Tap chi Hoa hgc, T. 48 (4), Tr. 397 - 403,2010
NGHIEN CLfU PHAN LAP THANH PHAN VA HOAT TINH CUA CAC CURCUMINOIT TRICH TLf cCl NGHE VANG {CURCUMA LONGA L.)
Den Tda soan 28-7-2008
PHAN THI H O A N G ANH*', LE XUAN TIEN', NGUYEN THI MAC PHUONG', TRAN THI VIET HOA', TRAN V A N SUNG^ PHAN THANH SON NAM'
'Trudng Dai hgc Bach Ichoa, DHQG-HCM
^Vien Hoa hgc, Vien Khoa hgc vd Cong nghe Viet Nam
ABSTRACT
Curcuminoid extracted from yellow turmeric is a mixture of 3 constituents: curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC). Previous published papers mainly concentrated on the biological activities of curcuminoid mixture. In this work:, we have separated three constituents of curcuminoid, characterized and comparatively studied antioxidant, antibacterial and cytotoxic acvitities of these curcuminoids. Curcumin exhibited stronger DPPH scavenging activity (ICso = 55.083) than the mixture, DMC. BDMC and the reference antioxidant, trolox. Meanwhile, the mixture showed the antibacterial property against Staphylococcus aureus - (MIC = 62.5 mgi ml) better than each components. The mixture and its three constituents also .,:?:;
demonstrated good abilities in suppressing the proliferation ofHep-G2 and RD cell-lines.
I - DAT VAN DE
Curcuminoit, thanh ph&i chinh trong cu nghe vang Curcuma longa L., tuf lau da diro'c biet den co nhiiu hoat tmh sinh hoc manh va da dang nhu hoat tuih khang khuin, khang oxy hoa, khang viem, khang ung thu... [1].
Curcuminoit thuang mai trich tdr nghe hien nay gom 3 thanh phan chfnh, curcumin [1,7-bis- (4-hydroxy-3-metoxyphenyl)-hepta-1,6-diene- 3,5-dione ~77%], demethoxycurcumin (DMC -17%) va bis-demethoxycurcumin (BDMC - 3 % ) [1]. Da CO nhieu c6ng trtnh nghien ciiu phan tach cac thanh phan trong h6n hgp curcuminoit bang phuong phap sSc ky ldp mong (TLC) va sac ky cot (CC) [2 -8]. Tuy nhien, cac ket qua c6ng bo diu cho do phan giai thap va do tinh khiet cua curcumin thu dugc chi khoang 80%, va cung chi dijtng lai 6 viec tach curcumin, chua d i cap den DMC va BDMC.
Trong nghien cdu nay, chiing toi khao sat phuang phap tach cac thanh phan trong h5n hgp curcuminoit dya vao TLC va CC. Cac thanh phSn sau khi tach se dugc kiim tra do tinh khie't bang HPLC, dong thdi xac dinh cSiu tnic bang pho MS, NMR. Hoat tfnh khang oxy hoa, khang khuan, khang te' bao ung thu cua cac thanh phfin nay cung dugc nghien ciiu. Theo hiiu biet cua chung t6i, viec tach 3 thanh phan trong h6n hcjp curcuminoid o dang tinh khiet, khao sat va so sanh hoat tfnh khang oxy hoa, khang khu&, khang te' bao ung thu cua cac thanh ph& tren chua dugc cong bo trudc day trong cac tap chf chuyen nganh trong nu6c.
^ II - THUC NGHIEM 1. Nguyen lieu
Bgt curcuminoit tho mua cua Vien Dugc lieu, Bg Y te, Ha Ngi.
Ban mong silieagel 60G F254 (Merck) cho sac ky Idrp mong va bgt silieagel 60G F254 (Merck) 0,063 - 0,2 mesh cho sac ky cot.
Cac dung mOi: metanol 99,9%, diclorometan 99,5%, cioroform 99,7%, axeton 99,7% (Shantou Vilong Chemical Factory).
2. Phuang phap nghien ciiru
Bgt curcuminoit tho dugc kit tinh lai 3 Mn trong h6n hgp metanol : nude d 60°C. H6n hgp sau ket tinh dugc Igc hiit chan khong thu dugc chat ran 1. Nude cai dem co quay dudi ap suat thap thu dugc chat ran 2.
Tach cac thanh phSn trong chat ran 1 va chat rin 2 bang sac ky cot. Qua trinh dugc thuc hien vdi cot silieagel 0,063 - 0,200 mesh, kich thudc 13x85 cm, he dung moi giai ly dugc lua chgn la CH2Cl2:metanol vdi do phan cue tang dan Mn lugt la CH2Cl2:metanoi = 100:0, 99,5:0,5, 99:1, 98:2, 95:5 va cudi ciing 90:10. Cac phan doan hung dugc kiem tra bang sac ky ban mong vdi he dung moi CH2Cl2:metanol 98:2, ggp^chung nhung phan doan cung Rf va co quay loai dung mdi.
Xac dinh ca'u tnic va phan tfch mdt so tfnh chat hda ly cac thanh phSn sau khi phan lap dugc:
Do diem chay (tren may Electrothermal 9100, thuc hien tai BM Hiru ca, khoa Hda hoc, Trudng DHBK TP. HCM): Curcumin: 179,5 -
183,5°C; DMC: 166,1 - 170,5°C; BDMC: 213,2 - 215,5°C.
Do phd UV-Vis (may do JENWAY 6505 UV-Vis Spectrophotometer, thuc hien tai Bd mdn Hiru ca, Khoa Hda hgc, DHBK TP. HCM):
Imax cua curcumin, DMC va BDMC lan lugt la 425,418 va 414 nm.
Phan tfch HPLC (thuc hien tai trung tam Dao tao va phat trien sac ky, TP. HCM): cot sac ky pha dao CI8 (250 x 4,6 mm, 5 cm), pha ddng axetonitril:H3P04 0,05% /55:45 (v/v), nhiet do cot 40°C, tdc dd ddng 0,8 ml/phut va diu dd UV-Vis d 422 nm: IR cua curcumin, DMC va BDMC lan lugt la 10,635; 9,462 va 8,616 phiit.
Phd MS do tren may HP 5989 MS Engine (Vien Hda hgc, Vien Khoa hgc va Cdng nghe
Viet Nam): curcumin [M-^H]^ = 369, BDMC [M+H]^ = 309, DMC [M+HT = 339.
Phan tfch 'H va "C-NMR (thuc hien tai Vien Hda hgc, Vien Khoa hgc va Cdng nghe Viet Nam), dung mdi DMS0-d6 do tren may Bruker AV 500, 500 MHz cho 'H va 125 MHz cho "C.
Hoat tinh khang oxy hoa: (thuc hifn tai Trung tam Sam va Dugc lieu TP. HCM)
Hdn hgp curcuminoit ban dau va cac thanh phan curcumin, DMC va BDMC thu dugc sau khi phan lap, dinh danh dugc khao sat hoat tfnh khang oxy hda bang phuang phap bat gdc tu do DPPH va dinh lugng MDA. Xac dinh % hoat tfnh chdng oxy hda HTCO% va IC50.
HTC0% = [(ODc - ODT) / ODC] x 100.
ODc: Mat dd quang cua chung, 0 0 ^ : Mat dd quang cua miu thii.
Hoat tinh khang khuan, khang nam:
(thuc hien tai Bd mdn Vi sinh, DH Y Duoc TPHCM)
Xac dinh hoat tfnh khang khuin vdi mdt sd ddng vi khuan gram (-1-): Streptococcus hemolyticus, Staphylococcus aureus, gram (-):
Pseudomonas aeruginosa, Salmonella typhi.
Shigella dysenteria, va hoat tfnh khang ufci Candida albicans theo phuong phap pha loang trong ban thach, b cac ndng dd hoat chat 15,625; 31,25;'62,5; 125; 250; 500;'lOOO'mg/ml, xac dinh ndng dd lie c h i tdi thieu MIC.
Hoat tinh khang ung thu: (thuc hien tai Phong sinh hgc thuc nghiem, Vien Hda hgc cac hpfp chat thien nhien, Vien Khoa hgc va Cdng nghe Viet Nam).
Xac dinh hoat tfnh khang ung thu ddi vdi 2 ddng te bao Hep-G2 {Hepatocellular carcinoma - Ung thu gan) va RD (Rhabdo sarcoma - ung thu mang tim) dugc cung d p bdi vien VSDT TU.
Phuang phap xac dinh: Theo phuang phap cua Skehan & CS (1990) va Likhiwitayawuid &
CS (1993), chat chuin chung duang tfnh: dung cha't chuin cd kha nang diet te' bao: Ellipiticin, Vinblastine hoac Taxol pha trong DMSO.
Dgc tren may ELISA d budc sdng 495-515 nm, xac dinh CS% (% te' bao sdng sdt) va IC50.
Ill - KET QUA VA THAO LUAN 1. Ket qua ket tinh curcuminoit tho trong
hdn hgp metanol: nude
Hieu suat qua trtnh kit tinh kha thip, khoang 40%. Sac ky ban mong cho thiy sau 3
lin ke't tinh da loai phin Idn nhua va BDMC, dieu nay cung phu hap vdi kit qua phan tfch HPLC (bang 1): ham lugng curcumin sau kit tinh lin 3 tang len dang ke (td 72,52% miu thd den 91,87% sau kit tinh), ham lugng DMC va dac biet BDMC giam rd ret, phan nay chu ye'u nim trong nude cli.
Bang I: Kit qua phin tfch HPLC
i
Dien tfch pic
% Dien tfch
Curcuminoit ban diu Curcumin
702334 72,52
DMC 147411
15,22
BDMC 118729 12,26
Curcuminoit sau kit tinh Curcumin
1139909 91,87
DMC 93561 7,54
BDMC 7329
0,59 2. Ket qua sac ky ban mong
Td ke't qua khao sit 2 he dung mdi CHjCtCHsOH va CH2Cl2:CH30H vdi cac ti le khac nhau, chiing tdi chgn he CH2Cl2:CH30H (98:2 v/v) la he dung mdi tach tdt nhit 3 thanh phan trong hdn hgp curcuminoit vdi gia tri Rf cua curcumin, DMC va BDMC lin lugt la 0,525;
0,313 va 0,188.
3. Ket qua tach b^ng sac ky cot
Kit qua cho thiy sic ky cot vdi he dung mdi da chgn tach rit tdt cac thanh phin trong hdn
hgp curcuminoit. Chat ran sau kit tinh chu yiu la curcumin (>77%), phin nhd DMC (-7%) va BDMC (-3,5%), con trong nude cai, BDMC cd ham lugng Idn nhl't -39%, ke la DMC (-27%) va cudi cung la curcumin (-17%).
4. Ke't qua phan tich pho cong hudng tiir hat nhan
Do dich chuyin hda hgc cua 3 thanh phin curcuminoit trong phd 'H va '^C-NMR dugc trtnh bay trong bang 2 va 3.
Bang 2: Dd dich chuyen hda hgc trong phd 'H-NMR cua curcuminoit Vitri
1 2 , 2 ' 3 , 3 ' 4 , 4 ' 5 , 5 ' 6 , 6 ' 7 , 7 ' 8 , 8 ' 9 , 9 ' 10, 10'
OCH, 8, 8'-OH (*): dp diet
Curcumin 6,06 (s, IH) 6,75 (d, I„^=16,0Hz, 2H) 7,55 (d, I„^-16,0Hz, 2H)
7,31 (d, I^ea,=l,5Hz, 2H)
6,8
(d, J„„,„=8,0Hz, 2H) 7,15(dd,J„^„=8,0Hz,
J^eu>-l,5Hz, 2H) 3,84 (s, 6H)
9,65 (s,2H)
1 chuyin hoa hpc cua 'H tai vi tri
BDMC 6,04 (s, IH) 6,68 (d, I^3„=16,0Hz, 2H)
7,54 (d, J„3„=16,0Hz)
7,56 (d, J„rtho=8,5Hz) 6,82 (d, I„^o=8,5Hz, 2H) 6,82 (d, Jo^„=8,5Hz, 2H)
7,56{d,J„^„=8,5Hz)
10,057 (s,2H) C7.
DMC 6,05 (s, IH) 6,68 (d, J^=16Hz, IH) 6,75 (d, J^an=16Hz, IH) 7,54 (d, I„,„-16Hz,lH) 7 , 5 5 ( d , I ^ = 1 6 H z , l H ) 7,56 (d, I„^„=8,5Hz) 7,31 (d, 1^,^=1,5Hz,lH) 6,82(d,I„^„=8,5Hz,lH)(*)
6,82 (d, J„^„=8,5Hz) 7,56 (d, J„^„-8,5Hz) 7,14 (dd, I„^„=8,5Hz, J,,,,
=l,5Hz, IH) 3,84 (s, 3H) 10,06 (s,lH) 9,06 (s,lH)
399
Bang 3: Do dich chuyen hda hgc trong phd '^C-NMR cua curcuminoit Vitri
1 2 , 2 ' 3 , 3 ' 4 , 4 ' 5 , 5 ' 6 , 6 ' 7 , 7 ' 8 , 8 ' 9 , 9 ' 10, 10'
OCH3 8, 8'-OH
Curcumin 100,841 183,206 121,110 140,707 126,360 111,375 148,007 149,358 115,727 123,110 55,708
BDMC 100,927 183,200 120,813 140,363 125,849 130,314 115,930 159,788 115,930 130,314 100,927
DMC 100,91 183,28; 183,14 120,85; 121,08 140,38; 140,71 125,38; 126,38 130,33; 111,31 115,94; 148,03 159,80; 149,36 115,94; 115,73 130,33; 123,17
55,72
Bang 4: Bang so sinh do dich chuyen hda hgc trong pho 'H va '^C-NMR cua DMC vdi cac so lieu cdng bd trong [8]
Vitri 1 2 , 2 ' 3 , 3 ' 4 , 4 ' 5 , 5 ' 6 , 6 ' 7 8 , 8 ' 9 , 9 ' 10,10'
OCH3 8 , 8 ' -
OH
'H-DMC (theo [8]) 6,02 (s, IH)
6,67 (d, J=15,8Hz, 2H) 7,53 (d, J=15,8 Hz,
2H)
7 , 3 1 ( d , J = l , 6 H z , 2 H ) -
-
6,81(d,J=8,3Hz,2H) 7,13 (dd,J„«,„=8,3 Hz, J ^ , , = l , 6 H z , 2 H )
3,82 (s, 3H) -
'H-DMC theo phin tich 6,05 (s, IH) 6,68 (d, J^=16Hz, IH) 6,75 (d, J^=16Hz, IH) 7 , 5 4 ( d , J ^ = 1 6 H z , l H ) 7,55 (d, J^=16Hz,lH) 7,56 (d, J„rthc=8,5Hz,lH) 7,31(d,J„,^=l,5Hz, IH) 6,82 (d, J^o=8,5Hz,lH) 6,82(d,J^o=8,5Hz,2H) 7,56(d,J„^„=8,5Hz,lH) 7,14(dd,J<^„-8,5Hz,4,^
=l,5Hz, IH) 3,84 (s, 3H) 10,06 (s,lH) 9,06 (s,lH)
''C-DMC theo [8]
100,9 183,2; 183,1 120,8; 121,1 140,4; 140,7 125,8; 126,4 130,4; 112,2 115,7; 148,0 159,8; 149,8 115,9; 115,7 123,1; 123.2
55,7
"C-DMC theo phan tfch
100,91 183,28; 183,14 120,85; 121,08 140,38; 140,71 125,38; 126,38 130,33; 111,31 115,94; 148,03 159,80; 149,36 115,94; 115,73 130,33; 123,17
55,72
7
R"] R2
5. Ket qua xac dinh mgt sd hoat tinh sinh hgc a) Hoat tinh Ichdng oxy hoa
+ Hoat tfnh khang gdc tu do DPPH
Curcumin:
BDMC:
DMC:
Ri = R2 = OCH3
Ri= H, R2 = OCH3
C u r c u m i n o i d s C u r c u m i n B D M C D M C T r o l o x
6 0 0 8 0 0 N o n g <3p ( | x . g / m l )
Hinh 4: Yi6\. qua xac dinh hoat tmh khang g6c tu do DPPH cua h6n hop curcuminoit ban d^u, - : " ' curcumin, DMC, BDMC va chat chiing trolox , | Hinh 4 cho tha'y cac thanh phfin phan lap duac d6u eo hoat tinh khang goc tu do DPPH manh hon chat chiing trolox, mot din xuat cua vitamine E, cu the la : Curcumin (IC5o= 53,5 mg/ml) > h6n hop curcuminoit {\C^o= 109,5 mg/ml) -> DMC (ICso^^ 134,8 mg/ml) >> BDMC (ICgo^ 693 mg/ml) >
trolox. - I + Hoat tfnh khang oxy hoa theo phuong phap MDA
- C u r c u m i n o i d s - C u r c u m i n - D M C - B D M C - T r o l o x
N & n g d p ( f i g / m l )
Hinh 5: K6't qua xac dinh hoat tinh khang Cj^y hoa theo phuong phap MDA
Ket qua cho thay, h6n hop curcuminoit, curcumin, DMC va BDMC deu co hoat tfnh khang su peroxide hoa lipid mang te bao tuong duong nhau va rnanh hon so v6i trolox.
b) Hoat tinh khdng khuan, khdng nam
H6n hop curcuminoit va cac thanh phfo co hoat tfnh khang khudn, khang nam kha thap vdi
cac vi khuan da khao sit, chi cd hoat tfnh tuang ddi cao vdi Staphylococcus aureus, trong dd hoat tfnh cua hdn hgp curcuminoid > DMC > BDMC - curcumin.
Bang 5: Ndng dd lie che tdi thieu MIC (mg/ml)
~~" •—-—____IIoat chit Vi khuin ~~~~" —___
Salmonella typhi Shigella dysenteria Pseudomonas aeruginosa Streptococcus hemolyticus
Staphylococcus aureus Candida albicans
Curcumin
>1000
>1000 1000
>1000
>1000 1000
DMC 1000 1000 1000 1000 125 250
BDMC 1000 1000 1000 1000 1000 1000
Hdn hgp curcuminoit 1000
1000 1000 250 62,5
250
c) Hoat tinh Ichdng te'bdo ung thu
Bang 6: Kit qua xac dinh CS% trong khao sat hoat tfnh khing t i bao ung thu cua curcumin, DMC, BDMC va hdn hgp curcuminoid vdi 2 ddng t i bao Hep-G2 va RD
STT 1 2 3 4 5 6
Ky hieu miu DMSO Chiing (-I-) Curcumin
Hdn hgp curcuminoit
BDMC DMC
T/C miu
TK TK TK TK TK
Ddng te b a o - C S % Hep-G2
100+0,0 3,5±0,01 21,33±0,7
20,8±0,1 11,2±0,5 22,75±0,58
RD 100±0,0 2,2±0,0 64,2+0,89 43,75+0,89
31,7±1,3 30,8±1,1
Kit luln
Duong tfnh Duong tfnh vdi 1 ddng
Duong tfnh ca 2 ddng Duong tfnh ca 2 ddng Duong tfnh ca 2 ddng
STT 2 3 4 5 6
Bang 7: Ke't qua xac
Ky hieu miu Chiing (+) Curcumin Hdn hap curcuminoit
BDMC DMC
dinh gia tri IC50 Ddng te bao - IC50 (mg/ml)
Hep-G2 0,3 2,919 2,908 2,508 3,025
RD 0,175
>5 4,414
3,88 2,469
Ke't luan Duang tfnh Duong tfnh vdi 1 ddng
Duong tinh ca 2 ddng Duong tfnh ca 2 ddng Duong tfnh ca 2 ddng Hdn hgp curcuminoit va cic thanh phin cd vdi ddng Hep-G2, va DMC cd hoat tfnh khang hoat tfnh Idling ung thu ddi vdi ddng t i bao ung thu tdt nhit ddi vdi ddng RD. Tuy nhien Hep-G2 manh hon so vdi ddng RD. So vdi 2 hoat tfnh khang ung thu cua hdn hgp thanh phin con lai, curcumin cd hoat tfnh khang curcuminoit va cac thanh phin ddi vdi cac ddng ung thu thip han vdi ca 2 ddng t i bao khao sat. t i bao khao sat vin chua cao so vdi cac chat
BDMC cd hoat tfnh khing ung thu tdt nha't ddi chiing. ' • '
T A I LIEU THAM K H A O ' H
1. Bharat B. Aggarwal, Anushree Kumar, Manoj S. Aggarwal and Shishir Shishodia, Phytopharmaceuticals in Cancer Chemoprevention, 349 - 387, CRC Press (2005).
2. N. Janaki, & J. L. Bose. Journal of the Indian Chemical Society, 44(11), 985 - 986 (1967).
3. Y. N. Krishnamurthy, A. G. Mathew, E. S.
Nambudiri, S. Shivashankar, Y. S. Lewis &
C. P. Natarajan. Tropical Science, 18(37), 37 - 46 (1976).
4. V. S. Govindarajan. CRC Critical Reviews
in Food Science and Nutrition, 12(3), 199 - 301 (1980).
5. A. Janben & T. H. Gole. Journal of the Indian Chemical Society, 44, 985 - 986 (1984).
6. T. Osawa, Y. Sugiyama, M. Inayoshi & S.
Kawakishi. Bioscience, Biotechnology and Biochemistry, 59, 1609 - 1612 (1995).
7. K. Srinivasan, K. Sambaiah, & N.
Chandrasekhara. Food Chemistry, 43, 271 - 274 (1992).
8. L. Peret-Almeida, A. P. F. Cherubino, R. J.
Alves, L. Dufosse, M. B. A Gldria, Food Research International, 38, 1039 - 1044 (2005).
\ i i '