luan van thac sy hoa hoc 2

8/8/2019 Luan Van Thac Sy Hoa Hoc 2

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I HC THI NGUYN

TRNG I HC S PHM ---------------------------------

NG C TRNG

NGHIN CU HO HC V NHN DNGMT S NHM CHT C TRONG CY

CH RNG CA ( PHYLLANTHUS URINARIA L., EUPHORBIACEAE)

Chuyn ngnh : Ho hu c M s : 60.44.27

LLUUNN VVNN TTHHCC SS HHOO HHCC

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

THI NGUYN - 2008


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LI CM N

Ti xin chn thnh by t lng cm n su sc ca mnh ti PGS.TS Phm Vn Thnh- N gi 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 tr ng cm n TS. Nguyn Quyt Tin, TS. Phm Th Hng Minh, Th.S . V Anh Tun, Th.S H a Vn Thao nhng ngi thy

ng vin v gip tng bc i ca ti trong qu trnh nghin cu thchin lun vn.

Xin chn thnh cm n phng hot cht sinh hc ca trng i hcY

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 vto 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, Banlnh 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


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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 cngb trong bt k cng trnh no khc.

Tc gi

NG C TRNG


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DANH M CCC CH VIT TT DNG TRONG LU NVN

SKLM : S ck lpmng UV : Ultraviolet spectrocopy

MS : Mass Spectroscopy

EI-MS : Electron Impact Mass Spectroscopy

LC-MS : Liqud chromatography - Mass Spectroscopy

FT-IR : Fourier Transform Infrared Spectroscopy

NMR : Nuclear Magnetic Resonance1H-NMR : 1H-Nuclear Magnetic Resonance13C-NMR : 13C- Nuclear Magnetic Resonance

DEPT : Distortionless Enhancement by Polarisation Transfer

COSY : Correlated Spectroscopy

HSQC : Heteronuclear Spectroscopy- Quantum Coherence

HMBC : Heteronuclear multiple - Bond Correlation

HIV : Human Immunodeficiency Virus

vC : n vCacbon


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DDAANNHH MMCC CC CC BBNNGG

Trang

Bng 2.1: Khi lngchttngs cchittngphn oncacy ch rngca (Phyllanthus urinaria L)....26

Bng 2.2: Pht hi ncc nhm ch ttrong cy ch rng ca27 Bng 2.3: Ktquth hottnh sinh h c cadch chitth t cy ch rng

ca (Phyllanthus urinaria L)........29

Bng 2.4: S liuph 13

C-NMR (CDCl 3, 125Mhz) c achtPH-1 trong cy chrng ca (Phyllanthus urinaria L).......30

Bng 2.5: Ph1H-NMR v 13C- NMR ca cht PE-3...32 Bng 3.1: S liu ph13C-NMR (CDCl 3, 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 ca5-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


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DDAANNHH MMCC CC CC HHNNHH

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 phn

phng php khuych tn trn thch....................................................28

Hnh 3.1 : Ph FT-IR ca -sitosterol (PH-1).......................................................36 Hnh 3.2 : Ph1H- NMR ca -sitosterol (PH-1)..................................................37

Hnh 3.3 : Ph13

C- NMR v ATP ca -sitosterol (PH-1)....................................38 Hnh 3.4 : Ph1H-NMR- DMSO ca PE-1...........................................................42 Hnh 3.5 : Ph13C-NMR- DMSO ca PE-1..........................................................43 Hnh 3.6 : Ph1H-NMR- DMSO ca PE-2...........................................................46 Hnh 3.7 : Ph13C-NMR- DMSO ca PE-2..........................................................47 Hnh 3.8 : Ph1H-NMR- AcetoneD6 ca PE-3.....................................................50 Hnh 3.9 : Ph13C-NMR- AcetoneD6 ca PE-3....................................................51

DDAANNHH MMCC CC CC SS

S 2.1: Quy trnh ngm chit mu...................................................................26


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MC LC

Li cm nLi cam oan Danh mc cc ch vit tt dng trong lun vnDanh mc cc bng Danh mc cc hnh v s M U........................................................................................................1CHNG I.TNG QUAN................................................................................31.1. M t thc vt............................................................................................31.2. Tc dng sinh hc ca chiPhyllanthus ........................................................41.2.1 Mt s cng dng ca chiPhyllanthus ......................................................41.2.2 Mt s tc dng dc l ca chiPhyllanthus .............................................51.3 Tnh hnh nghin cu ho hc thc vt ca chiPhyllanthus ..........................61.3.1 Mt s i din ca nhm tecpenoit..........................................................6

1.3.2 Mt s i din ca khung axit..................................................................81.3.3 Mt s i din ca Lignan.......................................................................91.3.4 Mt s i din ca khung flavonoit........................................................111.3.5 Mt s hp cht phenolic khc................................................................121.3.6 Mt s hp cht nhm ankaloit...............................................................151.4 Tnh h nh nghin cu ha hc ca loiPhyllanthus urinaria L....................16CHNG II.THC NGHIM..........................................................................232.1. i tng v phng php nghin cu......................................................232.1.1. Thu mu cy, xc nh tn khoa hc v phng php x l mu..............232.1.2. Phng php phn lp cc hp cht t cc dch chit..............................232.1.3. Phng php kho st v xc nh cu trc ho hc cc hp cht............24


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2.2. Dng c, ho cht v thit b nghin cu...................................................242.2.1. Dng c v ho cht..............................................................................24

2.2.2. Thit b nghin cu................................................................................252.3. Cc dch chit t cy ch rng ca(Phyllanthus urinaria L)..................252.3.1. Cc dch chit.......................................................................................252.3.2. Kho st nh tnh cc dch chit............................................................272.3.3. Th hot tnh sinh hc...........................................................................272.4. Phn lp, tinh ch cc cht t....................................................................292.4.1. Dch chit n-hexan ................................................................................29

2.4.2. Dch chit trong etylaxetat (PE).............................................................312.4.2.1 Cht PE-1............................................................................................312.4.2.2 Cht PE-2............................................................................................312.4.2.3 Cht PE-3............................................................................................32CHNG III.THO LUN KT QU NGHIN CU....................................343.1. Nguyn tc chung....................................................................................343.2. Xc nh nh tnh cc nhm cht thin nhin............................................343.3. Phn lp v nhn dng cc hp cht..........................................................353.3.1. -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.................................................................................................... 55DANH MC CC CNG TRNH CNG B LIN QUAN N LUN VN.... .56TI LIU THAM KHO..................................................................................57PH LC.......................................................................................................62


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S ha bi Trung tm Hc liu i hc Thi Nguyn http://www.lrc-tnu.edu.vn

1

M U

T xa n nay, nhng cy thuc dn gian vn ng vai tr ht scquan trng trong i sng hng ngy ca con ngi. T trc khi c s ra ca thuc ty, nhiu loi cy c trong t nhin c s dng trong dgian cha bnh v rt c hiu qu. Rt nhiu loi bnh tt c ckhi nh tho dc.

Ngy nay nhng hp cht t nhin c phn lp t cy c

ng dng rng ritrong rt nhiu ngnh cng nghip, nng nghip, chngc dng sn xut thuc cha bnh, thuc bo v thc vt, lm nguliu cho ngnh cng nghip thc phm v m phm v.v... Mc d cng nghtng hp ho dc ngy nay pht trin mnh m, to ra cc bit dc khnhau s dng trong cng tc phng, cha bnh. iu gp phn lm tntui 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 bitdc rt kh tng hp. Mt khc vic dng thuc nam hu nh khnggy ratc dng ph.

C nhiu b mn khoa hc nghin cu v cy thuc ra i. Vicnghin 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 mon mun lm thuc cha bnh.

Cy ch rng ca l mt cy thuc c s dng t lu trn thgii cng nh Vit Nam. Trong Y hc dn tc cy ny c nhn dn dnglm thuc cha nhiu loi bnh nh: au vim hng, inh ru, mn nhvim da l nga, sn hu huyt au bng, tr em ta li, chm m, bn

vim gan, rt c hiu qu.


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2

Thc vt ch rng ca c nhiu ng dng quan trng nhng gn mi c cc nh khoa hc cc nc quan tm chn lm i tng nghi

cu, cn nc ta hin c rt t cng trnh nghin cu v thnh phn ha hcv dc l hc ca cy ch rng ca.

Vi mc ch nghin cu v tm hiu thnh phn ha hc cc hp chc hot tnh sinh hc ca cych rng ca, gp phn lm tng thm shiu bit v ngun thc vt lm thuc phong ph v qu gi ca VitNam.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 hahc v nh n d ng mt s nhm ch t c trong cy ch rng ca(Phyllanthus urinaria L ., Euphor biaceae).


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3

CHNG I

TNG QUAN

1.1 M t thc vtCy ch rng ca cn gi ld ip 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 h

cc tnh thnh trong c nc, in hnh nh: Thi Nguyn, Bc Ninh, NghAn, H Giang, Trn th gii loi cy ny mc nhiu cc nc Chu nh: Trung Quc, n , Malaysia, Philippin, Inonexia, Myanma, ThiLan, Chu M nh: Brazil, Argentina,[5].

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


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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. Ho

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 ngnQu 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 d ngsinh h ccachi Phyllanthus 1.2.1 Mt s cng dng ca chi Phyllanthus Cc loi cy thuc chiPhyllanthus (Euphorbiaceae) c s dng

rng ri trong y hc dn tc ca nhiu nc cha bnh thn, bnh ting, bnh vim gan B[7]. Cc loi c dng lm thuc nhiu hn c 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 etThonn) [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 lm4 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, chinhiu ln ung trong ngy (thuc rt ng), liu trnh 30-40 ngy. Khu phnn hng ngy phi hn ch mui, tng m (tht, c, trng, u ph).

Ring cy ch rng ca (Phyllanthus urinaria L) trong y hc dntc c nhn dn dng cha au vim hng, inh ru, mn nht, vimda, l nga, sn hu huyt au bng, tr em ta li, chm m, cha bn

gan, st, rn rt cn rt c hiu qu.


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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 quanhnm nhng tt nht l vo ma h. Thng dng ti c khi phi kh, ngung 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 davi liu lng khng hn ch.1.2.2 Mt s tc dng dc l ca chi Phyllanthus

Nm 1961, phng ng YVinVi trng Vit Nam, nghin cu tcdng khng sinh ca cy ch rng ca thy kt qu tc dng khng sinnh sau: T cu trng (0,5 cm),Typhi (0,9 cm), Flexneri (1,1cm), Sonnei (0cm), Shiga (1cm), Subtilis (0,4 cm), Coli (0 cm).

Nm 1977, mt nhm bc s Vit Nam, khoa Tiu ho, Gan, Mt sdng 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 quxt nghim HBsAg (+). Sau mt thi gian iu tr, kt qu xt nghim m tnc coi l khi. T l t 26/98 bnh nhn. Ngoi ra, thuc cn gip c thngi dng sn xut khng th chng HBsAg (59/98 ngi). Liu iu

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 caPhyllanthus amarus vPhyllanthus niruri t kt qu m tnh 22/37 bnh nhn sau 30 ngy. Cc tcgi cn chng minhPhyllanthus amarus c cha cht lm c ch men pelymerase DNA ca virus vim gan siu vi B [5].


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Nm 2002, Nguyn B Kinh v cng s cng b kt qu nghin culm sng ti Bnh vin Thanh Nhn (H Ni), s dng ch phm LIV/9

(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. Thucc tc dng lm gim v sch HBsAg ca bnh nhn [6].

Nhng cng trnh nghin cu ha hc gn y v cc loiPhyllanthus pht hin mt vi lignan, flavonoit v tanin thy phn c tc dng bo vgan, c kh nng lm sch phn ln cc khng nguyn HBsAg, c ch mn

HIV transcriptase ngc [21].Cc th nghim in vitro ca cy ch vi khng nguyn HBsAg v

vi tn thng gan do cac bontetraclorit gy nn chng minh cy ch ckh chng virus vim gan B. Cy ch rng ca c tc dng khng khuni vi t cu vng, trc khun m xanh, trc khunColi, Shigelladysenteriae, 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 PhyllanthusCho n nay ngi ta pht hin chiPhyllanthus L. h thu du

(Euphorbiaceae) c 700 loi, gm t nhng cy thn tho n cy bi hay cyg 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 lPhyllanthusurinaria 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 loiPhyllanthus flexuosus phn lp c 5 tritecpen mi, cutrc 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-en-3,15,24-triol v hai cht bit l: tritecpen diol betulin [33]; lup-20(29)-

en-3,15-diol [41].


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Theo [39 ], t loiPhyllanthus oxyphyllus tch c: 29-nor -3,4-seco -friedelan tritecpen (axit 29- nor -3,4- seco -friedelan-4(23),20(30)-dien-3-

oic) v mt secquitecpen mi l guaiane secquitecpen (5-hydroxy-6,9-epoxyguaiane). Nhng hp cht ny c nh gi l c kh nng chng oxiha tt nng th nghimIC 50 = 0.017 0.001 mM.

HOOCHO

O

1. 29-nor-3,4-seco-friedelane tritecpen 2. Guaiane secquitecpen

T loiPhyllanthus reticulates phn lp c mt hp cht mi l21-hydroxyfriedel-4(23)-en-3- one v mt vi triterpenoit khc, bit l:friedelin, sitosterol, friedelan- 3-ol, glochi donol v 21-hydroxyfriedelan - 3- on [47].

Theo ti liu [46], t loiPhyllanthus plyanthus Chu Phi cc tc gi tm ra c mt s tritecpenoit:phyllanthol, phyllanthone, (20 S)-3-acetoxy-24-methylenedammaran-20- ol, -amyrin acetate, lupenone v (20 S)-3-acetoxy-24-methylenedammaran-20-ol.

H

HO

CH 3 OCOH

H

H

3. (20S)-3-acetoxy-24-methylenedammaran-20-ol


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T loiPhyllanthus acidus tch c hai pentacyclic tritecpenoit, l: phyllanthol v olean-12-en- 3-ol (-amyrin) [35].

T loiPhyllanthus niruri tch c mt acyclic tritec pen, cu trcca n c d on da trn cc phng php phn tch phl:3,7,11,15,19,23-hexamethyl-2 Z ,6 Z ,10 Z ,14 E ,18 E ,22 E -tetracoshexen-1-ol [10].

T loiPhyllanthus watsonii tch c hp cht tritecpen mi: 26-Nor-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 axitT loiPhyllanthus amarus phn lp c axit thiobarbituric, axit

ascorbic [42 ]. T dch chit n-hexan cng ca loiny Malaysia tchc axit oleanolic v axit ursolic [16], axit amariinic, axit repandusinic A,axit geraniinic B v mt cht l 1-O-galloyl-2,4-dehydrohexahydroxydiphenoyl -glucopyrano z elaeocarpusin [26].

T loiPhyllanthus emblica tch c axitphyllaemblic [52], axitascorbic [23].

T loiPhyllanthus fraternus tch c axit indole-3-butyric, axit 1-naphthaleneacetic [31] v axit trichadenic B c cu trc nh axit 3-hydroxy-D: A-friedooleanan-27-oic, c xc nh da vo phng php phn tch X-ray [38].

HO

H

HOOC

4. Axit trichadenic B


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1.3.3 Mt s i din ca Lignan Trong loi Phyllanthus discoides ngi ta tch c phyllanthin

C14H17O2N, phyllantin C 13H15O3N [27] v phyllantidin C 13H15O2N [20].

Trong loi Phyllanthus niruri L c phyllanthin C 24H34O6,

hypophyllanthin C 24H30O7, niranthin C 24H30O7 (cu trc ca niranthin cch ra l (+)3,4,3,4,9,9-hexamethoxy- 8:8-butyrolignan), nirtetralin C 24H30O7 v phyllteralin C 24H34O6 [8]. Da trn cc d liu ph khi lng v ph cnghng t ht nhn, cu trc canirtetralin v phylt etralin c ch ra l 1-

phenyl tetralins.

CH 2 OCH 3

CH 2 OCH 3

OCH 3

OCH 3

H

H

RO

R 1 O

R 2

CH2OCH3

CH2OCH3

O

OCH3

H

H

H3CO

H3CO

H

O

5 6

5a. Phyllanthin R= R 1 =CH 3, R 2 =H Hypophyllantin

5b. Niranthin R+R 1= -CH 2 -R 2 = OCH 3

CH 2 OCH 3

CH 2 OCH 3

OCH 3

OCH 3

H

H

O

O

H

CH 2 OCH 3

CH 2 OCH 3

OCH 3

H

OCH 3

OCH 3

H

H

H 3 CO

7. Nirtetralin 8. Phyllteralin


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1.3.4 Mt s i din ca khungflavonoitT dch chit etanol loiPhyllanthus emblica L phn lp cquecxetin

v catechin. y l hai cht c hot tnh chng oxi ha tt[49].

O

O

HO

OH

OH

OH

OH

OHO

OH

OH OH

OH**

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 5loi 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 loiPhyllanthus sellowianus phn lp crutin v isoquercitrin

l hai flavonoit chim thnh phn ch yu trong cy ny [19].

O

OH

HO

O -Ramnozo -glucozo

OH

OH

O

O

O

OH

OHHO

OH

13. Rutin 14. isoquercitrin

Theo ti liu [36], t loiPhyllanthus niruri tch c 2 flavanon:

8-(3-metyl-but-2-enyl)-2-phenyl chroman-4-on ( 15 ) v 2-(4-hydroxyphenyl)-


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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.

O

O

1

2

34

5

6

7

1'

2'

3'

4'

5'

6'

1''2''

3''

4a

8a8

O

O

1

2

34

5

6

7

1'

2'

3'

4'

5'

6'

1''2''

3''

4a

8a8

OH

15 16

1.3.5 Mt s hp cht phenolic khc T loiPhyllanthus niruri Linn tch c mt s hp cht phenolic

l: a xit gallic, axit ellagic, ellagitannin, ()-epicatechin, (+)- gallocatechin, ()-epigallocatechin, ()-epicatechin 3- O-gallat v ()-epigallocatechin 3- O -gallat[28]. Hp chtellagitannin cng c tch t loiPhyllanthus amarus [26].

Cc tc gi i Loan phn lp v nhn dng mt s hp chphenolic 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 chittrong metanol v sau c chit ln lt vietyl ete, etyl axetat, butanolv nc. Phn on etyl axetat cho thy c hm lng ln nht ca hp ch 1,1-diphenyl-2-picrylhydrazyl (DPPH) so vi c bn phn on. Phn onetyl axetat c tch bngsephadex v thu c 6 hp cht,chng c nhndng l: geraniin,quercetin-3- -D-glucopyranozit, kaempferol 3- -D-glucopyranozit, isocorilagin, quercetin , v kaempferol. Mt s hp chttrnln u tin c tch t loiPhyllanthus emblica. Cc hp cht tch,c lm sch v em kho st kh nng chng oxi ha. Kt qu cho thtrong s 6 cht trn th geraniin l cht c kh nng chng oxi ha tt nht (

nng IC 50 = 4.7 v 65.7 M, th nghim s oxi ha lipit).


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13

T loiPhyllanthus 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].

COOH

OHHO

OH

OHO

OH

O

OH

OH

OH

OH

OH

OH

C

O

17 . Axit shikimic 18 . Epigallocatechin-3-gallat

HO

HO OH HO OH

OH

COOC

O

OH

OO

OH

OH

OH

OH

OC

O

19. Proanthocyanidins

Theo L.Yeap Foo, Herbert Wong [24 ], cng t loi ny tch cmt sn phm t tannin thy phn (hay ellagitannin c bit) lphyllanthusiin D c cu trc c xc nh l: 1-galloyl-2,4-(acetonyl-dehydrohexahydroxydiphenoyl) -3,6-hexahydroxydiphenoylglucopyranozit.

Theo ti liu[25] cng t loi ny tch c amariin, l mt sn phmthy phn cadi-dehydrohexahydroxydiphenoyl (DHHDP), cu trc ca n

c xc nh l 1-galloyl-2,4: 3,6-bis-DHHDP-glucopyranosit trong phn


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vng cycloh exenetrione ca DHHDP lin kt vi nguyn t O-3 v O- 4 ca

phn ng glucoz, cng vi: geraniin, corilagin, 1,6-digalloylglucopyranozitcng nh rutin v quercetin-3-O-glucopyranozit c phn lp t

Phyllanthus amarus .

Cng t loi ny tch c axit amariinic- l mt dng ellagitannincngvi 1-O-galloyl-2,4-dehydrohexahydroxydiphenoyl-glucopyrano z 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 cDHHDP c gn vo O-4 ca phn ng glucoz .

T loiPhyllanthus polyphyllus L i Loan,Yew-Min Tzeng v cc

cng s tch c benzenoit, axit 4-O-methylgallic [51] , hp chtny chyu c to thnh t s chuyn haaxit gallic v n c nhiu trong ruvang , ch, trong nhiu loitri cy. Cng t loi ny tch c3

arylnaphthalide lignan c tn l: phyllamyricin C, justicidin B v diphyllin.T phn dch chit etylaxetat ca loiPhyllanthus niruri L, Vng

nh Bng, T Sn, Bc Ninh phn lp c hai chtl: 2,4,6-

xyclohepta-trien-2,3,7-trihydroxy-5-ethoxy-1-on ( 20 ) v 5,7,8 trihidroxi-9,10- peoxi- 4- ethoxy benzo[2,3-d] coumarin ( 21 ), hai cht ny cho n nay

cha thy thng bo c mt trong cc loiPhyllanthus [4].

O

HO

HO

OH

O - CH 2 -CH 3

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


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O

HO

HO

O O

OH

O

OCH 2 CH 3

21. 5,7,8 - trihidroxi-9,10- peoxi- 4- ethoxy benzo[2,3-d] coumarin

Hot tnh chng oxy ha ca5,7,8 trihidroxi-9,10- peoxi- 4- ethoxy

benzo[2,3-d] coumarin cng vi quercetin lm i chng c th bng phng phpPotterat trong cng mt iu kin. Cho thy cht ny th hinhot tnh chng oxy ha yu hn so vi quercetin ( nng 25 mg/l vn c phn ng dng tnh) [4].1.3.6 Mt s hp cht nhm ankaloit

T loiPhyllanthus amarus tch c 2 ankaloit mi l isobubbialine

v epibubbialine cng vi 3 ankaloit bit l phyllanthine, securinine vnorsecurinine . Cu trc ca chng c xc nh trn c s phn tch cc dliu 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 hpcht mi thuc nhm ankaloit l niruroidine, cu trc ca n c lm sng tda trn cc phng php ph[9].

T loiPhyllanthus simplex ln u tin phn lp c mt ankaloit lsimplexine (14-hydroxy-4-methoxy-13,14- dihydronorsecurinine) v mt ankaloit bit l phyllanthine [32]. Cht ny v ang c s dng lm thucTy Ydng vin nn ( sn phm c tn lsimplexin) , ph bin trn th trng.

Ngoi nhng nhm hp cht trn, t loiPhyllanthus anisolobus tch c hai -lacton: menisdaurilide v aquilegiolide [43].


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1.4 Tnh hnh nghin cu ha hc ca loi Phyllanthus urinaria L

T loiPhyllanthus 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 loiPhyllanthus

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

HO

glucozo - O

22 . -Amyrin 23. Glucosid

HO HO 24 . -Sitosterol 25 . Stigmasterol

T loiPhyllanthus urinaria L tch c mt s axit hu c nh:Axit succinic, axit ferulic, axit dotriancontanic, axit hecxacosanoic [11], [53].


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H3CO

HO

HO

OH

O

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[1,1-biphenyl]-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 loiPhyllanthus urinaria L[48].

HO

O

O O

O

OH

O

O

27

HO

OH 3 C HO

OH

O

28

T loiPhyllanthus urinaria L phn lp c 4 lignan mi

l 5-demethoxyniranthin ( 29a. C23

H30

O6), urinatetralin ( 30a .


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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].

R 1 O

R 2 O

OMe

OMe

OMe

OMe

R1O

R2O

OR2

OR 1

OMe

OMe

29a. R1 + R 2 = CH 2 30a. R1 + R 2 = CH 2

29b. R1 = R 2 =Me 30b. R1 = R 2 =Me

R 1 O

R 2 O

O

O

O

O

OO

O

OMe

MeO

OO

31a. R1 = R 2 =Me 32

31b. R1 + R 2 = CH 2


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O

O

OMe

OMe

OMe

OMe

OMe

MeO

O

OMe

OMe

OMe

OMe

O

33 34

O

O

OMe

OMe

OMe

OMe

OMe

OO

MeO

MeO

OMe

OMe

35 36

O

OMe

OMe

OMe

OMe

O

O

O

O

OMe

OMe

OMeMeO

37 38

Cng tloi Phyllanthus urinaria L Zhang L. Z. v cng s tchc mt cht mi l ellagitannin [54].


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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 cyStachys. annua [7].

OHO

HO

OOH

OCH 3

39. 4-methoxyscutellarein

T dch chit axeton ca loiPhyllanthus urinaria L cc nh khoa hci Loan tch c geraniin v 1,3,4,6-tetra-O-galloyl- -D-gluco z [13].

Cc th nghim in vitro ch ra hai cht ny c hot tnh chng li vi r bnh mn rp ngoi da v khng gy c.

T loi ny ngi ta cn tch c axit ellagic, axit 3, 3,4- tri-O- methylellagic, axit gallic, 4-O-brevifolincarbonyl-1-O-galloyl-3,6-

O-hexahydroxydiphenoyl-D- glucopyrano z , methyl brevifolincarboxylate[5], [53].

COOH

HO

OH

OH

O

HO

HO

OH

O

O

O

OCH 3

40 . Axit gallic 41. Methyl brevifolincarboxylate


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Cht methyl brevifolin cacboxylat cng c tch bng phng php sk ct t dch chit n- butanol ca cy ny huyn Lng, Ngh An [7].

Theo ti liu [37], t loiPh yllanthus urinaria L i Loan tm c cc hp cht sau: phyllanthin ( 42 ), phyltetralin ( 43 ), trimetyl-3,4-dehydrochebulate ( 44 ), metylgallat ( 45 ), rhamnocitrin ( 46 ), methyl

brevifolincarboxylat ( 47 ), -sitosterol-3- O --d-glucopyranosid ( 48 ),quercitrin ( 49 )v rutin ( 50 ).

C

O

OH

OH

O

OH

O

1

2

3

45

6

7

89

10

1'

2' 3'

4'

5'6'

H 3 C

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 cmt cch k lng v kh nng chng oxi ha v kh nng lm thuc khngvim. Cc cht(44) , (45) v (47) c kh nng chng oxi ha tt ( IC 50 = 9.4,9.8 v 8.9 M, cc ln th nghim ring r). Cn trong cc th nghim in vitrokhc v hot tnh khng vim th ngoi tr(48), cc cht cn li u c hottnh khng vim ca o. Ti liu ny ln u tin thng bo th nghim v khnng khng vim ca loiPhyllanthus urinaria v d o cc cht cphn lp t cyPhyllanthus urinaria c th c s dng lm thuc khngvim trong iu tr.

T loiPhyllanthus urinaria L tch cphyllurine (51) [44], cutrc caphyllurine nh sau:


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+

COO -

NH 2

OH NH 2

51. Phyllurine

Cht ny (51 ) c nhn dng l tng t nhphyllanthurinolacton(52 ). Trn c s cc tc gi xut gi thuyt c s chuyn ha qua ligia(51) v (52) .

Theo ti liu [18], t loi ny phn lp c phyllanthurinolacton (52), cu trc ca n nh sau:

O OO

CH2

NNH

O

O

OH

HO

OH

OH2N

O

O

5

H

52. Phyllanthurinolacton


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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 Huynng H v Thnh Ph Thi Nguyn.

Cy ch rng ca cn gi l cy dip h chu, dn a phng gi ldip he thi, cc nh khoa hc khoa Sinh trng HSP Thi Nguyn xcnh 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 600C cho ti khikh hon ton. Mu kh c nghin nh v ngm chit trong etanol 90o nhit phng nhiu ln, lin tc trong 10 ngy.

Sau khi ct loi dung mi, cn c n di dng xir c chit lnlt bng cc loi dung mi c phn cc tng dn: n-hexan, etylaxetat, n- butanol, metanol. Cc dch chit c ui kit dung mi bng thit b cquay nhit 500C di p sut thp. Cc cn th c phn chia bng sck ct vi cc h dung mi ra gii c phn cc tng dn phn lp ccht c phn cc gn ging nhau, kt tinh phn on v kt tinh li troh 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 khnhau ca cy ch rng ca chng ti phi hp s dng cc phng phsc k v kt tinh li trong dung mi thch hp cc phng php gm:


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- 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 trng: mu sc, mi v, h sR f , im nng chy, ghi cc loi ph nh: phhng ngoi (FT-IR), ph cng hng t ht nhn1H- NMR, ph13C- NMR,

ph khi lng (MS) v cc ph phn gii cao. Cc s liu ph thc nghimca 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 k ttinh

lil ho ch t loi tinh khitphn 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 ph

c trong cc dich chit, cc phn on chy ct v kim tra s b sch csn phm thu c.

Cc h dung mi khai trin SKLM:

1. n-Hexan CHCl 3 85:15 H A2. Cloroform etylaxetat 90:10 H B

3. Cloroform metanol 90:10 H C

4. Cloroform metanol 50:10 H D

5. Toluen etylaxetat axit formic 5:4:1 H E


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Cc bn SKLM sau khi sy kh c soi di n t ngoi (UV-BIOBLOCK ) bc sng = 254nm v 365nm. Thuc th hin mu l

vanilin 1% trong dung dch metanol-H 2SO 4 5%, sau sy trn 1000C .Cc gi tr R f trong h dung mi trin khai c biu thc:

2 .2.2. Thit b nghin cu- Nhit nng chy o trn my Boetus (c), hoc trn my

Eletronthermal IA 9200.- 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 trnmy MS-Engine-5989- HP ion ho bng va chm eletron

(EI- MS) 70ev v s dng ngn hng d liu DATABASE/WILLEY 250L. - Ph1H-NMR v 13C-NMR ghi trn my Bruker 500MHz, ni chun

TMS, dung mi CDCl 3, 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, nghinh c ngm chit kit bng etanol nhit phng cho n khi thu dch khng mu. Dch chit c ct loi ht dung mi p sut gim dng cao kh, xc nh khi lng cn kh, sau thm nc vo cn v llt 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 Na2SO 4, lc v ct kitdung mi bng c quay di p sut gim nhit 500 C. Cn c sykh v cn xc nh trng lng. Nh vy t ton b phn trn mt t c

cy ch rng ca thu nhn c 4 phn on l n-hexan, etylaxetat,

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


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butanol v metanol vi cc k hiu tng ng l: cn trong n-hexan (PH), cntrong etylaxetat (PE), cn trong butanol (PB) v cn trong metanol (PM).

S 2.1: Quy trnh ngm chit mu

1. Etanol

2. Cn trong nc

n-Hexan EtOAc BuOH MeOH

Bng 2.1: Khi lng cht tng s c chit tng phn on ca cy ch rng ca ( Phyllanthus urinaria L)

Khi lngnguyn liu kh

(g)

Khi lngcn cn tng

s (g)

Kh i lng cn chi t (g)

n-Hexan Etylaxetat n-Butanol Metanol

1230 302,05 37,01 24,95 26,14 50,28

MU KH

Cn n-Hexan(PH)

Cn BuOH (PB)

Cn EtOAc (PE)

Cn MeOH (PM)

PE-1 PE-2 PE-3

PH-1


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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 thuc 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 Nhm cht Thuc th

c hiu Hin tng

Kt

qu 1 ng kh Felinh Cho kt ta mu gch++

2 Ankaloit Dragendorf Mu vng da cam +

3 Steroit Liberman-Bourchar Mu xanh vng ++

4 Flavonoit Xianidin T hng n ++

5 Polifenol FeCl 3 Xanh thm ++

6 Cumarin Axit v kim Kt ta bng ++

7 Glycozit tim Kelle-Kiliani Khng c hin tng g -

8 Saponin To bt Bt bn trong axit ++

Ghi ch: Du (+) : Phn ng dng tnh, (++): Phn ng dng tnhrt 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.


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Hnh 2.1 : ng knh vng c ch xung quanh ging thch (mm)

theo phng php khuych tn trn thch


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Cc chng vi sinh vt gm i din cc nhm:* Vi khun Gr(-) Escherichia coli* Vi khun Gr(+)Staphylococcus auresu * Vi khun:Salmonella typhiChng ti tin hnh th cc ng dng lm thc phm chc nng c

dch chit cy ch rng ca ti C s sn xut kinh doanh thuc thn phm, thc phm chc nng Y hc c truyn Thi Nguyn.

Tin hnh kim trahot 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)

Dch chit ng knh vng c ch xung quanh ging thch (mm)

Staphylococcusauresu E.coli Salmonella typhi

Cn nc 34 23 40Cn EtOAc 29 18 32

2.4. P hn lp, tinh ch cc cht t cy ch rng ca ( Phyllanthusurinaria L)

2.4.1. Dch chit n-hexanT 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 csilicagel. H dung mi ra gii l n-hexan : etylaxetat vi t l theo tngdn ca dung mi phn cc t 0 n 100% etylaxetat, kim tra cc phn o bng sc k lp mng, cc phn on ging nhau em gp li v ct loi dumi thu c cht sch.


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* Cht PH -1 ( -Sitosterol)Tin hnh phn chia nh sc k ct bng hdung mi n-hexan -

etylaxetat (9:1). Sau khi ct loi dung mi, cn thu c kim tra bng sc lp mng trong h dung mi A, pht hin n bng vanilin 1% trong dung dchmetanol-H 2SO 4 5%, kt tinh li trong n-hexan thu c 19 mg cht rn, cnhit nng chy 139-141 0C, R f = 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,C 3); 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).

Ph1H-NMR (500MHz, CDCl 3); (ppm): 0,68 ppm (3H, s, CH 3-18);1,01 ppm (3H, s, CH 3-19); 0,81 ppm (3H, d, j 7Hz, CH 3-26); 0,88 ppm (3H, d, j

7Hz, CH 3-27); 0,83 ppm (3H, t, 7,32Hz, CH 3-29); 0,92 ppm (3H, d, j 10Hz,

CH 3-21); 3,52 ppm (1H, m, H-3 ); 5,42 ppm (1H, d, j 5Hz,H-6). Ph13C-NMR (500MHz, CDCl 3); (ppm): Xem bng 2.4.

Bng 2.4 : S liu ph13

C-NMR (CDCl 3, 125Mhz) ca cht PH-1trong cy ch rng ca ( Phyllanthus urinaria L)

STTca C

13C- NMR (-ppm) STTca C 13C- NMR (-ppm)

1 37,2 t 16 28,3 t2 31,7 t 17 56,2 d3 71,8 d 18 11,9 q4 42,3 t 19 19,4 q

5 140,8 s 20 36,2 d6 121,7 d 21 18,8 q7 31,9 t 22 33,9 t8 31,9 d 23 26,1 t9 50,1 d 24 45,8 d

10 36,5 s 25 29,2 d11 21,1 t 26 19,1 q12 39,8 t 27 19,4 d13 42,3 s 28 23,1 t14 56,8 d 29 11,9 q15 24,3 t - - -


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2.4.2 Dch chit etylaxetat (PE)T 5g cn chit etylaxetat ca cy ch rng ca (k hiu PE)

tin hnh tch cc cht trn sc k ct silicagel, ra gii ct sc k bng hdung mi cloroform : metanol c t l theo tng dn ca dung mi phncc, t 0-100% metanol, kim tra cc phn on trn sc k lp mng, thucth pht hin (FeCl3 + K 3[Fe(CN) 6]) 1% sau gp cc phn on gingnhau, ui ht dung mi v kt tinh li thu c 3 cn th. Tinh ch hai cu 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 micloroform : metanol (90:10). Nhng phnon c cng R f =0,8 trong h dung miC c gp li, thu c cn th. Tinhch 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.Ph1H-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).

Ph13C- NMR (500MHz, DMSO); (ppm): Cho cc pic C = 177,91 ppm (C 1);C = 151,70 ppm (C 2); C = 124,33 ppm (C 3); C =109,62 ppm (C 4);C = 162,13 ppm (C 5); C = 55,89 ppm (C 6).

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 uidung mi thu c mt cht rn kt tinh. Tinh ch li bng cloroform thc 40 mg tinh th hnh kim mu vng c nhit nng chy 249-250 oC.

Ph1H-NMR (500MHz, DMSO); (ppm): Cho mt pic 6,91ppm.

Ph13C-NMR (500MHz, DMSO); (ppm): C = 120,48 ppm (C 1);


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C = 108,73 ppm (C 2 v C 6); C = 145,40 ppm (C 3 v C 5); C = 137,98 ppm(C 4); C = 167,46 ppm (C 7).

2.4.2.3 Cht PE -3 (Kampherol)

Sau khi ra gii ct bng h cloroform : metanol (90:10) thu chaicht 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 vidung 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 csilicagel ra gii bng h dung mi toluen : etylaxetat : axit fomic (5:4:1) thuc 29mg cht rn mu vng (c R f =0,4 trong h E), nng chy 276-278 oC.

Ph LC- MS; m/z(%): Cho p ic ion phn tM+ m/z =286,9 v M - m/z =285,0.

Cc s liu ca ph1H-NMR (500MHz, DMSO); (ppm) v ph13C-NMR (500MHz, DMSO); (ppm) (xem bng 2.5):

Bng 2.5: Ph1H-NMR v 13C- NMR ca cht PE-3

Vtr C

13

C-NMR(-ppm)

H-NMR(-ppm, J-Hz)

Vtr C

13

C-NMR(-ppm)

H-NMR(-ppm, J-Hz)

2 147,03 __ 10 104,15 __

3 136,52 __ 1 / 123,32 __


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4 176,58 __ 2 / 130,448,15 (1H,dd,

J=8,2; 2,2)

5 157,78 __ 3 / 116,327,01(1H,dd,

J=8,2;2,2)

6 99,176,26 (1H, d,

J=2,1)4 / 160,16 __

7 164,98 __ 5 / 116,32 7,01(1H,dd,J=8,2; 2,2)

8 94,496,52 (1H, d,

J=2,1)6 / 130,44

8,15 (1H,dd,

J=8,2; 2,2)

9 162,30 __ __ __ __


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34

CHNG III

THO LUN KT QU NGHIN CU

3.1. Ng uyn tc chung phn lp c cc hp cht trong bt k mt thc vt no m khn

lm nh hng ti thnh phn ho hc c trong n th trc khi ngm chi bng dung mi hu c, mu thc vt phi c a i kh men ngay sau kthu mu v sy kh iu kin thch hp.

V nguyn tc vic ngm chit mu thc vt c th tin hnh theo cch ph bin sau.

1. Chit v phn lp cc hp cht t mu thc vt bng cc loi dunmi 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 ctng dn nh phng php 1 thu c cc dch chit c cha cc hp chc phn cc tng i ging nhau.

Vic chit mu thc vt ch rng ca (Phyllanthus urinaria L) cthc hin theo phng n 2(S 2.1).

Cc dch chit th em th hottnh sinh hc. iu gip cho vicnh hng tm kim cc hot cht trong nhng dch chit chottnh mnh vicc chngvi khu n thnghim.3.2. Xc nh nh tnh cc nhm cht thin nhin

Phn tch nh tnh cc hp cht hu c thin nhin trong cc munghin cu l vic lm cn thit trc khi tin hnh phn lp v xc nh c trc ca cc cht c th.


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xc nh nh tnh cc nhm hp cht hu c c hot tnh sinh lcao c trong thc vt thng dng hn c l phn tch bng cc phn ng

trng i vi mi nhm cht nu ra bng 2.2. T kt qu ch ra bny c th nhn thy trong cyPhyllanthus urinaria L t nht c cha ti 7nhm hp cht thin nhin quan trng l: ng kh, poliphenol,flavonoit, cumarin, steroit, saponintritecpen v ankaloit. Khng pht hin thyglycozit tim c trong cc mu th. T kt qu phn tch nh tnh cng s bcho thy cy ch rng ca l mt loi thc vt cha nhiu cht c hot t

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 khcnhau ca cy ch rng c a

Cc dch chit t cy ch rng ca (Phyllanthus urinaria L) u lnhng hn hp phc tp cha cc hp cht khc nhau. phn lp tng chra khi hn hp, s dng cc phng php sc k ct nh: Ct vi ch

hp ph silicagel, vi cc h dung mi ra gii thch hp v thng phi lli nhiu ln. Vic tinh ch cc cht thng dng phng php kt tinh trong dung mi hoc h dung mi thch hp. Nh s thu c cc hp chc tinh khit cao, p ng cc nhu cu kho st tnh chtha l vquang ph ca chng. l nhng yu t quan trng trong qu trnh nhdng v xc nh cu trc ha hc ca cc cht phn lp c t cc

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 schgm 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

R f = 0,72.


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Hnh 3.1 : Ph FT-IR ca -sitosterol (PH-1)


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37

Hnh 3.2 : Ph1H- NMR ca -sitosterol (PH-1)


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38

Hnh 3.3 : Ph13C- NMR v ATP ca -sitosterol (PH-1)


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Cht PH-1 l nhng tinh th hnh kim, khng mu, im nng chy

139-141 0C. Khi trn ln vi cht chun cho mt hn hp c nhit nnchy 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 ph1H-NMR cho = 3,52 ppm c trng cho proton trong

nhm CH lin kt vi nhm hidroxyl.

Ph13C- NMR cng nhn thy = 71,7 ppm c trng cho cacbon linkt trc tip vi oxy ca nhm hidroxyl.

Trong phn t cn c mt ni i th hin bi cc c trng ph FT-

IR 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 ph1H- NMR cng nhn c tn hiu = 5,35 ppm c trng cho H trong lin kt =C-

H v tn hiu ca 2 cacbon mang ni i vi = 140,7 ppm v = 121,5 ppmt c th quy kt hp chtPH-1 c mt ni i gia v tr C-5 v C-6.

So snh ph1H- NMR v ph13C- NMR ca cht PH-1 vi ph ca -

Sitosterol thy hon ton tng t nhau (bng 3.1). T cc kt qu phn tcthnh 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 honton c th cho php quy kt cht PH-1 phn lp c t cy ch rngca l -sitosterol.


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Bng 3.1: S liu ph13C-NMR (CDCl 3, 125Mhz) ca PH-1 trong cy

ch rng ca ( Phyllanthus urinaria L) v ph ca -sitosterol [15]

Cht PH-1 -Sitosterol [15]V tr C ca C (ppm) V tr C ca C (ppm)

1 37,2 1 37,22 31,7 2 31,63 71,8 3 71,74 42,3 4 42,35 140,8 5 140,76 121,7 6 121,77 31,9 7 31,98 31,9 8 31,99 50,1 9 50,1

10 36,5 10 36,511 21,1 11 21,212 39,8 12 39,813 42,3 13 42,314 56,8 14 56,715 24,3 15 24,316 28,3 16 28,217 56,2 17 56,118 11,9 18 11,919 19,4 19 19,420 36,2 20 36,121 18,8 21 18,922 33,9 22 34,023 26,1 23 26,124 45,8 24 45,825 29,2 25 29,226 19,1 26 19,827 19,4 27 19,528 23,1 28 23,029 11,9 29 12,0


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41

2

34

5

101

67

89

1312 17

16

21 22 24

2829

26

27

19

18

HO

11

14 15

20 23 25

-Sitosterol

3.3.2. 5-Hydroxymetylfufural (PE-1)

Cht PE-1 c tch t cn etylaxetatc R f = 0,8 (trong h dung micloroform : 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 iuny 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. Ph13C- NMR cho tn hiu = 177,91 ppm, chnh l chuyn dch ha hcca 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 vhiro (=C-H), c th quy kt = 124,31 ppm l C-3 cn = 109,62 ppm lca 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 hc151,70 ppm v 162,13 ppm l

cc nguyn t cacbonC-2 v C- 5. C mt nhm CH2 vi = 55,89 ppm. Chai 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.


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42

Hnh 3.4 : Ph 1H-NMR-DMSO c a PE-1


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43

Hnh 3.5 : Ph 13

C-NMR-DMSO c a PE-1


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44

Bng 3.2: S liu ph NMR ca PE-1 v s liu ph NMR trongphn mm ACD/NMR ca hidroxymetylfufural

V tr C Ph ca cht PE-1

Phn mm ACD cahidroxymetylfufural.

C- NMR

(ppm)

H-NMR

(ppm)ACD/ 13C-NMR ACD/ 1H-NMR

1 177,91 9,54 179,55 9,53

2 151,70 - 156,33 -3 124,31 7,48 121,65 7,41

4 109,62 6,60 112,74 6,68

5 162,13 - 160,22 -

6 55,89 4,50 56,63 4,70

Phn tch ph1H-NMR, HMBC cho thy tn hiuH-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 ( =

7,48 ppm) 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 109,62ppm v 162,13 ppm.

So snh ph NMR ca cht PE-1 vi phn mm ACD/1H-NMR vACD/ 13C- NMR cho thy s tng t gn nh hon ton vi ph ca5-

hidroxymetylfufural (xem bng 3.2).


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Tcc kt qu phn tchcc loi ph ca hp cht PE-1nh: ph khi, ph NMR vi cc k thut o1D v 2D, c so snh vi phn mm m phng

ACD/NMR, c th khng nh cht PE-1 l 5-hidroxymetylfufural.

1

2

34

56 O

C

O

H

HO

5-Hidroxymetylfufural

Ngy nay cht5-hidrox ymetylfufural c phn lp t mt s cythuc dn gian khc nh t cy a hong, cy trinh n hong cung n c vaitr trong qu trnh chuyn ho cht trong c th sinh vt. Trong cy ch rng ca (Phyllanthus urinaria ), cht5-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, tinhth hnh kim mu vng c nhit nng chy 249-250 oC.

Ph1H- 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.Ph13C- NMR cho thy cc chuyn dch ha hc = 120,48

ppm, = 137,98 ppm v = 167,46 ppm, tng ng vi chuyn dchhahc ca cc nguyn t cacbon C1, C 4 v C 7 trong axit gallic. C 2 tn hiu = 108,73 ppm v = 145,40 ppm, mi tn hiu ng vi 2 nguyn tcacbon. Cc tn hiu ny ph hp vi chuyn dch ha hc ca C2 v C 6

( = 108,73 ppm) v ca C3 v C 5 ( = 145,40 ppm).


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46

Hnh 3.6 : Ph 1

H-NMR-DMSO c a PE-2


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47

Hnh 3.7 : Ph 13

C-NMR-DMSO c a PE-2


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48

So snh ph NMR ca cht PE-2 vi phn mm ACD/1H-NMR vACD/ 13CNMR cho thy s tng t gn nh hon ton vi ph ca axit galli

(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 chtPE-2 c tch ra t dch chitetylaxetat ca cy ch rng ca c nhn dng l axit gallic.

1

COOH

OHHO

OH

2

3

4

5

6

7

Axit gallic

C th coi axit gallic l mt n phn tham gia vo vic to ra cctanin thu phn v tham gia vo cc chuyn ho khc tora cc lignan trong

cy ch rng ca.


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51

Hnh 3.9 : Ph 13

C-NMR-AcetoneD6 c a PE-3


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Bng 3.4 : S liu ph NMR ca PE-3 v s liu ph trong phn mm

ACD/NMR ca cht kampherol

V tr C Ph ca cht PE-3

Ph n m m ACD ca ch tkampherol

C- NMR

(ppm)1H-NMR (ppm)

ACD/ C-

NMRACD/ 1H-NMR

2 147,03 - 146,45 -

3 136,52 - 135,60 -

4 176,58 - 175,80 -

5 157,78 - 157,63 -

6 99,176,26 (1H, d,

J=2,1)98,25 6,28

7 164,98 - 163,90 -

8 94,496,52 (1H, d,

J=2,1)93,45 6,54

9 162,30 - 159,13 -

10 104,15 - 103 -

1 123,32 - 121,65 -

2 / 130,448,15 (1H, dd,

J=8,2; 2,2)129,40 8,10

3 / 116,32 7,01(1H, dd,J=8,2;2,2)

115,35 7,00

4 160,16 - 159,10 -

5 / 116,327,01(1H, dd,

J=8,2; 2,2)115,35 7,00

6 / 130,448,15 (1H, dd,

J=8,2; 2,2)129,40 8,10


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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 tngtc xa vi C-4 (160,16 ppm) v C-6 (130,44 ppm), H-6 tng tc xa vi C-

5 (116,32 ppm) v C-1 (123,32 ppm), H- 6 c tng tc xa vi C-5 (157,78ppm) v C-7 (164,98 ppm). Cn H- 8 c tng tc xa vi C-7 (164,98 ppm) vC-9 (162,30 ppm).

Qua vic phn tch cc loi ph ca hp cht PE-3 c so snh vichng trnh m phng ph ACD/1H-NMR v ACD/ 13CNMR cho thy stng thch kh tt vi sai s cho php c th khng nh hp cht PE-3

chnh l kampherol.

C

O

OH

OH

OH

HO

O

1

2

3

45

6

7

89

10

1'

2'3'

4 '

5'

6/

Kampherol hay 3,5,7,4 / -tetrahidroxi flavon

Kampherol l mt flavonoit c trong nhiu thc vt, n khng c vc tc dng sinh hc quan trng. Ln u tin cht ny c phn lp t cch 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 cny ti b mn Vi sinh Trng i hc Y khoa i hc Thi Nguyn. Mu

th gm c:


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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 tnhvi vi sinh vt kim nh.Cthl:

- Dch chit trong n cv d ch chitetylaxetat c acy ch rngca uc ho t tnh kh m nhvicc khu n Staphylococcusauresu (Tcu

vng) , E.coli (Thckhun ngrut) v Salmonella typhi (Th ng hn).- Dchchitncc ho ttnh mnhh n so v i d ch chitetylaxetat.

- Dch chitncc ho ttnh mnhnhtvikhun Salmonella typhi(Th ng hn).


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55

KT LUN

1. Ln u tin, cy ch rng ca (Phyllanthus urinaria L) mc hoang tiThi Nguyn c nghin cu sng lc ha thc vt, thit lp c quy trnhngm chit mu hp l thu c 4 dch chit chn lc.

2. Tthn 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 lcht: ng kh, ank aloit, 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 chittngcacy ch rngca (Phyllanthus urinaria L) cho thy c tc d ng khng cc khu n Staphylococcusauresu (T cuvng) , E.coli (Thc khun ngrut) v Salmonella typhi (Th ng hn).


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DANH MC CC CNG TRNH CNG B

LIN QUAN N LUN VN

1. Ng c Trng, Phm Vn Thnh (2008), Nghin cu ha hc thcvt cy ch rng ca(Phyllanthus urinaria L.) mchoang t iThi Nguyn(Chemical studies on the constituents of Phyllanthus urinaria L. growing

wildly in Thai Nguyen province), Tp ch Khoa hc& Cng ngh ihc

Thi Nguyn, s2 (46), tr 75-79.


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TI LIU THAM KHO

A. Ti liu ting Vit 1. Huy Bch v cng s (2004),Cy thu c 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 Y-

Dc, 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 nm43, tr.12-15.

4. Nguyn Vn u, Trn Th Thu H (2007), Ngin cu ha thc vt cych rng ca(Phyllanthus uticulatus niruri L). Tp ch dc hc, s369 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 ThngDong v ng tc gi (2001), Kho st cng dng lmthuc ca mt s loiPhyllanthus 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 shp cht phenolic t cy ch rng ca,Tp ch Dc hc, s 371 nm47, 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

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