR, 1 H-NMR, 13 C-NMR, EI-MS, FAB-MS, COSY, NOESY, HMQC, HMBC 등을이용해구조분석결과, HD 1 의분자식은 C 15 H 14 O 6 으로화학명은 3,3',4',5,7-pentahydroxyflavane 인 (+)-catechin 으로판명

48 분리된 (+)-catechin을쥐에급여한결과, 대조군에비해혈중 alcohol분해능이높았으며간장중의 ADH와 ALDH활성도유의적으로증가하였고또한사람을대상으로한실험에서도 (+)-catechin의 alcohol분해능이대조군에비해높게나타남 CCl 4 로유도한쥐에 (+)-catechin을급여한결과혈중 GOT와 GPT 활성이대조군에비해유의적으로낮아졌고간조직검사결과 (+)-catechin 투여에의해간조직보호효과가확인

49 이상과같은결과로부터헛개나무열매의조추출물과분획물은간해독 및 alcohol 분해활성이있는것으로확인되었으며, 분리한활성물질인 HD 1 은분자량 ( ), C 15 H 14 O 6 분자식을갖고있는 (+)-catechin 으로확인되었 다. 분리된 (+)-catechin 을 CCl 4 로유도한마우스에투여한결과혈중 GOT 와 GPT 활성이낮아져간조직보호효과가있음이확인되었고마우스와사람 에게 (+)-catechin 을 alcohol 과같이섭취시 ADH 와 ALDH 효소활성을증진시 킴으로서 alcohol 분해증진효과를확인할수있었으나분획물이나조추출물 의 alcohol 분해능과비교하면그활성이그다지높지않게나타나앞으로기 능성소재로서이용시추출수율이나경제적측면에서분리물질인 (+)- catechin 보다는조추출물을이용하는것이더욱효율적이라판단된다. 또한 앞으로헛개나무열매로부터세계최초로분리한 (+)-catechin 을이용한간기 능개선또는혈중 alcohol 대사개선식품개발이기대되며차후신제품응용 을위한지속적인연구를수행한다면기능성소재로서그가치가매우높을 것으로판단된다.

50 Alcohol 의분해과정 Alcohol Alcohol dehydrogenase H 2 Acetaldehyde Aldehyde dehydrogenase H 2 Acetate Carbon dioxide Water

Statistical Data of Dementia.

Screening of Prolyl Endopeptidase Inhibitors from Medicinal Plants. Lee Si-young, Lee Jung-han, Paik Young-Sook College of Environment and Applied Chemistry, Kyung Hee University. Statistical Data of Dementia.