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TẠP CHÍ KHOA HỌC, Đại học Huế, Số 63, 2010
TẠP CHÍ KHOA HỌC, Đại học Huế, Số 63, 2010
TM TT
Quy trnh chit xut stigmasterol ton phn t bt dc liu ry (Alocasia odora
(Roxb.) C. Koch) trn soxhlet trong 5 gi trong dung mi chloroform c thc hin v kim
sot bng phng php sc k lp mng khi trin khai trn h dung mi n-hexan/ethyl axetat
(8:2), xc nh vt bng thuc th Liebermann-Burchard. Hm lng stigmasterol c xc
nh bi phng php HPLC nhanh chng v c nhy cao, trn ct C8 vi nhit ct 300C
1, h dung mi pha ng l acetonitril/m phosphate (93:7) pH 4,8 vi tc dng 1ml/pht,
th tch tin 20l, bc sng pht hin ti 195nm. Phng php HPLC cho tng thch cao vi
cc thng s sc k c RSD < 2%. Khong tuyn tnh ca nng stigmasterol c kho st
t 10300ppm (r = 0,9995). Phng php c thm nh cho chnh xc v ng thch
hp vi t l phc hi ca stigmasterol t bt ry t 95,2%, gii hn nh lng l 10ppm.
I. t vn
Cy ry (Alocasia odora (Roxb.) C. Koch) hay cn gi l mn bc h, ry bc
h, bc h; i khi cn gi l ry di, d vu. Thn r ry c dng lm thuc trong
phm vi kinh nghim nhn dn, ch yu cha bnh ngoi da nh m ay n nga, l
kh cha, sn da chy nc (nu nc tm ra), xt vo ni b l han gy nga ty;
nhiu ni dng vi tc dng gim au do vo c, au xng khp, iu tr gan nhim
m [1], [3].
Phytosterol l mt trong nhng thnh phn ha hc ca cy ry c hot tnh sinh
hc v hm lng cao. Stigmasterol l mt trong nhng thnh phn chnh ca
phytosterol, c tc dng gim lng cholesterol trong mu, khng vim, dng trong
tng hp progesterone, v l tin cht ca vititam D3 [3], [4], [6].
Hin nay, xu hng v yu cu tiu chun ha cht lng dc liu theo WHO
ni chung cng nh Vit Nam ni ring l phi xy dng c quy trnh xc nh hm
lng hot cht hoc cht c trng chim t l ln trong dc liu [2], [7]. V vy,
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Dung dch th: cn thu c sau khi thu phn c ha tan trong 2ml MeOH.
Dung dch chun: ha tan 20mg stigmasterol trong 100ml MeOH. Cc dch c
lc siu m 5 pht ho tan, sau lc qua mng lc 0,45m trc khi bm vo my.
Quy trnh phn tch HPLC c xy dng, ti u ho cc thng s t l pha
ng, pH, tc dng v bc sng pht hin v thm nh tnh tng thch h thng,
tuyn tnh, chnh xc, ng (t l phc hi) v gii hn nh lng (LOQ).
3. Kt qu v bn lun
Kt qu kho st cc h dung mi trin khai trn TLC (Hnh 1) cho thy trong
cc h dung mi pha ng kho st, h n-hexan/ethyl axetat (8:2) cho peak stigmasterol
tch r rng vi cc vt k cn, c h s phn b Rf =0,64 nm trong khong thch hp
(0,3 0,7). Peak stigmasterol cho mu xanh lc c trng ca nhm phytosterol vi
thuc th Liebermann-Burchard.
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Hnh 2. Sc k mu stigmasterol (a) v mu dch chit thu phn trn ct C8, nhit ct
300C 1, pha ng ACN/m phosphate 0,025M (93:7), pH=4,8, tc dng 1ml/pht, th tch
tim 20l, max=195nm.
Ln o
Thi gian
lu (pht)
H s phn
gii(1)
S a l
thuyt
H s bt
i xng
Din tch
peak
14,71
1,57
10712
1,10
10573308
14,64
1,55
10701
1,13
10568175
14,62
1,56
10671
1,11
10584571
46
14,63
1,56
10718
1,09
10581461
14,64
1,55
10679
1,10
10582939
14,59
1,57
10726
1,09
10578733
Mean
14,64
1,56
10701
1,10
10578198
RSD (%)
0,26
0,57
0,2
1,36
0,06
(1)
Hnh 3. Sc k mu dch chit thu phn (a) v dch chit thu phn thm chun (b)
Hnh 5. th biu din khong tuyn tnh ca nng stigmasterol (ppm) tng ng din tch
peak o c ti 195nm vi R = 0,9995
- chnh xc
chnh xc c kho st bng s lp li hm lng stigmasterol xc nh trn
6 mu th vi lng cn v iu kin sc k chn, kt qu c trnh by trong Bng 2.
Bng 2. Kho st chnh xc ca phng php
Mu o
Lng bt (g)
Hm lng (%)
5,0039
0,0089
5,0053
0,0089
5,0017
0,0087
5,0008
0,0085
5,0042
0,0089
5,0056
0,0089
Trung bnh
5,00358
0,0089
RSD(%)
1,85
- ng
ng c xc nh bng t l phc hi khi thm mt lng chun chnh xc
vo 6 mu bt ry, tin hnh phn tch hm lng stigmasterol tng ca 6 mu dc liu
thm chun vi iu kin chy sc k chn, kt qu c trnh by trong Bng 3.
Bng 3. Kho st ng ca phng php
Mu o
Lng bt
(g)
thu hi
Rev(%)
5,0039
50
154
95,4
5,0053
50
154
94,9
5,0017
50
150
94,8
5,0008
50
149
94,7
5,0042
50
154
95,7
5,0056
50
154
95,3
Trung bnh
95,2
RSD(%)
1,63
(*)
S : stigmasterol
Kt qu cho thy phng php c ng cao (t l phc hi 95,2%) vi RSD
l 1,63%, ph hp vi yu cu chung v nh gi ng trong phng php xc nh
hm lng hot cht trong dc liu bng HPLC [7], [8].
- Gii hn nh lng (LOQ)
Nguyn tc xc nh gii hn nh lng trong HPLC l pha long dn dung
dch chun n nng thp nht v tin hnh sc k. LOQ c xc nh ti nng
m chiu cao ca peak hot cht gp 10 ln so vi nhiu ng nn v c
chnh xc c chp nhn [8]. Kt qu LOQ ca stigmasterol l 10ppm.
4. Kt lun
Chng ti xy dng c quy trnh chit xut stigmasterol ton phn (dng t
do v kt hp) bng chit Soxhlet sau 5h vi chloroform, hiu xut chit c kim sot
bng pht hin vt stigmasterol trong dch chit thu phn trn TLC vi h dung mi
trin khai l n-hexan/ethyl axetat (8:2), pht hin bng thuc th Liebermann-Burchard.
Quy trnh nh lng stigmasterol trong dch chit thu phn c thc hin bng
HPLC trn ct C8, nhit ct 300C 1, pha ng ACN/m phosphate 0,025M (93:7),
pH 4,8, tc dng 1ml/pht, th tch tim 20l, max=195nm. Phng php phn tch
cho tnh tng thch h thng cao (RSD < 2%) v c nh gi thm nh tt, tnh c
hiu, tuyn tnh (10 - 300ppm), chnh xc v ng t yu cu vi gii hn
pht hin l 10ppm. Hm lng stigmasterol trong bt dc liu ry kh l 0,0089%.
Do , phng php ny c th p dng nh gi cht lng ca cy ry ni ring v
cc dc liu cha phytosterol ni chung.
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SUMMARY
A procedure to extract virtually total stigmasterol from Alocasia odora (Roxb.) C. Koch
powder on Soxhlet extraction in 5h by chloroform was carried out and controlled by TLC
identification with a mobile phase of n-hexan/ethyl acetate (8:2), detected by LiebermannBurchard reagent. The amount of stigmasterol was determined by a fast and sensitive high
performance liquid chromatography (HPLC) assay on a C8 column (300C 1) using mobile
phase of 93:7 (v/v) acetonitril/phosphate buffer pH 4.8 at mobility of 1ml/min, injection of 20l
with detection at 195nm. The method was validated highly with all RSDs less than 2%. The
linearity of stigmasterol concentration is in the range of 10300 ppm and found to be linear (r
= 0,9995). The method proved to be sufficiently precise and accurate with the mean percent
recovery of stigmasterol from powder being 95,2%, LOQ of HPLC assay is 10ppm.
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