TRNG I HC LC HNG

KHOA CNG NGH HA THC PHM

-----E D-----

BO CO
NGHIN CU KHOA HC

TI:

TCH, TNG HP V KHO ST HOT TNH

SINH HC CA CC DN XUT CURCUMIN T
BT CURCUMINOID THNG PHM.

CAO TH KIM ANH

LU TH NGC VNH
 TRNG I HC LC HNG
KHOA CNG NGH HA THC PHM
-----E D-----

BO CO
NGHIN CU KHOA HC

TI:

TCH, TNG HP V KHO ST HOT TNH

SINH HC CA CC DN XUT CURCUMIN T
BT CURCUMINOID THNG PHM.

Sinh vin thc hin : CAO TH KIM ANH

LU TH NGC VNH
Gio vin hng dn : ThS. HONG MINH HO
ThS. PHAN TH HONG ANH

BIN HA, THNG 11/2010

 LI CM N

Chng ti xin chn thnh cm n Thy Hong Minh Ho, Khoa Cng Ngh
Ha -Thc Phm, Trng i hc Lc Hng v C Phan Th Hong Anh, B Mn
K thut Hu c, Trng i hc Bch Khoa, Tp.HCM hng dn tn tnh
chng ti trong sut qu trnh thc hin ti nghin cu khoa hc.

Xin cm n phng th nghim ha hu c, Trng i Hc Bch Khoa v

phng th nghim Trng i hc Lc Hng h tr dng c, my mc thit b.

Cm n gia nh v ngi thn ng vin v to iu kin tt nht cho

chng ti hon thnh ti nghin cu khoa hc.

Bin Ha, ngy 22 thng 11 nm 2010

 MC LC
Trang

Trang ph ba
Li cm n
Mc lc
Danh mc cc t vit tt
Danh mc cc hnh

Danh mc bng biu

Danh sch cc s

M U ....................................................................................................................1
CHNG 1: TNG QUAN...................................................................................4
1.1 Curcuminoid............................................................................................4

1.1.1 c im v s phn b ca cy ngh ........................................4

1.1.1.1 c im cy ngh ...........................................................4

1.1.1.2 S phn b ca cy ngh ..................................................5

1.1.2 Thnh phn ha hc trong thn r ca cy ngh..........................5

1.1.3 Cu trc ca cc dn xut curcuminoid .......................................6

1.1.4 Tnh cht ha l ca curcuminoid ................................................9

1.1.4.1 L tnh .............................................................................9
1.1.4.2 Ha tnh ...........................................................................9
1.1.5 Hot tnh sinh hc ca curcuminoid..........................................17

1.2 Cc nghin cu v imin v dn xut imin-curcumin ............................20

CHNG II: NI DUNG V PHNG PHP NGHIN CU.....................25
2.1 Ni dung nghin cu .............................................................................25
2.2 Phng php thc hin..........................................................................26
2.2.1 Phn lp curcumin......................................................................26
2.2.1.1 Quy trnh phn lp curcumin .........................................26
2.2.1.2 Kt tinh bt Curcuminoid thng phm.........................26
2.2.1.3 Sc k bn mng (TLC) .................................................27

2.2.1.4 Sc k ct.......................................................................29
2.2.2 Tng hp cc dn xut imin curcumin ...................................29
2.2.2.1 Tng hp 3,5- Difluorophenylhydrazinocurcumin ........29
2.2.2.2 Tng hp 4-Fluorophenylhydrazinocurcumin................34
2.2.3 Phn tch tinh khit v cu trc ca cc dn xut va tng
hp...........................................................................................38

2.2.3.1 Ph t ngoi kh kin (UV-Vis).....................................38

2.2.3.2 Khi ph (MS)................................................................39
2 2.3.3 Ph cng hng t ht nhn (NMR) ..............................39
2.2.4 Kho st hot tnh sinh hc........................................................39
2.2.4.1 nh gi hot tnh khng oxy ho..................................39
2.2.4.2 nh gi hot tnh khng ung th ..................................41

CHNG 3: KT QU V THO LUN.........................................................43

3.1 Phn lp curcumin.................................................................................43

3.1.1 Kt tinh curcumin ......................................................................43

3.1.2 Sc k ct phn lp curcumin ....................................................44
3.2 Tng hp dn xut 3,5-Difluorophenylhydrazinocurcumin .................46
3.2.1 Theo di phn ng .....................................................................46
 3.2.2 Sc k ct phn lp sn phm ....................................................48
3.2.3 Nhn danh cu trc ...................................................................49
3.2.3.1 Tnh cht vt l c trng...............................................49

3.2.3.2 Bin lun cu trc ha hc ca 3,5-Difluorophenylhydr-

azinocurcumin ...............................................................50
3.3 Tng hp dn xut 4-Fluorophenylhydrazinocurcumin ......................52
3.3.1 Theo di phn ng .....................................................................52
3.3.2 Sc k ct phn lp sn phm ....................................................54
3.3.3 Nhn danh cu trc ....................................................................55

3.3.3.1 Tnh cht vt l c trng..................................................55

3.3.3.2 Bin lun cu trc ca 4-Fluorophenylhydrazino-

curcumin .......................................................................................57

3.4 Kt qu kho st hot tnh sinh hc .....................................................59

3.4.1 Hot tnh khng oxy ha theo phng php DPPH...................59

3.4.2 Hot tnh khng t bo gy c .................................................60
Kt lun v kin ngh...........................................................................................62

Danh mc ti liu tham kho

Ph lc
 DANH SCH CC T VIT TT

BDMC: Bisdemethoxycurcumin.

Cur: Curcumin.

DMC: Demethoxycurcumin.

3,5-DFPHC: 3,5 Difluorophenylhydrazinocurcumin.

4-FPHC: 4-Fluorophenylhydrazinocurcumin.

HTCO: Hot tnh khng oxi ho

IC50: Nng hot cht c ch 50% vi khun, vi nm, t
bo ung th hoc gc t do (half maximal (50%)
inhibitory concentration).

CS: Kh nng sng st ca t bo nng no ca cht

th tnh theo % so vi i chng.
DPPH: Cht th 1,1-diphenyl-2-picrylhydrazyl.

MS: Khi ph (Mass spectrometry).

NMR: Ph cng hng t ht nhn (Nuclear magnetic

resonance spectroscopy).

TLC: Sc k bn mng (Thin layer chromatography).

 DANH SCH CC HNH

Hnh 1.1 Cy ngh vng Curcuma longa L. ..............................................................5

Hnh 1.2 Cng thc ha hc chung ca curcuminoid................................................7

Hnh 1.3 ng phn cis trans curcumin .................................................................8

Hnh 1.4 Cc ng phn enol ceton ca curcumin .................................................8

Hnh 1.5 Qu trnh tautomer ha ca cc hp cht curcuminoid ..............................8

Hnh 1.6 Cc dng phn ly ca curcuminoid ...........................................................10

Hnh 1.7 Phn hy di tc dng ca nh sng .......................................................11

Hnh 1.8a S phn hy ca curcumin trong mi trng kim.................................12

Hnh 1.8b S phn hy ca curcumin trong mi trng kim ................................12

Hnh 1.9 Phn ng cng H2 ................................................................................................................................ 13

Hnh 1.10 Phn ng imin ha ..................................................................................14

Hnh 1.11 Phn ng ca curcuminoid vi gc t do ...............................................15
Hnh 1.12 Phn ng to phc vi kim loi ..............................................................16
Hnh 1.13 Anion dicetonat .......................................................................................16
Hnh 1.14 Cng thc cu to ca curcumin.............................................................17
Hnh 1.15 Phn ng tng hp hydrazinocurcumin ..................................................20

Hnh 1.16. Phn ng tng hp hydrazinobenzoylcurcumin ....................................20

Hnh 1.17 Phn ng tng hp mt s dn xut imin t curcuminoid......................22
Hnh 1.18 3-Nitrophenylpyrazolecurcumin .............................................................22

Hnh 1.19 Hydrazinocurcumin.................................................................................22

Hnh 1.20 Cng thc cu to ca curcuminsemicarbazone ....................................23

Hnh 3.1 TLC kim tra hn hp curcuminoid sau cc ln kt tinh..........................44
Hnh 3.2 Sc k ct phn lp curcumin ...................................................................45
Hnh 3.3 TLC phn on tinh t sc k ct .............................................................45

Hnh 3.4a Phn ng 3,5-Difluorophenylhydrazinocurcumin...................................46

Hnh 3.4b Phn ng tng hp 3,5-Difluorophenylhydrazinocurcumin ..................47
Hnh 3.5 Theo di bn mng bng TLC ..................................................................47

Hnh 3.6 Sc k ct th ...........................................................................................48

Hnh 3.7 Sc k ct tinh...........................................................................................48
Hnh 3.8 TLC ca 3,5-Difluorophenylhydrazinocurcumin .....................................49

Hnh 3.9 Ph UV-Vis ca 3,5-Difluorophenylhydrazinocurcumin ...................................... 50

Hnh 3.10 Cng thc phn t ca 3,5-Difluorophenylhydrazinocurcumin,
C27H22N2F2O4 (M=477,04) ....................................................................51
Hinh 3.11a Phn ng tng hp 4-Fluorophenylhydrazinocurcumin .......................53
Hnh 3.11b Phng trnh phn ng tng hp 4-Fluorophenylhydrazinocurcumin .53
Hnh 3.12 TLC ca tng hp 4-Fluorophenylhydrazinocurcumin ..........................54
Hnh 3.13 Sc k ct th ..........................................................................................54

Hnh 3.14 Sc k ct tinh.........................................................................................54

Hnh 3.15 TLC ca 4-Fluorophenylhydrazincurcuin...............................................56

Hnh 3.16 Ph UV-Vis ca 4-Fluorophenylhydrazinocurcumin trong dung mi
CH2Cl2................................................................................................................................................................................... 56
Hnh 3.17 Cu trc ca 4-Fluorophenylhydrazinocurcumin, C27H23N2FO4
(M=458 .v.C)......................................................................................57
Hnh 3.18 Hot tnh khng oxy ha ca Cur, 3,5-
Difluorophenylhydrazinocurcumin, 4-Fluorophenylhydrazino-
curcumin v Vitamin C theo phng php DPPH ..................................59
Hnh 3.19 th tnh gi tr IC50 ca mu.............................................................60
 DANH SCH CC BNG

Bng 1.1 Thnh phn ha hc cc cht trong thn r ngh .......................................6

Bng 1.2 Cu trc cc thnh phn ca curcuminoid..................................................7

Bng 3.1 Kt qu kt tinh bt curcuminoid .............................................................43

Bng 3.2 Mt s tnh cht ca 3,5-Difluorophenylhydrazinocurcumin ..................50
Bng 3.3 D liu ph NMR ca 3,5-Difluorophenylhydrazinocurcumin trong
dung mi CDCl3 ................................................................................................................................... 51
Bng 3.4 Mt s tnh cht ca 4-Fluorophenylhydrazinocurcumin ........................57
Bng 3.5 D liu ph NMR ca 4-Fluorophenylhydrazinocurcumin .....................58
Bng 3.6 Gi tr IC50 trong th nghim hot tnh khng oxy ho DPPH ................60
Bng 3.7 Kt qu xc nh gi tr IC50 trong kho st hot tnh khng
ung th ca 3,5-Difluorophenylhydrazinocurcumin v 4-Fluoropheny-
lhydrazinocurcumin vi 3 dng t bo Hep-G2, Lu v RD ........................61
 S

S 2.1 Quy trnh thc nghim.............................................................................25

S 2.2 Quy trnh phn lp cur .............................................................................26
S 2.3 Quy trnh tng hp 3,5-Difluorophenylhydrazinocurcumin ...................30
S 2.4 Quy trnh tng hp 4-Fluorophenylhydrazinocurcumin.........................34
 1

M U
L do chn ti
T lu con ngi bit s dng ngh nh l mt loi gia v, thuc gia truyn
v cha c nhiu bnh. Ngy nay nhu cu ca con ngi ngy pht trin, nn
ngh c ng dng rng ri hn trong cc lnh vc nh m phm, dc phm,
thc phm...

Thnh phn chnh trong c ngh l hn hp curcuminoid bao gm curcumin 1,

2, 3, tng i tr v khng gy c i vi ngi ngay c khi dng vi liu lng
ln, chnh v tnh an ton nn curcuminoid ng dng ph bin, c bit l trong lnh
vc dc phm. Nhng nghin cu gn y chng t curcuminod c nhiu hot
tnh quan trng nh: cht chng ung th, chng oxi ha, chng vim khp, chng
thoi ha, chng thiu mu cc b v khng vim...Ngoi ra, trong mt s nghin
cu ti Trng Y Khoa Havard cho bit dn xut curcuminoid cn c tc dng
trong vic iu tr HIV, Alzheimer
nc ta, tuy tr lng ngh rt di do nhng vic tng hp dn xut
curcuminoid v nghin cu hot tnh sinh hc vn cn hn ch.
Nhng nghin cu gn y cho thy dn xut imin ca curcumin c hot tnh
sinh hc tng ng k, c bit c ngha quan trng trong lnh vc y dc. Chnh
v l do trn m nhm chng ti nghin cu v ti "Tch, tng hp v kho st
hot tnh sinh hc ca cc dn xut curcumin t bt curcuminoid thng
phm.
Tng quan lch s nghin cu ca ti

Tnh hnh nghin cu trong nc

L Xun Tin[4], Nghin cu tng hp hydrazincurcumin v isoxaxolcurcumin-
Kho st hot tnh sinh hc ca chng.

Trn Thanh V[6], Tng hp v xc nh hot tnh sinh hc ca dn xut pheny-

lhydrazinocurcuminoid.
 2

ng Th M L, Th Xun Vui[2], iu ch v kho st hot tnh sinh hc

ca cc dn xut imin 2-hydrazinobenzothiazolcurcumin v 2,4-
Difluorophenylhydrazinocurcumin t curcumin[2].

Tnh hnh nghin cu ngoi nc

S. Mishra cng cc cng s[13] tng hp c 2 dn xut: hydrazinocurcumin
(HC) v 3-Nitrophenylpyprazolcurcumin c kh nng c ch s pht trin ca
Plasmodium falciparum cao hn so vi cur.
C. Selvam v cng s [18] tng hp v kho st hot tnh sinh hc ca mt s
dn xut imincurcumin. Kt qu cho thy dn xut hydrazinocurcumin v
isoxazolcurcumin khng vim cao hn cur tng ng.
J.S.Shim v cng s[20] cng tng hp c mt s dn xut
hydrazinocurcumin (HC) v hydrazinobenzoylcurcumin (HBC).
Nhm chng ti cng tin hnh nghin cu v to dn xut imin ca curcumin,
nhng tnh ni bt ca ti l to ra c hai dn xut imin v kho st hot
tnh sinh hc ca hai dn xut ny.

Mc tiu ti
- Tng hp, phn lp v kho st hot tnh sinh ca cc dn xut:
3,5-Difluorophenylhydrazinocurcumin v 4-Fluorophenylhydrazinocurcumin. Sau
kho st hot tnh sinh hc ca cc dn xut va tng hp c.

i tng nghin cu v phm vi nghin cu

- i tng nghin cu : Bt curcuminoid thng phm ca Vin Dc Liu
H Ni c chit xut t ngh vng (Curcuma Longa L.).

- Phm vi nghin cu: Curcumin.

- Lnh vc nghin cu: Tng hp, phn lp v kho st hot tnh sinh hc cc
dn xut ca curcumin.

Phng php nghin cu

- Phn lp curcumin t bt curcuminoid thng phm.
 3

- Tng hp cc dn xut ca curcumin va phn lp c.

- Sc k ct phn lp sn phm.
- Kho st hot tnh sinh hc ca dn xut trn.

9 Hot tnh khng oxi ha.

9 Hot tnh khng ung th.

Nhng ng gp mi ca ti v nhng vn cha lm c

Nhng ng gp ca ti:
Tng hp c hai dn xut 4-Fluorophenylhydrazinocurcumin (4-FPHC) v
3,5-Difluorophenylhydrazinocurcumin (3,5-DFPHC). Xc nh cu trc v mt s
tnh cht vt l, th hot tnh khng ung th v khng oxi ha ca chng.

Nhng vn cha lm c:
- Phn lp cc dn xut curcumin (cur) vi hiu sut thp.

- Cha th nghim hot tnh sinh hc vi cc chun t bo khc nhau

nhm nng cao kh nng ng dng.
 4

CHNG I

TNG QUAN
1.1 Curcuminoid
1.1.1 c im v s phn b ca cy ngh [11]
1.1.1.1 c im cy ngh
Ngh cn gi l ngh vng, ut kim, khng hong, khinh lng (Ty).
Ngh l cy tho mc sng lu nm, c tn khoa hc l Curcuma longa L., thuc h
gng. Cao khong 0,60 n 01 mt. Thn r thnh c hnh tr hoc hi dt, khi b
hoc ct ngang c mu vng cam sm do c cha cht mu curcumin. L hnh tri
xoan thon nhn hai u, hai mt u nhn di ti 45cm, rng ti 18cm, l khum
hnh mng rng, u trn mu xanh lc nht, l non hp hn, mu hi tm nht.
Cm hoa mc t gia cc l ln thnh hnh nn tha, cnh hoa ngoi mu xanh lc
vng nht, chia thnh ba thy. C ngh c v ng, cay, mi thm hc, tnh m.
cha tinh du (3-5%) mu vng nht, thm, ngoi ra cn c tinh bt, canxi oxalat v
cht bo.
Ton b phn trn mt t tn li vo ma ng cc tnh pha Bc v ma
kh cc tnh pha Nam. Cy mc li vo gia ma xun, c hoa sau khi ra l.
Hoa mc trn nhng thn ca nhng chi nm trc. Nhng thn ra hoa th nm
sau khng mc li na v phn thn r ca chng tr thnh nhng "c ci" gi, sau
1 2 nm b thi, cho nhng nhnh non ny chi thnh cc c th mi.
 5

Hnh 1.1: Cy ngh vng Curcuma longa L.

1.1.1.2 S phn b ca cy ngh

Ngh c ngun gc nguyn thy t n , y n c trng vng ng
bng v trn cc o. Ngi n dng mt loi tinh cht t ngh ra mt trong
vic cha vim kt mc. T thi xa xa, cy ngh c trng nhiu ni v sau
tr nn hoang di, trc ht l Trung Quc. Ngy nay, ngh l mt cy trng quen
thuc khp cc nc vng nhit i, t Nam n ng Nam v ng .
Vit Nam, ngh c tr lng kh di do v c trng khp cc a
phng, t vng ng bng ven bin n vng ni cao trn 1500m. mt s ni
thuc huyn Qun B, Yn Minh, ng Vn, Lm ng, Mo Vc (H Giang), Sn
H, Phong Th (Lai Chu). Bn cnh ngun cung cp do trng trong nhn dn,
mt s a phng pha bc, ngh mc hoang di c tnh tr lng n 1000 tn.
1.1.2 Thnh phn ha hc trong thn r ngh [11]
Thnh phn ha hc chnh quan trng nht ca thn r ngh l curcuminoid
(6%), l thnh phn to mu vng cho ngh, trong lng curcumin chim khong
70-80% khi lng. Trong thn r ngh cn cha tinh du (2-7%) vi cc thnh
phn chnh l artumeron, zingberen, borneol.
 6

Ngoi ra cn c nhng thnh phn vi hm lng thp hn nh

demetoxycurcumin, bisdemetoxycurcumin, dihydrocurcumin, phytosterol, cc acid
bo v polysaccharid v mt s thnh phn khc nh bng 1.1

Bng 1.1: Thnh phn cc cht trong thn r ngh [12,11]

Thnh phn n v Khi lng/100g

Nc g 6,0

Nng lng thc phm kcal 390

Protein g 8,5

Cht bo g 8,9

Carbohydrat g 69,9

Calcium g 0,2

Phosphorous mg 260

Sodium mg 30

Potassium g 2

Iron g 47,5

Thiamine mg 0,09

Riboflavin mg 0,19

Niacin mg 4,8

Acid ascorbic mg 50

1.1.3 Cu trc ca cc dn xut curcuminoid [11,12,13]

Curcuminoid l nhm cht mu chit xut t thn r cy ngh vng (Curcuma
longa L.). Curcuminoid l nhng dn xut diarylheptan gm curcumin,
 7

demetoxycurcumin, bisdemetoxycurcumin v gn y cn tm thy c mt t

cyclocurcumin. Chng l nhng hp cht phenolic, hu ht cc dn xut u khc
nhau nhm th trn gc phenyl.

Lng curcumin trong bt ngh khong 3-6%. Hn hp curcuminoid khong

77% curcumin (cur), 17% demetoxycurcumin (DMC), 3% bisdemetoxycurcumin
(BDMC)[8].

Hnh 1.2: Cng thc ha hc chung ca curcuminoid[7]

Bng 1.2: Cu trc cc thnh phn ca curcuminoid [7]

Hp cht R1 R2 CTPT M (.v.C)

Cur OCH3 OCH3 C21H20O6 368

DMC OCH3 H C20H18O5 338

BDMC H H C19H16O4 308

Danh php quc t ca:

- Curcumin: 1,7-bis-(4-hydroxy-3-methoxyphenyl)-hepta-1,6-diene-3,5-dione
- Demetoxycurcumin: 1-(4-hydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-hepta-
1,6-diene-3,5-dione.
- Bisdemetoxycurcumin: 1,7-bis-(4-hydroxyphenyl)-hepta-1,6-diene-3,5-dione
Ngoi thnh phn chnh l curcuminoid nh trn, trong thn r ngh cn c mt s
ng phn hnh hc ca chng, nh ng phn cis trans cur.
 8

Hnh 1.3: ng phn cis trans curcumin

Curcuminoid l nhng -diceton tn ti dng ng phn tautomer: cis

diceton hoc trans diceton v dng enol (cis enol). Trong dung dch, cc hp
cht -diceton nh vy tn ti di dng hn hp ceton v enol do qu trnh
tautomer ha. Trong cu trc ca enol c lin kt hydro.

Hnh 1.4: Cc ng phn enol ceton ca curcumin

Hnh 1.5: Qu trnh tautomer ha ca cc hp cht curcuminoid

Cc phn t curcuminoid cha h thng lin hp m rng vi cc vng phenyl
nm cui mch (trong cu dng enol). Ngoi ra, curcuminoid cn cha cc nhm
carbonyl, metoxy v hydroxyl, gp phn to nn hot tnh sinh hc a dng ca
 9

curcuminoid. Vi nhng c im cu trc trn, curcuminoid l nhng polyphenol

c xp vo cc hp cht khng oxy ha dng phenolic.

1.1.4 Tnh cht ha l ca curcuminoid

1.1.4.1 L tnh [9,13]
L dng bt mu vng cam hunh quang, khng mi, bn vi nhit ,
khng bn vi nh sng. Khi dng dung dch cur d b phn hy bi nh sng v
nhit , tan trong cht bo, etanol, metanol, diclometan, aceton, acid acetic bng v
hu nh khng tan trong nc mi trng acid hay trung tnh ( tan <10 mg
250C). Tan trong mi trng kim to dung dch mu mu ri ng tm, tan trong
mi trng acid c mu ti.
Cc thng s l tnh c trng ca curcuminod[4] :

Thng s Cur DMC BDMC

im chy (oC) 184 172 222

UV Vis trong etanol (nm) 429 424 419

1.1.4.2 Ha tnh
Qu trnh in ly theo pH
S in ly theo pH ca cur bng phng php HPLC cho kt qu sau
- pH < 1: dung dch cur c mu do cur trng thi proton ha H4A+.

- pH = 1 7: cur trng thi trung tnh H3A. Trong khong pH ny cur kh tan
trong nc v to huyn ph mu vng.

- pH > 7,5: dung dch cur c mu v cur tn ti di cc dng H2A-, HA2-,

A3-. Gi tr hng s phn ly proton pKa1, pKa2, pKa3 ca cur (to thnh H2A-, HA2-,
A3-) c xc nh ln lt l 7,8; 8,5 v 9,0.
 10

Hnh 1.6: Cc dng phn ly ca curcuminoid[20]

Phn ng phn hy

- Phn hy di tc dng ca nh sng[20]

Di tc dng ca nh sng, cur phn hy thnh vanillin, acid vanillic,

aldehyd ferulic, acid ferulic.

Cur cng khng bn ngay c khi c v khng c mt ca oxy. Khi c mt

oxy v nh sng, cur b phn hy to thnh 4-Vinylguaialcol v vanillin .
CHO CH CH2
HO OH
hv,O2
H3CO OCH3 +
O O OCH3 OCH3
H
OH OH

CH3 CO CH3 + CO2

 11

Cur c th b vng ha trong iu kin khng c mt ca oxy

Hnh 1.7: Phn hy di tc dng ca nh sng

- Phn hy trong mi trng kim

Tonnesen v Karlsen (1985) nghin cu qu trnh kim phn hy ca cur,

sn phm ca qu trnh phn hy trong khong pH t 7 n 10 c xc nh bng
HPLC. Sn phm ca qu trnh phn hy l acid ferulic v feruloylmetan.
 12

Hnh 1.8a: S phn hy ca cur trong mi trng kim

Sau Feruroyl metan tip tc b phn hy thnh vanilin v aceton. Acid

ferulic b phn hy thnh vinylguaialcol v CO2.

Hnh 1.8b: S phn hy ca cur trong mi trng kim[10]

Phn ng cng H2

Trong hp cht cur c cha cc hydrocarbon cha no, do c kh nng tham

gia phn ng cng 1,2 hoc 3 phn t H2 to thnh cc dn xut dihydrocurcumin,
 13

tetrahydrocurcumin v hexahydrocurcumin, khi c mt xc tc kim loi hay oxit

kim loi (Ni, PtO2), cc sn phm ny cng l cc cht khng oxy ho.[16]

Hnh 1.9: Phn ng cng H2 [5]

Phn ng imin ha
Cur l hp cht diceton nn c th cho phn ng vi cc amin bc nht
(RNH2), hydroxylamin (NH2OH), hydrazin (NH2-NH2), semicarbazid
(NH2NHCONH2) to thnh cc dn xut imin (base Schiff) hoc dn xut imin
tng ng.[3,6,15].
 14

C ch phn ng:

Hnh 1.10: Phn ng imin ha[14]

Giai on u ca phn ng l s tn cng ca i in t t do trn
nguyn t nitrogen ca amin vo nguyn t carbon mang mt phn in tch
dng ca nhm carbonyl. Phn ng xy ra theo c ch cng hp i nhn thng
thng, hnh thnh hp cht trung gian cha ng thi hai nhm chc anion
alkoxid v cation ammonium. Hp cht trung gian ny chuyn ho nhanh thnh
sn phm trung gian bn hn l carbinolamin. Phn ng ny cn mt lng nh
acid lm xc tc, gip cho cn bng chuyn dch v pha tch nc t hp cht
trung gian carbinolamin, sinh ra dng proton ho ca imin hoc cc dn xut ca
imin. Cui cng l giai on tch proton, hnh thnh sn phm l imin hoc cc
dn xut ca imin.[14]

Trong c ch ca phn ng imin ho, pH ca mi trng ng vai tr quan

trng. Nu mi trng phn ng qu acid, ton b amin b proton ho. Cc
amin b proton ho khng c tnh i nhn nn chng khng phn ng vi cc
nhm ceton. Ngc li, gim mi trng acid s lm gim qu trnh tch nc to
imin. iu kin pH thch hp cho phn ng imin ho l khong pH ~ 4,5.[15]
 15

Phn ng ca nhm hydroxyl trn vng benzen

Cc cp electron cha lin kt ca oxy nhm hydroxyl lin hp mnh vi vng
benzen lm cho nguyn t hydro ca nhm hydroxyl tr nn linh ng hn. iu
ny gii thch tnh acid v kh nng phn ng vi cc gc t do ca cur.
Ngoi ra, phn ng vi cc gc t do cn lin quan n s chuyn nguyn t
hydro ca nhm metylen carbon gia mch, lm gim hay mt hot tnh ca cc
gc ny. Hai hng phn ng ny gp phn gii thch tnh chng oxy ha mnh ca
curcuminoid khi ng dng n trong ngnh dc.

Hnh 1.11: Phn ng ca curcuminoid vi gc t do[19]

Phn ng to phc
Phc kim loi bao gm ion kim loi trung tm lin kt vi cc phn t khc
bng lin kt cng ha tr. Curcuminoid vi cu trc -diceton trong mi trng
 16

acid hay trung tnh nm di dng h bin ceton enol i xng v n nh, lm
cho curcuminoid c kh nng to phc vi nhiu ion kim loi khc nhau: Mn2+,
Fe2+, Cu2+, Ga3+, [4]
Curcuminoid c kh nng cho i mt hay nhiu cp electron
t do trn cc nguyn t oxy ca cu trc ceton enol v cht nhn l cc ion kim
loi vi lp v electron cn trng. Do vy, lin kt cng ha tr gia curcuminoid
v kim loi c hnh thnh. S lin kt ny lm thay i nng lng trng tinh
th ca ion kim loi, lm thay i s phn b electron trong phn t. Kt qu l
phc ca kim loi v curcuminoid s c nhiu mu sc khc nhau.

Hnh 1.12: Phn ng to phc vi kim loi [5]

Proton metyl trong dng ceton v proton hydroxyl trong dng enol ca -
diceton c tnh acid v khi cc proton ny b phn ly s lm cho -dicetonat tch
in m. -dicetonat mang in m ny ng vai tr l tc nhn to phc mnh, c
kh nng to phc vi tt c kim loi chuyn tip v c cc kim loi phn nhm
chnh.

Hnh 1.13: Anion dicetonat

 17

1.1.5 Hot tnh sinh hc ca curcuminoid [8,9]:

Hot tnh sinh hc ch yu ca curcuminoid l khng oxy ho, khng khun, khng
virus v khng mt s loi ung th.
Nhiu cng trnh nghin cu th nghim cc nc trn th gii khng
nh t lu rng hot cht cur c tc dng hu dit t bo ung th vo loi mnh.
Ti M, i Loan, ngi ta tin hnh th lm sng dng cur iu tr ung th v
kt lun rng cur c th km hm s pht tc ca t bo ung th da, d dy, rut,
vm hng, d con, bng quang. Cur cn l cht b cho d dy, rut, gan, mt, lc
mu, lm sch mu, iu tr vt thng, chng vim khp, d ng, nm, chng vi
khun c hiu lc. T nm 1993, cc nh khoa hc thuc i hc Harvarrd (M)
cng b 3 cht c tc dng km hm t bo HIV-1, HIV-1-RT v 1 trong 3 cht
l cur.
Cur c gi tr hot tnh sinh hc cao l do trong cng thc cu to ca cur c
cc nhm hot tnh sau:

Hnh 1.14. Cng thc cu to ca curcumin

- Nhm parahydroxyl: hot tnh chng oxi ho.

- Nhm ceton: khng vim, khng ung th, chng t bin t bo.

- Nhm lin kt i: khng vim, khng ung th, chng t bin t bo.
Hot tnh khng oxy ha
 18

Oxy khng th thiu i vi vi sinh vt i kh. Oxy tham gia vo nhiu qu

trnh sinh ha hc trong c th.. Trong cc qu trnh , oxy to ra nhng tiu phn
trung gian gi l cc gc t do.
Khi nhn mt in t u tin, oxy to ra gc superoxid. y l gc t do quan
trng nht ca t bo. T gc superoxid (O2), nhiu gc t do v cc phn t khc
nhau ca oxy c kh nng phn ng cao c to ra nh: HO (gc hydroxyl),
H2O2, 1O2 (oxy n bi), LO (gc lipoxid), LOO (gc lipoperoxid), RO (gc
alkoxid), LOOH. Tn chung ca cc gc ny l cc dng oxy hot ng. Ngoi ra
trong c th cn c nhng dng gc t do hot ng khc c cha nit, clo
Trong c th lun tn ti nhng hp cht c kh nng loi b cc dng oxy
hot ng trn, v c gi l cc cht khng oxy ha. Tiu biu l enzym
superoxid dismutase (SOD), glutathion (GSH), enzym glutathion peroxydas (GSH
Px), enzym catalas v nhng phn t nh nh tocopherol, ascobat
Trong c th lun tn ti s cn bng gia cc dng oxy hot ng v cc dng
khng oxy ha. l trng thi c bn ca cn bng ni mi. Do nh hng ca
nhiu yu t tc ng t bn ngoi hay bn trong c th, lm cho cn bng ny di
chuyn theo hng gia tng cc dng oxy hot ng. Trng thi sinh l ny c
gi l stress oxy ha. Oxy kt hp vi m, oxy ha thnh phn t bo v cc phn t
sinh hc. Khi b stress oxy ha, cc gc t do to thnh c kh nng phn ng rt
cao vi cc hp cht sinh hc s gy tn thng m.
Curcuminoid l hp cht t nhin tch t cy ngh vng, c tnh cht khng
oxy ha kh cao, ngn cn s to thnh cc gc t do nh superoxid, hydroxyl
C ch qu trnh khng oxy ha ca curcuminoid l ngn cn s peroxid ha cc
lipid trong c th.
Phn ng peroxid ha lipid l phn ng dy chuyn v xy ra theo c ch gc
t do, gm cc bc sau:
Giai on khi mo: Di tc dng ca cc gc t do, nguyn t hydro b tch
ra khi cc acid bo cha bo ha (lipid).
 19

SH + R S + RH
S + O2 SOO

Phn ng dy chuyn: Gc t do peroxyl va hnh thnh tn cng cc acid bo

k cn.

SOO + SH SOOH + S
Qu trnh tip din dn n s tch ly cc peroxid bo trong mng t bo, lm
mng t bo khng n nh v cho php s xm nhp ca cc ion c hi. Gc t do
peroxyl tn cng cc ion cng nh cc protein mng t bo.
Hot tnh khng ung th

Ung th l mt cn bnh nan y t trc n nay. Yu cu ca thuc tr ung th

l c ch s pht trin v bit ha cc t bo ung th nhm chn ng s pht trin
v di cn ca ung th. Tuy nhin nhc im chung ca i a s cc thuc tr ung
th hin nay l khng c s chn lc cao, u d b ln thuc v h s an ton
gim dn theo thi gian s dng, ngoi ra chng c th gy c cho cc t bo
lnh v gy ra cc tc dng ph nh: rng tc, bun nn... V vy vic tm ra mt
hot cht c tc dng chng ung th ngun gc t t nhin v tng hot tnh ca cc
hot cht ang l vn c quan tm hng u.
Cur l cht hy dit t bo ung th vo loi mnh nht theo c ch t hy dit
tng phn cc t bo c tnh[4].. C tc dng km hm t bo ung th c ba giai
on khi pht, tin trin v giai on cui. Kt qu l cc t bo ung th b v hiu
ha nhng khng gy nh hng n t bo lnh tnh, ng thi ngn nga s hnh
thnh t bo ung th mi.
Ngoi ra cur cn c tc dng loi b cc men gy ung th, bt cc gc t do
gy ung th. Bi vy, cur c th gip c th va phng nga va chng ung th
mt cch hiu qu .
 20

1.2 Cc nghin cu v imin v dn xut imin-curcumin

Mt trong cc hng nghin cu v cur hin nay l nng cao kh nng ng dng v
hot tnh da trn cc nguyn tc sau:

- To phc vi cc kim loi chuyn tip.

- Tng hp cc hp cht imin.

- Encapsule ho cur.

- To cc dn xut cur vi glucose, glycin, alanin, acid acetic

Trong cc hng nghin cu trn th cc nghin cu v cc dn xut imin ngy

cng th hin nhiu tim nng cho ngnh dc phm.

J.S.Shim v cng s[20] tng hp c mt s dn xut hydrazinocurcumin

(HC) v hydrazinobenzoylcurcumin (HBC).

Hnh 1.15. Phn ng tng hp hydrazinocurcumin

Hnh1.16: Phn ng tng hp hydrazinobenzoylcurcumin

 21

Kt qu nghin cu cho thy HC c hot tnh c ch t bo BAEC (bovine

aortic endothelial cell), ngn chn tin trnh angiogenesis (mt trong cc tin trnh
pht trin ca ung th) cao hn 30 ln so vi cur. Ngoi ra HC cn c kh nng c
ch mt s dng t bo: HT29, NH3T3, Chang.
Ring HBC th hin hot tnh c ch dng t bo BAEC mnh hn cur nhng
km hn HC. HBC c hot tnh c ch mnh ln t bo HCT15, nng 40M
HBC th hu nh ton b lng t bo HCT15 b c ch sau hn 48 gi. Tuy nhin,
cng nh HC, HBC khng th hin hot tnh c ch i vi APN (amino peptidase
N).
Kt qu th nghim cng cho thy HBC c kh nng cn tr qu trnh pht
trin ca t bo ung th rut kt (rut gi HCT15 colon cancer cells) bng cch
kt hp vi nhm chc nng trung gian khc l Ca2+/CaM. Ca2+/CaM l mt protein
a chc nng, bn thn n khng c hot tnh no c bit nhng nhng nghin cu
gn y cho thy n c lin quan v thng c nhng biu hin bt thng i vi
mt vi loi t bo ung th. V vy, n c xem nh mt cht mang l tng cho
cc cht khc trong Ca2+ ng vai tr l cu ni gn kt nhng cht c hot tnh
sinh hc vi CaM. Kt qu nghin cu cho thy HBC c th gn trc tip ln
protein Ca2+/CaM vi tng thch cao.
[18]
C. Selvam v cng s tng hp mt s dn xut imincurcumin v qua
kho st cc hot tnh sinh hc ca cc dn xut ny cho thy dn xut HC v
isoxazolcurcumin khng vim cao hn cur tng ng.
 22

Hnh 1.17. Phn ng tng hp mt s dn xut imin t curcuminoid[18]

S.Mishra cng cc cng s[13] tng hp c 2 dn xut:
Hydrazinocurcumin v 3-Nitrophenylpyprazolcurcumin c kh nng c ch s pht
trin ca Plasmodium falciparum cao hn so vi cur.

Hnh 1.18. 3-nitrophenylpyrazolcurcumin

Hnh 1.19. Hydrazinocurcumin

Curcuminsemicarbazone (CSC) c tng hp t phn ng th mt nguyn t

oxy ca nhm diceton trong cur bng nhm semicarbazone[18]. Kt qu nghin cu
 23

cho thy hp cht ny th hin hot tnh chng peroxy ha lipid ca microsome gy
ra bi bc x trong th nghim TBARS tng t nh cur. C hai cht u th
hin hot tnh c ch qu trnh peroxy ha ny mi liu s dng, iu ny chng
t rng cu trc diceton khi lin kt vi cc nhm chc khc vn khng mt i hot
tnh sinh hc ca cur.

Hnh 1.20. Cng thc cu to ca curcuminsemicarbazone [13]

Cur th hin hot tnh bt gc DPPH nhanh hn nhiu so vi CSC. iu ny
c l gii l do c nhm th ht in t semicarbazone nn lm gi tr th oxy
ha ca nhm phenolic trong CSC hn trong cur.

Nhm tc gi o Hng Cng, L Hi Li (i hc Nng) [1] tng hp

thnh cng dn xut pyrazole gia phenylhydrazin v cur, dn xut isoxazol gia
hydroxylamin v cur. Nghin cu hot tnh khng khun, khng nm, c ch gc t
do DPPH ca chng, kt qu cho thy hai dn xut ny u c kh nng khng oxy
ha v khng 4 chng vi khun E.Coli, P.aeruginosa, B.subtillis, S.aureus. Tuy
nhin cc hot tnh sinh hc ca hai dn xut ny u km hn so vi cur.
Tc gi L Xun Tin (i Hc Bch Khoa TPHCM)[4] tng hp thnh
cng isoxazolcurcumin (IOZ) v hydrazinocurcumin (HC) t cur. Qua kho st cc
hot tnh sinh hc ca 2 dn xut ny cho thy IOZ v HC c tnh khng oxy ha
tng t nh cur. Trong IOZ v HC c kh nng gy c cho dng t bo ung
th Hep-G2 cao hn cur ring HC cho hot tnh cao gp 2 ln so vi cur.
Tc gi ng Th M L, Th Xun Vui (i Hc Nng Lm )[2], tng
hp thnh cng 2-Hydrazinobenzothiazolcurcumin (HBTC), 2,4- Difuorophenyl-
hydrazinocurcumin (DFPHC) t cur, qua kho st hot tnh sinh hc ca 2 dn xut
 24

ny cho thy chng c tnh khng oxy ha tng ng cur, DFPHC v cur c hot
tnh gy c i vi dng t bo Hep-G2, khng gy c t bo RD, LU v khng
lm tng hot tnh gy c i vi 3 dng t bo kho st.
 25

CHNG 2

NI DUNG V PHNG PHP NGHIN CU

2.1 Ni dung nghin cu

Bt curcuminoid

Phn lp curcumin

Kim tra tinh khit,

cu trc

Tng hp dn xut

Tinh ch dn xut

Kim tra tinh khit,

cu trc

Xc nh cc hot tnh
sinh hc

S 2.1: Quy trnh thc nghim

T nguyn liu ban u l bt curcuminoid thng phm ca Vin Dc Liu

H Ni c chit xut t ngh vng (Curcuma Longa L.). Bao gm c 3 thnh
 26

phn chnh Cur, DMC, BDMC. Tin hnh phn lp cur bng 2 bc: Kt tinh bt
curcuminoid thng phm v sc k ct phn lp cur tinh.

- Kim tra tinh khit ca cur tinh bng TLC.

- To cc dn xut 3,5-Difluorophenylhydrazinocurcumin (3,5-DFPHC) v 4-

Fluorophenylhydrazinocurcumin (4-FPHC). Phn lp dn xut bng sc k ct v
xc nh tinh khit, cu trc bng TLC, NMR, MS.

- Kho st hot tnh sinh hc ca cc dn xut phn lp c.

2.2 Phng php thc hin

2.2.1 Phn lp cur

2.2.1.1 Quy trnh phn lp cur

Bt curcuminoid

Kt tinh li H dung mi: MeOH:H2O

Thi gian 24 gi, 5oC

Tinh th

Sc k ct - Silica gel 60G, 0.063-0.200mm

- Dm: Diclometan:eter du (90:10)

Curcumin
tinh

S 2.2: Quy trnh phn lp cur

2.2.1.2 Kt tinh bt curcuminoid thng phm

Mc ch: loi b nha v gim hm lng DMC, BDMC v lm giu hm
lng cur.
 27

Ha cht, nguyn liu v dng c:

- Ha cht: MeOH, nc ct.

- Nguyn liu: Bt curcuminoid thng phm.

- Dng c: Erlen, lc ht chn khng, bp in, ni, nhit k 100oC, t .

Phng php:

- Kt tinh ln 1:
Cho bt curcuminoid vo erlen, va lng MeOH vo lc u 50oC
cho n khi hn hp trong, cho nc ct vo hn hp trn (500C) n khi chuyn
sang c, lc lin tc khi hn hp trong tr li th dng. dung dch ngui nhit
phng, kt tinh 5oC trong t khong 24 gi. Sau ly ra, tin hnh lc
ht chn khng. Thu phn trn giy lc v tip tc kt tinh.
- Kt tinh ln 2 v 3: Tin hnh tng t nh ln kt tinh 1.

2.2.1.3 Sc k bn mng (TLC)

Mc ch
- Xc nh cc thnh phn c trong bt curcuminoid thng phm.

- Xc nh h dung mi chy ct phn lp cur tinh.

Nguyn tc: Da trn s tng tc khc nhau gia cc thnh phn trong hn
hp vi dung mi v pha tnh, do mi thnh phn c phn cc khc nhau.
Nguyn liu, dng c, ha cht

- Nguyn liu: bt curcuminoid thng phm sau khi kt tinh 3 ln.

- Dng c: ct sc k, h bi, vi qun, bn mng silica gel 60G, Merck

20x20cm, n cn, bnh sc k, kp, giy lc, my sy, gi .

- Ha cht: MeOH, aceton, diclometan, nc, H2SO4 10%.

Phng php:
- Chun b
 28

Hot ha bn mng silica gel bng cch sy 1gi 110oC sau cn

bng trong bnh ht m 30 pht.
Ko ng vi qun bng ngn la n cn, ra vi qun 3 ln bng aceton.

- Chm mu ln bn mng
Ha tan mt t mu curcuminoid vo chai bi bng metanol.
Dng ng vi qun ht mt t mu ha tan trong chai bi chm ln
bn mng.
Khong cch vt chm cch b di 0,7cm hai b hai bn 0,5cm v
khong cch gia cc vt chm cch nhau 0,6 cm. Kch thc ca vt
chm va phi. V vy nn vic la chn dung mi ha tan mu rt
quan trng.

- Trin khai bn mng

Chun b bnh trin khai: ra sch, sy kh.

Pha h dung mi trin khai vo bnh ln lt theo cc t l xc nh.
t mt tm giy lc vo bnh trin khai.
Bo ha dung mi trong bnh trin khai t 15-20 pht.

Dng kp cho bn mng chm mu vo bnh, y np bnh li.

Theo di vch dung mi di chuyn n cch mp trn ca tm bn
mng khong 0,2cm th ly bn mng ra khi bnh trin khai.

Sy nh bn mng ui ht dung mi.

- Pht hin vt

Qua n UV ( bc sng = 254nm hoc 365nm).

Qua mu sc t nhin.

Phun thuc th H2SO4 10% trong H2O ri h nh trn bp in.

Qua hi Iod.
Nhn xt v nh gi cc vt trn cc bn mng chn dung mi chy ct
 29

ph hp.

2.2.1.4 Sc k ct (SKC)
Mc ch: Phn lp cc thnh phn trong hn hp ban u.

- Pha ng: Kho st trc bng TLC v chn h dung mi diclometan:eter

du ha (90:10)

- Pha tnh: Silica gel 60G, 0.063-0.200mm, Merck.

- Ct thy tinh =30mm.

- Mu c np vo ct theo phng php np mu t.

Tin hnh

- Bc 1: Chun b
Sy silicagel 110oC trong 4 gi, cn bng m trong bnh ht m
trong 30 pht. Ngm qua m vi dung mi chy ct.
Ct c ra sch, sy kh, kp thng gc trn gi, cho bng gn vo
y ct.

- Bc 2: Nhi ct
Cho t t lng silica gel vo becher cha sn dung mi chy ct,
lng dung mi va sao cho hn hp khng qu st.
Tin hnh cn bng ct trong 2-4 gi.

Bc 3: Cho bt curcuminoid sau khi kt tinh 3 ln ln b mt

silica gel ca ct.

- Bc 4: Trin khai ct.

- Bc 5: Thu v kim tra cc phn on thu c cur tinh khit.

2.2.2 Tng hp cc dn xut imin-curcumin.

2.2.2.1 Tng hp 3,5- Difluorophenylhydrazinocurcumin
a) Quy trnh tng hp:
 30

Dung mi : CH3OH Curcumin

iu chnh pH ~5 (CH3COONa)

3,5-Difluorophenylhy-
drazin hydrochloride

Imin ha Khuy trn hon lu

Nhit 60oC

Loi dung mi C gim p

Ra chit Dung mi

CH3COOC2H5:H2O

Phn lp
Sc k ct

Kim tra TLC, MS,

MNR

Kho st hot
tnh sinh hc
S 2.3: Quy trnh tng hp 3,5-DFPHC

b) Phng php thc hin

Ha cht, nguyn liu, dng c
- Nguyn liu: Cur tinh, 3,5-Difluorophenylhydrazin hydrochloride 97%
(CTPT: C6H6F2N2 .HCl, M=180,58)
 31

- Ha cht: Acid acetic, natri acetat, metanol, diclometan, hexan, etyl acetat
(Chemsol Vina).

- Dng c: Bnh cu 2 c, nhit k 100oC, bp in, b chng ct hon lu.

iu kin phn ng:

- Phn ng xy ra thch hp theo t l 1:1(mol/mol)

- Khi lng cur tham gia phn ng l 200mg

- Khi lng 3,5- Difluorophenylhydrazin hydrochloride 97% l 100mg

- Nhit phn ng 600C.

Tin hnh

- Cho 3,5-Difluorophenylhydrazin hydrochloride (100mg) vo bnh cu hai c

v ha tan bng MeOH, sau iu chnh PH~5 bng natri acetat. Tip theo cho
cur (200mg) vo lc u. un hn hp hon lu trn bp khuy t trong iu kin
kn nh sng, y l iu kin c bit quan trng nhm trnh xy ra mt s phn
ng ph gy kh khn trong qu trnh phn lp sn phm.
- Theo di phn ng v thng xuyn kim tra pH, khong pH rt quan trng
cho phn ng xy ra nhanh hay chm, lun lun gi pH~5.

- Duy tr n nh cc iu kin phn ng v kim tra im dng phn ng

bng TLC h dung mi diclometan:etyl acetat (96:4)

c) X l mu
Mc ch: Loi b bt tp cht sau phn ng ra khi hn hp.
Ha cht, nguyn liu v dng c

- Ha cht: Etyl acetat (Chemsol Vina), nc ct, Na2SO4 khan.

- Nguyn liu: Hn hp sau phn ng

- Dng c: Phu chit, gi , bnh xt nc ct.

Tin hnh
 32

- C quay chn khng p sut thp nhm loi dung mi cn li sau phn ng.
Ha tan hn hp sau khi c quay bng etyl acetat, cho hn hp trn vo phu chit,
t t cho nc vo v lc u, sau hn hp phn thnh 2 lp, x van chit b lp
di v ly lp etyl acetat pha trn.

- Cho Na2SO4 khan vo lp etyl acetat nhm loi nc, c quay loi b etyl
acetat, thu c cao th ha tan vi diclometan v hp ph silica gel, c quay gim
p loi dung mi.

d) Phn lp sn phm bng phng php sc k ct

Phn lp sn 3,5-DFPHC bng cch chy ct vi nhiu h dung mi khc
nhau. Qua kho st mt lot cc h dung mi nh diclometan:hexan,
diclometan:eter du ha, diclometan:etyl acetat, etyl acetat:eter du ha, hexan:etyl
acetat . Kt qu cho thy h hexan:etyl acetat c kh nng tch tt nht.
Sc k ct th

- Ha cht, nguyn liu, dng c

Ha cht: Hexan (Chemsol Vina), etyl acetat (Chemsol Vina), silica

gel 60G 0,04-0,06mm

Nguyn liu: Cao sn phm th sau phn ng tng hp 3,5-DFPHC

Dng c: ct sc k, h bi.
- iu kin sc k ct th

Silica gel 60G 0,04-0,06mm, Merck c hot ho 110oC, 4 gi

trc khi nhi ct.

Khi lng silica gel np ct: 25g.

ng knh trong ca ct: 2cm.

Chiu cao ca silica gel trong ct: 16 cm.

Vi h dung mi trin khai thch hp hexan:etyl acetat (80:20)

- Sau khi chy ct th phn lp c 3 phn on (hnh 3.8)
 33

Phn on 1: H 1-29: khng kho st

Phn on 2: H 30-34 bn trn u sn phm v vt sn phm b

vng u, vt mu xanh pha di sn phm (=365nm).

Phn on 3: H 35-54 sn phm vng di v 1 vt di sn phm

mu xanh (=365nm).
Thu phn on 3 lm bay dung mi kh cn c khi lng 76mg v hp
th silica gel chy ct tinh.
Sc k ct tinh

- Ha cht, nguyn liu, dng c

Ha cht: Hexan, etyl acetat, silica gel 60G, 0,04-0,06mm

Nguyn liu: Sn phm t sc k ct th phn on 3.

Dng c: Ct sc k, h bi.
- iu kin sc k ct tinh

Silica gel 60G, 0,04-0,06mm, Merck c hot ho 110oC, 4 gi

trc khi nhi ct.

Khi lng silica gel np ct l 4g.

Ct sc k: ng knh 1cm, chiu cao silica gel trong ct: 8 cm.

H dung mi trin khai ct hexan:etyl acetat (90:10)

e) iu kin sc k bn mng (TLC) kim tra qu trnh sc k ct
- TLC silica gel 60G F254 (Merck) c hot ho 110oC, 1 gi. Bn mng
8x3cm, chiu cao trin khai bn mng 6cm.

- Hin mu TLC vt mu tm hoc mu xanh di n UV (254nm hoc

365nm).
- Cho mt vt duy nht khi gii ly vi h dung mi diclometan:etyl acetat
(96:4), phun thuc th, hin hnh bng H2SO4 10%, sy bn 1100C cho mt vt
vng cam.
 34

2.2.2.2 Tng hp 4-Fuorophenylhydrazinocurcumin

a) Quy trnh tng hp

Dung mi CH3OH Chnh v

PH~5 (bng CH3COONa)
Curcumin

4-Fluorophenylhydrazin
hydrochloride

- Khuy trn hon lu

Imin ha
- Nhit 60oc

Loi dung
mi - C quay

Ra chit - Dung mi: Etyl acetat:H2O

Phn lp - Sc k ct

Xc nh - TLC, MS, NMR

cu trc

Kho st
hot tnh

S 2.4: Quy trnh tng hp 4-FPHC

b) Phng php thc hin

Nguyn liu, ha cht v dng c
 35

- Nguyn liu: Cur tinh, 4-Fluorophenylhydrazin hydrochloride 95% (CTPT:

C6H7FN2.HCl, M=162,59).

- Ha cht: Acid acetic, natri acetat, metanol, diclometan, etyl acetat (Chemsol
Vina).
- Dng c: Bnh cu 2 c, nhit k 100oC, bp in, b chng ct hon lu.

iu kin phn ng:

- Kho st cc t l s mol khc nhau gia cur v 4-Fluorophenylhydrazin

hydrochloride th thy t l phn ng 1:2 (mol/mol) l thch hp, vi t l 1:1
(mol/mol) phn ng xy ra rt chm, thi gian phn ng ko di n 80 gi m vt
sn phm li nht, trong khi sn phm ph sinh ra nhiu nm v tr trn sn
phm v pha di cur.

- Khi lng cur tham gia phn ng l 150mg

- Khi lng 4- Fluorophenylhydrazin hydrochloride 95% l 149mg

- Nhit phn ng 600C .

Tin hnh

- Cho 4- Fluorophenylhydrazin hydrochloride 95% (149mg) vo bnh cu hai

c v ha tan bng MeOH, iu chnh pH~5 bng natri acetat, sau cho cur
(150mg) vo lc u.

- un hn hp hon lu trn bp khuy t v bt kn, trnh nh sng l iu

kin c bit quan trng ca phn ng nhm trnh xy ra cc phn ng ph gy kh
khn trong vic tch sn phm.

- Lun lun duy tr pH~5. V vy thng xuyn kim tra pH iu chnh kp

thi. pH c vai tr quan trng quyt nh n qu trnh phn ng xy ra nhanh hay
chm.
- Duy tr n nh cc iu kin phn ng v kim tra im dng phn ng
bng TLC h dung mi gii ly diclometan:etyl acetat (96:4)

c) X l mu sau phn ng
 36

Mc ch: Loi b bt tp cht ra khi hn hp sau phn ng

Ha cht, nguyn liu, dng c

- Ha cht: Etyl acetat (Chemsol Vina), nc ct, mui Na2SO4 khan.

- Nguyn liu: Cao th 4-FPHC sau phn ng.

- Dng c: Bnh xt nc ct, phu chit, gi .

Thc hin tng t nh qu trnh x l mu 3,5-DFPHC.

d) Phn lp sn phm bng phng php sc k ct

Sc k ct th

- Nguyn liu, ha cht, dng c

Ha cht: Hexan v etyl acetat (Chemsol Vina), silica gel.

Nguyn liu: Cao th sau phn ng tng hp 4-FPHC

Dng c: Gi , h bi, ct sc k.
- iu kin sc k ct th

Silica gel 60G, 0,04-0,06mm c hot ho 110oC, 4 gi trc khi

nhi ct.

Khi lng silica gel np ct 25g

ng knh ct: 2cm

Chiu cao ca silica gel trong ct: 16cm

Dung mi trin khai TLC h hexan:etyl acetate (90:10, 80:20).

- Sau khi chy ct th tch c 3 phn on (hnh 3.15)

Phn on 1: Khng kho st.

Phn on 2: Sn phm c vt bn nm ngay trn u, v 1 vt xanh

nm di sn phm.

Phn on 3: Sn phm m v cur.

Thu phn on 2 ui dung mi, kh v cn c khi lng 50mg.
 37

Sc k ct tinh
S dng phn on 2 phn lp sn phm.

- Ha cht, nguyn liu, dng c.

Ha cht: Hexan, etyl acetat, silica gel 60G, 0,04-0,06mm

Nguyn liu: Phn on 2 ca sc k ct th.

Dng c: Ct sc k ng knh 1cm, h bi.

- iu kin sc k ct tinh

Hp th mu vi silica gel kh

Khi lng silica gel msilica gel=10g.

ng knh ct 0,8cm

Chiu cao ca silica gel trong ct l 7cm

H dung mi chy ct l hexan:etyl acetat (90:10)

Sau khi chy ct tinh ta vn khng tch c vt bn trn u sn phm v
vt xanh di sn phm. Tin hnh th nhiu h dung mi khc nhau cng khng
tch c nn tin hnh sc k iu ch (SKC).

e) Sc k iu ch
Nguyn liu, ha cht v dng c

- Nguyn liu: 4-FPHC thu c t sc k ct tinh.

- Ha cht: Etyl acetat, diclometan.

- Dng c: Bnh gii ly c kch thc 22x1,3x21cm, thc k 20cm, phu lc,
bt ch.

iu kin sc k iu ch

- TLC silica gel 60G F254 (Merck) 10x20cm, chiu cao trin khai bn mng
8,5cm
 38

- Dng bt ch vch nh cc mc xut pht cch mp di 1cm v mc tin

tuyn cch mp trn 0,5cm. Cha hai bn l mi bn 1cm. Chun b bnh gii ly
pha sn 100ml h dung mi diclometan:etyl acetat (96:4), t 1 tm giy lc dc
theo thnh bnh c chiu di bng chiu di bnh. y np bnh li bo ha dung
mi n nh khong 20 pht.

Tin hnh:
- Ha tan 39mg mu bng lng ti thiu diclometan (100%).
- Dng vi qun ht mu pha sn v ko trn bn mng thnh mt ng
di, u, dc theo mc xut pht. Khong 10mg mu p dng cho mt bn mng
c kch tht 10x20cm.

- Sy ui dung mi, t vo bnh gii ly chun b sn. Khi dung mi ln

n mc tin tuyn, khng nn bn lu trong bnh, ly bn ra khi bnh gii ly,
v s khuch tn v s bay hi ca dung mi c th lm cc vt va tch c s b
tri di ra.

- Vt sn phm hin mu xanh di n UV (=365nm). Dng bt ch vch

khoanh vng ni c cha sn phm trn bn. Ct ly phn ny, co ly lp silica
gel c cha mu, gii hp vi dung mi diclometan (100%). Lc b silica gel, thu
phn dung mi c cha mu v sy ui ht dung mi.

f. iu kin sc k bn mng kim tra qu trnh sc k ct

- TLC silica gel 60G F254 (Merck) c hot ho 110oC, 1 gi. Bn mng
8x3cm, chiu cao trin khai bn mng 6cm.
- Hin mu TLC vt mu tm hoc mu xanh di n UV (254nm/365nm).

- Cho mt vt duy nht khi gii ly vi h dung mi diclometan:etyl acetat

(96:4), phun thuc th, hin hnh bng H2SO4 10%, sy bn 1100C cho mt vt
vng cam.

2.2.3 Phn tch tinh khit v cu trc ca cc dn xut va tng hp

2.2.3.1 Ph t ngoi kh kin (UV-Vis). (Hnh 3.9 v 3.16)
 39

Ph t ngoi kh kin c o bng my HACH DR5000 ti phng th

nghim trung tm lc Ho du, i hc Bch Khoa Tp. H Ch Minh. phn gii
1nm.

2.2.3.2. Khi ph (ph lc 3 v 9)

Ph MS c phn tch ti Trung tm phn tch cng ngh cao Hon V,
112A Lng Th Vinh, Phng Tn Thi Ha, Q. Tn Ph, Tp. H Ch Minh.

2.2.3.3. Ph cng hng t ht nhn (ph lc 1,2,4,5)

- Ph NMR c phn tch ti Vin Ho hc, Vin Khoa hc & Cng ngh
Vit Nam, H Ni. My phn tch: my Bruker AV 500, 500 MHz cho 1H v 125
MHz cho 13C.

2.2.4 Kho st hot tnh sinh hc

2.2.4.1 nh gi hot tnh khng oxy ho (ph lc 10)
Phng php nh bt gc t do bng th nghim DPPH
(Thc hin ti phng th nghim trung tm lc Ho du, i hc Bch Khoa Tp. H
Ch Minh.)
- Nguyn tc
Cc cht nghin cu c tc dng khng oxi ha theo c ch bt gc t do s
chuyn gc t do DPPH (1,1-diphenyl-2-picrylhydrazyl) t mu tm sang mu vng
nht. Xc nh kh nng bt gc t do ca cht nghin cu bng phng php o
hp thu ca mu ti bc sng = 517nm. Ascorbic acid c s dng lm cht i
chiu.

Phng php tin hnh

- Thc hin vi mu th:
Ho tan DPPH trong dung mi metanol nng 6mM.

Ho tan mu trong DMSO 8 nng khc nhau 500; 100; 50; 10; 5; 1; 0.5;
0.1 M
 40

Cho 100l dung dch DPPH nng 6mM vo 2800l metanol, sau b
sung 100l mu th (thc hin ln lt cho tng nng ).

Dung dch c lc u, thc hin phn ng iu kin nhit phng

trong thi gian 30 pht, ri em mu o hp thu bc sng = 517nm.

Mi nng mu c th nghim lp 2 ln tnh gi tr trung bnh

- Thc hin vi mu i chiu v mu trng:

Ho tan ascorbic acid (vitamine C) trong DMSO 8 nng khc nhau 500;
100; 50; 10; 5; 1; 0.5; 0.1 M

Mu i chiu (ascorbic acid): Cho 100l dung dch DPPH nng

6mM vo 2800l metanol, sau b sung 100l dung dch ascorbic acid
Mu trng: Cho 100l DMSO vo 2900l metanol.
C hai dung dch c lc u, thc hin phn ng iu kin nhit
phng trong thi gian 30 pht, ri em mu o hp thu bc sng =
517 nm.

Mi nng cht i chiu c th nghim lp 2 ln ly gi tr trung bnh.

Tnh kt qu:

Phn trm bt gc t do DPPH ca cht nghin cu tnh theo cng thc:

Q (%) = 1
( A Ac ) 100

( A 0 A )
c

Trong : A l hp thu ca dung dch cha mu th

A0 l hp thu ca DPPH khi khng c mu

Ac l hp thu ca dung dch cha cht i chiu

 41

%Q Phn trm bt gc t do DPPH

Hot tnh khng oxy ha tnh theo tr s IC50 c suy ra t th ca nng

C v %Q.

2.2.4.2 nh gi hot tnh khng ung th

(Thc hin ti Phng Sinh hc Thc nghim, Vin Ho hc cc Hp cht Thin
Nhin, Vin Khoa hc v Cng ngh Vit Nam)
Phng php thc hin

Theo phng php ca Skehan & CS (1990) v Likhiwitayawuid & CS

(1993) hin ang c p dng ti Vin nghin cu ung th Quc gia ca M
(NCI) v trng i hc Dc, i hc Tng hp Illinois, Chicago, M.

Cc dng t bo th nghim:

- Hep-G2 (Heptatocellular carcinoma ung th gan).

- Lu (Lung cancer ung th phi).

- RD (Rhabdosarcoma - ung th mng tim ).

Mi trng nui cy t bo: DMEM (Dulbeccos Modified Eagle Medium) hoc

MEME (Minimum Essential Medium with Eagles salt). C b xung L- glutamine,
Sodium piruvat, NaHCO3, PSF (Penixillin- Streptomycin sulfate- Fungizone);
NAA (Non-Essential Amino Acids); 10% BCS (Bovine Calf Serum), Tripsin-
EDTA 0,05%; DMSO (Dimethyl Sulfoside); TCA (Trichloro Acetic acid); Tris
Base; PBS (Phosphate Buffered Saline); SRB (Sulfo Rhodamine B); Acid Acetic.

Cc dng c dng 1 ln: Bnh nui cy t bo, phin vi lng 96 ging, pipet
pasteur, cc u tup cho micropipet

Cht chun chng dng tnh:

Dng cht chun c kh nng dit t bo: Ellipithine, Vinblastine hoc Taxol pha
trong DMSO.

- c trn my ELISA bc sng 495-515nm

 42

Mt quang c o trn my ELISA bc sng 495-515 nm.

Tnh kt qu:

Gi tr CS: l kh nng sng st ca t bo nng no ca cht th

tnh theo % so vi i chng. Da trn kt qu o c ca chng OD (ngy 0),
DMSO 10% v so snh vi gi tr OD khi trn mu tm gi tr CS(%) theo cng
thc:

OD (mu) OD (ngy 0)

CS% = x 100

OD (DMSO) OD (ngy 0)

Gi tr CS% sau khi tnh theo cng thc trn, c a vo tnh ton Excel
tm ra % trung bnh lch tiu chun ca php th c lp li 3 ln theo
cng thc ca Ducan nh sau: lch tiu chun

(xi - x )2

n-1

Cc mu c biu hin hot tnh (CS < 50%) s c chn ra th nghim tip
tm gi tr IC50

Gi tr IC50 : dng gi tr CS ca 10 thang nng , da vo chng trnh

Table curve theo thang ga tr logarit ca ng cong pht trin t bo v nng
cht th tnh gi tr IC50. Cng thc: 1/y=a+blnX

Trong Y: nng cht th; X: Gi tr CS (%).

 43

CHNG 3

KT QU V THO LUN
3.1 Phn lp cur
3.1.1 Kt tinh curcuminoid
Bng 3.1: Kt qu kt tinh bt curcuminoid

Lng dung mi
Khi lng Khi lng Hiu sut (%)
Ln kt (ml)
STT trc kt tinh sau kt tinh
tinh Tng
CH3OH H2O (g) (g) Ring
cng

1 45 8 0,5001 0,3402 68,02

1 2 34 9 0,3420 0,2623 76,69

40,37
3 25 5 0,2623 0,2019 76,97

1 50 10 0,5012 0,3321 66,26

4
2 2 36 8 0,3321 0,2501 75,30
42,57
3 27 6 0.2501 0,2134 85,32

1 48 8 0.5026 0,3503 69,69

3 2 33 8 0,3503 0,2712 77,41 44,68

3 25 7 0,2712 0,2246 82,81

 44

Hnh 3.1: TLC kim tra hn hp curcuminoid sau cc ln kt tinh

Ch thch
M: mu cha kt tinh I: kt tinh ln 1 II: kt tinh ln 2 III: kt tinh ln 3

1: Cur 2: DMC 3: BDMC 4: Nha

Nhn xt:

Qua kt qu bng nh lng cho qu trnh kt tinh cho thy hiu sut ca
tng ln kt tinh tng i cao vo khong 68-84%. Nhng hiu sut cui cng qua
3 ln kt tinh li khng cao vo khong 40-44%.
Qua kho st TLC h dung mi diclometan:metanol (98:2) cho thy nh qu
trnh kt tinh m ta loi b c phn ln nha, DMC, BDMC gp phn lm
tng ng k hm lng cur.
Vy t 1,539g bt curcuminoid thng phm qua 3 ln kt tinh thu c
0,6399g cur th v hiu sut trung bnh c 3 ln kt tinh l 42,54%.

3.1.2 Sc k ct phn lp cur

- Khi lng mu np ct m0 = 1 g
 45

- Dung mi ra gii diclometan:eter du ha (90:10)

Lng silica gel np vo ct m = 50g, chiu cao tng ng ca silica gel
trong ct l 19cm.
ng knh trong ca ct: 3cm

a) b)

Hnh 3.2: Sc k ct phn lp cur Hnh 3.3: TLC phn on tinh t SKC
a) Ngoi nh sng 1: Hn hp curcumonoid ban u

b) Di n UV 365 nm 2: Curcumin tinh thu c qua sc k ct

Khi lng curcumin tinh thu c m1 = 0,6108 g
Hiu sut tch ct:

m1 0 . 6108
H = x 100 % = x 100 % = 61 . 08 %
m0 1

Nhn xt:

Hiu sut tch ct tng i cao: 61,08 %.

Kt qu kim tra nh tnh cur thu c bng TLC h diclometan:etyl acetat
(96:4) cho vt trn, r cho thy tinh khit ca sn phm tch c l kh cao.
 46

3.2. Tng hp dn xut 3,5-Difluorophenylhydrazinocurcumin

3.2.1 Theo di phn ng

Hnh 3.4a: Phn ng 3,5-DFPHC

Duy tr n nh cc iu kin phn ng v kim tra im dng phn ng

bng TLC. Quan st qu trnh phn ng cho thy, trong thi gian 14 gi u phn
ng xy ra tng i chm, d on c xut hin sn phm ngay trn u cur nhng
vt rt m. Phn ng bt u xy ra nhanh dn t gi 16 n gi th 20, khong
thi gian ny cho thy sn phm m ln rt r.
Tuy nhin sau 21gi theo quan st TLC cho thy sn phm khng m thm
m bt u thy sn phm ph sinh ra trn u sn phm v pha di cur. Ti thi
im 21gi nn dng phn ng v tin hnh SKC phn lp sn phm.
Phng trnh phn ng:
 47

Hnh 3.4b Phn ng tng hp 3,5-DFPHC

Theo di phn ng bng TLC h dung mi diclometan:etyl acetat (96:4)

1 2

16gi 21gi

1: Thi gian phn ng 16gi 2: Thi gian phn ng 21gi

Hnh 3.5. Theo di bn mng bng TLC

 48

3.2.2 Sc k ct phn lp sn phm

Hnh 3.6: Sc k ct th Hnh 3.7: Sc k ct tinh

Khi lng cur tinh tham gia phn ng: mcur = 200mg
Khi lng 3,5-DFPHC tinh tnh c theo phng trnh phn ng
M 3,5 DFPHC 476
mtinh = x mCur = x 200 = 259 mg
M Cur 368

Hiu sut th ca qu trnh phn lp 3,5-DFPHC

m0 125
H% = x100% = x100% = 46,3%
mhh 270

Hiu sut SKC tinh

m1 25
H% = x100 % = x100 = 20 %
m0 125
 49

Hiu sut ca ton b qu trnh phn lp sn phm

m1 25
H% = x100% = x100% = 9,65%
mtinh 259

Vi: m0: Khi lng 3,5-DFPHC t SKC th.

m1: Khi lng 3,5-DFPHC t SKC tinh
mhh: Khi lng hn hp sau tng hp

mtinh: Khi lng 3,5-DFPHC tinh tnh theo l thuyt.

Nhn xt:
T 300mg nguyn liu ban u bao gm cur v 3,5-DFPH qua qu trnh SKC
thu c 125mg dn xut th vi hiu sut t 46,3%, 25mg khi lng tinh vi
hiu sut 20% v hiu sut ton b qu trnh 9,65%.

3.2.3 Nhn danh cu trc

3.2.3.1 Tnh cht vt l c trng

- TLC h dung mi diclometan:etyl acetat (96:4)

II I m 1 2 1 2
a b c d

Hnh 3.8 TLC ca 3,5-DFPHC

b. Di n UV a. SKC th c. Di hi Iod d. Dng tinh th
I: phn on 2; II: phn on 3; m: mu; 1: Cur; 2: DFPHC
 50

Ph UV-Vis ca 3,5-DFPHC trong dung mi CH2Cl2

Hnh 3.9: Ph UV-Vis ca 3,5-DFPHC trong dung mi CH2Cl2

Bng 3.2: Mt s tnh cht ca 3,5-DFPHC

Tnh cht 3,5-DFPHC

Hnh dng Tinh th v nh hnh, mu vng nht

max UV-Vis (CH2Cl2) 379

Nhit nng chy(oC) 191-203oC

Rf TLC (diclometan:etyl acetate=96:4)

0,59

3.2.3.2 Bin lun cu trc ca 3,5-DFPHC (ph lc 4, 5, 6, 7, 8, 9)

Ph MS chy ch Positive cho mi m/z [M+H]+ = 477,07 cho bit khi lng
phn t ca 3,5-DFPHC l 476 (.v.C) tng ng vi cng thc phn t l
C27H23N2FO4. (M=476).
 51

Hnh 3.10: Cng thc phn t ca 3,5-DFPHC, C27H22N2F2O4 (M=477,04)

Bng 3.3: D liu ph NMR ca 3,5-DFPHC trong dung mi CDCl3
13 1
V tr C (ppm) H (ppm)

1 101,74 6,797 (s,1H)

2 142,80 -

2 152,15 -

3 117,71 7,008 (d, Jtrans = 16,5Hz, 1H)

3 112,64 6,745 (d, Jtrans = 16,5Hz, 1H)

4 131,48 7,146 (d, Jtrans = 16,5Hz, 1H)

4 133,55 7,098 (d, Jtrans = 16,5Hz, 1H)

5 129.52 -

5 128,71 -

6 108.13 7,087 (s, Jmeta = 2Hz, 1H)

6 108,71 6,935 (s, Jmeta =2Hz, 1H)

7 146,85 --

7 146,81 --

8 146,60 --

8 145,99 --
 52

7-OCH3 56,03 3,936 (s,3H)

7-OCH3 56,16 3,925 (s,3H)

8-OH -- 5,772 (s,1H)

8-OH -- 5,712 (s,1H)

9 114,83 6,935 (d, Jorto = 8Hz, 1H)

9 114,57 6,919 (d, Jorto = 8Hz, 1H)

10 120,95 7,008 (d, Jorto = 8,5Hz, 1H)

10 120,87 7,017 (d, Jorto = 8Hz, 1H)

a 141,65 -

b
108,29+108,13+108,07 m (7,146+7,113)
b

c
164,18+164,08+162,10 -
c

d 102,99 -

(s: singlet, d: doublet, dd: doublet doublet, m:multiple).

Nhn xt: Theo kt qu phn tch cu trc ho hc trn kt hp vi ph DEPT (ph
lc 6a, 6b), HMBC (ph lc 7a, 7b, 7c), HSQC (ph lc 8a, 8b), c th khng nh
tng hp thnh cng dn xut 3,5-DFPHC.

3.3 Tng hp dn xut 4-Fluorophenylhidrazinocurcumin

3.3.1 Theo di phn ng
 53

Hnh 3.11a: Phn ng tng hp 4-FPHC

Duy tr n nh cc iu kin phn ng v kim tra im dng phn ng
TLC. Quan st qu trnh phn ng cho thy, trong thi gian 40 gi u phn ng
xy ra tng i chm, mc du theo d on c xut hin sn phm chng trn u
cur nhng vt rt m. Phn ng bt u xy ra nhanh dn t ting th 55 n ting
62, khong thi gian ny cho thy sn phm m ln rt r.
Tuy nhin sau 66 gi theo quan st TLC cho thy sn phm khng m thm
ln m bt u thy sn phm ph sinh ra trn u sn phm v pha di cur. Mc
d vt cur vn cn m nhng nht hn so vi cur ban u nhiu. V vy ngay thi
im 66 gi c th dng phn ng v tin hnh SKC phn lp sn phm.

Hnh 3.11.b. Phng trnh phn ng tng hp 4-FPHC

Theo di phn ng bng TLC h dung mi diclometan:etyl acetat (96:4)
 54

1 2 3

Hnh 3.12: TLC ca tng hp 4-FPHC

1:Thi gian phn ng 29h 2: Thi gian phn ng 35h 3: Thi gian phn ng 65h

3.3.2 Sc k ct phn lp sn phm

Hnh 3.13: SKC th Hnh 3.14: SKC tinh

Khi lng Cur tinh tham gia phn ng: mcur = 150mg.
Khi lng 4-FPHC tinh tnh c theo phng trnh phn ng.
 55

M 4 FPHC 458
m tinh = xmcur = x150 = 187mg
M cur 368

Hiu sut th ca qu trnh phn lp 4-FPHC

m0 115
H % = x 100 % = x 100 % = 42 ,14 %
m hh 280

Hiu sut ca qu trnh phn lp sn phm t ct tinh

m1 39
H% = x100 % = x100 % = 34 %
m0 115

Hiu sut ca sc k iu ch
m2 18
H% = x100% = x100% = 46%
m1 39

Hiu sut ca ton b qu trnh tng hp v phn lp

m1 18
H% = x100% = x100% = 9,62%
mhh 187

Vi: m0: Khi lng 4-FPHC t SKC th.

m1: Khi lng 4-FPHC t SKC tinh

mhh: Khi lng hn hp sau tng hp

m2: Khi lng 4-FPHC t SKC
mtinh: Khi lng 4-FPHC tinh tnh theo l thuyt.
Nhn xt: T 299mg nguyn liu ban u bao gm cur v 4-FPH qua qu trnh
SKC th thu c 115mg 4-FPHC hiu sut t 42,14%, SKC tinh thu c 39mg
tng ng hiu sut t 34%, sc k iu ch thu c 18mg tinh khit t hiu
sut 9,62%.

3.3.3 Nhn danh cu trc

3.3.3.1. Tnh cht vt l c trng

TLC h dung mi diclometan:etyl acetate (96:4).
 56

m 2 3 1 2 1 2

a) b) c)

Hnh 3.15. TLC ca 4-FPHC

a. Hin mu = 365nm; m: mu sau phn ng; 2,3: Tng ng phn on 2 v 3

b. Hin mu di n UV ( 365nm): 1: Cur, 2: 4-FPHC tinh

c. Hin mu di hi Iod; 1:Cur; 2: 4-FPHC.

Ph UV-Vis ca 4-FPHC

Hnh 3.16. Ph UV-Vis ca 4-FPHC trong dung mi CH2Cl2

 57

Bng 3.4: Mt s tnh cht ca 4-FPHC

Tnh cht 4-FPHC

Hnh dng Tinh th v nh hnh, mu ng vng

max UV-Vis (CH2Cl2) 367

Nhit nng chy(oC) 101,4oC

RfTLC (diclometan:etyl acetate = 96:4) 0,34

3.3.3.2 Bin lun cu trc ca 4-FPHC (ph lc 1,2,3)

Ph MS chy ch Positive cho mi m/z[M+H]+ = 457,17 cho bit khi
lng phn t ca 4-FPHC l 458 (.v.C), tng ng vi cng thc phn t l
C27H23N2FO4 (M=476).

Hnh 3.17. Cu trc ca 4-FPHC, C27H23N2FO4 (M=458 .v.C)

 58

Bng 3.5. D liu ph NMR ca 4-FPHC

13 1
V tr C (ppm) H (ppm)

1 100,65 6,795 (s,1H)

2 151,63 -

2 142,86 -

3 118,34 7,026 (d, Jtrans = 16,5Hz, 1H)

3 113,23 6,654 (d, Jtrans =16Hz, 1H)

4 130,90 7,138 (d, Jtrans = 16Hz, 1H)

4 132,87 7,063 (d, Jtrans =16Hz, 1H)

5 129,93 -

5 129,13 -

6 108,20 7,082 (s, Jmeta = 1,5Hz, 1H)

6 108,95 6,889 (s, Jmeta =1Hz, 1H)

7 146,97 --

7 146,59 --

8 146,02 --

8 142,86 --

7-OCH3 56,05 3,927 (s, 3H)

7-OCH3 56.16 3,903 (s, 3H)

8-OH -- 5,705 (s,1H)

8-OH -- 5,753 (s, 1H)

9 114,96 6,912 (d, Jorto = 8Hz, 1H

9 114,72 6,903 (d, Jorto = 8Hz, 1H)

10 120,96 6,972 (d, Jorto = 8Hz, 1H)

 59

10 120,77 7.014 (d, Jorto = 8,5Hz, 1H)

a 135,9

b 127,33
m (7,519+7,509+7,491)
b 127,26

c 116,35
m (7,212+7,207+7,182+7,177)
c 116,17

d 163,13

(s: singlet, d: doublet, dd: doublet doublet, m:multiple)

Nhn xt: Theo kt qu phn tch cu trc ho hc trn, c th khng nh: tng
hp thnh cng dn xut 4-FPHC tinh khit.

3.4. Kt qu kho st hot tnh sinh hc

3.4.1 nh gi hot tnh khng oxy ha theo phng php DPPH (Ph
lc 10)

110
100 Cur
90 DFPHC
80 FPHC
70
Vitamine C
% Q

60
50
40
30
20
10
0
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0
logC

Hnh 3.18: Hot tnh khng oxy ha ca Cur, 3,5-DFPHC, 4-FPHC v Vitamin C
theo phng php DPPH
 60

Bng 3.6: Gi tr IC50 trong th nghim hot tnh khng oxy ho DPPH

STT Mu th hot tnh IC50 (M)

1 Cur 18,5 0,6

2 3,5-DFPHC 48,7 4,4

3 4-FPHC 15,6 0,4

Mu i chiu Ascorbic acid 64,3 1,0

Nhn xt: Qua kt qu kho st hot tnh khng oxy ha c sp xp theo

th t gim dn sau 4-FPHC > Cur > 3,5-DFPHC > vitamin C.

3.4.2 Hot tnh khng t bo gy c.

Hnh 3.19: th tnh gi tr IC50 ca mu

 61

Bng 3.7: Kt qu xc nh gi tr IC50 trong kho st hot tnh khng ung th ca

3,5-DFPHC v 4-FPHC vi 3 dng t bo Hep-G2, Lu v RD

Dng t bo

ST K hiu Gi tr IC50 (g/ml)

Kt lun
T mu Hep-G2 Lu RD

1 Chng (+) 0,31 0,42 0,22 Dng tnh

3,5 - Dng tnh vi 2 dng

2 4,75 >5 3,35
DFPHC Hep-G2 v RD

4 4 - FDHC >5 >5 3,41 Dng tnh vi dng RD

Nhn xt: Qua gi tr IC50 trong kho st hot tnh khng ung th ca 3,5-DFPHC
v 4-FPHC cho thy u th hin hot tnh gy c i vi t bo ung th RD,
khng gy c i vi t bo LU. Ngoi ra 3,5-DFPHC cn th hin hot tnh gy
c vi dng t bo ung th Hep-G2. Tuy nhin hot tnh gy c t bo ung th
ca 3,5-DFPHC v 4-FPHC khng cao hn so vi cht chng.
 62

KT LUN V KIN NGH

Kt lun

Lun vn t c nhng kt qu sau:

1. Phn lp c cur tinh t hn hp curcuminoid 3 thnh phn.

2. Tng hp thnh cng 4-Fluorophenylhydrazincurcumin (4-FPHC) v 3,5-Di-
fluorophenylhydrazincurcumin (3,5-DFPHC). Xc nh cu trc v mt s tnh cht
vt l ca chng.
3. Nghin cu, so snh hot tnh sinh hc ca cur, 4-FPHC, 3,5-DFPHC. Cc
dn xut 3,5-DFPHC v 4-FPHC u th hin c tnh i vi dng t bo ung th
Hep-G2, LU, RD. Th hin hot tnh khng oxy ha, th t c th sp xp gim dn
4-FPHC >Cur >3,5-DFPHC >Vitamin C.
Theo cc nghin cu trong nc cng b v vic to dn xut imin t cur,
ch c nhm tc gi o Hng Cng, L Hi Li (i hc Bch Khoa Nng)
nghin cu tng hp phenylhydrazinocurcumin, isoxazolcurcumin, tc gi L Xun
Tin (i hc Bch Khoa Tp H Ch Minh) nghin cu quy trnh tng hp dn xut
hydrazinocurcumin, isoxazolcurcumin. Lun vn ny gp phn cung cp thm
thng tin v xy dng quy trnh tng hp dn xut ca 4-FPHC v 3,5-DFPHC v
nghin cu hot tnh sinh hc ca chng, lm phong ph thm cc cng trnh
nghin cu v vic tng hp dn xut imin ca cur trn th gii cng nh trong
nc.

Kin ngh

1. Tip tc th nghim hot tnh sinh hc ca 3,5-DFPHC v 4-FPHC trn cc

chng t bo ung th khc nhau.
2. Tip tc tng hp cc dn xut imin ca cur vi cc cc hp cht d vng
khc.
 TI LIU THAM KHO

PHN TING VIT

[1]. o Hng Cng, L Hi Li (2006), Nghin cu phn ng amin ho -

dixeton ca curcumin, Ho hc v ng dng, 2, Trang 35-38.

[2]. ng Th M L, Th Xun Vui (2009), iu ch v kho st hot tnh

sinh hc ca cc dn xut imine 2-hydrazinobenzothiazolcurcumin v 2,4-
difluorophenylhydrazinocurcumin t curcumin, Lun vn tt nghip i hc,
Trng ai hoc Nng Lm Tp. HCM, Trang 22-31

[3]. L Xun Tin (2008), Nghin cu tng hp Hyrdrazinocurcumin v

Isoxaxolcurcumin Kho st hot tnh sinh hc ca chng, Lun vn thc s,
Trng ai hc Bch Khoa Tp. HCM, Trang 20-55.

[4]. Phan Thanh Sn Nam, Trn Th Vit Hoa (2007), Ho hu c, NXB i hc

Quc Gia Tp. HCM, Chng 13.

[5]. Thi Don Tnh (2005), C s ho hc hu c, Tp 2, NXB Khoa hc v K

thut, H Ni, Trang 62-85.

[6]. Trn Thanh V (2009), Tng hp v xc nh hot tnh sinh hc ca dn xut

phenylhydrazinocurcumin, ti tt nghip i hc, Trng ai hc Kinh t
Tp. HCM, Trang 24-35.
PHN TING ANH

[7]. Aggarwal B.B., Kumar A., Aggarwal M.S., Shishodia S (2005), Curcumin
derived from turmeric (Curcuma longa), A spice for all seasons, CRC Press
LLC.

[8]. Ajaikumar B.,Kunnumakkara Preetha Anand., Robert A. Newman., Bharat B.

Aggarwal ( 2007), Bioavailability of curcumin, problems and promises, Mol
Pharm.

[9]. Bharat B. Aggarwal., et al (2006), Curcumin - Biological and Medicinal

Properties.

[10]. Chattopadhyay I., Biswas K., Bandyopadhyay U., Banerjee R. K (2004),

Turmeric and curcumin: Biological actions and medicinal applications,
Current Science, 87.

[11]. Goel Ajay, Aggarwa A.B.K Bharat B (2007), Curcumin as Curecumin:

From kitchen to clinic.

[12]. Ishita Chattopadhyay., et al ( 2004), Turmeric and curcumin: Biological

actions and medicinal applications

[13]. Mishra S., Karmodiya K., Surolia N., Surolia A (2008), Synthesis and
exploration of novel curcumin analogues as anti-malarial agents, Bioorganic
& Medicinal Chemistry, 16, pp. 2894-2902

[14]. Phan T., See P., Lee S., Chan SY (2001), Protective effects of curcumin
against oxidative damage on skin cells in vitro, Its implication for wound
healing, J-Traum, 51, pp. 927-931.
[15]. Reddy A.P., Lokesh B.R (1992), Studies on spice principles as antioxidants
in the inhibition of lipid peroxidation of rat liver microsomes, Molecular and
Cellular Biochemistry, 111, pp. 117-124.

[16].Runothayanun P., Wirachwong P (2005), Development of

tetrahydrocurcuminoid liposomes as an ingredient for cosmetic products, 7th
ASCS conference Bangkok, Thailand.

[17]. Sabiri Dutta., Subhash Padhye K., Indira Priyadasini and Chris Newton
(2005), Antioxidant and antiproliferative activity of curcumin
semicarbazone, Bioorganic and Medicinal Chemistry Letters, 15, pp. 2738
2744.

[18]. Selvam C., Jachak S.M., Thilagavathi R., Chakraborti A.K (2005), Design,
synthesis, biological evaluation and molecular docking of curcumin analogues
as antioxidant, cyclooxygenase inhibitory and anti-inflammatory agents,
Bioorganic & Medicinal Chemistry Letter, 15, pp. 1793-1797.

[19]. Shen L., Ji H-F (2007), Theoretical study on physicochemical properties of

curcumin, Spectrochimica Acta Part A, Molecular and Biomolecular
Spectroscopy, 67, pp. 619-623

[20]. Shim J.S., et al (2002), Hydrazinocurcumin, a novel synthetic curcumin

derivative, is a potent inhibitor of endothelial cell proliferation, Bioorganic &
Medicinal Chemistry, 10, pp. 2987-2992
PH LC
Kt qu o hp thu bc sng 517nm (A) v phn trm bt gc t do (Q%)

1. i vi mu Vitamin C
A1(nm) A2(nm) Atb (nm)
Blank 1.835 1.821 1.828
Chun 0.123 0.088 0.1055

Nng A1 A2 %Q1 %Q2 %Qtb

(M)
500 0.123 0.088 98.984 101.02 100
100 0.401 0.357 82.845 85.399 84.122
50 1.294 1.353 31.001 27.576 29.289
10 1.95 2.001 -7.083 -10.04 -8.563
5 1.718 1.75 6.3861 4.5283 5.4572
1 1.723 1.699 6.0958 7.4891 6.7925
0.5 1.793 1.759 2.0319 4.0058 3.0189
0.1 1.777 1.824 2.9608 0.2322 1.5965

2. i vi mu curcumin

A1 (nm) A2 (nm) Atb (nm)

Blank 1.621 1.611 1.616
Chun 0.11 0.117 0.1135

Nng
(M) A1 A2 %Q1 %Q2 %Qtb
500 0.12 0.148 99.567 97.704 98.636
100 0.119 0.099 99.634 100.97 100.3
50 0.319 0.265 86.323 89.917 88.12
10 1.092 1.104 34.875 34.077 34.476
5 1.199 1.26 27.754 23.694 25.724
1 1.382 1.29 15.574 21.697 18.636
0.5 1.448 1.313 11.181 20.166 15.674
0.1 1.481 1.501 8.985 7.6539 8.3195
 3. i vi mu 3,5-DFPHC

A1 A2 Atb
Blank 2.35 2.325 2.3375
Chun 0.261 0.261 0.261

Nng (M) A1 A2 %Q1 %Q2 %Qtb

500 0.165 0.171 104.62 104.33 104.48
100 0.465 0.514 90.176 87.816 88.996
50 1.215 1.225 54.057 53.576 53.817
10 1.426 1.532 43.896 38.791 41.344
5 2.135 2.046 9.752 14.038 11.895
1 2 2.046 16.253 14.038 15.146
0.5 2.066 2.113 13.075 10.811 11.943
0.1 2.121 2.132 10.426 9.8965 10.161

4. i vi mu 4-FPHC

A1 A2 Atb
Blank 2.178 2.178 2.178
Chun 0.096 0.075 0.0855

Nng (M) A1 A2 %Q1 %Q2 %Qtb

500 0.144 0.146 97.204 97.109 97.157
100 0.14 0.129 97.395 97.921 97.658
50 0.131 0.227 97.826 93.238 95.532
10 1.428 1.399 35.842 37.228 36.535
5 1.529 1.692 31.016 23.226 27.121
1 2.007 1.787 8.172 18.686 13.429
0.5 1.709 1.833 22.413 16.487 19.45
0.1 1.78 1.892 19.02 13.668 16.344

Ph lc 10: Kt qu o hp thu bc sng 517nm (A) v phn trm bt gc t do

(Q%)