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MC LC
Trang LI CM N MC LC DANH MC CC K HIU, CH VIT TT DANH MC CC BNG DANH MC CC S DANH MC CC HNH i v vi vi vii

M U TNG QUAN
1. I CNG V CY MP NG 1.1 M t cy 1.2 Phn b v sinh thi 1.3 Y hc dn gian ca cy mp ng 1.3.1 R 1.3.2 Thn 1.3.3 L 1.3.4 Hoa 1.3.5 Tri 1.3.6 Ht 2. CC NGHIN CU V MP NG 2.1 Cc cng trnh nghin cu trong nc 2.1.1 Thnh phn ha hc 2.1.2 Tc dng dc l 5 5 5 6 6 6 7 7 7 7 2 2 4

2 2.2 Cc cng trnh nghin cu trn th gii 2.2.1 Thnh phn ha hc 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. Mt s triterpene trong cy mp ng 2.2.2.1. Cc triterpene glycoside c c lp t ht mp ng 2.2.2.2 Cc triterpene glycoside c c lp t tri mp ng 2.2.2.3 Cc triterpene glycoside c c lp t l v dy mp ng 2.2.3. Tc dng dc l 8 8 8 9 9 9 9 10 10 10 12 16 19

THC NGHIM
1. NGUYNLIU 2. NH TNH CC HP CHT HU C TRONG TRI MP NG 2.1 Kho st s hin din ca cc hp cht alkaloid 2.1.1 Thuc th alkaloid 2.1.2 nh tnh alkaloid 2..2. Kho st s hin din ca cc hp cht flavonoid 2.2.1 Thuc th flavonoid 2.2.2 nh tnh flavonoid 2.3. Kho st s hin din ca cc hp cht anthraglycoside 2.3.1 Thuc th anthraglycoside 2.3.2 nh tnh anthraglycoside 2.4. Kho st s hin din ca cc hp cht sterol 2.4.1 Thuc th sterol 2.4.2 nh tnh sterol 23 23 24 24 24 25 25 25 25 25 25 26 26 26

3 2.5 Kho st s hin din ca cc hp cht saponin 2.5.1 Thuc th saponin 2.5.2 nh tnh saponin 2.6. Kho st s hin din ca cc hp cht ng kh 2.7. Kho st s hin din ca cc hp cht tanin 2.7.1 Thuc th tanin 2.7.2 nh tnh tanin 2.8. Kho st s hin din ca cc hp cht glycoside 2.8.1 Thuc th glycoside 2.8.2 nh tnh glycoside 3. TCH CHIT, C LP V TINH CH CC HP CHT 3.1. Thit b v ha cht 3.1.1. Thit b 3.1.2. Ha cht 3.2. Chit xut cc nhm hp cht 3.3. Phn lp v tinh ch cc hp cht 26 26 27 28 28 28 28 28 28 29 30 30 30 30 31 32

KT QU & THO LUN


1. KT QU NH TNH CC HP CHT HU C TRONG TRI MP NG
2. NHN DANH CU TRC CC CHT TINH KHIT

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2.1. Cht MC1 2.1.1 Kt qu phn tch tinh khit ca MC1 bng HPLC 2.1.2 Nhn danh cu trc ha hc ca MC1A v MC1B 2.1.2.1. Mu cht MC1A 2.1.2.2 . Mu cht MC1B 2.2. Hp cht MC6 2.3. Hp cht MC5

37 37 37 37 41 47 51

KT LUN

4 1. CC KT QU NGHIN CU T C 2. KIN NGH TI LIU THAM KHO 52 52 viii xi

PH LC
DANH MC CC PH LC

DANH MC CC K HIU, CH VIT TT


brs COSY d dd DEPT DMSO PE EtOAc EtOH HMBC HPLC HSQC IR J m MeOH mp MS : Broad singlet (NMR) : Correlation Spectroscopy : Doublet (NMR) : m c : Doublet of doublet (NMR) : Distortionless Enhancement by Polarization Transfer : Dimethyl sulfoxid : Petroleum ether : Ethyl acetate : Ethanol : Heteronuclear Multiple Bond Correlation : High Performance Liquid Chromatography : Heteronuclear Single Quantum Correlation : Infrared : Coupling constant : Multiplet (NMR) : Methanol : Melting point : Mass Spectroscopy

5 NMR ppm Rf s t TLC UV -Glc : Nuclear Magnetic Resonance : Parts per million : Retention factor : Singlet (NMR) : Triplet (NMR) : Thin Layer Chromatography : Ultra Violet : -glucosidase : Chemical shift

DANH MC CC BNG
Trang Bng 2.1: Kt qu sc k ct thng Bng 3.1: Kt qu nh tnh cc hp cht hu c trong tri kh qua Bng 3.2: D liu ph 1H, 13C-NMR; DEPT v HMBC ca MC1A Bng 3.3 : D liu ph 13C-NMR v DEPT ca MC1B Bng 3.4: D liu ph 1H, 13C-NMR, DEPT v HMBC ca MC1B Bng 3.5: D liu ph 13C-NMR v DEPT ca MC6 Bng 3.6: D liu ph 1H, 13C-NMR, DEPT v HMBC ca MC6 Bng 3.7: So snh d liu ph MC6 vi ti liu 34 36 39 42 45 49 49 50

DANH MC CC S
S 2.1: Quy trnh chit xut cc nhm hp cht ca tri mp ng S 2.2: Quy trnh phn lp v tinh ch cc hp cht t tri mp ng 32 33

DANH MC CC HNH
Hnh 1.1: Cy kh qua Hnh 1.2: Dy v l kh qua Hnh 1.3: Tri kh qua Hnh 1.4: Ht kh qua Hnh 1. 5: Gin kh qua (Ph Yn) Hnh 1.6: Var. charantia L. Hnh 1.7: Var. abbreviata Ser. Hnh 2.1: Qu trnh sy nguyn liu Hnh 2.2: Sc k ct Hnh 2.3: Sc k lp mng Hnh 3.1: Tinh th MC1 Hnh 3.2: TLC ca MC1, MC1A v MC1B Hnh 3.3: TLC ca MC1A cc h gii ly khc nhau Hnh 3.4: Cng thc cu to ca MC1A Hnh 3.5: TLC ca MC1B cc h gii ly khc nhau Hnh 3.6: Cng thc cu to ca MC1B Hnh 3.7: Mu cht MC6 Hnh 3.8: TLC ca MC6 Hnh 3.9: Cng thc cu to ca MC6 Hnh 3.10: Mu cht MC5 Hnh 3.11: TLC ca MC5 3 3 3 3 3 5 5 23 30 30 37 37 38 39 42 45 48 48 51 51 51

LI C M N
Em xin gi li cm n su sc n: PGS.TS Nguyn Ngc Hnh ThS. Phng Vn Trung Ngi thy truyn t cho em nhng kin thc chuyn mn v kinh nghim nghin cu, tn tnh hng dn, ch bo em trong sut thi gian em nghin cu ti phng Ha hp cht thin nhin- Vin Cng ngh Ha hc. Khoa Cng ngh Ha- Thc phm cng Thy, C v cc bn sinh vin trng i hc Lc Hng quan tm ng vin, to mi iu kin cho em trong qu trnh hc tp v nghin cu. ThS. Phan Nht Minh, cc anh ch trong phng Ha hp cht thin nhin, cng cc anh ch nghin cu vin trng i hc Cn Th, cc bn sinh vin n lm ti nghin cu tn tnh hng dn, gp , cung cp ti liu, to iu kin gip em lm tt cng vic ca mnh. Con knh gi n Gia nh lng bit n su sc. Xin chn thnh cm n! LC HNG, thng 11 nm 2009 L Th Minh Nguyt

LI M U
Mp ng hay cn gi l kh qua (Momordica charantia L.), thuc h Bu b Cucurbitaceae) c trng nhiu nc trn th gii, c bit l nhng vng c kh hu nhit i. Vit Nam, cy c trng hu ht cc tnh t ng bng n trung du v min ni. Mp ng c v ng, tnh hn nn trong dn gian thng dng tr cc bnh mn nht, gii nhit, tr phin, thanh tm, sng mt, gim au Khoa hc ngy nay chng minh dch chit tri mp ng c kh nng c ch khi u, c tc dng h tr men gan v iu tr bnh i tho ng Hin nay bnh i tho ng l mt trong cc bnh mn tnh, gy t vong cao, ng hng th ba trn th gii sau bnh tim mch v ung th. Vit Nam, t l mc bnh ngy mt gia tng, i hi cp thit phi tm ra cc loi thuc hiu qu, c tr. Mp ng c tc dng c bit nh vy nhng hin nay vn cn rt t cng trnh nghin cu v hp cht tr bnh i tho ng. V th, chng ti thc hin nghin cu ti : Gp phn kho st thnh phn ha hc ca tri mp ng.

TNG QUAN
1. I CNG V CY MP NG
1.1. M t cy
Tn khoa hc: Momordica charantia L. H: Bu b (Cucurbitaceae) Tn nc ngoi: Bitter melon, bitter gourd (Anh), bitter apple, wild cucumber, bitter cucumber, ampalaya (Philipines), balsam pear (M), karela (n ) Tn Vit Nam: Mp ng, kh qua, lng qua, cm l chi[3] Ngoi ra n cn c nhiu tn khc nh: Mp m, chua hao, da mt, hng c nng, hng dng, b t, li qua. M t cy mp ng: [3,21] Cy mp ng thuc loi dy leo, c i sng khong mt nm. ng knh dy khong 5-10mm, dy b di 5 7m, thn mu xanh nht c gc cnh, leo c nh c nhiu tua cun, ngn c lng t. L n, nhm, mc so le, di 5-10cm, rng 4-8cm, phin l mng chia lm 5 7 thy hnh trng, mp c rng ca u, mt di l mu xanh nht hn mt trn l, gn l ni r mt di, phin l c lng ngn. Hoa mc n c k l, hoa c v hoa ci cng gc, c cung di. Hoa c c i v ng rt ngn, trng gm nm cnh mng hnh bu dc, nhy 5 ri nhau. Hoa ci c i v trng hoa ging hoa c. Trng hoa mu vng nht, ng knh khong 2cm . Tri hnh thoi, di 8 15cm, gc v u thun nhn. Mt v c nhiu u li to nh khng u. Tri khi cha chn c mu xanh hoc xanh vng nht, khi chn c mu vng hng. V th Trung Quc, mp ng cn c tn l hng dng, hng c nng. Khi chn, tri nt dn ra t u, tch ra lm ba phn l chm o ht mu bn trong .
[16]

10 Ht dt, di 13 15mm, rng 7 8mm, hnh rng nga, tht t ngt hai u. V ht cng, quanh ht c mng mu nh mng ht gc.

Hnh 1.1: Cy mp ng

Hnh 1.2: Dy v l mp ng

Hnh 1.3:Tri mp ng

Hnh 1.4: Ht mp ng

Hnh 1.5: Gin mp ng (Ph Yn)

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1.2. Phn b v sinh thi


Mp ng c phn b vng nhit i v cn nhit i khp cc chu lc. T thi xa xa, mp ng c trng ln u tin ng n v Nam Trung Quc, c s dng nh l loi rau n qu giu cht st v vitamin C. Sau cy c du nhp sang chu Phi v chu M Latinh. Qun th mp ng trng tr nn rt phong ph vi cc ging cy a dng c to ra trong qu trnh chn ging v lai to.[29] Vit Nam, mp ng c trng hu ht cc tnh t Bc Trung Nam, t ng bng n trung du v min ni. mt s vng ni cao v lnh nh Sa Pa (Lo Cai), Ph Bng (H Giang)khng thy c mp ng.[15] Trn th gii, mp ng cng c mt hu ht cc nc nhit i nh n , Nam , ng Nam , Trung Quc, chu Phi v vng Caribbean.[29] Cy mp ng c bin sinh thi tng i rng, nhit thch hp cho cy sinh trng t 20 n 35oC, lng ma hng nm t 1500mm n 2500mm, cao n 1000m. Cy chu ng c nhiu iu kin t khc nhau nhng pht trin tt nht trn t thot thy tt, giu cht hu c. Mp ng c th trng quanh nm. Cy sinh trng nhanh trong ma ma m, ra hoa qu sau 7 8 tun gieo trng. Hoa th phn ch yu nh cn trng. Sau khi tri gi, cy s tn li v kt thc vng i sau 4 5 thng tn ti. Hin nay, cy vn cn tn ti hai qun th: Mc hoang v c trng trt. Loi trng trt rt phong ph v ging nhng u c xp chung vo chi mp ng (Momordica charantia L.). Tuy nhin, cn c vo kch thc, hnh dng, mu sc ca qu m chia mp ng thnh hai chng loi: Momordica charantia L. var. charantia L., tri to (ng knh > 5cm), mu xanh nht, gai t, t ng. Momordica charantia L. var. abbreviata Ser., tri nh (ng knh < 5cm), mu xanh m, gai nhn, v rt ng.

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Hnh 1.6: Var. charantia L.

Hnh 1.7: Var. abbreviata Ser.

1.3. Y hc dn gian ca cy mp ng
Hu ht cc b phn ca cy nh r, thn, l, hoa, tri, ht u c th dng lm thuc cha bnh. [4] 1.3.1 R R ti, sc nc ung, mi ngy mt thang, t 1000ml nc vi 60g r mp ng ti, sc cn 400ml, chia lm 2 3 ln ung, c th p dng cho mi dng bnh. R mp ng dng tr l, nht l amip. Ti n , dch r (cng nh l, tri) mp ng c dng tr bnh tiu ng, do c tc dng lm gim ng huyt. R mp ng cn c th tr bnh gan. [2] 1.3.2 Thn Dy mp ng c dng lm thuc cha vim xoang, chy nc mi c mi hi hoc bnh gan lm vng da. [13] 1.3.3 L L c v ng, tnh mt. n l non tr bnh nng bc trong mnh. Gi l vt nc, thm cht mui, ung tr bnh nng m man hoc tr mn nht, rm sy. Ngoi ra, l cn c th tr c rn cn, lm thuc nhun trng, h st. [2, 4]

13 1.3.4 Hoa Hoa mp ng c dng cha au d dy, l cp tnh , au mt. [4,15] 1.3.5 Tri Tri cn xanh c v ng, khi chn th t ng hn. Tri mp ng c tnh hn (mt), khng c. Tri xanh c tnh gii nhit, lm tiu m, nhun trng, b thn, nui can huyt, bt mt mi, gim stress, xoa du thn kinh, gii c, li tiu, lm bt au khp. Khi chn mp ng c tnh b thn, kin t, dng huyt, dit giun (sn, li), ng thi c tc dng lm sng mt, b tim, b mu, mt gan, rt thch hp vi nhng ngi au gan, au l lch.[22] Trung Quc, tri mp ng cn dng tr t qu tim, bnh st, kh ming, vim hng. n , dch tri mp ng c dng tr rn cn. Ngi ta cn dng bt tri mp ng hn cc vt thng (lm ko da non), tr vt lot c tnh. Thi Lan, dch tri c dng tr bnh v gan, l lch.[2] Vi tnh dit khun v chng oxi ha, tri mp ng lm da mn mng, tr mn trng c hay bnh vy nn, v ngay c vi vt thng do cn trng cn, nhim trng da. Mt khc, tri mp ng cn cung cp ngun nng lng di do v tng kh nng chu ng cho c th.[22] Ngoi cc cng dng trn, tri mp ng cn c dng tr nhiu bnh nh: Tr ho, st, kit l, d dy, au tc, i dt, ph thng do gan, mt au nhc, gii nhit, hi hp, bun phin, tm cho tr em tr rm sy, lm h ng huyt bnh nhn i tho ng type 2 (khng ph thuc insulin). [17] 1.3.6 Ht Ht c cht bo, v ng, hi ngt, tnh m, thanh nhit, gii c, gii cm, tr ho, li tiu. Ht cn cha rn cn, cha nht c sng ty, vt thng nhim trng, h st au hng v chng th thai.[13] Ti nhiu nc khc, ht mp ng c dng tr bnh i tho ng.[18, 22]

14 Ngoi ra, theo mt s nghin cu gn y cho bit cc hot cht trong ht mp ng cn c tc dng chng ung th, lm h huyt p, khng virus HIV[17]

2. CC NGHIN CU V MP NG
2.1 Cc cng trnh nghin cu trong nc
2.1.1 Thnh phn ha hc Cc tc gi Phm Vn Thanh, Phm Kim Mn, on Th Nhu, Nguyn Thng Dong, V Kim Thu, Nguyn Kim Phng v L Minh Phng ca Vin Dc liu thng k v kho st s b cc nhm hot cht chnh ca cy mp ng. Tuy nhin, cc tc gi ny cha c lp c cc hot cht c hot tnh di dng cht tinh khit cng nh cha xc nh cu trc ca cc hot cht ny, m ch nh lng theo cht G6, mt aglycon ca nhm glycoside .[14] Cc tc gi Nguyn Thanh Hng, Nguyn Ngc Hnh, Phng Vn Trung Vin cng ngh Ha hc c lp v nhn danh c hai hp cht t ht: Momordicoside A v Momordicoside B v bn hp cht t tri: Momordicoside K, Momordicoside L, 3-O[-D-glucopyranosyl]-stigmasta-5,25(27)-diene v 23-O--D-allopyranosyl 5,19epoxycucurbita-6,24-dien-3,22,23-triol 3-O--D-allopyranoside. [10] 2.1.2 Tc dng dc l Y hc c truyn v dn gian Vit Nam c nhiu kinh nghim cha bnh t mp ng, nhng ch dng nguyn liu th ban u hoc cc dng nc p, nc sc m thi. Ngy nay, trn th trng xut hin nhiu sn phm tri mp ng nhng a s dng thc phm chc nng nh tr ha tan, tr ti lc. in hnh l sn phm tr mp ng ca Vin Dc liu hay tr ti lc mp ng ca Cng ty Traphaco. Nhm tc gi Phm Vn Thanh, Phm Kim Mn, on Th Nhu v cng s sn xut ch phm Morantin t thnh phn glycoside ca tri mp ng dng to, mu trng v chng minh tc dng h ng huyt ca nhm glycoside trn th gy i tho ng thc nghim bng Alloxan. [14]

15 Cc tc gi Nguyn Th Nh, Nguyn Th Bay nghin cu tc dng h ng huyt ca mt bi thuc nam trn thc nghim lm sng, l sn phm tr ti lc m thnh phn mp ng qua chim 60%. Cc tc gi Mai Phng Mai, V Phng Nguyn cng thm d tc dng h ng huyt ca mt s bi thuc dn gian m hnh i tho ng bng streptozotocin trn chut nht, m thnh phn ca bi thuc cng c cha tri mp ng.

2.2 Cc cng trnh nghin cu trn th gii


2.2.1 Thnh phn ha hc [14, 32] Ngi ta tm thy khong hn 200 hp cht c trong cy mp ng v c thng k s b thnh cc nhm chnh nh sau: 2. 2.1. 1. Triterpene Triterpene glycoside: Momordicosides A, B, C, D, E, F1, F2, G, I, K, L. Cucurbitane triterpenoid 1. Momordicine II, momordicine III. Goyaglycosides -a, -b, -c, -d, -e, -f, -g, -h. Momordicin I, momordicin II.

Triterpene saponin: Goyasaponins I, II, III. Cc triterpene khc: Momordicin, momordicinin, momordicilin. Cucurbitane triterpenoid 3, cucurbitane triterpenoid 6. -amyrin, cycloartenol, erythrodiol, gypsogenin, karounidiol, multiflorenol,

oleanolic acid, squalene, taraxerol

16 2.2.1.2. Steroid Steroid glycoside: -sitosterol-3-O--glucoside; 3-O-[-D-glucosyl]-stigmasta-5,25(27)-diene. 3-O-[6-O-palmitoyl--D-glucosyl]-stigmasta-5,25(27)-diene; 3-O-[6-O-stearoyl--D-glucosyl]-stigmasta-5,25(27)-diene.

Sterol: Elasterol; lanosterol; momordenol; -sitosterol; -spinasterol; stigmasterol; stigmasta-5-ene-3,25-diol. 2.2.1.3. Protein p-insulin, v-insulin. Map-30. Momorcharin I, momorcharin II, -momorcharin, -momorcharin, -

momorcharin, -momorcharin, -momorcharin. Momordin, momordin A, momordin B. Ribosome-inactivating proteins 1, 2, 3, 4. Trypsin inhibitor mcti-I, trypsin inhibitor mcti-II, trypsin inhibitor mci-3. Cc protein khc: Alanine, -alanine, phenylanaline, arginine, asparagine, aspartic acid 2.2.1.4. Lipid Arachidic acid, capric acid, cholesterol, -elaeostearic acid, lauric acid, linoleic acid, linolenic acid, palmitic acid, palmitoleic acid, stearic acid. 2.2.1. 5. Carbohydrate D-galacturonic acid, -glucose, -glucose, inulin, mycose, pectin, trehalose, trehalose.

17 2.2.1. 6. Carotenoid -carotene, cryptoxanthin, lutein, lycopene, mutatochrome, phytofluene, rubixanthin, zeaxantin, zeinoxanthin Ngoi ra cn c cc thnh phn khc: Alkaloid: Charine, zeatin, zeatin riboside; monoterpene: p-cymene, menthol ; sesquiterpene: Nerolidol; sapogenin: Diosgenin; cc cht khong: Ca, Mg, Fe, Cu, Zn, P, N, I, F Trong nhiu nghin cu, ngi ta chng minh c rng c t nht ba nhm hp cht c tc dng lm gim lng ng huyt hoc c hot tnh khng i tho ng. l hn hp ca hai steroid glycoside gi l charantin, cc peptide ging insulin (pinsulin) v alkaloid. Cc hp cht ny ch yu tp trung tri mp ng. 2.2.2. Mt s Triterpene Glycosede c trong cy mp ng 2.2.2.1. Cc triterpene glycoside c c lp t ht mp ng [25, 26, 27,28,31,33] Momordicoside A L 3-O--gentiobioside ca cucurbit-5-en-3,22(S),23(R),24(R),25-pentaol. Cng thc chung: C42H72O15; M = 816 vC; mp = 181 187oC; []20D = +1.05o. Cng thc cu to:
OH
21 18 12 11 1 2 6" 5" 4" 13 14 15 10 5 4 5' 4' 1" 19 6 30 31 8 7 32 17 16 20 22 23

OH
24 25 26

OH OH
27

OH O O

6'

O
1'

OH
3" 2"

OH
3' 2'

HO

HO OH

OH

18 Momordicoside B -3-O--D-xylopyranosyl(14)-[-D-glucopyranosyl(16)]--D-glucopyranoside ca cucurbit -5-en-3 ,22(S),23(R),24(R), 25pentaol. Cng thc chung: C47H80O19; M = 948 vC; mp = 238 242oC; []20D= +6.15o. Cng thc cu to:
OH
21 18 12 11 1 2 13 14 15 10 5 4 19 6 30 31 8 7 32 17 16 20 22 23

OH
24 25 26

OH OH
27

6" 5" 4"

OH O O
4' 1" 2"

6' 5'

O
1' 2'

OH
3"

OH
3'

HO

OH O

OH

5''' 4'''

O
1''' 2'''

OH
3'''

HO

OH

Momordicoside C L 3-O--gentiobioside ca cucurbit-5-en-3 ,23,24,25-tetraol. Cng thc chung: C42H72O14; M = 800 vC; mp = 224 227oC; []20D = +13.9o. Cng thc cu to:

19
OH
21 18 12 11 1 2 6" 5" 4" 13 9 8 19 6 30 31 7 32 14 15 10 5 4 5' 4' 1" 2" 17 16 20 22 23 24 25 26

OH OH
27

OH O O

6'

O
1'

OH
3"

OH
3' 2'

HO

OH

HO

OH

Momordicoside D L 3-O--gentiobioside ca cucurbita-5,24-dien-3 ,22,23-triol. Cng thc chung: C42H70O13; M = 782 vC; mp = 199 203oC; []20D = -126o. Cng thc cu to:
OH
21 18 12 11 1 2 6" 5" 4" 13 9 10 5 4 5' 4' 1" 2" 19 6 30 31 8 7 32 14 15 17 16 20 22 23 24 25 26

OH

27

OH O O

6'

O
1'

OH
3"

OH
3' 2'

HO

OH

HO

OH

2.2.2.2. Cc triterpene glycoside c c lp t tri mp ng [25, 26, 27,28,31,33] Momordicoside F1 L 3-O--D-glucopyranoside ca 5,19epoxy-25-methoxy-5-cucurbita-6,23dien-3-ol. Cng thc chung: C37H60O8; M = 632 vC; kt tinh trong MeOHH2O (1:1) cho tinh th hnh kim, khng mu; mp = 198 203oC; []20D = -11o.

20 Cng thc cu to:


21 18 12 11 19 1 10 2 9 13 14 15 8 7 6 30 31 32 17 16 27 20 22 23 24 25 26

OMe

O
5 4

HO
4'

6' 5'

O
1'

OH
3' 2'

HO

OH

Momordicoside F2 L 3-O--D-allopyranoside ca 5,19-epoxy-5-cucurbita-6,23-dien-3,25-diol. Cng thc chung: C35H58O8; M = 618 vC; kt tinh trong acetone-nc cho tinh th hnh vy, khng mu; mp = 155 158oC. Cng thc cu to:
21 18 12 11 19 1 10 2 9 13 14 15 8 7 6 30 31 32 17 16 27 20 22 23 24 25 26

OH

O
5 4

HO
4'

6' 5'

O
1'

HO

3'

2'

OH OH

Momordicoside G L 3-O--D-allopyranoside ca 5,19 epoxy-25-methoxy-5-cucurbita-6,23dien-3-ol. Cng thc chung: C37H60O8; M = 632 vC; kt tinh trong CH3CN-H2O cho tinh th hnh kim, khng mu; mp = 183 187oC; []20D = -107.3o.

21 Cng thc cu to:


21 18 12 11 19 1 10 2 9 13 14 15 8 7 6 30 31 32 17 16 27 20 22 23 24 25 26

OMe

O
5 4

HO
4'

6' 5'

O
1' 2'

HO

3'

OH OH

Momordicoside I L 3-O--D-glucopyranoside ca 5,19 epoxy-25-methoxy-5-cucurbita-6,23dien-3,25-diol. Cng thc chung: C36H58O8; M = 618 vC; kt tinh trong MeOH 50% cho cht bt mu trng; mp = 210 216oC; []20D = -110o (C=1.00; MeOH). Cng thc cu to:
21 18 12 11 19 1 10 2 9 13 14 15 8 7 6 30 31 32 17 16 27 20 22 23 24 25 26

OH

O
5 4

HO
4'

6' 5'

O
1'

OH
3' 2'

HO

OH

Momordicoside K L 3-O--D-glucopyranoside ca 3, 7-dihydroxy -25-methoxy cucurbita-5,23dien-19-al. Cng thc chung: C37H60O9; M = 648 vC; kt tinh trong MeOH cho tinh th hnh kim, khng mu, v ng; mp = 236 237oC; []20D = 63.3o.

22 Cng thc cu to:


21 18 12 11 19 9 8 7 6 30 31 32 13 14 15 10 4 5 17 16 27 20 22 23 24 25 26

OMe

OHC 1
2 3

HO

HO
5' 4'

6'

O
1'

O OH
3' 2'

HO

OH

Momordicoside L Cn c tn l 7-O--D-glucopyranoside ca 3,7,25-trihydroxy-cucurbita-5,23dien-19-al. Cng thc chung: C36H58O9; M = 634 vC; kt tinh trong CHCl3-MeOH cho tinh th hnh kim, khng mu, v ng; mp = 227 232oC; []20D = +57.3o. Cng thc cu to:

21 18 12 11 19 9 8 7 6 30 31 32 13 14

20

22 23

24

25

26

17 16 27

OH

OHC 1
2 3 5

15 10 4

HO

HO
4'

6'

O
1'

O 5' OH
3' 2'

HO

OH

2.2.2.3. Cc triterpene glycoside c c lp t l v dy mp ng Cucurbitan triterpenoid I

23 L 3,7 ,23-trihydroxycucurbita-5,24-dien-7-O- -D-glucoside. Cng thc chung: C36H60O8; M = 620 vC; dng bt v nh hnh, []20D = +89o (C=0.43; MeOH). Cng thc cu to:
21 18 12 11 13 1 2 3 4 5 6 30 31 1' 2' 10 9 19 8 7 32 6' 14 15 17 16 24 20 22 23 25 26

OH

27

HO

OH
4'

5'

HO
3'

OH

OH

Cucurbitan triterpenoid II L 3,7 ,23-trihydroxycucurbita-5,(23E)-dien19-al. Cng thc chung:C30H48O4; M = 472 vC; dng bt v nh hnh, []26D = +58.0o (C=0.48; MeOH). Cng thc cu to sau:
21 18 12 11 13 1 2 3 4 5 10 9 19 8 7 32 14 15 17 16 27 24 20 22 23 25 26

OH

CHO
6

HO
30 31

OH

Cucurbitan triterpenoid III

24 L 3,7 -dihydroxy-25-methoxycucurbita-5,(23E)-dien-19-al. Cng thc chung: C31H50O4; M = 486 vC; dng bt v nh hnh; []26D = +48.9o (C=0.45; MeOH). Cng thc cu to:
21 18 12 11 13 1 2 3 4 5 10 9 19 8 7 32 14 15 17 16 27 24 20 22 23 25 26

OMe

CHO
6

HO
30 31

OH

Momordicine I L 3,7 ,23-trihydroxycucurbita-5,24-dien-19-al. Cng thc chung: C30H48O4; M = 472 vC; mp = 125 128oC ; []20D = +81.3o. Cng thc cu to:
21 18 12 11 17 13 9 8 7 6 30 31 32 14 15 10 5 4 16 24 20 22 23 25 26

OH

27

OHC
1 2 3 19

HO

OH

Momordicine II L 23-O--glucopyranoside ca 3,7,23-trihydroxycucurbita-5,24-dien-19-al. Cng thc chung: C36H58O9; M = 634 vC; kt tinh trong CHCl3 cho tinh th dng bt khng mu.

25 Cng thc cu to:


21 18 12 11 17 13 9 8 7 6 30 31 32 1' 14 15 10 5 4 6' 5' 16 24 20 22 23 25 26

27

OHC
1 2 3 19

OH
4'

O
2'

HO
3'

HO

OH

OH

OH

Momordicine III L 23-O- -glucopyranoside ca 3,7,23-trihydroxy-24-oxo-cucurbita-5,25dien-19-al. Cng thc chung: C36H56O10; M = 648 vC. Cng thc cu to:
O
21 18 12 11 17 13 9 8 7 6 30 31 32 1' 14 15 10 5 4 6' 5' 16 20 22 23 24 25 26

27

OHC
1 2 3 19

OH
4'

O
2'

HO
3'

HO

OH

OH

OH

2.2.3. Tc dng dc l Theo y hc hin i, mp ng c tc dng: Dit vi khun v virus, chng li cc t bo ung th, h tr c lc cho bnh nhn ung th ang cha bng tia x.

26 Chng cc gc t do l nguyn nhn gy lo ha v pht sinh cc bnh tim mch, tng huyt p, ri lon lipid mu, tn thng thn kinh, vim ng tit niu, i tho ng. Tng oxy ha glucose, ngn chn s hp thu glucose vo t bo. c ch hot tnh cc men tng hp glucose. C tc dng sinh hc ging insulin, gip c th tng tit insulin. C tc dng tt vi ngi mc bnh i tho ng type 2. H tr tng tc dng, gim liu v gim tc dng ph ca cc loi sulfamid tr i tho ng type 2. Dch chit tri mp ng c kh nng c ch khi u h tr men gan, cha c nhiu bnh nh i tho ng, lch, gan, khp, gout .[14, 30] Theo ti liu , cao MeOH 50% t tri mp ng cho tc dng h ng huyt 25% (liu dng 30mg/kg), cao butanol cho kt qu l 34% vi liu dng nh trn. Cc tc gi ny cho rng cc hp cht phn cc, tan nhiu trong butanol c kh nng lm gim ng huyt. C ch hot ng tng t insulin hoc thng qua s tit insulin t tuyn ty. Nghin cu in vivo trn th gy i tho ng thc nghim bng Alloxan cho thy, khi dng nc p tri mp ng vi liu dng 6ml/kg B.W cho kt qu ti u, lm gim lng ng mu th bnh thng sau 2 gi v tng tr li sau 3 gi. Tuy nhin th mc bnh, lng ng mu tip tc gim ti 4 gi sau khi cho ung ri mi bt u tng tr li .[33] Theo bo co ca i hc Y khoa Calcuta (n ), th nghim cho 6 bnh nhn i tho ng type 2 ung mi ngy mt ln 100ml nc sc mp ng ti. Sau 3 tun l ung thuc lin tc, o lng ng trong mu (khi i) gim c 54% so vi ban u. Sau 7 tun dng thuc, c 6 bnh nhn u khng thy ng trong nc tiu, lng ng trong mu nh ngi bnh thng.

Tc dng dc l ca charantin Gii thiu[32]

27 Charantin l mt hn hp 2 steroid glycoside c cng b l mt trong nhng hot cht c hot tnh khng i tho ng type 2, c chit tch v c lp t tri mp ng. Nm 1962, Lotlikar v Rao ln u tin c lp c charantin vi hm lng khong 0.01%. n nm 1965, Sucrow xc nh c y l mt hn hp 2 steroid glycosides (t l 1:1) gm 3-O-[-D-glucopyranosyl]-stigmasta-5,25(27)-diene v sitosterol-3-O--glucoside , vi cng thc ln lt nh sau:
29

28 21 20 17 16 13 9 14 10 3 1' 8 7 6 15 22 24 23 27 25 26

18 12 19 11

H OH
4' 6' 5' 3'

1 2

H O
2'

HO HO H H

5 4

OH H

CTPT: C35H58O6 (M = 574)


29 28 21 20 17 16 13 9 14 10 3 1' 8 7 6 15 22 24 23 27 25 26

18 12 19 11

H OH
4' 6' 5' 3'

1 2

H O
2'

HO HO H H

5 4

OH H

CTPT: C35H60O6 (M = 576) Nm 1966, Lotlikar v Rao a ra qui trnh chit xut charantin vi hm lng cao hn, ng thi cng b v hot tnh khng i tho ng ca hot cht ny, c phn lp t dch chit EtOH ca tri mp ng kh .

28 Sau n nm 1979, Pugazhenthi v Suryanarayana Murthy khng nh mt ln na charantin l hn hp 2 cht v hot tnh sinh hc ca n vn c th mt i trong qu trnh chit xut ko di [32]. Hot tnh khng i tho ng ca charantin Tin hnh kho st hot tnh khng i tho ng ca charantin trn th gy i tho ng thc nghim bng Alloxan: Chn th c con c v con ci vi trng lng c th vo khong 1.5 3kg, c gy i tho ng bng cch tim Alloxan qua tnh mch vi liu lng 200mg/kg. Ch c 4 trong 20 con cn sng sau 5 ngy c tip tc em i kho st. Charantin ha tan trong Tween 80 vi nng 0.3%. X l bng cch cho ung hoc tim qua tnh mch 5ml dung dch ny. Mc ng huyt gim dn t gi th nht n gi th t sau khi x l, nhng sau s t t ly li mc ban u. Hot tnh ca charantin c hiu lc hn 5 gi, cao nht gi th t, c ghi nhn l c tc ng nh nhau ngay c khi x l bng cch cho ung hoc tim qua tnh mch. Vi liu 50mg/kg cho ung lm gim 42% lng ng huyt gi th t v gim cn 28% gi th nm sau x l. Tuy nhin khi s dng vi liu lng 25mg/kg cho ung s cho kt qu gim ng huyt tng t ch vi liu dng 15mg/kg tim qua tnh mch. [32] Charantin cho hot tnh gim ng huyt cao hn Tolbutamide, mt loi thuc thng thng cha i tho ng, vi cng liu dng. Mc d cch thc thay i glucose huyt khi x l c 2 trng hp u ging nhau. Tuy nhin vic charantin c phi l cht duy nht trong tri c hot tnh gim ng huyt hay khng vn cn l vn ang c t ra. V gi s c c 50mg charantin cn ti hn 1.5kg tri ti. Trong khi theo cc nghin cu, ch vi liu dng 50 60ml np p tri hng ngy cho kt qu lm sng tt, chng t khng ph thuc hon ton vo rt t khi lng ca charantin c trong . MAP, mt protein khng siu vi khun, c c ch nhim virus HIV-1 t bo lympho T v bch cu n nhn to, n khng c vi t bo bnh thng khng b nhim. Ht v v tri cha mt cht nha, mt saponin glycoside, v nhng alkaloid gy nn v tiu chy. Nhiu protein c hot tnh dc l c phn lp t ht , nh cc protein momorcharin v momorcharin c tc dng c hi gan trn t bo gan chut c lp. Trung Quc, ngi ta phn tch c hai hot cht hn ch sinh sn l protein v protein t ht. Cc th nghim nui cy, ghp phi in vitro cho thy 2 hot

29 cht ny c tc dng c ch qu trnh lm dy c nguyn bo phi trc khi lm t v hnh thnh phi thai k u, t phi ngng pht trin, thoi ha phn hy dn n sy thai [13]. Nhng nghin cu gn y cho thy protein v protein cng c nh hng n s sinh sn ca phi chut nht trng v nh hng n vic tng hp phn t ln t bo ni m t cung; chng cng c kh nng c ch tng hp ADN, ARN v protein, lm cho s pht trin ca ni mc t cung b c ch [13].

30

THC NGHIM
1. NGUYN LIU
Mp ng do Trung tm Bo tn v Pht trin Dc liu min Trung cung cp, l loi mp ng cn non, ht cha pht trin. Tri c ct thnh lt mng, sy nhit di 60oC trn h thng sy nguyn liu n khi lng khng i. Tip , nguyn liu c xay nhuyn qu trnh tch chit c trit hn.

Hnh 2.1: Qu trnh sy nguyn liu

2. NHN DANH CC NHM HP CHT HU C TRONG TRI MP NG


gip cho vic nghin cu thnh phn ha hc v chit xut hot cht t cy thuc, trc ht cn phi tin hnh phn tch s b thnh phn ha hc chng ta c khi nim s b v mt nh tnh, t c th nh hng cho vic chit xut cng nh vic xc nh cu trc ha hc cc hp cht trong cy thuc. T mu cy, s dng k thut chit tch khc nhau c c cao chit ethanol ton phn hoc cc loi cao c tnh phn cc khc nhau. p dng cc phng php phn tch s b v ha- thc vt bit trong cao chit c th cha cc loi hp cht t nhin no. Trong bo co ny, em xin trnh by v phng php s dng thuc th hin mu hoc xut hin kt ta m em thc hin.

31

2.1 Kho st s hin din ca cc hp cht alkaloid [7, 11]


2.1.1 Thuc th alkaloid C rt nhiu thuc th cho phn ng mu hoc kt ta vi alkaloid. * Phn ng to kt ta c mu: Thuc th Mayer: Cng thc: 1.35g HgCl2 ha tan trong 100ml dung dch KI 5%. Du hiu: To kt ta v nh hnh mu trng vng.

Thuc th Dragendoff: Cng thc:

+ Dung dch A: 850mg Bismut nitrat trong 40ml H2O v 10ml acetic bng. + Dung dch B: Ha tan 8g KI trong 20ml H2O. Trn hai th tch bng nhau ca hai dung dch A v B lm thuc th. Du hiu: To kt ta c mu t vng cam n .

Thuc th Wagner: Cng thc: Ha tan 5g Iod trong 100ml dung dch KI 10%. Du hiu: Cho kt ta mu nu sng n nu en.

Thuc th Bouchardat: Cng thc: 2.5g Iod v 5.0g KI ha tan trong 10ml nc ct. Du hiu cho kt ta mu nu hoc mu vng m.

2.1.2 nh tnh alkaloid Th nghim: Tm 10g bt tri mp ng bng 5ml dung dch NH4OH 25%. y kn bng giy lc ri qua m. Sau em chit vi 25ml CHCl3. Dch CHCl3 c lc vi 10ml dung dch H2SO4 2%. Ly dch acid lm mu th. -Vi thuc th Wagner: Cho kt ta mu nu.

32 -Vi thuc th Mayer: Dung dch c mu trng c.

2.2 Kho st s hin din ca cc hp cht flavonoid [6, 7]


2.2.1 Thuc th flavonoid Trong dung dch, flavonoid to kt ta mu vng cam hoc mu vi acetate ch, to kt ta mu xanh lc, i khi mu nu vi FeCl3. Flavonoid c xc nh bi phn ng Shibata, cn gi l phn ng Cindin ca Willstater. Thuc th l tp hp cc ha cht gm: dung dch HCl m c, bt Mg kim loi, ru isoamyl [CH3(CH3)CHCH2CH2CH2OH]. 2.2.2 nh tnh flavonoid Th nghim: un hon lu 5g bt tri mp ng trong 50ml EtOH 95o trong 30 pht. Lc, ly 1ml dch EtOH vo ng nghim, thm vi git HCl m c, sau cho mt t bt Mg vo lc th thy dung dch c mu tm.

2.3 Kho st s hin din ca cc hp cht anthraglycoside


2.3.1 Thuc th anthraglycoside Anthraquinon (phn ngBotrager): Pha hu c ca dch chit anthraquinon s c mu khi c s hin din ca cht kim. Do vy dng kt hp phi c thy phn v chuyn sang dng oxy ha trc khi thc hin phn ng. Anthron v anthranol: Phn ng Schouteten: Cho hunh quang xanh vi natri borat. Phn ng to mu xm vi natri nitrodimethyl alanin. 2.3.2 nh tnh anthraglycoside [7 ] Th nghim: un hon lu 5g bt tri mp ng trong 30ml ether ethyl. Lc v lp li nhiu ln cho n khi dch ether khng cn mu. Tp trung dch lc v lc vi 50ml dung dch KOH 10%. Lp kim c trung ha vi dung dch HCl 25% n pH=7. Lc, ly phn kt ta trn giy lc, em ha tan bng EtOH 95o. Nh vi git dung dch KOH 10% vo dch EtOH thy c mu vng cam.

33 B sau khi loi cc cht tan trong ether c chit tip vi EtOH 95o. Nh vo dch EtOH vi git KOH 10% thy c mu cam.

2.4.Kho st s hin din ca cc hp cht steroid [6, 7]


2.4.1 Thuc th steroid Liebermann-Burchard: Anhydrid acetic: 20ml. H2SO4 m c: 1ml. Cho 1 git thuc th vo dch CHCl3, nu c sterol s c mu xanh nht, lc, hng hoc bn vng trong mt thi gian. Phn ng Rosenheim: Cho vi git dung dch acid tricloacetic 90% vo dch CHCl3, nu c sterol s xut hin mu tm, sau 20 pht chuyn sang mu xanh l. Salkowski: Dung dch tch lm 2 lp: Lp H2SO4 c mu xanh, lp CHCl3 c mu nu . 2.4.2 nh tnh steroid Th nghim: Ha tan 1g bt tri mp ng kh trong 20ml CHCl3. Lc, ly dch lc lm mu th. Vi mu cao, s dng 0.1g ha vo 30ml CHCl3, ly dch lc lm mu th. Vi thuc th Liebermann- Burchard: C mu hng . Vi thuc th Salkowski: Thm 0.5ml dung dch H2SO4 m c. Dung dch tch lm hai lp, lp H2SO4 (lp trn) c mu xanh v lp CHCl3 (lp di) c mu vi thuc th Salkowski.

2.5 Kho st s hin din ca cc hp cht saponin [7]


2.5.1 Thuc th saponin Cn c vo ch s to bt xc nh s hin din ca saponin.

34 Dc in ca Php nh ngha ch s to bt nh sau: Ch s to bt ca saponin l long ca nguyn liu bng nc c chiu cao bt 1cm sau khi lc trong ng nghim c kch thc xc nh, tin hnh trong iu kin qui nh. Cch tin hnh: Cn 1g bt dc liu cho vo erlen 500ml cha sn 100ml nc si. Tip tc cho nc trong erlen si nh trong 30 pht na. Lc ngui, thm nc ct cho n 100ml (thu c nc sc). Ly 10 ng nghim c chiu cao 16cm, ng knh 16mm. Cho vo cc ng nghim ln lt 1, 2, 3, 4, 10ml nc sc. Thm nc ct vo mi ng cho 10ml. Bt ming ng nghim ri lc theo chiu dc ca ng trong 15 giy. Mi giy lc 2 ln. yn trong 15 pht. Sau o chiu cao cc ct bt. Nu ct bt trong cc ng thp di 1cm th ch s bt l di 100, ngha l khng c saponin. Phn ng Liebermann: Cch thc hin: Ha tan mu bng 1ml anhydrid acetic, thm t t 0.3 0.5ml

H2SO4 m c. Du hiu:

+ Nu vng ngn cch c mu hng n tm th s b nhn nh c saponin triterpene. + Nu vng ngn cch c mu xanh l cy th s b nhn nh c saponin steroid. Phn ng Kahlenberg: Cch thc hin: Ha tan mu bng 0.5ml dung dch SbCl3 bo ha trong

CHCl3, khuy u, em soi UV. Du hiu:

+ Nu c hunh quang mu xanh th s b nhn nh c saponin triterpene. + Nu hunh quang mu vng th s b nhn nh c saponin steroid. 2.5.2 nh tnh saponin Th nghim: Cn 1g bt tri mp ng cho vo erlen 500ml cha sn 100ml nc si. Tip tc cho nc trong erlen si nh trong 30 pht na. Lc, ngui. Cho khong

35 1ml dch lc vo ng nghim nh v lc mnh trong 15 giy th thy rt nhiu bt (ct bt cao 6cm).

2.6 Kho st s hin din ca cc hp cht ng kh [11]


Th nghim: Acid ha 2g bt tri mp ng bng 20ml dung dch H2SO4 1%. Lc, c cn cn 5ml. Nh vo mu th 4 5 git thuc th Fehling A v 4 5 git thuc th Fehling B. un nh thy xut hin ta mu nu.

2.7 Kho st s hin din ca cc hp cht tanin [6, 7 ]


2.7.1 Thuc th tannin Stiasny: Formol (36%): 20ml. Dung dch HCl m c : 10ml. Dung dch gelatin mn: Gelatin: 2g. Dung dch NaCl bo ha: 10ml. Dung dch acetate ch bo ha cho kt ta mu vng nht. Dung dch FeCl3 1% trong nc to phc mu en. 2.7.2 nh tnh tanin Th nghim: Ly 5g bt tri, thm 100ml nc ct ri un si trong 10 pht. Lc, ly dch lc lm mu th: Ly 2ml dch lc, thm 2 4 git dung dch acetate ch bo ha thy xut hin kt ta mu vng nht. Ly 2ml dch lc, thm vi git dung dch gelatin mn, xut hin kt ta trng. Ly 2ml dch lc, thm 2ml dung dch FeCl3 1%, dung dch chuyn thnh mu nu en.

2.8 Kho st s hin din ca cc hp cht glycoside [6, 7 , 11]


2.8.1 Thuc th glycoside Thuc th tc dng ln phn aglycon.

36 Thuc th Tollen (xc nh theo ng kh trong glycoside): Cng thc: Pha 0.5ml dung dch AgNO3 10% vi 0.5ml dung dch NaOH 10%.

Sau nh t t dung dch NH4OH n khi tan kt ta. Du hiu: C Ag kt ta.

Thuc th Molish: Cng thc: Nh 1 2 git dung dch thymol 2% vo 1ml H2SO4 m c. Du hiu: Xut hin mu thm.

Thuc th Baljet: Cng thc: Pha dung dch acid picric 1% trong EtOH vi dung dch NaOH

10% trong nc theo t l th tch 1:1. Du hiu: Nh 3 4 git thuc th vo 1mg cht th, nu c mu vng cam

hoc hng xn l phn ng dng tnh. Thuc th Legal: Cng thc: Ha tan 1mg cht th trong 2 3 git pyridin, thm 1 git dung

dch natri nitroprussiat 0.5% mi pha, sau cho t t tng git dung dch KOH 2N. Du hiu: Xut hin mu tm.

2.8.2 nh tnh glycoside Th nghim: Ly 10g bt tri mp ng kh, loi cc cht khng phn cc bng ether petrol. Tip tc chit bng soxhlet vi dung mi EtOH 50o. Dch lc EtOH 50o c loi tp bng acetate ch cho n khi khng cn trm hin. Sau , loi acetate ch d bng Na2SO4 bo ha. C cn dch lc c cao glycoside th. Ha tan cao bng EtOH 95o v ly dung dch ny lm mu th. - Vi thuc th Tollen: Xut hin kt ta trng. - Vi thuc th Molish: C mu carmine.

37

3.TCH CHIT, C LP V TINH CH CC HP CHT


3.1. Thit b v ha cht
3.1.1 Thit b 0.63mm. Sc k bn mng TLC c thc hin trn bn silicagel 60 F254, MERCK trng sn. My c quay chn khng. My soi UV im chy (mp oC) c o trn my Electrothermal IA 9000 Series. Sc k ct thng dng silicagel 60, MERCK, ng knh ht: 0.04

Hnh 2.2: Sc k ct thng 3.1.2 Ha cht Petroleum ether, Trung Quc Diethyl ether, Trung Quc Chloroform, Trung Quc Ethyl acetate, Trung Quc

Hnh 2.3: Sc k bn mng (TLC)

38 Acetone, Trung Quc Methanol, Trung Quc Nc ct Ethanol 95o

3.2. Chit xut cc nhm hp cht


Tri mp ng sy kh, xay nh (4.5 kg), c chit vi ethanol 95o. Sau , c loi dung mi thu c cao dng st gi l cao tng (cao EtOH 95o) (537 g). Cao tng ha tan trong mt lng nc va st. dung dch ny c lc vi petroleum ether (PE), thu ly dch PE v c loi dung mi thu c cao PE. Pha nc cn li sau khi lc vi EP c lc tip tc vi ethyl acetate (EtOAc), dch EtOAc c loi dung mi thu c cao EtOAc (219 g). Pha nc cn li sau khi lc vi EtOAc c lc tip tc vi methanol (MeOH), dch MeOH c ui dung mi thu c cao MeOH (254 g).

39 Bt tri mp ng Chit vi ethanol 95o C ui dung mi Cao EtOH 95o Chit vi petroleum ether C ui dung mi Cao PE

Dch nc Chit vi ethyl acetate C ui dung mi

Cao EtOAc

Dch nc Chit vi methanol C ui dung mi

Cao MeOH

Dch nc

S 2.1: Quy trnh chit xut cc nhm hp cht ca tri mp ng

3..3. Phn lp v tinh ch cc hp cht


Sau khi thu c cc cao chit, chng ti s dng sc k bn mng kim tra xem trong mi loi cao chit c khong bao nhiu hp cht. So snh vi nhng hp cht phn lp c t tri mp ng trc y, chng ti pht hin trong cao EtOAc c nhiu hp cht. V vy, chng ti dng cao EtOAC phn lp cc hp cht s dng phng php sc k ct thng.

40

Cao EtOAc
Sc k ct thng Sc k ct thng Sc k bn mng Sc k bn mng

Phn on 10

Phn on 11

Tinh ch

Tinh ch

MC 1

MC 6

MC 5

MC1A & MC1B


S 2.2: Quy trnh phn lp v tinh ch cc hp cht t tri mp ng T cao EtOAc, tin hnh ln ct thng vi cc thng s sau: Khi lng cao: 219 g Khi lng silica gel: 600 g ng knh ct: 9 cm Chiu cao lp silica gel: 35 cm Dung mi n nh ct: Petroleum ether. Th tch ly mi phn on: 500ml Tng dn phn cc ca h dung mi gii ly bng cch tng dn t l CHCl3 trong h PE:CHCl3. Theo di ct bng sc k bn mng, hin vt bng cch phun dung dch H2SO4 10% trong EtOH, h nng trn bp in.

Phn on 14

Phn on

Phn on 1

Phn on 8

Phn on 9

41 Kim tra cc phn on bng TLC (h dung mi gii ly CHCl3:MeOH:H2O = 9:1; 85:15; 8:2; 7:3:0.5; 6:4:1). Gom cc phn on c Rf ging nhau, thu c tt c 14 phn on chnh. Bng 2.1: Kt qu sc k ct thng Phn on 1 2 3 4 5 6 7 8 9 10 11 12 13 14 H dung mi PE:CHCl3 = 8:2 PE:CHCl3 = 7:3 PE:CHCl3 = 6:4 PE:CHCl3 = 4:6 PE:CHCl3 = 3:7 PE:CHCl3 = 2:8 CHCl3 100% CHCl3:MeOH = 95:5 CHCl3:MeOH = 93:7 CHCl3:MeOH = 9:1 CHCl3:MeOH = 88:12 CHCl3:MeOH = 85:15 CHCl3:MeOH=8:2 CHCl3:MeOH=75:25 Kt qu th TLC Vt ko di Nhiu vt Vt ko di Nhiu vt Nhiu vt Vt ko di Vt ko di C 2 vt m Vt ko di C 2 vt m C 1 vt m Vt ko di Nhiu vt Vt ko di

42 Ti phn on 8 (dung mi gii ly CHCl3:MeOH = 95:5), chy sc k bn mng thu c hai vt m. Ra bng ethyl acetae v kt tinh li nhiu trong chloroform thu c hai cht . K hiu MC1v MC6 . Ti phn on 10 (dung mi gii ly CHCl3:MeOH = 9:1), chy sc k lp mng thu c hai vt m. Ra bng ethyl acetate, kt tinh li nhiu ln thu c mt cht tinh khit. K hiu MC5.

43

KT QU V THO LUN
1. KT QU NH TNH CC NHM HP CHT TRONG TRI MP NG
Qua kho st s b cc hp cht trong tri mp ng, kt qu chi tit nh sau: Bng 3.1: Kt qu nh tnh cc hp cht hu c trong tri mp ng TT 1 2 3 4 HP CHT FLAVONOID ACID BO ANTHRAGLYCOSIDE STEROID THUCTH Mg/HCl Nh trn giy lc v bc hi KOH 10% Liebermann Acetate ch 5 TANIN Gelatin FeCl3 Mayer 6 ALKALOID Dragendoff To bt Fehling A+B Thymol 20% v H2SO4 Tollen Ch thch: (+): t, (++): nhiu, (+++): rt nhiu HIN TNG Ta hng nht li vt khng kh Mu cam Xanh lc Vng nht Ta trng Mu en Ta trng Dung dch c mu cam To bt bn Ta nu Cho mu thm Kt ta trng KTLUN + + ++ ++ ++ ++ ++ + + ++ ++ +++ +++

7 8

SAPONIN NG KH

GLYCOSIDE

44

2. NHN DANH CU TRC CC CHT TINH KHIT


2.1. Hp cht MC 1
2.1.1 Kt qu phn tch tinh khit ca MC1 bng HPLC Kt qu phn tch bng HPLC cho thy MC1 l hn hp ca hai cht, k hiu MC1A v MC1B. Tin hnh xc nh cu trc ca 2 cht bng cc phng php ph. Cc hp cht trong MC1 c c lp bng HPLC iu ch, thu c 12mg MC1A v 9mg MC1B. Kim tra TLC ca MC1A, MC1B so vi MC1 u cho mt vt trn r mu tm, Rf = 0.25 (h gii ly CHCl3:MeOH = 9:1). Vy chng ti d on MC1 l charantin.

Hnh 3.1: Tinh th MC1

Hnh 3.2: TLC ca MC1, MC1A v MC1B

2.1.2. Nhn danh cu trc ha hc ca MC1A v MC1B 2.1.2.1. MC1B Cc c tnh ca MC1B Dng bt, mu trng, kt tinh trong MeOH. im nng chy: mp = 281 283oC. Sc k bn mng (TLC) hin mu bng dung dch H2SO4 10% trong EtOH:

45 Gii ly bng h CHCl3:MeOH = 9:1 cho vt trn mu tm c Rf = 0.25. Gii ly bng h CHCl3:MeOH = 85:15 cho vt trn mu tm c Rf = 0.37.

H gii ly CHCl3:MeOH = 9:1

H gii ly CHCl3:MeOH = 85:15

Hnh 3.3: TLC ca MC1B cc h gii ly khc nhau Nhn danh cu trc MC1B Ph 13C-NMR ( ph lc 1.1) cho bit MC1B c 35C. Trong c 2 mi vng ni i c = 140.11 v 121.98 ppm chng t trong cu trc ca MC1B c 1 ni i. Mi 140.11 bin mt trn ph DEPT (ph lc 1.2) chng t y l C t cp c ni i. ng thi trn ph DEPT (ph lc 1.2 v 1.2a) cn cho thy trong phn t MC1B c 6 nhm CH3, 12 nhm CH2 (trong c 1 nhm CH2OH ca phn ng 61.716 ppm), 14 nhm >CH, 3C t cp. Bng 3.2: D liu ph 13C-NMR v DEPT ca MC1B V tr C 1 2 3
13

C (ppm) 36.53 29.42 77.01

DEPT90 Bin mt Bin mt Mi dng

DEPT135 Mi m Mi m Mi dng

Kt lun CH2 CH2 >CHO

46 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 38.52 140.11 121.98 31.74 31.70 49.59 36.54 20.87 39.58 42.15 56.57 24.09 28.04 55.88 11.62 18.77 35.96 18.54 33.77 Bin mt Bin mt Mi dng Bin mt Mi dng Mi dng Bin mt Bin mt Bin mt Bin mt Mi dng Bin mt Bin mt Mi dng Bin mt Bin mt Mi dng Bin mt Bin mt Mi m Bin mt Mi dng Mi m Mi dng Mi dng Bin mt Mi m Mi m Bin mt Mi dng Mi m Mi m Mi dng Mi dng Mi dng Mi dng Mi dng Mi m CH2 >C=C CH= CH2 CH< CH< >C< CH2 CH2 >C< CH< CH2 CH2 CH< CH3 CH3 CH< CH3 CH2

47 23 24 25 26 27 28 29 1 2 3 4 5 6 25.90 45.69 28.98 19.54 19.08 22.88 11.72 100.93 73.36 76.22 70.00 76.75 61.72 Bin mt Mi dng Mi dng Bin mt Bin mt Bin mt Bin mt Mi dng Mi dng Mi dng Mi dng Mi dng Bin mt Mi m Mi dng Mi dng Mi dng Mi dng Mi m Mi dng Mi dng Mi dng Mi dng Mi dng Mi dng Mi m CH2 CH< CH< CH3 CH3 CH2 CH3 OCHO >CHOH >CHOH >CHOH >CHO CH2OH

Mi c trng cho v tr 1 ca ng 100.98 ppm v cc mi trong vng 61.72 n 77.00 ppm ca ni CO trn
13

C-NMR (ph lc 1.1 v 1.1b) chng t MC1B l 1

glycoside c 1 n v ng. Du hiu nh tnh trn TLC cho vt c mu tm hng khi phun dung dch H2SO4 v h nng cho php gi thit phn aglycon l 1 sterol. Phn ng c cc mi ppm : 100.98; 76.36; 76.75; 73.36; 70.00 v 61.72 trn
13

C-NMR (ph lc 1.1 v 1.1b) chng t y l ng glucose. Hng s tng tc spin

spin ca proton 1 v 2 trn 1H-NMR J = 8 Hz chng t 2 proton ny u v tr trc, v vy lin kt gia phn ng v aglycon theo kiu (ph lc 1.3 v 1.3b).

48 Vy MC1B c tt c 35 nguyn t carbon, 6 nguyn t oxy. Nh vy cng thc phn t l C35H60O6; phn t khi l 576 vC. T nhng s liu trn v so snh vi ti liu MC1B l -sitosterol-3-O-glucopyranoside.
29

[5,32]

, xc nh cu trc ha hc ca

28 21 20 17 16 13 9 14 10 3 1' 8 7 6 15 22 24 23 27 25 26

18 12 19 11

H OH
4' 6' 5' 3'

1 2

H O
2'

HO HO H H

5 4

OH H

Hnh 3.4: Cng thc cu to MC1B Bng 3.3: D liu ph 1H, 13C-NMR, DEPT ca MC1B
13 1

V tr C/H 1 2 3

H-NMR ppm

C-

(s H; dng mi; J = Hz) 1.80; 0.99 (2H, m) 1.80; 1.47 (2H, m) 3.43 (1H, m) 2.12 (1H, t, J = 11.5)

NMR ppm 36.53 29.42 77.00

Loi carbon CH2 CH2 CHO

2.37 (1H, dd, J = 3.0 v 13.0)

38.52

CH2

49 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 0.96 (1H, m) 1.05; 1.52 (2H, m) 1.25; 1.80 (2H, m) 1.10 (1H, d, J = 10) 0.67 (3H, d, J = 10) 0.93 (3H, m) 1.38 (1H, m) 0.91 (3H, m) 1.0; 1.32 (2H, m) 1.18 (2H, m) 1.41; 1.50 (2H, m) 1.17; 1.97 (2H, m) 5.32 (1H, s) 1.90; 1.51 (2H, m) 1.39 (1H, m) 0.91 (1H, m) 140.11 121.98 31.74 31.70 49.59 36.54 20.87 39.58 41.15 56.57 24.09 28.04 55.88 11.62 18.77 35.96 18.54 33.77 25.90 >C=C CH= CH2 CH< CH< >C< CH2 CH2 >C< CH< CH2 CH2 CH< CH3 CH3 CH< CH3 CH2 CH2

50 24 25 26 27 28 29 1 0.93 (1H, m) 1.61 (1H, m) 0.81 (3H, m) 0.80 (3H, m) 1.20; 1.22 (2H, m) 0.82 (3H, m) 4.32 (1H, d, J = 8.0) 2.90 (1H, m) OH2 = 4.84 3.12 (1H, m) OH3 = 4.85 3.02 (1H, m) OH4 = 4.82 3.07 (1H, m) 3.40; 3.64 (2H, m) OH6 = 4.38 45.69 28.98 19.54 19.08 22.88 11.72 100.93 CH< CH< CH3 CH3 CH2 CH3 OCHO

73.36

>CHOH

GLUCOSE

76.22

>CHOH

70.00

>CHOH

76.75

>CHO

61.71

CH2OH

2.1.2.2. MC1A Cc c tnh ca MC1A Dng bt, mu trng, kt tinh trong MeOH. im nng chy: mp = 271 276oC.

51 Sc k bn mng (TLC) hin mu bng dung dch H2SO4 10% trong EtOH: Gii ly bng h CHCl3:MeOH = 9:1 cho vt trn mu tm c Rf = 0.25. Gii ly bng h CHCl3:MeOH = 85:15 cho vt trn mu tm c Rf = 0.37.

H gii ly CHCl3:MeOH = 9:1

H gii ly CHCl3:MeOH = 85:15

Hnh 3.5: TLC ca MC1A cc h gii ly khc nhau Nhn danh cu trc MC1A Theo ti liu [5,32] th charantin l hn hp ca hai steroid l 3-O-[-Dglucopyranosyl] stigmasta-5,25(27)-diene v -sitosterol-3-O--glucoside. V th, theo chng minh trn th MC1A l 3-O-[-D-glucopyranosyl]-stigmasta5,25(27)-diene. Vy MC1A c tt c 35 nguyn t carbon, 6 nguyn t oxy, c hai ni i v tr C-5 v C-27. Nh vy cng thc phn t l C35H58O6, phn t khi l 574 vC.

52
29

28 21 20 17 16 13 9 14 10 3 1' 8 7 6 15 22 24 23 27 25 26

18 12 19 11

H OH
4' 6' 5' 3'

1 2

H O
2'

HO HO H H

5 4

OH H

Hnh 3.6: Cng thc cu to MC1A

2.2. Hp cht MC 6
Cc c tnh ca MC 6 - Khi lng thu c m = 20 mg - Dng bt, hi vng - Sc k bn mng (TLC) hin mu xm di n t ngoi v mu vng trong hi Iod, khng hin mu vi dung dch H2SO4 10% trong EtOH c Rf = 0.24 (CHCl3:MeOH = 85:15) - im chy mp= 250oC ( cht bt u chuyn sang mu en) . - Tan t trong Methanol, tan nhiu trong Dimetyl sulfoxide (DMSO)

53

Hnh 3.7: Mu cht MC6 Nhn danh cu trc ca MC 6

Hnh 3.8: TLC hp cht MC6

- Ph hng ngoi IR (KBr, max, cm-1) ( ph lc 2.1) cho: + Tn hiu hp thu ca nhm N-H 3112. + Tn hiu th hin s dao ng ca C-H 2986 - 2821. + Tn hiu c trng cho 2 lin kt i k nhau c UV 1990. + Tn hiu c trng cho 2 lin kt C=O 1717. + Tn hiu c trng cho lin kt C=C 1669. - Ph 13C-NMR (DMSO, ppm) (ph lc 2.2) kt hp vi ph DEPT (ph lc 2.3) (bng 2.5) cho bit c: + 2 tn hiu dng l carbon olefin (-CH=) 100.16 v 142.09 ppm. + 2 tn hiu l carbon t cp ca nhm cacbonyl (>C=O) 151.44 v 164.26 ppm.

54 Bng 3.4: D liu ph 13C-NMR v DEPT ca cht MC 6


13

V tr C/H

C-NMR

( ppm) 100.16 14.2.09 151.44 164.26

DEPT 90

DEPT 135

Nhm C

5 6 2 4

Tn hiu dng Tn hiu dng Bin mt Bin mt

Mi dng Mi dng Bin mt Bin mt

CH< CH< >C=O >C=O

- Ph 1H- NMR (DMSO, ppm) (ph lc 2.4 v 2.4a) cho cc mi tn hiu ca: + 1 tn hiu proton metin (-CH<) 5.43 - 5.45 (d, J = 7.5). + 1 tn hiu proton metin (-CH< 7.36 - 7.38 (d, J = 7.5). + 2 tn hiu proton 10.89 (2H, brs). - Ph HMBC (ph lc 2.6) cho thy s tng tc ca proton vi cc v tr carbon c th hin trong bng 3.5. Nh vy, MC 6 c cng thc phn t l C4H4O2N2, phn t khi l 112 vC; bt bo ha l 4, trong c: 1 ni C=C chim 1 bt bo ha, 2 ni C=O chim 2 bt bo ha v 1 bt bo ho ca vng. Bng 3.5: D liu ph 1H-NMR, 13C-NMR, DEPT v HMBC ca cht MC 6 V tr C/H 1 2 3 151.44
13

C-NMR

( ppm )

Nhm C >NH >C=O >NH

H-NMR ( ppm, J = Hz) 10.89 (1H, brs)

HMBC (H C)

10.89 (1H, brs)

55 4 5 6 162.26 100.16 142.09 >C=O CH= CH= 5.43 5.45 (1H, d, J = 7.5) 7.36 7.38 (1H, d, J = 7.5) H5 C6, C4 H6 C5, C2, C4

Bng 3.6: So snh d liu ph ca MC 6 vi ti liu [20] MC6 mp (oC) V tr C/H 5 6 2 4


13

Uracil 250oC
13

246 - 247oC C-NMR


1

H-NMR

C-NMR

H-NMR

100.16 142.09 151.44 164.26

5.43 - 5.45 (d, J = 7.5) 7.36 - 7.38 (d, J = 7.5)

101.78 143.46 153.57 167.36

5.63 (d, J = 7.6) 7.41 (d, J = 7.6)

Tm li, da vo cc kt qu ph

H-NMR,

13

C-NMR, HSQC, HMBC, cc c

trng vt l v so snh vi ti liu cng b [20], chng ti nhn danh cht MC6 l Pyrimidine-2,4-(1H, 3H)-dione (hay Uracil).

H
O H N
3 N
H

2
HN1

4
H N

4 5

2
O

Hnh 3.9: Cng thc cu to ca MC6 v tng quan ph HMBC

56

2.3. Hp cht MC5


(Cha nhn danh c cu trc ca MC5) Cc c tnh ca MC 5 - Khi lng thu c m=331 mg - Dng bt, mu trng - Sc k bn mng (TLC) khng hin mu di n t ngoi, hin mu bng dung dch H2SO4 10% trong EtOH c Rf = 0.36 (CHCl3:MeOH = 85:15) cho vt trn mu xm. - im chy mp= 194.6oC - Tan trong MeOH

Hnh 3.10: Mu cht MC5

Hnh 3.11: TLC ca MC5

57

KT LUN V KIN NGH


1. CC KT QU T C
Bc u kho st thnh phn ha hc ca tri mp ng loi tri nh, gai nhn, rt ng c trng Ph Yn chng ti t c cc kt qu sau: 1) 2) 3) Kho st s b thnh phn ha hc nhn thy: tri mp ng c saponin, steroid, T dch chit cn 95o, chng ti tin hnh tch ring 3 loi cao da trn phn T cao EtOAc, chng ti phn lp c ba cht tinh khit v nhn danh c alkaloid, glycoside, tanin, anthraglycoside, ng kh, acid bo. cc ca cc hp cht c trong tri: cao PE, cao EtOAc v cao MeOH. cu trc ca chng. Gm hai glycoside steroid l 3-O-[-D-glucopyranosyl]-stigmasta5,25(27)-diene (MC1A) v -sitosterol-3-O-glucopyranoside (MC1B) v mt l pyrimidine-2,4-(1H, 3H)-dione (hay Uracil) (MC6). Tt c cc cht c nhn danh cu trc bng cc phng php ph hin i nh IR, ph 13C-NMR, ph 1H-NMR v so snh vi ti liu cng b. Bng 4.1: Cc cht phn lp c DANH PHP CNG THC CU TO
29 28 21 22 20 17 16 13 9 14 10 3 1' 8 7 6 15 24 23 27 25 26

STT

3-O-[-D-glucopyranosyl]stigmasta-5,25(27)-diene
HO

18 12 19 11

H OH
4' 6' 5' 3'

1 2

H O
2'

HO

H H

5 4

OH H

29

28 21 22 20 17 16 13 9 14 10 3 1' 8 7 6 15 24 23 27 25 26

-sitosterol-3-Oglucopyranoside
HO

18 12 19 11

H OH
4' 6' 5' 3'

1 2

H O
2'

HO

H H

5 4

OH H

H N

pyrimidine-2,4-(1H, 3H)-dione

2
HN1

4 5

58

2. KIN NGH
- Tinh ch MC5 v nhn danh cu trc ca hp cht ny. - Tip tc kho st cao EtOAc phn lp cc hp cht cn li trong cao. - Kho st thnh phn ha hc cc cao PE, MeOH. - Th hot tnh ca cc hp cht phn lp c ng dng trong cha bnh.

59

TI LIU THAM KHO


[1] [2] [3] [4] [5] [6] [7] [8] [9] B Y T, Dc in Vit Nam, Nh xut bn Y hc, 2002 Bi Ch Hiu, Dc l tr liu thuc nam, 1999, trang 212. Tt Li, Nhng Cy Thuc v V Thuc Vit Nam, Nh xut bn Khoa Hc v K Thut, H Ni, 1982, trang 736-737. Hi n, Mn n v thuc, Nh xut bn Thun Ha, 2002, trang 161-162. Ng Vn Thu, Bi ging dc liu tp I, Nh xut bn B Y T v Gio dc o to, 1998. Nguyn Khc Qunh C, Bi ging chit xut dc liu, khoa Dc, Trng i hc Y Dc Tp.HCM, 2000. PGS.TS. Nguyn Kim Phi Phng, Cc phng php tch chit hp cht hu c, Nh xut bn i hc Quc gia TP. H Ch Minh, 2007. PGS.TS. Nguyn Kim Phi Phng, Ph NMR s dng trong phn tch hu c, Nh xut bn i hc Quc gia TP. H Ch Minh, 2005. PGS.TS. Nguyn Kim Phi Phng, Khi ph s dng trong phn tch hu c, Nh xut bn i hc Quc gia TP. H Ch Minh, 2005.

[10] Nguyn Minh c, Trn Th Vy Cm, Kho st ha hc cc cht c tc dng sinh hc t ht mp ng (Momordica charantia L.), Y Hc TP. H Ch Minh, 2002 Tp 6, Ph bn ca S 1. [11] [12] [13] [14] [15] TS Nguyn Ngc Hnh , Gio trnh cao hc Tch chit v c lp hp cht t nhin, 2002. TS. Nguyn Thanh Hng, Cc phng php ph trong ha hc hu c, Nh xut bn Khoa Hc v K Thut, 2007. Phm Th Th, Huy Bch, 101 Cy Thuc Vi Sc Khe Sinh Sn Ph N, Nh xut bn Khoa Hc v K Thut, H Ni, 2003, trang 151-156. Phm Vn Thanh, Phm Kim Mn, on Th Nhu, Nguyn Thng Dong, V Kim Thu, Nguyn Kim Phng, Tp ch Dc liu, tp 6, s 2+3, 2001, trang 48-54. T Duy Chn, Nhng phng thuc hay cha bnh bng hoa, Nh xut bn Ngh An, 1999,trang 161-255.

[16] T Duy Chn, Nhng phng thuc hay rau c tr bnh, Nh xut bn Ngh An, 1999, trang 293-297.

60 [17] Tn Cng, Nguyn Vn Phn, Nhng cy thuc nam thng dng d tm v Tcc bi thuc gia truyn, Nh xut bn Thanh Ha, 2000, trang 61-62. [18] Thi Xun Mi, Tho dc cha bnh (Nguyn Thanh Tng dch, BS Ngc Tm hiu nh), Nh xut bn Thanh Ha, 2002, trang 62. [19] [20] Trn Nam Hng, Y hc dn gian tr bnh ti nh, Nh xut bn Tng hp ng Thp, 1998, trang 104-105. V Th Nga, Gerhard Maas, Nguyn Kim Phi Phng, Nguyn Ngc Sng, Tuyn tp cc cng trnh hi ngh Khoa hc v Cng ngh Ho hc hu c ton quc ln th t, NXB i hc Quc gia H Ni, 2007, trang 465 469.

[21] V Vn Chi, T in Cy Thuc Vit Nam, Nh xut bn Y hc, 1999, trang 795. [22] [23] Vng Tha n, Phng v cha bnh bng mn n hng ngy, Nh xut bn Thun Ha, 2002, trang 35. Anjali Singh, Satya Parakash Singh, Ramesh Bamezai, Momordica charantia L. (Bitter Gourd) peel, pulp, seed and whole fruit extract inhibits mouse skin papillomagenesis, Toxicology Letters, Vol 94, 1998, pp. 37-46.

[24] Catherine Jilka, Beth Strifler, G. William Fortner, Esther F. Hays, and Dolores J. Takemoto, In Vivo Antitumor Activity of the Bitter Melon (Momordica charantia L.), Cancer Research, Vol 43, 1983, pp. 5151-5155. [25] Hikaru Okabe, Yumi Miyahara, Tasuo Yamauchi, Kazmoto Miyahara and Toshio Kawasaki ,Studies on the Constituents of Momordica charantia L. I. Isolation and Characterization of Momordicosides A and B, Glycosides of a Pentahydroxycucurbitane Triterpene, Chem. Pharm. Bull. ,Vol 28 (9), 1980, pp. 2753-2762. [26] Hikaru Okabe, Yumi Miyahara, and Tasuo Yamauchi, Studies on the Constituents of Momordica charantia L. II. Isolation and Characterization of Mimor Seed Glycosides, Momordicosides C, D and E, Chem. Pharm. Bull., Vol 29 (6), 1981, pp. 1561-1566. [27] Hikaru Okabe, Yumi Miyahara, and Tasuo Yamauchi, Studies on the Constituents of Momordica charantia L. III. Characterization of New Cucurbitacin Glycosides of the Immature Fruits. Structures of Momordicosides G, F1, F2, and I, Chem. Pharm. Bull. , Vol 30 (11), 1982, pp. 3977-3986. [28] Hikaru Okabe, Yumi Miyahara and Tasuo Yamauchi, Studies on the Constituents of Momordica charantia L. IV. Characterization of New Cucurbitacin Glycosides of

61 the Immature Fruits. Structures of the Bitter Glycosides, Momordicosides K and L, Chem. Pharm. Bull. ,Vol 30 (12), 1982, 4334-4340. [29] Holm, L. et al., A geographical atlas of wo ld weeds, 1979. [30] Mai Phng Mai, Nguyn Ngc Hnh, Nguyn Th Hnh, Hypoglycemic activity of Momordica charantia L. fruit extracts in streptozotoxin induced diabetic mice, Proceedings of the thirth Indochina Conference on Pharmaceutical Sciences, Bangkok, Thailand, 2003, May 20-23. [31] Mee Jung Jung et al., Isolation of Flavonoids and a Cerebroside from the Stem Bark of Albizzia julibrissin, Arch Pharm Res, Vol 27, No. 6, 2004, pp. 593-599. [32] M. M. Lotlikar, M. R. Rajarama Rao (1966), Pharmacology of a Hypoglycaemic Principles Isolated from the Fruits of Momordica charantia Linn, The Indian Journal of Pharmacy, Vol 28, No. 5, pp. 129-133. [33] Sabira Begum, Mansoor Ahmed, Bina S. Siddiqui, Abdullah Khan, Zafar S.Saify and Mohammed Arif, Triterpenes, a Sterol and a Monocyclic Alcohol from Momordica charantia L., Phytochemistry, Vol 44, No. 7, 1997, pp. 1313-1320.

[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.

62

PH LC

DANH MC CC PH LC
Trang Ph lc 1.1: Ph 13C-NMR ca MC1B PL1 PL2 PL3 PL4 PL5 PL6 PL7 PL8 PL9 PL10 PL11 PL12 PL13 PL14 PL15 PL16

Ph lc 1.1a: Ph 13C-NMR ca MC1B Ph lc 1.1b: Ph 13C-NMR ca MC1B Ph lc 1.2: Ph lc 1.2a: Ph lc 1.3: Ph 13C-NMR v DEPT ca MC1B Ph 13C-NMR v DEPT ca MC1B Ph 1H-NMR ca MC1B

Ph lc 1.3a: Ph 1H-NMR ca MC1B Ph lc 1.3b: Ph 1H-NMR ca MC1B Ph lc 1.3c: Ph 1H-NMR ca MC1B Ph lc 2.1: Ph lc 2.2: Ph lc 2.3: Ph lc 2.4: Ph lc 2.5: Ph lc 2.6: Ph IR ca MC6 Ph 13C-NMR ca MC6 Ph 13C-NMR v DEPT ca MC6 Ph 1H-NMR ca MC6 Ph HSQC ca MC6 Ph HMBC ca MC6

Ph lc 2.4a: Ph 1H-NMR ca MC6

63

Ph lc 1.1: Ph 13C-NMR ca MC1B

64

Ph lc 1.1a: Ph 13C-NMR ca MC1B

65

Ph lc 1.1b: Ph 13C-NMR ca MC1B

66

Ph lc 1.2: Ph 13C-NMR v DEPT ca MC1B

67

Ph lc 1.2a: Ph 13C-NMR v DEPT ca MC1B

68

Ph lc 1.3: Ph 1H-NMR ca MC1B

69

Ph lc 1.3a: Ph 1H-NMR ca MC1B

70

Ph lc 1.3b: Ph 1H-NMR ca MC1B

71

Ph lc 1.3c: Ph 1H-NMR ca MC1B

72

Ph lc 2.1: Ph IR ca MC6

73

Ph lc 2.2: Ph 13C-NMR ca MC6

74

Ph lc 2.3: Ph 13C-NMR v DEPT ca MC6

75

Ph lc 2.4: Ph 1H-NMR ca MC6

76

Ph lc 2.4a: Ph 1H-NMR ca MC6

77

Ph lc 2.5: Ph HSQC ca MC6

78

Ph lc 2.6: Ph HMBC ca MC6