B Y T

TRNG I HC DC H NI

---------

NGUYN TH PHNG DUNG

PHN LP TETRODOTOXIN T

PH TNG C NC

KHA LUN TT NGHIP DC S

H NI-2014

B Y T TRNG I HC DC H NI

NGUYN TH PHNG DUNG

PHN LP TETRODOTOXIN T

PH TNG C NC

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. TS. o Th Thanh Hin

2. ThS. Phng Minh Dng

Ni thc hin:

1. B mn Dc c truyn

2. Cng ty CPDP Mediplantex

3. Vin Kim Nghim Thuc Trung ng

H NI-2014

LI CM N

Li u tin, ti xin chn thnh cm n TS.o Th Thanh Hin ging vin b

mn Dc hc c truyn, ThS.Phng Minh Dng tng gim c cng ty c phn

dc phm Mediplantex trc tip hng dn, ch bo v to iu kin thun li

ti hon thnh kha lun ny.

Ti cng xin gi li cm n TS.Trn Vit Hng ph gim c Vin kim

nghim thuc Trung ng, thy gio TS.Bi Hng Cng v cc thy c trong b

mn Dc hc c truyn, DS.Dng Minh Tn v cc anh ch trong Vin kim

nghim gp , tn tnh gip mi mt ti hon thnh kha lun ny.

Xin trn trng cm n Ban gim hiu, cc thy c gio trng i hc Dc

H Ni to mi iu kin tt nht cho ti trong qu trnh hc tp v nghin cu.

Cui cng ti xin t lng bit n ti gia nh, bn b nhng ngi thng xuyn

ng vin, gip ti trong sut qu trnh hc tp v nghin cu, ti t c kt

qu nh ngy hm nay.

Chn thnh cm n!

H Ni, ngy 22 thng 05 nm 2014

Sinh vin

Nguyn Th Phng Dung

LI CM N

DANH MC CC K HIU, CH VIT TT

DANH MC CC BNG

DANH MC CC HNH V, TH

MC LC

T VN .................................................................................................... 1

CHNG 1: TNG QUAN .......................................................................... 2

1.1. C NC .................................................................................................. 2

1.1.1. V tr, phn loi ............................................................................... 2

1.1.2. c trng hnh thi ......................................................................... 3

1.1.3. Tp tnh sng- phn b ................................................................... 3

1.1.4. Thnh phn c t ........................................................................... 4

1.1.5. c tnh............................................................................................ 5

1.2. TETRODOTOXIN .................................................................................. 6

1.2.1. Tnh cht, cu trc ........................................................................... 7

1.2.2. Tc dng dc l- c tnh .............................................................. 9

1.2.3. ng dng iu tr ca TTX .......................................................... 11

1.3 MT S NGHIN CU CHIT XUT TTX ..................................... 12

CHNG 2: I TNG V PHNG PHP NGHIN CU ........... 14

2.1. NGUYN VT LIU, THIT B ........................................................ 14

2.1.1. Nguyn liu .................................................................................... 14

2.1.2 Thit b, dng c, ha cht: ............................................................ 14

2.2. NI DUNG NGHIN CU ................................................................. 15

2.2.1 Chit xut c t ton phn ............................................................ 15

2.2.2. Phn lp TTX ................................................................................. 15

2.2.3. nh tnh TTX ............................................................................... 15

2.2.4. nh lng TTX ............................................................................ 15

2.3. PHNG PHP NGHIN CU......................................................... 15

2.3.1. Phng php chit xut ................................................................. 15

2.3.2. Phng php phn lp TTX ........................................................... 17

2.3.3. nh tnh TTX ............................................................................... 19

2.3.4 nh lng TTX bng phng php LC-MS/MS .......................... 20

CHNG 3: THC NGHIM, KT QU V BN LUN ................... 22

3.1. THC NGHIM, KT QU ............................................................... 22

3.1.1. Chit xut ....................................................................................... 22

3.1.2 Phn lp: ......................................................................................... 28

KT LUN V KIN NGH .......................................................................... 32

TI LIU THAM KHO ................................................................................ 33

PH LC ......................................................................................................... 36

DANH MC CC K HIU, CH VIT TT

K hiu, ch vit tt Ni dung

CE Collision energy-nng lng bn ph

dc- Decarbamoyl-

Dd Dung dch

ESI Electron spray ionization-ion ha tia in

GTX Gonyautoxin

HPLC Sc k lng hiu nng cao

LC-MS/MS Sc k lng khi ph

MLD Mean lethal dose-liu gy cht trung bnh

NMR Nuclear Magnetic Resonance-cng hng t ht

nhn

PL Ph lc

PSP Paralytic Shellfish Poisoning-c t gy lit

TTX Tetrodotoxin

STX Saxitoxin

SRM Select reaction monitoring

DANH MC CC BNG

Bng 3.1: Kt qu chit xut ............................................................................ 25

Bng 3.2: Kt qu kho st nng dung mi chit ........................................ 26

Bng 3.3: Kt qu kho st pH ......................................................................... 27

Bng 3.4: Kt qu phn lp .............................................................................. 30

Bng 3.5: Kt qu nh lng .......................................................................... 30

DANH MC CC HNH V, TH

Hnh 1.1: Cc h c nc trong b c nc Tetraodontiformes ............................. 3

Hnh 1.2: Cng thc TTX v cc analog (TTXs)............................................... 5

Hnh 1.3: Cng thc ca nhm cht c PSP (saxitoxin v cc dn xut) ........ 5

Hnh 1.4: Cu trc phn t TTX (A) cu trc phng, (B) cu trc 3-D ............. 7

Hnh 1.5: C ch s epime ha nhm OH-C4 ca TTX .................................... 9

Hnh 1.6: TTX chn knh vn chuyn natri lm t lit thn kinh .................... 10

Hnh 2.1:S chit xut TTX t ph tng c nc.......................................... 16

Hnh 2.2: S phn lp theo phng php ca Maoquing Zhou .................. 18

Hnh 3.1: (A) dch chit qua giy lc 5m, (B) dch chit qua giy lc

0,45m .............................................................................................................. 24

Hnh 3.2: Sc k lp mng (Rf=0,2) ................................................................ 24

Hnh 3.3: th kho st nh hng ca nng dung mi chit n kt qu

chit xut .......................................................................................................... 26

Hnh 3.4: th kho st nh hng ca pH chy ct n kt qu chit xut 27

1

T VN

Vit Nam l mt nc nm trong khu vc nhit i gi ma, c vng ti nguyn

bin di do v tim nng khai thc bin rt ln. Trong nhng nm gn y, cng tc

iu tra ngun li bin c y mnh nhm nh gi hin trng ngun li hi sn

v xu hng bin ng ca chng, c nc cng l mt trong nhng i tng c

quan tm nghin cu [2].

C nc c coi l loi c th hai trong th gii ng vt c xng sng, ch ng

sau cc c vng. c cht chit t c nc l hn hp ca hn 10 analog, ch yu

trong s ny l tetrodotoxin thuc nhm c t thn kinh cc k nguy him, c tnh

bn nhit ln v kh nng gy t vong cao [10]. TTX c trin vng thay th morphin

gim au trong ung th v cai nghin [3], [13] nhng TTX c hm lng rt thp

trong c nc. C vi nghin cu v chit xut TTX nhng khng c cng b hoc

cng b khng y [4], [6], [20], [21].

Vi mc ch nghin cu TTX trong c nc, ti thc hin ti: Phn lp

tetrodotoxin t ph tng c nc c tin hnh quy m phng th nghim.

Mc tiu ca ti:

1. Nghin cu chit xut c t ton phn t ph tng c nc quy m phng

th nghim

2. Nghin cu phn lp tetrodotoxin t c t ton phn quy m phng th

nghim.

2

CHNG 1: TNG QUAN

1.1. C NC

1.1.1. V tr, phn loi [10]

Phn ngnh c s Craniata

Lp c xng Osteichthyes

Phn lp c vy tia Actinopterygii

Lin b c vc Percomorpha

B c nc Tetraodontiformes

+ Phn b c nc hm Ostracioidei

1. H c nc hm Ostraciidae

H ph c nc hm lc lng Aracaninae

H ph c nc hm Ostraciinae

+ Phn b c nc Tetraodontoidei

2. H c nc ba rng Triodontidae

3. H c nc Tetraodontidae

H ph c nc dt Canthigasterinae

H ph c nc trn Tetraodontinae

4. H c nc nhm Diodontidae

V tng th, b Tetraodontiformes cha 10 h cn sinh tn vi khong 430 loi v

khong 9 h tuyt chng [15]. Kt qu nghin cu nm 2013 ca Betancur-R v

cng s cho rng b C nc c quan h h hng gn vi nhnh cha b Lophiiformes

v h Caproidae [17]. Ngi ta cng c tnh rng b ny l hu du ca cc loi

sinh sng ven san h, xut hin khong 95 triu nm trc [15].

3

1.1.2. c trng hnh thi [10]

Hnh 1.1: Cc h c nc trong b c nc Tetraodontiformes

c im quan trng nhn bit v phn bit vi cc loi c khc l c nc

khng c vy bng v cc vy u khng c gai cng. Vy lng v vy hu mn nm

i din hoc gn i din vi nhau v chng nm cch xa vy ngc, gn vi vy

ui. Vy ui thng trn hoc bng hoc lm nng (tr c nc ba rng c vy ui

ch su). C nc khng c khe mang, mang ch cn l l mang v ngay sau l mang

l gc vy ngc. Thn c nc khng c vy. C nc nhm c gai sc nhn nh lng

nhm. C nc hm c lp gip cng lin kt vi nhau thnh hnh hp bao quanh c

th. Ming c nc b nhng rng kho; xng hm v xng gn hm gn lin vi

nhau thnh m cng thch nghi vi cc loi thc n c v cng. C nc khng c

xng sn v cc xng dm phn tht. D dy c c th co dn v nhiu loi

c nc c th ht c nhiu nc hoc khng kh lm phng trn bng ln nh

qu bng.

1.1.3. Tp tnh sng- phn b [10]

C nc phn b kh rng c v khng gian v kha cnh sinh thi. Mt s loi

a sng y, trong khi s khc sng cc rn san h c su t vi chc mt n

hng trm mt hay cc vng nc ven b, m ly, ca sng; thm ch mt s t loi

cn sng nc ngt, sng sui, h... C nc l loi n tp, sng n l hoc theo

n v thng khng di c.

4

Trn th gii c khong 246 loi c nc, bao gm c c nc nc mn v nc

ngt, chng sng cc khu vc bin nhit i hoc cn nhit i ca Thi Bnh

Dng, n Dng v i Ty Dng. Vit Nam c khong 67 loi c nc thuc

bn h ni trn. Chng phn b kh rng v c bt gp gn nh ton vng bin

Vit Nam .

1.1.4. Thnh phn c t

c cht chit t c nc l hn hp ca hn 10 analog, ch yu l TTX,

(chim ti 70% - 80% khi lng chit). Ba analog chnh khc l acid tetrodonic, 4-

epi TTX v 4-epi anhydrotetrodotoxin. So vi TTX, ba cht ny khng khc nhiu

v c tnh ha hc, nhng khc nhau ng k v c tnh sinh hc (c t tnh) [20].

Theo nghin cu ca Nguyn Hu Hong (Vin Nghin Cu Hi Sn) trn 5

loi c nc (Takifugu oblongus, Arothron stellatus, Arothron hispidus, Torquigener

brevipinnis, Lagocephalus sceleratus) c thu thp cc tnh ven bin Vit Nam

nm 2005 2006: thnh phn c t c nc l hn hp nhiu c t. Trong , nhm

c t TTXs (TTX v cc dn xut 4,9-anhydro TTX, 4-epi TTX) l thnh phn

chnh, chim t l 97,47%. Cc c t thuc nhm cht c thn kinh PSP l saxitoxin

v cc dn xut ca n (neoSTX, dcSTX, GTX6 v GTX5) ch chim t l rt nh,

khong 2,53% [5].

Theo nghin cu ca Chih-Yu Chen v Hong-Nong Chou (1998) nh lng

TTX v cc dn cht ca n trong hai loi c nc Diodon holocanthus v Takifugu

rubripes rubripes hn o pha bc i Loan c kt qu TTX chim 77%, 4-epi-

TTX 11%, cn li anhydro-TTX 12% [12].

Nm 2011, Xiao-Wu Chen v cng s xc nh cng thc ca TTX v 10

analog ca TTX v nh lng TTX (92,54%) v 4 analog: 5,6,11 trideoxy TTX

(2,81%,), 11-nor TTX-6(R/S)-ol (0,412%), 4,9-anhydroTTX (1,32%) v 5-

deoxyTTX (2,92%) c trong dch chit c nc [28].

5

Hnh 1.2: Cng thc TTX v cc analog (TTXs)

Hnh 1.3: Cng thc ca nhm cht c PSP (saxitoxin v cc dn xut)

1.1.5. c tnh

C nc c coi l loi c th hai trong th gii ng vt c xng sng, ch

ng sau cc c vng. c t trong c nc c thnh phn ch yu l TTX, thuc

nhm c t thn kinh cc k nguy him, c tnh bn nhit ln v kh nng gy t

vong cao [3]. Tuy nhin, khng phi loi c nc no cng c. c t trong cc loi

6

c nc khc nhau th khc nhau. Hm lng c t trong c th cn thay i theo

ma, vng a l v giai on pht trin ca c th [10].

Cc b phn khc nhau ca c nc c c tnh vi mc rt khc nhau. Mc

c ca a s cc loi c th c sp xp theo trt t gim dn ca mnh nh

sau: trng, tinh hon, gan, rut, da, tht [7], [8].

Theo giai on trng thnh v thun thc sinh dc, c nc c c tnh cao

giai on 5 i vi c c v giai on 6 i vi c ci. c tnh ca c nc thng

tng cao vo cc thng 2-3 v 7-9 trong nm, y cng l ma sinh sn ca c nc

[7].

Ti Vit Nam, ngi ta tin hnh phn tch c t ca 35 loi [7], [8], trong :

10 loi c c tnh rt mnh

7 loi c c tnh mnh

4 loi c c tnh nh

C 14 loi cha pht hin thy c t

1.2. TETRODOTOXIN

TTX l cht c chnh trong c t c nc, c pht hin Nht Bn nm

1909 [13], l mt c t thn kinh cc mnh cho ti nay khng c cht gii c, c

t tn theo loi c nc u tin pht hin thy c cha c t ny [18].

c t tetrodotoxin c tm thy nhiu nht cc loi thuc h

Tetraodontidae. Ngoi ra TTX cn cha nhng loi sinh vt bin khc nhau nh

bch tuc tua xanh Australian (Hapooloclaena maculosa); c m vt, c thn tin

(Ostracion spp), c mt trn i dng,, mt s loi gh nh gh Philippin, gh

mt (Eriphia spp, Carcinoscorpius rotudicauda), mt s loai c sn bin, hai

loi ch c (Harlequin). C nc vn l nguyn liu chnh tch chit v nghin

cu v TTX (Mosher, 1986) [8].

7

1.2.1. Tnh cht, cu trc

1.2.1.1 c im, tnh cht:

- Tn IUPAC: (4 R ,4a R ,5 R ,6 S ,7 S ,8 S ,8a R ,10 S ,12 S )-2-azaniumylidene-

4,6,8,12-tetrahydroxy-6-(hydroxymethyl)-2,3,4,4a,5,6,7,8-octahydro-1 H -

8a,10-methano-5,7-(epoxymethanooxy)quinazolin-10-olate

- Cng thc phn t: C12H17N3O8

- Phn t lng: 319,27 g/mol

- TTX tinh khit l bt tinh th khng mu, khng mi, sm mu khong 2200C

nhng khng phn hy [13].

- Nhit nng chy 2250C [14].

(A) (B)

Hnh 1.4: Cu trc phn t TTX (A) cu trc phng, (B) cu trc 3-D

- Cu trc lin quan n tnh cht :

+ tan: trong phn t TTX c mt vi nhm hydroxyl thn nc, khin n

khng tan trong cc dung mi hu c. Khung phn t ca TTX tng t nh cu

trc lng ca , khin rt kh hydrat ha, do vy n t tan trong nc. Do trong

phn t c nhm guanidin perhydroquinazolin (guanidin c tnh kim mnh), nn

TTX tan trong dung dch acid. TTX cng c cu trc ni este, nn d b cc dung

dch acid mnh phn hy, do cch duy nht gi TTX bn vng trong dung

dch l ha tan trong acid hu c yu [11], [19].

8

+ pKa (H2O) = 8,76; pKa (50% alcol) = 9,4 [24]

+ Cht phn cc mnh [18].

1.2.1.2. Cu trc TTX v cc analog [20]

c tnh C-4 c th d dng nhn thy t cu trc phn t ca TTX. C-4 l v tr

ortho ca nguyn t nit vi nhm OH v tr xch o v nguyn t H v tr trc.

Bi vy, cc hot tnh ha hc v sinh hc ca nhm hydroxyl C-4 l rt ng k.

Nu H+ c mt trong dung dch, nguyn t xy t nhm hydroxyl ca C-4 s kt hp

vi n, to ra cu trc B ha tr dng t cu trc A. Cu trc B mt phn t H2O to

thnh cu trc C vi C-4 ha tr dng.

Cu trc C c th tng tc vi H2O trong dung dch. H2O c th tn cng v tr

ni phn t H2O gc b loi b v to thnh cu trc E, hoc tn cng v tr i din

ni phn t H2O gc b loi b v to thnh cu trc D. Nu phn t H2O b loi khi

cu trc E, cu trc A gc ca TTX c to thnh. Cu trc D chuyn thnh cu trc

F sau khi H2O b loi b. S khc nhau gia cu trc F v cu trc A l v tr ca H

v OH hon i cho nhau. H trong C-4 ca cu trc A l trc v OH l xch o, trong

khi cu trc F nguyn t H trong C-4 l xch o v OH l trc.

TTX cu trc A c gi tetrodotoxin, l nng ch yu ca TTX thu c

t c nc t nhin. TTX cu trc F thng c gi 4-epi tetrodotoxin. Do nhm

hydroxyl C-4 gn vi nhm hydroxyl C-9 trong 4-epi tyetrodotoxin, phn t H2O

d dng b loi bi tng tc vi H+, to ra mt analog ca TTX cha lin kt ether,

c gi l 4-epi anhydrotetrodotoxin. Cc c tnh ha hc ca ba phn t

tetrodotoxin ny khc nhau khng ng k. Nhng chng khc nhau ng k v

hot tnh sinh hc. V d, c t ca TTX l 4500 n v chut/mg; ca 4-epi TTX l

710 n v chut/mg v ca 4-epi anhydrotetrodoxin ch l 92 n v chut/mg.

S quan trng ca nhm hydroxyl C-4 cn th hin ch: c tnh ca n gim

ng k khi n c thay th bng nhng nhm khc, nh H, CH3 hay CH3CO-. Do

, v l thuyt, cn gi nhm hydroxyl trong C-4 v tr xch o trong qu trnh

chit TTX. Bi vy, iu quan trng l la chn ng cc vt liu v thit b chit,

pH v nhit ca dung dch, thi gian chit

9

Hnh 1.5: C ch s epime ha nhm OH-C4 ca TTX

1.2.2. Tc dng dc l- c tnh

1.2.2.1. C ch [9]

TTX l cht c thn kinh rt c hiu, bao vy c hiu cng tch in ca cc

knh Natri nm trn b mt ca mng t bo thn kinh.

- Cu trc phn t vi nhm Guanidin tch in dng v vng Pyrimidin to ra s

tng cng hp nht cc h thng vng kt hp vi 5 nhm hydroxyl lm tng tnh

bn vng ca phc h lin kt ga TTX vi knh Natri ti phn a nc. S lin

kt ca TTX vi knh Natri rt nhy (Kd = 10 10nM). TTX c tc dng rt ging

tc dng ca hydrat Natri, khi xm nhp vo ming knh lin lin kt vi nhm

peptit glutamat, sau tht cht vng li khi peptit thay i cu hnh khng gian

ca na phn lin kt. Do vy phc h nhn dng thay i

- TTX tn cng vo cc in tch c tc dng m cng ca knh Na+ (hu qu th

hai xy ra trong c th khi dehydrat phc h nc-Na+). S bm cht ca

tetrodotoxin c nc lm bao vy s truyn dn xung thn kinh ti phc h Na+-

knh c gii thch r rng hn bi thi gian chim gi lu ca TTX so vi

hydrat- Na+ trong phc h. Hydrat natri c th o ngc lin kt trong thi gian

1nanogiy, trong khi TTX lin kt vi knh v tn ti trong thi gian c tnh

10

bng ly tha 10 ca giy l mt khong thi gian v cng ln, bi v 1 gi ch

bng 3600 giy.

Hnh 1.6: TTX chn knh vn chuyn natri lm t lit thn kinh

- Vi lng ln cc phn t TTX ny khng cho natri c c hi xm nhp vo

knh, s di chuyn natri b bao vy vi hiu lc cao v in th hot ng dc theo

mng thn kinh b dng li. Mt miligram TTX hoc t hn (mt lng nh nh

u kim), cng git cht mt ngi trng thnh.

1.2.2.2. Tc dng-c tnh

TTX tc dng c ln h thn kinh trung ng ln thn kinh ngoi vi, vi cc biu

hin sau [1], [18]:

- Tim mch: Mch nhanh, huyt p h, ri lon nhp tim.

- H hp: Kh th, do lit c h hp, da v nim mc xanh tm.

- Thn kinh:

+ Thn kinh trung ng: Chong vng, au u, co git. Khng c ri lon

thc.

+ Thn kinh ngoi vi: Lit a dy thn kinh, ri lon cm gic (d cm) li,

mi, mt, ngn tay, ngn chn, ri lon li ni, kh nut.

+ Thn kinh c vn v c trn: Lit, rung git cc c, c ng hn n, yu c

v lit chi di, lit vn ng nhn cu, c bit nguy him l lit c lin sn,

c ngc v c honh.

+ Mt phn x ty v phn x gn xng.

11

Ngoi ra, TTX cn gy c vi gan, tit niu, ni tit v h sinh sn, da v vn

mch (ra nhiu m hi., mt, mi, hng, tai, tng tit nc bt, ri lon v gic, bch

cu tng, min dch, ri lon chuyn ho nc v in gii, d ng v cc ri lon

khc

Tnh c ca TTX gp 10 n 100 ln so vi liu gy cht ca nhn c v gp

10000 ln so vi cyanid [13].

1.2.3. ng dng iu tr ca TTX [4]

Th nht, TTX c tc dng kch thch hot ng ca h tun hon, lm thay

i nhp tim, thay i trng lc c ca thnh mch, dn n s thay i ca huyt

p. S dng TTX vi liu lng 1,3.10-3 mg/kg trong vic phng nga c hiu qu

cc trng thi x cng ng mch v bo ch cc loi thuc c tr cha bnh huyt

p, ri lon nhp tim.

Th hai, TTX lm gim tnh thm ca mang t bo i vi vic vn chuyn

ion Na+ m khng nh hng n qu trnh vn chuyn ion K+, trong khi cc thuc

gy t khc li c tc dng i vi c hai knh vn chuyn ny. iu ny rt c

ngha trong vic nghin cu sinh l mng t bo.

Th ba, TTX c dng iu ch thuc gy t, gy m trong phu thut.

Ngoi nhng tc dng gy t ging thuc: novocain, procain, cocain, TTX c nhiu

u im hn kh nng gy m ti ch v tc dng mnh hn nhiu so vi thuc gy

t khc. V d: km hm hot ng ca h thn kinh bng cocain phi cn n nng

500mg, trong khi TTX ch cn 0,03mg (hiu qu tc dng ca TTX mnh gp

60.000 ln cocain).

Th t, TTX cn c dng nh thuc gim au, khi s dng TTX vi liu

lng rt nh c kh nng ct cn au ca bnh nhn ung th gan giai on cui.

Cng ty International Wex Technologies (Canada) s dng TTX ch ra tectin

(thuc gim au), Tocudin (thuc gy t), v Tetrodin (thuc cai nghin ma ty)-mt

vi thuc gip bnh nhn ung th vt qua c nhng cn au hoc gip con nghin

heroin ct cn. Cc th nghim ban u khi s dng thuc cho kt qu kh quan.

Theo cc chuyn gia, cc thuc ny c th ngn chn t bo thn kinh chuyn tn hiu

12

au n no, chng khc vi cc thuc gim au khc ch n khng gy ra tc dng

ph nh morphin, khng xung t vi cc loi thuc khc v cng khng gy nghin.

Vit Nam cng nghin cu thnh cng bit dc Thin Thanh Hon c cha ti

a 0,1mg TTX v mt s v thuc ng y khc.

Th nm, nghin cu ca Lesort cho thy TTX c kh nng lin kt vi protein

ca HIV, c bit l gp-120, do lm cho genom ca HIV khng th gn vo genom

ca ngi. V vy, HIV khng th pht trin thnh AIDS c. y l tn hiu ng

mng cho qu trnh i tm thuc iu tr cn bnh th k HIV-AIDS.

1.3 MT S NGHIN CU CHIT XUT TTX

Th gii:

- Nm 1964 tc gi Goto Toshio cng cng s a ra phng php chit xut

phn lp v tinh ch TTX nh sau: t 100 kg trng c nc chit bng nc, phn

lp bng cch cho qua ct trao i ion Amberlite IRC-60, than hot tnh v kt

tinh trong dung dch amoniac cho 1-2 gam c t th [27].

- Nm 2003 Maoquing Zhou v cng s tin hnh chit xut, phn lp TTX nh

sau: t 20kg trng c nc chit bng acid acetic, un nng, lm lnh, lc; tch

TTX bng s dng ct trao i ion cha nha D-152, diatomaceous silica-than

hot, c c v kt tinh thu c 1,2g c t ton phn c cha TTX [20]. c

t TTX tip tc c lm tinh khit bng cch tinh ch nhiu ln [21].

Vit Nam

- Nm 1994, L Quang Hun v cng s l nhm tc gi u tin chit xut v

phn lp, tinh ch TTX t c nc ca bin min Trung Vit Nam. Phng php

ny c s dng dung mi hu c chloroform, ether chit vi quy trnh cn

phc tp, nhiu giai on. Tuy nhin nhm tc gi mi ch tinh ch c dng

th, s lng t v cha nh gi chnh xc c tinh khit [6].

- Nm 2007, sn xut huyt thanh khng c t TTX, nhm nghin cu ca

tc gi L Vn Hip thuc Vin Vcxin v Sinh phm y t Nha trang (IVAC)

phn lp c TTX t ph tng c nc. Nghin cu thu c 19,21mg tinh

th TTX c tinh khit 80-85%, hiu sut 35-50%. Tuy nhin nghin cu mi

13

ch dng li quy m nh phc v cng vic iu ch huyt thanh khng TTX

[4].

14

CHNG 2: I TNG V PHNG PHP NGHIN CU

2.1. NGUYN VT LIU, THIT B

2.1.1. Nguyn liu

Nguyn liu chit xut-phn lp: ph tng cc loi c nc thu mua vng bin

Nha Trang Khnh Ha, Vit Nam

2.1.2 Thit b, dng c, ha cht:

Thit b:

- My xay GL-1903

- B iu nhit BCHI 461 water bath

- My ct quay Buchi RE111 Rotavapor Evaporator

- Bm ht chn khng model DOA-P504-BN

- Cn phn tch, cn k thut

- My sc k lng khi ph Thermo Scientific TSQ quantum Ultra

Dng c:

- Bnh nh mc 20ml, 50ml

- Bnh nn 100ml

- Cc thy tinh 100ml, 200ml

- B lc thy tinh, phu Buchner, pipet

- Vi lc, giy lc kch thc 5m; 0,45m

- Giy ch th vn nng

- ng mao qun chm sc k 10L

- Bn silica gel 60 GF254 Merck, kch thc 5 cm x 20 cm

- Bm tim cho HPLC

- Ct: Alltech Apollo C8 (25 cm x 4,6 mm; 5m)

- Ct trao i ion: ct thy tinh hnh tr, cao 20cm x ng knh trong 5cm,

cha nha trao i ion cation ammoniac acid yu

Ha cht:

- Acid acetic khan

- Ammoniac m c, Amoni acetat

15

- Ethanol, methanol, n-butanol

- Thuc th hin mu Weber 5-({2-[(Iodoacetyl)amino]ethyl}amin)-1-

naphthalenesulfonic axit

- Tetrodotoxin (Sigma Aldrich, 1mg/l, hm lng: 99%, SKS: 038K1804

- Nha trao i ion cation ammoniac acid yu

- Dung mi pha mu: hn hp dd a.acetic 0,1% (tt/tt) v MetOH (20:80)

- m acetat; dung dch trong nc ct cha 15mM amoni acetat v 15 mM

a.acetic

2.2. NI DUNG NGHIN CU

2.2.1 Chit xut c t ton phn

Tham kho quy trnh chit xut ca Maoquing Zhou (2003) [20], kho st mt

s thng s: nng dung mi, iu kin lc, pH dch chy ct trao i ion.

2.2.2. Phn lp TTX

Tham kho quy trnh ca Maoquing Zhou (2003) [21]: la chn s ln tinh ch

thu c TTX t yu cu (> 80%).

2.2.3. nh tnh TTX

nh tnh TTX trong dch chit bng sc k lp mng

inh tnh TTX trong tinh th phn lp bng ESI-MS, LC/MS/MS, NMR

2.2.4. nh lng TTX

nh lng TTX trong tinh th, dch chit bng phng php LC/MS/MS

2.3. PHNG PHP NGHIN CU

2.3.1. Phng php chit xut

Tham kho phng php chit xut ca Maoquing Zhou (2003) [20], kho st mt

s thng s, tm quy trnh chit xut ph hp vi iu kin phng th nghim:

16

Hnh 2.1:S chit xut TTX t ph tng c nc

Ngm chit:

S dng phng php ngm lnh:

+ Nghin nh 1kg ph tng, sau thm vo 1,5L dung dch acid acetic 0,1%,

khuy trn trong 2 gi, lc qua vi x thu c dch ngm chit u. Thm

1,5L nc kh ion vo b, khuy trn v lc nh trn. Qu trnh chit tin hnh

lp li 3 ln chit kit.

+ Gp cc dch chit li, un nng dch chit 800C trong 10 pht, sau lm

lnh, lc thu c dch lc trong.

17

- Qu trnh lm giu bng trao i ion v tch TTX:

+ iu chnh pH dch lc thu c v 7,5 bng dung dch ammoniac 10% (tt/tt).

Cho dch lc chy qua ct trao i ion vi tc 100ml/h.

+ Sau khi ton b dch lc chy qua ct trao i ion, ra ct vi nc kh ion

cho n khi khng cn protein trong nc ra

+ Ra gii ct trao i ion bng dung dch acid acetic 10% (tt/tt) vi tc

50ml/h, thu dch TTX.

- S tinh ch qua than hot:

+ Gp dch TTX thu c trn. iu chnh ti pH 8,5 bng dung dch amoniac

10% (tt/tt). Lc qua giy lc 0,45m

+ Duy tr pH 8,5 ca dch ra gii trong 2 gi. Trong khong thi gian ny, cho

dch ra gii chy qua ct silica diatomaceous - than hot.

+ Ra ct silica diatomaceous - than hot vi nc kh ion, ri ra gii bng

dung dch acid acetic 0,2% trong ethanol 20% cho n khi ht TTX (kim tra

bng TLC).

- C c v kt tinh:

+ C c dung dch thu c bng my ct quay nhit 100oC n khi cn

khong 300ml th ly t ngui, ly em sc k lp mng.

+ C tip cho ti khi cn 10ml th lm lnh dch c c ti 200C, iu chnh pH

9,0 bng dung dch ammoniac m c.

+ Cho vo t lnh, yn cho c t kt tinh. Lc ly c t tinh th ra 3 ln,

mi ln vi 5 ml vi nc kh ion. Sy trong t sy chn khng (60oC; p sut

khng qu 0,5 kPa; phosphor pentoxyd; 2 h) cho n khi lng khng i

thu c tinh th c t ton phn cha TTX

2.3.2. Phng php phn lp TTX

Phn lp bng phng php tinh ch nhiu ln [21]

1) Ha tan c t tinh th sy kh trong 10 ml dung dch acid acetic 5% (tt/tt).

2) iu chnh ti pH 9,0 bng dung dch amoniac m c.

3) Cho vo t lnh, yn cho TTX kt tinh.

18

4) Lc ly TTX tinh th, ra 3 ln, mi ln vi 5 ml vi nc ct.

5) Sy trong t sy chn khng (60oC; p sut khng qu 0,5 kPa; phosphor

pentoxyd; 2 h).

6) Ha tan TTX tinh th sy kh trong 10 ml dung dch acid acetic 5% (tt/tt),

ri tip tc tin hnh nh m t cc bc t 2 n.4

7) Sy TTX tinh th thu c trong t sy chn khng (60oC; p sut khng qu

0,5 kPa; phosphor pentoxyd) n khi lng khng i.

Hnh 2.2: S phn lp theo phng php ca Maoquing Zhou

Tnh hiu sut qu trnh phn lp =

Trong mt: khi lng TTX trc phn lp

ms: khi lng TTX sau phn lp

19

2.3.3. nh tnh TTX

A. Sc k lp mng:

nh tnh TTX bng sc k lp mng: da trn ti liu [20], [19] v thc nghim iu

kin sc k c xc nh:

- Bn mng: silica gel 60 GF254 Merck trng sn 110oC trong 30 pht

- Chun: Tetrodotoxin (Sigma Aldrich, 1mg/l, hm lng: 99%, SKS: 038K1804)

- Dung dch i chiu: Dung dch TTX trong dung dch acid acetic 5% (tt/tt) c nng

khong 50 g/ml.

- Dung dch th: ly dch chit c chm sc k

- Pha ng: n-butanol, acid acetic khan v nc (2:1:1)

- Thuc th hin mu: dung dch thuc th Weber

- Cch th: Chm ring bit ln bn mng 10 l mi dung dch, lm kh cc vt chm

bng lung khng kh mt. Trin khai cho n khi dung mi i c khong 10 cm.

Ly bn mng ra, lm kh bng lung khng kh mt. Phun dung dch thuc th

Weber hin mu.

B. Phng php khi ph ESI-MS-tim trc tip vo detecter khi ph

C s ca phng php MS l ph v cu trc phn mnh phn t trung ha

thnh cc mnh ion, cc gc mang in tch (c khi lng nh hn) bng cc phn

t mang nng lng cao. Qu trnh phn mnh c trng cho cu trc ca phn t v

ch ra s c mt ca nhng nhm chc c th, cung cp nhng thng tin hu ch v

cu to hoc nhn dng, khng nh cu trc cht phn tch. c tnh ni bt ca khi

ph l tnh chn lc v nhy cao.

Cch tin hnh: tham kho ti liu [15] v lm thc nghim

tin hnh tim trc tip 1 lng mu vo detector khi ph, xc nh mnh ion m

m/z = 320 v cc mnh con tng ng c trng ca TTX

Mu: Pha dung dch TTX 10ppm trong pha ng (ethanol)

Tim trc tip vo detecter vi tc 5L/pht.

20

C. Phng php LC-MS/MS

Sc k thu c ca dung dch th phi c pic vi thi gian lu v ph MS/MS

ging vi thi gian lu ca pic v ph trong sc k thu c ca dung dch chun

iu kin sc k: tng t iu kin nh lng TTX bng phng php LC-MS/MS

D. Phng php NMR

Qut ph xc nh cu trc ca cht phn lp

2.3.4 nh lng TTX bng phng php LC-MS/MS

Tham kho ti liu [18], [22] v lm thc nghim iu kin ca phng php

c xc nh

Chun b cc iu kin sc k

- Chun: Tetrodotoxin (Sigma Aldrich, 1mg/l, hm lng: 99%, SKS: 038K1804)

- Dung dch chun: Dung dch TTX chun trong dung mi pha mu, c nng chnh

xc khong 1g/ml.

- Dung dch th: Ha tan c t tinh th thu c phn chit xut trong dung mi

pha mu vi nng khong 1g/ml.

Cc iu kin sc k:

- Thit b: My sc k lng khi ph Thermo Scientific TSQ quantum Ultra

- Ct: Alltech Apollo C8 (25 cm x 4,6mm; 5m)

- Pha ng: m acetat Methanol (30:70)

(m acetat: dung dch amoni acetat 15mM, acid acetic 15mM)

- Tc dng: 500l/min

- Th tch tim: 10l

- iu kin khi ph

+ Ngun: ESI+

+ Kh: SG: 20, AG: 10

+ Th ion ha: 3200V

+ Nhit ha hi: 200oC

+ Nhit mao qun: 360oC

+ SRM: 320 162 vi mc nng lng CE = 35V

21

Cch tin hnh

Tim ring bit dung dch chun v dung dch th vo my sc k lng khi ph, ghi

li sc k v tnh din tch ca pic TTX. Mi mu th lm li 3 ln, ly kt qu

trung bnh

Tnh hm lng ca TTX phn lp, da vo nng TTX trong dung dch chun

v din tch ca pic TTX trong sc k thu c ca dung dch chun, dung dch

th.

Cng thc:

Hm lng TTX c tnh theo cng thc

X% = 100t

t

C

c

t

m

VC

S

S

Trong :

Sc, St: din tch peak trong sc k cho bi dung dch chun, th

Cc: Nng dung dch chun (g/mL)

mt: khi lng cht em nh lng (g)

Vt: th tch pha mu th (mL)

22

CHNG 3: THC NGHIM, KT QU V BN LUN

3.1. THC NGHIM, KT QU

3.1.1. Chit xut

Acid acetic l mt acid hu c yu c kh nng ha tan TTX v khng c nn

la chn acid acetic lm dung mi chit. Theo ti liu [20] TTX c chit bng dung

mi acid acetic nng 0,1%. Kho st nng dung mi chit xut 3 nng

0,1%; 0,5%; 1% v so snh cc ch tiu: khi lng c t ton phn thu c sau

khi chit xut v tinh khit

Trong qu trnh th nghim, nhn thy dch chit c nc tng i c gy kh

lc v khng thu c dch trong sut v vy tin hnh lc 3 ln qua 3 loi vt liu

lc: vi x, giy lc c kch 5 m; 0,45m thu c dch lc trong. d dng

cho qu trnh lc v khng gy tc: tin hnh ly tm (6000v/p) trong 20 pht sau khi

lc qua vi x. Sau khi lc th qua vi x, un nng 800C trong 5 pht thy dch vn

xut hin ta nn un 800C trong 10 pht to ta ht.

Theo quy trnh dch lc thu c t giai on ngm chit trc khi chy ct trao

i ion c iu chnh pH 7,5; kho st cc mi trng acid pH 5,5; pH 7,5 v

mi trng kim pH 9,0; ch tiu so snh: khi lng c t ton phn thu c sau

khi chit xut v tinh khit

Tin hnh:

- Ngm chit:

+ Nghin nh 1kg ph tng cho vo trong x, sau thm vo 1,5L dung mi

chit

Trng hp 1: acid acetic 0,1%

Trng hp 2: acid acetic 0,5%

Trng hp 3: acid acetic 1%

+ Khuy trn trong 2 gi, lc qua vi x thu c dch ngm chit u. Thm

1,5L nc kh ion vo b, khuy trn v lc nh trn. Tin hnh 3 ln chit

kit.

23

+ Gp cc dch chit li, un nng dch chit 800C trong 10 pht, sau lm

lnh

+ Lc th qua vi, ly tm (6000v/p) trong 20 pht thu ly dch ri tin hnh lc

ln lt qua giy lc kch thc 5m trn phu Buchner; 0,45m trn b lc thy

tinh c bm ht chn khng thu c dch lc trong.

- Qu trnh lm giu bng trao i ion v tch TTX:

+ iu chnh pH dch lc thu c v pH:

Trng hp 1: pH=7,5

Trng hp 2 : pH=5,5

Trng hp 3: pH=9,0

bng dung dch ammoniac 10% (tt/tt). Cho dch lc chy qua ct trao i ion vi

tc 100ml/h

+ Sau khi ton b dch lc chy qua ct trao i ion, ra ct vi nc kh ion

cho n khi khng cn protein trong nc ra. Ra gii ct trao i ion bng

dung dch acid acetic 10% (tt/tt) vi tc 50ml/h, thu dch TTX.

- S tinh ch qua than hot:

+ Gp dch TTX thu c trn. iu chnh ti pH 8,5 bng dung dch amoniac

10% (tt/tt). Lc qua giy lc 0,45m

+ Duy tr pH 8,5 ca dch ra gii trong 2 gi. Trong khong thi gian ny, cho

dch ra gii chy qua ct silica diatomaceous - than hot.

+ Ra ct silica diatomaceous - than hot vi nc kh ion, ri ra gii bng

dung dch acid acetic 0,2% trong ethanol 20% cho n khi ht TTX (kim tra

bng TLC).

- C c v kt tinh:

+ C c dung dch thu c bng my ct quay nhit 100oC n khi cn

khong 300ml th ly t ngui, ly em sc k lp mng.

+ C tip cho ti khi cn 10ml th lm lnh dch c c ti 200C, iu chnh pH

9,0 bng dung dch ammoniac m c.

24

+ Cho vo t lnh, yn cho c t kt tinh. Lc ly c t tinh th ra 3 ln,

mi ln vi 5 ml vi nc kh ion. Sy trong t sy chn khng (60oC; p sut

khng qu 0,5 kPa; phosphor pentoxyd; 2 h) cho n khi lng khng i

thu c tinh th c t ton phn c cha TTX.

Kt qu:

1. Nng dung mi chit: acid acetic 0,1%

Dch chy ct trao i ion c pH=7,5

Dch chit qua cc loi giy lc

Hnh 3.1: (A) dch chit qua giy lc 5m, (B) dch chit qua giy lc 0,45m

nh tnh TTX bng sc k lp mng dch chit c

Hnh 3.2: Sc k lp mng (Rf=0,2)

(C): dung dch chun TTX

(T): dung dch th

25

Dung dch th v dung dch chun u cho vt mu hng khi phun thuc th

hin mu, v tr vt ca dung dch th v dung dch chun l tng ng nhau.

Bng 3.1: Kt qu chit xut

Acid acetic 0,1%

Khi lng nguyn liu (g) 1001,1

Khi lng c t ton phn (mg) 29,8

Lng TTX trong c t ton phn (mg) 21,2

tinh khit (%) 71,1

Bn lun:

- La chn dung mi l acid acetic long ha tan TTX. Qu trnh un nng,

lm lnh dch chit nhm loi b ta scleroprotein (protein si), cht bo.

- Tin hnh lc th nhm loi cc tp ln gy nh hng n qu trnh lc v

chy ct. Dch lc th em ly tm loi cc tp c th gy tc, nh hng n

tc lc. Lc tip qua giy lc c kch 5 m vn cn kh c nn lc qua giy

lc 0,45m. Dch lc thu c kh trong sut thun li cho qu trnh chy ct

- Giai on chy qua ct trao i ion gip loi b c protein, peptid v cc

dn cht ca TTX. Nu khng loi c nhng cht ny lm tng nht ca

dch gy cn tr cho cc qu trnh kt tinh TTX sau ny.

- Giai on chy qua ct diatomaceous silica-than hot nhm loi acid amin

kim

- C c dch ri iu chnh pH dch 9,0 ri lm lnh kt tinh TTX, ra vi

nc lnh loi bt cc tp lng thp cn st li trong dch, sy kh n khi

lng khng i nh lng lng TTX c trong c t ton phn.

- Da vo phng php chit xut ca Maoquing Zhou (2003) v ty vo iu

kin phng th nghim tin hnh, chit ra 29,8mg c t ton phn c tinh

26

khit tng i 71,7%. Phng php ny c u im s dng cc dung mi an

ton.

2. Kho st nng dung mi chit cc nng : 0,1%; 0,5%; 1%

pH dch chy ct trao i ion pH=7,5

Bng 3.2: Kt qu kho st nng dung mi chit

Acid acetic

0,1%

Acid acetic

0,5%

Acid acetic

1%

Khi lng nguyn liu (g) 1001,1 1002,9 1002,1

Khi lng c t ton phn

(mg) 29,8 28,3 27,6

Lng TTX trong c t ton

phn (mg) 21,2 18,1 17,2

tinh khit (%) 71,1 63,5 62,3

Hnh 3.3: th kho st nh hng ca nng dung mi chit n kt qu chit xut

56.00%

58.00%

60.00%

62.00%

64.00%

66.00%

68.00%

70.00%

72.00%

26.5

27

27.5

28

28.5

29

29.5

30

Acid acetic 0,1% Acid acetic 0,5% Acid acetic 1%

Khi lng c t ton phn tinh khit (%)

27

Bn lun: Khi lng tinh th c t ton phn c cha TTX v tinh khit

gim theo tng ca nng acid acetic. nng acid acetic 0,1% khi lng

tinh th c t ton phn c cha TTX v tinh khit thu c l cao nht. Do

cng nng acid cao th ha tan nhiu tp hn, nh hng n qu trnh chit

xut TTX.

3. Kho st pH chy ct trao i ion: pH 5,5; pH 7,5; pH 9,0

Dung mi chit: acid acetic 0,1%

Bng 3.3: Kt qu kho st pH

pH 5,5 pH 7,5 pH 9,0

Khi lng nguyn liu

(g) 1001,6 1001,1 1002,5

Khi lng c t ton

phn (mg) 19,3 29,8 29,5

Lng TTX trong c t

ton phn (mg) 13,8 21,2 19,9

tinh khit (%) 71,5 71,1 67,5

Hnh 3.4: th kho st nh hng ca pH chy ct n kt qu chit xut

65.00%

66.00%

67.00%

68.00%

69.00%

70.00%

71.00%

72.00%

0

5

10

15

20

25

30

35

pH 5,5 pH 7,5 pH 9,0

khi lng c t ton phn tinh khit (%)

28

Bn lun:

-pH ca dch lc chy ct trao i c nh hng n kt qu chit xut. Vi pH

5,5 dch khi em c v kt tinh kh hn, dch nht hn do khng loi b c

protein, peptid khin TTX kh kt tinh hn nn khi lng tinh th TTX ton phn

gim i nhiu; vi pH 9,0 lng tinh th TTX ton phn thu c khng khc

nhiu pH 7,5 nhng tinh khit thp hn do pH 9,0 kh nng b hp ph

ct trao i ion ca TTX gim nn lng TTX trong dch ra gii gim i.

- pH tt nht thun li cho qu trnh chit xut l 7,5 lng tinh th c t ton

phn thu c c khi lng ln nht v tinh khit cao nht.

3.1.2 Phn lp:

c cht chit t c nc l hn hp ca hn 10 analog, ch yu l TTX, cn li

u c hm lng nh. Trong qu trnh chit xut mt phn cc analog c

loi b. Ba analog chnh khc l acid tetrodonic, 4-epi TTX v 4-epi

anhydrotetrodotoxin thng xut hin vi TTX do c c tnh ha hc tng i

ging nhau. S dng phng php tinh ch nhiu ln nhm loi cc analog c hm

lng thp. S dng dung mi acid acetic long 5% tinh ch nhiu ln theo s

(Hnh 2.2)

Cch tin hnh:

1) Ha tan c t tinh th sy kh trong 10 ml dung dch acid acetic 5% (tt/tt).

2) iu chnh ti pH 9,0 bng dung dch amoniac m c.

3) Cho vo t lnh, yn cho TTX kt tinh.

4) Lc ly TTX tinh th, ra 3 ln, mi ln vi 5 ml vi nc ct.

5) Sy trong t sy chn khng (60oC; p sut khng qu 0,5 kPa; phosphor

pentoxyd; 2 h).

6) Ha tan TTX tinh th sy kh trong 10 ml dung dch acid acetic 5% (tt/tt),

ri tip tc tin hnh nh m t cc bc t 2 n.4

7) Sy TTX tinh th thu c trong t sy chn khng (60oC; p sut khng qu

0,5 kPa; phosphor pentoxyd) n khi lng khng i.

29

Kt qu :

1. nh tnh TTX trong tinh th phn lp

- Da vo khi ph ESI-MS : Tim trc tip 1 lng mu dch chit vo detector

khi ph, Trn sc k ESI-MS, ch qut ton thang (TIC Total ion

chromatographic) xut hin mnh khi m/z = 320, ch MS/MS, bn ph mnh

m m/z = 320 vi mc nng lng CE = 35V, xut hin cc mnh con m/z = 302,

m/z = 162 c trng cho TTX (PL1)

- LC-MS/MS: Sc k thu c ca dung dch th c pic vi thi gian lu

(tR=6,8 pht) v ph MS/MS ging vi thi gian lu ca pic v ph trong sc k

thu c ca dung dch chun (PL5, PL6).

- NMR: da vo ph cng hng t xc nh c cu trc c trng ca TTX

(PL2,PL3)

2. nh lng TTX bng phng php LC/MS/MS

- Dung dch chun: Dung dch TTX chun trong dung mi pha mu, c nng

chnh xc 1g/ml.

Cch pha: tim khong 4mL dung mi pha mu vo l chun 1mg, lc k, ho

tan hon ton TTX, rt dung dch ra, cho vo bnh nh mc 20,0mL. Trng li 3

ln, mi ln 4mL dung mi pha mu. Gp dch vo bnh nh mc 20,0mL. Thm

dung mi pha mu cho n vch, lc siu m k, lc qua giy lc. Ht 1ml dung

dch ny cho vo bnh nh mc 50ml, lc k. Lc qua mng lc 0,45m ta thu

c dung dch chun c nng 1g/ml.

- Dung dch th:

Ha tan c t tinh th thu c phn chit xut trong dung mi pha mu vi

nng khong 1g/ml.

Cch pha dung dch th: cn chnh xc khong 2,03mg cht va phn lp theo quy

trnh, pha trong bnh nh mc 20mL. Thm 12mL dung dch acid acetic 0,1%, lc

siu m k, thm dung dch acid acetic 0,1% va , lc qua giy lc. Ht 1ml

dung dch ny, pha trong 10mL Methanol thu c dung dch 1. Ht tip 1mL

30

dung dch 1, pha long trong 10mL Methanol. Lc qua mng lc 0,45m thu c

dung dch tim sc k.

Tin hnh sc k LC-MS/MS cc iu kin nu

Kt qu:

Sau khi tin hnh phn lp theo quy trnh trn t 29.8mg c t ton phn cha

TTX thu c 20,7 mg TTX tinh th.

Bng 3.4: Kt qu phn lp

Ln tinh ch Khi lng tinh th

(mg) Hm lng TTX (X%)

1 25,7 73,9

2 22,5 78,7

3 20,7 82,6

Tin hnh nh lng hm lng Tetrodotoxin trong lng tinh th phn lp c

theo iu kin trn, ta c bng sau

Din tch peak dung dch chun: Sc = 161602,8

Bng 3.5: Kt qu nh lng

TT St X%

Ln 1 137251,9 83,7

Ln 2 135365,7 82,9

Ln 3 130998,1 81,1

Trung bnh 82,6

RSD 1,61

Hiu sut qu trnh phn lp: H=69,5%

31

Bn lun:

- Qu trnh phn lp ta tin hnh tinh ch nhiu ln loi cc analog ca TTX.

iu chnh pH 8-9 nhm kt tinh TTX sau lc, ra, sy. Sau khi tin hnh

phn lp thu c 20,7mg TTX tinh th vi tinh khit l 82,6% t yu cu.

- Hiu sut ca qu trnh chit xut phn lp l: 69,5%

- Quy trnh phn lp n gin, d thc hin, t tn km vi dung mi n gin,

r tin. Thi gian phn lp nhanh.

32

KT LUN V KIN NGH

KT LUN:

- Tin hnh chit xut c 29,8mg c t ton phn c tinh khit 71,1% t

ph tng c nc quy m phng th nghim tham kho theo quy trnh

Maoquing Zhou (2003) vi cc giai on:

1. Chit bng phng php ngm lnh

2. Lm giu bng phng php trao i ion v tch TTX

3. S tinh ch bng diatomaceous silica-than hot

4. C c v kt tinh

- Kho st c mt s yu t nh hng n qu trnh chit xut: nng dung

mi, pH dch trc khi chy ct trao i ion

- Tin hnh phn lp TTX quy m phng th nghim theo quy trnh Maoquing

Zhou (2003) t 29,8mg c t ton phn bng cch tinh ch nhiu ln thu c

20,7mg TTX tinh th vi tinh khit l 82,6% t yu cu. Hiu sut ca qu

trnh phn lp l: 69,5%

- Quy trnh chit xut-phn lp n gin v khng dng n dung mi hu c

c xc nh c tinh khit ca TTX tinh th thu c.

I. KIN NGH:

Do thi gian tin hnh ngn, iu kin quy m phng th nghim cn cha

y nn hiu sut chit xut phn lp cn cha cao; cha kho st ht cc

thng s khc ca quy trnh. Khi lng nguyn liu cn t nn cn cha nh

gi ht nhng hn ch ca quy trnh nn chng ti ngh:

1. Kho st cc thng s khc ca quy trnh: nhit , lng than hot, pH chy

ct than hot...

2. Tip tc nghin cu trn khi lng ln hn ci tin v hon thin qu

trnh

TI LIU THAM KHO

Ti liu ting vit

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2. V Vit H, Nguyn Hoi Nam, ng Vn Thi (2006), Hin trng ngun li

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3. Bi Th Thu Hin, Bi Trng Tm, Phm Quc Long (2009), Tetrodotoxin

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634.

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v hu cn ngh c, 385-402.

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10. Nguyn Hoi Nam, Ths.ng Vn Thi (2007), Mt s thng tin v c nc

bin Vit Nam, Tp ch thy sn, (1), 25-28.

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University. Department of Applied Biology and Chemical Technology, Hong

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Edition, CRC Press,Taylor & Francis, Boca Raton, FL 2005, p. 3-482.

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fossil and extant tetraodontiform fishes (Acanthomorpha, Tetraodontiformes),

Upper Cretaceous to Recent, Zoological Journal of the Linnean Society,

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22. Monrat Chulanetra, Nitat Sookrung, Potjanee Srimanote, Nitaya Indrawattana,

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PH LC

1. PH LC 1: Ph ESI-MS ca TTX

2. PH LC 2:Ph 1H-NMR ca cht phn lp

3. PH LC 3:Ph 13C-NMR ca cht phn lp

4. PH LC 4:Ph DEPT ca cht phn lp

5. PH LC 5: Sc k LC-MS/MS dung dch mu trng

6. PH LC 6: Sc k LC-MS/MS dung dch chun TTX nng

1g/ml

7. PH LC 7:Sc k LC-MS/MS dung dch th

PH LC 1: Ph ESI-MS ca TTX

PH LC 2:Ph 1H-NMR ca cht phn lp

PH LC 3:Ph 13C-NMR ca cht phn lp

PH LC 4:Ph DEPT ca cht phn lp

PH LC 5: Sc k LC-MS/MS dung dch mu trng

PH LC 6: Sc k LC-MS/MS dung dch chun TTX nng 1g/ml

PH LC 7: Sc k LC-MS/MS dung dch th