Nghiên Cứu Xây Dựng Quy Trình Bán Tổng Hợp Tetrahydrocurcuminoid Từ Nguồn Curcuminoid Chiết Xuất Trong Nước

7/23/2019 Nghin Cu Xy Dng Quy Trnh Bn Tng Hp Tetrahydrocurcuminoid T Ngun Curcuminoid Chit Xut Trong N

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BGIO DC V O TO BY T

TRNG I HC DC H NI

NGUYN THPHNG

NGHIN CU DNG QU TRNH

BN TNG HP TETRAHYDROCURCUMINOID

TNGUN CURCUMINOID CHIT XUTTRONG NC

LUN VNTHC SDC HC

H NI - 2014


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BGIO DC V O TO BY T

TRNG I HC DC H NI

NGUYN THPHNG

NGHIN CU DNG QU TRNH

BN TNG HP TETRAHYDROCURCUMINOID

TNGUN CURCUMINOID CHIT XUT

TRONG NC

LUN VN THC SDC HC

CHUYN NGNH: CNG NGHDC PHM V BO CH THUC

M S: 60720402

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

H NI2014


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LI CM N

Tri qua hai nm hc cao hc c sgip tn tnh ca cc thy gio, c

gio, gia nh cng bn b, ti hon thnh kha hc v Lun vn Thc sDchc ca mnh.

Vi tt csknh trng, trc tin ti xin by tlng bit n su sc n thy

gio PGS. TS. Nguyn nh Luyn trc tip hng dn, chbo 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 ThVn 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 bmn 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 bmti

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 vnThc s Dc hc.

H Ni, ngy 20 thng 08 nm 2014

Hc vin

Nguyn ThPhng


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MC LC

DANH MC CC K HIU, CC CHVIT TT

DANH MC CC BNG

DANH MC CC HNH V

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

Chng 1: TNG QUAN ......................................................................................... 2

1.1. TNG QUAN VCURCUMINOID .................................................................. 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 VTETRAHYDROCURCUMINOID ..................................... 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 vcc 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 cnghin 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


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Chng 3: KT QUNGHIN CU .................................................................. 22

3.1. TCH CC CURCUMIN I, II, III THN HP CURCUMINOID ............. 22

3.2. KHHA TNG CURCUMINOID THNH PHN .................................... 26

3.2.1. Khha curcumin I (curcumin) ...................................................................... 26

3.2.2. Khha curcumin II (DMC) .......................................................................... 34

3.3. KHHA 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. THTC DNG CHNG OXY HA CA CC CHT BN TNG HP

C ....................................................................................................................... 54

Chng 4: BN LUN ........................................................................................... 56

4.1. Vphng php tch tng thnh phn thn hp curcuminoid ....................... 56

4.2.Vphn ng khha tng curcuminoid thnh phn .......................................... 56

4.3. Vphn ng khha curcuminoid hn hp ...................................................... 58

4.4. V xc nh cu trc .......................................................................................... 59

4.5. Vtc dng sinh hc .......................................................................................... 63

KT LUN V KIN NGH ................................................................................ 64

1. Kt lun . 64

2. Kin ngh.. 64

TI LIU THAM KHO


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DANH MC CC K HIU, CC CH VIT TT

AcOH Acid aceticAOM Azoxymethan

APPH 2,2-azobis(2-amidinopropan)dihydroclorid

BDMC Bisdemethoxycurcumin13C-NMR 13C - Nuclear Magnetic Resonance spectroscopy

(Phcng hng tht 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 vcarbon

EtOH Ethanol

H Gi

HHC Hexahydrocurcumin

HIV Virus gy suy gim min dch ngi

(Human immunodeficiency virus)

H-NMR H - Nuclear Magnetic Resonance spectroscopy

Phcng hng tht 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 Phhng ngoi (Infrared spectroscopy)

KL, m Khi lng


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LDL Lipoprotein ttrng thp (Lowdensity lipoprotein)

MeOH Methanol

MS Phkhi lng (Mass spectrometry)

OD Mt quang (Optical density)

OHC Octahydrocurcumin

P p sut

Rf Hslu gi(Retension factor)

SKLM Sc k lp mng

sp, SP Sn phm

TBA Acid 2-thiobarbituricTHC Tetrahydrocurcumin

Tonc Nhit nng chy

T Thi gian

TLTK Ti liu tham kho

V Thtch

xt Xc tc


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DANH MC CC BNG

Bng 1.1. Mt stnh cht vt l ca cc curcuminoid .............................................. 2

Bng 1.2. Mt stnh 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 cnghin cu ............................................... 19

Bng 3.1. Kt qukho st nh hng ca dung mi, nhit v thi gian kt tinh

n qu trnh kt tinh curcuminoid ............................................................ 23

Bng 3.2. Kt qukt tinh curcuminoid 3 ln trong EtOH 96% .............................. 24

Bng 3.3. Kt qutch cc curcuminoid thnh phn thn hp curcuminoid bng

sc k ct silica gel vi hdung mi dicloromethan: methanol = 20:1 .... 25

Bng 3.4. Kt qukho st nh hng ca dung mi n hiu sut phn ng bn

tng hp tetrahydrocurcumin tcurcumin vi tc nhn khl H2/Pd/C .. 27

Bng 3.5. Kt qukho st nh hng ca tlxc tc n thi gian v hiu sut

phn ng bn tng hp THC tcurcumin vi tc nhn khl H2/Pd/C .. 28

Bng 3.6. Kt qu kho st nh hng ca acid n hiu sut phn ng bn tng

hp tetrahydrocurcumin tcurcumin vi tc nhn khl Zn/acid ........... 30

Bng 3.7. Kt qukho st nh hng ca nng acid n hiu sut phn ng bn

tng hp tetrahydrocurcumin tcurcumin vi tc nhn khl Zn/HCl ... 31

Bng 3.8. Kt qukho st nh hng ca t lmol km/curcumin n hiu sut

phn ng bn tng hp THC tcurcumin vi tc nhn khl Zn/HCl .... 31


tng hp THC II tDMC vi tc nhn khl H2/Pd/C ............................ 35


phn ng bn tng hp THC II tDMC vi tc nhn khl H2/Pd/C .... 36

Bng 3.11. Kt qukho st nh hng ca acid n hiu sut phn ng bn tng

hp THC II tDMC vi tc nhn khl Zn/acid ..................................... 38

Bng 3.12. Kt qukho st nh hng ca nng acid n hiu sut phn ng

bn tng hp THC II tDMC vi tc nhn khl Zn/HCl ...................... 39


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Bng 3.13. Kt qukho st nh hng ca tlmol km/curcumin II n hiu sut

phn ng bn tng hp THC II tCurcumin II vi tc nhn khl Zn/HCl

.................................................................................................................... 40

Bng 3.14. Kt qutch cc tetrahydrocurcuminoid thn hp sn phm bng sc

k ct silica gel vi hdung mi dicloromethan: methanol = 20:1 .......... 43


k ct silica gel vi hdung mi n-hexan: ethyl acetat............................. 45

Bng 3.16. Kt quphn tch phESI-MS (MeOH) ca curcumin ......................... 47

Bng 3.17. Kt quphn tch ph1H-NMR (500 MHz, DMSO-d6) ca curcumin . 47

Bng 3.18. Kt quphn tch ph13

C-NMR (125 MHz, DMSO-d6) ca curcumin................................................................................................................................... 48

Bng 3.19. Kt quphn tch phESI-MS (MeOH) ca DMC ............................... 48

Bng 3.20. Kt quphn tch ph1H-NMR (500 MHz, CDCl3& MeOD) ca DMC

................................................................................................................................... 49

Bng 3.21. Kt quphn tch ph13C-NMR (125 MHz, CDCl3& MeOD) ca DMC

................................................................................................................................... 49

Bng 3.22. Kt quphn tch phIR (KBr) ca THC (khthnh phn) ................. 50

Bng 3.23. Kt quphn tch phESI-MS (MeOH) ca THC (khthnh phn) .... 51

Bng 3.24. Kt quphn tch ph1H-NMR (500 MHz, DMSO-d6) ca THC (kh

thnh phn)................................................................................................. 51

Bng 3.25. Kt quphn tch phESI-MS (MeOH) ca THC (tch tkhhn hp)

................................................................................................................................... 52

Bng 3.26. Kt quphn tch ph1H-NMR (500 MHz, DMSO-d6) ca THC (tch

tkhhn hp) .......................................................................................... 52

Bng 3.27. Kt quphn tch phESI-MS (MeOH) ca THC II ............................. 53

Bng 3.28. Kt quphn tch ph1H-NMR (500 MHz, CDCl3) ca THC II .......... 53

Bng 3.29. Kt qunh gi tc dng dn gc t do ca cc mu ththeo phng

php DPPH ................................................................................................ 55


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DANH MC CC HNH 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 hbin cetoenol ca curcumin trong dung dch ........................... 3

Hnh 1.4. Dng tn ti ca curcumin theo pH dung dch............................................ 4

Hnh1.5. Sphn 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


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-NiCl2sdng 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 khha curcumin vi tc nhn H2/Pd/C ........................ 26

Hnh 3.4. S phn ng khha curcumin vi tc nhn Zn/H+ ............................ 29

Hnh 3.5. SKLM ca THC trn hdung mi CH2Cl2: MeOH = 20:1 ...................... 33

Hnh 3.6. SKLM ca THC trn hdung mi AcOEt: n-hexan = 7:3 ....................... 33

Hnh 3.7. S phn ng khha DMC vi tc nhn H2/Pd/C .............................. 34

Hnh 3.8. S phn ng khha DMC vi tc nhn Zn/H+ .................................. 37

Hnh 3.9. SKLM ca THC II trn hdung mi CH2Cl2: MeOH = 20:1 .................. 41

Hnh 3.10. S phn ng khha curcuminoid vi tc nhn H2/Pd/C ................ 42

Hnh 3.11. S phn ng khha curcuminoid vi tc nhn Zn/H+..................... 43

Hnh 3.12. SKLM ca THC trn hdung mi CH2Cl2: MeOH = 20:1 .................... 46

Hnh 3.13. SKLM ca THC II trn hdung miCH2Cl2: MeOH = 20:1 .................. 46

Hnh 3.14. Cu trc enol v lin kt hydro ca phn tcurcumin I ......................... 60

Hnh 3.15. Dng hbin ceto-enol ca tetrahydrocurcumin .................................... 61


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1

T VN

Txa xa, cngh c sdng phbin mt snc chu nh mt th

gia vchnh gip iu hng, to mi vv mu sc hp dn cho thc phm. Khng

nhng th, nghcn c bit n nh mt loi thuc qu dng trmn 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 curcuminoidtuy chchim tlnhnhng l nhm hot cht chnh

to nn cc tc dng sinh hc quan trng ca cngh. Bn cnh , khi xu hng ca

thgii ngy cng a chung nhng sn phm t thin nhin, vic pht trin nhng

hot cht c ngun gc tho dc ngy cng trthnh mi quan tm ln i vi ngnhDc Vit Nam, trong c nhm cht mu curcuminoid.

Curcuminoid l tn gi chung ca cc cht mang khung diarylheptadien c

phn lp t thn rcy Nghvng (Curcuma longa, hGng - Zingiberaceae), bao

gm curcumin I (curcumin), curcumin II (demethoxycurcumin), curcumin III

(bisdemethoxycurcumin) v mt scht 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 sdng ca cc curcuminoid bhn ch, c

bit trong mphm. Sn phm khha 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 v th, vic nghin cu kh ha curcuminoid thnh

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

vy, chng ti thc hin ti Nghin cu xy dng quy trnh bn tng hp

tetrahydrocurcuminoid tngun curcuminoid chit xut trong nc

Vi nhng mc tiu sau:

n tng hp tetrahydrocurcuminoid quy m phng th nghim;

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


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2

Chng1: TNG QUAN

1.1. TNG QUAN V CURCUMINOID

1.1.1. Cu trc ha hc v tnh chtCu trc ha hc:

Nhm cht mu curcuminoid l hn hp ca 3 cht - thnh phn quan trng nht

ca cnghvng (Curcuma longa, hGng - Zingiberaceae), c cng thc cu to

nh hnh 1.1.

Hnh 1.1. Cng thc cu to ca curcuminoid

Tnh cht vt l:

Mt stnh cht vt l ca 3 cht c trong curcuminoid thhin bng 1.1.[4]

Bng 1.1. Mt stnh cht vt l ca cc curcuminoid

Curcumin I(Curcumin) (1)

1,7-Bis-(4-hydroxy

-3-methoxyphenyl)-

hepta-1,6-dien-3,5-

dion

Curcumin II(Desmethoxy-curcumin)

(2)1-(4-hydroxyphenyl)-7-

(4-hydroxy-3-

methoxyphenyl)hepta-

1,6-dien-3,5-dion

Curcumin III(Bisdesmethoxy-

curcumin) (3)1,7-Bis-(4-

hydroxyphenyl) -hepta-

1,6-dien-3,5-dion

CTPT C21H20O 6 C20H18O5 C19H16O4

PTL 368 338 308

Dng thhnh

Tinh th hnh kim,mu vng.

Tinh th hnh kim, muvng cam.

Tinh thhnh kim, muvng cam tm.

imchy

(oC)

183 172 222

im chy ca curcuminoid: 172178


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3

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 dboxy 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 tvng cam n nu en.

Tnh cht ca nhm diceton:

Trong mi trng acid acetic, curcumin dphn ng vi cc hp cht c cu

trc kim (Y-NH2) nh hydroxylamin (Y = -OH), hoc phenylhydrazin (Y = C6H5-

NH-) to sn phm ln lt l 3,5-bis(3-methoxy-4-hydroxylstiryl)isoxazol v 3,5-

bis(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 hbin:

Curcumin v cc dn cht tn ti trong dung dch dng cn bng hbin cadng diceton i xng v dng cetoenol c n nh bng lin kt hydro ni phn

t. (Hnh 1.4) [13].

Hnh 1.3. Dng hbin ceto enol ca curcumin trong dung dch


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4

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

diceton; ngc li, pH > 8, dng enol chim u thhn27.Ty theo tng dung mi

thch hp, c thc trn 95% curcumin tn ti dng enol [13].

nh hng ca pH ti dng tn ti ca curcuminoid trong dung dch nc (Hnh1.4)[13].

ng hc ca phn ng thy phn curcumin trong dung dch nc trong khong

pH = 111 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 chyu 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 trpka l 7,8; 8,5 v 9,0.

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


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5

n nh:

Curcumin tng i n nh pH acid, nhng nhanh chng bphn hy pH>7.

Cc sn phm phn hy curcumin pH = 7 10 c xc nh bng phng phpHPLC. 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 tvng n vng nu) ri bphn hy thnh vanillin v aceton (Hnh 1.5) [13].

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 xquang hc, cc sn phm c xc nh ging nh cc sn

phm phn hy l acid ferulic, acid vanillic v vanillin [13].

Sn phmngng t


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6

1.1.2. Tc dng dc l

Curcuminoid c rt nhiu tc dng c chng minh, c li cho vic phng

v iu trbnh. Mt stc dng ni bt l:

- Tc dng chng oxy ha:

Peroxynitrit (ONOO-) l cht trung gian c tnh c tbo 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 tCurcuma longa, c tc

dng qut peroxynitrit. Cc hp cht trn th hin hot tnh qut peroxynitrit vi

IC50ln lt l 4,0; 6,4 v 29,7 m[2].

Trong cc nghin cu vsbo vqu trnh peroxy ha lipid, curcumin th

hin sc chmnh (1880%). Ty thuc liu dng m qu trnh peroxy ha lipid

bc ch. Qu trnh ny gy bi Fe-NTA (ferric nitrilotriacetat) v H2O2c o bng

s hnh thnh malonyl dialdehyd (MDA) l sn phm ca qu trnh oxy ha lipid

mng sinh hc trong cc vi ththn [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 vtbo thn kinh:

Bnh Alzheimer (sa st tr tu, gim tr nh, ri lon chc nng tui gi) bt

ngun tstch ttinh bt (Abeta), tn thngdo chng vim v soxy 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 tAbeta 42 tt hn Ibuprofen v Naproxen, ngn chn s

hnh thnh cht oligomer Abeta 42. Khi nui chut nht trng thnh Tg2576, stch

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 nhbao vy stch tv hnh thnh x in vitrov in vivo. C ch

tc dng ca curcumin l lm gim stch ttinh bt v chng oxy ha protein tbo

no. Curcumin c thddng lin kt vi cc kim loi oxy ha- khst v ng, cc


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7

kim loi ny tp trung trn vno, c khnng gy ngng kt tinh bt v oxy ha

protein. Ngoi ra c thngn chn tn thng vim bng cch ngn nga scm ng

kim loi ca NF- kappaB. Nhng dliu trn cho thy curcumin ph vskt t

cc thnh phn ca mng c hiu qu, ng thi ngn chn c tn thng do vimv oxy ha-khprotein trn vno. V thcurcumin tnghvng hon ton c ths

dng trn lm sng dphng v iu trbnh 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 chchyu l c chreceptor NF-[2].

Jayaraj Ravindran v ng nghip (2010) chng minh

Bisdesmethoxycurcumin c tc dng chng vim mnh hn curcumin (c chreceptor

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 th1 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 vddy, chng vim lot ddy- t trng

Cao nc hoc cao methanol nghvng cho thung, lm gim tit dch vv

tng lng cht nhy. Cho chut cng trng ung cao ngh, lm gim tit dch vv

bo vnim mc ddy, t trng.Nghkch thch sn sinh cht nhy thnh rut ca chut. Cho bnh nhn ung

bt nghngy 4 ln, trong 7 ngy thy c hiu qutt 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 vvt lot thng qua vic c chhot tnh


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8

MMP- 9 v kch thch hot tnh MMP- 2. Nhm S. Swarnakar nghin cu tc dng

ca curcumin tnghvng trn m hnh lot ddy gy bi indomethacin. Kt qu

cho thy curcumin chng lot mnh vi lot ddy cp tnh bng cch dphng s

suy kit glutathion, chng peroxy ha lipid, ti to lp biu m nhm chng li sbomn 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 ddy t trng [2][29].

Helicobacter pyloril vi khun gy vim lot trn tbo biu m thnh ddy.

Tbo biu m nhim vi khun ny dn ti shot ha yu tsao chp NF- kappaB

gy cm ng gen cytokin/chemokin- l gen kch thch gy vim, p ng gen ng t

bo (spht tn tbo). Curcumin c chshot ha NF- kappaB v spht tn t

bo, do hn chqu trnh to khng nguyn gy vim v ngn chn c spht

trin ca vi khun. V vy, curumin rt c tim nng trong iu trvim lot ddy

khi pht tHelicobacter pylori[2].

-Phng v iu trung 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 chcht gy ung th da,

rut gi, rut kt, gan chut, ng vt c v, c ch sinh si tbo u gm tbo

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 tbo theo c chdit cc tbo c tnh, v hiu hatbo ung th v ngn chn hnh thnh tbo ung th mi m khng lm nh hng

n cc tbo lnh tnh. Trong khi , nhiu thuc khi dit tbo c tnh cng dit

lun tbo lnh tnh, lm c thsuy kit. Nhng thnghim 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 sdng phbin trong cc chphm ngn chn

skhi pht, pht trin v di cn ca khi u [21].


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9

Curcumin c khnng loi bgc tdo, c chcc loi men v hot tnh ca

mt scht gy t bin tbo c khnng dn n ung th trong ung hay thc

n c chbin v bo qun khng m bo cht lng, gip c thngn nga ung

th mt cch tch cc. Mt sthnghim in vitrov in vivopht hin thy curcuminc khnng c chhot 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 trbng, kem nghc tc dng khng khun, khng nm, loi trt

chc hoi t bng, c tc dng ti to t chc v lin so. Cao ngh chit vi

cloroform 10% c dng ti chvng bnh nm, tt vi cc bnh nm da.

Nghc tc dng khng khun nhmt sthnh phn ha hc nh: Curcumin I

c tc dng c chin vitrospht trin mt sloi vi khun m trong in hnh l

trc khun lao Salmonella paratyphi. Cht ar- tumeron t tinh du v dch chit n-

hexan tl nghdit 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 shtrti to m ca collagen. Mt snghin 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 tdo gy vim lot, kt hp vi khnng

kch thch tng sinh tbo ca collagen htrng ktrong qu trnh iu trvt

thng m [2].

- Htrchc nng gan, mt

Curcumin cho chut cng trng n, c tc dng kch thch hot tnh men

arylhydroxylase l men phthuc vo cytocrom P450 ca ty lp thtrong tbo gan,

gip gii c v bo v tbo gan, chng vim nhim, hoi t, gip tbo gan hi

phc. A. Raysid chng minh, curcumin to ra tc dng ng hc i vi mt. Vi20mg curcumin c khnng lm co ngn ti mt 29% khi quan st trong 2 gi. Do

curcumin c tim nng trong iu trdphng shnh 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:


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10

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

vim gan , C. c bit, curcumin c nghin cu vtc dng c ch integrase

HIV- 1, c chprotease 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 tnghc tc dng c chstan hng cu gy bi hydrogen

peroxyd nng thp nhng khng c chnng cao, khng c tc dng lm

gim slng bch cu a nhn trung tnh vi nng thnghim [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 caoHPLC, h dung mi s dng l benzene v ethyl acetat vi phn cc tng dn.

Curcumin thu c vi hdung mi benzene : EtOAc (82:18 tt/tt), trong khi DMC v

DMC thu c tng ng vi hdung mi benzene : EtOAc (70:30 tt/tt) v (58:42

tt/tt). Cc phn on thu c tct 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 lbng phng php sc k ct silica gel vi hdung 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 thhai 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 sdng hu ht l

silica gel 60G vi cc hdung 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 chthu c

curcumin c hm lng 80%. Hn na khng tch c demethoxycurcumin v

bisdemethoxycurcumin.


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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 ttnc ct, hn hp c gi5oC trong

2h. Tinh thcurcumin 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. Hdung mi sdng 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 stnh cht vt l ca cc tetrahydrocurcuminoid thhin bng

1.2 [10].


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12

Bng 1.2. Mt stnh cht vt l ca cc tetrahydrocurcuminoid

THC I

(Tetrahydro-

curcumin)

[1,7-Bis-(4-hydroxy-

3-methoxyphenyl)-

hepta-3,5-dion]

THC II

(Desmethoxy-

tetrahydrocurcumin)[1-(4-hydroxyphenyl)-7-

(4-hydroxy-3-

methoxyphenyl)hepta-

3,5-dion]

THC III

(Bisdesmethoxy-

tetrahydrocurcumin)[1,7-Bis-

(4-hydroxyphenyl) -

hepta-3,5-dion]

CTPT C21H24O 6 C20H12O5 C19H20O4

PTL 372 342 312

Dng th

hnh

Bt kt tinh trng,

khng mi, khng v

Cht bn rn Cht

nhy khng muTinh thkhng mu

im chy

(0C)95 - 97 101 - 102

1.2.2. Ngun gc tetrahydrocurcumin:

Cc nghin cu cho thy, trong c thtetrahydrocurcumin, hexahydrocurcumin,

v octahydrocurcumin l nhng cht chuyn ha ca curcumin ng tiu ha.

Hexahydrocurcumin tm thy m gan cchut 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 tc nghin cu. Hot lc chng vim

ca curcumin, cc dn xut ca curcumin v phenylbutazon c thit lp theo tht

gim dn nh sau:

Natricurcumin THC Curcumin Phenylbutazon triethylcurcumin


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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 ctrn in vivov in vitro.

Cc nghin cu u chra rng THC c khnng 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-2-

picrylhydrazyl) v chra rng THC thhin khnng dn gc tdo DPPH cao nhtvi gi trIC50= 18,7 M. Hot tnh ny gim dn theo tht: THC > HHC = OHC >

curcumin > DMC > BDMC. Nghin cu cng chng minh rng THC c khnng

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 gicho

rng chnh sc mt ca nhm methoxyphenyl v c bit l shydro ha hni i

lin hp trong mch carbon trung tm lm tng hot tnh ca curcumin ln mt cch

ng k[24].

Mt nghin cu in vitroso snh tc dng chng oxy ha ca tetrahydrocurcumin

v cc curcuminoid khc c thc hin trn tbo hng cu thv 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 qucho thy rng tetrahydrocurcumin c

hot tnh chng oxy ha mnh nht trong tt ccc curcuminoid [19][22].

Tc dng c chca 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 qutt hn curcumin. Cc tc

gikt lun rng, THC c khnng dn cc gc tdo nh tert-butoxyl v peroxyl mt

cch trit [19].

Gii thch vc chtc 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. Hcho rng THC ng vai tr quan trng trong c chchng oxy ha


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14

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

phenolic ng vai tr dn cc gc tdo 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 sphn ct lin kt CC trong khung -dicetonc quan st thy trong qu trnh chng oxy ha. Nh vy, nhm -diceton ng vai

tr dn cc gc tdo 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

chspht trin tim n khc thng ACF (aberrant crypt foci) v stng nhanh t

bo. THC ph hp trong vic sdng chng li tc nhn gy ung th trong c thcng nh curcumin [18][34].

1.2.4. Tng quan vcc 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, tonc98 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, CHCl3MeOH) tcurcumin, trong 2v 3chim mt tlnh[15].

Hydro ha 1vi Pd/C thu c 4, 5, 6[15]. Dung dch 1trong 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.


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hexahydrocurcumin, octahydrocurcumin dng xt Pd/C

1.2.4.3.Phng php ca Su-Lin Lee v cng s (2005)

Tcurcumin 1, nhiu sn phm kh4,5,6c m ttrong 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 5t1l 30% v octahydrocurcumin 6l 91,8 %

di p sut cao v thi gian phn ng di. Mc d 5v 6c ththu c t3bng

phn ng khha vi NaBH4, nhng hiu sut rt thp [30]. S phn ng c th

hin trn hnh 1.8.


hexahydrocurcumin, octahydrocurcumin dng xt Pt/C


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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 ng1, 2v 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 LongaL.). 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 cctetrahydrocurcuminoid tng ng dng xc tc Zn-NiCl2s 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 di qu trnh phn ng cho thy, trong khong 30

pht u, kh hydro mi sinh thot ra mnh v sau khong thi gian ny cccurcuminoid 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 : NiCl2l 1 : 6 :

1,5. Cu trc ca sn phm c khng nh bi ph cng hng t ht nhn 1H v


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13C-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 NiCl2trong 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].


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Chng2: I TNG V PHNG PHP NGHIN CU

2.1. HA CHT V TRANG THIT

ti sdng mt snguyn liu, ha cht, dung mi v thit bca phng th

nghim Tng hp Ha dc, Bmn Cng nghip Dc, Trng i hc Dc H

Ni. Nguyn liu curcuminoid c ly tCng ty cphn 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

1 Acid acetic Merck

2 Acid hydrocloric Trung Quc

3 Acid sulfuric Trung Quc

4 Dicloromethan Trung Quc

5 Diethyl ether Trung Quc

6 Ethanol 96% Trung Quc

7 Ethanol tuyt i Trung Quc

8 Ethyl acetat Trung Quc

9 n-Hexan Trung Quc

10 Km bt Trung Quc

11 Methanol Trung Quc

12 Natri carbonat Trung Quc

13 Natri sulfat Trung Quc14 Nc ct Vit Nam

15 Palladium -carbon 5% Sigma-Aldrich

16 Silicagel 60 GF254 Merck

17 Than hot Vit Nam


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2.1.2. Thit b, my mc v dng cnghin cu

Bng 2.2. Thit b, my mc v dng cnghin cu

STT Thit b, dng c Xut x

1 Bn mng silicagel GF254 c

2 Bnh chit 250 mL c

3 Bnh cu 1 c, 2 c, 3 c50mL, 100mL, 250mL c

4 Blc ht chn khng Buchner Trung Quc

5 Bnh sc k Trung Quc

6 Cn kthut Sartorius P 2001S, nhy 10-2 Thy S

7 Cc c m100mL c8 Ct sc k c

9 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 M

13My o ph hng ngoi GX-Perkin Elmer (Vin Ha

hc - Vin Hn lm Khoa hc v Cng nghVit Nam) M

14

My o phcng hng tht nhn proton (1H-NMR)

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

hc v Cng nghVit Nam)

M

15My o phkhi lng Varian 320 Ms (Vin Ha hc

Vin Hn lm Khoa hc v Cng nghVit Nam)M

16 My khuy tc bphn gia nhit IKA c

17 My sy n hng ngoi c

18 My sinh kh nit c

19 My sinh kh hydro c

20 ng ong 25mL, 50mL, 100mL Trung Quc

21 ng nghim Trung Quc

22 Pipet chia vch 1mL, 5mL, 10mL Trung Quc


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23 Qubp cao su c

24 Tsy Memmert c

2.2.NI DUNG NGHIN CU

- Tch tng thnh phn trong hn hp curcuminoid.

- Bn tng hp tetrahydrocurcuminoid tcurcuminoid theo s sau:

Tin hnh phn ng khha curcuminoid vi hai tc nhn khkhc nhau l kh hydro

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

- S bkim tra tinh khit ca sn phm bng SKLM vi hdung mi thch hp

v o nhit nng chy.

-

Xc nh cu trc sn phm bn tng hp c bng cch o phhng ngoi (IR),phkhi lng (MS) v phcng hng tht nhn proton (1H-NMR).

- Thtc 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 tcurcuminoid bng phn ng khha 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.


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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 trhslu giRf

Dung mi ha tan: ethanol

Hdung mi chy sc k: Dicloromethan : Methanol = 20:1

2.3.4. c nh cu trc sn phm

Phhng ngoi(IR)

Phkhi lng (MS)

Ph cng hng t ht nhn proton (1H-NMR), Ph cng hng t carbon (13C-

NMR)

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

nh gi tc dng dn gc tdo 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.


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Chng3: KT QU NGHIN CU

3.1. TCH CC CURCUMIN I, II, III THN HP CURCUMINOID

Dng phng php tch thch hp thu c tng thnh phn ring bit:

Curcumin I, Curcumin II, Curcumin III. S tch c thhin hnh 3.1.

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 thkt tinh c lc ht chn khng, ra, kim tra s btinh khit bng

SKLM.

- Dch lc kim tra s bbng SKLM, ct quay chn khng n kit thu sn phm

th. Tip tc em kt tinh li ln 2,3 tng tnh ln 1.

HA TAN

KT TINH

- Curcuminoid

- Dung mi

LC

SC K CTDicloromethan :Methanol (20:1)

CURCUMIN IICURCUMIN I CURCUMIN III

CURCUMIN I


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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 qukho st

c trnh by trong bng 3.1.Bng 3.1. Kt qukho st nh hng ca dung mi, nhit v thi gian kt tinh

n qu trnh kt tinh curcuminoid

STTDung mi

kt tinh

Nhit

kt tinh(oC)

Thi gian

kt tinh(h)

KL

curcumin I(g)

Tonc

(oC)

1 MeOH tophng 24 1,08 182184

2 EtOH 96% to phng 24 1,02 182184

Nhn xt:

- Vi hai dung mi sdng l EtOH 96% v MeOH, khi kt tinh kt tinh

nhit phng trong 24h, tinh ththu c l curcumin I vi nhit nng chy phhp 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 thcurcumin I. Kt qukt tinh c trnh by

bng 3.2.


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

Ln kt tinh Vdung mi(ml) KL tinh th(g) Hiu sut (%)

1 250 1.02

2 150 0,26

3 50 0,08

Tng: 1,36 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 hdung 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,

thng 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 nhnn 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 bmt lp silicagel.

c 3: a cht phn tch vo ct

-

Ha tan mu vo mt lng ti thiu dung mi pha ng.- Hmc 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 thtch khong 40

ml.

-

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

bng SKLM vi hdung mi CH2Cl2 : MeOH = 20:1.


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c 5: Thu hi cc phn on

- Cc phn on c Rfging nhau c gp li, ct quay chn khng, sy kh v

cn nh lng.

Kt qutch c thhin bng 3.3.Bng 3.3. Kt qutch cc curcuminoid thnh phn thn hp curcuminoid bng

sc k ct silica gel vi hdung mi dicloromethan: methanol = 20:1

Phn onTng thtch

(ml)Thnh phn Khi lng (g)

1 - 18 720 Curcumin I 0,46

19 - 22 160 Curcumin I, Curcumin II 0,1323 - 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 chyu 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 qucho thy cc cht thu 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.


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Hnh 3.2. SKLM ca cc curcuminoid thnh phn

3.2. KH HA TNG CURCUMINOID THNH PHN

Chng ti thc hin phn ng khha vi hai tc nhn kh l kh hydro c

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

3.2.1. Khha 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 cy trn dung tch

250 ml, thm 20 ml dung mi, khuy tnhit 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


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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 bxc tc. Dch lc em ct

loi dung mi bng my ct quay chn khng nhit khong 50oC. Tinh chsn

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 tnh phn 3.1.

Kho st phn ng:

Chng ti tin hnh kho st cc yu tnh hng n phn ng, bao gm dung

mi phn ng v tlkhi 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 qukho st c trnh by trong bng 3.4.


tng hp tetrahydrocurcumin tcurcumin vi tc nhn khl H2/Pd/C

TTDung mi phn

ng

TlKLxt/curcumin

(%)

KL sp

(g)

Hp

(%)Tonc(

oC)

1 MeOH 10 0,65 64,33 95.098,0

2 EtOH tuyt i 10 0,62 61,36 95,098,0

3 EtOH 96% 10 0,59 58,39 95,098,5

Nhn xt:

- Vi cba dung mi sdng, nhit nng chy ca sn phm thu c u ph

hp vi nhit nng chy ca sn phm dkin (Tonc,THC= 95,097,0oC [18]).

- Dung mi l EtOH 96% cho hiu sut phn ng thp nht (58,39%). Khi sdng

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.


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Tcc 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 tlxc 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 tckho st nh hng ca tlxc tc n thi gian v hiu sut phn ng. Tin hnh

kho st vi cc tlkhi lng xc tc l 2%, 5%, 8% v 10% so vi curcumin .



phn ng bn tng hp THC tcurcumin vi tc nhn khl H2/Pd/C

TT KLcurcumin (g)

KL xt(g)

TlKL

xt/curcumin

(%)

tp(h)

KLsp(g)

Hp(%)

Tonc(oC)

1 1,00 0,02 2 24 0,57 56,41 95,098,0

2 1,00 0,05 5 18 0,66 65,32 95,098,0

3 1,00 0,08 8 15 0,63 62,35 96,098,5

4 1,00 0,10 10 12 0,62 61,36 96,098,0

Nhn xt:

-Nhit nng chy ca cc sn phm thu c tcc th nghim kho st cc t

lxc tc khc nhau khng c schnh lch ng k.

- Khi tng tlxc tc th thi gian phn ng gim, hiu sut phn ng cng thay

i. Vi tlxc 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 lxc tc l 5%,

thi gian phn ng l 18h. Tip tc tng tlxc tc th hiu sut phn ng gim.

Tcc d liu trn, chng ti la chn t lxc tc l 5%, vi thi gian phn

ng l 18h.

Tcc kt qukho st trn thy rng, iu kin thch hp nht cho phn ng

khha curcumin vi tc nhn l kh hydro c xc tc Pd/C 5% l:

- Dung mi: Ethanol tuyt i

- Tlkhi lng xt/curcumin: 5%


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- Thi gian phn ng: 18h.

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


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 cy 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 ttvo 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 hdung mi ethyl acetat: n-

hexan = 7:3 xc nh thi gian phn ng thch hp, so snh vi THC tinh khit thu

c phn ng khha 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 Na2CO3bo 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.


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- Tinh chsn phm bng phng php sc k ct silica gel vi hdung mi n-

hexan: ethyl acetat thay i t50:0 n 1:1. Cch tin hnh sc k ct tng tnh

trnh by mc 3.1.

Kho st phn ng

Chng ti tin hnh kho st cc yu tnh hng n phn ng, bao gm acid

phn ng, nng acid v tlmol 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 qukho st c trnh by trong bng 3.6.Bng 3.6. Kt qukho st nh hng ca acid n hiu sut phn ng bn tng

hp tetrahydrocurcumin tcurcumin vi tc nhn khl Zn/acid

TT AcidV acid

(ml)

KL km

(g)

tp

(h)

KL

sp (g)

Hp

(%)

Tonc

(oC)Ghi ch

1 Acid acetic bng 15 5 4 0 0

Khng

xut hin

vt THC

2Acid hydrocloric

20%5 5 4 0,58 57,40 95,0-98,0

3 Acid sulfuric 10% 5 5 4 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 SKLMvi hdung 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 sdng 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.


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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 qukho st c trnh by trong bng 3.7.


bn tng hp tetrahydrocurcumin tcurcumin vi tc nhn khl Zn/HCl

TTNng acid

(% m/V)

V acid

(ml)

KL

km (g)

tp

(h)

KL

sp (g)

Hp

(%)Tonc(

oC)

1 5 20 5 7 0,38 37,61 95,098,0

2 10 10 5 6 0,45 44,54 95,597,0

3 20 5 5 4 0,58 57,40 95,098,0

4 Acid c 3 5 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 schnh 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%).

Tkt qukho st trn, dung dch HCl 20% c la chn thc hin cc phn

ng kho st tip theo.

c. Kho st tlmol 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 tlgia smol km v smol curcumin

n hiu sut phn ng. Tin hnh kho st vi cc tlmol Zn/curcumin ln lt l

6/1, 12/1, 18/1, 24/1 v 28/1.


Bng 3.8. Kt qukho st nh hng ca tlmol km/curcumin n hiu sut

phn ng bn tng hp THC tcurcumin vi tc nhn khl Zn/HCl


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TT

KL

curcumin

(g)

KL

km

(g)

Tlmol

km/

curcumin

tp(h)KL sn

phm (g) Hp (%)Tonc(

oC)

1 1,00 1,06 6/1 6,5 0,42 41,57 95,598,52 1,00 2,11 12/1 5,5 0,51 50,47 95,098,0

3 1,00 3,17 18/1 4 0,61 60,37 95,098,0

4 1,00 4,23 24/1 4 0,59 58,39 95,597,5

5 1,00 5,00 28/1 4 0,58 57,40 95,098,0

Nhn xt:

-Nhit nng chy ca cc sn phm thu c tcc th nghim kho st cc t

lmol khc nhau khng c schnh lch ng k.

- Vi tlsmol Zn/curcumin l 6/1 th hiu sut phn ng thu c l thp nht

(41,57%). Khi tng dn tlsmol Zn/curcumin th hiu sut phn ng tng ln. Khi

tlmol tng n 18/1 th hiu sut phn ng tng ln khong 1,5 ln so vi tlmol

l 6/1, t 60,37%. Tip tc tng tlmol Zn/curcumin ln ln lt l 24/1, 28/1 th

hiu sut phn ng gim.

-

Tcc dliu trn, chng ti la chn tlmol Zn/curcumin thch hp cho phnng l 18/1.


khha curcumin bng Zn/acid l:

- Acid: HCl 20%

- Tlmol Zn/curcumin:18/1

- Thi gian phn ng: 4h

3.2.1.3.Kim tra tinh khit sn phmTHC

- 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 hdung mi dicloromethan: methanol =

20: 1 v hethyl acetat: n-hexan = 7: 3.

- Sau khi chy sc k, soi bn mng di n tngoi bc sng 254 nm, cc

cht thu cho mt vt gn, r, khng c vt ph(Hnh 3.5 v 3.6).Vi hdung mi


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dicloromethan: methanol = 20: 1 cho Rf THC= 0,55 v hethyl 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 vihdung mi dicloromethan: methanol = 20: 1. Kt qucho thy cc sn phm cho cc

vt ng nht trn bn mng hdung mi sdng.

- 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 9598oC.

3.2.1.4. Tm tt kt qu:

Tnh cht sn phm:

- Cm quan: Bt kt tinh mu trng.

-Nhit nng chy: 9598oC.

- Rf (CH2Cl2: MeOH=20:1) = 0,55.

- Rf(AcOEt: n-hexan=7:3) = 0,69.

Hnh 3.5. SKLM ca THC trn h dung

mi CH2Cl2: MeOH = 20:1

Hnh 3.6. SKLM ca THC trn h dung

mi AcOEt: n-hexan = 7:3


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- 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/HCltrong iu kin thch hp cho hiu

sut tt nht t 60,37%.

3.2.2. Khha 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 haDMC vi tc nhn H2/Pd/C

Tin hnh phnng:

Hot ha xc tc: Cho xc tc Pd/C 5% vo bnh cu ba cy trn dung tch 250

ml, thm 10 ml dung mi, khuy tnhit 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 hdung mi dicloromethan:

methanol = 20:1 xc nh thi gian phn ng thch hp.

X l hn hp phn ng:




methanol = 20:1. Cc bc tin hnh sc k ct tng tnh phn 3.1.Cc phn on

c kt quSKLM ging nhau c gp li, ct quay chn khng, sy kh v cn.


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Kho st phn ng:

Chng ti tin hnh kho st cc yu tnh hng n phn ng, bao gm dung

mi phn ng v tlkhi 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%.



tng hp THC II tDMC vi tc nhn khl H2/Pd/C

TT Dung mi phn ng TlKLxt/DMC (%)

KL sp (g) Hp(%)

1 MeOH tuyt i 10 0,42 69,17

2 EtOH tuyt i 10 0,39 64,22

3 EtOH 96% 10 0,36 59,29

Nhn xt:

-

Khi sdng 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.

Tcc 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 tlxc 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 tlxc 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.



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phn ng bn tng hp THC II tDMC vi tc nhn khl H2/Pd/C

TTKL DMC

(g)

KL xt

(g)

TlKL xt/DMC

(%)

tp

(h)

KL sp

(g)

Hp (%)

1 0,60 0,01 2 24 0,33 54,35

2 0,60 0,03 5 18 0,42 69,18

3 0,60 0,05 8 15 0,40 65,89

4 0,60 0,06 10 12 0,39 64,24

Nhn xt:

- Khi tng tlxc tc th thi gian phn ng gim, hiu sut phn ng cng thay

i. Vi tlxc 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 lxc tc l 5%,

thi gian phn ng l 18h. Tip tc tng tlxc tc th hiu sut phn ng gim.

Tcc d liu trn, chng ti la chn t lxc tc l 5%, vi thi gian phn

ng l 18h.


khha DMC vi tc nhn l kh hydro c xc tc Pd/C 5% l:

- Dung mi: Ethanol tuyt i

- Tlkhi lng xt/DMC: 5%



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


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 cy 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 ttvo 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 hdung mi ethyl acetat: n-

hexan = 7:3 xc nh thi gian phn ng thch hp, so snh vi THC II tinh khitthu c phn ng khha vi tc nhnH2/Pd/C.

X l hn hp phn ng

- Lc loi cht rn, thu dch lc.

- Trung ha dch lc bng dung dch Na2CO3bo ha n pH trung tnh, lc loi

ta.

-

Dch lc em ct loi bt mt phn dung mi, sau em chit vidicloromethan: Ln 1 chit vi 20 ml dicloromethan, thm 10 ml nc ct. Ln 2 v 3,

mi ln 20 ml dicloromethan.





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hexan: ethyl acetat thay i t50:0 n 1:1. Cch tin hnh sc k ct tng tnh

trnh by mc 3.1.

Kho st phn ng

Chng ti tin hnh kho st cc yu tnh hng n phn ng, bao gm

acid phn ng, nng acid v tlmol 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 qukho st c trnh by trong bng 3.11.Bng 3.11. Kt qukho st nh hng ca acid n hiu sut phn ng bn tng

hp THC II tDMC vi tc nhn khl Zn/acid

TT AcidV acid

(ml)KL km

(g)tp(h)

KLsp (g)

Hp(%)

Ghi ch

1 Acid acetic bng 10 2 4 0 0Khng

xut hinvt THC

2 Acid hydrocloric20% 3 2 4 0,35 57,65

3 Acid sulfuric 10% 3 2 4 0,27 44,47

Nhn xt:

- Khi thc hin phn ng vi acid acetic, kim tra hn hp phn ng bng SKLM

vi hdung mi ethyl acetat: n-hexan = 7:3 v so snh vi THC II thu c phnng khha 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 sdng 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.


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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 qukho st c trnh by trong bng 3.12.


bn tng hp THC II tDMC vi tc nhn khl Zn/HCl

TTNng acid

(% m/V)V acid(ml)

KL km(g)

tp(h)KL sp(g)

Hp (%)

1 5 5 2 7 0,21 34,592 10 3 2 6 0,28 46,12

3 20 2 2 4 0,36 59,30

4 Acid c 1 2 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%).

Tkt qukho st trn, dung dch HCl 20% c la chn thc hin cc phn

ng kho st tip theo.

b.

Kho st tlmol 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 tlgia smol km v smol DMC n

hiu sut phn ng. Tin hnh kho st vi cc tlmol Zn/DMCln lt l 6/1, 12/1,

18/1, 24/1 v 30/1.



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Bng 3.13. Kt qukho st nh hng ca tlmol km/curcumin II n hiu

sut phn ng bn tng hp THC II tCurcumin II vi tc nhn khl Zn/HCl

TT

KL DMC

(g)

KL km

(g)

Tlmol

km/ DMCt

p(h)

KL sn

phm (g) Hp (%)1 0,60 0,69 6/1 6,5 0,24 39,53

2 0,60 1,38 12/1 5,5 0,31 51,06

3 0,60 2,07 18/1 4 0,37 60,95

4 0,60 2,76 24/1 4 0,35 57,65

5 0,60 3,45 30/1 4 0,34 56,00

Nhn xt:

Khi thay i tlsmol Zn/DMC th hiu sut phn ng thay i:

- Vi t l smol Zn/DMC l 6/1 th hiu sut phn ng thu c l thp nht

(39,53 %). Khi tngdn tlsmol Zn/DMC th hiu sut phn ng tng ln. Khi t

lmol tng n 18/1 th hiu sut phn ng tng ln khong 1,5 ln so vi tlmol l

6/1, t 60,95%. Tip tc tng t lmol Zn/DMC ln ln lt l 24/1, 30/1 th hiu

sut phn ng gim.- Tcc d liu trn, chng ti la chn t lmol Zn/DMC thch hp cho phn

ng l 18/1.


khha DMC bng Zn/acid l:

- Acid: HCl 20%.

- Tlmol Zn/DMC: 18/1.

-

Thi gian phn ng: 4h.

3.2.2.3.Kim tra tinh khit ca sn phm THC II


bng SKLM trn bn mng silicagel GF254, hdung mi dicloromethan: methanol =

20: 1.


cht thu cho mt vt gn, r, khng c vt ph, Rf THC II= 0,225 (Hnh 3.9).


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H2/Pd/C 5% v Zn/HCl c chm chng trn cng mt bn mng v chy sc k vi

hdung mi dicloromethan: methanol = 20: 1. Kt qucho thy cc sn phm cho cc


3.3. KH HA HN HP CURCUMINOID

Sau khi xc nh iu kin kh ha thch hp nht cho tng curcuminoid thnh

phn, chng titin hnh kh ha hn hp curcuminoid.

3.3.1. Phn ng khha vi tc nhn khl kh hydro c xc tc Pd/C 5%

Cc thng s ca phn ng:

- Dung mi phn ng: Ethanol tuyt i.

- Tlkhi lng xt/curcumin II: 5%.


S phn ng: c th hin hnh 3.10.

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


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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 cy trn dung tch

500 ml, thm 50 ml dung mi, khuy tnhit 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 hdung mi dicloromethan: methanol = 20:1.

X l hn hp phn ng:

-




methanol = 20:1. Cc bc tin hnh sc k ct tng tnh trnh by phn 3.1.

- Dung mi chy qua ct c hng bng cc phn on thtch bng nhau V = 40

ml. Kt qutch c thhin 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.


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k ct silica gel vi hdung mi dicloromethan: methanol = 20:1

Phn on Tng thtch (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 chyu 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 khha vi tc nhn km trong mi trng acid

Cc thng s ca phn ng:

- Acid sdng: HCl 20%.

- Tlmol Zn/curcuminoid: 18/1.



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


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Tin hnh phn ng:

Ha tan 2,00 g curcuminoid vo 200 ml EtOH tuyt i trong bnh cu ba cy

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 ttvo 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 hdung 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 Na2CO3bo 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.





hexan: ethyl acetat thay i t50:0 n 1:1.

+Dung mi chy qua ct c hng bng cc phn on thtch bng nhau V = 40

ml. Kt qutch c thhin bng 3.15.

+

Cc phn on c kt qu kim tra SKLM ging nhau c gp li, ct quaychn khng, sy v cn nh lng.


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k ct silica gel vi hdung mi n-hexan: ethyl acetat

Phn on Tng thtch (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 tphn ng khha vi tc nhn km trong mi trng acid

chyu 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


bng SKLM trn bn mng silicagel GF254, hdung mi dicloromethan: methanol =

20: 1.


cht thu cho mt vt gn, r, khng c vt ph, v cho gi trRftrng vi Rfca

THC I v THC II thu c khi khha 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).


H2/Pd/C 5% v Zn/HCl c chm chng trn cng mt bn mng v chy sc k vi

hdung mi dicloromethan: methanol = 20: 1. Kt qucho thy cc sn phm cho cc



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3.4. XC NH CU TRC

xc nh cu trc sn phm, chng ti tin hnh ghi v phn tch phhng

ngoi (IR), phkhi lng (MS), phcng hng tht nhn proton (1H-NMR), ph

cng hng tht nhn carbon (13

C-NMR). Kt quphc trnh by c thnhsau:

3.4.1. c nh cu trc curcumin I (curcumin)

PhMS, 1H-NMR, 13C-NMR ca curcumin I c trnh by cc phlc 1,

2, 3, 4, v 5.

Kt quphn tch phMS, 1H-NMR, 13C-NMR ca curcumin I c trnh by

ln lt trong cc bng 3.16, 3.17v 3.18.

Hnh 3.12. SKLM ca THC trn h

dung mi CH2Cl2: MeOH = 20:1

Hnh 3.13. SKLM ca THC II trn h

dung miCH2Cl2: MeOH = 20:1


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Bng 3.16. Kt quphn tch phESI-MS (MeOH) ca curcumin

Cu trc ca curcuminKLPT(g/mol)

m/z

368,38

369,06 [M+H]+

391,04

[M+Na]+

Bng 3.17. Kt quphn tch ph1H-NMR (500 MHz, DMSO-d6) ca curcumin

Cu trc ca curcumin (ppm), dng tn hiu, vtr H

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

6,05 (1H, s, =CHenol, 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 = 1,5Hz, 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.


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Bng 3.18. Kt quphn tch ph13C-NMR (125 MHz, DMSO-d6) ca curcumin

Cu trc ca curcumin (ppm), vtr C

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)

PhMS, 1H-NMR, 13C-NMR ca curcumin II c trnh by cc phlc 6,

7, 8, 9v 10.

Kt quphn tch phMS, 1H-NMR, 13C-NMR ca curcumin II c trnh by

ln lt trong cc bng 3.19, 3.20v 3.21.

Bng 3.19. Kt quphn tch phESI-MS (MeOH) ca DMC

Cu trc ca DMCKLPT(g/mol)

m/z

338,35361,00

[M+Na]+


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Bng 3.20. Kt quphn tch ph1H-NMR (500 MHz, CDCl3& MeOD) ca DMC

Cu trc ca DMC (ppm), dng tn hiu, vtr H

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= 2,0Hz, 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 quphn tch ph13C-NMR (125 MHz, CDCl3& MeOD) ca DMC

Cu trc ca DMC (ppm), vtr C

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)


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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)

PhIR, MS, 1H-NMR ca tetrahydrocurcumin I (khthnh phn) c trnh

by cc phlc 11, 12, 13v 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 v3.24.

Bng 3.22. Kt quphn tch phIR (KBr) ca THC (khthnh phn)

Cu trc THC Nhm chcnh hp thc trng (,

cm-1)

-OH phenol 3486

=C-H thm 3072

-C-H no 2931; 2854

-C=O

(-diceton)1608

-C=C- 1513; 1455


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-C-O 1271; 1157; 1231

Bng 3.23. Kt quphn tch phESI-MS (MeOH) ca THC (khthnh phn)

Cu trc THC KLPT(g/mol)

m/z

372,41395,07

[M+Na]+

Bng 3.24. Kt quphn tch ph1H-NMR (500 MHz, DMSO-d6) ca THC (kh

thnh phn)

Cu trc THC (ppm), dng tn hiu, vtr H

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, H-7)


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PhMS, 1H-NMR ca tetrahydrocurcumin I (tch tkhhn hp) c trnh

by cc phlc 15, 16v 17.

Kt quphn tch phMS, 1H-NMR ca tetrahydrocurcumin I (tch tkhhn

hp) c trnh by ln lt trong cc bng 3.25v 3.26.Bng 3.25. Kt quphn tch phESI-MS (MeOH) ca THC (tch tkhhn hp)

Cu trc THCKLPT(g/mol)

m/z

372,41

373,09

[M+H]+

395,04

[M+Na]+

Bng 3.26. Kt quphn tch ph1H-NMR (500 MHz, DMSO-d6) ca THC (tch

tkhhn hp)

Cu trc THC (ppm), dng tn hiu, vtr H

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)


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3.4.4. c nh cu trc THC II

PhMS, 1H-NMR ca tetrahydrocurcumin II (tch ra tkhhn hp) c

trnh by cc phlc 18, 19, 20v 21.

Kt quphn tch phMS, 1H-NMR ca tetrahydrocurcumin II c trnh by

ln lt trong cc bng 3.27.

Bng 3.27. Kt quphn tch phESI-MS (MeOH) ca THC II

Cu trc THC IIKLPT(g/mol)

m/z

342,39

365,06

[M+Na]+

397,05

[M+MeOH+Na]+

Bng 3.28. Kt quphn tch ph1H-NMR (500 MHz, CDCl3) ca THC II

Cu trc THC II (ppm), dng tn hiu, vtr H

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)


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3.5. TH TC DNG CHNG 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-2-

picrylhydrazyl (DPPH) l cht to ra gc tdo c dng sng lc tc dng chng

oxy ha ca cc cht nghin cu. Hot tnh chng oxy ha thhin 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 thc

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 tdo DPPH ca mu thc tnh theo cng thc sau:

SC% = (OD trngOD mu th)/ ODtrng (%).

EC50c tnh theo gi trSC tng quan vi cc nng khc nhau ca cht

th, th nghim c lp li vi n = 3, l gi trnng ti mu dn c 50% s

gc tdo DPPH.Kt qu:

Kt qunh gi tc dng dn gc tdo ca cc mu thc trnh by bng

3.29.


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Bng 3.29. Kt qunh gi tc dng dn gc t do ca cc mu ththeo phng

php DPPH

STT Tn mu

Nng c hiu qutrung ha gc tdoDPPH

EC50(g/ml)128

g/ml32g/ml

8g/ml

2g/ml

0,5g/ml

1 Curcumin I 98 70 20 8,9 7 22,4

2 Curcumin hn hp 97,76 76,17 30 3,8 0 18,43

3 THC 95,5 86 40 9,5 0 13,22

4 SP khhn hp 96,43 88,1 47 0 0 9,76

Resveratrol 8,3

Nhn xt:

Kt qubng trn cho thy tt ccc mu thgm curcumin I, curcuminoid

hn hp, THC I, tetrahydrocurcuminoid hn hp (SP khhn hp) u c tc dng

chng oxy ha mnh (c so snh vi cht tham kho l resveratrolcht ny csdng nh l mt cht chng oxy ha hiu qu). Trong sn phm khhn hp

curcuminoid l tetrahydrocurcuminoid c hot tnh chng oxy ha mnh nht (EC50=

9,76 g/ml) v hot tnh ny gim dn theo thtsau: tetrahydrocurcuminoid > THC

> curcuminoid hn hp > curcumin I.


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Chng 4: BN LUN

4.1. Vphng php tch tng thnh phn thn 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 sgp rt nhiu kh khn, tn km vdung 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 thn 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 ddng hn v hiu sut tch cao.

-

Vic la chn dung mi rt quan trng trong qu trnh kt tinh, trong hu ht ccti liu u kt tinh bng hdung mi MeOH : H2O (5:1). Tuy nhin vi hdung mi

ny, tinh ththu 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 thdo vic thay i dung mi t ngt

khin cho c3 curcuminoid thnh phn u kt tinh trli. Khi thc hin kt tinh bng

2 dung mi l MeOH v EtOH 96%, c th thu c tinh thcurcumin I tinh khit,

trong MeOH cho hiu sut ln hnkhng nhiu, mt khc MeOH li c hn nhiu

so vi EtOH, mt khc EtOH l dung mi r, dkim, 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 trnn 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 docurcumin rt dboxy ha, dung mi pha ng dbay hi nnvic tch gp nhiu kh

khn.

4.2.Vphn ng khha tng curcuminoid thnh phn

Cc phn ng khha curcumin u khng chn lc. iu ny c thquan 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


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n kh khn trong vic khha 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, chc thng

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 khha curcumin vi tc nhn l kh hydro c xc tc c nhiu

tc ginghin cu. Trong phn ng ny, khha chn lc nhm alken, trnh to

cc sn phm phkhng mong mun, cn ch kim sot t lxc tc v thi gian

phn ng. Khi tin hnh kho st nh hng ca tlkhi lng xc tc so vi khi

lng curcumin, chng ti nhn thy khi tng t lxc tc ln cao hn 5% th hiu

sut phn ng gim. iu ny c thl do lng xc tc nhiu dn n khha thmmt hoc chai nhm ceton, to ra cc sn phm ph l monoalcol v dialcol. Hin

tng ny cng c thgp phi nu thi gian phn ng qu di.

Phng php ny c u im l sn phm tng i sch, iu kin khha

kh n nh; tuy nhin c nhc im l kh hydro to hn hp nvi 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 sdng acid acetic th thy phn ng khng xy ra. iu ny c thl do:

- Thnht, lc acid ca acid acetic yu, khng mnh hot ha km.

- Thhai, 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 acidhydrocloric v acid sulfuric.

thc hin tt phn ng khha vi km trong mi trng acid hydrocloric,

thu c hiu sut cao, t sn phm ph, cn lu mt svn sau:

kim sot tc phn ng v khha chn lc

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