• Nghien cifU - Ky thuat
^
Phan lap va thiet lap chat chuan acid clorogenic
Nguyen Thi Anh Nguyf t', Le Phan Kim Trang', Pham Dong Phuvng^"
'Bo mon Dtrpc lieu - Khoa Dupc - DH YDtrac Tp. Hd Chi Minh - Tnmg tdm Ddo tgo va Nghien cmi phdt frien thuoc nguon gdc Itf nhien - DH Y Dugc Tp. Hd Chi Minh 'E-mail: phamdongphuong 1954{dyaltoo.coin
Summary
Chlorogenic acid (CGA) also known as 5-0-caffeoylquinic acid (5-CQA), which is responsible for antibaderial. antioxidant, anticardnogenic activities, and hypoglycemic, hypolipidemic effects ofArtidioka (Cynara scolymusL.). was isolated ^m the plant leaves by MPLC with Cl 8 reversed phase silica gel cdmrn (0.04 ^0.063 mm), and prep-HPLC with a C18 Sunfire column (10 x 150 mm; 5 pm). detector-PDA (323 nm); mobile phase - acetonitiil (B) and fonvi'c acid 0.1% (A), flow rate - 3 ml/min. The isolated cWorogenfc add was preliminanly identified by TL C, HPLC-PDA and further structurally confirmed by IR, MS and NMR.
The HPLC quantitative analysis of chlorogenic acid was validated for system suitability, selectivity, linearity range and precision by independent GLP laboratories based on ICH guideline. Packaging, validating tiie sample homogeneity and interiaboratory testing to establish the assigned content were tiased on guidelines of ISO 13528 and ISO Guide 35.
Keywords: Chlorogenic add. Cynam cardunculus, Cynara scolymus, secondary standard.
Dat van de
Aetisb (Cynara cardunculus L.= C. scolymus L., Asteraeeae) dupe s u dyng ti> rat Idu ddi d nhieu nude trfin t h ^ gidi cung nhu dupc biet den nhieu d Vifit Nam do ed tdc dyng nhu thbng mgt, lpi tieu, dieu trj thieu ndng gan, mgt, ldm giam cholesterol .. va tdc dyng trfin cua actisd Id do cdc hogt chat cynarin, acid clorogenic vd flavonoid ^-*K Ngodi ra. acid clorogenic cbn cd tdc dyng hg lipid mau, chOng oxy hba. hg dudng huyet i^'. Qua nghien cuu eho thSy ham lupng cynarin trong Id actisd riit th^p (gin nhu khbng cd) nhung hdm lup'ng acid clorogenic r^t cao i^' vd dieu nay cung phu hpp vdi Dup'c d i l n Anh va Chdu Au chi quy djnh djnh lupng acid clorogenic vd djnh tinh cynarosid trong Id actisd bdng HPLC i^i. HiOn nay, trfin thj trudng cd rat nhieu che pham tCr actisd dung hb trt? dieu trj edc bgnh ve gan, mgt vd tieu hod. Tuy nhien, eh4t lupng cde c h l pham eb actisb dang luu hdnh trong nudc cb chat lupng khbng on dinh. Trong khi db, Dupc dien Vifit Nam IV chi quy dinh djnh lupng cynarin trong la vd cao actisb bdng phuong phdp UV-Vis ^K De ndng cao tieu ehuan chat lupng la vd cde bdn thdnh ph^m, thdnh pham cb actisb thi ngodi viOc djnh lupng cynarin cung can d | t vdn de nghifin cuu djnh lupng acid clorogenic bdng HPLC. Vigc phdn lgp vd thi6t lap chat chuan cynarin da dup'c cbng bo'»'. Bdi bdo nay trinh bdy ve phdn lap vd thiet ldp chat chuan acid clorogenic nhdm phye vy eho viOc dinh lupng acid clorogenic
tnang actisb cung n h u cdc sdn phSm tu' actisO trSn thj trudng vd cbn dup'c s u dyng 6i djnh lupng mpt so d u p c liOu cd chua hogt ch^t chinh Id add clorogenic, gbp ph^n chu dOng vd thay th^ cho vigc phai mua ch^t chu^n tCr nude ngodi vdi giS thanh cao.
D o i t u ^ n g v a p h u v n g p h a p n g h i e n cihi Nguyen ligu: La actisb t u o i (200 kg) dupc Uiu hdi tgi phudng 12, Tp. Dd Lgt vdo thdng 10/2014, n>a sgch, de rdo vd rpc bd gdn chlnh, l^y rifing ph^n phien Id (90 kg).
Hod c h a t - t h i e t bj
Hoa' chit COn 96%, doroform, ethyl acetat n-butanol dgt tieu chulin phdn tich (PA), acetonitnl (Merck), methanol (Merck), acid formic (Merck), ethanol cua hang J.T.Baker - My. Acid clorogenic c h u i n (Phytolab-DCpc. s6 lb 857).
Thiit bi: Sac ky Idp mdng (SKLM) dupc thi.fc hifin trfin bdn mdng silica gel F^^^ trdng s3n (Merck).
Sac kl cOt chdn khdng (VLC) thye hiOn trfin cOts^
ki (7 X 40 cm). Sac k j Idng d i l u c h l dp suit tmng binh (MPLC) Biorad (Biologic Duoflow Pathfinder 80, d i u dd Quadtec UV- Vis). Sdc ky ldng di^u chi dp s u i t cao (prep-HPLC) t i i n hdnh trfin mdy sic ky d i l u ehe Waters 2767, d i u db PDA 2996. Ph6 hOng ngoai (IR) d u p c do trfin mdy Nieolet iS500 NIR-FTIR (My). Cac phdp do trfin d u p ^ thyc hiOn tgi ViOn K i l m nghiOm thuOc Tp. HO Chl Minh. PM cOng hudng t u hgt nhdn (NMR) d u v c ghi trfin mdy T4>PCHi DU'(?C HQC - 11/2016 (S6 487 N A M 56)
• Nghien CIFU - Ky thuat
Bruker Avance 500 MHz. P h i * H-NMR d u g c do d 500 MHz; p h i "C-NMR d u o c do d 125 MHz vdi TMS d u p c dung Idm chat ehuan npi (TMS) tgi Vifin Hdn Idm \Cnoa hpc Vigt Nam - Hd NOi. May do khoi pho Micromass micro™ APi - Waters; May HPLC Waters 2695 /Mllance. d i u dd PDA 2996; May do pho UV U-2010 (Shimadzu) tgi Tmng tam Dao tao va Nghien cuu phdt friln tfiuoc nguon goc t y nhien - DH Y Dup'c Tp. Ho Chl Minh.
Phuvmg phdp nghien c u u
Chiit xuit vd phan Ipp: Phien Id t u o i actisd d u p c 6n djnh vd dh\et ndng vdi con 96% vd sau db Id hon hyp cOn 96% - n u d c (1:1). Djch chiet dupc cfi thu hOi dung mbi de &iu d u p c cao long vd l i e phdn b l l i n l u p l vdi doroform, ethyl acetat, n-butanol, thu h l l dung mfii dup'c cdc cao chilt tuong ung. Cao n-butanol d u p c then khai trfin tren s i c ky cOt chdn khbng (VLC), chpn edc phdn dogn don gian va cd chda acid dorogenic d l phdn lap t i l p bdng sdc ky Ibng dp s u i t trung binh (MPLC) va tinh c h l b i n g sac ky Ibng cao dp dieu ehe (prep- HPLC) a l thu d u p c a d d clorogenic tinh khllt.
Tham dinh va Oiiet Ipp chit chuin acid clorogenic phdn Ipp dwgv
Djnh tinh vd xdc djnh ciu trOc: b i n g p h i UV, IR, MS vd NMR.
ThCr tinh khiit b i n g SKLM {vdl 3 hO dung mbi sde ky khdc nhau) vd HPLC-PDA.
Xdy dpng quy trinh djnh lupng add dorogenic bing HPLC: Khdo sdt d i l u kiOn s i c ki, bao gOm:
pha dOng, thdi gian phan tfch, toe dfi dbng, budc sbng phdt hiOn, nhifit dO cOt 25 °C.
Ddnh gid phucmg phdp djnh tupng acid dorogenic bdng HPLC theo hudng din cOa ICH:
ddnh gid tinh tuang thfeh hO thong, tinh ddc higu, tinh tuyen ttnh, dO IdP lgi vd dO dung.
Ddng gdi vd ddnh gid dd ddng nhat Ip trong qua trlnh ddng gdi: Add clorogenic d u p c dbng gdi vao lp thuy tinh mdu ndu, cd Idp dOm teflon vd hdn kin frong Glove-Box chua khi nito, moi ong 10 mg, dbng thdnh 45 Ip d l ddnh gid dong nhat Id frong qud frinh dbng gbi. Ddnh gid k i t q u i theo ISO Guide 35*". C h i t c h u i n phdn lgp dupe ddng gdi tgi ViOn K i l m nghiOm t h u l c TR H i Chi Minh frong d i l u kifin dbng gdi tifiu c h u i n .
Xdc djnh gid tq in djnh. dd khdng dam bao do theo ISO 13528 <'°' vd gid tn cdng bi trdn phiiu kiim nghidm dua trdn kit qud d/nh lupng tai 2 phdng thi nghidm: K i t qud djnh lupng d u p c thyc hiOn tgi it n h i t 2 phdng ttif nghiOm dOc ldp dgt ehuin GLP, hodc ISO 17025, fr^ng dd cb it nhat mOt phdng thi nghiOm cd du t h i m q u y l n va y nghTa
phdp ly doi vdi Viet Nam. moi phdng se dup'c gui 6 Ip ngau nhien kem theo chat c h u i n , quy trinh phdn tich. Hai phbng thi nghiOm tham gia ddnh gid chat c h u i n gom: Khoa t h i l t lap chat chuan vd chat doi diieu vd Khoa Kiem nghiem vgt ly do ludng - ViOn Kiem nghiOm thuoc TP. Ho Chi Minh.
K e t qua n g h i e n CLPU
C h i l t x u i t va phan lap acid clorogenic tip Id actisd
Chiet xuat: Ti> 200 kg Id t u o i aetisb, rpc bb gan chinh thu dup'c 90 kg p h i l n la t u o i . Tien hdnh on djnh vd chiet ndng vbi 80 lit con 96% trong 15 phut, dp l i y djch chiet. Bd cbn Igi d u p c c h i l t l i n 2 tuong t y nhu fren vdi 90 lit hdn hp'p con - nude (1:1).
Todn bO d i d ! chiet con d u p c cb thu h l l dung mbi dudi dp s u i t giam thu d u p c 9 kg cao long aetisb (ALE).
Phan tdch cdc phan dopn bang phuxmg phap phan bo tdng-ldng: 9 kg cao Idng dupe lac phdn b l l i n l u p l vdi doroform, ethyl acetat, f^butanol, thu hoi dung mdi d l thu dLPpc cde eao tuong ung, bao g i m : cao doroform (150 g), cao ettiy! acetat (90 g) vd cao n-butanol (200 g). Cao n-butanol d u y c dung tdch phdn dogn gidu acid clorogenic trfin s i c ky cOt chan khbng.
Phan tdch phan dopn bang VLC: 100 g cao n-butanol (dp am 25%) dupc t r i l n khai trfin s i c ky cpt chdn khbng vdi cOt 7 x 40 cm, khoi lupng 600 g silica gel 0,04-0,063 mm. pha dOng ethyl acetat vd methanol vdl lupng methanol tdng dan. K i l m tra cdc phdn dogn trfin SKLM, gom cde phdn dogn cb thdnh phan giong nhau. K i t qua thu d u y c 9 phdn dogn, trong do phan doan 8, 9 cb chua acid clorogenic nhung it tgp hon phdn dogn 5; 6; 7 (hinh 1). Tien hdnh lgp Igi cpt VLC l i n 2 vdi cung d i l u kifin nhu frfin thu d u p c tong cOng 60 g phdn dogn 8. 9 vd d u p c s u dyng de phan ldp t i l p trfin cOt MPLC.
Phdn lap phdn dopn giau acid clorogenic bang MPLC: Tien hanh tren 37 cOt MPLC, m6i cOt t r i l n khai 1 g phdn dogn 8; 9 cua cOt VLC, cOt sac ky 2,5 >= 50 cm, 170 g silica gel RP18 (0,04 ^ 0,063 mm), pha dpng gom aeetonitril (dung mbi B) vd acid formic 0 , 1 % (dung mbi A) vdi chuong trinh gradient nhu sau: 8% B (4 phiit) 8 - 15 % B (70 phut), 15 % B (30 phut), 15 - 30 % B (90 phut). 30
% B (30 phut), 30 -100 % B (15 phut), t i e dO dbng 10 ml/phOt, the tich hdng 20 ml vd b u d c sbng phdt hiOn 323 nm. K i t qud cho t h i y eb 3 phdn dogn ehinh (hinh 2). Phan dogn 2 dupc kiem fra trfin HPLC-PDA cho friiy gom 2 pic cd thdi gian tuu g i n nhau, trong db ed 1 pic cb thdi gian luu vd
TAPCHi Dir<?C HQC-11/2016(S6487 NAM 56) 23
• Nghien ciyu - r\y umat
pho UV imng vm acid clorogenic chuan, gop tat ca cac ptiSn doan nay cua nhieu lan then khai, co duOT ap suit giam de loai aeetonitril, dong kho va thu dLP?c 5,0 g phan doan 2. Phan doan nay diroc tinh ch^ Sep tren cgt prep-HPLC.
Hinh 1: Kil qua phin Up tren c6l VLC AC : acK^ clorogenic chuan
Hinh 2: Kit qua phan Up trdn cpt MPLC
PD: phin doan AC: acid donjgenic Ttnh che acid clorogenic bang prep-HPLCPh&n doan 2 cua cOt MPLC Qisgc hoa tan trong nud'c v^ trien khai I|p Igi nhieu lan tren prep-HPLC trong cung dieu kien, cot Sunfire C18 (10 X 150 mm; 5 pm), pha dpng gom aeetonitril (B) vi acid formic 0,1% (A) vai chuang trinh gradient v6i ti le aeetonitril tang d^n tCr 8% - 30%
trong thdi gian 20 phut, toe dp dong 3 ml/phut, the tich tifem m§u 80 pi, buac song phat hipn 323 nm. Ket qua cho thay ed 2 pic chlnh (hinh 3) trong d6 pic 1 c6 thd'i gian luu va pho UV tnjng vd'i acid clorogenic chuin, ggp tat ci cac l4n hijng pic 1 sau nhi^u lln trien khai, eo dud'i ip suat giam de loai acetonitnl, ddng kho va thu dugc 1.000 mg acid clorogenic phSn lgp.
Hinh 3: Ket qua phan ldp trdn cgt prep-HPLC Djnh tinh v4 xae djnh cSu tnic
Ph6 UV-Vis. Pho UVcua add clorogenic c6 3
dinh hap thu aj/c dai tai 217,5; 241.2 va 325,7 nm
217.5 _ 3 2 B ^
9.48:
Hlnh 4: Phd UV-Vis cda add dorogenic phin l$p
Pho IR: Pho IR cua acid clotggenic phSn \iteho cac dinh hap thu dgc tmng cOa nhdm -OH (khoSng 3.300 cm'), nhom carbonyl (khoSng 1.700 cm'), vong tham (khoing 1.500 cm', 650- 900 em') (hlnh 5).
i^
:
\ /"'' n ^ VI
rf / y
Hlnh 5: Pho IR cOa add clorogenic phin lip
Ph6 MS: Pho kh6i ESI-MS (negative) cila diSlphan Up (cho minh m/z = 353) tuang i>ng v«
cdng thuc nguyen C„H„0,la cong thuc nguy*
ciia cac dSn chit acid caffeoylquinic (hlnh 6).
Hlnh 6: P/ri5 MS cua acid dorogerac phin lip
TAP CHi Dirpc HQC - 11/2016 (S6 487 NAM 51Nghien ciru - Ky thuat
Pho NMR: D u ligu ph6 'H va "C-NMR eua acid clorogenic phan lap trong bang 1 phu hap
vol d u lieu pho NMR cija acid clorogenic theo tai lieu " " .
VlM 1 2 3 4 5 6 7 V 2' 3' 4' 5' S
r
8' 9' 4-OH 30H 3'-0H 4'-0HBang 1: So sanh dO" Sdu phd 'H
J j 73.5 37,2 68,1 70,4 70,9 36,3 174,9 125.6 114,7 145,6 148,3 1157 121,4 114.3 1445 1657
NMR (CDpD, 500 MHz) va"C- NMR cua add domgenic phin lip vol add dorogenic theo tai lieu "i
Acid clorogenic phin /|p (DMSO) SOO MHz S„ppm(J,Hz)
1.96 (IH.m) 1.79(1H,m) 3,90(1H,ljiri|
3,56 (IH, bns) 5,07 (1H,m) 1,96 (1H,m)
7,03(1H,c(,2Hz)
6,77 (IH, If, 8 Hz) 6,9d(1H,(«;8,5;2H2)
6,15(1H,lf, 16Hzl 7,42(1H.i(, 16Hz) 4,90 ( 1 H , M 4,76 (IH.W) 9.14 (1H, s) 9,57 (1H,S)
COSY
H-4, H-2 H-3. H-5 H4. H-6
H4 H-3
HMBC
C-7 C.6'; C-r, C-9 C-7 C-6'; C-3'; C-4'; C-1'
C-9'; C-3, C4, C-1 C7, C-2, C-5, C-1
C-6', C-8'; C-3'. C4'
C-1'; C-3'; C4' C-2'; 0^'; C4' C-1'; C-9' C-r, C-9'
C-2'; C-4'
Acid dorogenic (DMSO, 400 MHi) S,.
73.5 37,2 68,0 70,9 70,3 36,2 175,0 125,6 114,3 145,0 148,4 115,8 121,4 145,6 114,8 165,8
S„ppm (J. Hz)
2,03(1 Km) 1,77 (1H,m) 3,90(1H,(»tO 3,56|1H,M 5,07(1H.«I/, 10 Hz; 6 Hz)
2,03 (IH. m)
7.03 ( 1 H . M
6.76 (IH.d, 8 Hz) 6,98(lH,(*);S;2Hz) 6,15(1H,((,16Hz) 7.42(1H,cf.16Hz)
9,19 (1H,s) 9,67 (1H,s)
Hinh 7: C^u tnic phdn tu ciia acid ckirogenic phan I0p Xdc djnh t ^ p c h i t lifin quan
Dtnh tinh bang sdc ky Idp mdng: Thyc hien vdi 3 hfi dung mfii khdc nhau cd dp phdn c y «
tang d i n cho t h i y acid clorogenic phdn lgp chi cho mpt vet duy nhat tren s i c ky do.
Djnh tinh liing HPLC - PDA: Ham lup'ng tgp cd trong acid dorogenic dieu c h i tfnh theo % dipn tich pic Id 0,45 %.
Bang 2: Kit qud danh gia dd tinh khiit cua add dorogenic phdn Ipp
STT Ten pic D^n tich dinh %DI§n tich dinh 1 Aod dorogenic
2 Tgp
11,788 12.356
99,55 0.45
Hinh 8: Sdc ky dS HPLC kiim tra dp tinh khiet cua acid clorogenic phan Ipp duoc Xdy d i ^ g qui trinh djnh l u ^ n g ham lu>pfng
c h i t d6i chieu
Diiu Idpn sac ky: Cpt Sunfire C18 (4,6 x 150 mm.
5 pm); pha dpng la acetonitril-acid fonnic 0 , 1 % (10:90); thdi gian phdn tich 12 phut. t6c dp dbng 1 ml/phut; b u d c sbng phdt hipn 323 nm; nhi6t dp
T A P C H I DU'OC H Q C - 11/2016 (SO 487 NAM 56)
n^i I I C I I V I X U '
cpt 25 Ki; the tich tiem m i u 5 pi; mdu thCr vd acid clorogenic c h u i n d i j p e hba tan trong methanol a nong dp 100 ppm (0.1 mg/ml). Ham lu-png acid clorogenic phdn lgp du'p'c xdc djnh bdng cdch so sanh vdl dipn tfch dinh cua acid clorogenic chuan.
Ddnh gia quy trinh djnh tuong theo huvng din cua ICH: Quy trinh djnh luwng bang HPLC- PDA da thiet lap dpt cdc yeu c i u sau day
Dat tinh tuong thich he thong (RSD% cua thdi gian ILTU R, vd dipn tfch dinh S < 2%; k'>1,0; 1.05 <
a 5 2,0; 0,8 < As < 1,5; Rs > 1,5).
Dat tinh ddc hieu (t^ cua acid clorogenic phdn lap Id 9,7 va pho UV cua mdu thO hoan todn taing khdp vdl acid dorogenic ehuin).
Khodng tuyen tinh cua add dorogenic Id 15,6- 1.000 pg/ml.
Dp /dp lpi cb RSD = 0,55% vd dp dung ed ti le phye hoi tCr 94.4-101%.
Dong goi vd danh gia dong nhat Ip Bdng goK Khbi lu'png ban dau: 1000 mg. Dbng gbi: 10 mg/1 Ip. Tong so lo: 76 Ip (760 mg)
Danh gia ddng nhit lp: So Ip ddnh gid:
g = •. + 1 = 10 lp. L i y ngau nhien 10 Ip, m6i lp djnh lupng 2 lan. Ket qua djnh lu'p'ng d u v c trinh bdy d bdng 3
B a n g 3: Kef qua djnh lugng acid dorogenic ddnh gia dong nhat Id Sothirt\r
1 2 3 4 5 6 7 8 9 10
Lin 1 (%) 92,55 92,68 92,81 92,19 92.24 92,42 92,44 92.10 92.22 92.20
Lan^C^) 92.25 92.47 92,66 92J3 92,49 92,68 92,59 92,26 92,31 92,50
Trung binh 92,400 92,575 92,735 92,210 92,365 92.550 92,515 92,180 92,265 92,350 _ DUng Hie nghigm Dixon de kiSm tra: xep dc ket qua theo thu l u t i n g dhn. xet 2 ket qua nhd nh^t va Idn nhat eua chudi d u ligu:
" » , = 92.18% => G,,„ = 0,057 < G „ „ = 0,466 x ^ = 92,735% => G.,^ = 0,288 < G „ „ = 0,466 Ket lugn: ket qua 92,18% va 92,735% khong tjat thud'ng, nghTa la ehu6i d u lieu tren khdng cd gid tri t}4t thud'ng.
Danh gia dong nhat giira cac lo theo ISO Guide 35 nhu sau:
B a n g 4 : Phin tich phuong sai mpt yeu to theo ANOVA
Nguonsaisi ^ T * * " * * ^ ^ ^ f T
^ phuong tpOo trung bmfi " ^Giira cdc f^KJm 0,017405 1 0.017405 0.40916 44138?3 Trong tifng nhom 0,76569 18 0.042538 Tong cpng 0,783095 19
F^ = 0 , 4 0 9 < F = 4 , 4 1 4 . D o d d . c d c Ip add c l o r o g e n i c d o n g n h a t t r o n g q u d t r i n h d d n g gix.
Ddnh gid chat chuan lien phdng thi nghiffn B a n g 5: Kit qua dfnh luong add domgenk
tpi hai phdng tiii nghiem So thti tp PTHI PTN2 ~
92,55 92,68 92,81 92.19 92,24
92,44 92,10 92,22 92,20 92,30
Tmng binh 92,48 92,35
PTN 1: Khoa Thiit tdp diit diuan - chit dii diiiu, W ^ Kih nghidm thuie TPHCM
PTN 2: Khoa V$t ly do ludng. V4n Kiim nghidm thuie TPHCU B d n g 6 : Kit qua ddnh gid lidn phdng thi nghi$m
ttieo ANOVA Nguon sai Tong tiinh B$c tu Binh f^uong
io tmng tunh GiOa cac
nhdm Trong tung nhbm
T6ng cpng 0,721025
1 0,049408 0,735663 4,96460}
0,671617 10 0.067162
Nhdn xdt: F^ = 0.736 < F^ = 4,965. Do db. m qua trung binh ciia 2 phdng thi nghi$m khdc niiau khbng eb nghTa, phuiyng phdp phdn tieh cb dO l(ip lai cao, ham lu'p'ng acid clorogenic phdn ldp dtipc khbng php thupe vao phdng thi nghipm tham gia danh gid.
Xdc dinh gid trj an d j n h , d $ khdng d i m bio do vd gid trj c d n g bo tren phleu kiem nghidm cua acid clorogenic
Bang 7: Kit qua tinh gid trj in djnh hdm lupng add domgenic
S'1,5X5"
X--S x-*S Tmng binh Dglechs X' mai s'mdi
X".
92.42 0,26 92,36 0 2
x;
0,37 92.03 92,76 92.39 0,22 92,39 0,24
X', 0,367 92,03 92,76 92,39 0,22 92,39 0,245
0,367
«*.
92.03 9176 92,39
oa
92J9 0j45 N h S n x e t : s a u 4 l l n I h a y d o i . s * = 0 , 2 4 5 ktidng d o i , X" = 9 2 , 3 9 % d u i y c c h p n .TAP C H i DUgiC HOC - 11/2016 (S6 487 NAM » l
• Nghien ci>u - Ky thuat
TLF ket qua tinh theo z-score, t i t ca gia trj deu c61 z I < 2. Tinh ket qud gid trj an djnh trdn 12 ket qua n h i / sau:- Hdm lu'png trung binh: 92,39 % {trdn nguydn trgng)
- Dp lech s: 0,22
- DO khdng ddm bdo do p: 0,088.
Ket luan: Hdm iuwng acid donagenic Id 92,39
% tfnh tren c h i p h i m nguyen trang, dd khbng dam bdo do = 0,086; dp Ipch = 0,22 (n = 12).
Ket luan
Ti> Id actisd (Cynara scolymus L.) da chiet xuat vd phdn Ipp d u v e 1 -000 mg acid clorogenic d i sd dgng Idm c h i t c^uan doi chieu. Chat chuan acid clorogenic phdn [dp d u p c tham djnh day du cde tidu chi cua c h i t d i u i n theo h u ^ n g ddn cua ISO 13528 va ISO 35. Ket qua cho t h i y acid clorogenic phdn lgp dat tieu c h u i n quoc gia (secondary standard) vdi ham luwng du'pe xdc dinh Id 92,39%
tinh theo nguydn trgng {n = 12; s = 0,22; p = 0,088).
C h i t c h u i n acid dorogenic da phan ldp vd tiilit ldp eb t h i dwpc Cmg dgng cho cdng vipc k i i m nghipm ehit lu'png nguydn lieu, cao bdn thdnh p h i m va tiidnh p h i m tCr actisd.
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{Ngdy nh$n bai: 21/09/2016 - Ngay phan bipn: 10/10/2016 - Ngay duy^t ddng: 01/11/2016)
Nghien cihi tinh da dang loai cua chi Aspergillus Fr.:Fr.
tren vj thuoc ngu vi tu {Fructus Schizandrae
r r
dang luu hanh o* mot so hieu thuoc dong du'crc
Tran Trinh Cong*, Nguyen Quynh Le
Bo mon Vi Sinh & Sinh hpc - Tnrang Dgt hpc Dtrpc Ha Nqi
'E-mail: congdhn%gmail com Summary
The A^iergillus speaes associated witii 20 samples of Fmdus Sdiizandrae cdleded from herijal dnigstores in Hand. Bac Ninh and Hsd Duong pmvinces were invedigated. The occurrence of the fungi was preliminarily deteded by cultirure on PDA medium. The fungi growing on the fruit samples were furiher identified after subculture on Czapek Dox agar medium and evaluated in reference to tiie keys by Pitt and Hoddng. Raper and Fennell.
and Samson et al.. Five Aspergillus spedes were isdated and indentified as Aspergillus n'iger, A. paradticus, A.
fumigatijs. A usftis and A aculeabjs. Of tiiese Aspergillus species. A niger was tiie nKist frequent spedes in ttie coBeded samples. For A. niger is known as tiie predominant odiraUixins producer in natijre and it was isolated from a remari(able high pementage of ttie tested samfdes, good sbx-age practices were recommend to prevent the fungal contamination in general and ocairrence ofochratoxins in tiie hertial drugs in particular
Keywords: Aspergillus, A. aculeatus. A. fiimigatijs, A. n'iger. A. parasiticus. A. ushjs, Fnjctus Schizandrae.
TAPCHi DCQC H Q C - 11/2016 (SO 487 N.AM 56)
