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B GIO DC V O TO

B Y T

TRNG I HC DC H NI

NGUYN TH PHNG

NGHIN CU

D NG QU TR NH

BN TNG HP TETRAHYDROCURCUMINOID
T NGUN CURCUMINOID CHIT XUT
TRONG NC
LUN VN THC S DC HC

H NI - 2014

B GIO DC V O TO

B Y T

TRNG I HC DC H NI

NGUYN TH PHNG

NGHIN CU

D NG QU TR NH

BN TNG HP TETRAHYDROCURCUMINOID
T NGUN CURCUMINOID CHIT XUT
TRONG NC
LUN VN THC S DC HC
CHUYN NGNH: CNG NGH DC PHM V BO CH THU C
M S : 60720402

Ngi hng dn khoa hc: PGS.TS. Nguyn nh Luyn

H NI 2014

LI CM N
Tri qua hai nm hc cao hc c s gip tn tnh ca cc thy gio, c
gio, gia nh cng bn b, ti hon thnh kha hc v Lun vn Thc s Dc
hc ca mnh.
Vi tt c s knh trng, trc tin ti xin by t lng bit n su sc n thy
gio PGS. TS. Nguyn nh Luyn trc tip hng dn, ch bo tn tnh v to
mi iu kin gip ti nghin cu v thc hin lun vn ny.
Ti cng xin gi li cm n chn thnh ti TS. Nguyn Vn Hi, ThS.
Nguyn Vn Giang, DS. Nguyn Th Vn v CN. Phan Tin Thnh gip v
to mi iu kin tt nht cho ti trong sut thi gian hc v thc hin Lun vn.
Ti cng xin c gi li cm n n cc thy c gio thuc b mn Cng
nghip Dc, cng nh cc thy c trong trng i hc Dc H Ni, cc thy c
ca Phng Sau i hc to iu kin thun li cho ti hon thnh lun vn tt
nghip ny v dy bo ti tn tnh trong sut hai nm hc.
Cui cng, ti xin gi li bit n su sc n gia nh ti, c bit l b m ti
v li cm n chn thnh n bn b ti, l ngun ng lc khng th thiu, lun
bn ti gip ti sut thi gian i hc v trong sut qu trnh thc hin ti
Lun vn Thc s Dc hc.
H Ni, ngy 20 thng 08 nm 2014
Hc vin

Nguyn Th Phng

MC LC
DANH MC CC K HIU, CC CH VIT TT
DANH MC CC BNG
DANH MC CC HNH V
T VN ............................................................................................................1
Chng 1: TNG QUAN .........................................................................................2
1.1. TNG QUAN V CURCUMINOID ..................................................................2
1.1.1. Cu trc ha hc v tnh cht ............................................................................2
1.1.2. Tc dng dc l...............................................................................................6
1.1.3. Phng php tch hn hp curcuminoid ........................................................10
1.2. TNG QUAN V TETRAHYDROCURCUMINOID .....................................11
1.2.1. Cu trc ha hc v tnh cht ..........................................................................11
1.2.2. Ngun gc tetrahydrocurcumin ......................................................................12
1.2.3. Tc dng dc l.............................................................................................12
1.2.4. Tng quan v cc phng php bn tng hp tetrahydrocurcuminoid ..........14
Chng 2: I TNG V PHNG PHP NGHIN CU .......................18
2.1. HA CHT V TRANG THIT B ................................................................18
2.1.1. Nguyn liu v ha cht nghin cu ...............................................................18
2.1.2. Thit b, my mc v dng c nghin cu ......................................................19
2.2. NI DUNG NGHIN CU ..............................................................................20
2.3. PHNG PHP NGHIN CU......................................................................20
2.3.1. Phng php tch tng thnh phn trong hn hp curcuminoid ....................20
2.3.2. Bn tng hp ha hc: ....................................................................................20
2.3.3. Kim tra tinh khit .....................................................................................21
2.3.4. Xc nh cu trc sn phm ............................................................................21
2.3.5. Th tc dng chng oxy ha ca cc cht bn tng hp c ......................21

Chng 3: KT QU NGHIN CU ..................................................................22


3.1. TCH CC CURCUMIN I, II, III T HN HP CURCUMINOID .............22
3.2. KH HA TNG CURCUMINOID THNH PHN ....................................26
3.2.1. Kh ha curcumin I (curcumin) ......................................................................26
3.2.2. Kh ha curcumin II (DMC) ..........................................................................34
3.3. KH HA HN HP CURCUMINOID .........................................................41
3.4. XC NH CU TRC ...................................................................................46
3.4.1. Xc nh cu trc curcumin I (curcumin) .......................................................46
3.4.2. Xc nh cu trc curcumin II (DMC) ............................................................48
3.4.3. Xc nh cu trc THC I (THC) .....................................................................50
3.4.4. Xc nh cu trc THC II ................................................................................53
3.5. TH TC DNG CH NG OXY HA CA CC CHT BN TNG HP
C .......................................................................................................................54
Chng 4: BN LUN ...........................................................................................56
4.1. V phng php tch tng thnh phn t hn hp curcuminoid.......................56
4.2.V phn ng kh ha tng curcuminoid thnh phn ..........................................56
4.3. V phn ng kh ha curcuminoid hn hp ......................................................58
4.4. V xc nh cu trc ..........................................................................................59
4.5. V tc dng sinh hc ..........................................................................................63
KT LUN V KIN NGH ................................................................................64
1. Kt lun . 64
2. Kin ngh ..64

TI LIU THAM KHO

DANH MC CC K HIU, CC CH VIT TT

AcOH

Acid acetic

AOM

Azoxymethan

APPH

2,2-azobis(2-amidinopropan)dihydroclorid

BDMC

Bisdemethoxycurcumin

13

13

C-NMR

C - Nuclear Magnetic Resonance spectroscopy

(Ph cng hng t ht nhn cacbon 13C)


CTCT

Cng thc cu to

CTPT

Cng thc phn t

DMC

Demethoxycurcumin

DMF

Dimethylformamid

DMSO

Dimethylsulfoxid

DPPH

1,1-diphenyl-2-picrylhydrazyl

vC

n v carbon

EtOH

Ethanol

Gi

HHC

Hexahydrocurcumin

HIV

Virus gy suy gim min dch ngi


(Human immunodeficiency virus)

H-NMR

H - Nuclear Magnetic Resonance spectroscopy

Ph cng hng t ht nhn proton


HPLC

Sc k lng hiu nng cao


(High-performance liquid chromatography)

Hp

Hiu sut phn ng

EC50

Nng ti c tc dng vi 50% i tng th


(Effective concentration at 50%)

IR

Ph hng ngoi (Infrared spectroscopy)

KL, m

Khi lng

LDL

Lipoprotein t trng thp (Low density lipoprotein)

MeOH

Methanol

MS

Ph khi lng (Mass spectrometry)

OD

Mt quang (Optical density)

OHC

Octahydrocurcumin

p sut

Rf

H s lu gi (Retension factor)

SKLM

Sc k lp mng

sp, SP

Sn phm

TBA

Acid 2-thiobarbituric

THC

Tetrahydrocurcumin

Tonc

Nhit nng chy

Thi gian

TLTK

Ti liu tham kho

Th tch

xt

Xc tc

DANH MC CC BNG
Bng 1.1. Mt s tnh cht vt l ca cc curcuminoid ..............................................2
Bng 1.2. Mt s tnh cht vt l ca cc tetrahydrocurcuminoid............................12
Bng 2.1. Nguyn liu v ha cht nghin cu.........................................................18
Bng 2.2. Thit b, my mc v dng c nghin cu ...............................................19
Bng 3.1. Kt qu kho st nh hng ca dung mi, nhit v thi gian kt tinh
n qu trnh kt tinh curcuminoid ............................................................23
Bng 3.2. Kt qu kt tinh curcuminoid 3 ln trong EtOH 96% ..............................24
Bng 3.3. Kt qu tch cc curcuminoid thnh phn t hn hp curcuminoid bng
sc k ct silica gel vi h dung mi dicloromethan: methanol = 20:1 ....25
Bng 3.4. Kt qu kho st nh hng ca dung mi n hiu sut phn ng bn
tng hp tetrahydrocurcumin t curcumin vi tc nhn kh l H2/Pd/C ..27
Bng 3.5. Kt qu kho st nh hng ca t l xc tc n thi gian v hiu sut
phn ng bn tng hp THC t curcumin vi tc nhn kh l H2/Pd/C ..28
Bng 3.6. Kt qu kho st nh hng ca acid n hiu sut phn ng bn tng
hp tetrahydrocurcumin t curcumin vi tc nhn kh l Zn/acid ...........30
Bng 3.7. Kt qu kho st nh hng ca nng acid n hiu sut phn ng bn
tng hp tetrahydrocurcumin t curcumin vi tc nhn kh l Zn/HCl ...31
Bng 3.8. Kt qu kho st nh hng ca t l mol km/curcumin n hiu sut
phn ng bn tng hp THC t curcumin vi tc nhn kh l Zn/HCl ....31
Bng 3.9. Kt qu kho st nh hng ca dung mi n hiu sut phn ng bn
tng hp THC II t DMC vi tc nhn kh l H2/Pd/C ............................35
Bng 3.10. Kt qu kho st nh hng ca t l xc tc n thi gian v hiu sut
phn ng bn tng hp THC II t DMC vi tc nhn kh l H2/Pd/C ....36
Bng 3.11. Kt qu kho st nh hng ca acid n hiu sut phn ng bn tng
hp THC II t DMC vi tc nhn kh l Zn/acid .....................................38
Bng 3.12. Kt qu kho st nh hng ca nng acid n hiu sut phn ng
bn tng hp THC II t DMC vi tc nhn kh l Zn/HCl ......................39

Bng 3.13. Kt qu kho st nh hng ca t l mol km/curcumin II n hiu sut


phn ng bn tng hp THC II t Curcumin II vi tc nhn kh l Zn/HCl
....................................................................................................................40
Bng 3.14. Kt qu tch cc tetrahydrocurcuminoid t hn hp sn phm bng sc
k ct silica gel vi h dung mi dicloromethan: methanol = 20:1 ..........43
Bng 3.15. Kt qu tch cc tetrahydrocurcuminoid t hn hp sn phm bng sc
k ct silica gel vi h dung mi n-hexan: ethyl acetat.............................45
Bng 3.16. Kt qu phn tch ph ESI-MS (MeOH) ca curcumin .........................47
Bng 3.17. Kt qu phn tch ph 1H-NMR (500 MHz, DMSO-d6) ca curcumin .47
Bng 3.18. Kt qu phn tch ph 13C-NMR (125 MHz, DMSO-d6) ca curcumin
...................................................................................................................................48
Bng 3.19. Kt qu phn tch ph ESI-MS (MeOH) ca DMC ...............................48
Bng 3.20. Kt qu phn tch ph 1H-NMR (500 MHz, CDCl3 & MeOD) ca DMC
...................................................................................................................................49
Bng 3.21. Kt qu phn tch ph 13C-NMR (125 MHz, CDCl3 & MeOD) ca DMC
...................................................................................................................................49
Bng 3.22. Kt qu phn tch ph IR (KBr) ca THC (kh thnh phn) .................50
Bng 3.23. Kt qu phn tch ph ESI-MS (MeOH) ca THC (kh thnh phn) ....51
Bng 3.24. Kt qu phn tch ph 1H-NMR (500 MHz, DMSO-d6) ca THC (kh
thnh phn).................................................................................................51
Bng 3.25. Kt qu phn tch ph ESI-MS (MeOH) ca THC (tch t kh hn hp)
...................................................................................................................................52
Bng 3.26. Kt qu phn tch ph 1H-NMR (500 MHz, DMSO-d6) ca THC (tch
t kh hn hp)..........................................................................................52
Bng 3.27. Kt qu phn tch ph ESI-MS (MeOH) ca THC II .............................53
Bng 3.28. Kt qu phn tch ph 1H-NMR (500 MHz, CDCl3) ca THC II ..........53
Bng 3.29. Kt qu nh gi tc dng dn gc t do ca cc mu th theo phng
php DPPH ................................................................................................55

DANH MC CC H NH V
Hnh 1.1. Cng thc cu to ca curcuminoid ............................................................2
Hnh 1.2. Phn ng amin ha -diceton ca curcumin ...............................................3
Hnh 1.3. Dng h bin ceto enol ca curcumin trong dung dch ...........................3
Hnh 1.4. Dng tn ti ca curcumin theo pH dung dch............................................4
Hnh1.5. S phn hy curcumin trong mi trng kim ............................................5
Hnh 1.6. Cng thc cu to ca tetrahydrocurcuminoid .........................................11
Hnh 1.7. S phn ng kh ha curcumin to thnh tetrahydrocurcumin,
hexahydrocurcumin, octahydrocurcumin dng xt Pd/C ............................15
Hnh 1.8. S phn ng kh ha curcumin to thnh tetrahydrocurcumin,
hexahydrocurcumin, octahydrocurcumin dng xt Pt/C .............................15
Hnh 1.9. S phn ng kh ha cc curcuminoid to thnh cc
tetrahydrocurcuminoid tng ng dng xc tc Zn-NiCl2 s dng k thut
siu m trong dung mi EtOH-H2O ...........................................................16
Hnh 3.1. S tch tng thnh phn trong hn hp curcuminoid ..........................22
Hnh 3.2. SKLM ca cc curcuminoid thnh phn ...................................................26
Hnh 3.3. S phn ng kh ha curcumin vi tc nhn H2/Pd/C ........................26
Hnh 3.4. S phn ng kh ha curcumin vi tc nhn Zn/H+ ............................29
Hnh 3.5. SKLM ca THC trn h dung mi CH2Cl2: MeOH = 20:1 ......................33
Hnh 3.6. SKLM ca THC trn h dung mi AcOEt: n-hexan = 7:3 .......................33
Hnh 3.7. S phn ng kh ha DMC vi tc nhn H2/Pd/C ..............................34
Hnh 3.8. S phn ng kh ha DMC vi tc nhn Zn/H+ ..................................37
Hnh 3.9. SKLM ca THC II trn h dung mi CH2Cl2: MeOH = 20:1 ..................41
Hnh 3.10. S phn ng kh ha curcuminoid vi tc nhn H2/Pd/C ................42
Hnh 3.11. S phn ng kh ha curcuminoid vi tc nhn Zn/H+.....................43
Hnh 3.12. SKLM ca THC trn h dung mi CH2Cl2: MeOH = 20:1 ....................46
Hnh 3.13. SKLM ca THC II trn h dung miCH2Cl2: MeOH = 20:1..................46
Hnh 3.14. Cu trc enol v lin kt hydro ca phn t curcumin I .........................60
Hnh 3.15. Dng h bin ceto-enol ca tetrahydrocurcumin ....................................61

T VN
T xa xa, c ngh c s dng ph bin mt s nc chu nh mt th
gia v chnh gip iu hng, to mi v v mu sc hp dn cho thc phm. Khng
nhng th, ngh cn c bit n nh mt loi thuc qu dng tr mn nht, lm
lin so, lm lnh vt thng, v c bit dng cha cc bnh c lin quan n d
dy. Ngy nay, cng vi s tin b ca khoa hc k thut, ngi ta pht hin ra
nhm cht mu curcuminoid tuy ch chim t l nh nhng l nhm hot cht chnh
to nn cc tc dng sinh hc quan trng ca c ngh. Bn cnh , khi xu hng ca
th gii ngy cng a chung nhng sn phm t thin nhin, vic pht trin nhng
hot cht c ngun gc tho dc ngy cng tr thnh mi quan tm ln i vi ngnh
Dc Vit Nam, trong c nhm cht mu curcuminoid.
Curcuminoid l tn gi chung ca cc cht mang khung diarylheptadien c
phn lp t thn r cy Ngh vng (Curcuma longa, h Gng - Zingiberaceae), bao
gm curcumin I (curcumin), curcumin II (demethoxycurcumin), curcumin III
(bisdemethoxycurcumin) v mt s cht khc. Cc curcuminoid c nghin cu v
chng minh vi cc tc dng dc l nh: chng li qu trnh ng mu, chng vim,
chng oxy ha v chng ung th...[2][8][9][27]. Tuy nhin, cc curcuminoid c mu
vng v rt kh ra sch, nn nhu cu s dng ca cc curcuminoid b hn ch, c
bit trong m phm. Sn phm kh ha ca curcuminoid l tetrahydrocurcuminoid c
nhng u im l khng mu nn c s dng rng ri hn trong m phm. Mt
khc tetrahydrocurcumin cn c chng minh c hot tnh chng vim, chng oxy
ha tt hn curcumin [18][19][22][24][25].
Chnh

th,

vic

nghin

cu

kh

ha

curcuminoid

thnh

tetrahydrocurcuminoid l mt hng c trin vng trong m phm v dc phm. Do


vy, chng ti thc hin ti Nghin cu xy dng quy trnh bn tng hp
tetrahydrocurcuminoid t ngun curcuminoid chit xut trong nc
Vi nhng mc tiu sau:

n tng hp tetrahydrocurcuminoid quy m phng th nghim;

Th tc dng chng oxy ha ca cc cht bn tng hp c.

Chng 1: TNG QUAN


1.1. TNG QUAN V CURCUMINOID
1.1.1. Cu trc ha hc v tnh cht
Cu trc ha hc:
Nhm cht mu curcuminoid l hn hp ca 3 cht - thnh phn quan trng nht
ca c ngh vng (Curcuma longa, h Gng - Zingiberaceae), c cng thc cu to
nh hnh 1.1.

Hnh 1.1. Cng thc cu to ca curcuminoid


Tnh cht vt l:
Mt s tnh cht vt l ca 3 cht c trong curcuminoid th hin bng 1.1. [4]
Bng 1.1. Mt s tnh cht vt l ca cc curcuminoid
Curcumin II
Curcumin III
Curcumin I
(Desmethoxy-curcumin)
(Bisdesmethoxy(Curcumin) (1)
(2)
curcumin) (3)
1,7-Bis-(4-hydroxy
1-(4-hydroxyphenyl)-71,7-Bis-(4-3-methoxyphenyl)(4-hydroxy-3hydroxyphenyl) -heptahepta-1,6-dien-3,51,6-dien-3,5-dion
methoxyphenyl)heptadion
1,6-dien-3,5-dion
CTPT

C21H20O 6

C20H18O5

C19H16O4

PTL

368

338

308

Dng th
hnh
im
chy
(oC)

Tinh th hnh kim, Tinh th hnh kim, mu Tinh th hnh kim, mu


mu vng.
vng cam.
vng cam tm.
183

172
im chy ca curcuminoid: 172 178
2

222

Tnh cht ha hc:


Tnh cht ca nhm polyphenol:
- Tan trong dung dch kim.
- Tc dng vi cc tc nhn oxy ha: nhm OH hot ha nhn thm, lm nhn
thm nhy cm i vi cc tc nhn oxy ha, nn curcumin rt d b oxy ha [1].
- Tc dng vi dung dch mui kim loi to phc cht c mu: vi Fe3+ to phc
mu xanh en, vi thic (Sn), km (Zn), ng (Cu), canxi (Ca), magnesi (Mg) to hp
cht c mu t vng cam n nu en.
Tnh cht ca nhm diceton:
Trong mi trng acid acetic, curcumin d phn ng vi cc hp cht c cu
trc kim (Y-NH2) nh hydroxylamin (Y = -OH), hoc phenylhydrazin (Y = C6H5NH-) to sn phm ln lt l 3,5-bis(3-methoxy-4-hydroxylstiryl)isoxazol v 3,5bis(3-methoxy-4-hydroxylstiryl)-1-phenylpirazol. Cc sn phm ny u c hot tnh
khng nm v chng oxy ha tt [3].

Hnh 1.2. Phn ng amin ha -diceton ca curcumin


Hin tng h bin:
Curcumin v cc dn cht tn ti trong dung dch dng cn bng h bin ca
dng diceton i xng v dng ceto enol c n nh bng lin kt hydro ni phn
t. (Hnh 1.4) [13].

Hnh 1.3. Dng h bin ceto enol ca curcumin trong dung dch
3

Trong dung dch nc, pH acid v trung tnh, curcumin tn ti ch yu di dng


diceton; ngc li, pH > 8, dng enol chim u th hn 27. Ty theo tng dung mi
thch hp, c th c trn 95% curcumin tn ti dng enol [13].
nh hng ca pH ti dng tn ti ca curcuminoid trong dung dch nc (Hnh
1.4) [13].
ng hc ca phn ng thy phn curcumin trong dung dch nc trong khong
pH = 1 11 c nghin cu bng phng php HPLC.
- pH < 1, dung dch nc ca curcumin c mu v tn ti dng ion H4A+.
- khong pH = 1 7, curcumin rt t tan trong nc. khong pH ny, dung
dch nc ca curcumin c mu vng v tn ti ch yu dng trung tnh H3A.
- pH > 7,5, dung dch c mu , curcumin tn ti cc dng ion H2A-, HA2- v
A3- ln lt tng ng vi cc gi tr pka l 7,8; 8,5 v 9,0.

Hnh 1.4. Dng tn ti ca curcumin theo pH dung dch

n nh:
Curcumin tng i n nh pH acid, nhng nhanh chng b phn hy pH>7.
Cc sn phm phn hy curcumin pH = 7 10 c xc nh bng phng php
HPLC. Cc sn phm to thnh ban u l acid ferulic v feruloylmethan, ngoi ra cn
c cc sn phm ngng t. Sau feruloylmethan nhanh chng b chuyn mu (ch
yu l t vng n vng nu) ri b phn hy thnh vanillin v aceton (Hnh 1.5) [13].

Sn phm
ngng t

Hnh1.5. S phn hy curcumin trong mi trng kim

c bit, curcumin khng bn di tc dng ca nh sng, nht l trong dung


dch. Sau khi chiu bc x quang hc, cc sn phm c xc nh ging nh cc sn
phm phn hy l acid ferulic, acid vanillic v vanillin [13].

1.1.2. Tc dng dc l
Curcuminoid c rt nhiu tc dng c chng minh, c li cho vic phng
v iu tr bnh. Mt s tc dng ni bt l:
- Tc dng chng oxy ha:
Peroxynitrit (ONOO-) l cht trung gian c tnh c t bo c to ra bi phn
ng gia anion superoxid (O2-) v oxyd nitric (NO). Cc diarylheptanoid, curcumin I,
curcumin II, curcumin III l nhng hot cht c phn lp t Curcuma longa, c tc
dng qut peroxynitrit. Cc hp cht trn th hin hot tnh qut peroxynitrit vi
IC50 ln lt l 4,0; 6,4 v 29,7 m [2].
Trong cc nghin cu v s bo v qu trnh peroxy ha lipid, curcumin th
hin s c ch mnh (18 80%). Ty thuc liu dng m qu trnh peroxy ha lipid
b c ch. Qu trnh ny gy bi Fe-NTA (ferric nitrilotriacetat) v H2O2 c o bng
s hnh thnh malonyl dialdehyd (MDA) l sn phm ca qu trnh oxy ha lipid
mng sinh hc trong cc vi th thn [2].
Theo nghin cu ca Khopde v cc cng s, tc dng chng oxi ha ca
curcuminoid gp t nht 10 ln cc cht chng oxy ha khc thm ch c vitamin E
[16].
- Bo v t bo thn kinh:
Bnh Alzheimer (sa st tr tu, gim tr nh, ri lon chc nng tui gi) bt
ngun t s tch t tinh bt (Abeta), tn thng do chng vim v s oxy ha protein
v no, c cha tr bng vic s dng cht chng vim v chng oxy ha.
Curcumin trong ngh vng c hot tnh chng vim v chng oxy ha mnh c th
ngn chn nhng tn thng ni trn gy ra vng cht xm trn v no. In vitro,
curcumin c ch s tch t Abeta 42 tt hn Ibuprofen v Naproxen, ngn chn s
hnh thnh cht oligomer Abeta 42. Khi nui chut nht trng thnh Tg2576, s tch
t tinh bt hon chnh, cc mnh nh du curcumin thy gim mc tch t
tinh bt lm gim gnh nng cho mnh. Nh vy curcumin trc tip lin kt nhng
loi s-amyloid nh bao vy s tch t v hnh thnh x in vitro v in vivo. C ch
tc dng ca curcumin l lm gim s tch t tinh bt v chng oxy ha protein t bo
no. Curcumin c th d dng lin kt vi cc kim loi oxy ha- kh st v ng, cc

kim loi ny tp trung trn v no, c kh nng gy ngng kt tinh bt v oxy ha


protein. Ngoi ra c th ngn chn tn thng vim bng cch ngn nga s cm ng
kim loi ca NF- kappaB. Nhng d liu trn cho thy curcumin ph v s kt t
cc thnh phn ca mng c hiu qu, ng thi ngn chn c tn thng do vim
v oxy ha-kh protein trn v no. V th curcumin t ngh vng hon ton c th s
dng trn lm sng d phng v iu tr bnh Alzheimer [2].
- Tc dng chng vim
Curcuminoid c hot tnh chng vim cao. Curcuminoid lm bt hot cc enzym
tham gia vo phn ng vim, thng qua c ch ch yu l c ch receptor NF- [2].
Jayaraj

Ravindran

ng

nghip

(2010)

chng

minh

Bisdesmethoxycurcumin c tc dng chng vim mnh hn curcumin (c ch receptor


NF- ). Khi cho tin hnh so snh tc dng chng vim vi curcumin, hispolon,
hispolon methyl ether, dehydroxy hispolon, hydroxy hispolon, methoxy hispolon
methyl ether, v methoxy hispolon, (hispolon c cu trc tng t curcumin nhng
thiu mt nhn thm, c tc dng chng vim) nhn thy tc dng chng vim nh
sau: Bisdesmethoxycurcumin = Hispolon > hispolon methyl ether > hydroxy hispolon
> Curcumin > methoxy hispolon methyl ether > methoxy hispolon > dehydroxy
hispolon. iu ny cho thy vic thay th 1 nhm methoxy cho 1 nhm hydroxy ti v
tr metha vng phenyl ca curcumin tng cng ng k tc dng chng vim
[14][31].
- Bo v d dy, chng vim lot d dy- t trng
Cao nc hoc cao methanol ngh vng cho th ung, lm gim tit dch v v
tng lng cht nhy. Cho chut cng trng ung cao ngh, lm gim tit dch v v
bo v nim mc d dy, t trng.
Ngh kch thch sn sinh cht nhy thnh rut ca chut. Cho bnh nhn ung
bt ngh ngy 4 ln, trong 7 ngy thy c hiu qu tt vi ri lon tiu ha do acid, do
y hi v do mt trng lc c.
Cc proteinase kim loi c bn (MMP - matrix metalloproteinase) ng vai tr
quan trng trong iu tit dch v v lm lnh vt thng sau vim. Curcumin y
nhanh qu trnh lm lnh vt lot v bo v vt lot thng qua vic c ch hot tnh

MMP- 9 v kch thch hot tnh MMP- 2. Nhm S. Swarnakar nghin cu tc dng
ca curcumin t ngh vng trn m hnh lot d dy gy bi indomethacin. Kt qu
cho thy curcumin chng lot mnh vi lot d dy cp tnh bng cch d phng s
suy kit glutathion, chng peroxy ha lipid, ti to lp biu m nhm chng li s bo
mn b mt thnh d dy do cc tn thng gy ra trong khoang d dy. S dng
curcumin ty theo liu qua ng ung hay ng tim phc mc u c th ngn
chn vim lot d dy t trng [2][29].
Helicobacter pylori l vi khun gy vim lot trn t bo biu m thnh d dy.
T bo biu m nhim vi khun ny dn ti s hot ha yu t sao chp NF- kappaB
gy cm ng gen cytokin/chemokin- l gen kch thch gy vim, p ng gen ng t
bo (s pht tn t bo). Curcumin c ch s hot ha NF- kappaB v s pht tn t
bo, do hn ch qu trnh to khng nguyn gy vim v ngn chn c s pht
trin ca vi khun. V vy, curumin rt c tim nng trong iu tr vim lot d dy
khi pht t Helicobacter pylori [2].
- Phng v iu tr ung th
Curcuminoid ngn chn bin i, khi pht u, pht trin u, xm ln, hnh thnh
mch v di cn. Nghin cu in vivo cho thy curcumin c ch cht gy ung th da,
rut gi, rut kt, gan chut, ng vt c v, c ch sinh si t bo u gm t bo
bch cu ,T, ung th biu m rut kt, biu b, v.
C nhng bo co v s tc ng ca curcuminoid i vi s tng sinh t bo
MCF7 (dng t bo gy ung th tuyn v ca con ngi). Theo
desmethoxycurcunin l cht c ch mnh nht MCF-7, tip theo l curcumin v
bisdesmethoxycurcumin [28].
Curcumin l cht gy c t bo theo c ch dit cc t bo c tnh, v hiu ha
t bo ung th v ngn chn hnh thnh t bo ung th mi m khng lm nh hng
n cc t bo lnh tnh. Trong khi , nhiu thuc khi dit t bo c tnh cng dit
lun t bo lnh tnh, lm c th suy kit. Nhng th nghim trn lm sng cho thy
khng c gii hn liu dng c tnh khi dng nhng liu ti 10 gam/ngy. Do ,
curcumin hin nay c nghin cu s dng ph bin trong cc ch phm ngn chn
s khi pht, pht trin v di cn ca khi u [21].

Curcumin c kh nng loi b gc t do, c ch cc loi men v hot tnh ca


mt s cht gy t bin t bo c kh nng dn n ung th trong ung hay thc
n c ch bin v bo qun khng m bo cht lng, gip c th ngn nga ung
th mt cch tch cc. Mt s th nghim in vitro v in vivo pht hin thy curcumin
c kh nng c ch hot tnh gy ung th ca cc cht ny [2][6][21].
- Dng ngoi: khng khun, chng nm, chng lnh vt thng v lin so
Trong iu tr bng, kem ngh c tc dng khng khun, khng nm, loi tr t
chc hoi t bng, c tc dng ti to t chc v lin so. Cao ngh chit vi
cloroform 10% c dng ti ch vng bnh nm, tt vi cc bnh nm da.
Ngh c tc dng khng khun nh mt s thnh phn ha hc nh: Curcumin I
c tc dng c ch in vitro s pht trin mt s loi vi khun m trong in hnh l
trc khun lao Salmonella paratyphi. Cht ar- tumeron t tinh du v dch chit nhexan t l ngh dit u trng mui Aedesaegyti [2].
Trong qu trnh lm lnh vt thng c vai tr quan trng ca s gii phng
chm cc cht chng oxy ha v s h tr ti to m ca collagen. Mt s nghin cu
to lin kt gia curcumin v collagen cho thy hot tnh chng oxy ha ca curcumin
c hiu qu trong vic qut sch cc gc t do gy vim lot, kt hp vi kh nng
kch thch tng sinh t bo ca collagen h tr ng k trong qu trnh iu tr vt
thng m [2].
- H tr chc nng gan, mt
Curcumin cho chut cng trng n, c tc dng kch thch hot tnh men
arylhydroxylase l men ph thuc vo cytocrom P450 ca ty lp th trong t bo gan,
gip gii c v bo v t bo gan, chng vim nhim, hoi t, gip t bo gan hi
phc. A. Raysid chng minh, curcumin to ra tc dng ng hc i vi mt. Vi
20mg curcumin c kh nng lm co ngn ti mt 29% khi quan st trong 2 gi. Do
curcumin c tim nng trong iu tr d phng s hnh thnh si trong ti mt, c th
dng trn lm sng thc y dng mt hoc y dng bn mt trong ti mt ra
ngoi. Ngoi vic lm tng lu lng mt, curcumin cn lm tng lng cholesterol v
acid mt do mt tit ra [26].
- Cc tc dng khc:

Curcumin t ngh vng c tc dng khng virus, c trin vng ln trong iu tr


vim gan , C. c bit, curcumin c nghin cu v tc dng c ch integrase
HIV- 1, c ch protease HIV- 1 v HIV- 2 trin khai cc cht chng HIV, cht
t mt trong tm mt xch ca qu trnh nhim HIV [26].
Curcumin chit t ngh c tc dng c ch s tan hng cu gy bi hydrogen
peroxyd nng thp nhng khng c ch nng cao, khng c tc dng lm
gim s lng bch cu a nhn trung tnh vi nng th nghim [7.
1.1.3. Phng php tch hn hp curcuminoid:
Nm 2002, Guaddadarangavvanahally K. Jayaprakasha v cng s tch ba
thnh phn trong hn hp curcuminoid bng phng php sc k lng hiu nng cao
HPLC, h dung mi s dng l benzene v ethyl acetat vi phn cc tng dn.
Curcumin thu c vi h dung mi benzene : EtOAc (82:18 tt/tt), trong khi DMC v
DMC thu c tng ng vi h dung mi benzene : EtOAc (70:30 tt/tt) v (58:42
tt/tt). Cc phn on thu c t ct sc k c c chn khng v kt tinh li. Hiu
sut tch tng ng ca cc cht curcumin, DMC v BDMC l 2,2; 4,46 v 3,4% [11.
Nm 2003, Vajragupta O. v cng s tch hn hp curcuminoid thu cc thnh
phn n l bng phng php sc k ct silica gel vi h dung mi CHCl3 : MeOH :
AcOH (98 : 5 : 2), bt curcumin c ha tan trong aceton. Theo phng php ny,
curcumin c ra gii trc, sau l hn hp curcumin vi DMC, tip theo l
BDMC tinh khit. DMC tip tc c tch ln th hai bng sc k ct silica gel vi h
dung mi ra gii CH2Cl2 : MeOH (95:5) [33].
Nm 2005, L. Peret-Almeida v cng s ci tin phng php tch cc
curcuminoid. Trong mt vi nghin cu trc y, cc tc gi thc hin tch cc
curcuminoid bng sc k lp mng v sc k ct. Pha tnh c s dng hu ht l
silica gel 60G vi cc h dung mi khc nhau bao gm benzen, ethyl acetat, ethanol,
cloroform, acid acetic, n-hexan v methanol trong sc k lp mng v benzen, nc,
toluen, v ethyl acetate trong sc k ct. Tuy nhin, do phn gii km ch thu c
curcumin c hm lng 80%. Hn na khng tch c demethoxycurcumin v
bisdemethoxycurcumin.

10

Trong nghin cu ny, cc tc gi thc hin tch tng curcuminoid ra khi


hn hp nh sau:
a. Kt tinh curcumin:
Vic kt tinh curcumin c thc hin bng cch ha tan mu trong methanol
60oC. Sau khi ha tan hon ton, thm t t nc ct, hn hp c gi 5oC trong
2h. Tinh th curcumin c tch ra khi dung dch ban u bng cch lc.
b. Tch cc cht bng sc k ct:
Dung dch sau khi kt tinh c ct quay, sau ha tan trong aceton, ri em
chy sc k ct. H dung mi s dng l CH2Cl2 : MeOH (99:1) [23].
1.2. TNG QUAN V TETRAHYDROCURCUMINOID
1.2.1. Cu trc ha hc v tnh cht
Tetrahydrocurcuminoid c cng thc cu to nh hnh 1.6.

Hnh 1.6. Cng thc cu to ca tetrahydrocurcuminoid


Cu to v mt s tnh cht vt l ca cc tetrahydrocurcuminoid th hin bng
1.2 [10].

11

Bng 1.2. Mt s tnh cht vt l ca cc tetrahydrocurcuminoid


THC II

THC III

(Desmethoxy-

(Bisdesmethoxy-

tetrahydrocurcumin)

tetrahydrocurcumin)

THC I
(Tetrahydrocurcumin)
[1,7-Bis-(4-hydroxy3-methoxyphenyl)hepta-3,5-dion]

[1-(4-hydroxyphenyl)-7-

[1,7-Bis-

(4-hydroxy-3-

(4-hydroxyphenyl)

methoxyphenyl)hepta-

hepta-3,5-dion]

3,5-dion]

CTPT

C21H24O 6

C20H12O5

C19H20O4

PTL

372

342

312

Dng th
hnh

Bt kt tinh trng, Cht bn rn Cht


khng mi, khng v

im chy
(0C)

nhy khng mu

Tinh th khng mu

95 - 97

101 - 102

1.2.2. Ngun gc tetrahydrocurcumin:


Cc nghin cu cho thy, trong c th tetrahydrocurcumin, hexahydrocurcumin,
v octahydrocurcumin l nhng cht chuyn ha ca curcumin ng tiu ha.
Hexahydrocurcumin tm thy m gan c chut ci v chut c, trong khi chut
c hnh thnh octahydrocurcumin nhiu hn l THC, cn chut ci THC li c
hnh thnh nhiu hn so vi octahydrocurcumin [12].
1.2.3. Tc dng dc l
1.2.3.1. Tc dng chng vim:
Curcumin v 4 cht tng hp tng t c nghin cu. Hot lc chng vim
ca curcumin, cc dn xut ca curcumin v phenylbutazon c thit lp theo th t
gim dn nh sau:
Natricurcumin THC Curcumin Phenylbutazon triethylcurcumin

12

Khi so snh curcumin v cc dn xut ca n trong cc m hnh vim cp v bn


cp th cc dn cht ca curcumin c tc dng lm gim vim cp tt hn curcumin
[22].
1.2.3.2. Tc dng chng oxy ha:
Hot tnh chng oxy ha ca THC c nghin cu c trn in vivo v in vitro.
Cc nghin cu u ch ra rng THC c kh nng chng oxy ha mnh hn curcumin,
cc curcuminoid khc v vitamin E [22][25].
Poorichaya S. v cng s nghin cu so snh hot tnh chng oxy ha ca
curcumin cng cc dn xut ca n i vi gc t do DPPH (1,1-diphenyl-2picrylhydrazyl) v ch ra rng THC th hin kh nng dn gc t do DPPH cao nht
vi gi tr IC50 = 18,7 M. Hot tnh ny gim dn theo th t: THC > HHC = OHC >
curcumin > DMC > BDMC. Nghin cu cng chng minh rng THC c kh nng
c ch mnh nht i vi AAPH (2,2-azobis(2-amidinopropan)dihydroclorid) - mt
tc nhn gy ra qu trnh peroxy ha lipid. L gii cho nhng iu ny, cc tc gi cho
rng chnh s c mt ca nhm methoxyphenyl v c bit l s hydro ha h ni i
lin hp trong mch carbon trung tm lm tng hot tnh ca curcumin ln mt cch
ng k [24].
Mt nghin cu in vitro so snh tc dng chng oxy ha ca tetrahydrocurcumin
v cc curcuminoid khc c thc hin trn t bo hng cu th v gan chut, s
dng acid linoleic l cht nn trong h ethanol/nc, c phn tch bng phng
php thiocyanat v phng php TBA. Kt qu cho thy rng tetrahydrocurcumin c
hot tnh chng oxy ha mnh nht trong tt c cc curcuminoid [19][22].
Tc dng c ch ca curcumin v tetrahydrocurcumin i vi qu trnh peroxy
ha lipid trn mng hng cu gy bi tert-butylhydroperoxid c nghin cu gn
y. Kt qu chng minh rng THC cho thy hiu qu tt hn curcumin. Cc tc
gi kt lun rng, THC c kh nng dn cc gc t do nh tert-butoxyl v peroxyl mt
cch trit [19].
Gii thch v c ch tc dng v c tnh chng oxy ha mnh hn ca THC so
vi curcumin, cc tc gi cho rng THC l cht chuyn ha chnh ca curcumin
ng tiu ha. H cho rng THC ng vai tr quan trng trong c ch chng oxy ha

13

ca curcumin trong c th, khi m curcumin c chuyn ha thnh THC. Nhm


phenolic ng vai tr dn cc gc t do trong giai on u ca qu trnh chng oxy
ha bng THC. Mt khc, nhm -diceton cng ng vai tr quan trng trong hot
tnh chng oxy ha ca THC, bi v s phn ct lin kt CC trong khung -diceton
c quan st thy trong qu trnh chng oxy ha. Nh vy, nhm -diceton ng vai
tr dn cc gc t do trong giai on sau ca qu trnh chng oxy ha [19][22].
1.2.3.3. Tc dng trn t bo ung th:
Nghin cu chng minh rng THC c tc dng mnh hn curcumin trong vic c
ch s pht trin t im n khc thng ACF (aberrant crypt foci) v s tng nhanh t
bo. THC ph hp trong vic s dng chng li tc nhn gy ung th trong c th
cng nh curcumin [18][34].
1.2.4. Tng quan v cc phng php bn tng hp tetrahydrocurcuminoid
1.2.4.1. Phng php ca Uehara v cng s (1987)
Cc tc gi tin hnh hydro ha curcumin trong h dung mi ethyl acetat/ acid
acetic 50oC v 3,3 at trn xc tc PtO2. Sau phn ng, ct loi dung mi thu c
cn gm mu vng. Ha tan cn trong iso propanol nng v kt tinh qua m 2
4oC. Lc, ra cht rn to thnh, tinh ch bng sc k ct silicagel, h dung mi ra
gii n-hexan/ethyl acetat thu c tinh th THC mu trng, tonc 98 99oC vi hiu
sut

30% [32].

1.2.4.2. Phng php ca Junko Ishida v cng s (2002)


Cc hp cht curcumin I (1), curcumin II (2), curcumin III (3) thu c bng sc
k ct (silica gel, CHCl3 MeOH) t curcumin, trong 2 v 3 chim mt t l nh
[15].
Hydro ha 1 vi Pd/C thu c 4, 5, 6 [15]. Dung dch 1 trong MeOH c kh
ha trong 4 h nhit phng, 1 atm vi xc tc Pd/C 10%. S phn ng nh trn
hnh 1.7.

14

Hnh 1.7. S phn ng kh ha curcumin to thnh tetrahydrocurcumin,


hexahydrocurcumin, octahydrocurcumin dng xt Pd/C
1.2.4.3. Phng php ca Su-Lin Lee v cng s (2005)
T curcumin 1, nhiu sn phm kh 4, 5, 6 c m t trong ti liu. Nhng iu
kin khc nhau ca xc tc Pt/C trong phn ng hydro ha cng c xem xt. Hiu
sut ti u ca hexahydrocurcumin 5 t 1 l 30% v octahydrocurcumin 6 l 91,8 %
di p sut cao v thi gian phn ng di. Mc d 5 v 6 c th thu c t 3 bng
phn ng kh ha vi NaBH4, nhng hiu sut rt thp [30]. S phn ng c th
hin trn hnh 1.8.

Hnh 1.8. S phn ng kh ha curcumin to thnh tetrahydrocurcumin,


hexahydrocurcumin, octahydrocurcumin dng xt Pt/C

15

1.2.4.4. Phng php ca Trn Khc V v cng s (2011)


Vic tng hp cc tetrahydrocurcuminoid c m t s di y. Cc
curcuminoid nguyn liu phn ng 1, 2 v 3 c phn lp t hn hp ba thnh phn
curcumin I (1), curcumin II (2) v curcumin III (3) c chit xut t c ngh vng
(Curcuma Longa L.). Hn hp gm ba thnh phn trn c sc k ct silicagel vi h
dung mi ra gii diclorometan : methanol tng dn t CH2Cl2 : MeOH (100 : 0 n
90 : 10) thu c curcumin I, II, III. Cc tetrahydrocurcuminoid c iu ch ch
yu t phn ng kh ha ni i lin hp 1,3-diceton s dng xc tc Pd/C trong dung
mi a nc [5] (xem hnh 1.9).

Hnh 1.9. S phn ng kh ha cc curcuminoid to thnh cc


tetrahydrocurcuminoid tng ng dng xc tc Zn-NiCl2 s dng k thut siu m
trong dung mi EtOH-H2O
s trn, cc curcuminoid (1, 2, v 3) c ha tan trong h dung mi
(ethanol : nc), sau ha tan mui NiCl2 v cui cng bt km c cho vo t t
ng thi tin hnh siu m. Theo d i qu trnh phn ng cho thy, trong khong 30
pht u, kh hydro mi sinh thot ra mnh v sau khong thi gian ny cc
curcuminoid dn dn b mt mu, chuyn ha thnh cc tetrahydrocurcuminoid. Qu
trnh chuyn ha c kim tra bng sc k bn mng, h dung mi n-hexan :
ethylacetat. trnh bt km nhanh b ng c b mt v nhiu l do dn n mt hot
tnh xc tc, lng km c cho vo thnh nhiu t trong khong thi gian 30 pht.
Qu trnh kh ha cc curcuminoid thnh cc tetrahydrocurcuminoid cao nht l 82%
i vi curcumin trong thi gian 2 gi vi t l mol ca curcumin : Zn : NiCl 2 l 1 : 6 :
1,5. Cu trc ca sn phm c kh ng nh bi ph cng hng t ht nhn 1H v
16

13

C-NMR. C ch chnh xc ca qu trnh kh ha ny vn cha r . Tuy nhin cc

cng b ch ra vai tr quan trng ca NiCl2 trong vic hot ha bt km gii phng
hydro mi sinh di tc dng ca nng lng siu m, cn s c mt ca nc gip
ha tan tt hn mui NiCl2 [5].

17

Chng 2: I TNG V PHNG PHP NGHIN CU


2.1. HA CHT V TRANG THIT
ti s dng mt s nguyn liu, ha cht, dung mi v thit b ca phng th
nghim Tng hp Ha dc, B mn Cng nghip Dc, Trng i hc Dc H
Ni. Nguyn liu curcuminoid c ly t Cng ty c phn dc phm Bc Ninh, c
thnh phn curcumin I 62,1%.
2.1.1. Nguyn liu v ha cht nghin cu
Bng 2.1. Nguyn liu v ha cht nghin cu
STT

Tn nguyn liu v ha cht

Ngun gc

Acid acetic

Merck

Acid hydrocloric

Trung Quc

Acid sulfuric

Trung Quc

Dicloromethan

Trung Quc

Diethyl ether

Trung Quc

Ethanol 96%

Trung Quc

Ethanol tuyt i

Trung Quc

Ethyl acetat

Trung Quc

n-Hexan

Trung Quc

10

Km bt

Trung Quc

11

Methanol

Trung Quc

12

Natri carbonat

Trung Quc

13

Natri sulfat

Trung Quc

14

Nc ct

15

Palladium -carbon 5%

16

Silicagel 60 GF254

17

Than hot

Vit Nam
Sigma-Aldrich
Merck
Vit Nam

18

2.1.2. Thit b, my mc v dng c nghin cu


Bng 2.2. Thit b, my mc v dng c nghin cu
STT

Thit b, dng c

Xut x

Bn mng silicagel GF254

Bnh chit 250 mL

Bnh cu 1 c, 2 c, 3 c 50mL, 100mL, 250mL

B lc ht chn khng Buchner

Trung Quc

Bnh sc k

Trung Quc

Cn k thut Sartorius P 2001S, nhy 10-2

Cc c m 100mL

Ct sc k

Giy lc

Vit Nam

10

a petri

Trung Quc

11

My cy quay chn khng Buchi B480

Thy S

12

My o nhit ng nng chy EZ-Melt

13

My o ph hng ngoi GX-Perkin Elmer (Vin Ha


hc - Vin Hn lm Khoa hc v Cng ngh Vit Nam)

Thy S

My o ph cng hng t ht nhn proton (1H-NMR)


14

Bruker AV-500 (Vin Ha hc - Vin Hn lm Khoa

hc v Cng ngh Vit Nam)


15

My o ph khi lng Varian 320 Ms (Vin Ha hc


Vin Hn lm Khoa hc v Cng ngh Vit Nam)

16

My khuy t c b phn gia nhit IKA

17

My sy n hng ngoi

18

My sinh kh nit

19

My sinh kh hydro

20

ng ong 25mL, 50mL, 100mL

Trung Quc

21

ng nghim

Trung Quc

22

Pipet chia vch 1mL, 5mL, 10mL

19

Trung Quc

23

Qu bp cao su

24

T sy Memmert

2.2. NI DUNG NGHIN CU

- Tch tng thnh phn trong hn hp curcuminoid.


- Bn tng hp tetrahydrocurcuminoid t curcuminoid theo s sau:

Tin hnh phn ng kh ha curcuminoid vi hai tc nhn kh khc nhau l kh hydro


c xc tc Pd/C 5% v km trong mi trng acid.

- S b kim tra tinh khit ca sn phm bng SKLM vi h dung mi thch hp


v o nhit nng chy.

- Xc nh cu trc sn phm bn tng hp c bng cch o ph hng ngoi (IR),


ph khi lng (MS) v ph cng hng t ht nhn proton (1H-NMR).

- Th tc dng chng oxy ha ca sn phm bn tng hp c bng phng php


DPPH.
2.3. PHNG PHP NGHIN CU
2.3.1. Phng php tch tng thnh phn trong hn hp curcuminoid
- Dng phng php kt tinh phn on
- Dng sc k ct vi h dung mi thch hp
2.3.2. Bn tng hp ha hc:
Bn tng hp tetrahydrocurcuminoid t curcuminoid bng phn ng kh ha s dng
cc tc nhn kh ha khc nhau:
-

Dng kh hydro vi xc tc Pd/C 5 %.

Dng kim loi trong mi trng acid.

20

2.3.3. Kim tra tinh khit


Kim tra s b tinh khit sn phm thu c bng phng php SKLM v
phng php o nhit nng chy.
SKLM: Tnh gi tr h s lu gi Rf

Dung mi ha tan: ethanol

H dung mi chy sc k: Dicloromethan : Methanol = 20:1

2.3.4. c nh cu trc sn phm


Ph hng ngoi(IR)
Ph khi lng (MS)
Ph cng hng t ht nhn proton (1H-NMR), Ph cng hng t carbon (13CNMR)
2.3.5. Th tc dng chng oxy ha ca cc cht bn tng hp c
nh gi tc dng dn gc t do DPPH (phng php DPPH) c thc hin
ti Phng Ha sinh ng dng, Vin Ha hc Vin hn lm khoa hc & cng ngh
Vit Nam.

21

Chng 3: KT QU NGHIN CU
3.1. TCH CC CURCUMIN I, II, III T HN HP CURCUMINOID
Dng phng php tch thch hp thu c tng thnh phn ring bit:
Curcumin I, Curcumin II, Curcumin III. S tch c th hin hnh 3.1.
H A TAN

- Curcuminoid
- Dung mi

KT TINH

CURCUMIN I

LC

S C K CT

CURCUMIN I

CURCUMIN II

Dicloromethan :
Methanol (20:1)

CURCUMIN III

Hnh 3.1. S tch tng thnh phn trong hn hp curcuminoid


Cch tin hnh
Bt curcuminoid c thnh phn curcumin I (62,1 %), curcumin II (24,6%), v
curcumin III (8,3%) u tin c kt tinh li 3 ln trong dung mi thch hp, cc
bc thc hin nh sau:
- Ha tan hon ton 3,00 g curcuminoid trong 250 ml dung mi 60oC, sau
kt tinh.
- Tinh th kt tinh c lc ht chn khng, ra, kim tra s b tinh khit bng
SKLM.
- Dch lc kim tra s b bng SKLM, ct quay chn khng n kit thu sn phm
th. Tip tc em kt tinh li ln 2,3 tng t nh ln 1.

22

Chng ti tin hnh kho st nh hng ca dung mi dng kt tinh n qu


trnh kt tinh.
Tin hnh kho st vi 2 dung mi l EtOH 96% v MeOH. Kt qu kho st
c trnh by trong bng 3.1.
Bng 3.1. Kt qu kho st nh hng ca dung mi, nhit v thi gian kt tinh
n qu trnh kt tinh curcuminoid
Dung mi
kt tinh

STT

Nhit
kt tinh
(oC)

Thi gian
kt tinh
(h)

KL
curcumin I
(g)

Tonc
(oC)

MeOH

to phng

24

1,08

182 184

EtOH 96%

to phng

24

1,02

182 184

Nhn xt:
- Vi hai dung mi s dng l EtOH 96% v MeOH, khi kt tinh kt tinh
nhit phng trong 24h, tinh th thu c l curcumin I vi nhit nng chy ph
hp vi nhit nng chy ca curcumin I trong cc ti liu c cng b..
- Dung mi s dng l MeOH cho khi lng tinh th curcumin I nhiu hn,
nhng khng ng k, mt khc MeOH li c hn nhiu so vi EtOH 96%.
T cc d liu trn cho thy dung mi EtOH 96% c nhiu u im hn, do
EtOH 96% c chn lm dung mi kt tinh, kt tinh ti nhit phng trong 24h.
Sau khi chn c cc iu kin kt tinh thch hp, tin hnh kt tinh 3 ln, lc,
ra, sy, cn nh lng thu c tinh th curcumin I. Kt qu kt tinh c trnh by
bng 3.2.

23

Bng 3.2. Kt qu kt tinh curcuminoid 3 ln trong EtOH 96%


Ln kt tinh

Vdung mi (ml)

KL tinh th (g)

250

1.02

150

0,26

50

0,08

Tng:

1,36

Hiu sut (%)

73,12

Nc ci cui cng l hn hp curcuminoid ba thnh phn, trong curcumin II v


III c lm giu, tin hnh sc k ct silica gel vi h dung mi CH2Cl2 : MeOH =
20:1 thu ly tng curcuminoid tng thnh phn.
Cc bc tin hnh sc k ct:

c 1: Chun b

Cn 100,00 g silicagel, ngm trong pha ng trong khong 30 pht.

Ct ra sch, sy kh, lt y ct bng bng, c nh ct sc k song song,

th ng gc vi trc ca gi .

c 2: Nhi ct
Cho dung mi n khong na chiu cao ct, m van, t t nhi lin tc

silicagel vo ct cho n ht lng silicagel cn nhi, ng thi g nh nn cc lp


silicagel. Ch khng mc dung mi xung thp hn mc silicagel trnh hin
tng kh ct.

t mt ming giy lc ln trn b mt lp silicagel.


c 3: a cht phn tch vo ct

Ha tan mu vo mt lng ti thiu dung mi pha ng.

H mc dung mi trong ct xung ngang mc giy lc, ri np ht dung dch

cht phn tch vo ct.

c 4: Trin khai sc k ct
Cho dung mi ra gii chy qua ct, hng cc phn on c th tch khong 40

ml.
-

Cc phn on c ct quay chn khng loi bt dung mi, sau kim tra

bng SKLM vi h dung mi CH2Cl2 : MeOH = 20:1.


24

c 5: Thu hi cc phn on
Cc phn on c Rf ging nhau c gp li, ct quay chn khng, sy kh v

cn nh lng.
Kt qu tch c th hin bng 3.3.
Bng 3.3. Kt qu tch cc curcuminoid thnh phn t hn hp curcuminoid bng
sc k ct silica gel vi h dung mi dicloromethan: methanol = 20:1
Phn on

Tng th tch
(ml)

Thnh phn

Khi lng (g)

1 - 18

720

Curcumin I

0,46

19 - 22

160

Curcumin I, Curcumin II

0,13

23 - 45

880

Curcumin II

0,61

46 - 52

280

Curcumin II, Curcumin III

0,29

53 - 55

120

Curcumin III

0,04

Nhn xt:
- Nc ci thu c sau khi kt tinh hn hp curcuminoid, em chy sc k ct,
thu c cc curcumin thnh phn, trong ch yu l curcumin I (0,46 g), curcumin
II (0,61 g), cn curcumin III thu c mt lng rt nh (0,04 g).
Kim tra tinh khit ca cc curcuminoid thnh phn:
Cc curcuminoid thnh phn sau khi tch, tin hnh kim tra tinh khit bng
SKLM trn bn mng silicagel GF254, h dung mi khai trin dicloromethan :
methanol (20:1). Kt qu cho thy cc cht th u cho mt vt gn, r, khng c vt
ph (Hnh 3.1). Trong Rf curcumin I = 0,8 ; Rf curcumin II = 0,46 v Rf curcumin III = 0,34.

25

Hnh 3.2. SKLM ca cc curcuminoid thnh phn


3.2. KH HA TNG CURCUMINOID THNH PHN
Chng ti thc hin phn ng kh ha vi hai tc nhn kh l kh hydro c
xc tc Pd/C 5% v km trong mi trng acid.
3.2.1. Kh ha curcumin I (curcumin)
3.2.1.1. Phn ng kh ha vi tc nhn kh l kh hydro c xc tc Pd/C 5%
S phn ng: c th hin hnh 3.3.

Hnh 3.3. S phn ng kh ha curcumin vi tc nhn H2/Pd/C


Tin hnh phn ng:
Hot ha xc tc: Cho xc tc Pd/C 5% vo bnh cu ba c y trn dung tch
250 ml, thm 20 ml dung mi, khuy t nhit khong 0-5oC trong 5 pht, ng
thi sc kh nit ui ht khng kh trong bnh phn ng. Ha tan 1,00 g curcumin
(2,71 mmol) trong 100 ml dung mi, cho dung dch thu c vo bnh phn ng trn.
Tip tc khuy hn hp nhit phng, ng thi sc kh hydro lin tc trong qu

26

trnh phn ng p sut kh quyn. Theo di hn hp phn ng bng SKLM vi h


dung mi dicloromethan: methanol = 20:1 xc nh thi gian phn ng thch hp.
X l hn hp phn ng:
Hn hp phn ng em lc ht chn khng loi b xc tc. Dch lc em ct
loi dung mi bng my ct quay chn khng nhit khong 50oC. Tinh ch sn
phm bng phng php sc k ct silica gel vi h dung mi dicloromethan:
methanol = 20:1. Cc bc tin hnh sc k ct tng t nh phn 3.1.
Kho st phn ng:
Chng ti tin hnh kho st cc yu t nh hng n phn ng, bao gm dung
mi phn ng v t l khi lng xc tc so vi curcumin.
a. Kho st dung mi phn ng
Tin hnh kho st vi 3 dung mi l MeOH, EtOH tuyt i v EtOH 96%.
Kt qu kho st c trnh by trong bng 3.4.
Bng 3.4. Kt qu kho st nh hng ca dung mi n hiu sut phn ng bn
tng hp tetrahydrocurcumin t curcumin vi tc nhn kh l H2/Pd/C
Dung mi phn

TT

ng

T l KL
xt/curcumin
(%)

KL sp

Hp

(g)

(%)

Tonc (oC)

MeOH

10

0,65

64,33

95.0 98,0

EtOH tuyt i

10

0,62

61,36

95,0 98,0

EtOH 96%

10

0,59

58,39

95,0 98,5

Nhn xt:
- Vi c ba dung mi s dng, nhit nng chy ca sn phm thu c u ph
hp vi nhit nng chy ca sn phm d kin (Tonc,THC = 95,0 97,0oC [18]).
- Dung mi l EtOH 96% cho hiu sut phn ng thp nht (58,39%). Khi s dng
dung mi l EtOH tuyt i cho hiu sut phn ng cao hn (61,36%).
- Dung mi l MeOH cho hiu sut phn ng cao nht (64,33%). Tuy nhin,
MeOH li c hn nhiu so vi EtOH.

27

T cc d liu trn cho thy dung mi EtOH c nhiu u im hn, do EtOH


tuyt i c chn lm dung mi cho cc phn ng kho st tip theo.
b. Kho st nh hng ca t l xc tc n thi gian v hiu sut phn ng
Sau khi chn c dung mi thch hp cho phn ng l EtOH tuyt i, tip tc
kho st nh hng ca t l xc tc n thi gian v hiu sut phn ng. Tin hnh
kho st vi cc t l khi lng xc tc l 2%, 5%, 8% v 10% so vi curcumin .
Kt qu kho st c trnh by trong bng 3.5.
Bng 3.5. Kt qu kho st nh hng ca t l xc tc n thi gian v hiu sut
phn ng bn tng hp THC t curcumin vi tc nhn kh l H2/Pd/C

TT

KL

KL xt

curcumin (g)

(g)

T l KL
xt/curcumin
(%)

tp

KL

Hp

(h)

sp(g)

(%)

Tonc (oC)

1,00

0,02

24

0,57

56,41

95,098,0

1,00

0,05

18

0,66

65,32

95,098,0

1,00

0,08

15

0,63

62,35

96,098,5

1,00

0,10

10

12

0,62

61,36

96,098,0

Nhn xt:
- Nhit nng chy ca cc sn phm thu c t cc th nghim kho st cc t
l xc tc khc nhau khng c s chnh lch ng k.
- Khi tng t l xc tc th thi gian phn ng gim, hiu sut phn ng cng thay
i. Vi t l xc tc l 2%, thi gian phn ng l 24h, hiu sut phn ng l thp nht
(56,41%). Hiu sut phn ng t c cao nht l 65,32% khi t l xc tc l 5%,
thi gian phn ng l 18h. Tip tc tng t l xc tc th hiu sut phn ng gim.
T cc d liu trn, chng ti la chn t l xc tc l 5%, vi thi gian phn
ng l 18h.
T cc kt qu kho st trn thy rng, iu kin thch hp nht cho phn ng
kh ha curcumin vi tc nhn l kh hydro c xc tc Pd/C 5% l:
- Dung mi: Ethanol tuyt i
- T l khi lng xt/curcumin: 5%

28

- Thi gian phn ng: 18h.


3.2.1.2. Phn ng kh ha vi tc nhn km trong mi trng acid
S phn ng: c th hin hnh 3.4.

Hnh 3.4. S phn ng kh ha curcumin vi tc nhn Zn/H+


Tin hnh phn ng:
Ha tan 1,00 g curcumin (2,71 mmol) vo 100 ml EtOH tuyt i trong bnh cu
ba c y trn dung tch 250 ml. Sc nit ui ht khng kh trong bnh phn ng,
thm acid vo bnh phn ng.
Cn bt km, thm t t vo hn hp phn ng thnh nhiu t trong thi gian
khong 30 pht. Khuy hn hp phn ng nhit phng, duy tr pH acid trong sut
qu trnh phn ng. Theo di phn ng bng SKLM vi h dung mi ethyl acetat: nhexan = 7:3 xc nh thi gian phn ng thch hp, so snh vi THC tinh khit thu
c phn ng kh ha vi tc nhn H2/Pd/C.
X l hn hp phn ng
- Lc loi cht rn, thu dch lc.
- Trung ha dch lc bng dung dch Na2CO3 bo ha n pH trung tnh, lc loi
ta.
- Dch lc em ct loi bt mt phn dung mi, sau em chit vi
dicloromethan: Ln 1 chit vi 30ml dicloromethan, thm 20ml nc ct. Ln 2 v 3,
mi ln 30ml dicloromethan.
- Lp dung mi hu c c gp li, thm 5,00g Na2SO4 khan, lc u, yn
khong 1h loi nc. Sau lc, dch lc em ct loi dung mi bng my ct quay
chn khng nhit khong 40oC.

29

- Tinh ch sn phm bng phng php sc k ct silica gel vi h dung mi nhexan: ethyl acetat thay i t 50:0 n 1:1. Cch tin hnh sc k ct tng t nh
trnh by mc 3.1.
Kho st phn ng
Chng ti tin hnh kho st cc yu t nh hng n phn ng, bao gm acid
phn ng, nng acid v t l mol Zn/curcumin.
a. Kho st acid
Tin hnh kho st vi 3 acid l: acid acetic bng, acid hydrocloric 20% v acid
sulfuric 10%.
Kt qu kho st c trnh by trong bng 3.6.
Bng 3.6. Kt qu kho st nh hng ca acid n hiu sut phn ng bn tng
hp tetrahydrocurcumin t curcumin vi tc nhn kh l Zn/acid
TT

Acid

V acid

KL km

tp

KL

Hp

(ml)

(g)

(h)

sp (g)

(%)

Tonc
(oC)

Ghi ch
Khng

Acid acetic bng

15

xut hin
vt THC

Acid hydrocloric
20%
Acid sulfuric 10%

0,58

57,40

95,0-98,0

0,50

49,49

95,0-98,5

Nhn xt:
- Khi thc hin phn ng vi acid acetic, kim tra hn hp phn ng bng SKLM
vi h dung mi ethyl acetat: n-hexan = 7:3 v so snh vi THC thu c phn ng
kh ha vi xc tc H2/Pd/C th trong hn hp phn ng khng thy xut hin vt
THC.
- Thc hin phn ng vi acid hydrocloric (H=57,40%) cho hiu sut phn ng
cao hn khi s dng acid sulfuric (H=49,49%).
T kt qu kho st trn, chng ti chn acid hydrocloric thc hin cc phn
ng kho st tip theo.
30

b. Kho st nng acid HCl


Sau khi chn c acid thch hp cho phn ng l acid hydrocloric, tip tc kho
st nh hng ca nng acid n hiu sut phn ng. Tin hnh kho st vi dung
dch acid hydrocloric 4 nng : 5%, 10%, 20% v acid c (khong 37%).
Kt qu kho st c trnh by trong bng 3.7.
Bng 3.7. Kt qu kho st nh hng ca nng acid n hiu sut phn ng
bn tng hp tetrahydrocurcumin t curcumin vi tc nhn kh l Zn/HCl
TT

Nng acid V acid

KL

tp

KL

Hp

Tonc (oC)

(% m/V)

(ml)

km (g)

(h)

sp (g)

(%)

20

0,38

37,61

95,098,0

10

10

0,45

44,54

95,597,0

20

0,58

57,40

95,098,0

Acid c

3,5

0,55

54,43

95,098,0

Nhn xt
- Nhit nng chy ca cc sn phm thu c t cc th nghim khc nhau
khng c s chnh lch ng k.
- Vi dung dch HCl 5%, hiu sut phn ng thu c thp nht (37,61%)
- Khi tng dn nng acid th hiu sut phn ng tng ln, n nng 20% th
hiu sut phn ng cao nht (57,40%). Tip tc tng n acid HCl c (nng
khong 37%) th hiu sut phn ng li gim (54,43%).
T kt qu kho st trn, dung dch HCl 20% c la chn thc hin cc phn
ng kho st tip theo.
c. Kho st t l mol km v curcumin
Sau khi la chn c iu kin thch cho phn ng l acid hydrocloric 20%,
chng ti tip tc kho st nh hng ca t l gia s mol km v s mol curcumin
n hiu sut phn ng. Tin hnh kho st vi cc t l mol Zn/curcumin ln lt l
6/1, 12/1, 18/1, 24/1 v 28/1.
Kt qu kho st c trnh by trong bng 3.8.
Bng 3.8. Kt qu kho st nh hng ca t l mol km/curcumin n hiu sut
phn ng bn tng hp THC t curcumin vi tc nhn kh l Zn/HCl

31

KL

KL

T l mol

curcumin

km

km/

(g)

(g)

curcumin

1,00

1,06

6/1

1,00

2,11

1,00

4
5

TT

tp (h)

KL sn

Tonc (oC)

phm (g)

Hp (%)

6,5

0,42

41,57

95,598,5

12/1

5,5

0,51

50,47

95,098,0

3,17

18/1

0,61

60,37

95,098,0

1,00

4,23

24/1

0,59

58,39

95,597,5

1,00

5,00

28/1

0,58

57,40

95,098,0

Nhn xt:
- Nhit nng chy ca cc sn phm thu c t cc th nghim kho st cc t
l mol khc nhau khng c s chnh lch ng k.
- Vi t l s mol Zn/curcumin l 6/1 th hiu sut phn ng thu c l thp nht
(41,57%). Khi tng dn t l s mol Zn/curcumin th hiu sut phn ng tng ln. Khi
t l mol tng n 18/1 th hiu sut phn ng tng ln khong 1,5 ln so vi t l mol
l 6/1, t 60,37%. Tip tc tng t l mol Zn/curcumin ln ln lt l 24/1, 28/1 th
hiu sut phn ng gim.
- T cc d liu trn, chng ti la chn t l mol Zn/curcumin thch hp cho phn
ng l 18/1.
T cc kt qu kho st trn thy rng, iu kin thch hp nht cho phn ng
kh ha curcumin bng Zn/acid l:
- Acid: HCl 20%
- T l mol Zn/curcumin:18/1
- Thi gian phn ng: 4h
3.2.1.3. Kim tra tinh khit sn phm THC
- Sau khi bn tng hp cc cht, tin hnh kim tra tinh khit ca cc sn phm
bng SKLM trn bn mng silicagel GF254: Sn phm c ha tan trong ethanol nng
chm sc k. Pha ng chy sc k l h dung mi dicloromethan: methanol =
20: 1 v h ethyl acetat: n-hexan = 7: 3.
- Sau khi chy sc k, soi bn mng di n t ngoi bc sng 254 nm, cc
cht th u cho mt vt gn, r, khng c vt ph (Hnh 3.5 v 3.6). Vi h dung mi
32

dicloromethan: methanol = 20: 1 cho Rf THC = 0,55 v h ethyl acetat: n-hexan = 7: 3


cho Rf THC = 0,69.
- Sn phm thu c t hai phn ng kh vi hai tc nhn kh khc nhau l
H2/Pd/C 5% v Zn/HCl c chm chng trn cng mt bn mng v chy sc k vi
h dung mi dicloromethan: methanol = 20: 1. Kt qu cho thy cc sn phm cho cc
vt ng nht trn bn mng h dung mi s dng.

Hnh 3.5. SKLM ca THC trn h dung

Hnh 3.6. SKLM ca THC trn h dung

mi CH2Cl2: MeOH = 20:1

mi AcOEt: n-hexan = 7:3

- ng thi, tin hnh o nhit nng chy ca sn phm bn tng hp c.


Kt qu o nhit nng chy cho thy cc cht u c im chy r rng trong
khong 95 98oC.
3.2.1.4. Tm tt kt qu:
Tnh cht sn phm:
- Cm quan: Bt kt tinh mu trng.
- Nhit nng chy: 95 98oC.
- Rf (CH2Cl2: MeOH=20:1) = 0,55.
- Rf (AcOEt: n-hexan=7:3) = 0,69.

33

- Hiu sut phn ng: Phn ng vi H2/Pd/C 5% trong iu kin thch hp cho
hiu sut tt nht t 65,32%. Phn ng vi Zn/HCl trong iu kin thch hp cho hiu
sut tt nht t 60,37%.
3.2.2. Kh ha curcumin II (DMC)
3.2.2.1. Phn ng kh ha vi tc nhn kh l kh hydro c xc tc Pd/C 5%
S phn ng: c th hin hnh 3.7.

Hnh 3.7. S phn ng kh ha DMC vi tc nhn H2/Pd/C


Tin hnh phn ng:
Hot ha xc tc: Cho xc tc Pd/C 5% vo bnh cu ba c y trn dung tch 250
ml, thm 10 ml dung mi, khuy t nhit khong 0-5oC trong 5 pht, ng thi
sc kh nit ui ht khng kh trong bnh phn ng. Ha tan 0,60 g DMC trong 50
ml dung mi, cho dung dch thu c vo bnh phn ng trn. Tip tc khuy hn hp
nhit phng, ng thi sc kh hydro lin tc trong qu trnh phn ng p sut
kh quyn. Theo di hn hp phn ng bng SKLM vi h dung mi dicloromethan:
methanol = 20:1 xc nh thi gian phn ng thch hp.
X l hn hp phn ng:
Hn hp phn ng em lc ht chn khng loi b xc tc. Dch lc em ct
loi dung mi bng my ct quay chn khng nhit khong 50oC. Tinh ch sn
phm bng phng php sc k ct silica gel vi h dung mi dicloromethan:
methanol = 20:1. Cc bc tin hnh sc k ct tng t nh phn 3.1. Cc phn on
c kt qu SKLM ging nhau c gp li, ct quay chn khng, sy kh v cn.

34

Kho st phn ng:


Chng ti tin hnh kho st cc yu t nh hng n phn ng, bao gm dung
mi phn ng v t l khi lng xc tc so vi DMC.
a. Kho st dung mi phn ng:
Tin hnh kho st vi 3 dung mi l MeOH, EtOH tuyt i v EtOH 96%.
Kt qu kho st c trnh by trong bng 3.9.
Bng 3.9. Kt qu kho st nh hng ca dung mi n hiu sut phn ng bn
tng hp THC II t DMC vi tc nhn kh l H2/Pd/C
TT

Dung mi phn ng

T l KL
xt/DMC (%)

KL sp (g)

Hp (%)

MeOH tuyt i

10

0,42

69,17

EtOH tuyt i

10

0,39

64,22

EtOH 96%

10

0,36

59,29

Nhn xt:
- Khi s dng dung mi phn ng l EtOH 96% cho hiu sut phn ng thp nht
(59,29%), s dng dung mi l EtOH tuyt i cho hiu sut phn ng cao hn
(64,22%).
- Dung mi l MeOH cho hiu sut phn ng cao nht (69,17%). Tuy nhin,
MeOH li c hn nhiu so vi EtOH.
T cc d liu trn cho thy dung mi EtOH c nhiu u im hn, do EtOH
tuyt i c chn lm dung mi cho cc phn ng kho st tip theo.
b. Kho st nh hng ca t l xc tc n thi gian v hiu sut phn ng
Sau khi chn c dung mi thch hp cho phn ng l EtOH tuyt i, chng
ti tip tc kho st nh hng ca t l xc tc n thi gian v hiu sut phn ng.
Tin hnh kho st vi cc t l khi lng xc tc l 2%, 5%, 8% v 10% so vi
DMC.
Kt qu kho st c trnh by trong bng 3.10.

35

Bng 3.10. Kt qu kho st nh hng ca t l xc tc n thi gian v hiu sut


phn ng bn tng hp THC II t DMC vi tc nhn kh l H2/Pd/C
KL DMC
(g)

TT

KL xt
(g)

T l KL xt/DMC
(%)

tp
(h)

KL sp
(g)

Hp (%)

0,60

0,01

24

0,33

54,35

0,60

0,03

18

0,42

69,18

0,60

0,05

15

0,40

65,89

0,60

0,06

10

12

0,39

64,24

Nhn xt:
- Khi tng t l xc tc th thi gian phn ng gim, hiu sut phn ng cng thay
i. Vi t l xc tc l 2%, thi gian phn ng l 24h, hiu sut phn ng l thp nht
(54,35%). Hiu sut phn ng t c cao nht l 69,18% khi t l xc tc l 5%,
thi gian phn ng l 18h. Tip tc tng t l xc tc th hiu sut phn ng gim.
T cc d liu trn, chng ti la chn t l xc tc l 5%, vi thi gian phn
ng l 18h.
T cc kt qu kho st trn thy rng, iu kin thch hp nht cho phn ng
kh ha DMC vi tc nhn l kh hydro c xc tc Pd/C 5% l:
- Dung mi: Ethanol tuyt i
- T l khi lng xt/DMC: 5%
- Thi gian phn ng: 18h.

36

3.2.2.2. Phn ng kh ha vi tc nhn km trong mi trng acid


S phn ng: c th hin hnh 3.8.

Hnh 3.8. S phn ng kh ha DMC vi tc nhn Zn/H+


Tin hnh phn ng:
Ha tan 0,60 g DMC vo 50 ml EtOH tuyt i trong bnh cu ba c y trn
dung tch 100 ml. Sc nit ui ht khng kh trong bnh phn ng, thm acid vo
bnh phn ng.
Cn bt km, thm t t vo hn hp phn ng thnh nhiu t trong thi gian
khong 30 pht. Khuy hn hp phn ng nhit phng, duy tr pH acid trong sut
qu trnh phn ng. Theo di phn ng bng SKLM vi h dung mi ethyl acetat: nhexan = 7:3 xc nh thi gian phn ng thch hp, so snh vi THC II tinh khit
thu c phn ng kh ha vi tc nhn H2/Pd/C.
X l hn hp phn ng
- Lc loi cht rn, thu dch lc.
- Trung ha dch lc bng dung dch Na2CO3 bo ha n pH trung tnh, lc loi
ta.
- Dch lc em ct loi bt mt phn dung mi, sau em chit vi
dicloromethan: Ln 1 chit vi 20 ml dicloromethan, thm 10 ml nc ct. Ln 2 v 3,
mi ln 20 ml dicloromethan.
- Lp dung mi hu c c gp li, thm 3,00g Na2SO4 khan, lc u, yn
khong 1h loi nc. Sau lc, dch lc em ct loi dung mi bng my ct quay
chn khng nhit khong 40oC.

37

- Tinh ch sn phm bng phng php sc k ct silica gel vi h dung mi nhexan: ethyl acetat thay i t 50:0 n 1:1. Cch tin hnh sc k ct tng t nh
trnh by mc 3.1.
Kho st phn ng
Chng ti tin hnh kho st cc yu t nh hng n phn ng, bao gm
acid phn ng, nng acid v t l mol Zn/DMC.

a. Kho st acid phn ng


Tin hnh kho st vi 3 acid l: acid acetic bng, acid hydrocloric 20% v acid
sulfuric 10%.
Kt qu kho st c trnh by trong bng 3.11.
Bng 3.11. Kt qu kho st nh hng ca acid n hiu sut phn ng bn tng
hp THC II t DMC vi tc nhn kh l Zn/acid
TT

Acid

V acid
(ml)

KL km
(g)

tp
(h)

KL
sp (g)

Hp
(%)

Ghi ch
Khng
xut hin
vt THC

Acid acetic bng

10

Acid hydrocloric
20%

0,35

57,65

Acid sulfuric 10%

0,27

44,47

Nhn xt:
- Khi thc hin phn ng vi acid acetic, kim tra hn hp phn ng bng SKLM
vi h dung mi ethyl acetat: n-hexan = 7:3 v so snh vi THC II thu c phn
ng kh ha vi tc nhn H2/Pd/C th trong hn hp phn ng khng thy xut hin
vt THC II.
- Thc hin phn ng vi acid hydrocloric (H=57,65%) cho hiu sut phn ng
cao hn khi s dng acid sulfuric (H=44,47%).
T kt qu kho st trn, chng ti chn acid hydrocloric thc hin cc phn
ng kho st tip theo.

38

a. Kho st nng acid


Sau khi chn c acid thch hp cho phn ng l acid hydrocloric, tip tc kho
st nh hng ca nng acid n hiu sut phn ng. Tin hnh kho st vi dung
dch acid hydrocloric 4 nng : 5%, 10%, 20% v acid c (khong 37%).
Kt qu kho st c trnh by trong bng 3.12.
Bng 3.12. Kt qu kho st nh hng ca nng acid n hiu sut phn ng
bn tng hp THC II t DMC vi tc nhn kh l Zn/HCl

TT

Nng acid
(% m/V)

V acid
(ml)

KL km
(g)

tp (h)

10

20

Acid c

KL sp
(g)

Hp (%)

0,21

34,59

0,28

46,12

0,36

59,30

3,5

0,31

51,06

Nhn xt
Khi thay i nng acid phn ng, hiu sut phn ng cng thay i:
- Vi dung dch HCl 5%, hiu sut phn ng thu c thp nht (34,59%)
- Khi tng dn nng acid th hiu sut phn ng tng ln, n nng 20% th
hiu sut phn ng cao nht (59,30%). Tip tc tng n acid HCl c (nng
khong 37%) th hiu sut phn ng li gim (51,06%).
T kt qu kho st trn, dung dch HCl 20% c la chn thc hin cc phn
ng kho st tip theo.
b. Kho st t l mol km v DMC:
Sau khi la chn c iu kin thch cho phn ng l acid hydrocloric 20%,
chng ti tip tc kho st nh hng ca t l gia s mol km v s mol DMC n
hiu sut phn ng. Tin hnh kho st vi cc t l mol Zn/DMCln lt l 6/1, 12/1,
18/1, 24/1 v 30/1.
Kt qu kho st c trnh by trong bng 3.13.

39

Bng 3.13. Kt qu kho st nh hng ca t l mol km/curcumin II n hiu


sut phn ng bn tng hp THC II t Curcumin II vi tc nhn kh l Zn/HCl

TT

KL DMC
(g)

KL km
(g)

T l mol
km/ DMC

tp (h)

KL sn
phm (g)

Hp (%)

0,60

0,69

6/1

6,5

0,24

39,53

0,60

1,38

12/1

5,5

0,31

51,06

0,60

2,07

18/1

0,37

60,95

0,60

2,76

24/1

0,35

57,65

0,60

3,45

30/1

0,34

56,00

Nhn xt:
Khi thay i t l s mol Zn/DMC th hiu sut phn ng thay i:
- Vi t l s mol Zn/DMC l 6/1 th hiu sut phn ng thu c l thp nht
(39,53 %). Khi tng dn t l s mol Zn/DMC th hiu sut phn ng tng ln. Khi t
l mol tng n 18/1 th hiu sut phn ng tng ln khong 1,5 ln so vi t l mol l
6/1, t 60,95%. Tip tc tng t l mol Zn/DMC ln ln lt l 24/1, 30/1 th hiu
sut phn ng gim.
- T cc d liu trn, chng ti la chn t l mol Zn/DMC thch hp cho phn
ng l 18/1.
T cc kt qu kho st trn thy rng, iu kin thch hp nht cho phn ng
kh ha DMC bng Zn/acid l:
- Acid: HCl 20%.
- T l mol Zn/DMC: 18/1.
- Thi gian phn ng: 4h.
3.2.2.3. Kim tra tinh khit ca sn phm THC II
- Sau khi bn tng hp cc cht, tin hnh kim tra tinh khit ca cc sn phm
bng SKLM trn bn mng silicagel GF254, h dung mi dicloromethan: methanol =
20: 1.
- Sau khi chy sc k, soi bn mng di n t ngoi bc sng 254 nm, cc
cht th u cho mt vt gn, r, khng c vt ph, Rf THC II = 0,225 (Hnh 3.9).
40

- Sn phm thu c t hai phn ng kh vi hai tc nhn kh khc nhau l


H2/Pd/C 5% v Zn/HCl c chm chng trn cng mt bn mng v chy sc k vi
h dung mi dicloromethan: methanol = 20: 1. Kt qu cho thy cc sn phm cho cc
vt ng nht trn bn mng h dung mi s dng.

Hnh 3.9. SKLM ca THC II trn h dung mi CH2Cl2: MeOH = 20:1


3.3. KH HA HN HP CURCUMINOID
Sau khi xc nh iu kin kh ha thch hp nht cho tng curcuminoid thnh
phn, chng ti tin hnh kh ha hn hp curcuminoid.
3.3.1. Phn ng kh ha vi tc nhn kh l kh hydro c xc tc Pd/C 5%
Cc thng s ca phn ng:
- Dung mi phn ng: Ethanol tuyt i.
- T l khi lng xt/curcumin II: 5%.
- Thi gian phn ng: 18h.
S phn ng: c th hin hnh 3.10.

41

Hnh 3.10. S phn ng kh ha curcuminoid vi tc nhn H2/Pd/C


Tin hnh phn ng:
Hot ha xc tc: Cho 0,1 g xc tc Pd/C 5% vo bnh cu ba c y trn dung tch
500 ml, thm 50 ml dung mi, khuy t nhit khong 0-5oC trong 5 pht, ng
thi sc kh nit ui ht khng kh trong bnh phn ng. Ha tan 2,00 g
curcuminoid trong 200 ml ethanol tuyt i, cho dung dch thu c vo bnh phn
ng trn. Tip tc khuy hn hp nhit phng, ng thi sc kh hydro lin tc
trong qu trnh phn ng p sut kh quyn. Trong sut thi gian phn ng, theo di
hn hp phn ng bng SKLM vi h dung mi dicloromethan: methanol = 20:1.
X l hn hp phn ng:
- Hn hp phn ng em lc ht chn khng loi b xc tc. Dch lc em ct
loi dung mi bng my ct quay chn khng nhit khong 50oC. Tinh ch sn
phm bng phng php sc k ct silica gel vi h dung mi dicloromethan:
methanol = 20:1. Cc bc tin hnh sc k ct tng t nh trnh by phn 3.1.
- Dung mi chy qua ct c hng bng cc phn on th tch bng nhau V = 40
ml. Kt qu tch c th hin bng 3.14.
- Cc phn on c kt qu kim tra SKLM ging nhau c gp li, ct quay
chn khng, sy v cn nh lng.

42

Bng 3.14. Kt qu tch cc tetrahydrocurcuminoid t hn hp sn phm bng sc


k ct silica gel vi h dung mi dicloromethan: methanol = 20:1
Phn on

Tng th tch (ml)

Thnh phn

Khi lng (g)

1 - 36

1440

THC I

0,79

36 - 38

120

THC I, THC II

0,07

39 - 48

400

THC II

0,31

49-50

80

THC II, THC III

0,07

51-53

120

THC III

0,04

Nhn xt:
- Sn phm thu c ch yu l THC I (chim > 61,72%), tip theo l THC II (
chim > 24,21%), THC III chim mt lng rt nh (3,13%).
3.3.2. Phn ng kh ha vi tc nhn km trong mi trng acid
Cc thng s ca phn ng:
- Acid s dng: HCl 20%.
- T l mol Zn/curcuminoid: 18/1.
- Thi gian phn ng: 4h.
S phn ng: c th hin hnh 3.11.

Hnh 3.11. S phn ng kh ha curcuminoid vi tc nhn Zn/H+

43

Tin hnh phn ng:


Ha tan 2,00 g curcuminoid vo 200 ml EtOH tuyt i trong bnh cu ba c y
trn dung tch 500 ml. Sc nit ui ht khng kh trong bnh phn ng, thm 10
ml acid HCl 20% vo bnh phn ng.
Cn 3,95 g bt km, thm t t vo hn hp phn ng thnh nhiu t trong thi
gian khong 30 pht. Khuy hn hp phn ng nhit phng, duy tr pH acid trong
sut qu trnh phn ng. Theo di phn ng bng SKLM vi h dung mi ethyl acetat:
n-hexan = 7:3.
X l hn hp phn ng:
- Lc loi cht rn, thu dch lc.
- Trung ha dch lc bng dung dch Na2CO3 bo ha n pH trung tnh, lc loi
ta.
- Dch lc em ct loi bt mt phn dung mi, sau em chit vi
dicloromethan: Ln 1 chit vi 50ml dicloromethan, thm 30ml nc ct. Ln 2 v 3,
mi ln 50ml dicloromethan.
- Lp dung mi hu c c gp li, thm 10,00g Na2SO4 khan, lc u, yn
khong 1h loi nc. Sau lc, dch lc em ct loi dung mi bng my ct quay
chn khng nhit khong 40oC.
- Tinh ch sn phm bng phng php sc k ct silica gel vi h dung mi nhexan: ethyl acetat thay i t 50:0 n 1:1.
+ Dung mi chy qua ct c hng bng cc phn on th tch bng nhau V = 40
ml. Kt qu tch c th hin bng 3.15.
+ Cc phn on c kt qu kim tra SKLM ging nhau c gp li, ct quay
chn khng, sy v cn nh lng.

44

Bng 3.15. Kt qu tch cc tetrahydrocurcuminoid t hn hp sn phm bng sc


k ct silica gel vi h dung mi n-hexan: ethyl acetat
Phn on

Tng th tch (ml)

Thnh phn

Khi lng (g)

1 - 29

1200

THC I

0,74

30 - 32

120

THC I, THC II

0,08

33 - 41

360

THC II

0,28

41-44

160

THC II, THC III

0,08

45-47

120

THC III

0,03

Nhn xt:
- Sn phm thu c t phn ng kh ha vi tc nhn km trong mi trng acid
ch yu l THC I (chim > 61,15%), tip theo l THC II ( chim > 23,14%), THC III
chim mt lng rt nh (2,48%).
3.3.2. Kim tra tinh khit ca sn phm
- Sau khi bn tng hp cc cht, tin hnh kim tra tinh khit ca cc sn phm
bng SKLM trn bn mng silicagel GF254, h dung mi dicloromethan: methanol =
20: 1.
- Sau khi chy sc k, soi bn mng di n t ngoi bc sng 254 nm, cc
cht th u cho mt vt gn, r, khng c vt ph, v cho gi tr Rf trng vi Rf ca
THC I v THC II thu c khi kh ha tng thnh phn curcumin I v curcumin II
tng ng: Rf THC I = 0,55; Rf THC II = 0,225 (Hnh 3.12 v Hnh 3.13).
- Sn phm thu c t hai phn ng kh vi hai tc nhn kh khc nhau l
H2/Pd/C 5% v Zn/HCl c chm chng trn cng mt bn mng v chy sc k vi
h dung mi dicloromethan: methanol = 20: 1. Kt qu cho thy cc sn phm cho cc
vt ng nht trn bn mng h dung mi s dng.

45

Hnh 3.12. SKLM ca THC trn h

Hnh 3.13. SKLM ca THC II trn h

dung mi CH2Cl2: MeOH = 20:1

dung miCH2Cl2: MeOH = 20:1

3.4. XC NH CU TRC
xc nh cu trc sn phm, chng ti tin hnh ghi v phn tch ph hng
ngoi (IR), ph khi lng (MS), ph cng hng t ht nhn proton (1H-NMR), ph
cng hng t ht nhn carbon (13C-NMR). Kt qu ph c trnh by c th nh
sau:
3.4.1. c nh cu trc curcumin I (curcumin)
Ph MS, 1H-NMR, 13C-NMR ca curcumin I c trnh by cc ph lc 1,
2, 3, 4, v 5.
Kt qu phn tch ph MS, 1H-NMR,
ln lt trong cc bng 3.16, 3.17 v 3.18.

46

13

C-NMR ca curcumin I c trnh by

Bng 3.16. Kt qu phn tch ph ESI-MS (MeOH) ca curcumin


KLPT
(g/mol)

Cu trc ca curcumin

m/z
369,06 [M+H]+

368,38

391,04
[M+Na]+

Bng 3.17. Kt qu phn tch ph 1H-NMR (500 MHz, DMSO-d6) ca curcumin


(ppm), dng tn hiu, v tr H

Cu trc ca curcumin

3,83 (6H, s, 2 O-CH3)


6,05 (1H, s, =CH enol, H-4)
6,75 (2H, d, J = 15,5 Hz, H-2, H-6)
6,82 (2H, d, J = 8,0 Hz, H-5, H-5)
7,15 (2H, dd, J1 = 8,0 Hz, J2 = 1,5
Hz, H-6, H-6)
7,31 (2H, d, J = 1,5 Hz, H-2, H-2)
7,55 (2H, d, J = 16,0 Hz, H-1, H-7)
9,64 (2H, s, 2 -OH phenol)
Ghi ch: : chuyn dch ha hc; s: singlet; d: doublet, dd: doublet ca doublet.

47

Bng 3.18. Kt qu phn tch ph 13C-NMR (125 MHz, DMSO-d6) ca curcumin


(ppm), v tr C

Cu trc ca curcumin

55,68 (C-8, C-8)


100,79 (C-4)
111,35 (C-2, C-2)
115,70 (C-5, C-5)
121,08 (C-2, C-6)
123,08 (C-6, C-6)
126,32 (C-1, C-1)
140,67 (C-1, C-7)
147,97 (C-3, C-3)
149,33 (C-4, C-4)
183,18 (C-3, C-5)

3.4.2. c nh cu trc curcumin II (DMC)


Ph MS, 1H-NMR, 13C-NMR ca curcumin II c trnh by cc ph lc 6,
7, 8, 9 v 10.
Kt qu phn tch ph MS, 1H-NMR, 13C-NMR ca curcumin II c trnh by
ln lt trong cc bng 3.19, 3.20 v 3.21.
Bng 3.19. Kt qu phn tch ph ESI-MS (MeOH) ca DMC
KLPT
(g/mol)

Cu trc ca DMC

m/z

361,00
338,35

48

[M+Na]+

Bng 3.20. Kt qu phn tch ph 1H-NMR (500 MHz, CDCl3 & MeOD) ca DMC
(ppm), dng tn hiu, v tr H

Cu trc ca DMC

3,94 (3H, s, O-CH3)


5,80 (1H, s, =CH enol, H-4)
6,47 (1H, d, J = 16,0 Hz, H-2)
6,48 (1H, d, J = 15,5 Hz, H-6)
6,84 (2H, d, J = 8,5 Hz, H-3, H-5)
6,90 (1H, d, J = 8,0 Hz, H-5)
7,07 (1H, d, J = 2,0 Hz, H-2)
7, 11 (1H, dd, J1 = 8,0 Hz, J2 = 2,0
Hz, H-6)
7,44 (2H, d, J = 8,5 Hz, H-2, H-6)
7,57 (1H, d, J = 16,0 Hz, H-1)
7,59 (1H, d, J = 16,0 Hz, H-7)

Bng 3.21. Kt qu phn tch ph 13C-NMR (125 MHz, CDCl3 & MeOD) ca DMC
(ppm), v tr C

Cu trc ca DMC

56,02 (C-8)
101,21 (C-4)
110,14 (C-2)
115,25 (C-5)
116,02 (C-3, C-5)
121,16 (C-2)
121,58 (C-6)
123,00 (C-6)

49

126,89 (C-1)
127,58 (C-1)
130,10 (C-2, C-6)
140,69 (C-1)
140,80 (C-7)
147,40 (C-3)
148,35 (C-4)
159,29 (C-4)
182,75 (C-5)
183,43 (C-3)

3.4.3. c nh cu trc THC I (THC)


Ph IR, MS, 1H-NMR ca tetrahydrocurcumin I (kh thnh phn) c trnh
by cc ph lc 11, 12, 13 v 14.
Kt qu phn tch ph IR, MS, 1H-NMR ca tetrahydrocurcumin I (kh thnh
phn) c trnh by ln lt trong cc bng 3.22, 3.23 v 3.24.
Bng 3.22. Kt qu phn tch ph IR (KBr) ca THC (kh thnh phn)
Cu trc THC

Nhm chc

nh hp th
c trng (,
cm-1)

-OH phenol

3486

=C-H thm

3072

-C-H no

2931; 2854

-C=O
(-diceton)
-C=C-

50

1608
1513; 1455

-C-O

1271; 1157; 1231

Bng 3.23. Kt qu phn tch ph ESI-MS (MeOH) ca THC (kh thnh phn)
KLPT
(g/mol)

Cu trc THC

m/z

395,07
372,41

[M+Na]+

Bng 3.24. Kt qu phn tch ph 1H-NMR (500 MHz, DMSO-d6) ca THC (kh
thnh phn)
(ppm), dng tn hiu, v tr H

Cu trc THC

2,54-2,68 (4H, m, 2 -CH2, H-2, H-6)


2,74 (4H, m, 2 -CH2, H-1, H-7)
3,68; 5,72 (2H, s, H-4)
3,73 (6H, s, 2 O-CH3, H-8, H-8)
6,55-6,59 (2H, d, J = 8,0 Hz, H-6,
H-6)
6,66-6,68 (2H, d, J = 8,0 Hz, H-5,
H-5)
6,74-6,78 (2H, s, H-2, H-2)
8,69, 8,71 (2H, s, 2 -OH, H-7, H7)

51

Ph MS, 1H-NMR ca tetrahydrocurcumin I (tch t kh hn hp) c trnh


by cc ph lc 15, 16 v 17.
Kt qu phn tch ph MS, 1H-NMR ca tetrahydrocurcumin I (tch t kh hn
hp) c trnh by ln lt trong cc bng 3.25 v 3.26.
Bng 3.25. Kt qu phn tch ph ESI-MS (MeOH) ca THC (tch t kh hn hp)
KLPT
(g/mol)

Cu trc THC

m/z
373,09
[M+H]+

372,41
395,04
[M+Na]+

Bng 3.26. Kt qu phn tch ph 1H-NMR (500 MHz, DMSO-d6) ca THC (tch
t kh hn hp)
(ppm), dng tn hiu, v tr H

Cu trc THC

2,54-2,67 (4H, m, 2 -CH2, H-2, H-6)


2,73 (4H, m, 2 -CH2, H-1, H-7)
3,68; 5,72 (2H, s, H-4)
3,73 (6H, s, 2 O-CH3)
6,55-6,59 (2H, d, J = 8,0 Hz, H-6,
H-6)
6,64-6,66 (2H, d, J = 8,0 Hz, H-5,
H-5)
6,74-6,77 (2H, s, H-2, H-2)
8,65, 8,68 (2H, s, 2 -OH)

52

3.4.4. c nh cu trc THC II


Ph MS, 1H-NMR ca tetrahydrocurcumin II (tch ra t kh hn hp) c
trnh by cc ph lc 18, 19, 20 v 21.
Kt qu phn tch ph MS, 1H-NMR ca tetrahydrocurcumin II c trnh by
ln lt trong cc bng 3.27.
Bng 3.27. Kt qu phn tch ph ESI-MS (MeOH) ca THC II
KLPT
(g/mol)

Cu trc THC II

m/z
365,06
[M+Na]+

342,39
397,05
[M+MeOH+Na]+

Bng 3.28. Kt qu phn tch ph 1H-NMR (500 MHz, CDCl3) ca THC II


(ppm), dng tn hiu, v tr H

Cu trc THC II

2,51-2,62 (4H, m, 2 -CH2, H-2, H-6)


2,70-2,86 (4H, m, 2 -CH2, H-1, H-7)
3,86 (3H, s, 1 O-CH3)
3,85; 5,39 (2H, s, H-4)
5,49-5,50 (2H, s, 2 OH phenol)
6,66 (2H, m, H-3, H-5)
6,69 (1H, s, H-2)
6,73 (1H, d, J = 8,0 Hz, H-5)
6,82 (2H, d, J = 8,0 Hz, H-2, H-6)
7,02 (1H, d, J = 8,5 Hz, H-6)

53

3.5. TH TC DNG CH NG OXY HA CA CC CHT

N TNG HP

C
Phng php th:
nh gi tc dng dn gc t do DPPH phng php DPPH: 1,1-diphenyl-2picrylhydrazyl (DPPH) l cht to ra gc t do c dng sng lc tc dng chng
oxy ha ca cc cht nghin cu. Hot tnh chng oxy ha th hin qua vic lm gim
mu ca DPPH, c xc nh bng phng php o quang bc sng = 517 nm.
Cch tin hnh:
Pha dung dch DPPH c nng 1 mM trong Methanol (MeOH). Cht th c
pha trong DMSO 100% sao cho nng cui cng t c mt dy cc nng
128; 32; 8; 2; 0,5 g/ml. thi gian phn ng 30 pht 370C, c mt hp th
ca DPPH cha phn ng bng my c Genios Tecan bc sng 517 nm. % qut
gc t do DPPH ca mu th c tnh theo cng thc sau:
SC% = (OD trng OD mu th)/ ODtrng (%).
EC50 c tnh theo gi tr SC tng quan vi cc nng khc nhau ca cht
th, th nghim c lp li vi n = 3, l gi tr nng ti mu dn c 50% s
gc t do DPPH.
Kt qu:
Kt qu nh gi tc dng dn gc t do ca cc mu th c trnh by bng
3.29.

54

Bng 3.29. Kt qu nh gi tc dng dn gc t do ca cc mu th theo phng


php DPPH
Nng c hiu qu trung ha gc t do
DPPH
STT

Tn mu
128
g/ml

32
g/ml

8
g/ml

2
g/ml

0,5
g/ml

EC50
(g/ml)

Curcumin I

98

70

20

8,9

22,4

Curcumin hn hp

97,76

76,17

30

3,8

18,43

THC

95,5

86

40

9,5

13,22

SP kh hn hp

96,43

88,1

47

9,76

Resveratrol

8,3

Nhn xt:
Kt qu bng trn cho thy tt c cc mu th gm curcumin I, curcuminoid
hn hp, THC I, tetrahydrocurcuminoid hn hp (SP kh hn hp) u c tc dng
chng oxy ha mnh (c so snh vi cht tham kho l resveratrol cht ny c
s dng nh l mt cht chng oxy ha hiu qu). Trong sn phm kh hn hp
curcuminoid l tetrahydrocurcuminoid c hot tnh chng oxy ha mnh nht (EC50 =
9,76 g/ml) v hot tnh ny gim dn theo th t sau: tetrahydrocurcuminoid > THC
> curcuminoid hn hp > curcumin I.

55

Chng 4: BN LUN
4.1. V phng php tch tng thnh phn t hn hp curcuminoid
Kt tinh curcumin I:
- Vic tch ring tng thnh phn trong hn hp curcuminod nu tin hnh ngay
bng sc k ct silica gel s gp rt nhiu kh khn, tn km v dung mi, thi gian,
m hiu sut tch li thp, do vic kt tinh loi bt curcumin I trong hn hp c
ngha rt ln i vi qu trnh tch. Kt tinh c curcumin I t hn hp va thu c
curcumin I tinh khit va gim thiu c khi lng cht a ln ct, lm cho qu
trnh tch bng sc k ct c d dng hn v hiu sut tch cao.
- Vic la chn dung mi rt quan trng trong qu trnh kt tinh, trong hu ht cc
ti liu u kt tinh bng h dung mi MeOH : H2O (5:1). Tuy nhin vi h dung mi
ny, tinh th thu c l hn hp curcuminoid 3 thnh phn, v vn phi tin hnh sc
k ct tch ring 3 thnh phn ny, iu ny khng c ngha trong vic ti u ha
qu trnh tch bng sc k ct. iu ny c th do vic thay i dung mi t ngt
khin cho c 3 curcuminoid thnh phn u kt tinh tr li. Khi thc hin kt tinh bng
2 dung mi l MeOH v EtOH 96%, c th thu c tinh th curcumin I tinh khit,
trong MeOH cho hiu sut ln hn khng nhiu, mt khc MeOH li c hn nhiu
so vi EtOH, mt khc EtOH l dung mi r, d kim, nn vic chn EtOH l dung
mi kt tinh l hon ton hp l.
Sc k ct:
Khi nc ci c lm giu 2 thnh phn curcmin II v III sau khi kt tinh bt
curcumin I, vic sc k ct tr nn n gin hn. Mc d vy, do hm lng curcumin
III trong hn hp qu t nn vic tch thnh phn ny cng rt kh khn. Mt phn do
curcumin rt d b oxy ha, dung mi pha ng d bay hi nn vic tch gp nhiu kh
khn.
4.2.V phn ng kh ha tng curcuminoid thnh phn
Cc phn ng kh ha curcumin u khng chn lc. iu ny c th quan st
thy trn SKLM. Cc sn phm ph ca phn ng c th l sn phm hydro ha mt
ni i hoc sn phm kh ha c nhm chc ceton khng mong mun. iu ny dn

56

n kh khn trong vic kh ha chn lc nhm alken to THC cng nh tinh ch sn


phm. Tinh ch sn phm bng phng php sc k ct rt tn km, ch c th ng
dng trong nghin cu quy m nh, kh p dng c quy m sn xut ln.
Phn ng kh ha vi H2/Pd/C:
Phn ng kh ha curcumin vi tc nhn l kh hydro c xc tc c nhiu
tc gi nghin cu. Trong phn ng ny, kh ha chn lc nhm alken, trnh to
cc sn phm ph khng mong mun, cn ch kim sot t l xc tc v thi gian
phn ng. Khi tin hnh kho st nh hng ca t l khi lng xc tc so vi khi
lng curcumin, chng ti nhn thy khi tng t l xc tc ln cao hn 5% th hiu
sut phn ng gim. iu ny c th l do lng xc tc nhiu dn n kh ha thm
mt hoc c hai nhm ceton, to ra cc sn phm ph l monoalcol v dialcol. Hin
tng ny cng c th gp phi nu thi gian phn ng qu di.
Phng php ny c u im l sn phm tng i sch, iu kin kh ha
kh n nh; tuy nhin c nhc im l kh hydro to hn hp n vi khng kh
khong nng rt rng (4,1-74,2%), cn ch vn an ton khi thc hin phn
ng. Phng php ny kh ng dng quy m ln trong iu kin ca Vit Nam.
Phn ng kh ha vi Zn/acid:
Khi s dng acid acetic th thy phn ng khng xy ra. iu ny c th l do:
-

Th nht, lc acid ca acid acetic yu, khng mnh hot ha km.

Th hai, km phn ng vi acid acetic to ra mui km acetat, dng mui km

ny c th to phc vi curcumin, lm nh hng n kh nng phn ng ca


curcumin. Cn dng mui km clorid hay km sulfat kh to phc vi curcumin hn.
Trong khi , phn ng kh ha xy ra d dng hn trong mi trng acid
hydrocloric v acid sulfuric.
thc hin tt phn ng kh ha vi km trong mi trng acid hydrocloric,
thu c hiu sut cao, t sn phm ph, cn lu mt s vn sau:
kim sot tc phn ng v kh ha chn lc trn nhm alken, lng km nn
c cho vo t t thnh nhiu t trong qu trnh phn ng. Nu cho km qu nhanh

57

c th kh lun nhm chc ceton, to nhiu sn phm ph, lm gim hiu sut phn
ng v kh tinh ch sn phm.
-

T l km so vi curcumin cng nh hng nhiu n phn ng. Nu lng

km khng , phn ng xy ra khng hon ton, curcumin cn d kh loi b sau


phn ng. Nhng nu lng km d qu nhiu c th kh lun nhm chc ceton, to
nhiu sn phm ph. iu ny cng xy ra nu thi gian phn ng ko di.
-

Nng acid s dng cng c nh hng n phn ng. Kt qu kho st cho

thy, khi s sng acid long (5%, 10%), hiu sut phn ng thp. iu ny c th do
nng acid long, km c hot ha km, kh nng kh ha gim. Nhng nu s
dng acid HCl c, km c hot ha mnh, c th kh c nhm ceton, lm gim
hiu sut phn ng to THC. m bo phn ng xy ra tt, cn duy tr mi trng
acid trong sut qu trnh phn ng.
-

Nhm OH phenol rt d b oxy ha bi oxy trong khng kh, do cn phi

sc nit ui ht khng kh trong bnh trc khi tin hnh phn ng.
Phng php kh ha curcumin vi tc nhn l km trong mi trng acid hin
nay cha c ti liu no cng b. Phng php ny c u im l n gin, r tin,
hiu sut tt, c th ng dng trong sn xut quy m cng nghip.
4.3. V phn ng kh ha curcuminoid hn hp
Tng t nh phn ng kh ha curcuminoid thnh phn, phn ng kh ha
curcuminoid hn hp l phn ng khng chn lc.
Tuy nhin da vo vic kho st cc iu kin ca phn ng kh ha cc
curcuminoid thnh phn, p dng vo kh ha curcuminoid hn hp th phn ng kh
ha ny c kim sot tr nn kh chn lc nhm alken hn.
Phn ng kh ha vi H2/Pd/C:
Khi t l xc tc v thi gian phn ng c kim sot, th phn ng kh ha
xy ra chn lc hn, khng to sn phm ph khng mong mun. Sn phm thu c
l hn hp 3 thnh phn khng mu. Tng t phn ng c nhc im l kh hydro
to hn hp n vi khng kh khong nng rt rng (4,1-74,2%), cn ch vn
an ton khi thc hin phn ng. u im ca phn ng kh ha vi H2/Pd/C l sn

58

phm tng i sch, iu kin kh ha kh n nh tuy nhin phng php ny kh


ng dng quy m ln trong iu kin ca Vit Nam.
Phn ng kh ha vi Zn/acid:
Tng t nh phn ng kh ha vi H2/Pd/C, phn ng kh ha curcuminoid
hn hp vi Zn/acid c kim sot cht ch t l km so vi curcuminoid, cng nh
lng km cho vo phn ng tng t v thi gian phn ng, phn ng xy ra chn
lc hn trn nhm alken. Sn phm thu c l hn hp 3 thnh phn khng mu.
Phng php kh ha curcuminoid vi tc nhn l km trong mi trng acid hin
nay cha c ti liu no cng b. Phng php ny c u im l n gin, r tin,
hiu sut tt, c th ng dng trong sn xut quy m cng nghip.
4.4. V xc nh cu trc
4.4.1. V cu trc ca nguyn liu curcumin I
Trn ph khi lng (ESI-MS, MeOH) xut hin pic phn t [M+H]+ (m/z =
369,06) v [M+Na]+ (m/z = 391,04), ph hp vi s khi ca phn t cht d kin,
ng vi khi lng phn t curcumin (M = 368,38 ; CTPT C21H20O6).
Ph cng hng t ht nhn proton (1H-NMR) ca curcumin c cc tn hiu
cho php nhn bit dng proton v s lng proton, c th nh sau:
- Tn hiu singlet ti = 3,83 ppm c trng cho 6 proton ca 2 nhm methoxy
(H-8, H-8).
- Hai nhn thm cho cc tn hiu ti vng 7,14-7,56 ppm, trong quan st thy
tng tc spin-spin meta v ortho ca cc proton trn mi nhn thm. Ch ng hn, H5 tng tc ortho vi H-6 nn cho tn hiu doublet (d) vi hng s J = 8,0 Hz. Trong
khi , H-6 c thm tng tc meta vi H-2 ngoi tng tc ortho vi H-5 nn c
tn hiu dd vi J1=1,5 Hz, J2= 8,0 Hz. H-2 cho tn hiu doublet ti 7,31 ppm vi hng
s tng tc meta J = 1,5 Hz.
- Cc hydro-anken trong phn t curcumin tn ti dng trans. iu ny th
hin hng s tng tc spin-spin gia cc cp: H-1 v H-2; H-6 v H-7 l 15,5-16,0
Hz.

59

- Tn hiu singlet duy nht ti = 9,64 ppm c trng cho 2 proton ca 2 nhm
OH phenol.
- Tn hiu singlet ti = 6,05 ppm ng vi 1 proton cho thy: phn t curcumin
hu nh tn ti dng enol trong dung mi o ph (DMSO-d6) (~ 100%). Lin kt
hydro ni phn t ca dng ny m bo tnh i xng ca phn t (xem hnh 3.14).
iu ny cng quan st thy thng qua s ng nht ca cc cp tn hiu phn tch
trn (H-2 v H-2, H-5 v H-5, H-6 v H-6, 2 OH phenol, 2 O-CH3).

Hnh 3.14. Cu trc enol v lin kt hydro ca phn t curcumin I


Dung mi DMSO cho tn hiu ti 2,50 ppm.
Ph 13C-NMR ca curcumin tch c cho cc tn hiu carbon ph hp vi cu
trc d kin (xem bng 3.18) v ti liu tham kho [17].
Kt qu phn tch cc ph MS, 1H-NMR,

13

C-NMR cho php chng ti kh ng

nh cu trc ca nguyn liu curcumin I tch c t hn hp curcuminoid.


4.4.2. V cu trc ca nguyn liu curcumin II
Trn ph khi lng (ESI-MS, MeOH) xut hin pic phn t [M+Na]+ (m/z =
361,00), ph hp vi s khi ca phn t cht d kin, ng vi khi lng phn t
curcumin II (M = 338,35, CTPT C20H18O5).
Ph cng hng t ht nhn proton (1H-NMR) ca curcumin II cho thy: phn
t ny tn ti ch yu dng enol (H-4 cho tn hiu singlet ti 5,80 ppm) vi cc
hydro-anken dng trans (J = 16,0 Hz); ch c tn hiu ca 1 nhm methoxy ( = 3,94
ppm, singlet). Chnh v th trn ph ca curcumin II quan st thy tch r rt cc b tn
hiu ca 2 nhn thm, khc vi trng hp curcumin I. Ch ng hn, H-2 cho tn hiu
doublet ti 7,07 ppm vi Jmeta = 2,0 Hz, trong khi H-2 c tn hiu ti 7,44 ppm vi
Jortho = 8,5 Hz. B tn hiu cc proton cn li u ph hp vi cu trc curcumin II. Tn
hiu ca dung mi xut hin ti 7,32 (CHCl3) v 3,39 ppm (MeOH).

60

Ph 13C-NMR ca curcumin II cho thy: ch c 2 cp carbon (C-2 v C-6; C3 v C-5) l tng ng t, cc tn hiu ca cc carbon cn li u tch r rt v
ph hp vi cu trc phn t d kin (xem bng 3.21) v ti liu tham kho [17].
Kt qu phn tch cc ph MS, 1H-NMR,

13

C-NMR trn cho php chng ti

kh ng nh cu trc ca nguyn liu curcumin II tch c t hn hp curcuminoid.


4.4.3.V cu trc ca sn phm tetrahydrocurcumin I
Kt qu phn tch ph MS v 1H-NMR cho thy, cu trc tetrahydrocurcumin
kh thnh phn v tetrahydrocurcumin tch t kh hn hp l hon ton ng nht
(xem cc bng 3.23, 3.24, 3.25 v 3.26).
Ph hng ngoi (IR)
Trn ph hng ngoi ca sn phm xut hin cc di hp th c trng cho tng
nhm chc, c th nh sau:
- Di hp th c trng cho nhm chc -diceton (O=C-CH2-C=O) xut hin vi
s sng l 1608 cm-1. S sng ca cc nhm chc C=O ny gim mnh so vi nhm
ceton thng thng (c max = 1715 cm-1) l do phn t tetrahydrocurcumin tn ti
dng h bin ceto-enol (keto-enol), to ra lin kt hydro ni phn t (xem hnh 3.15)

Hnh 3.15. Dng h bin ceto-enol ca tetrahydrocurcumin


- Cc lin kt no -C-H ca sn phm kh ha 2 ni i cho di hp th c trng
ti s sng 2931 v 2854 cm-1.
- Hai nhn thm cho cc di hp th c trng ti s sng 3072 cm-1 (=C-H
thm) v 1513, 1455 cm-1 (-C=C- thm).
- Di hp th c trng cho nhm -OH phenol xut hin vi cng tng i
mnh, vi s sng l 3486 cm-1.
Ph khi lng (MS)
Trn ph xut hin pic phn t [M+Na]+ (m/z = 395,07), ph hp vi khi
lng phn t tetrahydrocurcumin (M=372,41), ng vi CTPT C21H24O6.

61

Ph cng hng t ht nhn proton (1H-NMR)


Ph ca sn phm kh ha c cc tn hiu cho php nhn bit dng proton v
s lng proton, c th nh sau:
- Cc tn hiu ti = 2,54 2,68 ppm c trng cho 8 proton cc v tr C1, C2,
C6, C7 ca mch heptan. Kt qu ny chng t rng c hai lin kt i alken trong phn
t curcumin u c kh ha hon ton thnh alkan.
- Tn hiu ti = 3,73 ppm c trng cho 6 proton ca 2 nhm methoxy trn
nhn thm.
- Cc tn hiu ti = 6,55 6,78 ppm c trng cho 6 proton ca nhn thm.
Khc vi b tn hiu 2 nhn thm ca nguyn liu curcumin, b tn hiu 2 nhn thm
ca THC khng hon ton ng nht: H-2 v H-2 cho tn hiu singlet ti 6,74 v
6,77 ppm; H-5 v H-5 cho tn hiu doublet ti 6,65 v 6,66 ppm (Jortho = 8,0 Hz); H6 v H-6 cho tn hiu ti 6,55 v 6,58 ppm (vi Jortho = 8,0 Hz).
- Tn hiu ti = 8,69 v 8,71 ppm c trng cho 2 proton ca 2 nhm OH
phenol khng tng ng t.
- ng ch l, trn ph quan st thy tn hiu singlet ti = 5,72 ppm. Tn hiu
ny gy ra bi hin tng h bin ceto-enol, ng vi proton dng enol (=CH-O) ca
C4. Tuy nhin, tn hiu ny khng cho gi tr s lng proton trn 1 (0,5 proton), do
ch c mt t l phn trm sn phm nht nh (~ 50%) dng h bin ceton-enol,
phn cn li tn ti dng diceton. Proton dng ceto (-CH2) ca carbon ny cho tn
hiu ti = 3,68 ppm. iu ny cng ph hp vi kt qu ph IR phn tch trn.
Kt qu phn tch cc ph IR, MS, 1H-NMR ph hp vi ti liu tham kho [22],
cho php chng ti kh ng nh sn phm thu c l tetrahydrocurcumin (1,7-bis(4hydroxy-3-methoxyphenyl)heptan-3,5-dion).
4.4.3. V cu trc ca sn phm tetrahydrocurcumin II
Trn ph khi lng (ESI-MS, MeOH) xut hin pic phn t [M+Na]+ (m/z =
365,06) v [M+MeOH+Na]+ (m/z = 397,05), ph hp vi s khi ca phn t cht d
kin, ng vi khi lng phn t tetrahydrocurcumin II (M = 342,39, CTPT
C20H22O5).

62

Ph cng hng t ht nhn proton (1H-NMR) ca sn phm kh ha c cc


tn hiu ph hp vi cu trc phn t d kin THC II. ng ch l ph ca THC II
c o trong CDCl3, nn tn hiu ca 2 nhm OH phenol c mt ti trng mnh hn
(ti 5,49 v 5,50 ppm, singlet) so vi trng hp ca curcumin v THC (o trong
DMSO-d6). Dng enol (=CH) ca THC II tn ti t l khng cao (~38%) ti = 5,39
ppm, dng ceto (-CH2-) cho tn hiu proton xen ph vi tn hiu ca nhm methoxy ti
3,85-3,86 ppm.
Kt qu phn tch cc ph MS, 1H-NMR ph hp vi ti liu tham kho [22], cho
php chng ti kh ng nh sn phm tetrahydrocurcumin II.
4.5. V tc dng sinh hc
kh ng nh tc dng chng oxy ha ca cc sn phm thu c sau khi kh
ha so vi nguyn liu trc kh, chng ti tin hnh th tc dng chng oxy ha vi
cc mu th curcumin, curcuminoid hn hp, THC v sn phm kh hn hp
tetrahydrocurcuminoid. Kt qu cho thy tt c cc mu th u cho tc dng chng
oxy ha mnh. THC thu c sau khi kh ha vn gi c hot tnh chng oxy ha
ca curcumin, thm ch tc dng cn tt hn (EC50 THC = 13,22 g/ml < EC50 Curcumin =
22,74 g/ml), c bit sn phm kh hn hp l tetrahydrocurcuminoid cn c tc
dng mnh hn so vi THC (EC50 SP kh hn hp = 9,76 g/ml < EC50 THC = 13,22 g/ml),
iu ny c ngha rt ln trong thc tin, l cho php thu sn phm l hn hp
tetrahydrocurcuminoid va khng mu m vn gi c hot tnh chng oxy ha,
thm ch cn tt hn so vi curcuminoid, gip cho vic n gin ha c vic tch v
kh ha tng curcuminoid thnh phn.

63

KT LUN V KIN NGH


1. Kt lun
- phn lp v chng minh c cu trc ca hai thnh phn curcumin v
demethoxycurcumin trong hn hp curcuminoid.
- chn c iu kin kh ha hai thnh phn curcumin v
demethoxycurcumin trong hn hp.
- xy dng c quy trnh bn tng hp tetrahydrocurcuminoid t
curcuminoid quy m phng th nghim, tch ring v chng minh c cu trc ca
hai thnh phn l THC v tetrahydrodemethoxycurcumin trong hn hp sn phm
tetrahydrocurcuminoid.
- th tc dng chng oxy ha ca THC, hn hp tetrahydrocurcuminoid bn
tng hp c.
2. Kin ngh
Trn y ch l nhng nghin cu kho st bc u chng ti thc hin la
chn phng php thch hp nht c th ng dng cho sn xut quy m cng nghip.
tip tc pht trin cc nghin cu t c, chng ti c mt s kin ngh sau:
- Tip tc nghin cu kho cc yu t nh hng n phn ng nh: kho st vi
cc tc nhn kh khc nh nhm, st, thic trong mi trng acid. Kho st nh hng
ca cc yu t nh dung mi, nhit , thi gian phn ng nng cao hiu sut phn
ng.
- Nghin cu tinh ch sn phm bng phng php kt tinh, n gin, t tn km
hn.
- Tip tc nghin cu nng cp quy trnh tng hp curcuminoid ln quy m
pilot.

64

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PH LC
Ph lc 1:

Ph MS ca curcumin

Ph lc 2:

Ph 1H-NMR ca curcumin

Ph lc 3:

Ph gin 1H-NMR ca curcumin

Ph lc 4:

Ph 13C-NMR ca curcumin

Ph lc 5:

Ph gin 13C-NMR ca curcumin

Ph lc 6:

Ph MS ca DMC

Ph lc 7:

Ph 1H-NMR ca DMC

Ph lc 8:

Ph gin 1H-NMR ca DMC

Ph lc 9:

Ph 13C-NMR ca DMC

Ph lc 10:

Ph gin 13C-NMR ca DMC

Ph lc 11:

Ph IR ca THC I (kh thnh phn)

Ph lc 12:

Ph MS ca THC I (kh thnh phn)

Ph lc 13:

Ph 1H-NMR ca THC I (kh thnh phn)

Ph lc 14:

Ph gin 1H-NMR ca THC I (kh thnh phn)

Ph lc 15:

Ph MS ca THC I (tch t kh hn hp)

Ph lc 16:

Ph 1H-NMR ca THC I (tch t kh hn hp)

Ph lc 17:

Ph gin 1H-NMR ca THC I (tch t kh hn hp)

Ph lc 18:

Ph MS ca THC II (tch t kh hn hp)

Ph lc 19:

Ph 1H-NMR ca THC II (tch t kh hn hp)

Ph lc 20:

Ph gin 1H-NMR 1 ca THC II (tch t kh hn hp)

Ph lc 21:

Ph gin 1H-NMR 2 ca THC II (tch t kh hn hp)