NGHltN COTU CHI^TXUAT, PHAN LAP VA TINH CHE ACID ARISTOLOCHIC I TCT Dl/aC LJEU PHONG KY
TRANMINHNGOC
Vi$n Du^c U^tt
NGUYEN THI LAN PHlfONG, NGUYEN TUAN ANH, TRINH VAN LAU
Vien Kiem nghiem thuoc Trung mmg
l.Datvinde
Acid aristolochic, mgt tac nhan gay suy than v i ung thu, li cic chit dugc tim thiy trong mgt sd ciy fhude bg Mgc huong (Aristolochiaceae). Hien nay, da cd nhidu cong hinh nghidn ciiu cdng bd trong cac cay thudc chi Aristoiochia va Asarum cd cac acid aristolochic I, H, III va IV Uong dd acid aristolochic I (AA I) la thanh phan cd dgc tmh cao v i chiim him lugng cao hon so vdi cic acid aristolochic khac. Trong sd cac cay thudc hai chi nay, Phdng ky l i dugc lieu dugc phat hien thiy cd cac acid aristolochic trong dd chit yeu la AA 1 va li dugc li?u dugc sir dyng rgng rai di dieu tri benh trong y hoc c^ truyen d nude ta.
Theo chi dgo cua BO Y te, cac Trung tam Kidm nghi?m Dugc - My phim vi cac phdng Kiem tra chit lugng ciia doanh nghi?p sin xuat thuoc ddng dugc phii tien hinh kiem nghiem cac acid aristolochic I trong cic mlu dugc lieu thugc hg Aristolochiaceae, neu phit hi^n dugc acid aristolochic 1 trong cac mau dugc li?u phii td chiic tieu huy. Doi vdi cac benh vien y hgc cd truyin, Bg Y te da yeu cau tam ngimg sir dyng va nh^p vio cac dugc lieu ndi tren, lay miu v i niem phong cic dugc lieu cdn lai de giii kiem nghiem acid aristolochic I. Van de dat ra hien nay ddi vdi he thdng kidm nghiem hi Trung uong den dia phuong, cic phdng Kiem nghiem cua cac co sd san xuat li thieu chat chuan acid aristolochic 1. Chat chuan nay hien nay c6 gii cao, vi phai dat mua vdi thdi gian tuong ddi lau. Xuat phit tir nhu cau thyc te va tinh cap thiet cua cdng tac kidm tra chit lugng dugc Ueu, chiing tdi da tien hinh de tii nghien ciru chiet xuit, phan lap v i tinh che acid aristolochic I hi dugc U^u Phong ky lim chit chuan di kidm tra chat lugng dugc U?u.
2. T h ^ nghifm 2.1. Nguyen lifu
Vi dugc li^u Phdng ky dugc thu mua tren phd Lan
Ong, Ha Ndi. Kiem tra su cd mat ciia acid aristolochic I, so bd xac dinh ham lugng AA I. Su dung nhimg dugc Ueu cd ham lugng tren 0,02% dimg lim nguyen lieu chiet xuat.
2.2. Phuang tien nghien cuu 2.2.1. Thietbi, dungcu
Cac thiit hi, dyng cy cua Vien Kiim nghiem thudc Trung uong (da dugc hieu chuan theo ISO/lEC 17025 va GLP) va A^en Hda hgc - Vien Khoa hgc va Cdng nghe Viet Nam, gdm:
- May sic ky Idng higu nang cao Shimadzu UFLC 10 ATvp, detector diod array SPD-MlOAvp;
- May do phd IR: 670-FTIR NICOLET- NEXUS;
May do phd khdi ESI-MS: THERMO FINNIGAN LCQ ADVANTAGE MAX;
- May cdng hudng tii Bniker Avance AM500 FT- NMR;
- May quang phd tir ngoai kha kien (UV-VIS) HP 8453;
- May do diem chay Electrothemal IA 6304;
- May cat quay chan khdng Heidolph;
- Cac dung cy thuy tinh cin tbidt cho qui trinh thuc ng^em.
2.2.2. Hoa chdt, dung moi
Cac dung mdi, hda chat diing trong nghien ciiu de tai dat cip do tinh khiet phin tich (PA) va tinh khiet sac ky (dimg cho HPLC).
- Loai tinh khiet phan tich (PA): n-hexan, cloroform, ethyl acetat, aceton, methanol, ether diu hda (40 - dO^C), ethanol 75%, ethanol tuyet ddi, acid phosphoric...
- Loai tinh khiet sac ky (HPLC): acetonitril Z3, Phmmgphdp nghien cuu
2.3.1. Phuangphdp chiet xuat. phan lap va tinh cheAA I tie duac lieu Phong ky
T»p chi KI£M N G H I $ M T H U 6 C - S6 4.2011; Tap 9.(34) 25
- Chiet xuit: AA I thudng de tan trong ethanol, do vay dung eth^iol 75% lam dung mdi 66 chidt xuat ket hgp vdi lie sidu am. Dich chiet sau khi cat thu hdi dung mdi tiep tyc dugc loai tap bang n-hexan va tien hanh chidt long - long bang dung mdi etbylacetat. Kiem tra AA I chiet dugc bang SKLM [4] so sanh vdi chuan AA I. Xac dinh him lugng AA I trong can ethylacetat tiiu dugc bing HPLC [3].
- Phan lip: Sir dung phuong phip sac ky cgt qua 2 budc vdi hai pha ffiih hap phu la sUica gel loai 63 - 200 iim va 40 - 63 jun de phan lap cac thanh phin boat chit. Khao sit lua chgn dung mdi nia giai va kidm tra cac phan doan bing phuong phip SKLM [4], Xic dinh ham lugng AA I phan lap dugc bang HPLC [3].
- Tinh che: Su dyng phuong phap sac ky cgt pha dio vdi pha tinh C18 de tinh che AA I hi san pham cua qui trinh phan lap dd thu dugc chat chuan cd ham lugng phii hgp. Kiem tra AA I tinh chd dugc bang phuong phip SKLM [4] va HPLC [3].
2.3.2. Xdc dinh cdu true, nhgn dgng vd danh gid do tinh khiet cua chdt chiet duac
- Xac dinh ciu tnic v i nhin dang: Tir chat tinh khidt da tinh che dugc, xac dinh ciu tnic bang cac phuong phap hi?n dai nhu: xac dinh didm chiy, UV-VIS, IR, NMR ('H, '3c, DEPT), LC-MS.
- Dinh tmh, dinh lugng va danh gia do tinh khidt:
Sir dung phuong phap SKLM [4] va HPLC [3] so sanh vdi AA I chuin de dinh tinh, dinh lugng va danh gia do tmh khidt cua chat chiet dugc.
3. Ket qua va ban luan
3.1. Quy trinh chik xuSt, phan lap vd tinh chi/LA I 3.1.1. Chiet xuat
Dua vao tmh chit cua AA I v i kit qui khao sat cho thay vdi phuong phap chidt AA I hi dugc lieu bing each lac sieu am vdi dung mdi ethanol 75%, loai tap bing n-hexan rdi tidp tuc chiit AA 1 bing ethylacetat da dua ra mgt quy hinh chidt xuit dn dinh (RSD=4,18%), sin phim chiet cd him lugng AA I khoang 3,44%.
Quy trinh da sir dyng ethanol mdt loai dung mdi it dgc, re tidn, de kiem lam dung mdi chidt xuit. Sir dyng n-hexan li hgp ly de logi tap m i khdng lam mit di lugng hoat chit cin chidt xuit,
3.1.2. Phan lap
Dd phan lap AA I chiing tdi da sii dyng phuong phap sic ky cdt vdi pha tmh la silica gel qua 2 giai doan:
- Giai doan 1 sii dung silica gel cd kich thudc hat 63 - 200 pm vdi tfiinh phin dung mdi nia giai li hdn hgp clorofonn - methanol vdi dg phin cue tang dan va hiing phan doyn cd chiia AA I.
- Giai doan 2 sii dyng silica gel cd kich thudc hat 40 - 63 ^m vdi dung mdi nia giai l i hdn hgp cloroform - methanol - nude co dg phin cue ting din da tich dugc AA I ra khdi cac tiianh phin khac.
CJuy trinh phan lap da xay dyng dugc de thyc hien, dn dinh vi sin pham phan lap cho him lugng AA I khoang 81,43%.
3.1.3. Tinh che
San pham cua qua trinh phan ISp dugc tinh chi qua cdt sic ky pha dao YMC-RP18 vdi dung mdi nia giii li hdn hgp methanol - nude (2:1) thu dugc sin pham li acid aristolochic I co dang bgt v6 dinh hinh mau vang, him lugng 94,2% (HPLC) [3]. Hieu suit cua qua hinh tinh che la 83,7%.
3,2. Xdc dinh cau trAc, nh^n dgng vd danh gid dp tinh khiit c^a chdt chiet d^c
San pham cudi ciing ciia qua ttinh tinh chi dugc phin tich bing cac thdng sd:
- Tinh chit: Bgt vd dinh hinh mau ving.
- Nhiet dd ndng chay: 28l'*C phu hgp vdi nhiet dd ndng chay ciia AA I da cdng bd [1].
- Phd hip tiiu tii ngoai khi kidn (UV-VIS) va phd bdng ngogi (IR) ddu triing voi cic phd ciia chat chuin acid aristolochic I.
- Phd cdng hudng tii hat nhan:
+ Phd ' H-NMR cho tin hieu cua 10 proton bao gom hai tin hidu suiglet d 5jj 7,82 va 8,58 ppm ciia hai protcffl vdng thorn d vi hi H-2 v i H-9, hai tin hieu doublet vdi dd dich chuydn hda hgc d 5,^ 8,65 (d, J = 8,0 Hz, H-5), v i 7,37 (d, / = 8,0 Hz, H-7) v i mgt proton cho tin hieu triplet d 5^ 8,65 (t, 7 = 8,5 Hz, H-6), tui hieu singlet ciia methylen proton d 5^^ 6,49 (2H, s, H-12) va pic cua metiioxy proton d 6j, 4,03 (OCH3).
+ Tren phd'^C & DEPT - NMR cho tin hieu cua 17 carbon ti-ong dd pic ciia carbonyl carbon d 5f. 167,7 ppm va 9 pic ciia carixm bic bon, 5 methylen carbon, 1 ethylen carbon va pic cua metiio^Qfl carbon d 5^, 56,3 ppm.
T9P chf KlfeM NGHI$M THU6C - S6 4.2011; T^p 9.(34)
Vay cic sd lieu phd NMR chi tidt d i dugc xiy dung va dugc duara la chinh xac va hoin toin phu hgp vdi cau tnic cua phin tii AA I (8-methoxy-6-nitrophenanthro- [3,4-d]-l,3-dioxole-5-carboxylic acid) sau day:
- Pho khoi: Phd khdi cua chat tinh che dugc cho pic minh ion phin tir m/z 340 [M-H]" va 342 [M+H]"". Nhu v%y, phin tii lugng la 341 phii hgp vdi khdi lugng phin tii cua AA I cd cdng thiic phan hi C[7Hj,NO.j.
- Ket qua xac dinh ham lugng AA I trong chat tinh che dugc bing phuong phip HPLC l i 94,2%.
Tur phin tich cic tiidng sd vat ly nhu tinh chit, nhi^t do ndng chiy vi phan tich dii lieu phd UV-VIS, IR, NMR v i MS kit hgp so sinh vdi tii lieu tham khio cd the khang dinh chit chiet dugc li acid aristolochic I vdi ham lugng 94,2%.
4. Kit luan
Da xay dung dugc qui trinh chiet xuit, phan lap vi tmh che acid aristolochic I tii dugc lieu Phdng ky dugc thu mua trdn thi trudng. Cic quy hinh niy ddu dd tiin hinh, tiet kiem thdi gian, su dyng cic logi dung mdi re tidn, dd kiim vi it dgc.
Da tien hinh xic minh ciu true ciia acid aristolochic I tinh che dugc dya tren phan tich cac thdng sd hda ly nhu tinh chat, nhiet do ndng chiy va cac phuong phip phd cdng hudng hi hat nhin, phuong phip phan tich khdi phd, phuong phip quang pho hong ngoai. Ket qui thuc nghiem da khang dinh cdng thiic phan tir ciing nhu cau true cua acid aristolochic I tinh chd dugc la phu hgp vdi cic kdt qua ngfaien ciiu da dugc cdng bd.
Tii li^u tham khao
1. Anencyclopedia of chemicals, dmgsandbiological: "7%eMercfe/«rfer", Thirteenth edition. Part one, pages 134.
i. J. De Pascual Teresa, Julio .G Urones and Alvaro Fernandez, (March, 1983), "i4n Aristolochic Acid derivative from Aristoiochia Longd', Phytochemistiy Volume 22, Issue 12, pages TlAS-llAl.
3. The Japanese Pharmacopoeia XIV.
4, Vien Kiem nghiem thuic Trung uong (2011), Thudng quy ky thu^t: ^"Phdt hi^n acid aristolochic 1 trong duoc lieu (VKN/TQKT-DD/08)".
SUMM/iRY
The extraction, isolation, purification of Aristolochic Acid I from Phong Ky medicinal plants bought in the market The extraction of Aristolochic Acid 1 are used by Ethanol 75%, Ethyl acetate and eliminated by n-Hexane. Aristolochic Acid I was isolated and purified by Chromatographic Column Method using Silica gel (the particle size of the Silica gel is in the range of 40 - 63 pm in the first step and 63 - 200 pm in the second step) and reverse phases (RP18) as station phases with the suitable resolution solvent-systems.
The Aristolochic Acid 1 structure of final products is confirmed by IR, MS and NMR Spectrography. The content of Aristolochic Acid! obtained is 94.2% (determined by HPLC). This product can be used to control the quality of medicinal plants and drugs which are suspected to contain the Aristolochic Acid I.
T9P chf \iltM NGHIEM THU6C - So 4.2011; Tap 9.(34)
