Nghien CIFU - Ky thuat
Xay dyng quy trinh dinh lu-ong dong thoi daidzein va genistein trong hat dau nanh / {Glycine max (L.) Merr., Fabaceae)
Thai Thi Cam*, Huynh Ngoc Thuy
Khog Duac, DH YDuac TP Hd ChlMinh 'E-mgil: thaicam06(a),gmail. com
Summary
An HPLC/PDA method was established for simultaneous determination of daidzein (D) and genistein (G) in soya (Glycine max L): Column - Restek 18 (250x46 mm, 05 pm), Detector 2996 PDA, mobile phase:
acid acetic 0.1%- methanol (52-48), flow rate: 1.2 ml/min, injection volume: 10 pi. The method was validated showing good accuracy with the average of recoveries in range of 99-102% (with RSD 0.10% and 0.42% for D and G, respectively). The contents and repeatabilities for daidzein and genistein in the samples were 0.0056
% (RSD 1.54%), 0.0071 % (RSD 1.61%,), respectively LOD, LOQ of D and G were 0.05pg/mi and 0.15pg/
ml In practice, the method proved simple, time-saving but quite precise, and so, applicable to detenmination of main compounds in soya and other related species. Application of the method to samples collected In different areas of the Central and South Vietnam showed a significant diversity in contents of these two compounds.
Keywords: Glycine max, soya, daidzein, genistein, HPLC/PDA.
Dat van de
IVIdt trong nhOng nguin phytoestrogen quan trpng la cac isoflavon trong hat dau nanh {Glycine max (L.) IVIerr, Fabaceae) i'-^'. Phytoestrogen trong dau nanh cd ciu true hda hpc rit gin vdi estrpgen, cac hoat chat cd tac dung estrogen trong hat dau nanh chu ylu la daidzein, genistein i'-"icd t h i giup lam giam muc dp nghiem trpng va tin s l xuit hien cua cac trieu chCmg kinh nguyet va man kinh nhu bic hoa tung can, toat nhilu m l hdi va d l m l hdi v l dem, phdng ngua benh tim mach ^^i va loang xuang trong khi khdng gay ra cac tac dung phu len he sinh san nhu trong lieu phap estrogen dai han, khdng gay tang nguy ca ung thu da con hay ung thu vu PI. Viet Nam da quang cao nhilu san pham thuc pham chuc nang chua isoflavon dau nanh.
Tuy nliien chat lugng thuc su cua cac chi phim nay vin chua kilm soat dupc do thilu phuang phap kilm nghiem, khdng cd chit chuan d l dii chilu. Dllu dd da gay nhilu khd khan cho cdng tac kiem tra, danh gia chit lugng cua dugc lieu va cac chi phim cd chiJ-a dau nanh. Nhim muc dich gdp pliin kilm soat chit lugng nguyen lieu diu vao va diu ra cua cac san phim cd chua dau nanh dang luu hanh tren thi trudng, chiing tdi dat vin d l xay dung quy trinh dinh lugng ddng thdi genistein, daidzein trong hat dau nanh bing phuang phap HPLC/PDA vdimuc tieu khao sat quy trinh dinh lugng cd dp I n dinh, dp lap lai dat yeu cau
cua phep dinh lugng, tu dd chuin hoa diiu kien HPLC cho quy trinh dinh lugng, xay dung va tham d|nh quy trinh dinh lugng thanh phin chinh trpng dau nanh. D l tai hpan t i t cd t h i be sung va nang cao tieu chuan kilm nghiem cho DDVN-V bang phuang phap dinh lugng da xay dung dugc.
Ddi tu'O'ng va phu'O'ng phap nghien
CLPU
Nguyen lieu
Dau nanh dung trong khao sat quy trinh dinh lugng dugc thu mua tai huyen Due Trpng, tinh Lam Odng vao thang 2/2013, miu nay dugc luu gii> tai BM. Dugc lieu Khoa Dugc - DH. Y Dugc Tp. H i Chi Minh. Cac miu dau nanh d l khao sat so sanh ham lugng thanh phin chinh dugc thu hai tai nhUng tinh thanh khac nhau: Lam Ding, Bin Tre, Cin Thg, An Giang, D i e Lie, Binh Thuan, Vinh Phuc, Hung Yen... cung thdi dilm thang 2/2013. Dugc lieu dau nanh Glycine max da thu hai dat cac chi tieu v l dp i m , ty le chat chilt dugc va tap chit.
Sau khi thu thap, miu dugc phgi khd, xay min, qua ray chp miu dugc dong nhit dung d l kilm nghiem. T i t ca cac chi tieu thi> tinh khiit diu dugc liy kit qua la gia tri trung binh cua 3 lan thu dpc lap, cd kit qua on dinh.
- Chit dii chilu daidzein vdi dp tinh khiit tren HPLC la 99,77% cua Sigma, genistein dp tinh khiit
56 T A P C H I D U O r :
Nghien cCru - Ky thuat
99,66% do BM. Dugc lieu, Khoa Dugc - DH Y Dugc TP. H i Chi Minh cung cip.
Dung moi hoa chit, thilt bj
Cac hda chit, dung mdi thulc thu dat tieu chuin tinh khiit dung trong kilm nghiem dugc lieu; May HPLC Waters 2695 (Alliance); D i u dd PDA 2996; Cdt sac ky: Restek C,^ (250 x 4,6 mm;
5 Mm) va mdt s l dung cu thdng thuib'ng khac.
Phu'O'ng phap nghien ciru
Khao sat dieu kien HPLC: Khao sat budc sdng phat hien, khao sat each thirc triln khai HPLC tren miu thip vdi each thirc rira giai ding ddng (isocratic), khao sat pha ddng triln khai miu thif trong cung diiu kien sic ky vd'\ he pha ddng thay ddi nhim tim pha ddng thich hgp cd t h i phan tich daidzein va genistein dat cac yeu ciu v l thdng s l sac ky, khao sat t i c dp ddng lua chpn tic dp ddng phii hgp. Tif kit qua ciia khao sat diiu kien lya chpn mdt phugng phap ru'a giai tdi uu de dinh lugng cac thanh phin chinh trpng hat dau nanh.
Xay dung quy trinh chuan bj mau i3inh luong
Khao sat: Dung mdi chilt, thiji gian ehilt xuit, s l lin ehilt, phuang phap ehilt (ed leai tap hpae khdng)... vdi cae diiu kien dun hdi luu tren bip each thuy a 95°C trong 20 phut. Cac khao sat dugc danh gia dua tren kit qua sie ky Icng hieu nang cao. Sau eae bude khao sat, dua ra quy trinh chilt xuit d l dinh lugng cae hoat ehit trong dau nanh.
Ktiao sat xay dung phuong phap tham dinh danh gia quy trlnh dinh luong cac chat chlnh trong dau nanh
Xay dung duiD-ng tuyin tinh, tim miln gia tri (khoang ap dung), tim gitji han dudi va tim gidi han tren, tinh tuang thieh he thing, tinh chpn Ipc (tinh dac hieu), dp lap lai (dp ehinh xac), dp hii phuc theo diing yeu ciu eiia phep dinh lugng.
(fng dung quy trinh da xay du'ng de djnh luong cac thanh phan chlnh trong dau nanh
Tiln hanh chilt xuit va dinh lugng ehit chinh trong cae miu duge lieu thu hai tai cac viing dia ly khac nhau theo quy trinh da xay dung.
Kit qua va ban luan
Khao sat diiu kien sac ky Qk djnh lu'p'ng daidzein va genistein trong dau nanh
Daidzein cd bude sdng hip thu cue dai la 248,5 nm, genistein ed bude sdng hip thu cue dai 260,3 nm. Trong cung dllu kien sic ky, d budc sdng 260 nm cd su phii hgp v l dien tich dinh cua 2 chit daidzein va genistein, cac thdng so s i e ky d i u dat nen dugc chpn lam bude sdng
phat hien eho ca 2 pie ehinh.
o l tai da tiln hanh khao sat chuang trinh rira giai dang ddng vdi he dung mdi nude acid acetic 0,1% (A) - methanol (B) vdi nhilu ty le khac nhau nhu: 55 (A) - 45 (B), 54 (A) - 46 (B), 53 (A) - 47 (B), 52,5 (A) - 47,5 (B), 52 (A) - 48 (B)... T i c dp ddng 1,2 ml/phut; Lugng mau tiem 10 pi; Nhiet dp cpt 30°C. Kit qua: Cac pic tach t i t nhung thc'i gian luu edn dai, pic keo dudi va khdng dat dp tinh khiit, rieng ehl ed ty le dung mdi 52 (A) - 48 (B) pie dep, dinh then nhpn, thdi gian luu ngin, pic dat dp tinh khiit.
Kit qua lira chgn dieu kien khai trien sic ky May HPLC \/\feters 2695(Allianee); D i u dd PDA 2996; Cpt sie ky: Restek C,g (250x4,6 mm; 5 |jm).
Budc sdng phat hien 260 nm; Chuang trinh ru'a giai dang ddng vdi ty le pha dpng: nude acid acetic 0,1 % - methanol (52^8). T i c dp ddng 1,2 ml/phiit;
Lupng miu tiem 10 pi; Nhiet dp cdt 30 °C; Thai gian phan tieh 20 phiit.
Kilm tra dp tinh khiit 2 chit daidzein va genistein dung lam chit d i i chilu dinh lugng va'\
dllu kien s i c ky vifa tim dugc. Kit qua danh gia dp tinh khiit eua daidzein va genistein eho thiy dp tinh khiit (purity angle) nhd han dp tinh khiit theo ly thuylt (purity threshold) chifng td cae ehit dam bae dp tinh khiit cho phan tieh dinh lugng (bangi).
Bang 1: Kit qua danh gia do tinh khiit ciia daidzein va genistein bang HPLC/PD
Stt Ten pic
1 Daidzein 2 Genistein
Thoi gian iwu 9,084 14,916
Bo tinh iihiet 0,042 0,100
Bo tinh khiet ly thuyet
0,337 0,490
% Dien tich dinh 99,77 % 99,66 %
Xay dipng quy trinh chuan bi mau de djnh lu'p'ng
Sau khi khao sat eae diiu kien chilt xuit, qui trinh chuin bi miu chilt hoat chit daidzein va genistein tif duge lieu dau nanh duge thuc hien nhu sau:
Can ehinh xac 1 g bdt dupc lieu da dugc xay din kich thudc ding nhit vao binh nut mai, tiln hanh chilt xuit vai methanpl 3 lin (30 ml/20 phiit, 20 ml/20 phiit, 20 ml/20 phiit) bang phuang phap dun hdi luu tren bip each thuy 95 °C, Ipc ndng qua giiy ipc. Gdp cac dich Ipe, loai bdt dung moi cho vao binh dinh mipc 50 mi, dllu chinh methanol din vach. Dieh methanol dugc Ipe qua mang Ipe milipore cellulose acetat 0,45 pm tru(D'c khi triln khai HPLC theo dllu kien da lua chpn.
TAPrTijni[r'r"^r '•"-"'":"^^AMM*A«^^^
57• Nghien
CLFU- Ky thuat
T h i m djnh quy trinh djnh lu'p'ng Do dac hieu - Tinh chgn Igc
imsfflassixsaxji^aasi
s k ky So k4t qua khao sat do i!ac hieu
PhoUVcLiapicD trong m i u thCf
Hinh 1 : Kit qua khao sat do dac hieu ciia quy tririh anh luung Sac ky 66 phan tich miu thif
bkg HPLC
Pho UV cua pic G trong miu thif
Quy trinh dinh lugng dac hieu vdi c h i t c i n dinh lugng, pha ddng khdng cd pic trung vdi pic c i n dinh lugng, khi them e h i t ddi c h i l u , pie cua c h i t e i n dinh lugng tang eao dp h i p thu. P h i UV ciia D va G trong m i u thif tren s i c ky d l HPLC cho t h i y khdng bi pie tap nao xen phu (hinh 1).
Tinh tuyen tinh va miin gia tri
Daidzein t u y i n tinh trong khoang n i n g dp tif 0,15 1000 pg/ml; genistein t u y i n tinh trong khoang ndng dp tif 0,15 - 1 0 0 0 pg/ml. LOD eua D va G la 0,05 pg/ml. LOQ eua D va G la 0,15 pg/ml.
B a n g 2: Kit qua khao sat tuyin tinh cua daidzein va genistein
Khoang tuyen tinh Phwang trinh _ (pg/ml) twung quan hoi quy
LOD LOQ pg/ml pg/ml Daidzein 0,15-1000 y = 46745x 0,m 0,05 0,15 Genistein 0,15-1000 y = S0353x 0,999 0,05 0,15
Tuong thich he thong
Bang 3: Kit qua khao sat tuung thich he thing lAn bom (n = 6j tR Dien tich pic k' As N TB 13,98 7169253 8,98 1,53 6181,997 SD 0.02 56531.63 0,01 0,007 56,0874 RSD (%) 0,20 0,79 0,22 0,47 0,91
Sau 6 l i n tiem m l u thif, RSD cua cac thdng s l thdi gian luu (t^), dien tich pic (S), s l dia ly t h u y l t (N) d i u nhd han 2 %. Cae thdng s l k'. As d i u ed trung binh n i m trong gidi han eho phep (k' > 1,0;
0,8 < a < 1,5) (bang 3).
D o chinh xac: K i t qua d u g e trinh bay trong bang 4.
58
Bang 4: Kit qua khao sat do chinh xac cua phuung phap anh luung Miu
Daidzein
Daidzein
Genistein
Genistein Chuin TBkhoi lu'O'nq can
SD RSD (%)
Chuan TB khoi lycng can
SD RSD (%)
Khoi Iwang Dien tich pic Ham Iwang % mau(mg) (mAUxmin) /dum lieu 0,04 mg/ml 2534079
1000 63307,67 976 1,54 0,04 mg/ml 2724228
1000 87066,83 1404,3
1,61
0,0056
0,00711
P h u a n g phap ed dp ehinh xac vdi RSD eua daidzein la 1,54% va RSD ciia genistein la 1,61%, dat yeu c i u cho qui trinh dinh lugng cac hgp chat trong cao c h i l t tCf d u g e lieu.
Do hoi phuc
Tif k i t qua eua khao sat dp ehinh xac, cd t h i xac dinh ham lugng % trpng bdt d u g e lieu cua D la 0,0056% va G la 0,0071%. T i l n hanh tham dinh dp h i i phuc cua quy trinh dinh lugng thu d u g e k i t qua trinh bay treng bang 5, 6.
B a n g 5: Kit qua khao sat do hdi phuc ciia phuurig phap dmh luurig daidzein Chuan them vao
80%
100%
120%
Lwangthem Lwong tim thay (mg) (mg) 0,0408
0,0408 0,0408 0,0510 0,0510 0,0510 0,0612 0,0612 0,0612
0,0879 0,0899 0,0905 0,0985 0,0997 0,0989 0,1104 0,1102 0,1102
Tyle(%) hoi phuc
95,75 97,95 98,54 96,55 97,72 96,96 98,37 98,26 98,18
TAPCHIDU'Qfi'':
Nghien CIPU - Ky thuat
Bang 6: Kit qua khao sat do hdi phuc cila phuung phip dmh lugng genistein Chuan them
vao
80%
100%
120%
Lwgng them (mg) 0,0522 0,0522 0,0522 0,0652 0,0652 0,0652 0,0782 0,0782 0,0782
Lwong tim thay (mg) 0,1156 0,1186 0,1172 0,1359 0,1401 0,1409 0,1635 0,1635 0,1647
Tyle(%)h6i phuc 98,47 101,02 99,91 104,20 107,42 108,05 113,96 113,96 114,79 Quy trinh dinh lugng dat dp hdi phue cap cho ca phan tich D (RSD 0,1) va G (RSD 0,42), ty le h i i phuc trung binh treng khoang 9 9 - 1 0 0 %.
U'ng d u n g q u y t r i n h d j n h lu'p'ng du'O'c lieu dau nanh if cac v u n g dja ly khac nhau
D'ng dung quy trinh dinh lugng va quy trinh chilt xuat vifa xay dung d u g c d l dinh lugng cae m i u d u g c lieu dau nanh d u g e thu hai tai cac viing dia ly khac nhau. K i t qua d u g c trinh bay d bang 7.
Bang 7: Kit qua arih lugng daidzein va genisteiri frong cac miu da thu thap
Mau Dau nanh Ninh Binh Oau nanh Thai Binh Oau nanh Vinh Phiic Oau nanh Hu-ng Yen Oau nanh Thanh Hoa Oau nanh Binh Thuan Oau nanh Ojc Lac Oau nanh Lam B6ng Oau nanh Bin Tre Oau nanh Can Tho Oau nanh An Giang Oau nanh Bac Lieu
Daidzein
'„
8,859 8,866 8,874 8,868 8,877 8,865 8,781 8,823 8,781 8,790 8,814 8,775
Ham lmmg
%/dl 0,0011 0,0009 0,0057 0,0124 0,0017 0,0035 0,0131 0,0057 0,0099 0,0197 0,0144 0,0076
Genistein t.
13,962 13,943 13,965 13,967 13,992 13,966 13,829 13,884 13,826 13,836 13,868 13,825
Ham luung
%/dl 0,0012 0,0003 0,0059 0,0117 0,0015 0,0042 0,0150 0,0072 0,0107 0,0230 0,0207 0,0077 Ket qua tren cho thay ham lugng daidzein va genistein thay doi r i t khac nhau theo viing dia ly.
Bau nanh t r i n g tai tinh C i n Tha cho ham lugng daidzein va genistein cao n h i t trong 12 m i u d u g c chpn. C i n cd them nhu'ng khao sat de theo ddi ham lugng cac hoat c h i t theo miia, theo vung dia ly de thu hai dung miia, bao dam hieu qua d l l u tri ciia nguyen lieu sir dung.
Kit luan
Da xay dung d u g e quy trinh dinh lupng d i n g thdi daidzein va genistein la nhu'ng c h i t c h i l m ham lugng kha eao trong dau nanh vdi thdi gian
phan tich d u d i 20 phiit, quy trinh c h u i n bi m i u d a n gian, k i t qua cd dp lap lai cap ehp t h i y quy trinh c h i l t I n dinh, chinh xac vdi RSD cua daidzein va genistein d i u < 2%. T h i m dinh quy trinh dat d u g e tinh chpn Ipc, tinh t u a n g thich he t h i n g va dat eae yeu e i u cua mpt phep phan tich dinh lugng trong gidi han vai cac thdng s l ed RSD < 2%. K i t qua dinh lugng daidzein va genistein trpng cac m i u dau nanh thu hai tai cac tinh thanh khac nhau ehe t h i y hai e h i t nay ed thay dei va khae nhau dang ke tiiy theo vung dia ly, Quy trinh dinh lugng b i n g HPLC vifa xay dung ed the sir dung d l b l sung chuyen luan DDVN V n h i m tang eudng viee dam bao nang cao chat lugng va hieu qua d l l u tri tif d u g c lieu dau nanh,
Tai lieu tham khao
1. 0 0 Tat Lei (2006), NhOng cay thudc va vi thudc Viet Nam, Nha xuit ban Y hpc, tr. 930-932.
2. Vien Dugc lieu (2003), Cay thudc va ddng vat lam thuoc a Viet Nam, tap 1, Nha XB Khoa hoc ky thuat, tr.
765-768.
3. Vo Van Chi (2012), TUdien cay thudc Viet Nam (Bo m&i), tap 1, Nha XB. Y hoc, tr. 914-916.
4. Ajay K. Dixit, J. I. X. Antony, Navin K. Sharma and Rakesh K.Tiwari (2011), "Soybean constituents and their functional benefits". Opportunity, Challenge and Scope of natural products in IVIedicinai Chemistry, pp 367-383.
5. David J. A. Jenkins et al. (2000), "Effect of soy protein foods on low-density lipoprotein oxidation and Ex Vivo Sex Hormone receptor activity-A Controlled crossover trial". Metabolism, 49 (4), pp 537-543.
6. Kenneth D. R. Setchell, PhD. (2001), Soy isoflavons—Benefits and risks from nature's selective estrogen receptor modulators (SERMs), J. of the American College of Nutrition, 20 (5), pp 354-362.
7. Kim E. H., Chung J. I. (2006), "Analysis of phenolic compounds and isoflavons in soybean seeds {Glycine max (L.) IVIerill) and sprouts grown under different conditions", Eur Food Technol, 222, pp 201-208.
8. Kim Jin - Ae, Hong Seung - Beom, Chung III - Min (2007), "Comparison of isoflavons composition in seed, embryo, cotyledon and seed coat of cooked - with - rice and vegetable soybean (Glycine max L) varieties". Food Chemistry, 102, pp 738-744.
9. Mitani Kurie, Narimatsu Shizuo, Kataoka Hiroyuki (2003), "Determination of daidzein and Genistein in soybean foods by automated on-line in-tube solid-phase microextraction coupled to high-performance liquyd chromatography, J. of Chromatography A., 986, pp 169- 177.
10. Revital Cohen, Yevgenia Orlova, Marina Kovalev et al. (2008), "Structural and functional properties of amylose complexes with genistein", J. Agric. Food Chem., 56, pp 4212-4218,
{NgaynhSnbai 12/10/2016-Ngay phan bien: 17/11/2016 - Ngay duyet dang: 02/12/2016)
