Nghien cuu Y hpc Y Hpc TP. Hb Chi Minh * T|p 20 * So 6 * 2016
THIET LAP MOT CHAT CHUAN DOI CHIEU TAXOID
TU" LA THONG DO LA DAI (Taxus wallichiam Zucc.) TRONG 6 LAM D 6 N G ma Hodng Oanh**, TrdnNggc Bdo Trdn*% LiBdngKhuattg***',Hd Dieu Ly**', Bdi The Vinh**', Trdn Cong Luan****^, Nguyen Phttimg Dung**"^
TOMTAT
Ma ddu: Hiin nay, hai dugc pham duoc sie dung nhieu vd him qud nhcA tiang hda tri ung thu buong tribig ung thu vu vd ung thu phm Id Taxol® (paclitaxel) vd Taxota^(docdaxel)^'^K Cd hai hoqt chM ndy dew dugc chi^
xuai tie vo thdn vd Id cua cdy Thong do. Tuy nhiin, ngmn Thong do tiong tu nhien kha hian, vi tM, Trung tam tiong vd che bien cdy thuoc Dd Lat dd nghiin cieu, nhdn giohg vd tiong thdrdi cong Thdng do la ddi de chiei lay paclitaxel vd cdc hgp chat taxoid ldm nguyen liiu sdn xudi thuoc tri ung thi^^K Dii vdy, Thdng do la ddi van chiea dugc nghien cieu ddy du vicdc hgp chdl taxoid ldn can taxol deldm ca s& die lieu, gop phan ieng dung tiong kiem nghiem nguon nguyen lieu taxol cua Viet Nam. Do do, di tdi ndy dugc thuc hiin nham muc Heu phdn lap, Hnh chthgp chcd toxoid cd tiong phdn doqn chiii Thong do Id ddi vd thiii lap chat chuan ddi chim phuc vu cho cang tdc kian nghiem dugc lieu vd chi'phdm lien quan.
Boi tuang vd phuang phdp nghien dha Boi tuang nghien dht: 1.000 mg phdn doqn giau taxoid dugc phdn lap tie Thong do Id ddi tiong & Ldm EJdng dugc trinh bdy tiong Saddla phucmg phdp nghiin deu. Phuang phdp nghien cdu: Phdn lap vd xdc dinh cdu triic hgp chdl tu nhien: Hen hdnh phdn lap taxoid co tiong phdn doqn gidu taxoid (dugc chiii tdch tie nhihig nghiin chi truac d&y) bang phiecmg phdp sdc ky cot trung dp, Hnh chi'lqi bdng phieang phdp tdi kH tinh vd xdc dinh cau true hai chdi phan lap dugc bang cdc phuang phap pho'nghiem UV'Vis, IR, MS, NMR. Sau do, do Hnh khiei cua chdl phdn lap dugc xdc dinh bang ky thuqt HPLC pha ddo v&i dau do PDA. Thiii lap chfft chu^n thiin nhiin theo hu&ng ddn cua WHO, ASEAN vd ISO/IEC15328: danh gid chdi chuan bang dinh Hnh dm tiin cdc gid tri: pho'UV-Vis, IR, MS, NMR, dinh lugng bang HPLC; d&ng gm, ddnh ga dong nhai, ddnh gid lien phong thi nghiem (ba phong thi nghiim dqt Hiu diuan GLP).
K^ qud: Tie 1.000 mg phdn doqn TWddphan lap duac 268 mg TW. Dua vdokeiqudphdn tidi pho UV- Vis, IR, MS, NMR 1-D vd 2-D 0H-, "C-, DEPT, HSQC, COSY, HMBC) dd xdc dfnh dugc cSu tiuc ciia hop chdi ndy Id 10-deacetyl taxinin B, co do Hnh khi^ tiin 98%. Theo hu&ng ddn cita WHO, ASEAN vd ISO/IEC 15328,158 mg chai chuan dm chieu 10-deacetyl taxinin B dugc thiei lap dqt do dong nhai khi ddng gdi vd do tinh khiii 98,57%.
Kei ludn: De tdi dd phan lap mgt taxoid la 10-deacetyl taxinin B tie Id Thong do Id ddi (Taxus wdlichiana Zucc.) ti'dng a Lam Dong. 10-deacetyl taxinin B dugc thiei lap ldm chai chudn dm chim dqt do tinh Idiiei 98,57%.
Tit khoa: Taxus wallichiana, 10-deacetyl taxinin B, reference standard.
ABSTRACT
ESTABUSHMENT OF REFERENCE STANDARD 10-DEACETYL TAXININ B FROM THE NEEDLES OF Taxus wallichiana Zucc. CULTIVATED IN LAM DONG PROVINCE
Hua Hoang Oanh, Tran Ngoc Bao Tran, Le Dang Khuong, Ha Dieu Ly, Bui The Vinh, Tran Cong Luan, Nguyen Phuong Dung * Y Hoc TP. Ho Chi Minh * Vol. 20 - No 6 - 2016:44 - 49
* Khoa Y hpc co truyen - Dgi hpc Y Duoc TP. Ho Chi Minh ** Khoa Dugc - DH Y Dugc TP. Ho Chf Minh
*** Vien kiem thuoc TP. Ho Chi Minh ""Trung tam Sam va Dupe lieu TP. HCM '*' Tdcgid lien lac: ThS.DS. Hiia Hoang Oanh DT 0909623235 Email: [email protected]
Y Hpc TP. H53 Chi M i n h * Tap 20 * So 6 * 2016 Nghien ciru Y hpc
Background - Objectives: Nowadays, Taxol® (paclitaxel) and Taxoter^ (docetaxel) are two pharmaceuticals that used widely and most Actively to tieat the ovarian canca, breast canca and lung cancer^^>. Both active ingredimts are extiadedfrom the barks and leaves of the natural Taxus spp. However, the populaHons of Taxus spp. in natural environmmt are very rare, hmce, the Cmtafor Cultivating and Processing medidnal plants in Lam Dong have successfully researched and planted successfully Taxus wallichiana Zucc.
(Taxaceae) in order to extiad paclitaxel and taxoid compounds as raw materials for production of anH-canca drugs'8'. Though, Taxus wdlichiana Zucc. In Vietnam is not completely studied on the taxoid compounds which structures are similar to paclitaxel to establish databases cantribuHng to quality contiol. Tha^ore, the aim of this thesis was to isolate, purify taxoid compound from Taxus wallidiiana Zucc, and thm estid>lish it as a r^ermce standard using in quality contiol.
Materials and Methods: Materials: 1.000 mg of taxmd-containing fraction extiadedfrom the needles of Taxus wallichiana Zucc cultivated in Lam Dong province shown in Figure 1 in experimental. Methods: IsolaHon and idmHfication of chemical consHtnent taxoid TWwas isolated and purified in taxoid-containingfracHonfrom the previous studies by Medium Pressure Liquid Chromatography (MPLC), and crystallized in solvmt The purified compourui was elucidated chemical structure by means of UV-Vis, IR, MS, NMR spectioscopy; the purity (^ TWwas determined by reversed phase HPLC with PDA detector. Establishment of r^ence standard according to the guidelines of WHO, ASEAN and ISO/IEC 15328: Basing on the data of infrared spedrometry, ultiaviolet spedrometry, mass spedrometry, nuclear magneHc resonance spedrometry and high performance liquid diromatography, the quality and quanHty c^TWwere evaluated. In addiHon, the validation of method and the assessment of inta-laboratories wae conducted to set up the assigned values of the established refamce standard.
Results: From 1.000 mg of the initid taxoid-containing fraction, TWwas identified as 10-deacetyl taxinin B (268 mg) vnth ova 98% purity. Thm, 158 mg of TW (98.57% purity) was established as r^aence standard according to the guidelines of WHO, ASEAN and ISO/IEC 15328.
Conclusiom 10-deacetyl taxinin B was successfully isolated from the needles of Taxus wallichiana Zucc.
Cultivated in Lam Dong province, and it was established as the refamce Standard and the assigned value reached 98.57% purity.
Key words: Taxus wdlichiana, 10-deacdyl taxinin B, refermce standard.
M 6 D A U hormon va thuoc khang hormon, ... Trong do, nhom thuoc duoe sii dung pho bioi trong hoa tri Theo bao cao ciia IMS Health vao ngay y^^ khSi u 6 vii, tuyen Uen Uet va phoi la nhom 05/05/2015, tong toan bo chi phi cho thi tnrong ^g^ ^^ ^ ^ ^ ^^^ ^^^^j ^ ~ , pj,g ^ ^ j ^ thuoc ung thu toan cau la 75 ty USD nam 2009 p ^ ^ ^ , ^^ ^^^f^^jf ^(^ ^ ^g a^, j g ^ g g^
thi Sen nam 2014 da can moc 100 ty USD, tang y.^_ „ ^ ^ ^ . ^^ ^^ ^^ ^^ ^^^^ ^gj ^ . p ^ 10,3% so vod nam 2013. Con so nay du doan se p^^^^^f ^ g j j <i„„<, ^ ^ jhay co trong Thong khong dimg lai ma con co the tang len d m 147 ty ^^ ^ ^ ^ ^^.^^ wallichiam Zucc.) duoc trong 6 USD vao nam 2018m. TM tarimg thuoc ung thu ^ j . j j j ^ „ , ^ ^.^^ .^^^ ^^ ,, ^^ ^g^ ^^^
la mpt thi t r « m g nhieu tiem nang, da va dang ^ ^ ^ ^ ^ ^ ^ ^ ^ ^^ ^„ ^ , ^ . ^^ ^^^
phat trim rat da dang vm nhieu nhom thuoc tac ^^ ^.^ j » ^ ^^ ^ ^ ^ , ^ dong len te bao ung thu theo nhieu co che khac ^ ^ ^ ^ » ^ ^ ^ ^ ^^^^ ^^ ^ ^ ^ ^ nhau nhu nhom thuoc khang chuyen hoa, nhom „•„ \7;AI. V T , „ \r n- u - ' ^ •- . -
" -' cua Viet Nam. Muc heu nghien a m nay la phan thuoc alkvl hoa, nhom khang sinh tri ung thu, , - „ „ i „i *• u "• *_• -^ -^ i . ~
" ^ ' . , , , . l^P ^^ "^^ " ^ C3U true mot SCP hop chat tu nhom alkaloid thuc vat, nhom enzym, nhom
nhien tir la Thong do la dai (Taxus wallichiana
45
N g h i e n ciru Y hpc Y Hpc TP. H b Chi M i n h * T^p 20 * So 6 * 2016 Zucc); buoc c^u thiet lap chat chuaii doi chieu
taxoid phan ]^p dugc, co the dimg trong ki&n tra chat lupng dupe lieu va cac che pham lien quan.
Cao methanol 100%
PE-Ace (100:5)
So rfo 1: Quy trinh chiei xual phdn doqn TW.
Phtrong phap nghien ciht Phdn lap hgp chat
Phan doan TW duoc chiet tach bang phuong phap sac ky cot trung ap (MPLC) de tach TW vao nhiing phan doan khac nhau. Ket. tinh TW dupe tinh che bang each cho tai ket tinh nhieu Jan vai DCM va PE de thu duac chat tinh khiet;
kiem tra l^i bang sac ky lop mong, dugc gop lai thanh ket tinh TW. Sau do, TW dupe kiem tra do tinh khiet bang HPLC pha dao voi ^ u do PDA.
Xdc dinh cau true
Dua vao cac ket qua phan tich pho UV-Vis, IR, MS, NMR da xac dinh duoc hpp chat TW la 10-deacetyl taxinin B.
Bieu kien sac ky long hieu ndng cao He thong MPLC Combiflash Companion TS (So sen 207C20178), dau do UV - K2600 (Merck Knauer). Cot trung ap RediSep Column tu nhoi (30 x 3 cm) voi chat hap phu la silica gel c6 hat 40 - 6 3 [im.
He thong HPLC phan tich CBM - 20A (Ma E(3/01/TC/035) ctia hang Shimadzu, dau do diod quang SPD - M20A; cot Phenomenex C18 (250 x
EOlUtlNG-IWUtJNGIHAPNGHIENajtJ Doi tirgng nghien ciiu
Phan do?n TW co khoi lupng 1.000 mg chiet til sac ky cot co dien eua nhiing nghien ciki truoc day. Qui trinh chiet xuat dupe trinh bay trong so d o i .
PE-Ace (90:10)
Phan doan TW TW
20AC; bp phan khu khi dung moi true tiqi DGU - 2OA5R. Cac dieu kien tiai hanh HPLC phan tich duoc tiinh bay trong bang 1.
Bdng 1: Diiu Men sac ky tiong phdn lap vd ddnh gid do Hnh khiei hgp chdi TW
Dilu ki$n sic ky Pha d$ng Nhi#t 6Q CQt Bade sdng phdt hi^n
Dilu ki^n sac ky Pha dpng Nhi^t 6^ c$t
MPLC PE-Aceton
(84:16) 25''C 280 nm 40 ml/phCil
1 ml/Ian 10% (g/100ml)
HPLC ACN-nydc (70:30)
25°C 280 nm 1 ml/phut
10 Ml 0,1% (g/100ml) Thiei lg.p chdi chuan
Hpp chat 10-deacetyl taxinin B sau khi phan lap da dupe xac djnh cau true bang cac ket qua phan tich pho nghiem UV-Vis, IR, MS, NMR Hpp chat nay co dp tinh khiet cao tren 98% khi danh gia bang phuong phap HPLC, dupe dung lam nguyen lieu de thiet lap chat chuan dieu che qua cae buoc sau day:
Djnh lugng va tham dfnh qui trinh dfnh lugng bang HPLC
Vl khong CO chat chuan doi diieu 10-deacetyl taxinin B, ket qua djnh luong 10-deaeetyl taxinin
Y Hpc TP. H o Chi M i n h * Tap 20 * So 6 * 2016 Nghien cihi Y hpc
qui ve 100% dien tich pic tren sac ky do. Qui trinh dinh lupng dupe thain dinh bao gom khao sat tinh phu hpp he thong, tinh chpn loe, khoang tuyen tinh va dp lap lai.
ThiB lap chat chuan 10-deacetyl taxinin B Qui trinh thiet lap chat chuan 10-deacetyl taxinin B gom cac buoc: dong goi, danh gia dp dong nhat giiia cac Ip, danh gia ket qua dinh lirpng lien phong thi nghiem tai ba phong thi nghiem dat GLP, xac dinh gid tri an dinh tren phieii ki&n nghiem*^*"'.
KET QUA V A BAN L U A N Phan lap hpp chat
Tit 1.000 mg phan doan giau hpp chat TW ban dau, sau khi dupe phan tach bang l ^ thuat MPLC va thm: hien tinh che da thu dupe 268 mg hpp chat TW (dp tinh khiet > 98%).
Xac dinh cau tnic Hop chdi TW
Hop chat TW la ket tinh tring, hinh ldm dai, dang xop, tan ^ t trong ACN, aceton; it tan trong MeOH, EtOH, PE va EtOAc.
Tren pho IR ciia TW cho cac tin hieu cua nhom - O H t u do (3.495 cm'), nhom C=0 ciia ester (1,737 cm-^), nhom C-O-C eiia ester (1,168 cm-^), nhom carbonyl C=0 (1,665 cm-^). EJieu nay phii hop vdd cau tnic taxoidP''^ Pho UV - Vis ciia TW cho dinh hap thu cpc d?i 6 buoc song 274 nm, phu hpp voi taxoid^'^i. Pho MS-ES+ trong MeOH cho thay ion co m/z = 645,2; do la manh [M-Na]+ do vly TW co khoi lupng phan tu la 622 Dalton, co kha nang tuong ling voi cong thiic phan tu ciia mpt hpp chat taxoid la 10 - deacetyl taxinin B (C35H42O10)'''. So sanh dii Heu p h o ' H NMR va ^^C NMR ciia TW vol cae tai lieu da dirpe cong bo thi dii Ueu pho ciia TW hoan toan trung khop v6i dii lieu pho ciia 10-deacetyl taxinin Bti'">
Bdng 2. So sdnh phd'^H-NMR (do tiong CDCh; 500 MHz) cua TWvalO- deacetyl taxinin B^IV
c
1 2 3 5 6a 6b 7 9 10 14a 14b 16 17 18 19 20a 20b 22 23 Me Me Me 2', 6' 3', 5' 4'
TW SH(J,HZ)
2,23, s 5,60, d (6) 3,32, d (6) 5,35, s 1,76, m 2,08, m 5,38, m 5,83, d (10) 5,30, d (10) 2,38, d (20) 2,86, dd (7,20)
1,23, s 1,80, s 2,24, s 1.02, s 4,90,5 5,42, s 6,44, d (15,5) 7,69, d (15,5) 2,08, m 2,08, m 2,08, m 7,78, d (7)
7,42, m l,M,m
10-deacetyl taxinin B
«/,(J,tt?) 2,20, m 5,60, dd (6,3,2)
3,33, d (6) 5,30, m 1,76, m 2,09, m 5,38, m 5,80, d(10) 5,28, d(10) 2.38, d( 20) 2,86, dd (20,6)
1,24, s 1,82, s 2,23, s 1,02, s 4,90, s 5,42, s 6,45, d (16)
7,6,d(16)
7,80 7.42 7.42
Hirih 1. Cau true hoa Jwc cua 10-deacetyl taxinin B Tu cac ket qua pho nghiem UV-Vis, IR, MS, NMR, hpp chat TW dupe xac dinh la 10-deacetyl taxinin B. Chat nay da dupe tim thay trong loai Taxus cuspidata Sibe et Zucc vao nam 1994'"'.
Ddnh gid do tinh khiei cua TW
Dp tinh khiet ciia TW dupe danh gia bang phuong phap HPLC phan tfch: TW co dp tinh khiet tren 98%.
47
Nghien clhi Y hpc Y Hoc TF. Hb Chi Minh * T | p 20 * So 6 * 2016
,1 HJAMuM'
(a) (b) Hinh 2- Dp Hnh Idiiei sackycuaTW
(a): Sac kydo2 chieu kian tra do tinh Idtiel tqi Aisoimi (b) SSck^dd3 ch^ (200 - 500 nm). DKU hen tiin hdnh sac Thiet lap chat chuan doi chieu
Dinh luang vd th^m djnh qui trinh djnh Ittgng bang HPLC
Bdng 3: KH qud tham dinh qui trinh dinh lugng 10- deacdyl taxinin B bang HPLC
Tmh chpn ipc BQ I$P I ^ (n=6) Tinh tuy^n tinh B6 diing (n=6)
RSD(tR) = 0,114%<1%
RSD(Spfc) = 0,128%<2%
D9t RSD = 0,117%<2%
R-'s 0,9998 > 0,995 98,57%
ky dugc trinh bdy trong bdng 1.
dupe dinh lupng 2 Ian de tranh sai so va dimg ph^p kiem Dixon de l o ^ gia tri bat thutmg trong ket qua dinh lupng.
Bdng 4: K^ qud phdn Hdi phmmg sai ANOVA mgt ymto
Nhan xet: qui trinh dinh lupng bang HPLC dat cac yeu cau ve tinh phu hpp he thong, tinh chpn loc, khoang tuym tmh va dp lap lai.
Ham lupng 10-deacetyl taxinin B dupe tinh Iheo phirong phap HPLC qui ve 100% dien tich pic tren sac ky do. Ket qua: ham lupng 10- deaeetyi taxinin B la 98,57% tinh tren che pham nguyen trang.
Thiei lap chat chuan IG-deacetyl taxinin B Bdnggoi vd ddnh gid do dong nhdi giiia cdc lo
Khoi lugng TW dupe dong goi: 158 mg. Khoi lupng dong goi: 5 mg/1 Ip. So Ip: 30. Dieu kien dong goi: trong buong Glove-box chiia 99, 99%
khi nito 6 nhiet dp phong (25°Q va dp am 30 - 40%. Danh gia dp dong nhat giua cac Ip sau khi dong goi: so lo danh gia (g) dupe chpn ngau nhien, so lupng Ip dupe tinh bang cong
GiOa cac nhdm Trong cCing
ntwm Tfing cgng
T&ng binh phuong
0,004 0,012 0,016
B^ctv do
1 10 11
Binh phuong
0,004 0,001
FB 3.648
F, 4.9611
Nh|n x6fc Fm = 3,648 < FK = 4,964. Nhu v|y, cac Ip TW dat dp dong nhat sau khi dong Ip.
Bdnh gid kei qud djnh heang lien phong ihi
Bang 5: KIOTI tra Hnh phii hap cm he thShg vi M qua dmh luang TW tqi 3 phdng thi nghiSm (n^)
S 6 d n g 1 2 3 4 6 6 H^m luvng trung btnh
%RSD (phai nho hon 2%) PTN1«
98.81 98.80 98,87 98.78 98.82 98.80 98,81 0.031
pro 2*
98,69 98,70 98,65 98.75 98.69 98,72 98,70 0.032
P T « S 98,65; 98.85; 98.6«!
98.631 93.63 98,6* 98,64 0.009
r ?
I
??'•
*PTN 1: KhoaThiitlSp didi dimn-diaid6idiieU,Viin Ki^ nghiim thuoc Tp. Ho Chi fAinh.
PTN 2: Khoa Vqt ly do Irnng, V^n Kian nghian thuoc Tp.
HS Chi Minh.
PTN 3: Phong Ddm bdo chdi lugng, Cdng ty cd'ph&t Duac
Y Hpc TP. H o CW M i n h * Tap 20 • So 6 * 2016 Nghien cthi Y hpc
Xac ^inh gia tri an dinh
Bdng 6: Kil qua tinh gid tri an dinh hdm luang TW (n = 18)
5 = 1.5xs*
X - S x + 6 Trung binh
€ ) 5 l ^ s X m d i s m6i
Xo
98.716 0,078 98.695 0.082
X i 0,122 98,573 98,817 98.716 0.073 98,715 0,082
X a 0,124 98,591 98,838 98,715 0,073 98,715 0,082 Tir ket qua tren, tinh z-score. Tat ca 18 gia tri dinh luong deu co < 2.
Nhir vay, ham luong TW la 98,715% tinh Iheo nguyen trang voi dp lech 0,078 va dp khong dam bao do p=l,25 x 0,078 x V l 8 = 0,413. Cac Ip TW dupe de xuat bao quan 6 nhiet dp 2 - 8'C, tranh anh sang.
KETLUAN
Ttr 1.000 mg phan doan giau taxoid chiet tu- la Thong do la dai (Taxus wallichiana Zucc.), bang phucmg phap MPLC va ky thult tai ket tinh da phan lap dupe 268 mg hpp chat TW. Qua bien giai cac dii heu pho (UV, IR, MS, NMR) cung vol viec so sanh cac d u lieu tfiam khao, cau tnic ciia TW dupe xac dinh la 10 - deacetyl taxinin B co dp tinh khiet cao (tren 98%), dii dfeu kien de thiet lap chat ehuah doi chieu.
Hpp ehat 10-deacetyl taxinin B da phan lap dupe su dung lam nguyen Ueu de thiet lap ehat chuan doi ehieii theo huong dan ciia WHO va ASEAN. Lo chat chuan dong goi dupe danh gia dp dong nhat va dirpc tham dinh tai ba phong thi nghiem dat tieu chu&i GLP. Chat chuan 10- deacetyl taxinin B eo dp tinh khiet cao 98,57%
(tinh tr&i che phain nguyen trang), eo the dupe su dung trong kiem nghiem dupe lieu Thong do
la dai Vi|t Nam (Taxus wallidiiana Zucc.) va eae ehe pham lien quan.
Lai cam tm: Nhom tdc gid xin chan thanh cam an Trung tam tr^ng m chtbieh cay thuoc Da Lat; Tmng lam Sdm va Duqc Ueu Tp. Ho Chi Mink da cung c^ duac lieu vd cao chiit tir la Thong do Id dai (Taxus walUchiana Zucc.) trong cr Lam Dong Chiing t6i ding xin to long biet mi sdu sac deh phong Hoa - hap chdi thiin nhiin thuoc Viin Cong nghe Hoa hpc TP. HCM vd Viin Kii'm nghiem thuoc Tp. HS Chi Minh da ho trg viic thuc hien de tdi
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Ngay nhdn bdi bdo: 30/07/2016 Ngdy phdn bien nhqn xet bdi bdo- 31/08/2016 Ngdy bdi bdo dugc dang: 25/11/2016
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