Luan Van 08 SP HH NDT

I HC THI NGUYN TRNG I HC S PHM ------------------------------
NG C TRNG
NGHIN CU HO HC V NHN DNG MT S NHM CHT C TRONG CY CH RNG CA (PHYLLANTHUS URINARIA L., EUPHORBIACEAE)
LUN V N TH C S HO HC
THI NGUYN - 2008
I HC THI NGUYN TRNG I HC S PHM ---------------------------------
NG C TRNG
NGHIN CU HO HC V NHN DNG MT S NHM CHT C TRONG CY CH RNG CA (PHYLLANTHUS URINARIA L., EUPHORBIACEAE)
Chuyn ngnh : Ho hu c M s : 60.44.27
LUN V N TH C S HO HC
Ngi hng dn khoa hc: PGS.TS PHM VN THNH
THI NGUYN - 2008
LI CM N
Ti xin chn thnh by t lng cm n su sc ca mnh ti PGS.TS Phm Vn Thnh - Ngi thy tn tnh hng dn, ng vin v gip ti trong sut qu trnh hc tp, nghin cu v thc hin lun vn. Ti xin chn trn trng cm n TS. Nguyn Quyt Tin, TS. Phm Th Hng Minh, Th.S. V Anh Tun, Th.S Ha Vn Thao nhng ngi thy ng vin v gip tng bc i ca ti trong qu trnh nghin cu thc hin lun vn. Xin chn thnh cm n phng hot cht sinh hc ca trng i hc Y Thi Nguyn v c s sn xut kinh doanh thuc thnh phm ca Bc s Hong Sm, phng nghin cu cu trc - Vin Ha hc tn tnh gip v to mi iu kin thun li ti hon thnh cc k hoch nghin cu. Nhn dp ny, ti cng xin chn trng cm n Ban Gim hiu, Ban lnh o Khoa Ha, Khoa Sau i hc - Trng i hc S phm Thi Nguyn to mi iu kin thun li cho ti hon thnh bn lun vn ny.
Thi Nguyn, thng 9 nm 2008 Tc gi
Ng c Trng
LI CAM OAN Ti xin cam oan y l cng trnh nghin cu ca ring ti, cc s liu, kt qu nu trong lun n ny l trung thc v cha tng c ai cng b trong bt k cng trnh no khc.
Tc gi
NG C TRNG
DANH MC CC CH VIT TT DNG TRONG LUN VN
SKLM UV MS EI-MS LC-MS FT-IR NMR

1
: Sc k lp mng : Ultraviolet spectrocopy : Mass Spectroscopy : Electron Impact Mass Spectroscopy : Liqud chromatography - Mass Spectroscopy : Fourier Transform Infrared Spectroscopy : Nuclear Magnetic Resonance : 1H-Nuclear Magnetic Resonance : 13C- Nuclear Magnetic Resonance : Distortionless Enhancement by Polarisation Transfer : Correlated Spectroscopy : Heteronuclear Spectroscopy- Quantum Coherence : Heteronuclear multiple - Bond Correlation : Human Immunodeficiency Virus : n v Cacbon
H-NMR C-NMR
13
DEPT COSY HSQC HMBC HIV vC
DANH MC CC BNG Trang Bng 2.1: Khi lng cht tng s c chit tng phn on ca cy ch rng ca (Phyllanthus urinaria L)....26 Bng 2.2: Pht hin cc nhm cht trong cy ch rng ca27 Bng 2.3: Kt qu th hot tnh sinh hc ca dch chit th t cy ch rng ca (Phyllanthus urinaria L)........29 Bng 2.4: S liu ph 13C-NMR (CDCl3, 125Mhz) ca cht PH-1 trong cy ch rng ca (Phyllanthus urinaria L).......30 Bng 2.5: Ph 1H-NMR v 13C-NMR ca cht PE-3...32 Bng 3.1: S liu ph
13
C-NMR (CDCl3, 125Mhz) ca PH-1 trong cy ch
rng ca (Phyllanthus urinaria L) v ph ca -sitosterol [15]...40 Bng 3.2: S liu ph NMR ca PE-1 v s liu ph NMR trong phn mm ACD/NMR ca 5-hidroxymetylfufural................................................44 Bng 3.3: S liu ph NMR ca PE-2 v s liu ph NMR trong phn mm ACD/NMR ca axit gallic....................................................................48 Bng 3.4: S liu ph NMR ca PE-3 v s liu ph trong phn mm ACD/NMR ca cht kampherol...........................................................52
DANH MC CC HNH Trang Hnh 1.1: Cy ch rng ca (Phyllanthus urinaria L, Euphorbiaceae............. 3 Hnh 2.1: ng knh vng c ch xung quanh ging thch (mm) theo phng phng php khuych tn trn thch....................................................28 Hnh 3.1: Ph FT-IR ca -sitosterol (PH-1).......................................................36 Hnh 3.2: Ph 1H-NMR ca -sitosterol (PH-1)..................................................37 Hnh 3.3: Ph 13C-NMR v ATP ca -sitosterol (PH-1)....................................38 Hnh 3.4: Ph 1H-NMR-DMSO ca PE-1...........................................................42 Hnh 3.5: Ph 13C-NMR-DMSO ca PE-1..........................................................43 Hnh 3.6: Ph 1H-NMR-DMSO ca PE-2...........................................................46 Hnh 3.7: Ph 13C-NMR-DMSO ca PE-2..........................................................47 Hnh 3.8: Ph 1H-NMR-AcetoneD6 ca PE-3.....................................................50 Hnh 3.9: Ph 13C-NMR-AcetoneD6 ca PE-3....................................................51
DANH MC CC S S 2.1: Quy trnh ngm chit mu...................................................................26
MC LC Li cm n Li cam oan Danh mc cc ch vit tt dng trong lun vn Danh mc cc bng Danh mc cc hnh v s M U ........................................................................................................1
CHNG I. TNG QUAN ................................................................................3
1.1. M t thc vt ............................................................................................3 1.2. Tc dng sinh hc ca chi Phyllanthus........................................................4 1.2.1 Mt s cng dng ca chi Phyllanthus ......................................................4 1.2.2 Mt s tc dng dc l ca chi Phyllanthus .............................................5 1.3 Tnh hnh nghin cu ho hc thc vt ca chi Phyllanthus ..........................6 1.3.1 Mt s i din ca nhm tecpenoit ..........................................................6 1.3.2 Mt s i din ca khung axit..................................................................8 1.3.3 Mt s i din ca Lignan .......................................................................9 1.3.4 Mt s i din ca khung flavonoit........................................................11 1.3.5 Mt s hp cht phenolic khc ................................................................12 1.3.6 Mt s hp cht nhm ankaloit ...............................................................15 1.4 Tnh hnh nghin cu ha hc ca loi Phyllanthus urinaria L....................16
CHNG II. THC NGHIM..........................................................................23
2.1. i tng v phng php nghin cu ......................................................23 2.1.1. Thu mu cy, xc nh tn khoa hc v phng php x l mu..............23 2.1.2. Phng php phn lp cc hp cht t cc dch chit ..............................23 2.1.3. Phng php kho st v xc nh cu trc ho hc cc hp cht ............24
2.2. Dng c, ho cht v thit b nghin cu ...................................................24 2.2.1. Dng c v ho cht ..............................................................................24 2.2.2. Thit b nghin cu................................................................................25 2.3. Cc dch chit t cy ch rng ca (Phyllanthus urinaria L)..................25 2.3.1. Cc dch chit .......................................................................................25 2.3.2. Kho st nh tnh cc dch chit ............................................................27 2.3.3. Th hot tnh sinh hc ...........................................................................27 2.4. Phn lp, tinh ch cc cht t ....................................................................29 2.4.1. Dch chit n-hexan ................................................................................29 2.4.2. Dch chit trong etylaxetat (PE) .............................................................31 2.4.2.1 Cht PE-1............................................................................................31 2.4.2.2 Cht PE-2............................................................................................31 2.4.2.3 Cht PE-3............................................................................................32
CHNG III. THO LUN KT QU NGHIN CU....................................34
3.1. Nguyn tc chung ....................................................................................34 3.2. Xc nh nh tnh cc nhm cht thin nhin............................................34 3.3. Phn lp v nhn dng cc hp cht ..........................................................35 3.3.1. -sitosterol (PH-1).................................................................................35 3.3.2. 5-Hydroxymetylfufural (PE-1) ...............................................................41 3.3.3. Axit gallic (PE-2) ..................................................................................45 3.3.4. Kampherol (PE-3) .................................................................................49 3.4. Th hot tnh sinh hc ..............................................................................53 KT LUN.................................................................................................... 55
DANH MC CC CNG TRNH CNG B LIN QUAN N LUN VN .... .56 TI LIU THAM KHO ..................................................................................57 PH LC .......................................................................................................62
M U
T xa n nay, nhng cy thuc dn gian vn ng vai tr ht sc quan trng trong i sng hng ngy ca con ngi. T trc khi c s ra i ca thuc ty, nhiu loi cy c trong t nhin c s dng trong dn gian cha bnh v rt c hiu qu. Rt nhiu loi bnh tt c cha khi nh tho dc. Ngy nay nhng hp cht t nhin c phn lp t cy c c ng dng rng ri trong rt nhiu ngnh cng nghip, nng nghip, chng c dng sn xut thuc cha bnh, thuc bo v thc vt, lm nguyn liu cho ngnh cng nghip thc phm v m phm v.v... Mc d cng ngh tng hp ho dc ngy nay pht trin mnh m, to ra cc bit dc khc nhau s dng trong cng tc phng, cha bnh. iu gp phn lm tng tui th con ngi, song nhu cu s dng cy c lm thuc cng ngy cng tng ln, c khoa hc hin i soi sng, v trong chng c cha nhng bit dc rt kh tng hp. Mt khc vic dng thuc nam hu nh khng gy ra tc dng ph. C nhiu b mn khoa hc nghin cu v cy thuc ra i. Vic nghin cu cy thuc gip cho chng ta hiu r v thnh phn v cu trc ha hc, hot tnh sinh hc, tc dng dc l ca cy thuc. Trn c s cc nghin cu c th to ra cht mi c hot tnh sinh hc cao nh mong mun lm thuc cha bnh. Cy ch rng ca l mt cy thuc c s dng t lu trn th gii cng nh Vit Nam. Trong Y hc dn tc cy ny c nhn dn dng lm thuc cha nhiu loi bnh nh: au vim hng, inh ru, mn nht, vim da l nga, sn hu huyt au bng, tr em ta li, chm m, bnh vim gan, rt c hiu qu.
S ha bi Trung tm Hc liu i hc Thi Nguyn
http://www.lrc-tnu.edu.vn
Thc vt ch rng ca c nhiu ng dng quan trng nhng gn y mi c cc nh khoa hc cc nc quan tm chn lm i tng nghin cu, cn nc ta hin c rt t cng trnh nghin cu v thnh phn ha hc v dc l hc ca cy ch rng ca. Vi mc ch nghin cu v tm hiu thnh phn ha hc cc hp cht c hot tnh sinh hc ca cy ch rng ca, gp phn lm tng thm s hiu bit v ngun thc vt lm thuc phong ph v qu gi ca Vit Nam. Chng ti chn cy ch rng ca (Phyllanthus urinaria L.) lm i tng nghin cu cho cng trnh nghin cu ny. Tn ti l: Nghin cu ha hc v nhn dng mt s nhm cht c trong cy ch rng ca (Phyllanthus urinaria L., Euphorbiaceae).
3 CHNG I
TNG QUAN
1.1 M t thc vt Cy ch rng ca cn gi l dip h chu, dip ho thi, lo nha chu, trn chu tho, cam kim, rt t, khao ham (Ty), prakphle (Campuchia). Tn khoa hc l: Phyllanthus urinaria L, thuc h thu du (Euphorbiaceae). Phn b a l: Cy ch rng ca phn b rng khp cc vng nhit i v cn nhit i. Vit Nam, cy ch rng ca thng thy mc t nhin hu ht cc tnh thnh trong c nc, in hnh nh: Thi Nguyn, Bc Ninh, Ngh An, H Giang, Trn th gii loi cy ny mc nhiu cc nc Chu nh: Trung Quc, n , Malaysia, Philippin, Inonexia, Myanma, Thi Lan, Chu M nh: Brazil, Argentina,[5].
Hnh 1.1: Cy ch rng ca (Phyllanthus urinaria L, Euphorbiaceae)
c im sinh trng v pht trin: Cy ch rng ca l mt loi c mc hng nm, cao chng 30 cm, thn gn nh nhn, mc thng ng, mang cnh, thng c mu hi ta. Cy c v hi ngt. L mc so le lng h hay c cung rt ngn. Hoa mc k l, n tnh, mu nu. Hoa c v hoa ci cng gc. Hoa c u cnh, hoa ci cui cnh. Hoa khng c cung hoc c cung rt ngn. Qu nang khng cung hnh cu hi dt. ng knh qu c th t ti 2mm. Ht ba cnh hnh trng mu nu nht c vn ngang [5]. 1.2. Tc dng sinh hc ca chi Phyllanthus 1.2.1 Mt s cng dng ca chi Phyllanthus Cc loi cy thuc chi Phyllanthus (Euphorbiaceae) c s dng rng ri trong y hc dn tc ca nhiu nc cha bnh thn, bnh tiu ng, bnh vim gan B[7]. Cc loi c dng lm thuc nhiu hn c l 3 loi Phyllanthus urinaria L, Phyllanthus niruri L v Phyllanthus amarus Schum et Thonn [4]. Mt s bi thuc dng cy ch ng (Phyllanthus amarus Schum et Thonn) [6]: Cha vim gan do virus: Ch ng sao kh 20g, sc nc 3 ln. Trn chung cc nc sc. Thm 50g ng, un si cho tan ng. Chia lm 4 ln ung trong ngy. Khi kt qu xt nghim HBsAg (-) th ngng thuc. Cha x gan c trng th nng: Ch ng sao kh 100g sc nc 3 ln. Trn chung nc sc, thm 150g ng, un si cho tan ng, chia nhiu ln ung trong ngy (thuc rt ng), liu trnh 30-40 ngy. Khu phn n hng ngy phi hn ch mui, tng m (tht, c, trng, u ph). Ring cy ch rng ca (Phyllanthus urinaria L) trong y hc dn tc c nhn dn dng cha au vim hng, inh ru, mn nht, vim da, l nga, sn hu huyt au bng, tr em ta li, chm m, cha bnh gan, st, rn rt cn rt c hiu qu.
Mt s bi thuc dng cy ch rng ca (Phyllanthus urinaria L) [5], [6]: Nhn dn ta thng dng ton cy hi v lm thuc, ma thu hi quanh nm nhng tt nht l vo ma h. Thng dng ti c khi phi kh, ngy ung 20-40g cy ti, sao kh, sc c, ung thay nc. Cha suy gan (do st rt, sn l, l amip, mt, nhim c): Ch rng ca sao kh 20g, cam tho t sao kh 20g. Sc nc ung hng ngy. i vi bnh ngoi da: Cy ti d nt vi mt t mui p ngoi da vi liu lng khng hn ch. 1.2.2 Mt s tc dng dc l ca chi Phyllanthus Nm 1961, phng ng Y Vin Vi trng Vit Nam, nghin cu tc dng khng sinh ca cy ch rng ca thy kt qu tc dng khng sinh nh sau: T cu trng (0,5 cm), Typhi (0,9 cm), Flexneri (1,1cm), Sonnei (0 cm), Shiga (1cm), Subtilis (0,4 cm), Coli (0 cm). Nm 1977, mt nhm bc s Vit Nam, khoa Tiu ho, Gan, Mt s dng bi thuc gia truyn ca Lng y Trn Xun Thin gm 3 v l ch ng, xuyn tm lin, qu dnh dnh iu tr cho nhng ngi c kt qu xt nghim HBsAg (+). Sau mt thi gian iu tr, kt qu xt nghim m tnh c coi l khi. T l t 26/98 bnh nhn. Ngoi ra, thuc cn gip c th ngi dng sn xut khng th chng HBsAg (59/98 ngi). Liu iu tr trung bnh 4-5 thng [6]. Nm 1988 cc tc gi Blunberg v Thiogarajan cng b iu tr 37 bnh nhn vim gan siu vi B bng ch rng ca Phyllanthus amarus v Phyllanthus niruri t kt qu m tnh 22/37 bnh nhn sau 30 ngy. Cc tc gi cn chng minh Phyllanthus amarus c cha cht lm c ch men pelymerase DNA ca virus vim gan siu vi B [5].
Nm 2002, Nguyn B Kinh v cng s cng b kt qu nghin cu lm sng ti Bnh vin Thanh Nhn (H Ni), s dng ch phm LIV/94 (ch rng ca l mt trong 3 thnh phn chnh ca thuc) iu tr cho cc bnh nhn vim gan mn tnh trong 2 nm (2001-2002) t kt qu tt. Thuc c tc dng lm gim v sch HBsAg ca bnh nhn [6]. Nhng cng trnh nghin cu ha hc gn y v cc loi Phyllanthus pht hin mt vi lignan, flavonoit v tanin thy phn c tc dng bo v gan, c kh nng lm sch phn ln cc khng nguyn HBsAg, c ch mnh HIV transcriptase ngc [21]. Cc th nghim in vitro ca cy ch vi khng nguyn HBsAg v vi tn thng gan do cacbontetraclorit gy nn chng minh cy ch c kh chng virus vim gan B. Cy ch rng ca c tc dng khng khun i vi t cu vng, trc khun m xanh, trc khun Coli, Shigella dysenteriae, S. flexneri, S. shigae, Moraxella v khng nm i vi Aspergillus fumigatus [1]. 1.3 Tnh hnh nghin cu ho hc thc vt ca chi Phyllanthus Cho n nay ngi ta pht hin chi Phyllanthus L. h thu du (Euphorbiaceae) c 700 loi, gm t nhng cy thn tho n cy bi hay cy g nh. Thnh phn ho hc ca chi ny rt phong ph v a dng [11]. Vit Nam c 44 loi, cc loi ng c ch nhiu hn c l Phyllanthus urinaria L. (ch rng ca), Phyllanthus niruri L. (ch thn xanh) v Phyllanthus amarus Schum et Thonn (ch ng hay dip h chu ng). 1.3.1 Mt s i din ca nhm tecpenoit T loi Phyllanthus flexuosus phn lp c 5 tritecpen mi, cu trc c ch ra l: olean-12-en-3,15-diol; lup-20 (29)-en-3,24-diol; olean-12-en-3,24-diol; oleana-11:13(18)-dien-3,24-diol v olean-12-en3,15,24-triol v hai cht bit l: tritecpen diol betulin [33]; lup-20(29)en-3,15-diol [41].
Theo [39], t loi Phyllanthus oxyphyllus tch c: 29-nor-3,4seco-friedelan tritecpen (axit 29-nor-3,4-seco-friedelan-4(23),20(30)-dien-3oic) v mt secquitecpen mi l guaiane secquitecpen (5-hydroxy-6,9epoxyguaiane). Nhng hp cht ny c nh gi l c kh nng chng oxi ha tt nng th nghim IC50 = 0.017 0.001 mM.
O
HOOC
HO
1. 29-nor-3,4-seco-friedelane tritecpen
2. Guaiane secquitecpen
T loi Phyllanthus reticulates phn lp c mt hp cht mi l 21-hydroxyfriedel-4(23)-en-3-one v mt vi triterpenoit khc, bit l: friedelin, sitosterol, friedelan-3-ol, glochidonol v 21-hydroxyfriedelan - 3 - on [47]. Theo ti liu [46], t loi Phyllanthus plyanthus Chu Phi cc tc gi tm ra c mt s tritecpenoit: phyllanthol, phyllanthone, (20S)-3acetoxy-24-methylenedammaran-20-ol, -amyrin acetate, lupenone v (20S)3-acetoxy-24-methylenedammaran-20-ol.
HO H H
H CH3OCO H
3. (20S)-3-acetoxy-24-methylenedammaran-20-ol
T loi Phyllanthus acidus tch c hai pentacyclic tritecpenoit, l: phyllanthol v olean-12-en-3-ol (-amyrin) [35]. T loi Phyllanthus niruri tch c mt acyclic tritecpen, cu trc ca n c d on da trn cc phng php phn tch ph l: 3,7,11,15,19,23-hexamethyl-2Z,6Z,10Z,14E,18E,22E-tetracoshexen-1-ol [10]. T loi Phyllanthus watsonii tch c hp cht tritecpen mi: 26Nor-D:A-friedooleanane tritecpen cng vi lupenyl palmitate, friedelin, epifriedelanol, glochidone, glochidonol, lupeol, lup-20(2) [29]. 1.3.2 Mt s i din ca khung axit T loi Phyllanthus amarus phn lp c axit thiobarbituric, axit ascorbic [42]. T dch chit n-hexan cng ca loi ny Malaysia tch c axit oleanolic v axit ursolic [16], axit amariinic, axit repandusinic A, axit geraniinic B v mt cht l 1-O-galloyl-2,4-dehydrohexahydroxydiphe noyl -glucopyranoz elaeocarpusin [26]. T loi Phyllanthus emblica tch c axit phyllaemblic [52], axit ascorbic [23]. T loi Phyllanthus fraternus tch c axit indole-3-butyric, axit 1naphthaleneacetic [31] v axit trichadenic B c cu trc nh axit 3-hydroxyD: A-friedooleanan-27-oic, c xc nh da vo phng php phn tch Xray [38].
HOOC
HO
4. Axit trichadenic B
1.3.3 Mt s i din ca Lignan Trong loi Phyllanthus discoides ngi ta tch c phyllanthin C14H17O2N, phyllantin C13H15O3N [27] v phyllantidin C 13H15O2N [20]. Trong loi Phyllanthus niruri L c phyllanthin C 24H34O6, hypophyllanthin C 24H30O7, niranthin C24H30O7 (cu trc ca niranthin c ch ra l (+)3,4,3,4,9,9-hexamethoxy-8:8-butyrolignan), nirtetralin C24H30O7 v phyllteralin C24H34O6 [8]. Da trn cc d liu ph khi lng v ph cng hng t ht nhn, cu trc ca nirtetralin v phyltetralin c ch ra l 1phenyl tetralins.
H RO CH2OCH3
H H3CO CH2OCH3
R1O H R2
CH2OCH3
H3CO H H
CH2OCH3
OCH3 OCH3
O O
OCH3
5 5a. Phyllanthin R= R 1 =CH3, R2 =H 5b. Niranthin R+R1= -CH2 -R2 = OCH3
6 Hypophyllantin
H
H O CH2OCH3
CH2OCH3
O H H
CH2OCH3
H3CO H OCH3 H
CH2OCH3
OCH3 OCH3
OCH3 OCH3
7. Nirtetralin
8. Phyllteralin
10
Cng t phn dch chit n-hexan ca loi ny, ngi ta tch c triacontanal v triacontanol [40]. Cc cht hypophyllanthin, phyllanthin cng c phn lp trong cn n-hexan bng phng php sc k lng trung p (MPLC) t loi Phyllanthus niruri L vng nh Bng, T Sn, Bc Ninh nc ta [4], y l hai lignan c chng minh l c hot tnh bo v gan trn m hnh gy c t bo gan bng CCl4 v D- GalN/ TNF- [3], [45]. Theo ti liu [34], t loi Phyllanthus niruri tch c 4 diarylbutane lignan mi, chng c coi l sn phm chuyn ha thuc h thng 5 -desmethoxy niranthin. Trong hai cht (9a) v (9b) l sn phm chuyn ha t axit veratrylidene piperonylidene succinic, (10a) c chuyn ha t axit veratrylidene 3,4,5-trimethoxy benzylidene succinic v (10b) c chuyn ho t (10a).
CH2OR
RO
CH2OMe
CH2OR
RO OMe
CH2OMe
OMe OMe
OMe
OMe
9a. R=H; 9b. R=CH3
10a. R=H; 10b. R=CH3
T loi Phyllanthus polyphyllus L tch c cc lignan l arylnaphthalide, phyllamyricin C, justicidin B v diphyllin [51]. T loi Phyllanthus anisolobus tch c ichtyotoxic lignan justicidin B v lignan-glucozit phyllanthostatin A [43].
11
1.3.4 Mt s i din ca khung flavonoit T dch chit etanol loi Phyllanthus emblica L phn lp c quecxetin v catechin. y l hai cht c hot tnh chng oxi ha tt [49].
OH HO O OH
HO
* *
OH OH OH
OH
OH OH O
11. Quecxetin
12. Catechin
Cng v vn nghin cu kh nng chng oxi ha, cc nh khoa hc kho st kh nng ny t dch chit methanol ca 5 loi phyllanthus c bit t n l: Phyllanthus debilis, Phyllanthus debilis, Phyllanthus urinaria, Phyllanthus virgatus, Phyllanthus maderaspatensis, Phyllanthus amarus. Tt c cc dch chit ny u cho kt qu tt [22]. T loi Phyllanthus sellowianus phn lp c rutin v isoquercitrin l hai flavonoit chim thnh phn ch yu trong cy ny [19].
OH HO O OH
HO
OH
OH
O -Ramnozo -glucozo OH O
OH
13. Rutin
14. isoquercitrin
Theo ti liu [36], t loi Phyllanthus niruri tch c 2 flavanon: 8-(3-metyl-but-2-enyl)-2-phenyl chroman-4-on (15) v 2-(4-hydroxyphenyl)-
12
8-(3-metyl-but-2-enyl)-chroman-4-on (16). Hai cht trn c cc tc gi nhn nh l c kh nng lm thuc cha bnh Gt (Gout) v bnh thn.
3'' 3' 1''
3'' 3'
2''
1 8 8a
O
2' 1' 2 6'
4' 5'
7 6
1''
2'' 1 8 8a
O
OH
2' 1' 2 6'
4' 5'
7 6
4a 5
4a 5
15 1.3.5 Mt s hp cht phenolic khc
16
T loi Phyllanthus niruri Linn tch c mt s hp cht phenolic l: axit gallic, axit ellagic, ellagitannin, ()-epicatechin, (+)-gallocatechin, ()epigallocatechin, ()-epicatechin 3-O-gallat v ()-epigallocatechin 3-O-gallat [28]. Hp cht ellagitannin cng c tch t loi Phyllanthus amarus [26]. Cc tc gi i Loan phn lp v nhn dng mt s hp cht phenolic v nghin cu hot tnh chng oxi ha ca cc chng t loi phyllanthus emblica L [50]. Nguyn liu sau khi lm kh c ngm chit trong metanol v sau c chit ln lt vi etyl ete, etyl axetat, butanol v nc. Phn on etyl axetat cho thy c hm lng ln nht ca hp cht 1,1-diphenyl-2-picrylhydrazyl (DPPH) so vi c bn phn on. Phn on etyl axetat c tch bng sephadex v thu c 6 hp cht, chng c nhn dng l: geraniin, quercetin-3--D-glucopyranozit, kaempferol 3--Dglucopyranozit, isocorilagin, quercetin , v kaempferol. Mt s hp cht trn ln u tin c tch t loi Phyllanthus emblica. Cc hp cht tch, c lm sch v em kho st kh nng chng oxi ha. Kt qu cho thy trong s 6 cht trn th geraniin l cht c kh nng chng oxi ha tt nht ( nng IC50 = 4.7 v 65.7 M, th nghim s oxi ha lipit).
S ha bi Trung tm Hc liu i hc Thi Nguyn http://www.lrc-tnu.edu.vn
13
T loi Phyllanthus amarus tch c axit shikimic (3- and 5-O-gallat), flavan-3-ol (epigallocatechin 3-gallat), proanthocyanidin v hydrolysable tannin (corilagin, casuariin, geraniin) [17], [14].
OH OH
COOH
HO
O OH
OH
O
HO OH OH
C O OH
OH
OH
17. Axit shikimic

OH HO
18. Epigallocatechin-3-gallat
HO
OH
HO
OH OH OC O O O OH CO
C O OH
OH
OH
19. Proanthocyanidins Theo L.Yeap Foo, Herbert Wong [24], cng t loi ny tch c mt sn phm t tannin thy phn (hay ellagitannin c bit) l phyllanthusiin D c cu trc c xc nh l: 1-galloyl-2,4-(acetonyldehydrohexahydroxydiphenoyl) -3,6-hexahydroxydiphenoylglucopyranozit. Theo ti liu [25] cng t loi ny tch c amariin, l mt sn phm thy phn ca di-dehydrohexahydroxydiphenoyl (DHHDP), cu trc ca n c xc nh l 1-galloyl-2,4: 3,6-bis-DHHDP-glucopyranosit trong phn
14
vng cyclohexenetrione ca DHHDP lin kt vi nguyn t O-3 v O-4 ca phn ng glucoz, cng vi: geraniin, corilagin, 1,6-digalloylglucopyranozit cng nh rutin v quercetin-3-O-glucopyranozit c phn lp t Phyllanthus amarus. Cng t loi ny tch c axit amariinic - l mt dng ellagitannin cng vi 1-O-galloyl-2,4-dehydrohexahydroxydiphenoyl-glucopyranoz elaeocarpusin, axit repandusinic A v axit geraniinic B [26]. Cu trc ca axit amariinic c chng minh bng cht oxi ha m phn vng cyclohexentrione ca DHHDP c gn vo O-4 ca phn ng glucoz. T loi Phyllanthus polyphyllus L i Loan, Yew-Min Tzeng v cc cng s tch c benzenoit, axit 4-O-methylgallic [51], hp cht ny ch yu c to thnh t s chuyn ha axit gallic v n c nhiu trong ru vang , ch, trong nhiu loi tri cy. Cng t loi ny tch c 3 arylnaphthalide lignan c tn l: phyllamyricin C, justicidin B v diphyllin. T phn dch chit etylaxetat ca loi Phyllanthus niruri L, Vng nh Bng, T Sn, Bc Ninh phn lp c hai cht l: 2,4,6xyclohepta-trien-2,3,7-trihydroxy-5-ethoxy-1-on (20) v 5,7,8 trihidroxi9,10- peoxi- 4- ethoxy benzo[2,3-d] coumarin (21), hai cht ny cho n nay cha thy thng bo c mt trong cc loi Phyllanthus [4].
HO O O - CH2 -CH3 HO OH
20. 2,4,6- xyclohepta-trien-2,3,7-trihydroxy-5-ethoxy-1-on
15
OH HO
OCH2CH3
HO O O
21. 5,7,8 - trihidroxi-9,10- peoxi- 4- ethoxy benzo[2,3-d] coumarin Hot tnh chng oxy ha ca 5,7,8 trihidroxi-9,10- peoxi- 4- ethoxy benzo[2,3-d] coumarin cng vi quercetin lm i chng c th bng phng php Potterat trong cng mt iu kin. Cho thy cht ny th hin hot tnh chng oxy ha yu hn so vi quercetin ( nng 25 mg/l vn cn phn ng dng tnh) [4]. 1.3.6 Mt s hp cht nhm ankaloit T loi Phyllanthus amarus tch c 2 ankaloit mi l isobubbialine v epibubbialine cng vi 3 ankaloit bit l phyllanthine, securinine v norsecurinine. Cu trc ca chng c xc nh trn c s phn tch cc d liu ph UV, IR, ph khi lng v ph NMR [30]. T loi Phyllanthus niruroides bang Florida nc M cc tc gi: Babady-Bila, Thomas E. Gedris v Werner Herz, phn lp c mt hp cht mi thuc nhm ankaloit l niruroidine, cu trc ca n c lm sng t da trn cc phng php ph [9]. T loi Phyllanthus simplex ln u tin phn lp c mt ankaloit l simplexine (14-hydroxy-4-methoxy-13,14-dihydronorsecurinine) v mt ankaloit bit l phyllanthine [32]. Cht ny v ang c s dng lm thuc Ty Y dng vin nn (sn phm c tn l simplexin), ph bin trn th trng. Ngoi nhng nhm hp cht trn, t loi Phyllanthus anisolobus tch c hai -lacton: menisdaurilide v aquilegiolide [43].
16
1.4 Tnh hnh nghin cu ha hc ca loi Phyllanthus urinaria L T loi Phyllanthus urinaria L. tch c mt s triterpen nh: stigmasterol, daucosterol, sigmasterol-3-O- -glucosid, -sitosterol, glucosid, lupeol acetat, -amyrin, lup-20 (29)-en-3 -ol [5], [53]. Cht
-sitosterol cng c phn lp t dch chit n-hexan ca loi Phyllanthus
niruri L. tnh Bc Ninh nc ta [4]. Cu trc mt s hp cht in hnh c xc nh nh sau:
HO
glucozo- O
22. -Amyrin
23. Glucosid
HO
HO
24. -Sitosterol
25. Stigmasterol
T loi Phyllanthus urinaria L tch c mt s axit hu c nh: Axit succinic, axit ferulic, axit dotriancontanic, axit hecxacosanoic [11], [53].
17
HO
HO
OH
H3CO
26. Axit ferulic Gn y, cng t loi cy ny, nhm cc nh khoa hc Trung Quc phn lp c 5 hp cht, l: ester mono-[2-(4-carboxy-phenoxycarbonyl)vinyl] terephthalat, axit (E)-3-(5-hydroperoxy-2,2-dihydroxy[1,1biphenyl]-4-yl)-2-propenoic, axit 3,4,5-trihydroxybenzoic, axit succinnic (hay axit butanedioic) v axit 2,3,4,5,6-pentahydroxybenzoic . Trong 25 v 26 l hai hp cht mi, ln u tin c phn lp t loi Phyllanthus urinaria L [48].
O O O HO O O
OH
27
H3C O HO O
HO
OH
28 T loi Phyllanthus urinaria L phn lp c 4 lignan m i l 5-demethoxyniranthin (29a. C 23 H30 O 6 ), urinatetralin (30a.
18
C 22 H24 O 6 ), dextrobursehernin (31a. C21H22O6), urinaligran (32. C22H24O7) , cng vi 9 lignan bit l: phyllanthin (29b), niranthin (33), phyltetralin (30b), hypophyllanthin (34), nitetralin (35), lintetralin (36), isolintetralin (37), heliobuphthalminlactone (31b) v virgatusin (38) [12].
R1O
R1O
OMe
OMe OMe R2O
OMe
R2O
OMe OMe
OR2
OR1
29a. R1 + R2 = CH2 29b. R1 = R2 =Me
30a. R1 + R2 = CH2 30b. R1 = R2 =Me

OMe MeO
O R1O O R2O
O O O
O O
31a. R1 = R2 =Me 31b. R1 + R2 = CH2
32
19
O
MeO
OMe OMe
O
OMe OMe
O OMe
OMe OMe
OMe
OMe
33
O OMe OMe O OMe
34
MeO OMe OMe MeO
OMe OMe
O O
35
O OMe OMe O
36
MeO
OMe
O
OMe OMe
OMe O
OMe
37
38
Cng t loi Phyllanthus urinaria L Zhang L. Z. v cng s tch c mt cht mi l ellagitannin [54].
20
Ring loi Phyllanthus urinaria L c thu hi huyn Lng, Ngh An -Vit Nam cc nh khoa hc Vit nam ln u tin phn lp c 4-methoxyscu tellarein. Cht ny ln u tin c phn lp t cy Stachys. annua [7].
OCH3
HO
HO OH O
39. 4-methoxyscutellarein T dch chit axeton ca loi Phyllanthus urinaria L cc nh khoa hc i Loan tch c geraniin v 1,3,4,6-tetra-O-galloyl--D-glucoz [13]. Cc th nghim in vitro ch ra hai cht ny c hot tnh chng li vi rt bnh mn rp ngoi da v khng gy c. T loi ny ngi ta cn tch c axit ellagic, axit 3, 3,4- triO- methylellagic, axit gallic , 4-O-brevifolincarbonyl-1-O-galloyl-3,6O-hexahydroxydiphenoyl-D- glucopyranoz, methyl brevifolincarboxylate [5], [53].
OCH3 O
COOH
HO
OH O
HO OH
OH
HO O
40. Axit gallic
41. Methyl brevifolincarboxylate
21
Cht methyl brevifolin cacboxylat cng c tch bng phng php sc k ct t dch chit n-butanol ca cy ny huyn Lng, Ngh An [7]. Theo ti liu [37], t loi Phyllanthus urinaria L i Loan tm c cc hp cht sau: phyllanthin (42), phyltetralin (43), trimetyl3,4-dehydrochebulate (44), metylgallat (45), rhamnocitrin (46), methyl brevifolincarboxylat (47), -sitosterol-3-O--d-glucopyranosid (48), quercitrin (49)v rutin (50).
H3C
8 9 7 6 5
OH
1
O
2'
3'
4'
2 3
1' 6' 5'
OH
10
C O
OH
46. Rhamnocitrin (hay kampferol-7-methylete) Cu trc cc hp cht (44) v (47) c xc minh bng phng php ph khi lng v ph cng hng t. Cc hp cht trn c nghin cu mt cch k lng v kh nng chng oxi ha v kh nng lm thuc khng vim. Cc cht (44), (45) v (47) c kh nng chng oxi ha tt ( IC50 = 9.4, 9.8 v 8.9 M, cc ln th nghim ring r). Cn trong cc th nghim in vitro khc v hot tnh khng vim th ngoi tr (48), cc cht cn li u c hot tnh khng vim cao. Ti liu ny ln u tin thng bo th nghim v kh nng khng vim ca loi Phyllanthus urinaria v do cc cht c phn lp t cy Phyllanthus urinaria c th c s dng lm thuc khng vim trong iu tr. T loi Phyllanthus urinaria L tch c phyllurine (51) [44], cu trc ca phyllurine nh sau:
22
OH NH2
+
NH2
COO-
51. Phyllurine Cht ny (51) c nhn dng l tng t nh phyllanthurinolacton (52). Trn c s cc tc gi xut gi thuyt c s chuyn ha qua li gia (51) v (52). Theo ti liu [18], t loi ny phn lp c phyllanthurinolacton (52), cu trc ca n nh sau:
CH2 H N O
5 OH
O H2N O O HO
NH O O OH
52. Phyllanthurinolacton
23 CHNG II
THC NGHIM
2.1. i tng v phng php nghin cu 2.1.1. Thu mu cy, xc nh tn khoa hc v phng php x l mu Nguyn liu nghin cu gm ton b phn trn mt t ca cy ch rng ca, thu hi vo thng 11 nm 2007 ti vng gip ranh gia Huyn ng H v Thnh Ph Thi Nguyn. Cy ch rng ca cn gi l cy dip h chu, dn a phng gi l dip he thi, cc nh khoa hc khoa Sinh trng HSP Thi Nguyn xc nh tn khoa hc l Phyllanthus urinaria L.. H thu du (Euphorbiaceae). Mu cy ti sau khi thu hi gm c thn, l v qu c em sy 800C trong 10 pht dit men, sau sy kh nhit 60 0C cho ti khi kh hon ton. Mu kh c nghin nh v ngm chit trong etanol 90 o nhit phng nhiu ln, lin tc trong 10 ngy. Sau khi ct loi dung mi, cn c n di dng xir c chit ln lt bng cc loi dung mi c phn cc tng dn: n-hexan, etylaxetat, nbutanol, metanol. Cc dch chit c ui kit dung mi bng thit b ct quay nhit 500C di p sut thp. Cc cn th c phn chia bng sc k ct vi cc h dung mi ra gii c phn cc tng dn phn lp cc cht c phn cc gn ging nhau, kt tinh phn on v kt tinh li trong h dung mi thch hp thu c cc cht sch . 2.1.2. Phng php phn lp cc hp cht t cc dch chit pht hin, phn lp c nhng hp cht sch t cc dch th khc nhau ca cy ch rng ca chng ti phi hp s dng cc phng php sc k v kt tinh li trong dung mi thch hp cc phng php gm:
24
- Sc k lp mng (SKLM). - Sc k ct silicagen, thng dng silicagen Merck 63- 200nm. - Kt tinh phn on v kt tinh li. 2.1.3. Phng php kho st v xc nh cu trc ho hc cc hp cht Cc cht kt tinh phn lp ra c xc nh nhng hng s vt l c trng: mu sc, mi v, h s Rf, im nng chy, ghi cc loi ph nh: ph hng ngoi (FT-IR), ph cng hng t ht nhn 1 H- NMR, ph 13C- NMR, ph khi lng (MS) v cc ph phn gii cao. Cc s liu ph thc nghim ca cc cht sch c dng nhn dng cu trc ho hc ca chng. 2.2. Dng c, ho cht v thit b nghin cu 2.2.1. Dng c v ho cht Cc loi dung mi dng ngm chit mu l cc loi tinh khit (pure), cn cc loi dung mi dng sc k ct, sc k lp mng, dng kt tinh li l ho cht loi tinh khit phn tch (PA). Sc k lp mng dng bn mng nhm DC Alufolien Kiesegel 60 F254 trng sn, dy 0,2mm c s dng xc nh s b s thnh phn c trong cc dich chit, cc phn on chy ct v kim tra s b sch ca sn phm thu c. Cc h dung mi khai trin SKLM: 1. n-Hexan CHCl3 2. Cloroform etylaxetat 3. Cloroform metanol 4. Cloroform metanol 5. Toluen etylaxetat axit formic 85:15 90:10 90:10 50:10 5:4:1 H A H B H C H D H E
25
Cc bn SKLM sau khi sy kh c soi di n t ngoi (UVBIOBLOCK) bc sng = 254nm v 365nm. Thuc th hin mu l vanilin 1% trong dung dch metanol-H2SO4 5%, sau sy trn 1000C . Cc gi tr Rf trong h dung mi trin khai c biu thc:
Rf =
chiu di di chuyn ca cht th chiu di di chuyn ca dung mi
2.2.2. Thit b nghin cu - Nhit nng chy o trn my Boetus (c), hoc trn my Eletronthermal IA9200. - Ph hng ngoi ghi trn my IMPACT - 410 dng vin nn KBr. - Ph LC/MS ghi trn my ESI-LC/MS 1100 MSD Trap spectrometer. - Ph khi ghi trn my MS-Engine-5989-HP ion ho bng va chm eletron (EI- MS) 70ev v s dng ngn hng d liu DATABASE/WILLEY 250L. - Ph 1H-NMR v 13C-NMR ghi trn my Bruker 500MHz, ni chun TMS, dung mi CDCl3, DMSO, aceton-D6. 2.3. Cc dch chit t cy ch rng ca (Phyllanthus urinaria L) 2.3.1. Cc dch chit Ton b phn trn mt t ca cy ch rng ca phi kh, nghin nh c ngm chit kit bng etanol nhit phng cho n khi thu c dch khng mu. Dch chit c ct loi ht dung mi p sut gim n dng cao kh, xc nh khi lng cn kh, sau thm nc vo cn v ln lt chit vi cc loi dung mi: n-hexan, etylaxetat, ui ht nc v chit bng butanol, tip tc ui ht nc v chit bng metanol. Cc dch chit ni trn c lm khan bng Na2SO4, lc v ct kit dung mi bng c quay di p sut gim nhit 50 0 C. Cn c sy kh v cn xc nh trng lng. Nh vy t ton b phn trn mt t ca cy ch rng ca thu nhn c 4 phn on l n-hexan, etylaxetat,
26
butanol v metanol vi cc k hiu tng ng l: cn trong n-hexan (PH), cn trong etylaxetat (PE), cn trong butanol (PB) v cn trong metanol (PM). S 2.1: Quy trnh ngm chit mu
MU KH
1. Etanol 2. Cn trong nc n-Hexan

Cn n-Hexan (PH)
EtOAc
Cn EtOAc (PE)
BuOH
Cn BuOH (PB)
MeOH
Cn MeOH (PM)
PH-1
PE-1
PE-2
PE-3
Bng 2.1: Khi lng cht tng s c chit tng phn on ca cy ch rng ca (Phyllanthus urinaria L) Khi lng Khi lng Khi lng cn chit (g)
nguyn liu kh cn cn tng n-Hexan Etylaxetat n-Butanol Metanol s (g) (g) 1230 302,05 37,01 24,95 26,14 50,28
27
2.3.2. Kho st nh tnh cc dch chit Trn c s dng cc thuc th c hiu pht hin cc nhm hp cht thin nhin c hot tnh sinh l cao trong thc vt [2] chng ti thu c kt qu th nh tnh vi cc nhm cht, kt qu y c ch ra bng 2.2: Bng 2.2: Pht hin cc nhm cht trong cy ch rng ca STT 1 2 3 4 5 6 7 8 Nhm cht ng kh Ankaloit Steroit Flavonoit Polifenol Cumarin Glycozit tim Saponin Thuc th c hiu Felinh Dragendorf Liberman-Bourchard Xianidin FeCl3 Axit v kim Kelle-Kiliani To bt Hin tng Cho kt ta mu gch Mu vng da cam Mu xanh vng T hng n Xanh thm Kt ta bng Khng c hin tng g Bt bn trong axit Kt qu ++ + ++ ++ ++ ++ ++
Ghi ch: Du (+): Phn ng dng tnh, (++): Phn ng dng tnh rt r; (-): Khng c. 2.3.3. Th hot tnh sinh hc Th hot tnh vi sinh vt kim nh. Th nh tnh theo phng php khuych tn trn thch, s dng khoang giy lc tm cht th theo nng tiu chun.
28
Hnh 2.1: ng knh vng c ch xung quanh ging thch (mm) theo phng php khuych tn trn thch
29
Cc chng vi sinh vt gm i din cc nhm: * Vi khun Gr(-) Escherichia coli * Vi khun Gr(+) Staphylococcus auresu * Vi khun: Salmonella typhi Chng ti tin hnh th cc ng dng lm thc phm chc nng ca dch chit cy ch rng ca ti C s sn xut kinh doanh thuc thnh phm, thc phm chc nng Y hc c truyn Thi Nguyn. Tin hnh kim tra hot tnh khng khun ca dc liu ti b mn Vi Sinh trng i hc Y Thi Nguyn. Kt qu thc nghim thu c (xem hnh 2.1). Bng 2.3: Kt qu th hot tnh sinh hc ca dch chit th t cy ch rng ca (Phyllanthus urinaria L) ng knh vng c ch xung quanh ging thch (mm) Dch chit Staphylococcusauresu Cn nc Cn EtOAc 34 29 E.coli 23 18 Salmonella typhi 40 32
2.4. Phn lp, tinh ch cc cht t cy ch rng ca (Phyllanthus urinaria L) 2.4.1. Dch chit n-hexan T 2g cn dch n-hexan ca ton b phn trn mt t ca cy ch rng ca. K hiu (PH) c tin hnh phn lp cc cht trn sc k ct silicagel. H dung mi ra gii l n-hexan : etylaxetat vi t l theo tng dn ca dung mi phn cc t 0 n 100% etylaxetat, kim tra cc phn on bng sc k lp mng, cc phn on ging nhau em gp li v ct loi dung mi thu c cht sch.
30
* Cht PH-1 (-Sitosterol) Tin hnh phn chia nh sc k ct bng h dung mi n-hexan etylaxetat (9:1). Sau khi ct loi dung mi, cn thu c kim tra bng sc k lp mng trong h dung mi A, pht hin n bng vanilin 1% trong dung dch metanol-H2SO4 5%, kt tinh li trong n-hexan thu c 19 mg cht rn, c nhit nng chy 139-1410C, Rf = 0,72 (trong h dung mi A). Tin hnh o ph cht PH-1 thu c cc thng tin ph nh sau: Ph FT-IR (KBr); (cm-1): max = 3450 (cm-1) cho mt vn hp th rng (H32,C3); mt vn hp th 3010- 1650 (cm-1) (lin kt i). Ph EI-MS; m/z(%): 414 [M+] (20); 413[M-H]+(41); 398(28); 397(100); 395(32); 383(11); 361(11); 257(3); 255(6,3); 151(5,6); 139(11). Ph 1H-NMR (500MHz, CDCl3); (ppm): 0,68 ppm (3H, s, CH3-18); 1,01 ppm (3H, s, CH3-19); 0,81 ppm (3H, d, j 7Hz, CH3-26); 0,88 ppm (3H, d, j 7Hz, CH3-27); 0,83 ppm (3H, t, 7,32Hz, CH3-29); 0,92 ppm (3H, d, j 10Hz, CH3-21); 3,52 ppm (1H, m, H-3); 5,42 ppm (1H, d, j 5Hz,H-6). Ph NMR (500MHz, CDCl3); (ppm): Xem bng 2.4. Bng 2.4: S liu ph 13C-NMR (CDCl3, 125Mhz) ca cht PH-1 trong cy ch rng ca (Phyllanthus urinaria L) STT ca C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
13 13
C-
C-NMR (-ppm) t t d t s d t d d s t t s d t
37,2 31,7 71,8 42,3 140,8 121,7 31,9 31,9 50,1 36,5 21,1 39,8 42,3 56,8 24,3
STT ca C 16 17 18 19 20 21 22 23 24 25 26 27 28 29 -
13
C-NMR (-ppm) t d q q d q t t d d q d t q -
28,3 56,2 11,9 19,4 36,2 18,8 33,9 26,1 45,8 29,2 19,1 19,4 23,1 11,9 -
31
2.4.2 Dch chit etylaxetat (PE) T 5g cn chit etylaxetat ca cy ch rng ca (k hiu PE) c tin hnh tch cc cht trn sc k ct silicagel, ra gii ct sc k bng h dung mi cloroform : metanol c t l theo tng dn ca dung mi phn cc, t 0-100% metanol, kim tra cc phn on trn sc k lp mng, thuc th pht hin (FeCl3 + K3[Fe(CN)6]) 1% sau gp cc phn on ging nhau, ui ht dung mi v kt tinh li thu c 3 cn th. Tinh ch hai cn u thu c hai cht: PE-1v PE-2. Cn th ba c tch tip bng h dung mi toluen : etylaxetat : axit formic (5:4:1) thu c cht PE-3. 2.4.2.1 Cht PE-1(5-hidroxymetylfurfural) Ra gii ct bng h dung mi cloroform : metanol (90:10). Nhng phn on c cng Rf =0,8 trong h dung mi C c gp li, thu c cn th. Tinh ch li cn ny trong CHCl3 thu c 8 mg mt cht du st mu nu. Tin hnh o ph cht PE-1 thu c cc thng tin ph nh sau: Ph LC- MS; m/z(%): Cho pic M+ = 126,8. Ph 1H-NMR (500MHz, DMSO); (ppm): H = 7,48 ppm (1H,d, CH-3); H= 4,50 ppm (2H, d, CH 2 -6); H= 6,60 ppm (1H,d, J=3,6 Hz, CH-4); H= 9,54 ppm (1H,s,CHO-1). Ph 13C- NMR (500MHz, DMSO); (ppm): Cho cc pic C = 177,91 ppm (C1); C = 151,70 ppm (C 2 ); C = 124,33 ppm (C 3 ); C =109,62 ppm (C 4 ); C = 162,13 ppm (C5); C = 55,89 ppm (C6). 2.4.2.2 Cht PE-2 (axit gallic) Cht PE-2 cng c tch t cn etylaxetat. Nhng phn on tip theo ging nhau c cng R f = 0,7 (trong h dung mi C) c gp li, em ct ui dung mi thu c mt cht rn kt tinh. Tinh ch li bng cloroform thu c 40 mg tinh th hnh kim mu vng c nhit nng chy 249-250oC. Ph 1H-NMR (500MHz, DMSO); (ppm): Cho mt pic 6,91ppm. Ph 13C-NMR (500MHz, DMSO); (ppm): C = 120,48 ppm (C1);
32
C = 108,73 ppm (C 2 v C6); C = 145,40 ppm (C 3 v C5); C = 137,98 ppm (C4); C = 167,46 ppm (C7). 2.4.2.3 Cht PE-3 (Kampherol) Sau khi ra gii ct bng h cloroform : metanol (90:10) thu c hai cht PE-1 v PE-2. Tip tc ra gii ct bng h dung mi chloroform : metanol (50:10), thu c mt mt hn hp c mu vng c cng R f = 0,65 (trong h dung mi D). Hn hp ny cho mu cam vi phn ng xianiin, mu xanh en vi dung dch FeCl3 1% iu ni ln trong hn hp ny c flavonoit. Hn hp ny tip tc c phn chia bng phng php sc k ct silicagel ra gii bng h dung mi toluen : etylaxetat : axit fomic (5:4:1) thu c 29mg cht rn mu vng (c R f =0,4 trong h E), nng chy 276278oC. Ph LC- MS; m/z(%): Cho pic ion phn t M+ m/z =286,9 v M- m/z =285,0. Cc s liu ca ph 1H-NMR (500MHz, DMSO); (ppm) v ph 13CNMR (500MHz, DMSO); (ppm) (xem bng 2.5): Bng 2.5: Ph 1H-NMR v 13C-NMR ca cht PE-3 V tr C 2 3
13
C-NMR
H-NMR
13
C-NMR
H-NMR
(-ppm) 147,03 136,52
(-ppm, J-Hz) tr C __ __ 10 1/
(-ppm) 104,15 123,32
(-ppm, J-Hz) __ __
33
176,58
__
2/
130,44
8,15 (1H,dd, J=8,2; 2,2) 7,01(1H,dd, J=8,2;2,2) __
157,78
__ 6,26 (1H, d, J=2,1)
3/
116,32
99,17
4/
160,16
164,98
__
5/
116,32
7,01(1H,dd, J=8,2; 2,2) 8,15 (1H,dd, J=8,2; 2,2) __
94,49
6,52 (1H, d, J=2,1) __
130,44
162,30
__
__
34 CHNG III
THO LUN KT QU NGHIN CU

3.1. Nguyn tc chung phn lp c cc hp cht trong bt k mt thc vt no m khng lm nh hng ti thnh phn ho hc c trong n th trc khi ngm chit bng dung mi hu c, mu thc vt phi c a i kh men ngay sau khi thu mu v sy kh iu kin thch hp. V nguyn tc vic ngm chit mu thc vt c th tin hnh theo 2 cch ph bin sau. 1. Chit v phn lp cc hp cht t mu thc vt bng cc loi dung mi c phn cc tng dn: ete du ha hoc n-hexan, cloroform, etylaxetat, metanol hoc etanol vv... 2. Chit tng bng cc ancol (metanol, etanol) hay h dung mi ancol : nc. Sau sng lc cc hp cht bng cc loi dung mi c phn cc tng dn nh phng php 1 thu c cc dch chit c cha cc hp cht c phn cc tng i ging nhau. Vic chit mu thc vt ch rng ca (Phyllanthus urinaria L) c thc hin theo phng n 2 (S 2.1). Cc dch chit th em th hot tnh sinh hc. iu gip cho vic nh hng tm kim cc hot cht trong nhng dch chit c hot tnh mnh vi cc chng vi khun th nghim. 3.2. Xc nh nh tnh cc nhm cht thin nhin Phn tch nh tnh cc hp cht hu c thin nhin trong cc mu nghin cu l vic lm cn thit trc khi tin hnh phn lp v xc nh cu trc ca cc cht c th.
35
xc nh nh tnh cc nhm hp cht hu c c hot tnh sinh l cao c trong thc vt thng dng hn c l phn tch bng cc phn ng c trng i vi mi nhm cht nu ra bng 2.2. T kt qu ch ra bng ny c th nhn thy trong cy Phyllanthus urinaria L t nht c cha ti 7 nhm hp cht thin nhin quan trng l: ng kh, poliphenol, flavonoit, cumarin, steroit, saponintritecpen v ankaloit. Khng pht hin thy glycozit tim c trong cc mu th. T kt qu phn tch nh tnh cng s b cho thy cy ch rng ca l mt loi thc vt cha nhiu cht c hot tnh sinh hc cao, c li cho sc khe con ngi. 3.3. Phn lp v nhn dng cc hp cht c trong cc dch chit khc nhau ca cy ch rng ca Cc dch chit t cy ch rng ca (Phyllanthus urinaria L) u l nhng hn hp phc tp cha cc hp cht khc nhau. phn lp tng cht ra khi hn hp, s dng cc phng php sc k ct nh: Ct vi cht hp ph silicagel, vi cc h dung mi ra gii thch hp v thng phi lp li nhiu ln. Vic tinh ch cc cht thng dng phng php kt tinh li trong dung mi hoc h dung mi thch hp. Nh s thu c cc hp cht c tinh khit cao, p ng cc nhu cu kho st tnh cht ha l v quang ph ca chng. l nhng yu t quan trng trong qu trnh nhn dng v xc nh cu trc ha hc ca cc cht phn lp c t cc i tng nghin cu ni trn. Vic phn lp cc thnh phn ha hc t cy ch rng ca c thc hin nh s 2.1 v thu c cc hp cht sch gm cc nhm cht: tritecpenoit, furan, axit gallic v flavonoit. 3.3.1. -sitosterol (PH-1) T cn ca dch chit n-hexan phn chia trn sc k ct silicagel, ra ri bng h dung mi n-hexan : etylaxetat (9:1) thu c cht PH-1 c Rf = 0,72.
36
Hnh 3.1: Ph FT-IR ca -sitosterol (PH-1)

37
Hnh 3.2: Ph 1H-NMR ca -sitosterol (PH-1)

38
Hnh 3.3: Ph 13C-NMR v ATP ca -sitosterol (PH-1)

39
Cht PH-1 l nhng tinh th hnh kim, khng mu, im nng chy 139-1410C. Khi trn ln vi cht chun cho mt hn hp c nhit nng chy khng thay i. Cht PH-1 trong ph EI-MS cho mnh ion phn t M+ = 414, nh vy c ngha khi lng phn t bng 414 vC s ng vi cng thc phn t C29H50O. Trong phn t c mt nhm OH th hin ph FT-IR vi vn hp th rng = 3380cm-1. Mt khc ph 1H-NMR cho = 3,52 ppm c trng cho proton trong nhm CH lin kt vi nhm hidroxyl. Ph 13C-NMR cng nhn thy = 71,7 ppm c trng cho cacbon lin kt trc tip vi oxy ca nhm hidroxyl. Trong phn t cn c mt ni i th hin bi cc c trng ph FTIR c vn 3010 cm-1 c trng cho dao ng ho tr ca lin kt C-H v vn 1650 cm-1c trng cho dao ng ho tr ca lin kt i C=C. Trong ph 1HNMR cng nhn c tn hiu = 5,35 ppm c trng cho H trong lin kt =CH v tn hiu ca 2 cacbon mang ni i vi = 140,7 ppm v = 121,5 ppm t c th quy kt hp cht PH-1 c mt ni i gia v tr C-5 v C-6. So snh ph 1H-NMR v ph 13C-NMR ca cht PH-1 vi ph ca Sitosterol thy hon ton tng t nhau (bng 3.1). T cc kt qu phn tch thnh phn ho hc, tnh cht vt l, tnh cht quang ph nht l da trn c s so snh cc s liu ph ca cht PH-1 vi ph ca cht -sitosterol hon ton c th cho php quy kt cht PH-1 phn lp c t cy ch rng ca l -sitosterol.
40
Bng 3.1: S liu ph 13C-NMR (CDCl3, 125Mhz) ca PH-1 trong cy ch rng ca (Phyllanthus urinaria L) v ph ca -sitosterol [15] Cht PH-1 V tr C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 ca 13C (ppm) 37,2 31,7 71,8 42,3 140,8 121,7 31,9 31,9 50,1 36,5 21,1 39,8 42,3 56,8 24,3 28,3 56,2 11,9 19,4 36,2 18,8 33,9 26,1 45,8 29,2 19,1 19,4 23,1 11,9 -Sitosterol [15] V tr C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 ca 13C (ppm) 37,2 31,6 71,7 42,3 140,7 121,7 31,9 31,9 50,1 36,5 21,2 39,8 42,3 56,7 24,3 28,2 56,1 11,9 19,4 36,1 18,9 34,0 26,1 45,8 29,2 19,8 19,5 23,0 12,0
41
28 21 19 12
11 20
29 27
25
22
23
24 26
17 13
14
16
15
18 1 2 HO 3 4 10 5 6 9 8 7
-Sitosterol 3.3.2. 5-Hydroxymetylfufural (PE-1) Cht PE-1 c tch t cn etylaxetat c Rf= 0,8 (trong h dung mi cloroform : etylaxetat (9:1). N l cht du st mu nu. Ph LC-MS cho bit cht PE-1 c pc ion phn t M+ = 126,8 iu ny cho thy cht PE-1 c khi lng phn t 126 vC. Cc d liu ph NMR cng xc nhn nhng thng tin quan trng. Ph
13
C-NMR cho tn hiu = 177,91 ppm, chnh l chuyn dch ha hc
ca cacbon cacbonyl. chuyn dch ha hc = 124,31 ppm v = 109,62 ppm l chuyn dch ca hai nguyn t cacbon cha no lin kt vi hiro (=C-H), c th quy kt = 124,31 ppm l C-3 cn = 109,62 ppm l ca C-4. T ph NMR k thut DEPT cho thy c mt s loi cc bon nh sau: hai cacbon bc 4 chuyn dch ha hc 151,70 ppm v 162,13 ppm l cc nguyn t cacbon C-2 v C-5. C mt nhm CH2 vi = 55,89 ppm. C hai nhm CH (metin) cc dch chuyn ha hc 109,62 ppm v 124,31 ppm. Theo ph HSQC th nguyn t H-1 c dch chuyn ha hc 9,54 ppm, cc nguyn t H-6a,b c dch chuyn ha hc 4,50 ppm, nguyn t H-3 c dch chuyn ha hc 7,48 ppm. Cn nguyn t H-4 c dch chuyn ha hc 6,60 ppm.
42
Hnh 3.4: Ph 1H-NMR-DMSO ca PE-1

43
Hnh 3.5: Ph 13C-NMR-DMSO ca PE-1
44
Bng 3.2: S liu ph NMR ca PE-1 v s liu ph NMR trong phn mm ACD/NMR ca hidroxymetylfufural Ph ca cht PE-1 V tr C
13
Phn mm ACD ca hidroxymetylfufural. ACD/13C-NMR 179,55 156,33 121,65 112,74 160,22 56,63 ACD/1H-NMR 9,53 7,41 6,68 4,70
C- NMR (ppm)
H-NMR (ppm) 9,54 7,48 6,60 4,50
1 2 3 4 5 6
177,91 151,70 124,31 109,62 162,13 55,89
Phn tch ph 1H-NMR, HMBC cho thy tn hiu H-1 ( = 9,54 ppm) l chuyn dch ha hc ca hitro lin kt vi nhm C=O c tng tc xa vi C-2 dch chuyn ha hc 151,70 ppm v C-3 124,31 ppm, H-3 ( = 7,48ppm) c tng tc xa vi cc cacbon C-2 151,70 ppm v C-4 109,62 ppm, H-4 (1H, d, j=3,7Hz; = 6,60 ppm) c tng tc xa vi C-3 v C-5 cc dch chuyn ha hc 124,31 ppm v 162,13 ppm. Cn cc nguyn t H-6a,b c tng tc xa vi C-4 v C-5 cc dch chuyn ha hc 109,62 ppm v 162,13 ppm. So snh ph NMR ca cht PE-1 vi phn mm ACD/1H-NMR v ACD/13C-NMR cho thy s tng t gn nh hon ton vi ph ca 5hidroxymetylfufural (xem bng 3.2).
45
T cc kt qu phn tch cc loi ph ca hp cht PE-1nh: ph khi, ph NMR vi cc k thut o 1D v 2D, c so snh vi phn mm m phng ACD/NMR, c th khng nh cht PE-1 l 5-hidroxymetylfufural.
H 6 HO 4 3 5 O 2 1 C O
5-Hidroxymetylfufural Ngy nay cht 5-hidroxymetylfufural c phn lp t mt s cy thuc dn gian khc nh t cy a hong, cy trinh n hong cung n c vai tr trong qu trnh chuyn ho cht trong c th sinh vt. Trong cy ch rng ca (Phyllanthus urinaria), cht 5-hidroxymetylfufural ln u tin c phn lp v nhn dng cu trc phn t. 3.3.3. Axit gallic (PE-2) Cht PE-2 cng c tch t cn etylaxetat, l cht rn kt tinh, tinh th hnh kim mu vng c nhit nng chy 249-250oC. Ph 1H-NMR cho tn hiu 7,092 ppm ng vi 2 proton c trng cho chuyn dch ha hc ca cc proton vng thm, c quy kt cho cc proton cc v tr C-2 v C-6. Ph
13
C-NMR cho thy cc chuyn dch ha hc = 120,48
ppm, = 137,98 ppm v = 167,46 ppm, tng ng vi chuyn dch ha hc ca cc nguyn t cacbon C 1, C4 v C7 trong axit gallic. C 2 tn hiu = 108,73 ppm v = 145,40 ppm, mi tn hiu ng vi 2 nguyn t cacbon. Cc tn hiu ny ph hp vi chuyn dch ha hc ca C 2 v C6 ( = 108,73 ppm) v ca C 3 v C5 ( = 145,40 ppm).
46
Hnh 3.6: Ph 1H-NMR-DMSO ca PE-2
47
Hnh 3.7: Ph 13C-NMR-DMSO ca PE-2
48
So snh ph NMR ca cht PE-2 vi phn mm ACD/1H-NMR v ACD/13CNMR cho thy s tng t gn nh hon ton vi ph ca axit gallic (xem bng 3.3). Bng 3.3: S liu ph NMR ca PE-2 v s liu ph NMR trong phn mm ACD/NMR ca axit gallic
Nh vy, c th quy kt cht PE-2 c tch ra t dch chit etylaxetat ca cy ch rng ca c nhn dng l axit gallic.
7 COOH 1 6 2 5 4 OH 3 OH
HO
Axit gallic C th coi axit gallic l mt n phn tham gia vo vic to ra cc tanin thu phn v tham gia vo cc chuyn ho khc to ra cc lignan trong cy ch rng ca.
49
3.3.4. Kampherol (PE-3) Cht PE-3. T cn etylaxetat ngoi cc cht PE-1 v PE-2 trnh by trn chng ti cn thu c mt hn hp c mu vng, cho mu cam vi phn ng Xianiin, mu xanh en vi dung dch FeCl3 1% iu ni ln trong hn hp ny c flavonoit. Bng phng php sc k ct silicagel hn hp thu c ny, ra gii bng h dung mi toluen : etylaxetat : axitfomic (5:4:1) thu c 29mg cht rn mu vng (c R f = 0,4 trong h dung mi E) v nng chy 276-278oC. Ph khi lng LC-MS cho pic ion phn t M+ m/z =286,9 v M- m/z =285,0 ng vi M = 286 vC v ph hp vi cng thc C 15H10O6. Phn tch ph 13C-NMR (DEPT) cho thy hp cht PE-3 c tng cng 15 nguyn t cacbon trong c 9 C bc 4, c 6 cacbon bc 3 (c 6 nhm CH), khng cha cc cacbon bc 1 v bc 2. C th quy kt cc tn hiu = 176,58 ppm l ca cacbon cacbonyl C-4; dch chuyn ha hc = 147,03 ppm l ca cacbon C-2; = 136,52 ppm l tn hiu ca C-3, l nhng nguyn t cacbon olefin, C-2 lin kt vi oxi v tr s 1 cn C-3 lin kt vi nhm OH v nhm C=O. Tn hiu = 157,78 ppm v = 164,98 ppm tng ng vi chuyn dch ho hc ca C-5 v C-7, cn = 160,16 ppm l tn hiu ca C-4/ v 3 nguyn t cacbon ny lin kt trc tip vi cc nhm hiroxyl. Theo ph HSQC ca hp cht PE-3 th tn hiu cng hng proton ti 8,15 ppm (dd, J=8,2 v 2,2) l ca H-2/ v H-6/ v ti 7,01 ppm (dd, J=8,2 v 2,2) l tnh hiu ca H-3/ v H-5/, cn cc tn hiu = 6,26 ppm v 6,52 ppm l ca cc nguyn t H-6 v H-8.
50
Hnh 3.8: Ph 1H-NMR-AcetoneD6 ca PE-3
51
Hnh 3.9: Ph 13C-NMR-AcetoneD6 ca PE-3
52
Bng 3.4: S liu ph NMR ca PE-3 v s liu ph trong phn mm ACD/NMR ca cht kampherol Ph ca cht PE-3 V tr C
13
Phn mm ACD ca cht kampherol ACD/13CNMR 146,45 135,60 175,80 157,63 98,25 163,90 93,45 159,13 103 121,65 129,40 115,35 159,10 115,35 129,40 ACD/1H-NMR 6,28 6,54 8,10 7,00 7,00 8,10
C- NMR (ppm)
H-NMR (ppm) 6,26 (1H, d, J=2,1) 6,52 (1H, d, J=2,1) 8,15 (1H, dd, J=8,2; 2,2) 7,01(1H, dd, J=8,2;2,2) 7,01(1H, dd, J=8,2; 2,2) 8,15 (1H, dd, J=8,2; 2,2)
2 3 4 5 6 7 8 9 10 1/ 2/ 3/ 4/ 5/ 6/
147,03 136,52 176,58 157,78 99,17 164,98 94,49 162,30 104,15 123,32 130,44 116,32 160,16 116,32 130,44
53
T ph HMBC cho thy H-2( = 8,15 ppm) c tng tc xa vi cc nguyn t cacbon C-1 (123,32 ppm) v C-3 (116,32 ppm), H-3( = 7,01 ppm) c tng tc xa vi C-2 (130,44 ppm) v C-4(160,16 ppm), H-5 tng tc xa vi C-4 (160,16 ppm) v C-6 (130,44 ppm), H-6 tng tc xa vi C5 (116,32 ppm) v C-1 (123,32 ppm), H-6 c tng tc xa vi C-5 (157,78 ppm) v C-7 (164,98 ppm). Cn H-8 c tng tc xa vi C-7 (164,98 ppm) v C-9 (162,30 ppm). Qua vic phn tch cc loi ph ca hp cht PE-3 c so snh vi chng trnh m phng ph ACD/1H-NMR v ACD/13CNMR cho thy s tng thch kh tt vi sai s cho php c th khng nh hp cht PE-3 chnh l kampherol.
8 9 7 6 5 OH 10 C 4 O 3 OH
5'
6/
1 O 2
1'
2'
3'
4'
HO
OH
Kampherol hay 3,5,7,4/-tetrahidroxi flavon Kampherol l mt flavonoit c trong nhiu thc vt, n khng c v c tc dng sinh hc quan trng. Ln u tin cht ny c phn lp t cy ch rng ca (phyllanthus urinaria) thu hi ti Thi Nguyn. 3.4. Th hot tnh sinh hc Cn c vo thc t v ang c ng dng lm thuc ca cy ch rng ca, chng ti tin hnh th hot tnh sinh hc ca dch chit ca cy ny ti b mn Vi sinh Trng i hc Y khoa i hc Thi Nguyn. Mu th gm c:
54
Mu 1: Dch chit tng s trong nc nng. Mu 2: Dch chit trong EtOAc ( loi b ht phn cht tan trong n-hexan). Kt qu th hot tnh vi vi sinh vt kim nh ca dch chit cy ch rng ca nu trong bng 2.3, cho bit cc dch chit th u c hot tnh vi vi sinh vt kim nh. C th l: - Dch chit trong nc v dch chit etylaxetat ca cy ch rng ca u c hot tnh kh mnh vi cc khun Staphylococcusauresu (T cu vng), E.coli (Thc khun ng rut) v Salmonella typhi (Thng hn). - Dch chit nc c hot tnh mnh hn so vi dch chit etylaxetat. - Dch chit nc c hot tnh mnh nht vi khun Salmonella typhi (Thng hn).
55
KT LUN
1. Ln u tin, cy ch rng ca (Phyllanthus urinaria L) mc hoang ti Thi Nguyn c nghin cu sng lc ha thc vt, thit lp c quy trnh ngm chit mu hp l thu c 4 dch chit chn lc. 2. T thn v l cy ch rng ca (Phyllanthus urinaria L), bng phng php phn tch nh tnh khng nh trong cy ch rng ca c nhng lp cht: ng kh, ankaloit, steroit, flavonoit, poliphenol, cumarin v saponin. 3. phn lp v xc nh c cu trc ha hc ca 4 hp cht l: Sitosterol, 5-hidroxymetylfufural, axit gallic v kampherol. 4. th hot tnh sinh hc ca cc dch chit tng ca cy ch rng ca (Phyllanthus urinaria L) cho thy c tc dng khng cc khun Staphylococcusauresu (T cu vng), E.coli (Thc khun ng rut) v Salmonella typhi (Thng hn).
56
DANH MC CC CNG TRNH CNG B LIN QUAN N LUN VN
1. Ng c Trng, Phm Vn Thnh (2008), Nghin cu ha hc thc vt cy ch rng ca (Phyllanthus urinaria L.) mc hoang ti Thi Nguyn (Chemical studies on the constituents of Phyllanthus urinaria L. growing wildly in Thai Nguyen province), Tp ch Khoa hc & Cng ngh i hc Thi Nguyn, s 2 (46), tr 75-79.
57
TI LIU THAM KHO

A. Ti liu ting Vit 1. Huy Bch v cng s (2004), Cy thuc v ng vt lm thuc Vit Nam, NXB Khoa hc v K thut, H Ni. 2. Nguyn Vn n (1997), Cc phng php nghin cu cy thuc, NXB YDc, Tp. H Ch Minh. 3. Nguyn Vn u, Lu Hong Ngc, Nguyn nh Chung (2003), Phn lp mt s hot cht t cy ch rng ca, Tp ch Dc hc, s 9 nm 43, tr.12-15. 4. Nguyn Vn u, Trn Th Thu H (2007), Ngin cu ha thc vt cy ch rng ca (Phyllanthus uticulatus niruri L). Tp ch dc hc, s 369 nm 47, tr.15-18. 5. Tt Li (1997), Nhng cy thuc v v thuc Vit Nam, NXB Y hc, H Ni, tr.65-67. 6. Nguyn Thng Dong v ng tc gi (2001), Kho st cng dng lm thuc ca mt s loi Phyllanthus Vit Nam, Tp ch dc liu, 6 (2+3), tr.72-75. 7. Trn nh Thng v cng s (2007), Phn lp v xc nh cu trc mt s hp cht phenolic t cy ch rng ca, Tp ch Dc hc, s 371 nm 47, tr.14-17. B. Ti liu ting Anh 8. Anjaneyulu (1973), A. S. R et al, Tetrahedron, 29(1), p.1291. 9. Babady-Bila (1996), Niruroidine, a norsecurinine-type alkaloid from Phyllanthus niruroides, Phytochemistry, 41(5), p.1441-1443. 10. Bikram Singh, P.K. Agrawal and R.S. Thakur (1989), An acyclic triterpene from Phyllanthus niruri, Phytochemistry, 28(7), p.1980-1981.
58
11. Calixto J. B., Santos A. R. (1998), A review of the plants of the genus Phyllanthus, Pharmacology, 4(18), p.225-258. 12. Chia-Chuan Chang, Yu-Chin Lien (2003), Four lignans were isolated from Phyllanthus urinaria L, Phytochemistry, 63(7), p.825-833. 13. Chien-Min Yang, Hua-Yew Cheng (2007), Geraniin from Phyllanthus urinaria L, Ethnopharmacology, 110(3), p.555-558. 14. Farah Naaz, Saleem Javed, M.Z. Abdin (2007), Hepatoprotective effect of ethanolic extract of Phyllanthus amarus Schum. et Thonn, Ethnopharmacology, 113(3), p.503-509. 15. Goat J.L., Akihisa T. (1997), Analysis of steroit frest ed., Phytochemistry, p.324. 16. Hasenah Ali (2006), -Amylase inhibitory activity of some Malaysian plants used to treat diabetes, Ethnopharmacology, 107(3), p.449-455. 17. Herbert Kolodziej (2005), Tannins and related compounds induce nitric oxide synthase and cytokines gene expressions in Leishmania major, Bioorganic, 13(23), p.6470-6476. 18. Hirotaka S., Masayoshi I. (2005), Direct observation of a target cell of the leaf-closing factor by using novel fluorescence-labeled phyllanthurinolactone, Tetrahedron Letters, 46(33), p.5537-5541. 19. Hnatyszyn, J. Mio, G. Ferraro (2002), The hypoglycemic effect of Phyllanthus sellowianus fractions, Phytomedicine, 9(6), p.556-559. 20. Horii Z. (1972), Et all, Tetrahedron Letters, p.1887. 21. Jangfang Quian-Cutrone (1996), J. Nat. Prod., (59), p.196-199. 22. Kumaran, Karunakaran (2007), In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India, Food Science, 40(2), p.344-352.
59
23. Kumar Roy, H. Dhir, A. Sharma (1992), Modification of metal-induced micronuclei formation in mouse bone marrow erythrocytes", Toxicology Letters, 62(1), p.9-17. 24. L. Yeap Foo (1992), Phyllanthusiin D, an unusual hydrolysable tannin from Phyllanthus amarus, Phytochemistry, 31(2), p.711-713. 25. L. Yeap Foo (1993), Amariin, a di-dehydrohexahydroxydiphenoyl hydrolysable tannin from Phyllanthus amarus, Phytochemistry, 33(2), p.487-491. 26. L. Yeap Foo (1995), Amariinic acid and related ellagitannins from Phyllanthus amarus, Phytochemistry, 39(1), p.217-224. 27. Manske R. H. F (1973), The Alkaloids chemistry and physiology, Phytochemistry, (15), p.428. 28. Masturah Markom, Masitah Hasan (2007), Extraction of hydrolysable tannins from Phyllanthus niruri L, Technology, 52(3), p.487-496. 29. Matsunaga, Tanaka (1992), 26-Nor-D:A-friedooleanane triterpenes from Phyllanthus watsonii, Phytochemistry, 32(1), p.165-170. 30. Peter J. Houghton, Tibebe Z. Woldemariam (1996), Two securinega-type alkaloids from Phyllanthus amarus, Phytochemistry, 43(3), p.715-717. 31. Rajasubramaniam, Pardha Saradhi (1997), Rapid multiplication of Phyllanthus fraternus: a plant with anti-hepatitis viral activity, Industrial Crops and Products, 6(1), p.35-40. 32. Rajkishor S. Negi, Thawra M. Fakhir (1998), An alkaloid from Phyllanthus simplex, Phytochemistry, 27(9), p.3027-3028. 33. Reiko Tanaka, Shunyo Matsunaga (1998), Triterpenes from the stem bark of Phyllanthus flexuosus, Phytochemistry, 27(11), p.3563-3567.
60
34. Satyanarayana, Venkateswarlu (1991), Isolation, structure and synthesis of new diarylbutane lignans from Phyllanthus niruri, Tetrahedron, 47(42), p.8931-8940. 35. Sengupta, Mukhopadhyay (1966), Terpenoids and related compoundsVII : Triterpenoids of Phyllanthus acidus skeels, Phytochemistry, 5(3), p.531-534. 36. Shakil, Pankaj (2008), Nematicidal prenylated flavanones from Phyllanthus niruri, Phytochemistry, 63(3), p.759-764. 37. Shih-Hua Fang, Yerra Koteswara Rao (2008), Anti-oxidant and inflammatory mediator's growth inhibitory effects of compounds isolated from Phyllanthus urinaria, Ethnopharmacology, 116(2), p.333-340. 38. Shunyo Matsunaga, Reiko Tanaka, (1988), Revised structure of trichadenic acid B, Tetrahedron Letters, 29(37), p.4751-4754. 39. Sutthivaiyakit, Nakorn N. (2003), A new guaiane sesquiterpene from the roots of Phyllanthus oxyphyllus, Tetrahedron, 59(50), p.9991-9995. 40. Syamasundar, Bikram Singh (1985), Antihepatotoxic principles of Phyllanthus niruri herbs, Ethnopharmacology, 14(1), p.41-44. 41. Tanaka, K. Masuda and S. Matsunaga (1993), Lup-20(29)-en-3,15diol and ocotillol-II, Phytochemistry, 32(2), p.472-474. 42. Thales R. Cipriani, Caroline G. Mellinger (2008), Acidic heteroxylans from medicinal plants and their anti-ulcer activity, CarbohydratePolymers, 74(2), p.274-278. 43. Torben L. Bachmann, Felipe Ghia (1993), Lignans and lactones from Phyllanthus anisolobus, Phytochemistry, 33(1), p.189-191. 44. Toshimasa Ishida (1998), Phyllurine, leaf-opening substance of a nyctinastic plant Phyllanthus urinaria L, Tetrahedron Letters, 39(52), p.9731-9734.
61
45. Van der B.D.A.(1991), Viettlink A. J., Plant Biochemistry, 6, p.47-70. 46. Vuyelwa J. N., Neil R. C. (2008), Triterpenoids from the African tree Phyllanthus polyanthus, Phytochemistry Letters, 1(1), p.11-17. 47. Wai-Haan Hui, Man-Moon Li (1976), Tritecpenoit isolated from Phyllanthus reticulates , Phytochemistry, 5(15), p.797-798. 48. Wanxing Wei, Yuanjiang Pan (2005), Carboxylic acids from Phyllanthus urinaria, Chemistry of Natural Compounds, 41(1), p.17-20. 49. Xiaoli Liu, Chun Cui, M. Zhao (5/2008), Antioxidant activity of methanolic extract of emblica fruit, Journal of Food, 21(3), p.219-228. 50. Xiao Liu, Chun Cui, M. Zhao (8/2008), Identification of phenolics in the fruit of emblica, Food Chemistry, 109(4), p.909-915. 51. Yerra Koteswara Rao (2006), Anti-inflammatory activities of constituents isolated from Phyllanthus polyphyllus, Ethnopharmacology, 103(2), p.181-186. 52. Ying-Jun Zhang (2006). A novel highly oxygenated norbisabolane from the roots of Phyllanthus emblica, Faculty of Pharmaceutical Sciences, Japan, p.1-14. 53. Zhang L. Z., Guo Y. J. (2000), Studies on chemical constituents of Phyllanthus urinaria L, Zhongguo Zhong Yao Za Zhi, 10(25), p.615-617. 54. Zhang L. Z., Guo Y. J. (2004), Isolation and indentification of a novel ellagitannin from Phyllanthus urinaria L, Yao Xue Xue Bao, 2(39), p.119-112.
62
PH LC
1. Ph EI-MS ca -Sitosterol (PH-1) 2. Ph FT-IR ca -Sitosterol (PH-1) 3. Ph 1H-NMR ca -Sitosterol (PH-1) 4. Ph 13C-NMR ca -Sitosterol (PH-1) 5. Ph LC-MS ca hydroxymetylfufural (PE-1) 6. Ph 1H-NMR v ACD/1 H-NMR ca hydroxymetylfufural (PE-1) 7. Ph 13C-NMR v ACD/13C-NMR ca hydroxymetylfufural (PE-1) 8. Ph 13C-NMR v DEPT ca hydroxymetylfufural (PE-1) 9. Ph HMBC ca hydroxymetylfufural (PE-1) 10. Ph HSQC ca hydroxymetylfufural (PE-1) 11. Ph 1H-NMR v ACD/1H-NMR ca axit gallic (PE-2) 12. Ph 13C-NMR v ACD/13C-NMR ca axit gallic (PE-2) 13. Ph LC-MS ca kampherol (PE-3) 14. Ph 1H-NMR v ACD/1H-NMR ca kampherol (PE-3) 15. Ph 13C-NMR v ACD/13C-NMR ca kampherol (PE-3) 16. Ph 13C-NMR v DEPT ca kampherol (PE-3) 17. Ph COSY ca kampherol (PE-3) 18. Ph HMBC ca kampherol (PE-3) 19. Ph HSQC ca kampherol (PE-3) 20. Kt qu xc nh tnh nng dc, kt qu th hot tnh khng khun ca cc dch chit th t cy ch rng ca
63
64
65
Hnh 3.2: Ph 1 H-NMR ca -sitosterol (PH-1)
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
TRNG H Y KHOA TN
CNG HO X HI CH NGHA VIT NAM

c lp - T do - Hnh phc
B MN VI SINH
KT QU KIM TRA DC TNH KHNG KHUN CA DC LIU
Ngy kim tra: 19/05/2008. Kt qu: ng knh vng c ch xung quanh ging thch (mm) Dc liu Staphylococcus aureus E.coli Salmonella typhi cn EtOAc 34 23 40 (Trng) cn nc 29 18 32 (Trng) - Kt lun: Cc dch chit th u c hot tnh vi vi sinh vt kim nh. C th l: - Dch chit trong nc v dch chit etylaxetat ca cy ch rng ca u c hot tnh kh mnh vi cc khun Staphylococcusauresu (T cu vng), E.coli (Thc khun ng rut) v Salmonella typhi (Thng hn). - Dch chit nc c hot tnh mnh hn so vi dch chit etylaxetat. - Dch chit nc c hot tnh mnh nht vi khun Salmonella typhi (Thng hn).
Thi Nguyn, ngy 20 thng 05 nm 2008 Ph trng b mn Vi sinh
Ts. Nguyn ng Trung

Luan Van 08 SP HH NDT

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Luan Van 08 SP HH NDT

Uploaded by

Copyright:

Available Formats

I HC THI NGUYN TRNG I HC S PHM ------------------------------

THI NGUYN - 2008

I HC THI NGUYN TRNG I HC S PHM ---------------------------------

Ngi hng dn khoa hc: PGS.TS PHM VN THNH

THI NGUYN - 2008

Thi Nguyn, thng 9 nm 2008 Tc gi

DANH MC CC CH VIT TT DNG TRONG LUN VN

SKLM UV MS EI-MS LC-MS FT-IR NMR

DEPT COSY HSQC HMBC HIV vC

C-NMR (CDCl3, 125Mhz) ca PH-1 trong cy ch

DANH MC CC S S 2.1: Quy trnh ngm chit mu...................................................................26

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

Hnh 1.1: Cy ch rng ca (Phyllanthus urinaria L, Euphorbiaceae)

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

5 5a. Phyllanthin R= R 1 =CH3, R2 =H 5b. Niranthin R+R1= -CH2 -R2 = OCH3

S ha bi Trung tm Hc liu i hc Thi Nguyn

9a. R=H; 9b. R=CH3

10a. R=H; 10b. R=CH3

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

3'' 3' 1''

2' 1' 2 6'

2' 1' 2 6'

15 1.3.5 Mt s hp cht phenolic khc

17. Axit shikimic

S ha bi Trung tm Hc liu i hc Thi Nguyn

20. 2,4,6- xyclohepta-trien-2,3,7-trihydroxy-5-ethoxy-1-on

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

niruri L. tnh Bc Ninh nc ta [4]. Cu trc mt s hp cht in hnh c xc nh nh sau:

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

OMe OMe R2O

29a. R1 + R2 = CH2 29b. R1 = R2 =Me

30a. R1 + R2 = CH2 30b. R1 = R2 =Me

31a. R1 = R2 =Me 31b. R1 + R2 = CH2

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

40. Axit gallic

41. Methyl brevifolincarboxylate

S ha bi Trung tm Hc liu i hc Thi Nguyn

1' 6' 5'

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

chiu di di chuyn ca cht th chiu di di chuyn ca dung mi

1. Etanol 2. Cn trong nc n-Hexan

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

(-ppm) 147,03 136,52

(-ppm) 104,15 123,32

S ha bi Trung tm Hc liu i hc Thi Nguyn

8,15 (1H,dd, J=8,2; 2,2) 7,01(1H,dd, J=8,2;2,2) __