MUC LUC LOLCAM ON MUC LUC DANH MUC CAC KY HIEU, CHU VIET TAT DANH MUC CAC BANG DANH MUC CAC SO BO. DANH MUC CAC HiNH MO DAU TONG QUAN 1. DAI CUONG VE CAY MUOP DANG 1.1 Mo ti cay 1.2 Phan bé va sinh thai 1.3 Y hoc dan gian ctia cay muép ding 13.1 RE 13.2 Than 13.314 1.3.4 Hoa 1.3.5 Trai 13.6 Hat 2. CAC NGHIEN CUU VE MUGP BANG 2.1 Cae eng trinh nghién eiru trong nude 2.1.1 Thanh phdn héa hoc 2.1.2 Tae dung duge ly Trang vi vi AaAwwaaaauas2.2 Cie cng trinh nghién etru trén 2.2.1 Thanh phan héa hoc 2.2.1.1, Triterpene 2.2.1.2. Steroid 2.2.1.3. Protein 2.2.1.4. Lipid 2.2.1.5. Carbohydrate 2.2.1.6. Caroteniod 2.2.2. Mét s6 triterpene trong cay musp ding 2.2.2.1. Céc triterpene glycoside duge o6 lip tir hat mudp ding 2.2.2.2 Cée triterpene glycoside durge ¢6 lap tr tréi mudp ding 2.2.2.3 Cac triterpene glycoside duge c6 lap tir i va day mur6p ding 2.2.3. Tae dung duage ly THUC NGHIEM 1. NGUYENLIEU 2. BINH TINH CAC HOP CHAT HOU CO TRONG TRAI MUGP DANG 2.1 Khao sit su hign din cia céc hgp chit alkaloid 2.1.1 Thudc thir alkaloid 2.1.2 Dinh tinh alkaloid 2..2. Khao sat su hign din ca cdc hgp chat flavonoid 2.2.1 Thuéc thir flavonoid 2.2.2 Dinh tinh flavonoid 2.3. Khio sit sy hign dign cia cdc hop chit anthraglycoside 2.3.1 Thude thir anthraglycoside 2.3.2 Dinh tinh anthraglycoside 2.4, Khao sat sy hign dign cia cdc hgp chat sterol 2.4.1 Thudc thir sterol 2.4.2 Dinh tinh sterol Ce eo em wm & 10 10 10 12 16 19 2B 2B 4 4 24 25 25 25 5 25 25 26 26 263 2.5 Khao sat sy hign dign ciia cac hyp chat saponin 2.5.1 Thude thir saponin 2.5.2 Dinh tinh saponin 2.6. Khao sat sy hign dign ciia cde hop chit dudmg khir 2.7. Khao sat sy hign dign cla cde hgp chat tanin 2.7.1 Thuée thir tanin 2.7.2 Dinh tinh tanin 2.8. Khao sat su hign dign cia cdc hgp chat glycoside 2.8.1 Thudc thir glycoside 2.8.2 Dinh tinh glycoside 3. TACH CHIET, CO LAP VA TINH CHE CAC HOP CHAT 3.1. Thiét bj va hoa chat 3.1.1. Thiét bj 3.1.2. Héa chat 3.2. Chiét xudt cdc nhém hop chat 3.3. Phan lip va tinh ché cdc hgp chat KET QUA & THAO LUAN 1. KET QUA BINH TINH CAC HGP CHAT HOU CO TRONG TRAI MUGP DANG 2: NHANDANH CAUTRUC CAC CHAT TINH KHIET 2.1. Chat MCL 2.1.1 Két qua phan tich 46 tinh khiét cia MC1 bing HPLC 2.1.2 Nhan danb cau triie héa hoc eda MCLA va MC1B 2.1.2.1. Mau chat MCLA 2.1.2.2 . Mau chat MCIB 2.2. Hyp chit MC6 2.3. Hgp chat MCS KET LU. 26 26 21 28 28 28 28 28 28 29 30 30 30 30 31 32 36 37 37 37 37 41 47 314 1. CAC KET QUA NGHIEN CUU DAT DUGC 2. KIEN NGH] TALLIEU THAM KHAO PHY LUC DANH MUC CAC PHU LUC DANH MUC CAC kY HIEU, CHU’ VIET TAT brs road singlet (NMR) COSY _: Correlation Spectroscopy d : Doublet (NMR) ad : Dam die dd : Doublet of doublet (NMR) DEPT _: Distortionless Enhancement by Polarization Transfer DMSO: Dimethyl sulfoxid PE : Petroleum ether EtOAc: Ethyl acetate EtOH Ethanol HMBC : Heteronuclear Multiple Bond Correlation HPLC: High Performance Liquid Chromatography HSQC _ : Heteronuclear Single Quantum Correlation R : Infrared J : Coupling constant m : Multiplet (NMR) MeOH —_ : Methanol mp : Melting point MS : Mass Spectroscopy 52 52. viii xi5 NMR : Nuclear Magnetic Resonance ppm : Parts per million Re : Retention factor s : Singlet (NMR) t : Triplet (NMR) TLC : Thin Layer Chromatography UV : Ultra Violet a-Gle : a-glucosidase hhemical shift DANH MUC CAC BANG Bang 2.1: Két qua sic ky cét thuéng Bang 3.1: Két qua dinh tinh cac hgp chat hitu co trong trai kho qua Bang 3.2: Dit ligu phd 'H, “C-NMR; DEPT va HMBC cia MCIA Bang 3.3 : Dit ligu phd "C-NMR va DEPT cla MCIB Bang 3.4: Dit ligu pho 'H, "C-NMR, DEPT va HMBC cia MCIB Bang 3.5: Dit ligu pho "C-NMR va DEPT cia MC6_ Bang 3.6: Dir ligu phd 1H, 13C-NMR, DEPT va HMBC cia MC6 Bang 3.7: So sinh dit ligu phd MC6 v6i tai ligu DANH MUC CAC SO DO So dé 2.1: Quy trinh chiét xuat cdc nhém hgp chit cia trai muép ding So d6 2.2: Quy trinh phan lip va tinh ché cdc hop chat tir trai mudp ding Trang 4 36 39 42 45 49 49 50 32 33Hin 1.1: Hinh 1.2: Hinh 1.3: Hinh 1.4: Hinh 1. 5: Hinh 1.6: Hinh 1.7: Hinh 2.1: Hinh 2.2: Hinh 2.3: Hinh 3.1: Hinh 3.2; Hinh 3.3: Hinh 3.4: Hinh 3.5: Hinh 3.6; Hinh 3.7: Hinh 3.8: Hinh 3.9: Hinh 3.10) Hinh 3.11 6 DANH MUC CAC HINH Cay khd qua. Day va la khd qua ‘Trai khé qua Hat khé qua Gian khé qua (Pha Yén) ‘Var. charantia L. Var. abbreviata Ser. Qué trinh say nguyén ligu Sac ky cot Sac ky lép mong Tinh thé MCL TLC etia MCI, MCIA va MCIB TLC ciia MCLA 6 cc hé giai ly khac nhau Céng thite cu tao cia MC1A TLC etia MCIB 6 céc hé giai ly khac nhau Céng thite cu tg0 cia MCIB ‘Miu chat MC6 TLC cia MC6 Céng thitc céu tao cia MC6 }: Mau chat MCS : TLC cia MCS 23 30 30 3 37 38, 39 a2 45 48 48 SI SI SILOI CAM ON Em xin giti Idi cam on sau sic dén: PGS.TS Nguyén Ngoc Hanh ThS. Phing Vin Trung ‘Ngusi thay da truyén dat cho em nhiing kién thite chuyén m6n va kinh nghiém nghién citu, tan tinh huéng dan, chi bao em trong sudt thoi gian em nghién citu tai phng Héa hop chat thién nhién- Vign Cong nghé Héa hoe. Khoa Céng nghé Héa- Thye phim cing Thay, C6 va céc ban sinh ‘vién tring Dai hoc Lac Héng da quan tam déng vién, tao moi diéu kign cho em trong qué trinh hoc tap va nghién ciru. ‘ThS. Phan Nhat Minh, cdc anh chj trong phong Héa hgp chat thién nhién, ciing céc anh chi nghién eiru vién trig Dai hoc Cin Tho, céc ban sinh vién lam dé tai nghién ciru da tin tinh huéng din, gdp ¥, cung cp tai ligu, tao diéu kign giup em lim tot céng viée ciia minh. Con kinh giti dén Gia dinh long biét on sau si Xin chan thanh cim on! LAC HONG, thang 11 nam 2009 LO Thi finh Nguyét8 LOI MO DAU Mudp ding hay cén goi la qua (Momordica charantia L.), thugc hg Bau bi - Cucurbitaceae) duge tring 6 nhiéu nude trén thé gidi, dac biét la nhiing ving c6 khi héu mhigt déi. O Viét Nam, céy duge trang 6 hau hét cdc tinh tir dong bang dén trung du va mien ni, Mudp ding ¢6 vi ding, tinh han nén trong din gian thudng ding dé trj cdc bénh myn hot, gidi nhigt, tri phién, thanh tm, sing mit, gidm dau... Khoa hoc ngay nay da ching minh dich chiét trai mudp ding cé kha nang ire ché khdi u, ¢6 tic dung hé try men gan va diéu tri bénh dai thao duong... Hign nay bgnh déi théo during 14 m6t trong cae bénh man tinh, gay tt vong cao, img hang thir ba trén thé gidi sau bénh tim mach va ung thu. O Viét Nam, ti I mic bénh ngay mét gia ting, ddi hoi cp thiét phai tim ra céc loai thuéc higu qua, dac tri. Musp ding cé tic dung dic biét nhu vay nhung hign nay van con rat it c6ng trinh nghién ciru vé hop chat trj bénh dai thao dudng. Vi thé, ching toi thyc hign nghién ctru dé tai : “Gép phan khdo sdt thanh phdn héa hoc cia trai mwép ding”.9 TONG QUAN 1. DAI CUONG VE CAY MUOP DANG 1.1, M6 ta cay ‘Tén khoa hoe: Momordica charantia L. Ho: Bau bi (Cucurbitaceae) ‘Tén mudc ngoai: Bitter melon, bitter gourd (Anh), bitter apple, wild cucumber, bitter cucumber, ampalaya (Philipines), balsam pear (MY), karela (An D9). ‘Tén Viét Nam: Muép dang, khé qua, Inong qua, cam 1é chi..." Ngoai ra n6 cén cé nhiéu tén khéc nhu: Muép mi, chua hao, dua mat, hing cé nuong, héng duong, bé dat, lai qua. |"! M6 ta cay muép ding: """! Cay mudp ding thugc loai diy leo, 6 déi séng khoding mét nim. Dung kinh day Khoang 5-10mm, day bd dai 5 — 7m, than mau xanh nhat c6 géc canh, leo duge nhi c6 nhiéu tua cudn, 6 nggn cé long to. Lé don, nhém, moe so le, dai $-10em, réng 4-8em, phién Id mong chia lam 5 ~ 7 thiy hinh trimg, mép cé rng cua déu, mat duéi la mau xanh nhat hon mat trén 14, gan la néi rd 6 mat didi, phién 14 06 long ngin. Hoa moe don die & ké 14, hoa dye va hoa cai cling géc, c6 cudng dai. Hoa dye c6 dai va éng rat ngin, trang gdm nim cénh méng hinh bau duc, nhuy 5 rdi nhau. Hoa c4i cé dai va trang hoa giéng hoa dye. Trang hoa miu vang nhat, duéng kinh khoang 2em ‘Trai hinh thoi, dai 8 — 15cm, géc va dau thuén nhon. Mat v6 c6 nhiéu u 1bi to nho khOng déu. Trdi khi chua chin c6 mau xanh hodc xanh vang nhat, khi chin cé mau vang hong. Vi thé & Trung Quéc, muép dng cén cé tén ld hong duong, héng ¢O nuong. Khi chin, trai nit dan ra tir dau, tich ra lam ba phan dé 1 chim do hat mau dé bén trong10 Hat det, dai 13 — 15mm, réng 7 ~ 8mm, hinh ring ngya, thit dt ngot 6 hai dau. Vo ‘hat cig, quanh hat cé mang mau dé nhu mang hat gic.1.2. Phan bé va sinh thai Musp ding duge phan bé & ving nhiét déi va cAn nhiét déi khp cac chau luc. Tir théi xa xua, mudp dang duge tring ln dau tién 6 Déng An va Nam Trung Quéc, duge sit dyng nhw [a loai rau an qua gidu chat sit va vitamin C. Sau dé cdy duge du nh§p sang chau Phi va chau M¥ Latinh. Quan thé muép ding tréng da tro nén rit phong phi v6i cdc giéng cay da dang dugc tao ra trong qua trinh chon gidng va lai tao."! 6 Vigt Nam, muép ding duge trong 6 hau hét céc tinh tir Bac Trung Nam, tir dong bang dén trung du va mién nai. G mét sé ving ndi cao va lanh nhu Sa Pa (Lao Cai), Pho ‘Bang (Ha Giang)...khéng thy c6 muép ding.!"*) Trén thé gidi, mudp dang cing cé mat 6 hdu hét cdc nude nhiét d6i nhu An DO, Nam A, Déng Nam A, Trung Quéc, chau Phi va ving Caribbean. Cay mudp ding cé bign dé sinh thai tuong d6i rong, nhiét d6 thich hop cho cay sinh truéng tir 20 dén 35°C, lugng mua hang nam tir 1500mm dén 2500mm, 46 cao dén 1000m. iy chiu dung duge nhiéu diéu kién dat khéc nhau nhung phat trién t6t nhdt trén at thoat thiy t6t, giau chat hitu co. Musp ding cé thé tring quanh nm. CAy sinh truéng nhanh trong mia mua 4m, ra hoa qua sau 7 — 8 tuan gico trdng. Hoa thy phin chi yéu nhé c6n tring. Sau khi trai gid, cay sé tan Iyi va két thie vong di sau 4 — 5 thang ton tai. Hign nay, edy vin con ton tai & hai quan thé: Moc hoang va duge tring trot. Logi tréng trot rit phong pha vé giéng nhung déu duge chung vdo chi muép ding (Momordica charantia L..). Tuy nhién, cn cit vio kich thud, hinh dang, mau sic ca qua ma chia mup dang thanh hai ching logi: — Momordica charantia L. var. charantia L.., tei to (dung kinh > Sem), miu xanh nhat, gai ti, it ding. — Momordica charantia L. var. abbreviata Ser., trai nh6 (duémg kinh < Sem), mu xanh dam, gai nhon, vj rat ding.12 Hinh 1.6: Var. charantia L. Hinh 1.7: Var. abbreviata Ser. 1.3. Y hge dan gian cita cay muép ding Hau hét céc b6 ph§n ciia cay nhw ré, than, la, hoa, trai, hat déu c6 thé ding lim thude chia bénh. 1.3.1 RE RE tuoi, sc nude udng, mdi ngiy mét thang, tir 1000ml nude voi 60g ré mudp ding tuoi, sic con 400ml, chia lam 2 — 3 lin udng, c6 thé dp dung cho moi dang bénh. R& mudp ding ding tri ly, nhat la amip. Tgi An D6, dich ré (cing nhu 14, trai) mudp ding urge ding trj bénh tigu dudng, do c6 tie dung lim giam dung huyét. RE mudp ding con 6 thé trj bénh gan, 1.3.2 Than Day musp ding duge ding lam thud chita viém xoang, chay nuée mii c6 mai héi ‘hodc bénh gan kim vang da. "7! 1.3.3 La LA cé vj dang, tinh mat. An la non trj bénh néng bite trong minh. Gid 14 vit nude, thém chit mudi, udng tri bénh néng mé man hodc tri mun nhot, rom sy. Ngoai ra, ld con 6 thé tri duge ran can, lam thuéc nhudn truéng, ha sét. 4!1.3.4 Hoa Hoa musp ding duge ding dé chita dau da day, ly cap tinh , dau mat. '' 13.5 Trai Trai con xanh c6 vj ding, khi chin thi it ding hon. Trai muép dang cé tinh han (mat), khong dc. Tréi xanh c6 tinh gidi nhigt, lam tiéu dém, nhugn trang, bé than, nudi can huyét, bét mét méi, giam stress, xoa diu thin kinh, gidi dc, Igi tiéu, lam bét dau hop. Khi chin muép dang cé tinh bé than, kign ty, duéng huyét, digt giun (sin, lai), dng théi cé tae dung Lim sang mit, bé tim, bé mau, mat gan, rit thich hop véi nhing ngudi dau gan, dau la lich! G Trung Quéc, trai muép ding con ding dé tri d6t qui tim, bénh sét, khé miéng, viém hong. O An D6, dich trai mudp ding duge ding trj rin cin, Ngudi ta con ding bot trai muép ding dé han cac vét thuong (Lim kéo da non), tri vét loét ac tinh. G Thai Lan, dich trai duge diing tri bénh vé gan, 1a léch.”! V6i tinh digt khudn va chéng oxi héa, trai muép ding lam da min mang, tri myn trimg ca hay bénh vay nén, va ngay ca véi vét thuong do cén tring cn, nhiém tring da, Mat khdc, trai mudp ding con cung cdp ngudn nang lugng doi dao va ting kha nang chju dung cho co thé"! Ngoai cac céng dung trén, trai muép ding con duge ding dé tri nhiéu bénh nhu: Tri ho, sot, kiét ly, da day, dau tire, dai dat, phi thing do gan, mat do dau nhirc, giai nhiét, hoi ‘h6p, budn phién, tim cho tré em tri rém say, lim ha duéng huyét 6 bénh nhén dai thio dung type 2 (khéng phy thude insulin). "7! 1.3.6 Hat Hat c6 chat béo, vj dang, hoi ngot, tinh dm, thanh nhiét, gidi d6c, gidi cam, trj ho, loi sin, chita nhot déc sung tay, vét thuong nhiém tring, ha sot dau hong va chéng thy thai"! Tai nhiéu nude khac, hat mudp ding duge ding dé trj benh dai théo dudng."* tigu, Hat con chita ri4 Ngoai ra, theo mét so nghién ctu gan day cho biét cac hogt chat trong hat musp ding cdn cé téc dung chéng ung thu, lam ha huyét 4p, khang virus HIV...!"7) 2. CAC NGHIEN CUU VE MUGP DANG 2.1 Cac cong trinh nghién ciru trong muse 2.1.1 Thanh phan héa hoc Cac téc gid Pham Van Thanh, Pham Kim Man, Doan Thi Nhu, Nguyén Thuong Dong, Va Kim Thu, Nguyén Kim Phugng va Lé Minh Phugng ciia Vign Duge ligu di théng ké va khdo sat sor b@ cic nhém hoat chat chinh cia céy muép dang. Tuy nhién, cdc tac gid nay chua cé lap durge cde hoat chat c6 hoat tinh duéi dang chat tinh khiét cing nhu chua xac dinh cau tric cua céc hoat chat nay, ma chi dinh lugng theo chat G6, mot aglycon cia nhém glycoside 4) Cae tae gid Nguyén Thanh Hong, Nguyén Ngoc Hanh, Phing Van Trung 6 Vi cong nghé Héa hoc dai ¢6 lap va nhdn danh duge hai hop chat tir hat: Momordicoside A ‘va Momordicoside B va bén hgp chat tir tréis Momordicoside K, Momordicoside L, 3-O- [B-D-glucopyranosyl]-stigmasta-5,25(27)-diene vi 23-O-f-D-allopyranosyl 5,19 ‘epoxycucurbita-6,24-dien-3,22,23¢-triol 3-O-B-D-allopyranoside. !"") 2.1.2 Tée dung durge ly Y hoe cé truyén va dan gian Vigt Nam da cé nhiéu kinh nghigm chita bénh tir mup ding, nhung chi & dang nguyén ligu thé ban dau hode cdc dang nude ép, nude sic ma théi. Ngay nay, trén thj trréng da xuat hign nhiéu sin pham trai muép ding nhung da sé 6 dang thye phim chite nang nhur tri hoa tan, tra tii loc. Dién hinh 1a sin phim tri muép ding cia Vién Duge ligu hay tra tii loc muép ding ciia Cong ty Traphaco. Nhdm téc gid Pham Vin Thanh, Pham Kim Man, Doan Thi Nhu va eOng sy d% sin xuit ché phim Morantin tir thanh phan glycoside ciia trai muép dang dang to, mau tring va chimg minh téc dung ha duéng huyét ctia nhém glycoside trén thé gay dai théo duémg ‘thyc nghigm bang Alloxan,"“!15 Cac tac gid Nguyén Thi Nhu, Nguyén Thj Bay da nghién ciru tac dung ha duéng huyét cia m6t bai thuée nam trén thye nghiém lim sang, dé 1a sin phim tra tii lye ma thanh phan muép dang qua chiém 60%. Cac tée gid Mai Phuong Mai, V6 Phing Nguyén ciing di thm dé tac dung ha dung huyét ctia mét s6 bai thude dan gian & mé hinh dai thao duéng bang streptozotocin trén chu6t nhit, ma thanh phan ctia bai thude cting cé chira trai mudp ding. 2.2 Cée edng trinh nghién eiru trén thé giéi 2.2.1 Thanh phan héa hge "4 Ngudi ta da tim thdy khoang hon 200 hgp chat cé trong cay mudp dang va duge théng ké so bd thanh ede nhém chinh nhur sau: 2.21. Triterpene — Triterpene glycoside: © Momordicosides A, B,C, D, E, Fl, F2, G, I, K, L. © Cucurbitane triterpenoid 1 ‘* Momordicine II, momordicine III. © Goyaglycosides -a, -b, -c, -d, -¢, -f,-g, -h. © Momordicin I, momordicin IL Triterpene saponin: Goyasaponins I, IL, II. — Cie triterpene khée: Momordicin, momordicinin, momordicilin. Cucurbitane triterpenoid 3, cucurbitane triterpenoid 6. © Pramyrin, cycloartenol, erythrodiol, gypsogenin, karounidiol, multiflorenol, oleanolie acid, squalene, taraxerol...16 2.2.1.2, Steroid Steroid glycoside: B-sitosterol-3-O-f-glucoside; 3-O-[B-D-glucosyl]-stigmasta-5,25(27)-diene. 3-0-[6°-O-palmitoyl--D-glucosyl}-stigmasta-5,25(27)-diene; 3.0-[6’-O-stearoyl-P-D-glucosy]]-stigmasta-5,25(27)-diene. Sterol: Elasterol; lanosterol; momordenol; f-sitosterol; a-spinasterol; stigmasterol; stigmasta-S-ene-3B,25-diol. 2.2.1.3. Protein — p-insulin, v-insulin, — Map-30. — Momorcharin 1, momorcharin Il, -momorcharin, f-momorcharin, A- ‘momorcharin, ¢-momorcharin, y-momorcharin, — Momordin, momordin A, momordin B. Ribosome-inactivating proteins 1, 2, 3, 4 ‘Trypsin inhibitor meti-I, trypsin inhibitor meti-II, trypsin inhibitor mei-3. Cée protein khée: Alanine, B-alanine, phenylanaline, arginine, asparagine, aspartic acid. 2.2.1.4. Lipid Arachidic acid, capric acid, cholesterol, a-elaeostearic acid, lauric acid, linoleic acid, linolenic acid, palmitic acid, palmitoleic acid, stearic acid. 2.2.1. 5. Carbohydrate D-galacturonic acid, a-glucose, f-glucose, inulin, mycose, pectin, trehalose, a- trehalose.7 2.2.1. 6. Carotenoid B-carotene, cryptoxanthin, lutein, lycopene, mutatochrome, phytofluene, rubixanthin, zeaxantin, zeinoxanthin. .. Ngoii ra cén e6 cc thinh phan khéc: Alkaloid: Charine, zeatin, zeatin riboside. ‘monoterpene: p-cymene, menthol ; sesquiterpene: Nerolidol; sapogenin: Diosgenin; céc chat khoang: Ca, Mg, Fe, Cu, Zn, P,N, I, F... Trong nhiéu nghién ciru, nguéi ta da chimg minh duge ring cé it nhat ba nhhém hgp. chat c6 tée dung lam giam Iugng dudng huyét ho%e c6 hoat tinh khang dai thio dudng. D6 1a hin hop ctia hai steroid glycoside goi 14 charantin, cdc peptide gidng insulin (p- insulin) va alkaloid, Cac hgp chat nay chit yéu tap trung & trai muép dang. 2.2.2. M@ts6 Triterpene Glycosede cé trong cy muép ding 2.2.2.1. Cie triterpene glycoside duge cé Ifip tic hat murép ding 79). + Momordicoside A — LA3-0-B-gentiobioside eta cucurbit-5-en-38,22(S),23(R),24(R),25-pentaol. — Céng thie chung: CsyH20,s; M = 816 dvC; mp= 181 — 187°C; [a= +1.05°. — Céng thite cdu tao:18 Momordicoside B -3-0-f-D-xylopyranosyl(—>4)-[B-D-glucopyranosyl(1+6)]}-f-D-glucopyranoside cia cucurbit -5-en-3 f,22(S),23(R),24(R), 2Spentaol. = Céng thire chung: CsHw Os; M= 948 ': mp = 238 — 242°C; [a]"p= 46.15°. = Céng thite edu tao: “© Momordicoside C — La3-0-f-gentiobioside cia cucurbit-5-en-3 B,23,24,25-tetraol. — Céng thie chung: CHy20,4; M = 800 dvC; mp =224—227°C; faf"")= 13.9". = Céng thite céu tao:19 oH, oO i & Momordicoside D — La3-O-f-gentiobioside ciia cucurbita-5,24-dien-3 P,22,23-triol. = Céng thire chung: CigH;Oi3; M = 782 dvC; mp = 199 203°C; [af"p=-126" — COng thite cdu tao: 2.2.2.2. Cée triterpene glycoside duge c6 ldp tit trai mudp ding °* 709) Momordicoside F; = LA 3-O-f-D-glucopyranoside iia 5,19-epoxy-25-methoxy-5B-cucurbita-6,23- dien-3B-ol, = Céng thire chung: Cs7HygOs; M = 632 dvC; két tinh trong MeOH-H,0 (1:1) cho tinh thé hinh kim, khéng mau; mp = 198 — 203°C; [a]"°p = -11°.20 — Céng thitc cau tao: © Momordicoside F; — LA3-O-f-D-allopyranoside cia 5,19-epoxy-5-cucurbita-6,23-dien-3B,25-diol. — Céng thire chung: CssHssOs; M = 618 dvC; két tinh trong acetone-nude cho tinh, thé hinh vay, khéng mau; mp = 155 - 158°C, = Céng thite cau tao: “ Momordicoside G — La 3-O-f-D-allopyranoside cla 5,19 —epoxy-25-methoxy-5P-cucurbita-6,23- dien-3B-ol. = Céng thire chung: Cy-Hgj05; M = 632 dvC; két tinh trong CH;CN-H,0 cho tinh thé hinh kim, khong mau; mp = 183 — 187°C; [a]*°)= -107.3°.21 = Céng thite cfu tao: Momordicoside I — LA 3-O-f-D-glucopyranoside cla 5,19 -epoxy-25-methoxy-5B-cucurbita-6,23- dien-3f,25-diol. — Céng thie chung: C;gHs¢Os; M = 618 dvC; két tinh trong MeOH 50% cho chat bot mau tring; mp = 210 — 216°C; [a}"p = -110° (C=1.00; MeOH). = COng thite céu tao: & Momordicoside K — La 3-O--D-glucopyranoside cia 3B, 7P-dihydroxy -25-methoxy cucurbita-5,23- dien-19-al, — Céng thire chung: Cx;HsoOo; M = 648 dvC; két tinh trong MeOH cho tinh thé hinh kim, khéng mau, vi ding; mp = 236 — 237°C; [a]°p= 63.3°.22 — Céng thite cu tao: Momordicoside L — Cdn o6 tén li 7-O-B-D-glucopyranoside cia 3B,7B,25-trihydroxy-cucurbita-5,23- dien-19-al = Céng thire chung: CsgHssOs; M = 634 dvC; két tinh trong CHCls-MeOH cho tinh. thé hinh kim, khéng mau, vj ding; mp = 227 — 232°C; [a]""p=+57.3°. — COng thite cdu tao: 2.2.2.3. Cée triterpene glycoside duege c6 Idp tit Id va day muép ding © Cucurbitan triterpenoid I23 — LA3B,7 B,23-trihydroxycucurbita-5,24-dien-7-O- B-D-glucoside. = COng thire chung: CssHqo0s; M = 620 dvC; dang bt v6 dink hinh, [al"*y= +89° (C=0.43; MeOH), — Céng thite cu tao: 4“ Cucurbitan triterpenoid II — 1A3B,7 B.23-trihydroxycucurbita-5,(23E)-ien-19-al. — Céng thite chung:CyHyO«; M = 472 dvC; dang bot v6 dinh hinh, [a}p= +58.0° (C=0.48; MeOH). = Céng thie céu tao sau: Cucurbitan triterpenoid IT24 — LA 3B,7 B-dihydroxy-25-methoxycucurbita-5,(23E)-dien-19-al. = Céng thire chung: Cy1Hy04; M = 486 dvC; dang bot v6 dink hinh; [a}""p = +48.9° (C=0.45; MeOH). — Céng thite cfu tao: % Momordicine I — LA3B,7 B,23é-trihydroxyeueurbita-5,24-dien-19-al = Céng tite chung: CayHyOx; M= 472 dvC; mp = 125 128°C ; [af*>= +81.3°. — Céng thite cdu tao: Momordicine IT = 1423-0 -f-glucopyranoside ciia 3B,79,23é-trihydroxycucurbita-5,24-dien-19-al. — Céng thite chung: CsgHs4Oo; M = 634 dvC; két tinh trong CHCI, cho tinh thé dang bot khéng mau.25 — CGng thite céu tao: + Momordicine Hl — LA 23-0- B-glucopyranoside ciia 38,7B,23é-trihydroxy-24-oxo-cucurbita-5,25- dien-19-al. = COng thre chung: CygHssO,0; M = 648 dvC. — Céng thitc cau tao: 2.2.3, Tae dung durge ly Theo y hoc hién dai, musp dang cé tac dun; — Diét vi khuan va virus, chéng lai cdc té bao ung thu, hd try dic lye cho bénh nhan ung thu dang chita bang tia xa.26 — Chéng cdc géc ty do — la nguyén nhin gay lio héa va phat sinh cdc bénh tim mach, tang huyét 4p, réi logn lipid mau, tén thuong than kinh, viém dudng tiét nigu, dai thio dudng. — Ting oxy héa glucose, ngin chin sy hap thu glucose vao té bao. Uc ché hoat tinh cdc men ting hgp glucose. — Cé tac dung sinh hoe giéng insulin, gitip co thé ting tiét insulin. — Cé te dung t6t véi nguéi mac bénh dai théo during type 2. Hé trg ting tac dung, giim liéu va gidm tée dung phy cia cdc logi sulfamid trj dai théo dudmg type 2. Dich chiét trai muép dang cé kha ning tre ché khéi u hé try men gan, chita duge nhiéu bénh nhu di théo dudng, lach, gan, khép, gout ...."4%" Theo tai ligu , cao MeOH 50% tir trai musp ding cho tac dung ha dudng huyét 25% (ligu ding 30mg/kg), cao butanol cho két qua la 34% véi ligu ding nhu trén. Cac tac gid nay cho rang cde hgp chat phan eye, tan nhiéu trong butanol ¢6 kha nang lam giém dudng huyét. Co ché hoat dng tong ty insulin hode théng qua sy tiét insulin tir tuyén tuy. Nghién ciru in vivo trén thé gay dai thdo duéng thye nghiém bing Alloxan cho thay, khi ding nude ép trai mudp ding vai liéu ding 6ml/kg B.W cho két qua téi wu, lam giam Iugng dudng mau 6 thé binh thudng sau 2 gid va ting tré Iai sau 3 gid. Tuy nbién 6 tho mic bénh, lugng duéng mau tiép tuc gidm t6i 4 gig’ sau khi cho uéng rdi méi bat dau ting torial Theo bao eo cita Dai hoc Y khoa Calcuta (An D6), da thir nghigm cho 6 bénh nhan ai thao dudng type 2 uéng mdi ngay mét Lin 100ml nude sic mudp ding tuoi. Sau 3 tuan 18 udng thudc lién tuc, do lugng dung trong mau (khi d6i) di gidm duge 54% so véi ban dau. Sau 7 tuan ding thudc, ca 6 bénh nhin déu khéng thay dudng trong nude tieu, lugng dudng trong miu nhu ngudi binh thudng. * ‘Tae dung duge ly cita charantin Gidi thigu27 Charantin 1a mét hén hgp 2 steroid glycoside durge céng bé 1a m6t trong nhiing hoat chat c6 hoat tinh khang dai thao duéng type 2, duge chiét tach va c6 lip ttt trdi mudp ding. Nam 1962, Lotlikar va Rao lan dau tién c6 lap duge charantin véi ham long khong 0.01%, Dén nim 1965, Sucrow da xc dinh duge day 1a mt hon hgp 2 steroid glycosides (ti 1G 1:1) gdm 3-O-[B-D-glucopyranosyl]-stigmasta-5,25(27)-diene va B- sitosterol-3-O-f-glucoside , v6i cOng thite lin lugt nhur sau: CTPT: CasHoOs (M = 576) Nam 1966, Lotlikar va Rao da dua ra qui trinh chiét xuat charantin voi ham lugng cao hon, ding thi céng bé vé hoat tinh khang dai th4o duvng cla hoat chat nay, duge phan lap tir dich chiét EtOH. cia trai mudp ding khé .28 ‘Sau 6 dén nim 1979, Pugazhenthi va Suryanarayana Murthy da khang dinh mét lan nita charantin la hén hgp 2 chit va hoat tinh sinh hgc cia né vin c6 thé mit di trong qua trinh chiét xudt kéo dai"! Hoat tinh khang dai théo durimg cia charantin Tién hanh Khao sat hoat tinh khang dai thao dung cia charantin trén thé gay dai théo duéng thye nghiém bang Alloxan: — Chon thé & cd con dye va con céi v6i trong long eo thé vao khodng 1.5 ~ 3kg, duge gay dai thio duing bing cach tiém Alloxan qua finh mach véi ligu lrong 200mg/kg. Chi cé 4 trong 20 con con sing sau 5 ngiy duge tiép tuc dem di khao sat. — Charantin hoa tan trong Tween 80 véi ndng dé 0.3%. Xir ly bang cach cho wong hoje tiém qua tinh mach Sml dung dich nay. Mire dung huyét gidm dan tir gid thir nhat én gig thir tr sau khi xi ly, nhung sau 6 sé tr ti léy lai mite ban du, — Hoat tinh cia charantin c6 higu Ic hon 5 gid, cao nhit & gid thir tu, durge ghi nhan 1a ¢6 tac d6ng nhu nhau ngay ca khi xir ly bing cach cho uéng hodc tiém qua tinh mach. V6i ligu SOmg/kg cho udng lam gidm 42% hrong dudng huyét 6 gid thi tur va giém con 28% 6 gid thir nim sau xit ly. Tuy nhién khi sir dung véi liéu lugng 25mg/kg cho udng sé cho két qua giam durong huyét tong ty chi véi ligu ding 15mg/kg tiém qua tinh mach, ) Charantin cho hoat tinh giam duéng huyét cao hon Tolbutamide, mét loai thuéc théng thuéng dé chita dai thio dudng, véi cing liéu ding. Mac di cach thite thay déi glucose huyét khi xi ly & ca 2 trudng hop déu gidng nhau. Tuy nhién vige charantin c6 phai 1a cht duy nht trong tréi_cé hoat tinh giam dung huyét hay khéng van cén la van dé dang duge dit ra. Vi gia sir dé c6 duge 50mg charantin cin ti hon 1.5kg trai twoi, Trong khi theo cde nghién citu, chi véi ligu ding 50 ~ 60ml nudp ép trai_ hang ngay da cho két qua lam sang tt, chimg t6 khéng phu thudc hoan to’in ‘yao rit it khdi lrong ciia charantin 06 trong dé. MAP, m6t protein khang siéu vi khuan, c6 we ché nhiém virus HIV-1 6 té bao lympho T va bach cau don nhan to, né khéng 6c véi té bao binh thuéng khéng bj nhiém. Hat vi v6 tréi_ chira mot chét nha, mt saponin glycoside, va nhiing alkaloid gay nén va tiéu chay. Nhiéu protein c6 hoat tinh dirge ly duge phan lap tir hat , nhu céc protein a-momorcharin va B-momorcharin cé téc dung dgc hai gan trén té bao gan chudt 6 lap. G Trung Quéc, ngwai ta da phan tich duge hai hoat chat han ché sinh san la o— protein va [protein tir hat. Cac thi nghiém nudi cy, ghép phéi in vitro cho thay 2 hoat29 chat nay cé tée dung tte ché qua trinh lam day dae nguyén bao phdi trude khi lam t6 va hinh thanh phéi 6 thai ky dau, tir 46 phdi ngimg phat trién, thoai héa phan hay din dén siy thai "1 Nhimg nghién citu gin day cho thiy a-protein va f-protein cing cé anh huéng dén sur sinh san cia phi chudt nhit tring va anh hung dén viée téng hgp phan tir lén té bao ‘n6i mé tir cung; ching ciing cé kha nang ite ché ting hop ADN, ARN va protein, lam cho sur phat cla ni mac tir cung bi tre ché !"”!30 THUC NGHIEM 1, NGUYEN LIEU Musp ding do Trung tam Bao tén va Phat trién Duge ligu mién Trung cung cp, la loai muép dang cén non, hat chua phat trién. Tréi duge cat thanh lat méng, say 6 nhiét 46 dui 60°C trén hé théng say nguyén ligu dén khdi luong khong déi. Tiép 46, nguyén ligu duc xay nhuyén dé qua trinh tach chiét dugc trigt dé hon. ‘Hinh 2.1: Qua trinh say nguyén ligu 2. NHAN DANH CAC NHOM HOP CHAT HUU CO TRONG TRAI MUOP DANG Bé gitip cho vige nghién ctu thanh phan héa hoc va chiét xudt hoat chat tr cdy thude, truée hét can phai tién hanh phan tich so b6 thinh phan héa hgc dé ching ta cd khai nigm so bo vé mat dinh tinh, tir d6 6 thé dinh huéng cho vige chiét xuat cing nhu vige xdc dinh cu trie héa hoe cdc hgp chit trong cy thude. Tir mau cay, sir dung k¥ thuat chiét tich khdc nhau dé cé duge cao chiét ethanol toan phan hoje cic logi cao cé tinh phin cye khéc nhau. Ap dung cdc phuong phap phan tich so b6 vé héa- thye vat dé biét trong cao chiét cé thé chita cdc loai hgp chat ty nhién nao. Trong bio céo nay, em xin trinh bay vé phuong phap sir dung thudc thir hign mau hode xuat hign két tia ma em da thye hin.31 2.1 Khao sat sw hign dign cua cac hop chit alkaloid"! 2.1.1 Thuéc thir alkaloid Cé rit nhiéu thudc thir cho phan img mau hode két tia véi alkaloid. * Phan tmg tao két tia cé mau: ‘Thudc thir Mayer: (Céng thite: 1.35g HgCl héa tan trong 100ml dung dich KI 5%. Dau higu: Tao két ta v6 dinh hinh mau tring vang. — Thudc thir Dragendoff: © Céng thite: + Dung dich A: 850mg Bismut nitrat trong 40m! va 10ml acetic bang. + Dung dich B: Hoa tan 8g KI trong 20ml H20. ‘Tron hai thé tich bing nhau cia hai dung dich A va B dé lam thudc thir. © Dau higu: Tao két tita c6 mau tir vang cam dén do. — Thuéc thir Wagner: © Céng thire: Héa tan Sg lod trong 100ml dung dich KI 10%. © Dau higu: Cho két tia mau nau sang dén nau den. — Thuéc thir Bouchardat: Céng thite: 2.5g lod va 5.0g KI hda tan trong 10ml nude eat Dau higu cho két tia mau nau hogc mau vang dam. 2.1.2 Dinh tinh alkaloid Thi nghiém: Tam 10g b6t trai muép ding bing Sml dung dich NH,OH 25%. Day kin bing gidy loc rdi dé qua dém. Sau 46 dem chiét v6i 25ml CHCl. Dich CHCl duge lic voi 10ml dung dich H)SO, 2%. Lay dich acid lam miu thir. -VGi thudée thir Wagner: Cho két tia mau nau.32 -Véi thuéc thir Mayer: Dung dich cé mau tring duc. 2.2 Khao sat sy hién dign cua cac hop chit flavonoid “7 2.2.1 Thudc thir flavonoid Trong dung dich, flavonoid tao két tia mau ving cam hoje mau dé v6i acetate chi, tao két tia mau xanh luc, d6i khi miu néu d6 véi FeCl, Flavonoid duge xée dinh béi phan (mg Shibata, con goi la phan img Cindin cha Willstater. Thudc thir li tap hop cdc héa chat gém: dung dich HCI dam dic, bot Mg kim loai, rutgu isoamyl [CH3(CH,)CHCH,CH;CH,OH]. 2.2.2 Dinh tinh flavonoid ‘Thi nghigm: un hoan Iu Sg bot tréi muép dang trong 50ml EtOH 95° trong 30 phit. Loc, ay Iml dich EtOH vao 6ng nghiém, thém vai giot HCI dam dac, sau dé cho ‘mét it bot Mg vao Lic thi thay dung dich cé mau tim. 2.3 Khao sit sw hign dign cia ede hgp chit anthraglycoside 2.3.1 Thuéc thir anthraglycoside Anthraquinon (phan imgBotrager): Pha hitu co ciia dich chiét anthraquinon sé cé mau dé khi ¢6 sy hign dign cia chat kiém. Do vay dang két hop phai duge thy phan va chuyén sang dang oxy héa truéc khi thyc hién phan img. Anthron va anthranol: — Phan tmg Schouteten: Cho huynh quang xanh v6i natri borat. — Phan tmg tgo mau xém véi natri nitrodimethy! alanin, 2.3.2 Dinh tinh anthraglycoside "! ‘Thi nghigm: Dun hoan hu 5g bot tréi mudp ding trong 30ml ether ethyl. Loe va 1p lai nhiéu lin cho dén khi dich ether khong cdn mau. Tép trung dich loc va Kic voi 50ml dung dich KOH 10%. Lép kiém duge trung héa véi dung dich HCl 25% dén pH=7. Loe, lay phan két tia trén gidy loc, dem hda tan bing EtOH 95°. Nho vai git dung dich KOH 10% vao dich EtOH thay cé mau ving cam.33 Ba sau khi da loai céc chat tan trong ether dirge chiét tiép voi EtOH 95°. Nho vao dich EtOH vai gigt KOH 10% thy c6 mau d6 cam. 2.4.Khao sit sy hign dign ciia céc hgp chat steroid "! 2.4.1 Thudc thir steroid — Liebermann-Burchard: — Anhydrid acetic: 20ml. H,80, dam die: Iml. Cho 1 giot thuée thir vio dich CHCIs, néu cé sterol sé cé mau xanh nhat, luc, hong hoge do bén viing trong mét thai gian. — Phan img Rosenheim: Cho vai git dung dich acid tricloacetic 90% vao dich CHCh, néu cé sterol s& xuat hign mau tim, sau 20 phiit chuyén sang mau xanh lo. = Salkowski: Dung dich tach lam 2 Iép: Lép H:SO, ¢6 mau xanh, lsp CHCl; ¢6 mau nau dd. 2.4.2 inh tinh steroid ‘Thi nghiém: Hoa tan 1g bét trai muép ding khé trong 20m! CHC1. Loc, liy dich Ige Lim mau thir. V6i mau cao, sir dung 0.1g hda vao 30ml CHCl, lay dich Ige lam mau thir. © V6i thudc thir Liebermann- Burchard: Cé mau hing do. ‘© Voi thude thir Salkowski: Thém 0.5ml dung dich H.SO, dim dic. Dung dich tach Lim hai Iép, lp HySO, (l6p trén) c6 mau xanh va lop CHCI; (Iép dudi) cé mau 46 v6i thudc thir Salkowski. 2.5 Khao sat sy hign dign cia cdc hgp cht saponin ™ 2.5.1 Thuéc thir saponin Can ett vao chi s6 tao bot dé xc dinh sy hign dign cia saponin.34 Duge dién cia Phap dinh nghia chi s6 tao bot nhu sau: Chi s6 tao bot cia saponin la 46 lo’ing cia nguyén ligu bing nude dé c6 chiéu cao bot 1cm sau khi bic trong éng nghiém ¢6 kich thuée xéc dinh, tién hanh trong diéu kign qui dinh. Cich tién hank: Can 1g b6t duge ligu cho vao erlen 500ml chira sin 100ml nude si. Tiép tue cho nuée trong erlen s6i nhe trong 30 phiit nita. Loc dé ngudi, thém nude cAt cho dén 100ml (thu duge nude sic). Lay 10 dng nghiém c6 chiéu cao 16cm, dung kinh 16mm. Cho vao cdc éng nghiém lan lugt 1, 2, 3, 4, ...10ml nude sic. Thém nude ct vao méi dng cho du 10m. Bit migng éng nghiém rai lac theo chiéu doc ciia éng trong 15 gidy. Mdi gidy lic 2 Min, Bé yén trong 15 phit. Sau dé do chiéu cao cc eGt bot. Néu Ot bot trong cac dng thap duéi Icm thi chi sé bot ld diréi 100, nghia la khéng cé saponin. — Phan img Liebermann: © Céch thye hign: Hoa tan mau bing Iml anhydrid acetic, thém tir ti 0.3 — 0.5ml H,SO, dim die. ° Daw higu: + Néu vong ngan cach cé mau hong dén 46 tim thi sob nh§n dinh cé saponin triterpene. +Néu vong ngin céch e6 mau xanh i edy thi sob} nhn dinh c6 saponin steroid. — Phan img Kahlenberg: © © C&ch thye hign: Hda tan mu bing 0.5ml dung dich SbCl, bao hda trong CHCh, khuay déu, dem soi UV. © Dau higu: + Néu cé6 huynh quang mau xanh thi so’b6 nbn dinh c6 saponin triterpene. + Néu huynh quang mau vang thi sorb nhén dinh ¢6 saponin steroid. 2.5.2 Dinh tinh saponin Thi nghigm: Can 1g bot trai mup ding cho vio erlen 500ml chita sin 100ml nude sdi. Tiép tue cho nude trong erlen s6i nhe trong 30 phit nia. Loc, dé ngudi. Cho khoang35 Im dich Ige vio dng nghiém nhé va lic manh trong 15 gidy thi thay rat nhidu bot (c6t bot cao 6cm), 2.6 Khao sat sy hign dign ciia cdc hgp chat dong khir'") ‘Thi nghiém: Acid héa 2g bét trai mudp ding bing 20m! dung dich H»SO4 1%. Loc, 6 can con Sml. Nhé vio mau thir 4 ~5 giot thude thir Fehling A va 45 gigt thude thir Fehling B. Bun nhg thay xuat hién tia mau dé nau. 2.7 Khao sit sy hign dign cia céc hgp chat tanin 7! 2.7.1 Thuéc thir tannin Stiasny: Formol (36%): 20ml. Dung dich HCI dam dic: 10ml. Dung dich gelatin man: Gelatin: 2g Dung dich NaCI bio héa: 10m1 Dung dich acetate chi bao hoa cho két tia mau vang nhat. Dung dich FeCl; 1% trong mde tq0 phite miu den. 2.7.2 Dinh tinh tanin ‘Thi nghiém: Lay 5g bét trai, thém 100ml nude cat réi dun s6i trong 10 phit. Loc, Jay dich loc lam mau thir: Lay 2ml dich loc, thém 2 — 4 gigt dung dich acetate chi bio héa thay xuat hign két tia miu ving nhat. Lay 2ml dich loc, thém vai git dung dich gelatin main, xudt hign két tua tring. Lay 2ml dich Igc, thém 2ml dung dich FeCl; 1%, dung dich chuyén thanh mau nau den. 2.8 Khao sat sy hign dign cia cdc hgp chat glycoside 7+"! 2.8.1 Thudc thir glycoside ‘Thuéc thir tac dung lén phan aglycon.36 — Thudc thir Tollen (xée dinh theo dug khir trong glycoside): © Céng thite: Pha 0.5ml dung dich AgNO; 10% véi 0.5m dung dich NaOH 10%, ‘Sau dé nho tir tir dung dich NH,OH dén khi tan két tia © Dau higu: C6 Ag két tia. Thuéc thir Molish: Céng thre: Nho 1 ~2 gigt dung dich thymol 2% vao Iml HsSO, dm ac. du higu: Xuat hin mau do thim, ‘Thudc thir Baljet: © Céng thire: Pha dung dich acid pierie 1% trong EtOH véi dung dich NaOH 10% trong nude theo ty 1é thé tich 1:1, Dau higu: Nho 3 — 4 gigt thudc thir vao Img chat thir, néu cé mau vang cam hoi héng xin la phan img duong tinh. — Thuéc thir Legal: © — Céng thire: Héa tan Img chat thir trong 2 — 3 giot pyridin, thém 1 giot dung dich natri nitroprussiat 0.5% méi pha, sau dé cho tir tir timg giot dung dich KOH 2N. © Du higu: Xuat hign mau do tim. 2.8.2 Dinh tinh glycoside ‘Thi nghiém: Lay 10g bét trai muép ding khé, logi cc chat khong phan cyc bing ether petrol. Tiép tuc chiét bing soxhlet voi dung méi EtOH 50°, Dich loc EtOH 50° duge loai tap bang acetate chi cho dén khi khéng cn trim hign. Sau dé, loai acetate chi dur bing Na,SO, bao héa. C6 can dich loc duge cao glycoside thé. Héa tan cao bing EtOH 95° va ld dung dich nay lim miu thi. = V6i thudc thir Tollen: Xuat hin két tia tring. ~ V6i thude thir Molish: Cé mau 46 carmine.a 3.TACH CHIET, CO LAP VA TINH CHE CAC HOP CHAT 3.1. Thiét bj va hoa chat 3.L1 Thiét bj = Biém chay (mp °C) durge do trén may Electrothermal IA 9000 Series. - Sic ky c6t thutng cing silicagel 60, MERCK, dudmg kinh hat: 0.04 — ~ Sie ky bin méng TLC duge thye hign trén ban silicagel 60 Fasg, MERCK tring sin, = May cé quay chan khong. = May soi UV Hinh 2.2: Se ky cOt thudng Hinh 2.3: Sic ky ban méng (TLC) 3.1.2 Hoa chit = Petroleum ether, Trung Quéc - Diethyl ether, Trung Quéc = Chloroform, Trung Quéc - Ethyl acetate, Trung Quéc38 Acetone, Trung Quéc Methanol, Trung Quéc Nuse cat Ethanol 95° 3.2. Chiét xudt cdc nhom hgp chat Trai musp dang say kh6, xay nhé (4.5 kg), duge chiét véi ethanol 95°, Sau dé, ¢6 Joai dung moi thu duge cao dang sét goi ld cao ting (ea EtOH 95°) (537 g). Cao téng héa tan trong m6t lugng nude vita di sét. dung dich nay duge lic voi petroleum ether (PE), thu lly dich PE va c6 loai dung mdi thu duge eao PE. Pha nude con Iai sau khi lie voi EP duge lic tiép tue vai ethyl acetate (EtOAc), dich EtOAc c6 logi dung mai thu duge cao EtOAc (219 g). Pha nuéc cén Iai sau khi lic voi EtOAc duge lic tiép tuc véi methanol (MeOH), dich MeOH ¢6 dudi dung méi thu due eao MeOH (254 g).39 BOt trai mudp ding CChiét vi ethanol 95° ¢ C6 dudi dung méi Cao EtOH 95” Chiét v6i petroleum ether C6 dudi dung méi + ’ Cao PE Dich née 1 véi ethyl acetate C6 dudi dung moi I l Cao EtOAc Dich nuée Chiét voi methanol C6 dudi dung moi Cao MeOH Djch née So dé 2.1: Quy trinh chiét xu4t cde nhém hgp chit ciia trai muép ding 3.3. Phin lp va tinh ché cdc hgp chat Sau khi thu duge céc cao chiét, ching t6i da sir dung sic ky ban méng dé kiém tra xem trong méi loai cao chiét cé khoang bao nhiéu hgp chit. So sinh véi nhimg hgp chat 43 phin 1p duge tir tdi murdp ding trade day, chiing t6i phat hign trong cao EtOAc 6 nhiéu hgp chat. Vi vay, ching t6i da ding cao EtOAC dé phan lap cac hgp chat sir dung phuong phap sic ky cét thuéng.Cao EtOAc — Sie ky cot thudng = Sie ky bin méng “ 2 2 s z x : 2 : : z 5 5 é s é 3 é é 3 a & & a & & & = = = = z z z conc | Tinh ché MCI mco | | mcs MCIA & MCIB ‘So dé 2.2: Quy trinh phan lap va tinh ché cde hyp chat tir tr4i muép ding Tir cao EtOAc, tién hanh lén eGt thug véi cde thing s6 sau: - Khéi lrong cao: 219 g = Khéi lugng silica gel: 600 g¢ = Duong kinh cét: 9 em = Chiéu cao lép silica gel: 35 cm = Dung méi én dinh c6t: Petroleum ether. ~ Thé tich ldy méi phan doan: 500ml Tang dan d6 phan cyc cia hé dung méi giai ly bing cach ting dan ti 1¢ CHCI; trong hé PE:CHCh. Theo doi c6t bang sie ky ban mong, hign vét bang céch phun dung dich HSO, 10% trong EtOH, ho néng trén bép dign.4 Kiém tra cde phan dogn bing TLC (hé dung méi gidi ly CHCl: MeOH:H,0 = 9:1; 85:15; 8:2; 7:3:0.5; 6:4:1), Gom cde phiin dogn c6 Rr giéng nhau, thu duge tit eé 14 phiin doan chinh. Bang 2.1; Két qua sic ky et thuong Phan dogn ‘HG dung méi Két qua thir TLC 1 Vét kéo dai 2 PE:CHCl; = 7:3 Nhidu vét 3 Vét kéo dai 4 PE:CHCI = 4:6 5 PE:CHCl = 3:7 Nhigu vét 6 PE:CHCI, = 2:8 Vit kéo dai 7 CHCI,; 100% Vét kéo dai 8 CHCIy:MeOH = 95:5 C62 vét aim 9 CHCls:MeOH = 93:7 Vet kéo dai 10 CHCI;:MeOH = 9:1 C62 vét agm i CHCl;:MeOH = 88:12 C61 vét dim 12 CHCIs:MeOH = 85:15 Vét kéo dai 13 CHCl;:MeOH=8:2 Nhigu vét 14 CHCls:MeQH=75:25 Vet kéo dai42 + Tai phan dogn 8 (dung moi giai ly CHCl: MeOH ), chay sic ky bin mong thu duge hai vét dim, Rita bang ethyl acetae va két tinh lai nl higu MC1va MC6. trong chloroform thu duge hai chat . Ki ¢ Tai phn doan 10 (dung méi giai ly CHCl:MeOH = 9:1), chay sic ky lop mong thu durge hai vét dam. Rita bing ethyl acetate, Jan thu duge mét chat tinh khiét. Ki higu MCS.4B KET QUA VA THAO LUAN 1. KET QUA DINH TINH CAC NHOM HOP CHAT TRONG TRAI MUOP BANG Qua khao sit so b6 cdc hop chat trong trai muép ding, két qua chi tiét nhur sau: Bang 3.1: Két qua djnh tinh cic hgp chit hitu co trong trai muép ding a HOP CHAT THUOCTHU | HIEN TUQNG | KETLUAN 1 FLAVONOID, Mg/HCldd | Tia héng nhat + 2 ACID BEO Nbo trey sly DE lai peckhong + 3 ANTHRAGLYCOSIDE KOH 10% Mau dé cam + 4 STEROID Liebermann Xanh ye + Acetate chi Vang nhat ot 5 TANIN Gelatin Tua tring + FeCl Mau den, + Mayer ‘Tua tring + : euemae Dragendost | Pune dich 06 + 7 SAPONIN Tao bot Tao bot bén +H 8 DUONG KHU" Fehling A+B Tia do nau + Thymol 20% | Cho mau do a 9 GLYCOSIDE yA H:SO. dd a Tollen Két tia tring Saal Chi thich: (+): it, (+4): niu, (4+): rét nhidu2. NHAN DANH CAU TRUC CAC CHAT TINH KHIET. 2.1. Hop chat MC 1 2.1.1 Két qua phan tich d@ tinh khiét cia MC1 bang HPLC Két qua phan tich bing HPLC cho thay MC1 1a hén hgp cia hai chat, ky higu MCLA va MCIB. Tién hanh xc dinh cau trac ciia 2 chat bang cic phuong phap phd. Cac hgp chat trong MC1 duge cé lip bing HPLC diéu ché, thu duge 12mg MC1A va 9mg MCIB. Kiém tra TLC cia MCIA, MCIB so véi MCI déu cho mét vét tron 13 ‘mau tim, Re= 0.25 (he giai ly CHCl:MeOH Vay ching téi dur don MCI 1a charantin, Hinh 3.1: Tinh thé MCL_—_‘Hinh 3.2: TLC cia MC1, MCIA va MCIB 2.1.2. Nhjin danh cu trite héa hgc cia MC1A va MCIB 2.1.2.1. MCIB 4 Cie dic tinh cia MCIB — Dang bét, mau tring, két tinh trong MeOH. — Biém néng chay: mp = 281 — 283°C. = Sie ky bin méng (TLC) hign mau bing dung dich H;SO, 10% trong EtOH:45 © Giai ly bing hé CHCl;:MeOH = 9:1 cho vét tron mau tim 6 Rr= 0.25, © Giai ly bang hé CHCls:MeOH = 85:15 cho vét tron mau tim ¢6 R= 0.37. Hé giai ly CHCIs:MeOH = 9:1 Hé gidi ly CHCl: MeOH = 85:15 Hinh 3.3: TLC cia MCLB 6 ede hé gidi ly khée nhau & Nhgn danh céu tric MCIB Phé “C-NMR ( phu lye 1.1) cho biét MCIB cé 35C. Trong 46 c6 2 mii & ving néi 446i 6 6 = 140.11 va 121.98 ppm chimg 16 trong cdu trie ciia MCIB €6 1 n6i déi. Mii 140.11 bién mat trén phd DEPT (phy lye 1.2) chitng t6 day 1 C uit cdp c6 néi doi. Dong ‘thoi trén ph DEPT (phy Iye 1.2 va 1.2a) cn cho thay trong phan tir MCIB 6 6 nhém— CHs, 12 nh6m -CH2- (trong 46 ¢6 1 nhém ~CH,OH eiia phin dudng 6 61.716 ppm), 14 nhém >CH-, 3C tir edp. Baing 3.2: Dir ligu pho “C-NMR va DEPT cia MCIB VitriC | “C(@ppm) | DEPT90 | DEPT135 Két luan 1 36.53 |Bién mit | Mai am ~CH- 2 29.42 |Bién mat — [Mai am —CH- 3 77.01 |Miiduong |Mai dong >CH-O4 38.52 Mai am ~CH- 5 140.11 Bién mat >C=C 6 121.98 Mai duong CH= 7 3.74 Mai am CH, 8 31.70 Mai duong ~CH< 9 49.59 Mai duong ~CH< 10 36.54 Bién mat >e< ul 20.87 Mai m CH 12 39.58 Mai am -CH- 1B 42.15 |Biénmat | Bién mat >C< 4 56.57 |Mai duong | Mai duong ~CH< 15 24.09 |Bién mat — [Mai am ~CH:~ 16 28.04 |Bién mit | Mai am CHy 7 35.88 |Mai duong {Mai duong ~CH< 18 11.62 Bién mat | Mai duong ~CH; 19 18.77 Bién mat | Mii duong —CH; 20 35.96 |Mai duong | Mai duong ~CH< 21 18.54 |Biénmét | Mai duong CH; 2 33.77 |Bién mat — | Mai am —CH23 25.90 |Biénmét | Mai am -CH- 24 45.69 Mai duong ~CH< 25 28.98 Mai duong ~CH< 26 19.54 Mai duong -CH, 2 19.08 Mai dong CH, 28 22.88 Bién mit = | Mai am ea 29 11.72 Bién mit | Mai duong ~CH; v 100.93 |Muidwong |Maiduong | O-CH-O r 73.36 |Midwong |Maiduong | >CH-OH x 76.22 |Maidwong |Maiduong | >CH-OH o 70.00 |Maiduong |Maiduong | >CH-OH s 76.75 |Mai dong | Mai duong >CH-O oe 61.72 |Biénmét | Mai am ‘CH;-OH Mai dic trung cho vj tri 1° eda dug 6 100.98 ppm va ede mili trong ving 61.72 dén 77.00 ppm ciia ndi C-O trén C-NMR (phy luc 1.1 va 1.1b) chimg to MCIB Li 1 glycoside cé 1 don vj dong. Dau higu dinh tinh trén TLC cho vét c6 mau tim héng khi phun dung dich H,SO, va ho néng cho phép gia thiét phan aglycon la 1 sterol. Phin dudng c6 cde mili & Spm : 100.98; 76.36; 76.75; 73.36; 70.00 va 61.72 trén C-NMR (phy luc 1.1 va 1.1b) chig t6 day 14 dudng glucose. Hing sé tuong tie spin spin cua proton 1° va 2° trén 'H-NMR J = 8 Hz chimg t6 2 proton nay déu & vj tri truc, vi ‘vay lién két gitra phin dumg va aglycon theo kiéu B (phy luc 1.3 va 1.3b).Vay MCIB cé tit ca 35 nguyén tir carbon, 6 nguyén tit oxy. Nhu vay céng thite phan 48 tir 1a CosHpOg, phan tr khdi la 576 dvC. Tir nhiing sé ligu trén va so sdnh véi tai ligu °*"!, xc dinh cdu tric héa hoc cia MCB la B-sitosterol-3-0--glucopyranoside. Hinh 3.4: Cong thire edu tgo MC1B Bang 3.3: Dir ligu phé 'H, “C-NMR, DEPT cia MCIB "c Vi tri “H-NMR 8 Loai tri ppm a ogi C/H | (sé. H; dang mii; J = Hz) carbon éppm 1 1,80; 0.99 (2H, m) 36.53 | -CH 2 1,80; 1.47 (2H, m) 29.42 | -CH> 3 3.43 (1H, m) 77.00 | -CH-O 2.12 (1H, t, = 11.5) 4 2.37 (IH, dd,J=3.0va | 38.52 | ~CHs~ 13.0)49 5 140.11 | >C=C 6 5.32 (IH, s) 121.98 | -CH= 7 1.90; 1.51 2H, m) 31.74 | CH 8 1.39 (1H, m) 31.70 | -CH< 9 0.91 (1H, m) 49.59 | -CH< 10 3654 | >C< uw 1.41; 1.50 2H, m) 2087 | -CH- 2 1.17; 1.97 GH, m) 39.58 | -CH- 13 41s | >c< 4 0.96 (1H, m) 36.57 | -CH< 15 1.05; 1.52 2H, m) 24.09 | -CH- 16 1.25; 1.80 2H, m) 28.04 | -CH.- 7 1.10 (1H, d, J= 10) 3588 | -CH< 18 0.67 (3H, d, J= 10) 11.62 | -CH; 19 0.93 (3H, m) 18.77 | -CHs 20 1.38 (1H, m) 35.96 | —CH< ai 0.91 GH, m) 18.54 | —CHs 22 1.0; 1.32 (2H, m) 33.77 | -CH 23 1.18 (2H, m) 25.90 | -CH-50 24 0.93 (IH, m) 45.69 | -CH< 25 1.61 (1H, m) 28.98 | -CH< 26 0.81 GH, m) 19.54 | -CHy 27 0.80 (3H, m) 1908 | -CH; 28 1.20; 1.22 (2H, m) 2288 | -CH- 29 0.82 (3H, m) 1172 | -cH, Vv] 432CH,4,3=80) | 10093 | O-CH-o 2.90 (1H, m) Y 7336 |>CH-OH OH = 4.84 3.12 (1H, m) s 7622 |>CH-OH g -OHy= 4.85 é & 3 3.02 (1H, m) So ly 70.00. |>CH-OH ~OHy = 4.82 x 3.07 (IH, m) 76.18 | >CH-O 3.40; 3.64 (2H, m) ° 61.71 |-CHyOH OH = 4.38 2.1.2.2, MCIA & Clie dae tinh cia MCIA — Dang bét, mau tring, két tinh trong MeOH. — Diém néng chay: mp = 271 — 276°C.sl — Sac ky bin méng (TLC) hign mau bing dung dich H2SO, 10% trong EtOH: © Giai ly bling hé CHCls:MeOH = 9:1 cho vét tron mau tim 6 R= 0.25. H6 gidi ly CHCl: MeOH He giai ly CHCI,: MeOH = 85:15 Hinh 3.5: TLC ctia MCIA 6 ede he giai ly khde nhau & Nhén danh céu trite MCIA Theo tai ligu [5,32] thi charantin 1a hén hop cia hai steroid ki 3-O-[B-D- glucopyranosy!] stigmasta-5,25(27)-diene va f-sitosterol-3-O-B-glucoside. Vi thé, theo chimg minh trén thi MCIA la 3-O-[B-D-glucopyranosyl]-stigmasta- 5,25(27)-diene. Vay MCA cé tat cd 35 nguyén tir carbon, 6 nguyén tit oxy, cd hai néi ddi 6 vi tri C-5 va C-27. Nhu vay cng thite phan tir li CssHs,Og, phan tir khdi ki 574 dvC.52 Hinh 3.6: Cong thire cfu tao MC1A 2.2. Hop chat MC 6 & Cae dic tinh cia MC 6 ~ Khéi lugng thu duge m = 20 mg ~ Dang b6t, hoi ving = Sic ky ban méng (TLC) hign mau xam duéi dén tir ngoai va mau vang trong hoi Jod, khéng hién mau véi dung dich H2SO, 10% trong EtOH c6 R= 0.24 (CHCl;:MeOH = 85:15) = Diém chay mp= 250°C ( chat bat dau chuyén sang mau den) . - Tan it trong Methanol, tan nhiéu trong Dimetyl sulfoxide (DMSO)53 Hinh 3.7: Miu chat MC6 Hinh 3.8: TLC hgp chat MC6 4 Nhdn danh céu tric cia MC 6 ~~ Phd hdng ngoai IR (KBr, Vmax, cm") ( phy lye 2.1) cho: + Tin higu hép thu cita nhém N-H 6 3112. + Tin higu thé hign syr dao dng clia C-H 6 2986 - 2821. + Tin higu dac trung cho 2 lign két d6i ké nhau c6 UV 6 1990. + Tin higu die trung cho 2 lién két C=O 6 1717. + Tin higu die trung cho lign két C=C 6 1669. ~ Pho “C-NMR (DMSO, dppm) (phy luc 2.2) két hgp véi phé DEPT (phy luc 2.3) (bang 2.5) cho biét 6: + 2 tin higu duong la carbon olefin (-CH=) 6 100.16 va 142.09 ppm. + 2 tin higu la carbon tir cp cia nhom cacbonyl (>C=O) & 151.44 va 164.26 ppm.54 Bang 3.4: Dir ligu pho "C-NMR va DEPT ciia chat MC 6 “C-NMR, Vi tri CH DEPT 90 DEPT 135 Nhéom @ ppm) 5 100.16 | Tinhiguduong | Mii duong ~CH< 6 142.09 | Tinhiguduong | Mai duong ~CH< 2 151.44 Bién mat Bién >C=0 4 164.26 Bién mat Bién >C=0 - Phé 'H- NMR (DMSO, dppm) (phy luc 2.4 va 2.4a) cho cfc mii tin higu cua: + 1 tin higu proton metin (-CH<) 6 5.43 - 5.45 (d, J=7.5). +1 tin higu proton metin (-CH< 6 7.36 - 7.38 (d, J= 7.5). +2 tin higu proton & 10.89 (2H, brs). ~ Phé HMBC (phy luc 2.6) cho thay su tuong tic cia proton véi cdc vi tri carbon duge thé hign trong bang 3.5. Nhu vay, MC 6 6 cng thie phin tir 1A CJH,O;No, phan tir kh6i 1a 112 dvC; 46 bat bao héa 1a 4, trong dé c6: 1 ndi C=C chiém 1 dG bt bio hoa, 2 ndi C=O chiém 2 46 bat bio héa va 1 d6 bat bao hoa cita vong. Bang 3.5: Dit ligu phd 'H-NMR, “C-NMR, DEPT vi HMBC ciia chit MC 6 Viti | C-NMR NhémC | 'H-NMR (6 ppm,J=Hz) | HMBC (HC) C/H | (ppm) 1 >N-H 10.89 (1H, brs) 2 | 15144 | >c-0 3 oN-H 10.89 (1H, brs)55 4 | 16226 | >c-0 5 | 100.16 | -CH= | 5.43-5.45(1H,d,J=7.5) | Hs CoC, 6 | 142.09 | -CH= | 7.36-7.38(1H,d,J=7.5) | He Cs, C, Cy Bang 3.6: So sinh dir ligu pho eéia MC 6 v6i tai ligu [20] MC6 Uracil mp (°C) 246 - 247°C 250°C Vitti | ay \ 1 \ oe C-NMR ‘H-NMR ‘C-NMR H-NMR 5 100.16 | 5.43-5.45(d,J=7.5) | 101.78 | 5.63 (d,1=7.6) 6 142.09 | 7.36-7.38(d,J=7.5) | 143.46 | 7.41 (4, 1=7.6) 2 151.44 153.57 4 164.26 167.36 Tom lai, dya vao cdc két qua phd 'H-NMR, ''C-NMR, HSQC, HMBC, cic dic trung vat ly va so sinh véi tai liu di céng bé [20], ching t6i nhin danh chat MC6 1a Pyrimidine-2,4-(1H, 3H)-dione (hay Uracil). H Q; N, O a 7 | HN, 6 A Hinh 3.9: Céng thire cau tgo cia MC6 va tong quan pho HMBC56 2.3. Hop chit MCS (Chua nhin danh duge cau tric ca MCS) + Che dite tinh cia MCS ~ Khéi lung thu duge m=331 mg ~ Dang bt, mau tring = Sac ky ban méng (TLC) kh6ng hién mau duéi dén tir ngoai, hién mau bing dung dich HzSO, 10% trong EtOH ¢6 Ry= 0.36 (CHCl: MeOH = 85:15) cho vét tron mau xém. - Diém chay mp= 194.6°C - Tan trong MeOH Hinh 3.10: Mau chit MCS Hinh 3.11: TLC cia MCS,37 KET LUAN VA KIEN NGHI 1. CAC KET QUA DAT DUQC Buc dau khao sat thanh phan héa hoc cia trai muép dang loai trai nhé, gai nhon, rit ding duge trong 6 Phi Yén ching t6i dat duge cdc két qua sau: 1) _ Khao sat so b@ than phan héa hge nhan thay: trai mudp ding co saponin, steroid, alkaloid, glycoside, tanin, anthraglycoside, durong Khir, acid béo. 2) Tirdjch chiét edn 95°, ching t6i tién hanh tach riéng 3 loai cao dya trén do phan ‘cuc cia cdc hop chit ¢é trong trdi: cao PE, cao EtOAc va cao MeOH. 3) Tircao EtOAc, ching t6i da phan lap duge ba chat tinh khiét va 43 nhan danh durge cdu tric cita ching. Gém hai glycoside steroid 1a 3-O-[B-D-glucopyranosyl]-stigmasta- 5,25(27)-diene (MC1A) va B-sitosterol-3-O-B-glucopyranoside (MC1B) va mét la pyrimidine-2,4-(1H, 3H)-dione (hay Uracil) (MC6). Tt ca cde chat duge nhén danh céu triie bing cde phuong phap phé hign dai nhu IR, phé 13C-NMR, phd 1H-NMR va so sanh véi tai ligu da cong bé. Bang 4.1: Cac chit da phan lip duge STT DANH PHAP CONG THUC CAU TAO 3-O-{-D-glucopyranosy!]- stigmasta-5,25(27)-diene ke B-sitosterol-3-O- B-glucopyranoside 3. | pyrimidine-2,4-(1H, 3H)-dione58 2. KIEN NGHI ~ Tinh ché MCS va nhan danh cau tric cua hgp chat nay. = Tiép tuc khao sat cao EtOAc dé phan lap cac hop chat can Iai trong cao. ~ Khao sit thanh phn héa hoe cde cao PE, MeOH. - Thir hogt tinh ciia cae hgp chat da phan lp duge dé img dung trong chifa bénh.(10) oy (2) U3} [4] U5] 16} 59 TAI LIEU THAM KHAO B6 Y Té, Due dién Viét Nam, Nha xuat bin Y hoc, 2002 Bai Chi Hiéu, Duge iy 17 ligu thudc nam, 1999, trang 212. at Li, Nhing Cay Thudc va Vi Thudc Viét Nam, Nha xuat ban Khoa Hoc va Ky Thuat, Ha N6i, 1982, trang 736-737. Hai An, Mén dn vj thuéc, Nha xuat ban Thuan Héa, 2002, trang 161-162. Ng6 Van Thu, Bai gidng dugc ligu tap I, Nha xuat ban BG Y Té va Gido duc Dao ta0, 1998, Nguyén Khac Quynh Cit, Bai gidng chiét xudt dugc liéu, Khoa Duge, Truong Dai gc Y Duge Tp.HCM, 2000. PGS.TS. Nguyén Kim Phi Phung, Cac phwong phdp tach chiét hop chat hitu co, Nha xuat ban Dai hoc Quéc gia TP. Hé Chi Minh, 2007. PGS.TS. 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[34] Mr. Jesada Pitiphanpong, Extraction of charantin from the fruits of Momordica charantia using high pressure solvent, A Thesis for the Degree of Master of Engineering in Chemical Engineering, Chulalongkorn University, Thailand,2004.Phy lye 1.1: Phy Tye Ila: Phy Iye 1.1b: Phy Tye 1.2: Phy Tye 12a: Phu lye 1.3: Phy luc 1.3a: Phy Iye 1.3b: Phy luc 1.3e: Phy lye 2.1: Phy lye 2.2: Phu Tye 2.3: Phy Iyc 2.4: Phy Iue 2.4a: Phu luc 2.5: Phy luc 2.6: 62 PHY LUC DANH MUC CAC PHU LUC Phé "C-NMR cia MCIB Phé "°C-NMR cia MCIB Pho "'C-NMR cia MCIB Phé '°C-NMR va DEPT cla MCIB Phé "C-NMR va DEPT cia MCLB Pho 'H-NMR cia MCIB Phé ‘H-NMR cia MCIB Pho 'H-NMR cla MCIB Pho 'H-NMR cia MCIB Phd IR cia MC6 Phé "C-NMR cia MC6 Phé ‘C-NMR va DEPT cia MC6 Phé 'H-NMR cla MC6 Pho H-NMR cia MC6 Phé HSQC cia MC6 Phé HMBC ctia MC6 Trang PLI PL2 PL3 PL4 PLS PL6 PLT PLS PL9 PLO PLL PLI2 PLI3 PLi4 PLIS PLI6oy 0308S aotearoa oz wid 63 Phy lye 1.1: Phd “C-NMR cia MCIB GaDETO-dOPW3ETIGO-aTOWst o se of sz oz st Phy lye 11a: Pho “C-NMR cia MCIB aD€ T9-CO®W3ETIGO-ET OW09 $9 OL $4 08 Se 06 $6 oot sor ort sit ozt set oet set ort s Phy Iye 1.1b: 65 Phé “C-NMR cia MCIB GaD€ 19-GOeW3ETOGD-a TOW66 DEPT9O MC1B-CDC13&MeOD-C13CPD &DEPT DEPT135 ne eS cisceD Phy Iyc 1.2: Phé “C-NMR va DEPT cia MCIB67 DEPT90 MC1B-CDC13&MeOD-C13CPD &DEPT c13cPp Phy lye 1.2a: Phé “C-NMR va DEPT cia MCIB68 Phy lye 1.3: Phé 'H-NMR cia MCIB HT-GO2W3€TOGD-aTOW“SSAA MMA nt 1 ey pn 13a: Phé "H-NMR cia MCIB70 MC1B-CDC13&MeOD-1H Baia a aie ara tei Gaertn re aeaiive era GHiteeC 6 Hae Sueeeta a feeetE 3 HL a WOR Mr AA Phy luc 1.3b: Pho ‘H-NMR cia MC1Bn V MC1B-CDC136MeOD-1H Phy luc 1,3¢: Phd "H-NMR ciia MCIBR fuse mnt ‘ance mon oF es ats z 3 Tea oa ‘008 trina 1738.2. eee os ose Phy luc 2.1: Phé IR cia MC6oet 002 ost oy 09 oot oz B Phy lye 2.2: Phé “C-NMR cita MC6 Ga9€T9-OSWd-99H14 MC6-DMSO-C13CPD &DEPT DEPT9O Wo gee sO Ww TT Teo TO We Td) GD NO Teo 50 We www OO Pm DEPT135 c13ceD Phy lye 2.3: Phé "C-NMR va DEPT cia MC6ou ot 15 Phy lyc 2.4: Phé "H-NMR cia MC6 HT-OSWd-90Hes 09 9 bs 99 88 OL 5 wd Phy lye 2.4a: 16 HT-OSWO-90n11 MC6-DMSO-HSQC - | 807570 65 6.0 55 5.0 4.5 4.0 5.5 3.0 2.5 2.0 ppm Phy luc 2.5: Phd HSQC cita MC68 ‘MC6-DMSO-HMBC Phy lye 2.6: Phé HMBC cita MC6