TOM TT

Mt phng phap c phat trin inh lng cac flavonoids quercetin va kaempferol trong nc tiu ngi s dung chit pha rn theo sc ky khi. Cht ni chun co dteri cua cht ang phn tich c thm vao trong cac mu trc khi chit. Gii han phat hin cua phng phap khoang: 10 pg trn ct va chinh xac cua phng phap inh lng trong mi mu nc tiu la 9,4% vi kaempferol va 7,34%
vi quercetin. Cac mu nc tiu c x ly vi -glucuronidase tng ro rt mc cua flavonoid c phat hin, cung c thm quan im rng kaempferol va quercetin la bai tit trong nc tiu nh cac glucuronide.

NI DUNG
I. GII THIU Flavonoids la mt nhom ln cua polyphenols t nhin vi mt pham vi dc tinh rng [1]. Vi du, co khng inh cho rng khu phn n giau flavonoid ti l nghich vi nguy c mc bnh tim mach vanh [2] va c tinh khng bi oxy hoa cua mt s flavonoid co th bao v chng lai cac loai bnh ung th nht inh [3, 4]. Quercetin va kaempferol la flavonoid ph bin trong thc phm va c c bit quan tm nh cht chng oxy hoa, do chung co mt nhom 3-hydroxyl vi kha nng oxy hoa tng i thp, ma khi bi oxy hoa vn khng thay i, do vy tranh c s lp lai cua s oxy hoa kh [5]. Khi mt ham lng ang k cua cac flavonoid c tiu thu hng ngay trong khu phn n phng Ty, mt s nghin cu da trn s hp phu, chuyn hoa va bai tit cua cac lp hp cht trong c th ngi. Nghin cu s hp phu cua flavoniod co s mu thun va cac tranh lun quanh vn chung hp thu nh cac aglycone hay glycosides mc du mt nghin cu gn y a co kha nng chng minh c cac glycoside flavonoid nh cac cu t binh thng cua huyt tng ngi [6]. S chuyn hoa cua cac flavonoid theo c ch hp phu co th co mt anh hng ln trn cac c tinh sinh hoc cua chung, va gn y [7], mt nghin cu a ng y rng s chuyn hoa cua kaempferol va quercetin trong con ngi co th khac so vi quan sat trong ng nghim s dung vi lap th gan chut, ni ma kaempferol c chuyn hoa thanh quercetin bi phan ng hydro hoa cua vng B nhng cac nghin cu ging nhau u khng phat hin c quercetin trong mu nc tiu. iu nay a khng inh rng s hydro hoa cua kaempferol thanh quercein co th khng xut hin trong c th ngi. Mt phng phap ban inh lng anh gia flavonoid trong nc tiu ngi theo ch n ung co thuc Ginkgo bilabo c phat trin gn y trong phong thi nghim cua chung ti [8]. Nghin cu gn y m ta mt phng phap nng cao inh lng kaempferol va quercetin trong nc tiu ngi ca trc va sau khi dung thuc Ginkgo bilabo s dung dn xut co cha Dteri cua cac hp cht nay nh cht ni chun va dung phng phap chit pha rn iu ch mu.

II. THI NGHIM 2.1. Hoa cht Hoa cht c chun bi t cac ngun sau: kaempferol, quercetin, sulphat (EC 3.1.6.1), -glucuronidase (EC 3.2.1.31), KH2PO4, CH3COOH, CH3COONa, Na2SO4 khan, C2D4O2 (98% Dteri), DCl (99,5% Dteri), C2H6O (99,5% Dteri), trifluoroacetic acid (TFA), va dung dich Dteri (99,9% Dteri) t Sigma Aldrich Chemical Co. (Poole, Dorset, UK). HPLC s dng methanol va acetolnitrile t BDH Merck (Poole, Dorset, UK). N-O-(bis)trimetylsilyl acetamide (BSA) t Fluka Chemical Co. (Poole, Dorset, UK). Tng ham lng quy inh cua thuc Ginkgo biloba la 28,8 mg flavonoid glycosides va c mua t Boots (Glasgow, UK). 2.2. iu ch cht ni chun co Dteri Dn xut Dteri cua kaempferol ([2H4] kaempferol) va quercetin ([2H5] quercetin) c iu ch nh sau: 10 mg cua quercetin hoc kaempferol c hoa tan trong hn hp cua 4 ml CH3COO2H va 4 ml cua dung dich 1:4 (v/v) cua 2HCl (37% w/w) trong D2O. Dung dich c un nong trong ng nghim 800C trong 4 ngay. Hn hp phan ng a lam ngui c lam bay hi di iu kin ap sut thp bi mt ham lng nho C2H5O2H (3x1 ml). Cht ba (khoang 8 mg) c chit vi ethyl acetate (2x4 ml); dich trich ly hn hp hu c c sy kh khi i qua mt ng pipette Pasteur vi Na2SO4 khan (khoang 2g) va dung mi bay hi di dong N2. Thanh phn ng vi cua dn xut dteri kaempferol va quercetin c anh gia bi NICI GC-MS vi TMS dng kim tra ion c chon. Bang 1 chi ra cac peak ng vi cho ca cht ni chun va flavonoid khng co dteri. i vi trng hp cua quercetin, s co mt cua nm nguyn t Si trong cu truc va s lng ln carbon M+1 va M+2 la nhiu. 2.3. Phn tich GC MS H thng HP5988A GC-MS c s dung trong phng phap ion ha ion m (NICI) vi methane nh cht khi phan ng c a vao cung cp mt ngun ion tai ap sut 133,3 Pa. Sc ky khi c gn vi mt ct mao quan Restek RTX 5 (30 m, 0.32 mm I.D., 0.5 m) vi Heli nh mt cht khi mang tai 68,9 Kpa. Nhit bung bm mu c iu chinh tai 2500C va dong di chuyn GC-MS tai 2800C. B phn iu nhit co chng trinh iu khin nhit nh sau: 1600C (1 phut), sau o tai 200C phut-1 la 2900C, va 3200C tai 50C phut-1. Ion c chon sau khi a kim tra c s dung cho inh lng. Kim tra cac ion nay nh sau: m/z 662 va 574 (tng ng vi TMS t dn xut quercetin va kaempferol), m/z 577 va 578 (tng ng vi dn xut ca
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kaempferol c doteri) , m/z 666 va 667 (tng ng vi dn xut quercetin c doteri), nhin hinh 1.

2.4. S thu thp va iu ch cac mu nc tiu Cac mu nc tiu c thu thp t nm ngi tinh nguyn khng co ch n king, 6 gi trc va sau khi ung mt vin thuc Ginkgo biloba. Cac mu nc tiu c gi trong tu lanh (tai -20 0C). Trc khi phn tich, dung dich nc tiu c quay ly tm (10000 vong, 5 phut) va lp trn b mt c em phn tich. S thuy phn enzym vi sulfat c thc hin vi vic a trong 1 ml nc tiu gm 10 n vi sulfat trong 0,25 ml dung dich m CH3COONa 1M (pH =5), mu c bao quan trong 1 gi tai 370C. S thuy phn vi glucuronidase c thc hin bi 100 n vi glucuronidase trong 0,25 ml dung dich m K2PO4 (pH=6,8) c a vao trong 1 ml nc tiu va bao quan 1 gi tai 370C. Cac mu glucuronidase a x ly c acid hoa vi 1 ml HCl 1M trc khi em chit pha rn (SPE). S thuy phn acid c thc hin 0,5 ml HCl 3M c a vao trong 1 ml nc tiu va c un nong 800C trong 1 gi. Cac mu nay c m pH=6,8 trc khi chit pha rn bi a vao mu 1 ml dung dich m phosphate 1M. Cac mu khng thuy phn c iu ch cho chit pha rn bi s thm vao 0,25 ml dung dich m CH3COONa 1M (pH=5) trong 1 ml dung dich nc tiu. Cht ni chun cha dteri (200 ng) c a vao trong tt ca cac mu trc khi chit pha rn.

2.5. Chit pha rn Hp ISOLUTE ENV+ (100 mg ml-1) ( Craw ford Scientific, Strathaven) c iu m bng cach nho giot lin tuc 1 ml cua methanol va 0,8 ml nc vi mt tc dong chay khoang 4 ml phut-1. Hp ng cac mu sau khi phn tich c lam sach bi khng khi va sau o ra vi 1,5 ml (3 x 0,5 ml) cua dung dich methanol trong nc 7% (c iu chinh n pH 3,5 vi TFA 0,01M) tai tc dong chay khoang 3,5 ml phut-1. Cui cung, cac hp ng mu c ngm 1 ml (2 x 0,5 ml) dung dich acetonitrile trong nc (8:2) tai tc dong chay khoang 3,5 ml phut-1. cac mu c phut khi lam kh bi dong N2 va cht ba con lai c tao dn xut bi 0,2 ml cua BSA, un nong cac mu trong cac ng nghim co np y kin tai 70 0 C trong 3 phut. Cac dung dich mu (1l) c nap vao GC-MS vi mt ng bm Hamilton 10 l. 2.6. S tai sinh thi nghim xac inh s tai sinh cua kaempferol va quercetin s dung phng phap chit pha rn, 1 ml cua nc ct c thm vi 50 ng cua ca quercetin va kaempferol va chit tng t nh phng phap chit pha rn s dung cho cac mu nc tiu. xac inh s tai sinh, 200 ng cua cht ni chun doteri c a vao trc hoc sau khi chit rn. S tai sinh c tinh toan bi s so sanh ti l din tich peak cha cht phn tich co dteri va khng dteri thu c khi thm cht ni chun trc va sau chit rn. 2.7. S hiu chun va inh lng ng cong hiu chun c chun bi cho quercetin va kaempferol bi hn hp co ham lng khac nhau (0 200 ng) cua quercetin hoc kaempferol khng co dteri vi ham lng khng i (100 ng mi cht) va sau tao dn xut nh m ta trn. Trong cac mu, ti l din tich gia cac peak cua quercetin va kaempferol khng c doteri (tng ng m/z 662 va 574) va c dteri (tng ng m/z 666 + m/z 667 va m/z 577 + m/z 578) c s dung cho inh lng (nhin hinh 1) vi s tham khao t cac ng cong hiu chun. 2.8. Phn tich thng k Y nghia gia cac nhom nghin cu c kim chng bng phng phap ghep cp Students t-test.

III. KT QUA VA THAO LUN Nghin cu gn y m t phng php GC-MS nh lng flavonoids trong nc tiu ngi s dng dn xut dteri ca kaempferol v quercetin nh cht ni chun. S dng chit pha rn trc khi phn tch ci thin nghin cu trc [8]. S ci thin ny cho php nh lng quercetin v kaempferol ti mc thp trong cc mu nc tiu t i tng trc khi tiu th thuc ca Gikgo biloba. Vi vy, nng ca kaempferol va quercetin t cac mu nay phn nh s ng gp ca mt ch n ung bnh thng va khng b sung. Hnh 1 tng ng cc cht kaempferol v quercetin c doteri. Tt c cc v tr proton trong quercetin l kch hot trao i vi dteri trong iu kin acid v 4 v tr hot ha trong kaempferol. T bng 1 ta c th thy n khng trao i hon ton vi cc proton do , quercetin l hn hp ca cc ng v 2H4 v 2H5, v kaempferol l hn hp ca cc ng v 2H3 v 2H4. Tim nng cho nhiu x xuyn m gia cc hp cht khng dteri v cc ng v dteri c accessed v c trnh by trong bng 1. Lng ln cc nguyn t hydro v silicon trong cu trc ca hp cht khng dteri trong cc peaks ng v M+1 v M+2 c cng cao. Tuy nhin, khng c s giao thoa ng k gia cc ion trong cc ng v dteri.

Phn ng ca quercetin v kaempferol tuyn tnh trn phm vi tp trung nghin cu. Kh nng lp li ca phng php c anh gia cho mu x ly -glucuronidase c phn tch nh 6 c s ring bit v c a ra R.S.D t hn 10% cho mt lng ca kaempferol v quercetin (tng ng 13.00 ng/ml 9.40 % v 4,45 ng /ml 7.34 %, n = 6) Chit pha rn s dng kt hp C18, cation v anion trao i c th s dng xc nh flavonoids trong nc tiu. Trong nghin cu ca chng ti, th nghim s b cho thy rng khi s dng hp ng mu C18 khng cho php loi b cc cht ni sinh khi c cht phn tch. iu ny ph hp vi kt qu ca Ishiiet al., [11], bo co ca h s dng anion mnh trao i trong cac hp ng mu cho khai thc naringin v naringenin t nc tiu ngi thay v hp ng mu C18, n khng hiu qu trong vic loi b cac cht anh hng. Nghin cu hin ti ca chng ti s dng pha polymeric (hp ng Isolute ENV+) a ra la chn tt hn hp ng C18, mc d n loi b khng hon ton cc cht ni sinh. Tuy nhin, chn lc kim tra ion l c s cho phn gii ca cc peak. Vic ti sinh flavomoids s dng phng php hin nay anh hng bi nhu cu di chuyn ca cc hp cht giao thoa. Do , s tai sinh thu c s tng ng gia cc cht phn tch v loi b cc cht nh hng. Tuy nhin, xt s ti sinh ca phenol cho hp ng C18 l khong 50% (cng ngh HPLC, chng nhn phn tch) chit xut polyphenol ti sinh thu c l hp l v ph hp vi yu cu cao hn cho pha polymeric ENV+ so vi pha C18. Kh nng ti sinh ca kaempferol v quercetin l tng t (gia S.D., n = 6) 76.6 10.5 % v 73.0 9.8 %). Hnh 2 v 3 a ra la chn ion in hnh cho cng mu nc tiu trc v sau khi x l vi -glucuronidase. Cc vt cho thy cc mu khng c x l ca quercetin v kaempferol mc thp, v kt qu vi cc peak nn ln. Trong tt c cc trng hp, gii quyt c bn cho cc peak cn quan tm t cac cht anh hng c ci thin.

Hnh 4 cho thy mc flavonoids trong mu nc tiu trc v sau khi tiu th thuc ca Ginkgo biloba. Mc flavonoids trc khi tiu th thuc cho tn hiu thp hn cc mu sau khi tiu th, nhng lng unconjugated ca flavonoids li cao hn (c bit l quercetin) c pht hin so vi nghin cu trc y ca chng ti [8]. Cc mc c tm thy trc khi tiu th thuc ch yu phn nh t ch n ung, s bin i gia cc c nhn l c k vng. Trong thc t, nghin cu gn y ca Hollman [12] ch ra rng kha nng tich luy sinh hoc ca quercetin t mt s loi thc phm rt khc nhau, ch c mt lng nh c tiu th. Trong cng mt nghin cu, kha nng tich luy sinh hoc t cc i l 1.39% ca lng tiu th. Cc mu x l bng -glucuronidase cho tn hiu (P< 0.05) gim i vi kaempferol v quercetin (hnh 4B), cho thy flavonoids c bi tit nh glucuronides. Lng kaempferol v quercetin bi tit trong nc tiu ch l mt phn nh so vi lng tiu th, iu l ph hp vi kt qu [13]. Trn thc t, mt nghin cu gn y [7] s dng HPLC v LC-MS th khng pht hin quercetin trong acid thuy phn trong nc tiu ngi sau khi tiu th bng ci xanh v c mt lng nh kaempferol. Mc d , sulphatase-x l mu cho thy lng ln kaempferol v quercetin so vi mu khng c x l, s khc nhau ny l khng c ngha thng k trn 5 mu. Tuy nhin, 2 trong s cc mu trc khi ung thuc cho thy quercetin tng r rt sau khi x l sulphatase, iu cho thy sulphatase tham gia qu trnh trao i cht. Mu acid thuy phn (hnh 4 B) cho thy quercetin c hm lng cao ng k (P<0.05, n = 5), cn kaempferol th khng c. Tuy nhin, iu kin x l mu aicd thuy phn khng c ti u ha v thi gian v nng . Hn th na acid thuy phn s lm mt cht phn tch khi vng pyran d b thy phn di cc iu kin khc nhau.

Lng flavonoids c chit xut t Ginkgo biloba chun (e.g.EGb 761) thng c biu din l Ginkgo flavone glycoside. o tng quan tng ham lng cua aglycones (quercetin, kaempferol, va isorhamnetin) c xac inh sau khi thuy phn cht trich ly tng ham lng glycoside flavonoid. iu nay c thc hin bng s chuyn hoa cac yu t c a vao tinh toan khi lng phn t cua ester glycoside flavonol coumaroyl. Haslert [14] chi ra rng chit xut Gonkgo biloba chun cha trung binh khoang 0,39% (w/w) cua kaempferol va 0,5% (w/w) cua quercetin. iu nay cung c goi la lam giau chit xut vi khoang 24 27% (w/w) cua glycoside Ginkgo flavone. Cac thuc c s dung trong nghin cu cha mt ham lng 28,8 mg tng glycoside flavonoid trn mi vin, va do o c lng mt ham lng cua ring kaempferol la 0,44 mg va quercetin la 0,50 mg nh la cac aglycones t do trong mi vin thuc. Kt qua cua chung ti phu hp, vi vy s hp phu quercetin va kaempferol bng ng ung la co th, nh a chi ra trc y [6].

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Khi thc hin cung nhau, cac kt qua u khng nh rng kha nng tac dung sinh hoc cua kaempferol va quercetin la thp, va con ng chinh cua s trao i cht la thng qua vic b sung cac acid glucuronic. Xet quang ph cua cac hoat tinh sinh hoc c a ra bi flavonoid va ch n ung rng rai cua no co mt nhu cu cho mt s hiu bit y u hn v s hp phu, trao i cht va bai tit cua nhom hp cht nay. Ung mt vin thuc cha 28 mg flavonoid co mt anh hng ro rt trn mc c s cua kaempferol va quercetin trong nc tiu, chi ra rng mc cao cua cac hp cht nay la khng hp thu t mt ch n ung binh thng. Phng phap GC MS hin ai nn co nhay u cho phep xac inh cac hp cht nay trong huyt tng.

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LI CAM N
Nghin cu nay c h tr bi mt hoc bng nghin cu nc ngoai (ORS) c cp t CVCP (UK).

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TAI LIU THAM KHAO

[1] E.Middlecton Jr., Int. J. Phamacongn. 34 (1996) 344. [2] M.G.L. Hertog, E.J.M. Feskens, P.C.H. Hollman, M.B. Katan, D. Kromhout, Lancet 342 (1993) 1007. [3] M.G.L. Hertog et al., Arch. Intern. Med. 155 (1995) 381. [4] S.M. Kuo, Crit. Rev. Oncog. 8 (1) (1997) 47. [5] H.P. Hendrickson, A.D. Kaufman, C.E. Lunte, J. Pharm.Biomed. Anal. 12 (3) (1994) 325. [6] C.A. Rice-Evans, N.J. Miller, G. Paganga, Free Radic. Biol. Med. 20 (1996) 933. [7] S.E. Nielsen, M. Kall, U. Justesen, A. Schou, L.O. Dragsted, Cancer Lett. 144 (1997) 173. [8] D.G. Watson, A.R. Pitt, Rapid Commun. Mass Spectrom. 112 (1998) 153. [9] C. Bannwart, T. Fotsis, R. Heikkinen, H.Adlercreutz, Clin. Chim. Acta 136 (1984) 165. [10] H. Adlercreutz, T. Fotsis, C. Bannwart, K. Wahala, G. Brunow, T. Hase, Clin. Chim. Acta 199 (1991) 263. [11] K. Ishii, T. Furuta, Y. Kasuya, J. Chromatogr. B 704 (1997) 299. [12] P.C.H. Hollman, J.M.P van Trijp, M.N.C.P Buysman, M.S.v.d. Gaag, M.J.B. Mengelers, J.H.M. de Vries, M.B. Katan, FEBS Lett. 418 (1997) 152. [13] P.G. Pietta, C. Gardana, P.L. Mauri, J. Chromatogr. B 693 (1997) 249. [14] A. Hasler, O. Sticher, J. Chromatogr. 605 (1992) 41. [15] O. Sticher, A. Hasler, B. Meier, Dtsch. Apoth. Ztg. 131 (1991) 1827.

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