B Y T TRNG I HC DC H NI NGUYN TH NHUNG NGHIN CU NH LNG FLAVONOID
V POLYSACCHARID TRONG CY U(A. carmichaeli Debx.)TRNG TNH H GIANG
KHA LUN TT NGHIP DC S H NI - 2014 B Y T TRNG I HC DC H NI NGUYN TH
NHUNG NGHIN CU NH LNG FLAVONOID V POLYSACCHARID TRONG CY U
(A.carmichaeli Debx.) TRNG TNH H GIANG KHA LUN TT NGHIP DC S Ngi
hng dn: 1. PGS.TS. Nguyn Tin Vng 2. ThS. Th Thanh Thy Ni thc hin:
1. B mn ha dc2. Khoa Y Dc, HQGHN H NI - 2014 LI CM N Trcht,emxin
chnthnh bytlng knh trngvbit nsusc ti PGS. TS. Nguyn Tin Vng - Ph
Vin trng Vin Php y Quc gia v ThS. Th Thanh Thy - Ging vin b mn Ha
Dc, hng dn tn tnh, ng vin v gip em trong sut qu trnh thc hin kha
lun. Emcngxingi li cmntiThS.Vc LiGingvinKhoaY Dc i hc Quc gia H Ni,
cc anh ch k thut vin b mn Ha Dc Trng i hc Dc H Ni v khoa Y dc Trng
i hc Quc gia H Ni, gip em trong sut qu trnh lm thc nghim.
XincmnBangimhiu,Phngoto,ccphngbantomi iu kin gip em hon thnh kha
lun. Cm n cc thy c trng i hc
DcHNiquantmdudtvtruynthkinthcchoemtrong5 nm hc va qua. Cui cng, em
xin gi li cm n ti gia nh v bn b lun ng h, ng vin v khch l em trong
qu trnh hc tp v lm kha lun. H Ni, thng 5 nm 2014 Sinh vin Nguyn Th
Nhung MC LC LI CM N MC LCDANHMC CH VIT TT DANH MC CC BNGDANH MC CC
HNH CHNG 1.TNG QUAN
................................................................ 2
1.1. Tng quan v cy u
.............................................................................
2 1.1.1.c im thc
vt..................................................................................
2 1.1.2.Phn b v trng hi
..............................................................................
3 1.1.3.B phn dng
........................................................................................
3 1.1.4.Thnh phn ha hc
..............................................................................
3 1.2.Tng quan v Flavonoid
............................................................................
4 1.2.1.nh ngha, cu trc v phn
loi........................................................... 4
1.2.2.Tnh cht
...............................................................................................
4 1.2.3.Cc phng php nh lng flavonoid trong dc liu
........................ 5 1.3.Tng quan v polysaccharid
......................................................................
7 1.3.1.nh ngha, cu trc v phn
loi........................................................... 7
1.3.2.Tnh cht
...............................................................................................
7 1.3.3.Phng php nh lng polysaccharidtrong dc liu
....................... 8 1.4.Tng quan v phng php quang ph hp th t
ngoi kh kin (UV-VIS) ...
..................................................................................................................
9 1.4.1.Nguyn tc
............................................................................................
9 1.4.2.Cc k thut nh lng bng phng php o quang
......................... 11 1.5.Thm nh phng php phn tch
........................................................ 13 1.5.1.
c hiu
.........................................................................................
13 1.5.2. tuyn tnh
.......................................................................................
13 1.5.3. lp li
.............................................................................................
13 1.5.4.
ng...............................................................................................
14 1.5.5.Gii hn pht hin (LOD)
....................................................................
14 1.5.6.Gii hn nh lng (LOQ)
.................................................................
15 CHNG 2. I TNG V PHNGPHP NGHIN CU 16 2.1. Nguyn vt liu, thit b
............................................................................
16 2.1.1.Nguyn vt liu
...................................................................................
16 2.1.2.Dng c thit b
...................................................................................
16 2.2.Ni dung nghin cu
................................................................................
17 2.2.1.Xy dng v thm nh phng php o quang nh lng flavonoid ton phn
trong l cy u
...........................................................................
17 2.2.2.Xy dng v thm nh phng php o quang nh lng polysaccharid ton
phn trong ph t
.............................................................. 17
2.3.Phng php nghin cu
.........................................................................
18 2.3.1.Xy dng phng php phn tch
........................................................ 18
2.3.2.Thm nh phng php phn
tch....................................................... 18
2.3.3.Phng php x l s liu
...................................................................
19 CHNG 3. THC NGHIM V KT QU ................................ 20
3.1.Xy dng v thm nh phng php nh lng Flavonoid ton phn trong l u
..................................................................................................
20 3.1.1.Xy dng phng php nh lng flavonoid ton phn trong l u .
...........................................................................................................
20 3.1.2.Thm nh phng php nh lng flavonoid ton phn trong l u ...
.................................................................................................
22 3.1.3.ng dng phng php nh lng xy dng v thm nh nh lng flavonoid
ton phn trong mu l u H Giang ................................ 27
3.2.Xy dng v thm nh phng php nh lng Polysaccharid ton phn trong ph
t
............................................................................................
28 3.2.1.Xy dng phng php nh lng polysaccharid ton phn trong ph t.
....................................................................................................
28 3.2.2.Thm nh phng php nh lng polysaccharid ton phn trong ph t..
...............................................................................................
30 3.2.3.ng dng phng php va xy dng xc nh hm lng polysaccharid ton
phn trong mu ph t H Giang ...................................... 35
BN LUN
.........................................................................................
37 KT LUN V KIN NGH
............................................................ 38 A.
KT LUN
.........................................................................................
38 B. KIN NGH
........................................................................................
40 TI LIU THAM KHO DANH MC CH VIT TT EtOH:Ethanol
MeOH:Methanol2,4-DNP:2,4- dinitrophenylhydrazin
UV-VIS:Ultraviolet-Visible (T ngoi-kh kin) HPLC:High Performance
Liquid Chromatography ( Sc k lng hiu nng cao) PA:Pure Analysis (
Tinh khit phn tch) PTN:Phng th nghim LOD:Limit of detection (gii hn
pht hin) LOQ:Limit of quanlity (gii hn nh lng) DANH MC CC BNG Bng
1.1. Mt s nghin cu nh lng flavonoid ton phn trong dc liu bng phng
php o quang
............................................................................................
6 Bng 1.2. Mt s nghin cu nh lng polysaccharid ton phn trong dc liu
bng phng php o quang s dng thuc th phenol-acid sulfuric
...................... 8 Bng 3.1. Cch pha dy chun quercetin o quang
.......................................... 21 Bng 3.2: Kt qu kho st
tuyn tnh gia nng v hp th ca dung dch chun quercetin
.....................................................................................................
24 Bng 3.3 : Kt qu lp li ca phng php nh lng flavonoid ton phn ...
25 Bng 3.4.Kt qu kho st ng ca phng php nh lng flavonoid ton phn
......................................................................................................................
26 Bng 3.5. Kt qu xc nh lch chun ca mu trng khi nh lng flavonoid
ton phn
..............................................................................................................
26 Bng 3.6. Kt qu xc nh hm lng flavonoid ton phn trong mu l u H
Giang
....................................................................................................................
27 Bng 3.7.Cch pha dy chun glucose o quang
............................................ 29 Bng 3.8: Kt qu kho
st tuyn tnh gia nng v hp th ca dung dch chun glucose
........................................................................................................
32 Bng 3.9. Kt qu lp li ca phng php nh lng polysaccharid ton phn
.... 33 Bng 3.10. Kt qu kho st ng ca phng php nh lng polysaccharid
ton phn
..............................................................................................................
34 Bng 3.11. Kt qu xc nh lch chun ca mu trng khi nh lng
polysaccharid ton phn
.......................................................................................
35 Bng 3.12. Kt qu xc nh hm lng polysaccharid ton phn trong mu Ph t
H Giang
...............................................................................................................
36 DANH MC CC HNH Hnh 1.1. Tiu bn cy u
..................................................................................
2 Hnh 1.2. Cnh mang hoa, qu ca cy
u......................................................... 2 Hnh
1.3. R c u, ph t
..................................................................................
3 Hnh 1.4. L ca cy u
.....................................................................................
3 Hnh 3.1. Ph hp th ca mu trng, mu chun quercetin v mu th
............... 23 Hnh 3.2: th biu din s ph thuc tuyn tnh ca hp th
v nng quercetin.
..............................................................................................................
24 Hnh 3.3. Ph hp th ca mu trng, mu chun glucose v mu th
.................. 31 Hnh 3.4. th biu din s ph thuc tuyn tnh ca hp
th v nng glucose.33 1 T VN Cy u (Aconitum sp.)lmt loi cy thuc qu c
slu i trongy hc c truyn dn tc vi tn v thuc l u (Radix aconiti) v ph
t hay c con ca cy u (Radix aconiti lateralis). u, ph t cha chbin ch
yudng ngoi xoa bp khi nhc u, mi tay chn, au khp, bong gn; ph t ch
(dim ph,bchph,hcph)yhcctruyncoilmtvthuchidngkhphong hn, dng trong
mt s triu chng nguy cp (try tim mch, ra nhiu m hi, chn
taygilnh)[3].CyuVitNam(Aconitumcarmichaeli)mchoangv c trng cc vng
ni cao nh H Giang, Lo Cai, Sa Pa [10], [11]. Trong cy u hot cht
chnh v c tc dng dc l mnh nht l alcaloid c ng dng nhiu trong y hc c
truyn. Ngy nay vi s pht trin ca khoa hc k thut v ang c nhiu cng
trnh nghin cu v tc dng sinh hc ca cc thnh phn khc trong cy u ng dng
trong y hc nh: flavonoid trong l v
polysaccharidtrongrccyu.Quanhiunghincuchothythnhphn flavonoid trong
l u c tc dng chng oxy ha v kh nng dn dp gc t do
[24],[27],[35],[37].Polysacharaidtrongrcuctcdng:chngoxyha, tng kh
nng min dch, c hot ng khng u tt, gim cholesterol mucho kt qu kh
quan[25], [28], [34], [40], [41], [42]. Song song vi vic nghin cu v
tc dng sinh hc ca cc thnh phn trong dc liu th vic nghin cu xy dng
cc phng php nh lng cc thnh phn ny cng rt cn thit cho cng tc kim tra
chtlngdcliu.TuynhintrongDcinVitNamchacccphng php nh lng hai thnh
phn flavonoid v polysaccharid trong cy u.Xut pht t cc yu cu thc t
trn, chng ti tin hnh thc hin ti "Nghin
cunhlngflavonoidvpolysaccharidtrongcyu(Aconitum carmichaeli Debx.)
trng tnh H Giang" vi cc mc tiu sau: 1.Xydngvthmnhphngphpoquangnhlng
flavonoid ton phn trong l cy u. 2.Xydngvthmnhphngphpoquangnhlng
polysaccharid ton phn trong ph t 2 CHNG 1.TNG QUAN 1.1. Tng quan v
cy uCy u Vit Nam c tn khoa hc l Aconitum carmichaeli Debx., thuc h
Hong lin Ranunculaceae [10], [11]. 1.1.1.c im thc vt [3], [5], [8],
[9], [15], [17], [18] Cy u thuc loi cy tho, mc hng nm, cao chng
0,6-1m. R c mp, hnh con quay, r ci to mang nhiu r nh (nn c tn l ph
t). Thn ng, hnh tr, t phn nhnh. L mc so le, c gn hnh chn vt, l ca
cy con hnh tim trn, crngcato,lgix3-5thy,mpkharngcanhn,haimtclngngn,
mt trn lc bng, mt di nht.Cm hoa mc ngn thn thnh chm, hoa lng tnh,
khng u, hoa to mu xanhlamhocxanhtmmcstnhau.Baohoagm5li,litrnthngv
cong hnhm chp kn trng hoa tiugim, nh nhiu, bu c 3 cha nhiu l non.
Qu gm 5 i mng, ht nhiu, trn mt c nhiu vy nh. Hnh 1.1. Tiu bn cy
uHnh 1.2. Cnh mang hoa, qu ca cy u 3 Hnh 1.3. R c u, ph t Hnh 1.4.
L ca cy u 1.1.2.Phn b v trng
hiulcycavngnim.CytrngVitNamthchnghicaovi
iukinkhhummtcavngnhitinicaonhSaPa,BcH,SnH, ng Vn, Qung B Cy a sng,
khi cn nh l cy chu bng [8], [9], [15].
uctrngbnghthocccon.Ngitathngthuhochkhicy trng c 1-2 nm, ct b r con,
ra sch t, phi hay sy kh. Nu cn thu ly
htthikhithuhtxongtiptcchmscthucvomahoanmsau. nc ta thu hi vo khong
thng 9, thng 10 khi cy ang ra hoa. Trng vo thng 1, thng 2[3], [8],
[18]. 1.1.3.B phn dng B phn dng ca cy u l r c [2], [8] : - u l r c
m ca cy u phi hay sy kh. -Phtlcnhnh(ccon)cacyu.Tphtcthchthnhdim ph,
hc ph,bch ph. 1.1.4.Thnh phn ha hcCc thnh phn ha hc trong cy u kh a
dng v phong ph. Theo mt stiliuthamkhottcbphncacyuuchaalcaloidtrong
4 alcaloidchnhlaconitin[3],[8],[9].TheoktqunghincucatcgiBi Hng Cng
[10], [12] trong mt s b phn ca cy cn c nhiu thnh phn nh: -R c c thm
polysaccharid, cht bo v sterol.-Thn cy c thm carotenoid.-L v hoa c
thm carotenoid, sterol v flavonoid. -Ht c thm cht bo v carotenoid.
1.2.Tng quan v Flavonoid 1.2.1. nh ngha, cu trc v phn loi [1], [13]
-Flavonoid l nhm hp cht t nhin ln thng gp trong dc liu ngun gc thc
vt, phn ln c mu vng.
-Vcutrchahc,flavonoidlnhmhpchtphenol,ccutokhungc bntheokiu C6-C3-C6
(khungcbngm2vngbenzenAvBnivinhau qua mt mch 3 carbon) v c chia lm
nhiu nhm khc nhau. -Phn loi flavonoid: c nhiu cch khc nhau phn loi
flavonoid + Cch phn loi thng dng l da vo v tr ca gc aryl (vng B) v
cc mcoxyhocamch3C,ngitachiaflavonoidthnhbanhmchnh: euflavonoid,
isoflavonoid v neoflavonoid.
+Cncthphnloi:biflavonoidlnhngflavonoiddimer,triflavonoid cu to bi 3
monomer flavonoid, flavolignan l nhng flavonoid m phn t c mt phn cu
trc lignan. 1.2.2.Tnh cht [1]
-Musc:Ccdnchtflavoncmuvngrtnhtckhikhngmu(trng hp cc nhm OH methyl
ho), flavonolvng nht nvng, chalconvauron
vngmncam.Ccchtthucnhmisoflavon,flavanon,isoflavanon,
flavanonol,leuco-anthocyanidin,flavan-3-oldokhngcniilinhipgia 5
vngBvinhmcarbonylnnkhngmu.Ccdnchtanthocyanidinthmu thay i tu theo
pH ca mi trng. Tuy nhin khi flavonoid trong cc b phn ca cy th mu sc
ca chng cn ph thuc vo hn hp vi cc sc t khc. - tan: tan ca cc
flavonoid khng ging nhau. Thng flavonoid glycosid v flavonoid
sulfat l nhng hp cht phn cc nn khng tan hoc t tan trong dung
mihuc,tanctrongncvtanttnhttronghnhpcnnc.Cc aglycon flavonoid th tan
c trong dungmi hu c, khng tan trong nc. Cc dn cht flavonoid c nhm
7-hydroxy thng d tan trong dung dch kim long. 1.2.3.Cc phng php nh
lngflavonoid trong dc liu1.2.3.1.Phng php cn -Nguyn tc: Chit xut v
tinh chthnh phn flavonoid trong dc liu sau em cn tnh ra hm lng
flavonoid c trong dc liu. -ng dng i vi nguyn liu giu flavonoid v
dch chit t tp cht, v d nh lng rutin trong hoa he hay nh lng
flavonoid trong tht qu m :
+nhlngflavonoidtrongthtqum(Prunusarmeniaca)bngphng
phpcn[23]:BtdcliuchitbngCHCl3trnbnhSoxhletloitp,sy kh. Chit bng
EtOH 96% trn bnh Soxhlet thu ly dch, sau bc hi dung mi.
ChitlibngncnngsauchitbngEtOAcbayhidungmi.Thuc cn. Cn cn xc nh lng
flavonoid ton phn. +nh lng rutin trong n hoa he (Sophora japonica)
[1]: loi tp bng HCl 0.5%, chit bng EtOH 96% nng, thy phn bng H2SO4
, quercetin rt t tan c lc v cn ri tnh ra rutin. -Phng php nyc uim l
thc hinnhanh, d thc hintuy nhinsai s mc phi ln.1.2.3.2.Phng php o
quang ph t ngoi - Nguyn tc: Cho flavonoid phn ng vi thuc th to cc
hp cht mu c hp th xc nh ri nh lng bng phng php o quang: phn ng
cyanidin; phn ng kt hp vi mui diazoni; to phc mu vi AlCl3, mui
titan, mui chrom 6 Bng 1.1. Mt s nghin cu nh lng flavonoid ton phn
trong dc liu bng phng php o quang STTTn loi Mu dc liu Thuc thCht
chun Bc sng TL TK 1Aconitum heterophyllum Bt c AlCl3/EtOH(20mg/ml);
AcOH Rutin415 nm[36] 2Trikarshika *Bt hn hp 2,4-DNP 1% ; KOH 1%/
MeOH 70% Quercetin415 nm[31] 3Chrysanthemum indicumL. Bt hoa kh
AlCl3 2% /MeOHQuercetin415nm[14] 4Raw propolis from northern
Croatia Keo ong nguyn liu 2,4-DNP 1%; KOH 1%/ EtOH 70% Naringenin
495nm [30] AlCl3 10% ; kali acetat 1M Quercetin415nm 5Romanian
Propolis Keo ongAlCl3 5% / MeOHGalangin425nm[32] 2,4-DNP 1% , KOH
10 %/ MeOH Pinocembrin486nm 6Crinum lactifolium Bt lAlCl3 5% / MeOH
cha 5% AcOH, natri citrat 1% Quercetin425nm[20] 7Cleistocalyx
operculatus N v lNaNO2 5% , AlCl3 10% ,NaOH 1M Catechin510 nm[22]
Ghi ch: (*)Trikarshika l hn hp kh ng lng ca thn r Zingiber
officinale, r c Aconitumheterophyllum v thn r Cyperus pangore.
1.2.3.3.Phng php sc k lng hiu nng cao (HPLC) -Nguyn tc: Da vo i lc
khc nhau ca cc cht khc nhau vi hai pha lun tip xc m khng ng tan vi
nhau, mt pha ng v mt pha tnh. Qu trnh sc 7 k xy ra da trn cc c ch:
hp ph, phn b, trao i ion hoc ry phn t. Thi gian lu camt cht tnh t
lc timmuvo ct nkhi cht rakhi ct t gi tr cc i cho pic trn sc k .
-Phngphpscklnghiunngcao(HPLC)cthngdngnhlng
flavonoidtrongdcliuchoktqutincyvchnhxccao.Tuynhinch nh lng c mt vi
cht c th, khng xc nh hm lng ton phn c. V
d:nhlngflavonoid(baicalein)trongvcyncnc(Oroxylumindicum) bng HPLC
[21]. 1.3.Tng quan v polysaccharid 1.3.1.nh ngha, cu trc v phn loi
[1] -Polysaccharidlnhnghpchtbaogmhngtrmnhngnghn monosaccharid kt hp
vi nhau bng lin kt glycosid. -Phn loi: +Phn t polysaccharid c th gm
mt hoc vi loi monome khc nhau, do
ngitachiarapolysaccharidngth(homopolysaccharid)vpolysaccharid d th
(heteropolysaccharid).+Tytheongungcngitaphnccpolysaccharidtnhinrabanhm
ln:Pytopolysaccharid(polysaccharidthcvt),Zoopolysaccharid(polysaccharid
ng vt) v Polysaccharid vi sinh vt. 1.3.2.Tnh cht [1]
Polysaccharidmangnhiutnhchtkhcmono-vdisaccharidnhkhngc phn ngkh,
khng c v ngt, thngkhng tan trong nc, khi ha tan d hnh thnh dung dch
keo. 8 1.3.3.Phng php nh lng polysaccharidtrong dc liu
Trongsnhiuphngphpsomunhlngcarbohydrat,phngphp dng thuc th
phenol-acid sulfuric l phng php n gin, nhanh chng v ng tin cy nht
xc nh hm lng carbohydrat ton phn trong mu. Phng php c th p dng nh
lng tt c cc loi carbohydrat: mono-, di- , oligosaccharid,v
polysaccharid;kclngngtrongproteoglycan,glycoproteinvglycolipid
[38], [39]. Phng php s dng thuc th phenol- acid sulfuric c s dng
rng ri v nhy v cch tin hnh n gin [39]. - Nguyn tc:
Trongphngphpnydungdchacidsulfuricmcthyphnpolysaccharid,
oligosaccharid, v disaccharid thnh cc monosaccharid. Sau cc
monosaccharidmt nc to thnh furfural (Pentose) hoc hydroxymethyl
furfural (hexose). Cc hp cht ny phn ng vi phenol to thnh hp cht mu
vng. i vi cc mu th c hm lng ng pentose cao s c cc i hp th ti 480
nm. ivimu th c hm lng ng ng hexose cao s c cc i hp th ti 490 nm. Mu
sc ca sn phm to mu n nh trong vi gi, v chnh xc ca phng php tng i
cao [35]. Phngphptomuvithucthphenol-H2SO4oquangphUV-VIS c s dng ph
bin inh lng polysaccharid trong dc liu ni chung cng nh trong r c cy
u ni ring. Bng 1.2. Mt s nghin cu nh lng polysaccharid ton phn
trong dc liu bng phng php o quang s dng thuc th phenol-acid
sulfuric STTTn loiMu dc liuBc sng nh lng Hm lngTLTK
1Aconitumcarmichaeli Bt c484 nm18,156 %[45] 2Aconitum coreanum Bt
r490nm4,62 %[43] 9 3Aconitum carmichaeli Ph t (c con) u (c m) R con
484 nm22,02 % 33, 53% 6,10% [44] 4Aconitum coreanum Bt r490
nm5,7%[34] 5Aconitum kusnezofi Bt r490nm6,0%[41] 6 Lentinus edodes
Qu th (fruiting body) 492 nm3,84%[16] 7rong m(Sargassum) Fucoidan
(Thn rong)490 nm50,63%[19] 1.4.Tng quan v phng php quang ph hp th t
ngoi kh kin (UV-VIS) 1.4.1.Nguyn tc [2], [6] 1.4.1.1.nh lut
Lambert- Beer Chiu mt chm nh sng n sc c bc sng v cng I0 qua dung
dchngnhtcnngCbdylpdungdchL.khiiquadungdch,mt
phnnhsngsbhpthli,mtphnb phnx,phncnlisiquadung dch vi cng I. Mi quan
h gia phn nh sng I v I0 c th hin bng nh lut Lambert-Beer C LI I.
.010 Trong : :l hp th ring ca dung dch. H s ny khng ph thuc vo
nng,chphthucvobnchtcachttanvbcsngca chm tia n sc chiu vo dung dch.
L : l b dy ca lp dung dch (cm) C : l nng ca dung dch -iu kin ng dng
nh lut + Chm tia sng phi n sc. + Dung dch phi nm trong khong nng
thch hp. + Dung dch phi trong sut. 10
+Chtthphibntrongdungdchvphibnditcdngcanh sng UV-VIS 1.4.1.2.Mt s i
lng thng dng- truyn qua truyn qua hay cn gi l thu quang (T): c trng
cho trong sut ( v mt quang hc) ca dung dch, c tnh theo cng thc:
0IIT T thng tnh ra phn trm. Mt cht c T=1 ( hay 100%), ngha l hon
ton khng hp th nh sng, ngi ta ni cht hon ton trong sut. - hp th hp
th hay cn gi l mt quang (A) , c tnh theo cng thc: C LTLog A . .1 i
vimt cht xc nh (c xc nh) otrn mt loi cuvet o c b dy L= 1cm, hp th t
l vi nng dung dch : C K A . L K . -H s hp th ring (cmE1% 1): cmE1%
1 chnh l hp th ca dung dch c nng 1% khi dng cuvet o c b dy 1cm. Vi
mt cht tan xc nh, ti mt bc sng o xc nh,cmE1% 1 l mt hng s. -H s hp
th phn t (M ) H s hp th phn tcn gi l h s hp th mol l hp th ca dung
dch c nng 1M, khi dng cuvet o c b dy 1cm. Cng nh cmE1% 1, vi mt cht
xc nh, trong iu kin o nht nh( bc sng, dung mi, nhit ), M l hng s.
Gia cmE1% 1 v Mc mi lin h : MEcmM 101% 1M l phn t lng ca cht tan.
11 1.4.2.Cc k thut nh lng bng phng php o quang [4], [6] 1.4.2.1.Xc
nh nng khi bit h s hp th ring Viccchtcmthpthringbittrcchnhxcvnnhmt
bcsngno,ngitacthomtquangcadungdchchttrong
dungmitngngtibcsngny.Thngthnglbcsngchp th cc i. Nng dung dch c tnh
theo cng thc: cmxxEAC1% 1Mun p dng k thut ny my quang ph phi c chun
ha v bc sng vgitrmtquang,dungdchphitrongsutvcnngnmtrongvng p ng ca
nh lut Lambert- Beer. 1.4.2.2.K thut so snh vi dung dch chunXc nh
mt quang ca dung dch th c nng Cx (cha bit) c tin hnh ng thi vi vic
xc nh mt quang ca dung dch chun c nng Cc
bitchnhxc,trongiukinthamnnhlutLambert-BeerthCxc tnh theo cng thc
Trong phng php so snh kt qu cng chnh xc khi nng Cx cng gn vi nng
dung dch chun Cc. 1.4.2.3.K thut ng chun xy dng ng chun ngi ta chun
b mt s dung dch chun c nng
Cskhcnhau,thngdng5-8dungdchchun.ohpthAscady chun v lp th A theo C.
o hp th Ax ca dung dch mu th v da vo ng chun xc nh c nng cht th Cx.
12 1.4.2.4.K thut thm chun hnchnhhngcatpchtngitaathmvodungdchmt lng
chnh xc cht chun lm cho nng dung dch tng thm mt lng C0. o mt quang
ca dung dch th (A) v dung dch th thm chun (A), nng ca dung dch th c
tnh theo cng thc: 1.4.2.5.K thut thm ng chun:Tin hnh bng cch thm
chnh xc nhng th tch ging nhau ca dung dch th vo dy chun cha nhng
lng khc nhau v chnh xc ca cht chun. o
hpthcadyrivngchunquanhgiamtquangvilngcht chun thm vo. Giao im ca ng
chun vi trc honh (nng ) cho ta nng ca cht cn nh lng. 13 1.5.Thm nh
phng php phn tch [6], [7] 1.5.1. c hiu
-nhngha:Lkhnngxcnhcchtphntchkhicmtccthnh
phnkhcnhtpcht,snphmphnhy,tdcViphpthnhlng phi a ra kt qu chnh xc v
hm lng hay hot lc ca cht phn tch c trong mu. -Cch xc nh: Thmvo sn
phmmt lng thch hp tp cht hoc t dc
riphntchchngminhrngktqunhlngkhngbnhhngbitp cht hoc t dc. 1.5.2.
tuyn tnh -nh ngha: tuyn tnh ca mt phng php phn tch l s ph thuc tuyn
tnh gia i lng o c (y) v nng cht cn phn tch (x) trong khong xc
nh.Ncbiuthbngphngtrnhhiquyy=ax+bvhstngquan tuyn tnh
r.-Cchxcnh:Chunb1dygm5-10dungdchchuncnngxi tng
dnx1,x2,xn.Tacpngyi:y1,y2,yn.Tinhnhphntchmubng phng php cn kho st,
xy dng phng trnh hi quy yi = axi +b v xc nh h s tng quan r.1.5.3.
lp li -nhngha:lplicamtphngphpphntchlmcthngnht gia cc kt qu th ring
bit theo quy trnh th nghim c p dng lp i lp li nhiu ln trn cng mt
mu, lp li c biu th bng lch chun tng i (RSD) hoc sai s tng i (r %).
-Cch xc nh: Tin hnh th nghim lp i lp li n ln, thu c cc kt quxi lx1,
x2, xn. Trung bnh cc kt qu l niixnX11 Ta tnh lp li theo cng thc
sau: + lch chun:1) (12 nX xSDnii 14 + lch chun tng i: (%) 100
XSDRSD+ Sai s tng i:(%) 100 XtnSDrtl tr s gii hn ph thuc vo xc sut
(p) v s bc t do (n-1) c tra trong bng phn b t (student); = 1- p
1.5.4. ng - ng ca mt phng php phn tch l mc st gn ca cc gi tr tm thy
trongphn tch so vi gi tr thc. -Cch xc nh: thng c xc nh bng 2 cch: +
Cch xc nh trn mu t to: T to mu vi cc thnh phn hot cht v t
dcctrongchphmvihmlnghotchtkhong90%,100%,110%hm
lngghitrnnhn.Tinhnhnhlnghotchttheophngphpphntch
cnnhgi.ngltlphntrmgiaktqunhlngsovihotcht cho vo. + Cch xc nh trn mu
th c thm chun: thm vo mu th mt lng
chtchun(khong10%,20%,30%sovilnghotchtcsnsaochotng
lnghotchtctrongmunmtrongkhongtuyntnh)ritinhnhnh lng theo phng php
phn tch ra. ng s c tnh bng t l phn trm gia lng cht chun tm c so vi
lng cht chun thm vo. 1.5.5.Gii hn pht hin (LOD) -nh ngha: Gii hn
pht hin l lng cht thp nht cn phn tch c th pht hin c v mt nh tnh.
-Cch xc nh: + Cch 1: o tn hiu lin tc t mu trng (B) v mu th (S).
Thit lp t s S/B. Nu S/B = 3/1 l chp nhn c. +Cch 2: o ln ca ng nn
bngcch phn tchmt smu trng. Gii hn pht hin c tnh theo cng
thc.aSDLOD3 15 Trong :SD l lch chun ca p nga l dc ca ng chun ( h s
a ca phng trnh ng chun y=ax+b) +Cch 3: Phn tch cc mu c nng bit sau
pha long cho ti khi tm c nng ti thiu no cn pht hin c bng phng php
xut. 1.5.6.Gii hn nh lng (LOQ) -nh ngha: l lng thp nht ca cht cn th
c trong mu th c th pht hin c v mt nh lng vi ng v chnh xc thch hp.
-Cch xc nh: +Cch1:olncangnnbngcchphntchmtsmutrng. Gii hn nh lng c
tnh theo cng thc. aSDLOQ10= 3,3 LOD Trong :SD l lch chun ca p nga l
dc ca ng chun(h s a ca phng trnh ng chun y=ax+b) +Cch 2: Phn tch cc
mu c nng bit sau pha long cho ti khi tm c nng ti thiu no cn c th xc
nh c cht th vi chnh xc v ng chp nhn c. 16 CHNG 2.I TNG V PHNGPHP
NGHIN CU 2.1. Nguyn vt liu, thit b 2.1.1.Nguyn vt liu 2.1.1.1.Dc
liu Dc liu: cy u (Aconitum carmichaeli ) trng tnh H Giang. -Ph t ly
khi cy ra qu v li, ra sch, phi sy kh, nghin thnh bt kch thc khong
1mm. -L ti cy u, ly khi cy ra qu phi sy kh, xay nh kch thc 2-5 mm.
2.1.1.2.Ha cht, dung mi -Cht i chiu :+ Cht chun PTN quercetin hm
lng 99,0%+ Cht chun PTN glucose hm lng 98,6%. -Dung mi, ha cht: +
EtOH 960, t tiu chun PA. + H2SO4 c, t tiu chun PA. + Phenol , t tiu
chun PA.+ AlCl3, t tiu chun PA. + NaOH, t tiu chun PA.+ Nc ct 1 ln,
t tiu chun PA. 2.1.2.Dng c thit b -My quang ph UV- VIS Cary 60. -My
siu m Utrasonic cleaner-MRC. -My o pH AL 20 pH- Aqualitic. -My ly
tm HSCEN- 204- MRC. -My lc xoay in Velp- Zx3. -Cn phn tch AUW 220.
17 -Phu lc G3 -Ni cch thy Memmert WNB10 -Micropipet100 - 1000l (5l)
Hirschman - c -Cc dng c thy tinh khc: Bnh nh mc, pipet, cc c
m,2.2.Ni dung nghin cu 2.2.1.Xy dng v thm nh phng php o quang nh
lng flavonoid ton phn trong l cy u 2.2.1.1.Xy dng quy trnh nh lng
flavonoid ton phn trong l cy u -Quy trnh chit: Chitflavonoid ton
phn t l cy u tin hnh nh lng. -La chn dung mi pha mu. -La chn iu kin
o quang: Kho st v la chn bc sng ph hp nh lng flavonoid ton phn
trong l u. -Kho st v la chn nng nh lng thch hp 2.2.1.2.
Thmnhphngphpoquangnhlngflavonoidtonphn trong l cy u -Thm nh phng
php phn tch xy dng vi cc ch tiu:+ c hiu + tuyn tnh+ lp li + ng +Gii
hn pht hin (LOD) +Gii hn nh lng (LOQ) 2.2.2.Xy dng v thm nh phng
php o quang nh lng polysaccharid ton phn trong ph t 2.2.2.1.Xy dng
quy trnh nh lng polysaccharid ton phn trong ph t -Quy trnh chit:
Chit polysaccharid ton phn tph t tin hnh nh lng . -La chn dung mi
pha mu. 18 -La chn iu kin o quang: Kho st v la chn bc sng ph hp nh
lng polysaccharid ton phn trong ph t. -Kho st v la chn nng nh lng
thch hp 2.2.2.2. Thm nh phng php o quang nh lng polysaccharid ton
phn trong ph t -Thm nh phng php phn tch xy dng vi cc ch tiu:+ c hiu
+ tuyn tnh+ lp li + ng, chnh xc +Gii hn pht hin (LOD) +Gii hn nh
lng (LOQ) 2.3.Phng php nghin cu 2.3.1.Xy dng phng php phn tch -Dng
phng php chit nng bng nc trn ni cch thy chit polysaccharid ton phn
t ph t, lc, ta polysaccharid ton phn bng EtOH 96% , ly tm tch ly
ta. -Dng phng php siu m s dng dung mi EtOH 96% chit flavonoid ton
phn t l u. -Dng phng php o quang s dng k thut ng chun nh lng
flavonoid ton phn trong l u v polysaccharid ton phn trong ph t.
2.3.2.Thm nh phng php phn tch 2.3.2.1. c hiu Tin hnh qut ph hp th
ca 3 mu: mu trng, mu th, mu chun. 2.3.2.2. tuyn tnh Tin hnh pha dy
dung dch quercetin chun v glucose chun o ph hp th ca cc dung dch
chun t xy dng ng hi quy tuyn tnh gia nng cht chun v hp th. Xc
nhphng trnh hi quyy=ax + b v h s tng quan r ( iu kin h s tng quan r
phi >0,99). 19 2.3.2.3. lp li Tin hnh x l 6 mu bt ph t i vi phn
tch polysaccharid v 6 mu l i vi phn tch flavonoidsong song cng iu
kin ( chit xut, o quang), xc nh kt qu inh lng theo ng chun xy dng.
Tnh lch chun tng i gia cc gi tr ca cc ln nh lng (iu kin RSD
