Nghien CLFU - Ky thuat
r r
Chiet xuat curcumin tir cu nghe vang
(Rhizoma Curcumae longae) va xay dyng bo dft lieu chuan cua curcumin de thiet lap
chat chuan chiet tir du^crc lieu
D a t v a n d e
Cil nghe vdng (Rhizoma Curcumae longae) cdn cd cdc ten gpi nhu khuang hodng, vj cay ddng, tinh binh, cd tdc dung hdnh khi, hoat huylt, lam tan mdu, tan (y va giam dau. Trong ddn gian,'-nghe vdng dugc diing de bdi len da non cho mau liln da khdng bj n h i l m khuan, nhilu ngudi dimg bdt nghe vdng mdt ong de chira benh viem loet da ddy - td trdng, cdc benh v l viem tiii mat, sdi mat'''
Thdnh phdn hod hpc cd trong nghe vdng Id hgp chit cureuminoid gdm curcumin, demethoxyeureumin vd bisdemethoxycureumin trong dd curcumin Id thdnh phdn chinh. Nhilu cdng trinh nghien ciPU thir nghiem d edc nudc tren t h i gidi dd nhdn thay curcumin cd t h i kim hdm s u phdt then cua mdt sd loai t l bdo ung thu'^' Curcumin cdn Id c h i t bo cho da day, rugt, gan, mat, Igc mdu, Idm sach mdu, dilu trj v i t thugng, chdng viem khdp, dj ipng, nam, chong vi khuan cd hieu luc.
Tai Viet Nam, cd rdt nhilu cdc san pham thude, thuc pham chirc ndng, my pham sCr dung curcumin Id thdnh phan chinh, tuy vdy viec kiem tra chdt lugng vdn chua dugc thuc hien do thilu ngudn chuan doi c h i l u .
Theo xu hudng vd yeu cau tieu chudn hod chit lugng thude ddng dugc theo WHO Id can cd chuan dugc lieu, vi vdy chiing tdi nghien ciru chiit xult curcumin tir cii nghe vdng vd xdy dung bd dir lieu cho chdt chiet curcumin nhdm hudng tdi thilt lap chdt ddi chilu curcumin phuc vu cho cdng tdc kilm tra chdt lugng cdc san pham cd chira curcumin luu hdnh tren thi trudng.
Doi tu'O'ng va phu'O'ng phap nghien
CLPU
Trjnh Hoang Dirong, Ha Dieu Ly
Vien Kiem nghiem Thuoc TP Ho Chi MinhM I U n g h i e n CIPU
Cu nghe vang khd do cdng ty Cafecontrol thu mua d Bdc Giang cung cdp ._ . ~.^_
T h i l t bj, d u n g c u , hoa chdt
Thilt bi vd dung cu dd d u g c hieu chuan theo ISO/IEC 17025 v d G L P
May cd quay Heidolph, be sieu dm Branson 3510.
May quang pho UV-Vis Shimadzu 1700, may hong ngoai Nicolet 760, mdy sdc ky Idng Shimadzu LC-20A vdi detector PDA, mdy phdn tich nhiet DSC Q20 (VKN T TP.HCM)
Mdy do cdng hudng tir hat nhdn Bruker 500 MHz (Trudng DHKHTN Tp.HCM)
C h i t chuan: Curcumin USP, demethoxyeureumin USP
Ethanol 90°, n- hexan, acetonitril (HPLC), acid acetic (PA).
Phu'O'ng phap nghien cii'u Chiet xuat, phan lap
Trich ly bdng sieu am, cao d u g c c h i i t vdi n- hexan de loai bd tinh dau vd k i t tinh lai thu san pham curcuminoidthd.
Tinh che
San pham curcuminnoid thd d u g c sdc ky cot tren silica gel vd tinh c h i bdng sdc ky Idng dilu c h i tren cdt C-18, thu d u g c curcumin tinh khilt
Xay dirng bg dir lieu chuan cua curcumin Phd hdng ngoai do tren mdy Nicolet, dap vien KBr
Phd tCr ngoai- kha kiin sir dung may do pho UV-Vis Shimadzu 1700
Xdc dinh ciu true: do tren mdy cpng hudng tip hat nhan Bruker 500MHz (^H NMR, '^C NMR) Dp tinh khiSt Su' dung mdy phdn tich nhiet DSC Q20 de do diem chay vd xdc dinh dp tinh khiet.
26 TAP CHi DUOC HQC - 8/2011 (SO 424 NAM 51)
N g h i e n
CLFU- K y t h u a t
Dinh Iwang: curcumin bdng HPLC, cdt C-18, detector PDA d bude sdng 210 - 450 nm. Sir dung chit chuan curcumin USP lam chuan ddi chieu.
Ket qua va ban luan C h i i t x u l t , phan lap
Qua tham khao quy trinh chiit x u l t curcumin tip cil nghe eiia mdt sd tdi lieu [4-6] vd bdng nghien ciru thue nghiem chiing tdi thu duge eao thd cd ham lugng ede cureuminoid tuang ddi cao theo quy trinh sau;
Cil nghe khd xay thdnh bpt (1 kg), trich ly bdng sieu am vdi hdn hgp ethanol 96° trong 15 phiit d nhiet dp phdng. Thu hdi dung mdi, eao ed dae duge chiit vdi n- hexan de loai bd tinh d i u . Ldp nude d l lanh k i t tinh thu san pham cureuminoid thd.
T i l n hdnh tinh che hdn hgp cureuminoid thd bdng sdc ky cdt vdi he dung mdi Id eloroform:
methanol (99:1) vd HPLC d i l u e h i tren cdt pha dao RP-18, pha ddng Id aeetonidril - dung dich acid acetic 0,03% (50: 50), tdc dd ddng 3,0 ml/
phiit, thu duge curcumin tinh khilt. Chat phdn Idp sau khi duoi dung mdi, s l y d dp s u i t giam va nhiet dd 60°C thu dugc tinh the mdu vdng ddm vdi hieu s u i t chiit khoang 2,2 %. Viec sir dung phuang phdp chiit x u l t bdng sieu dm '®' khdng phai gia nhiet ddn tdi giam duge dang ke ehi phi eung nhu Idm giam duge lugng curcumin phdn buy do nhiet dd
Xay d i f n g bo dCi' lieu chudn cua curcumin
RI=R2=OCII3: curcumin
Rl= H: R2= 0CU3 : demethoxyeureumin Rl=ll; R2=ll: bisdemethoxycureumin
Hinh 1: Ciu true curcumin 1.7-bis-(4-hydroxy- 3-methoxyphenyl)-1.6-heptadien-2, 5-dion
Tinh chat C h i t k i t tinh mdu vdng dam.
Xae dinh cau true: H]nh ^.
Quang pho tir ngoai: pha dung djch cd ndng dd 10 pg/ ml trong ethanol do trong dai bude sdng tir 210 d i n 450 nm thu dugc hai ct/c dai hap thu d 263 nm vd 426 nm (Hinh 2).
Retention Tone 9 US C omc aund N use Curciinuii SpFcUuni Oijcitlioi. Hunt
•M5i
1 '-..L.-^- 310 S3
/ /
4yuiJ
\ J26 83
, , , , ,
669 9^
1
Hinh 2: Phd UV cua curcumin
Pho hong ngoai: chdt chiit duge ghi phd IR trong KBr eho cdc dao ddng ddc trung d 3508,3; 1627,7, 1508,7; 1428, 9; 1282,8, 1206,4; 1153,6; 1114,9; 1026,8 vd 962,7 cm"^
phii hgp vdi phd curcumin chuan.
- Pho NMR: k i t qua phdn tich edng hudng tir hat nhdn (^H-NMR, ''^C-NMR) eho dir lieu phd ghi trong bang 1 dd khdng djnh e l u triic ciia c h i t chiit Id curcumin (hinh 3) irng vdi cdng thue C21H20O6, phdn tip lugng M= 368,4 so sanh vdi dir lieu phd theo tai lieu tham khao'^'
_J'...h
C l - H e O D - l H
1 |l
Cl-MeOD-13C
*NP«M^
Hinh 3: Phd 'H-NMR va '^C-NMR cua chit chiit curcumin trong MeOD
TAP CHi DUQC HQC - 8/2011 (SO 424 NAM 51)
27• Nghien CLFU - Ky thuat
Bang 1: S6 lieu phd ^H-NMR va '^C-NMR sd ghep J tinh bing Hz)
Vj tn SH
1 7,59 d (16,0) 2 6,65 d (16,0) 3
4 6,01 s 5
6 6,65 d (16,0) 7 7,59 d (16,0) 8
9 7,24 d (1,5) 10
Sc 142,1 124,6 184,8 111,9 184,8 124,6 142,1 131,1 116,9 149,5
cua curcumin trong MeOD (s6 lieu trong ngoac la hing
Vi tri SH
11
12 6,85 d (8,0) 13 7,14dd ( 1 , 5 v a 8 , 0 ) 14
15 7,24 d (1,5) 16
17
18 6,85 d (8,0) 19 7 , 1 4 d d ( 1 , 5 v a 8 , 0 ) IO/I6-OCH3 3,94 s
5c 150,5 124,3 128,6 131,1 116,9 149,5 150,5 124,3 128,6 56,5
-Do tinh khiet va nhiet do nong chay: do tren mdy phdn tich nhiet DSC Q20
C h i t chiit cho diem chay d 183,5°C phii hgp vdi nhiet dd ndng chay eiia curcumin la 183-184
°C vd dp tinh khilt do dugc Id 99,12 % (Hinh 4)
Hinh 4: Phd DSC xae dinh do tinh khiit cua chit chiit curcumin
Dinh Iwang: bdng phuang phdp sdc ky Idng hieu ndng cao
- Diiu kien sic ky:
Cdt: RP-18 (250 mm x 4,6 mm, S^im) hay cdt tuang duang
Detector: quang pho hap thu tip ngoai vdi ddy diod quang PDA d budc sdng 263 nm Pha ddng: acetonitril - dung dich acid acetic 0,03%
(50: 50)
Tdc dp ddng: 1,0 ml/ phut The tich tiem: 10 |.il
Dung dich ddi chiiu: Hda tan chinh xdc mdt lugng khoang 10 mg chudn ddi chieu curcumin USP trong binh dinh miPc 100 ml bdng methanol.
Pha lodng 5 ml dung dich ndy trong pha dgng vira dii 50 ml. Loc qua mang Igc 0,45 |.tm.
28
Dung dich thir: Hda tan chinh xdc mot lugng khoang 10 mg chat chiet curcumin trong binh djnh mipc 100 ml bdng methanol. Pha lodng 5 ml dung djch nay trong pha ddng vipa du 50 ml. Lge qua mdng Igc 0,45 |.tm
Khao sat tinh twang thich cua he thing sSc ky Vdi d i l u kien sdc ky da lua chgn, khao sat tinh tuang thich cua he thdng sdc ky thdng qua 6 Idn tiem lap lai mdu c h u i n . He thdng sdc ky dat yeu cau vdi dp lech chuan tuang ddi khdng qud 2%, he sd doi xipng khdng qud 1,5, dp phan giai giu'a curcumin vd demethoxyeureumin Idn han 2 (Hinh 5). • • ••• - ^ . x
Ket qua djnh lugng curcumin cd ham lugng Id 96,38 % tinh tren nguyen trang.
nnc Hinh 5: Sic ky dd HPLC cua curcumin va demethoxyeureumin
K§t luan
Quy trinh chiet x u l t curcumin tip cu nghe vdng bdng sieu dm sir dung ethanol 96 % va tinh c h i bdng sac ky cdt silica gel k i t hgp HPLC d i l u c h i tren pha dac RP 18 thu d u g c curcumin cd dp tinh khilt cao (>99%o), phuang phdp chiit dan gian d l dp dung cho hieu xudt chiet thu duge khoang 2,2%.
TAP CHI DUOC HQC - 8/2011 (SO 424 NAM 511
Nghien CLFU - Ky thuat
Bdng ede phuang phdp phd nghiem nhu UV- Vis, IR, NMR, d i l m chay vd do dd tinh khilt tren may phan tich nhiet DSC chiing tdi dd xay dung bd dir lieu chuan de xae dinh e l u triie curcumin.
Dinh luang curcumin b i n g HPLC tren opt pha dao RP 18 eho ham lugng 96,38% ±0,136 so vdi c h i t chuan curcumin USP dat yeu e l u chit lugng cua nguyen lieu ddi chilu.
Hop chat curcumin dang t i l p tue theo doi dp dn dinh de thiet lap chat ehuIn chiet tCr duge lieu, phuc vu edng tae kiem nghiem thude ddng duac cae san p h i m ed ngudn gde dugc lieu
Summary
A procedure for isolation of curcumin from the Rhizoma Curcuma longae by ultrasonic extraction with ethanol was reported. The cureuminoid residues were separated by column-chromatography (on silica gel) and preparative HPLC with RP-18 column. The pure curcumin was identified by UV, IR, melting point and NMR spectroscopic data The Impunty of the obtained compound was determined by DSC. The content of curcumin was 96.4% by HPLC in comparison with the reference Curcumin of USP. The product is under a further
study of the stability for use as a reference standard for quality control of the products containing curcurmin.
Keyword: Rhizoma Curcuma longae, curcumin, demethoxyeureumin
Tai lieu tham khao
1. Vo Van Chi, Td dien cay thuoc Viet Nam, NXB Y hoc, Tp, Ho Chi Minh, (1996),
2. M T, Huang, R,C, Smart, C Q, Wong, A,H, Conney, Cancer Res., (1988) 48, 5941
3. B. B, AgganA/al, A. Kumar, A, C, Bharti, Anticancer Res. (2003) Jan-Feb; 23(1 A), 363 - 98
4. Q A.EIiseo; D A. Joaquin. Method for obtaining apolar and polar extracts of curcuma and applications thereof. United States Patent 6440468.
5. V, G. Gaikar, D, V. Dandekar: Process for extraction of curcuminoids from curcuma species.
United States Patent 6224877,
6. Tran Trung Kien, Nghiem Xuan San, Phiing Lan Hud'ng, Pham Van Thiem, Nghien ciru tach curcumin tip cii nghe vang b i n g phuang phap trich ly sieu am. Tap chi hoa hoc, (2007) T. 45, 52 - 57
7. L, Peret-Almeida, A.P.F. Cherubino, R J, Alves, L, Dufosse, MB,A, Gloria, Separation and determination of physico-chemical charactenstics of curcumin, demethoxyeureumin and bisdemethoxycureumin. Food Research Intemational (2005) 38,1039-1044.
Xae dmh hoat chat ^wZ-kauran diterpenoid trong cay kho sam Bac Bo
(Croton tonkinensis Gagnep., Euphorbiaceae) bSng sac ky long hieu nang cao
Phan Minh Giang, Phan Tong Scrn
KJKKI Hod hgc, Truang dgi hgc Khoa hgc Tu nhien, Dai hgc Quoc gia Ha Ngi
Dat van de
Cay khd sdm B l e Bd (Croton tonkinensis Gagnep., Euphorbiaceae) dugc dimg phd biln trong y hgc ddn gian ciia Viet Nam, La cdy khd sdm Bdc Bd diing ehii'a ung nhpt. Id loet, viem mui, dau bung, tieu hda kem, ly vd viem loet da day ta trdng''' Mdt sd thdnh phan enf-kauran
diterpenoid chinh dd dugc phdt hien trong Id cay khd sdm Bdc Bd '^'^' Cae boat tinh sinh hgc lien quan d i n nhdm hgp chat ndy bao gdm khang ky sinh triing sdt ret Plasmodium falciparum, khang khuan dde biet Id khdng ehung khdng khdng sinh Staphylococcus aureus va khdng nam, chdng viem vd chong ung thu'^^'
TAP Clii DUQC HO C- 8 / 2 0 1 1 (.SO 424 NAM 51)