TP CH KHOA HC, i hc Hu, S 63, 2010

XY DNG QUY TRNH CHIT XUT V PHNG PHP NH LNG STIGMASTEROL TRONG CY RY (Alocasia odora (Roxb.) C. Koch) BNG SC K LNG HIU NNG CAO
Trn Hu Dng Trng i hc Y Dc, i hc Hu ng Th Ngc Hoa Trng i hc Khoa hc, i hc Hu

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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trong bi bo ny, chng ti gii thiu kt qu nghin cu phng php nh lng stigmasterol trong cy ry bng sc k lng hiu nng cao (HPLC). 2. Thc nghim nghin cu 2.1. X l mu nghin cu Thn r ry (Alocasia odora (Roxb.) thu hi vo thng 12 nm 2009, gt b v, ra sch bng nc, thi tng lt mng 2-5mm, sy m can, t trong t sy n kh, nghin mn, thu c bt ry, bo qun trong bnh nha kn. Chun stigmasterol 95% ca Sigma-aldrich (No: 027K5302), bn Silica gel F254 (Merck), my HPLC SHIMADZU LC-20A series 2000-PAD detector, ct Sulpenco C8 v C18 (150 x 4,6mm, 5m) v cc dung mi ho cht tinh khit (PA v HPLC). 2.2. Kho st h dung mi trin khai sc k lp mng nh tnh stigmasterol Dung dch th: bt ry khuy t trong 2 gi vi CHCl3/MeOH = 2:1(v/v), lc qua giy lc. Dch chit c thy phn bng HCl 1M/MeOH trong 45 pht 800C, sau trung ha bng KOH 20%/MeOH n pH > 7, chit trong ete du ho v loi nc bng Na2SO4 khan, c dch ete thu c cn. Ha tan cn trong 1ml CHCl3 chm sc k. Dung dch chun: cn 11.4mg stigmasterol ha tan trong 1ml CHCl3 Trin khai trn bn Silica gel F254 vi cc h dung mi khc nhau: n-hexan/ethyl acetate (9:1), n-hexan/ethyl acetate (8:2), n-hexan/ethyl acetate (7:3), CHCl3/MeOH (98:2) v CHCl3/CH3COCH3 (8:2) chn h pha ng thch hp nht [2]. Pht hin peak phytosterol bng thuc th Liebermann-Burchard. 2.3.Xy dng quy trnh chit stigmasterol ton phn bng phng php chit Soxhlet Cn 5g bt ry, ngm lnh 12 gi vi cc h dung mi chit CHCl3/MeOH (1:1), (2:1) v CHCl3 100%, chit Soxhlet vi cng h dung mi ngm lnh trong 3 gi, 650C chn h dung mi thch hp nht cho vic kho st thi gian chit kit stigmasterol ti 3, 4 v 5h chn thi gian chit ti u [6]. Hiu qu chit xut c nh gi bng tn hiu peak stigmasterol trn TLC vi thuc th Liebermann-Burchard sau khi dch chit c thy phn bng HCl 1M/MeOH trong 45 pht 800C. 2.4. Xy dng phng php nh lng stigmasterol trn HPLC Kho st cc iu kin ct sc k pha o (C8 v C18), cc h dung mi trin khai acetonitril (ACN): m phosphate 0,025M vi cc t l khc nhau (90:10, 93:7, 95:5), ti cc pH m khc nhau (5,3; 5,8; 6,5; 4,8) v thay i tc dng khc nhau (1,0; 1,2; 1,5ml/pht) trn my HPLC SHIMADZU LC-20A series 2000-PAD detector [6].
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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.

Hnh 1. Cc h dung mi trin khai trn TLC vi thuc th Liebermann-Burchard (a) CHCl3/CH3COCH3 (8:2), (b) CHCl3/MeOH (98:2), (c) n-hexan/ethyl acetate (9:1), (d) nhexan/ethyl acetate (8:2) v (e) n-hexan/ethyl acetate (7:3)

Kt qu kho st cc dung mi chit xut stigmasterol ton phn trong bt ry bng chit Soxhlet cho thy sau 3h chit xut, dch chit CHCl3 thu c hm lng hot cht nhiu nht, th hin bng cng peak stigmasterol m hn so vi tn hiu peak ca dch CHCl3/MeOH (1:1) v (2:1) trong cng iu kin chit xut. Thi gian chit kit ti u nht khi dng CHCl3 l 6h, th hin bng vic khng th pht hin vt ca stigmasterol trn b dc liu thu c. Qu trnh kho st cc thng s, iu kin sc k thch hp cho qu trnh nh lng stigmasterol bng HPLC c th hin trong Hnh 2. Khi trin khai phn tch phytosterol trn HPLC vi pha ng l ACN/m phosphate 0,025M (95:5), pH=5,8 [6], tc dng 1,2ml/pht, chng ti thy vi ct C8 nhit 300C 1 cho s phn gii gia cc peak hot cht ca nhm phytosterol tt hn so vi ct C18 trong cng iu kin.

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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.

Khi thay i t l ca ACN trong pha ng t 95% n 93%, s phn gii ca peak stigmasterol so vi hai peak hai bn ci thin rt r, tuy nhin khi h t l ACN xung 90%, peak stigmasterol b dnh vo peak ra trc . Do vy, chng ti chn t l ACN/m phosphate 0,025M (93:7) l thch hp nht kho st pH thch hp. Khi thay i ln lt cc pH 4,8; 5,3; 5,8; 6,5, kt qu phn tch cho thy khi h pH pha ng ACN/m phosphate 0,025M (93:7) xung th kh nng tch ca peak stigmasterol so vi cc peak hai bn rt ci thin, ti pH=4,8 cho kh nng tch peak stigmasterol tt nht, peak sc nhn v i xng, cc chn peak hu nh khng dnh vo chn peak trc v sau n. Khi thay i tc dng t 1,0; 1,2; 1,5ml/pht trn h pha ng ACN/m phosphat = 93:7, pH 4,8. Kt qu cho thy ti tc dng 1ml/pht, chn peak stigmasterol tch ri hon ton khi chn peak ra lin trc n, trong khi vi tc dng ln hn (1,2 v 1,5ml/pht), d thi gian phn tch c ngn li, nhng phn gii li gim. Cng qua kho st cc bc sng pht hin trn Detector PAD, chng ti nhn thy ti bc sng 195nm cho tn hiu peak l cao nht, ph hp vi s pht hin cc hot cht nhm phytosterol. 3.1. Thm nh phng php HPLC - Tnh tng thch h thng
Bng 1. Kho st tnh tng thch ca h thng sc k

Ln o 1 2 3

Thi gian lu (pht) 14,71 14,64 14,62

H s phn gii(1) 1,57 1,55 1,56

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S a l thuyt 10712 10701 10671

H s bt i xng 1,10 1,13 1,11

Din tch peak 10573308 10568175 10584571

4 5 6 Mean RSD (%)

(1)

14,63 14,64 14,59 14,64 0,26

1,56 1,55 1,57 1,56 0,57

10718 10679 10726 10701 0,2

1,09 1,10 1,09 1,10 1,36

10581461 10582939 10578733 10578198 0,06

: h s phn gii gia peak stigmasterol vi peak trc n.

Tnh tng thch ca h thng sc k c xc nh bi tin hnh tim lp li 6 ln trn mt mu th, vi cc iu kin chy sc k chn trn. Kt qu kho st tnh tng thch ca h thng c trnh by ti Bng 1. Cc s liu thu c cho thy h thng sc k chn la l tng thch cho vic phn tch nh lng stigmasterol trong bt dc liu ry (RSD 2%). - Tnh c hiu Da vo thi gian lu v hnh dng sc nhn, i xng ca peak stigmasterol thu c khi phn tch mu th v mu th thm chun (hnh 3), kt hp so snh ph UV ca peak mu th so vi ph stigmasterol chun thu c khi qut ph trn detector PDA t 190 400nm (Hnh 4) cho thy peak stigmasterol trong dung dch th c thi gian lu (14.64 pht) v ph UV tng ng vi peak stigmasterol chun. Phng php phn tch sc k trn cho tnh c hiu cao.

Hnh 3. Sc k mu dch chit thu phn (a) v dch chit thu phn thm chun (b)

Hnh 4. Ph UV mu stigmasterol chun (a) v mu dch chit thu phn (b) 47

- Tnh tuyn tnh Kt qu s kho st tuyn tnh ca nng dung dch stigmasterol chun theo din tch peak thu c ti bc sng 195nm (Hnh 5) cho thy khong nng tuyn tnh l (10 - 300ppm), vi phng trnh hi quy c dng y = 46914x + 128511, h s R = 0,9995, cho thy c s tng quan tuyn tnh cht ch gia nng hot cht vi din tch peak p ng.

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 1 2 3 4 5 6 Trung bnh RSD(%)

Lng bt (g) 5,0039 5,0053 5,0017 5,0008 5,0042 5,0056 5,00358

Hm lng (%) 0,0089 0,0089 0,0087 0,0085 0,0089 0,0089 0,0089 1,85

Hm lng stigmasterol t do ton phn c trong thn r ry l 0,009%, ph hp vi cc gi tr tham kho [5], [6] vi lch chun tng i RSD l 1,85%. Phng php c chnh xc tt.
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- 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 1 2 3 4 5 6

Lng bt (g) 5,0039 5,0053 5,0017 5,0008 5,0042 5,0056

Lng S(*) chun thm vo (ppm) 50 50 50 50 50 50 Trung bnh RSD(%)

Tng lng S(*) tm c (ppm) 154 154 150 149 154 154

thu hi Rev(%) 95,4 94,9 94,8 94,7 95,7 95,3 95,2 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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(*)

TI LIU THAM KHO

[1]. Tt Li, Nhng cy thuc v v thuc Vit Nam. NXB Y hc v TW, 2007. [2]. B Y t, Quy ch nh gi tnh an ton v hiu lc thuc c truyn, Tp ch Dc hc, s 5, (1996), 3. [3]. L Ngc Knh, Nghin cu thnh phn ho hc ca c ry thnh ph Hu, NXB Y hc Thc hnh, B Y t, s 521, 2005. [4]. L Ngc Knh, Tc dng h cholesterol trong huyt thanh th ca dch chit t thn r cy ry,Tp ch Dc hc, s 374, (2007), 11. [5]. L Th Bch Hin, Xy dng quy trnh chit xut phytosterol t thn r cy ry (Alocasia Odora Roxb.), Lun vn tt nghip, Trng i hc Y Dc Hu, 2010. [6]. Ellen Odeen Thesis, Investigation of phytosterols in Alocasia Odora Roxb. by reversed phase HPLC, A study made in Hue City, Vietnam, (2009), 23. [7]. World Health Organisation, General guidelines for Methodologies on Research and Evaluation of Traditional Medicine, 1998. [8]. Ludwing huber, Validation of Analytical Methods, Review and Strategy, (2001), 13.

EFFICIENT EXTRACTION AND DETERMINATION OF FERULIC ACID IN Alocasia Odora (Roxb.) C. Koch BY HPLC
Tran Huu Dung College of Medicine and pharmacy, Hue University Dang Thi Ngoc Hoa College of sciences, Hue University

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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