Đánh giá tác dụng ức chế enzym xanthin oxidase in vitro của cây nở ngày đất (gomphrena celosiodes mart )

Tail lieu,luan van thac si , luan an tien si of 169 I HC QUC GIA H NI KHOA Y DC V TH HOA NH GI TC DNG C CH ENZYM XANTHIN OXIDASE IN VITRO CA CY N NGY T (GOMPHRENA CELOSIODES MART.) KHểA LUN TT NGHIP I HC NGNH DC HC H Ni 2017 Tail lieu,luan van thac si , luan an tien si of 169 Tail lieu,luan van thac si , luan an tien si of 169 I HC QUC GIA H NI KHOA Y DC V TH HOA NH GI TC DNG C CH ENZYM XANTHIN OXIDASE IN VITRO CA CY N NGY T (GOMPHRENA CELOSIODES MART.) KHểA LUN TT NGHIP I HC NGNH DC HC Khúa: QH.2012.Y Ngi hng dn: ThS NG KIM THU TS BI THANH TNG H Ni 2017 Tail lieu,luan van thac si , luan an tien si of 169 Tail lieu,luan van thac si , luan an tien si of 169 LI CM N hon thnh khúa lun ny, tụi ó nhn c s giỳp quý bỏu ca cỏc th, cỏc thy cụ giỏo, bn bố v nhng ngi thõn ca tụi Trc ht, tụi xin by t lũng bit n sõu sc ti ThS ng Kim Thu, ngi thy ó hng dn tn tỡnh v quan tõm giỳp tụi sut quỏ trỡnh thc hin khúa lun Tụi cng chõn thnh bit n TS Bựi Thanh Tựng ó tn tỡnh hng dn, gii ỏp nhng thc mc v giỳp tụi quỏ trỡnh thc hin ti Tụi xin trõn trng cm n ng y, Ban Giỏm hiu v cỏc b mụn Khoa Y - Dc, i hc Quc Gia H Ni ó to iu kin thun li cho tụi thi gian hc v nghiờn cu Tụi xin gi li cm n chõn thnh ti: Cỏc thy cụ v anh ch k thut viờn b mụn Dc lý Dc lõm sng,b mụn Dc Liu, b mụn Y Dc hc c , Khoa Y-Dc, i hc Quc Gia H Ni ó giỳp v to iu kin cho tụi hon thnh khúa lun Li cui cựng, tụi xin cm n ti gia ỡnh, bn bố, ngi thõn - nhng ngi ó ng h, chia s, ng viờn tụi nhng lỳc khú khn v giỳp tụi hon thnh khúa lun ny H Ni, thỏng 5, nm 2017 Sinh viờn V Th Hoa Tail lieu,luan van thac si , luan an tien si of 169 Tail lieu,luan van thac si , luan an tien si of 169 MC LC LI CM N MC LC DANH MC CH VIT TT DANH MC BNG DANH MC HèNH T VN CHNG - TNG QUAN 1.1 Tng acid uric mỏu 1.1.1 Sinh chuyn húa acid uric 1.1.2 Vai trũ ca enzym XO sinh chuyn húa acid uric 1.1.3 Tng acid uric mỏu 10 1.2 Enzym XO v cỏc cht c ch enzym XO 11 1.2.1 Enzym XO 12 1.2.2 Cỏc thuc cú tỏc dng c ch enzym XO 13 1.3 N ngy t (Gomphrena Celosiodes Mart.) 17 1.3.1 N ngy t 17 1.3.2 c im thc vt v phõn b 18 1.3.3 Thnh phn húa hc 18 1.3.4 Tỏc dng dc lý 19 1.4 Cỏc phng phỏp ỏnh giỏ tỏc dng c ch enzym XO in vitro 19 Tail lieu,luan van thac si , luan an tien si of 169 Tail lieu,luan van thac si , luan an tien si of 169 1.4.1 Phng phỏp o quang 20 1.4.2 Phng phỏp o ỏp 21 1.4.3 Phng phỏp o s dng HPLC vi detector hunh quang 21 1.4.4 Mt s phng phỏp khỏc 21 CHNG - I TNG V PHNG PHP NGHIấN CU 22 2.1 i tng nghiờn cu 22 2.1.1 Dc liu nghiờn cu 22 2.1.2 Chun b cỏc mu nghiờn cu 22 2.2 Phng tin nghiờn cu 23 2.2.1 Húa cht v thuc th 23 2.2.2 Thit b v dng c 24 2.3 Ni dung nghiờn cu 24 2.4 Phng phỏp nghiờn cu 25 2.4.1 Nghiờn cu trin khai mụ hỡnh ỏnh giỏ tỏc dng c ch enzym XO in vitro ca dc liu 25 2.4.2 Nghiờn cu tỏc dng c ch enzym XO in vitro ca dch chit ton phn v cỏc phõn on dch chit ca cõy N ngy t 27 2.5 Phng phỏp x lớ s liu 30 CHNG - KT QU V THO LUN 31 3.1 Kt qu 31 3.1.1 Kt qu nghiờn cu trin khai mụ hỡnh ỏnh giỏ tỏc dng c ch enzym XO in vitro ca dc liu 31 Tail lieu,luan van thac si , luan an tien si of 169 Tail lieu,luan van thac si , luan an tien si of 169 3.1.2 Kt qu nghiờn cu tỏc dng c ch enzym XO in vitro ca dch chit ton phn v cỏc phõn on dch chit ca cõy N ngy t 35 3.2 Bn lun 38 3.2.1 V nghiờn cu trin khai mụ hỡnh ỏnh giỏ tỏc dng c ch enzym XO in vitro ca dc liu 38 3.2.2 V nghiờn cu tỏc dng c ch enzym XO in vitro ca dch chit ton phn v cỏc phõn on dch chit ca cõy N ngy t 39 KT LUN V XUT 41 TI LIU THAM KHO Tail lieu,luan van thac si , luan an tien si of 169 Tail lieu,luan van thac si , luan an tien si of 169 DANH MC CH VIT TT STT Tờn vit tt Tờn y ABTS 2,2-azino-bis (3-ethybenzothiazolin-6-sulphonat) AHP 2-amino-4-hydroxypteridin DMSO dimethyl sulfoxid ETDA ethylence diamine tetraacetic acid FAD dinucleotide adenine flavin FDA Cc qun lớ Thc phm v Dc phm Hoa K (Food and Drug Administration) HPLC sc kớ lng hiu nng cao (high-performance liquid chromatography) IC50 nng c ch 50% (the half maximal inhibitory concentration) IXP isoxanthopterin 10 NSAIDS 11 OD mt quang hc 12 SOD superoxide dismutase 13 XO xanthin oxidase thuc chng viờm khụng steroid (non steroidial anti inflammatory drugs) Tail lieu,luan van thac si , luan an tien si of 169 Tail lieu,luan van thac si , luan an tien si of 169 DANH MC BNG Bng 2.1 Bng b trớ thớ nghim kho sỏt nh hng ca nng enzym XO v c cht xanthin lờn s hỡnh thnh acid uric 26 Bng 3.1 Kt qu kho sỏt nh hng nng enzym XO v c cht xanthin lờn s hỡnh thnh acid uric 31 Bng 3.2 Kh nng c ch enzym XO in vitro ca Allopurinol 34 Bng 3.3 Kt qu nh tớnh flavonoid dch chit ethanol ton phn v cỏc phõn on dch chit ca cõy N ngy t 35 Bng 3.4 Kh nng c ch enzym XO in vitro ca dch chit ton phn v cỏc phõn on dch chit cõy N ngy t cỏc nng khỏc 36 Tail lieu,luan van thac si , luan an tien si of 169 Tail lieu,luan van thac si , luan an tien si of 169 DANH MC HèNH Hỡnh 1.1 S chuyn húa purin 10 Hỡnh 1.2 Cu trỳc enzym xanthin oxidase 12 Hỡnh 1.3 Cu trỳc húa hc Allopurinol 14 Hỡnh 1.4 Cu trỳc húa hc ca Febuxostat (TMX-67, TEI-6720) 15 Hỡnh 1.5 Cu trỳc húa hc ca Quercetin 17 Hỡnh 1.6 Cu trỳc húa hc ca Luteolin 17 Hỡnh 2.1 Cõy N ngy t 22 Hỡnh 2.2: Quy trỡnh chit xut dc liu 23 Hỡnh 2.3 S quy trỡnh thớ nghim 29 Hỡnh 3.1 th biu din nh hng ca nng enzym XO v c cht xanthin lờn hiu sut phn ng 32 Hỡnh 3.2 Kh nng c ch enzym XO in vitro ca Allopurinol 34 Hỡnh 3.3 Kh nng c ch enzym XO in vitro ca dch chit ton phn v cỏc phõn on dch chit N ngy t cỏc nng khỏc 37 Tail lieu,luan van thac si , luan an tien si of 169 Tail lieu,luan van thac si , luan an tien si 10 of 169 T VN Xanthin oxidase (XO) l mt enzym cú cu trỳc phc tp, c bit n cỏch õy hn 100 nm v c xem l enzym chỡa khúa chuyn húa purin Enzym XO xỳc tỏc cho phn ng oxy húa hypoxanthin thnh xanthin v phn ng oxy húa xanthin thnh acid uric õy l hai phn ng giai on cui cựng ca quỏ trỡnh chuyn húa cỏc base purin c th [29] Bt kỡ tỏc ng no nh hng n hot ng ca xanthin oxidase cng nh hng n s to thnh acid uric Acid uric l sn phm thoỏi giỏng cui cựng ca nucleoprotein cú cha nhõn purin Tng acid uric mỏu c xỏc nh l yu t nguy c ca gỳt [24] Nng acid uric mỏu cng cao thỡ nguy c mc gỳt cng ln [33] Bt kỡ yu t no lm tng quỏ trỡnh tng hp hoc gim s thi tr acid uric u cú th lm tng nng acid uric mỏu [24] Vỡ vy, vic s dng cỏc cht c ch enzym XO cn tr hỡnh thnh acid uric c th l mt mc tiờu m cỏc nghiờn cu ó v ang hng ti phũng v iu tr bnh gỳt Trờn th gii ó cú nhiu nghiờn cu phỏt trin thuc iu tr tng acid uric mỏu Bờn cnh vic phỏt trin cỏc thuc tng hp húa hc, thc vt v dc liu cng l mt ngun nguyờn liu ln c quan tõm giỳp quỏ trỡnh iu tr tng acid uric mỏu Cõy N ngy t cú tờn khoa hc l Gomphrena celosiodes Mart., h Dn (Amranthaceae) Ngi ta ó tỡm thy cỏc hp cht phenolic, flavonoid, saponin, sterol, terpen, tannin v coumarins thnh phn húa hc ca cõy N ngy t [12] Nhiu nghiờn cu cng ó ch kh nng chng oxy húa, kh nng c ch cao i vi lipid peroxidation ca acid linoleic [12][54] Ti mt s vựng ngi dõn ó s dng cõy N ngy t iu tr bnh gỳt Tuy nhiờn, hin cha cú nghiờn cu khoa hc no c cụng b v tỏc dng iu tr bnh gỳt cng nh kh nng c ch enzym XO ca cõy N ngy t Trờn thc th ú, gúp phn sng lc nhm tỡm kim cỏc dc liu cú kh nng c ch XO, bc u quỏ trỡnh xỏc nh dc liu tim nng iu tr gỳt, ti nghiờn cu: ỏnh giỏ tỏc dng c ch enzym xanthin oxidase in vitro ca cõy N ngy t (Gomphrena Celosiodes Mart.) c thc hin vi hai mc tiờu sau: Trin khai c mụ hỡnh ỏnh giỏ tỏc dng c ch enzym xanthin oxidase in vitro ca dc liu ỏnh giỏ c tỏc dng c ch enzym xanthin oxidase ca dch chit ton phn v cỏc phõn on dch chit ca cõy N ngy t Tail lieu,luan van thac si , luan an tien si 10 of 169 Tail lieu,luan van thac si , luan an tien si 35 of 169 2,12 % Gi nguyờn nng enzym l 0,025 U/mL, thay i cỏc nng xanthin ln lt l 150 mM, 250 mM, 500mM hiu sut phn ng cng tng dn t 82,17 1,34 %; 83,24 2,01 %; 85,13 2,34 % Khi nng enzym l 0,05 U/mL, gi nguyờn nng enzym v tng nng xanthin tng dn ln lt l 75 mM, 150 mM, 250 mM, 500mM hiu sut phn ng cng tng dn t 72,15 1,96 %; 86,46 1,67 %; 87,17 1,89 %; 87,24 2,22 % Cng thay i nng xanthin thnh cỏc nng 75 mM, 150 mM, 250 mM, 500mM, v o nng enzym 0,1 U/mL hiu sut ca phn ng cng tng dn t l thun vi m c xanthin l 75,46 1,84 %; 91,17 1,85 %; 93,15 1,95 %; 94,06 1,87 % nng enzym 0,2 U/mL v nng xanthin 75 mM hiu sut ca phn ng l 85,32 2,03 %, thay i nng xanthin thnh 150 mM, 250 mM, 500 mM v nng enzym gi nguyờn 0,2 U/mL hiu sut ca phn ng tng dn c kt qu ln lt l 96,28 1,94 %; 96,34 1,85 %; 97,05 1,95 % Kt qu trờn cho thy, nng enzym khụng i, nng xanthin tng dn thỡ hiu sut ca phn ng cng tng dn Nng enzym khụng i, nng xanthin tng t 75 mM lờn 150 mM cú khong tng hiu sut ln nht, thay i nng xanthin lờn 250 mM, 500 mM hiu sut phn ng cú mc tng khụng ỏng k Da vo kt qu trờn, nhúm nghiờn cu chỳng tụi ó chn nng enzym l 0,2 U/mL v nng xanthin l 150 mM cho cỏc thớ nghim mụ hỡnh nghiờn cu 3.1.1.2 Kt qu thm nh phng phỏp thụng qua ỏnh giỏ tỏc dng ca Allopurinol Allopurinol l mt cht cú tỏc dng c ch enzym XO in vitro v in vivo ó v ang c s dng lõm sng.Trong nhiu nghiờn cu ỏnh giỏ tỏc dng c ch enzym XO in vitro ó s dng Allopurinol lm cht i chng thm nh phng phỏp ỏnh giỏ tỏc dng c ch hot ng enzym XO in vitro ca dc liu bng phng phỏp o quang, nhúm nghiờn cu chỳng tụi tin hnh ỏnh giỏ tỏc dng ca cht ny Kt qu c biu din Bng 3.2 v Hỡnh 3.2: 33 Tail lieu,luan van thac si , luan an tien si 35 of 169 Tail lieu,luan van thac si , luan an tien si 36 of 169 Bng 3.2 Kh nng c ch enzym XO in vitro ca Allopurinol Nng (àg/mL) Phn trm c ch (I%) 0,5 12,5 1,25 24,4 2,5 41,5 66,7 10 92,6 12,5 98,9 Hỡnh 3.2 Kh nng c ch enzym XO in vitro ca Allopurinol Nhn xột: T Bng 3.2 v Hỡnh 3.2: Allopurinol th hin kh nng c ch enzym XO ph thuc vo nng , cỏc nng khỏc th hin s c ch rừ rt hot ng ca enzym XO, nng cng lờn cao, kh nng c ch cng ln Nng Allopurinol 0,5 àg/mL hiu sut c ch ch t 12,5 %, nhng tng nng Allopurinol lờn ln lt l 1,25 àg/mL, 2,5 àg/mL, àg/mL, 10 àg/mL hiu sut c ch cng tng t kt qu ln lt l 24,4 %, 41,5 %, 66,7%,92,6 % Ti nng 12,5 àg/mL kh nng c ch t 98,9 % 34 Tail lieu,luan van thac si , luan an tien si 36 of 169 Tail lieu,luan van thac si , luan an tien si 37 of 169 Theo kt qu trờn th, tỏc dng c ch enzym XO in vitro ca Allopurinol th hin qua giỏ tr IC50 l 3,37 0,24 àg/mL 3.1.2 Kt qu nghiờn cu tỏc dng c ch enzym XO in vitro ca dch chit ton phn v cỏc phõn on dch chit ca cõy N ngy t 3.1.2.1 Kt qu nh tớnh flavonoid ca dch chit ethanol ton phn v cỏc phõn on dch chit t cõy N ngy t nh tớnh flavonoid dch chit ethanol ton phn v cỏc phõn on dch chit Kt qu nh tớnh flavonoid dch chit dc liu c trỡnh by Bng 3.3 Bng 3.3 Kt qu nh tớnh flavonoid dch chit ethanol ton phn v cỏc phõn on dch chit ca cõy N ngy t Phõn on EtOH n-hexan EtOAc n-BuOH Phn ng Cyanidin ++ + + ++ +++ Phn ng vi kim ++ + ++ +++ Phn ng vi FeCl3 ++ + ++ +++ Phn ng diazo húa + _ + ++ Phn ng Ghi chỳ: (+) phn ng dng tớnh (-) phn ng õm tớnh Nhn xột: T kt qu nh tớnh Bng 3.3 cho thy cõy N ngy t cú cha thnh phn flavonoid Trong ú, flavonoid cú dch chit n-BuOH cho cỏc phn ng nh tớnh dng tớnh rừ nht sau ú l dch chit ethanol ton phn v dch chit EtOAc Dch chit n-hexan cho phn ng nh tớnh flavonoid khụng rừ rng 35 Tail lieu,luan van thac si , luan an tien si 37 of 169 Tail lieu,luan van thac si , luan an tien si 38 of 169 3.1.2.2 Kt qu ỏnh giỏ tỏc dng c ch enzym XO in vitro ca dch chit ethanol ton phn v cỏc phõn on dch chit t cõy N ngy t ỏnh giỏ nh hng ca dch chit ton phn v cỏc phõn on dch chit t cõy N ngy t lờn hot enzym XO in vitro Giỏ tr phn trm c ch I (%) ca dch chit ton phn v cỏc phõn on dch chit nng khỏc t cõy N ngy c trỡnh by Bng 3.4 v Hỡnh 3.3 Bng 3.4 Kh nng c ch enzym XO in vitro ca dch chit ton phn v cỏc phõn on dch chit cõy N ngy t cỏc nng khỏc Nng (àg/mL) I (%) IC50 (àg/m L) àg/mL 10 àg/mL 25 àg/mL 50 àg/mL 100 àg/mL Chng 0 0 - EtOH 8,69 0,67 15,85 1,15 30,24 1,32 51,05 0,94 78,66 1,12 47,37 0,26 n-Hexan 4,24 0,94 10,35 0,61 23,75 0,73 36,75 0,87 56,23 1.02 81,59 0,21 EtOAc 12,77 0,86 20,54 0,76 39,55 0,92 62,51 1,21 86,57 0,93 33,36 0,51 n-BuOH 12,47 1,17 25,57 1,29 45,68 1,03 68,37 1,11 88,56 0,74 27,39 0,31 Phõn on 36 Tail lieu,luan van thac si , luan an tien si 38 of 169 Tail lieu,luan van thac si , luan an tien si 39 of 169 Hỡnh 3.3 Kh nng c ch enzym XO in vitro ca dch chit ton phn v cỏc phõn on dch chit N ngy t cỏc nng khỏc Nhn xột: T kt qu Bng 3.4 v Hỡnh 3.3, cho thy tỏc dng c ch XO ca dch chit ton phn v cỏc phõn on dch chit t cõy N ngy t tng dn theo nng Dch chit ethanol ton phn t cõy N ngy t th hin tỏc dng c ch enzym XO in vitro vi giỏ tr IC50 tớnh c l 47,37 0,26 àg/mL Trong cỏc phõn on dch chit t cõy N ngy t, phõn on nBuOH th hin tỏc dng c ch enzym XO in vitro mnh nht vi I% nng cao nht 100 àg/mL l 88,56 0,74 %, giỏ tr IC50 tớnh c l 27,93 0,31 àg/mL Tip theo l phõn on EtOAc vi giỏ tr I% t 86,57 0,93 % nng cao nht 100 àg/mL, giỏ tr IC50 tớnh c l 33,36 0,51 àg/mL Phõn on n-hexan th hin tỏc dng c ch enzym XO in vitro yu vi giỏ tr IC50 tớnh c l 81,59 0,2 àg/mL Song song vi cỏc mu th, tin hnh tng t vi mu chng dng Allopurinol cho thy tỏc dng c ch enzym XO in vitro ca Allopurinol th hin qua giỏ tr IC50 l 3,37 0,24 àg/mL (Hỡnh 3.2) 37 Tail lieu,luan van thac si , luan an tien si 39 of 169 Tail lieu,luan van thac si , luan an tien si 40 of 169 3.2 Bn lun 3.2.1 V nghiờn cu trin khai mụ hỡnh ỏnh giỏ tỏc dng c ch enzym XO in vitro ca dc liu 3.2.1.1 V kho sỏt nh hng ca nng enzym XO v c cht xanthin lờn s hỡnh thnh acid uric Xu hng nghiờn cu v tỏc dng c ch enzym XO ca dc liu ang ngy cng phỏt trin, trờn th gii cng cú nhiu phng phỏp ỏnh giỏ hot enzym XO cng nh phng phỏp ỏnh giỏ tỏc dng c ch enzym XO, phng phỏp o quang da trờn nh vic lng acid uric l phng phỏp ph bin nht hin [52][44].Trong nc, ó cú mt s nghiờn cu ỏnh giỏ tỏc dng c ch enzym XO s dng phng phỏp ny, mt vi nghiờn cu ỏp dng o quang trờn h thng ELISA, cng cú nghiờn cu ó ỏp dng o quang trờn cuvet [5][9] hon thin hn mụ hỡnh ỏnh giỏ tỏc dng c ch enzym XO bng phng phỏp o quang da trờn vic nh lng acid uric s dng cuvet, nhúm nghiờn cu chỳng tụi ó tin hnh ti vi ni dung ỏnh giỏ tỏc dng c ch enzym XO bng phng phỏp o quang da trờn vic nh lng acid uric s dng cuvet Trong quỏ trỡnh bo qun, chỳng tụi luụn gi enzym XO nhit bo qun l 4oC Tuy nhiờn, quỏ trỡnh pha v tin hnh thớ nghim, enzym cú th b hot húa v gim hot Vỡ th, gim thiu ti a tỡnh trng ny, quỏ trỡnh pha v quỏ trỡnh lm thớ nghim cc ng enzym sau thao tỏc u c ngõm nc ỏ m bo nhit thp Enzym v c c cht u c pha trc dựng Trong mụ hỡnh ca chỳng tụi, chỳng tụi ó tin hnh kho sỏt ng hc enzym XO vỡ vy sau kho sỏt ó la chn enzym nng 0,2 U/mL v xanthin nng 150 mM cho cỏc thớ nghim tip theo ca mụ hỡnh, vỡ nhng nng ny enzym hot ng xỳc tỏc tt, hiu sut phn ng to acid uric cao 3.2.1.2 V thm nh phng phỏp thụng qua ỏnh giỏ tỏc dng ca Allopurinol Allopurinol l mt thuc ó c s dng rng rói lõm sng iu tr tng gỳt, cú tỏc dng h acid uric mỏu thụng qua c ch c ch hot ng ca enzym XO, nú thng c s dng lm cht i chng cỏc th nghim ỏnh giỏ tỏc dng c ch enzym XO Trong phng phỏp ny, Allopurinol ó th hin c ch enzym XO rừ rt vi giỏ tr IC50 l 3,37 0,24 àg/mL Gớa tr ny tng i sỏt vi nhng kho sỏt ó c tin hnh trc õy, vỡ vy cú th dựng i chng trc tip vi cỏc giỏ tr c ch IC50 ca dch chit ton phn v cỏc phõn on dch chit ca cõy thuc.[5][9][52] 38 Tail lieu,luan van thac si , luan an tien si 40 of 169 Tail lieu,luan van thac si , luan an tien si 41 of 169 3.2.2 V nghiờn cu tỏc dng c ch enzym XO in vitro ca dch chit ton phn v cỏc phõn on dch chit ca cõy N ngy t 3.2.2.1 V nh tớnh flavonoid ca dch chit ethanol ton phn v cỏc phõn on dch chit t cõy N ngy t Nghiờn cu ny ó ch s cú mt ca flavonoid dch chit ton phn v cỏc phõn on dch chit t cõy N ngy t thụng qua kt qu nh tớnh cỏc phn ng húa hc c trng xỏc nh s cú mt ca flavonoid Cú th nhn rng, hm lng flavonoid tng i cao dch chit ethanol ton phn v cỏc phõn on dch chit t cõy N ngy t, c bit flavonoid c xỏc nh cú mt nng cao nht phõn on n-BuOH th hin phn ng mónh lit nht cỏc phn ng húa hc c trng ó tin hnh thớ nghim ó cú nghiờn cu ch rng, flavonoid cú mt rng rói nhiu thc phm ung cú ngun gc thc vt [31] Mt s flavonoid c bit n nh cỏc cht chng oxy húa mnh bi chỳng ó th hin hot tớnh oxy húa cỏc gc t v ion húa ion kim loi [19][22] Ngoi hot tớnh chng oxy húa, ó cú bỏo cỏo ch flavonoid cũn cú tỏc dng c ch enzym khỏc nh cyclooxygenase v lipoxygenase [27] Mt s flavonoid c chng minh ó th hin tỏc dng c ch trờn enzym XO sn xut hydrogen peroxide v anion superoxide quỏ trỡnh oxy húa hypoxanthin thnh xanthin, sau ú xanthin thnh acid uric [41][43] Cỏc nghiờn cu v s c ch hot ng ca enzym XO in vitro ó cho thy mt s cht cú tỏc dng c ch enzym ny nh p-aminophenol dng oxy hoỏ, pyrogallol, bromoacetophenone, 6-pteridylaldehyde v hydroxymethylpterin Vic kim tra cu trỳc ca cỏc hp cht ny cho thy rng chỳng cú c im chung l gi cỏc nhúm hydroxyl hoc carbonyl Trong nghiờn cu ca mỡnh, Beiler v Martin ó ch cỏc hp cht ca flavonoid, giu cỏc nhúm hydroxyl v cú kh nng to thnh cỏc hp cht quinonoid cỏc h thng sinh hc, cú tỏc dng nh cht c ch xanthin oxidase Hot tớnh ca enzym XO ó b c ch bi mt nhúm flavonoid v cỏc hp cht cú liờn quan.[57] Cỏc flavonoid cú cu trỳc phng v liờn kt ụi C2=C3 hot ng c ch enzym XO mnh hn so vi cỏc flavonoid b methyl húa hay hydroxyl húa [58] 3.2.2.2 V ỏnh giỏ tỏc dng c ch enzym XO in vitro ca dch chit ethanol ton phn v cỏc phõn on dch chit t cõy N ngy t Kh nng c ch enzym XO in vitro ca dch chit ton phn v cỏc phõn on dch chit t cõy N ngy t c xỏc nh da vo giỏ tr IC50 Trong nghiờn cu ny, chỳng tụi ó ch rng dch chit ethanol ton phn v cỏc phõn on n-hexan, phõn on EtOAc, phõn on n-BuOH t cõy N ngy t th hin tỏc dng c ch enzym XO in vitro vi IC50 ln lt l 47,37 0,26 àg/mL; 81,59 0,2 àg/mL; 33,36 0,5 àg/mL; 27,93 0,3 39 Tail lieu,luan van thac si , luan an tien si 41 of 169 Tail lieu,luan van thac si , luan an tien si 42 of 169 àg/mL iu ny chng t, cao chit ethanol v cỏc phõn on cao chit n-hexan, cao chit EtOAc v cao chit n-BuOH u cha cht cú hot tớnh c ch enzym XO Trong ú, hm lng cỏc cht cú hot tớnh c ch enzym XO cú mt nhiu nht phõn on cao chit n-BuOH v phõn on EtOAc in hỡnh l cỏc hp cht flavonoid, c ỏnh giỏ l cú kh nng c ch hot ng ca enzym XO v hot tớnh chng oxy húa cao Kong v cng s (2002) ó cú nghiờn cu ch rng, vic c ch hot ng enzym XO khụng ch gim nng acid uric mỏu, m cũn gim s sn xut cỏc gc t [38] S c ch hot ng enzym XO c xem nh l mt c ch chng oxy húa ca cỏc hp cht polyphenol Nhiu nghiờn cu v enzym XO chng minh rng, hot ng ca enzym XO l nguyờn nhõn dn ti s to nhiu gc t Nờn vic b sung cỏc cht c ch enzym XO va cú tỏc dng c ch to thnh acid uric ngn nga bnh gỳt, va cú tỏc dng ngn chn li stress oxy húa l nguyờn nhõn gõy tn thng t bo v mụ c th [21][32].Vỡ vy, nhúm nghiờn cu chỳng tụi tin hnh ỏnh giỏ kh nng c ch enzym XO ca cõy N ngy t bi õy l mt dc liu r tin, d kim v kt qu nghiờn cu ó m hng mi cho vic s dng N ngy t lm ngun dc liu tim nng nghiờn cu cỏc cht c ch XO, nht l cỏc cht t phõn on n-BuOH v phõn on EtOAc 40 Tail lieu,luan van thac si , luan an tien si 42 of 169 Tail lieu,luan van thac si , luan an tien si 43 of 169 KT LUN V XUT KT LUN V nghiờn cu trin khai mụ hỡnh ỏnh giỏ tỏc dng c ch enzym XO in vitro ca dc liu ó tin hnh kho sỏt nh hng ca nng enzym XO v c cht xanthin lờn s hỡnh thnh acid uric Kho sỏt ny chỳng tụi ó la chn c nng enzym v nng c cht xanthin thớch hp cho mụ hỡnh,trong ú: nng enzym l 0,2 U/mL v nng xanthin l 150 mM cho cỏc thớ nghim mụ hỡnh nghiờn cu ó trin khai c mụ hỡnh ỏnh giỏ tỏc dng c ch enzym XO in vitro ca dc liu bng phng phỏp o quang bc súng 295 nm Bng phng phỏp ny nhúm nghiờn cu chỳng tụi ó xỏc nh c IC50 ca Allopurinol l 3,37 0,24 àg/mL V nghiờn cu tỏc dng c ch enzym XO in vitro ca dch chit ton phn v cỏc phõn on dch chit ca cõy N ngy t ó chng minh c s cú mt ca flavonoid dch chit ton phn v cỏc phõn on dch chit t cõy N ngy t, cng nh so sỏnh c nng ca flavonoid cỏc dch chit ú ó ỏnh giỏ c tỏc dng c ch enzym XO ca dch chit ton phn v cỏc phõn on dch chit t cõy N ngy t Kt qu nghiờn cu xỏc nh dch chit ethanol ton phn t cõy N ngy t c ch enzym XO in vitro vi giỏ tr IC50 l 47,37 0,26 àg/mL Phõn on dch chit n-BuOH th hin tỏc dng c ch enzym XO in vitro vi giỏ tr IC50 tớnh c l 27,93 0,31 àg/mL Tip theo l phõn on EtOAc vi giỏ tr IC50 tớnh c l 33,36 0,51 àg/mL Phõn on n-hexan th hin tỏc dng c ch enzym XO in vitro yu vi giỏ tr IC50 tớnh c l 81,59 0,2 àg/mL XUT T nhng kt qu thu c, nhúm nghiờn cu chỳng tụi xin xut : - Tip tc trin khai phng phỏp ỏnh giỏ tỏc dng c ch hot ng enzym XO bng phng phỏp o quang bc súng 295 nm - Kho sỏt kh nng c ch enzym XO ca cao chit t cõy N ngy mc in vivo - Nghiờn cu cỏc thnh phn húa hc v tỏch cỏc phõn on vi cỏc dung mụi khỏc cao chit t cõy N ngy t nhm 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2013 57.Beiler J M and Gustav J Martin, "The inhibition of xanthin oxidase by flavonoids and related compounds," J Biol Chem, vol 192, pp831-834, 1951 58.Suyun Lin; Guowen Zhang; Yijing Liao; Junhui Pan and Deming Gong, "Dietary Flavonoids as Xanthine Oxidase Inhibitors: Structure Affinity and StructureActivity Relationships," J Agric Food Chem, vol 63 (35), pp 77847794, 2015 Tail lieu,luan van thac si , luan an tien si 49 of 169 ... cứu: Đánh giá tác dụng ức chế enzym xanthin oxidase in vitro Nở ngày đất (Gomphrena Celosiodes Mart.) thực với hai mục tiêu sau: Triển khai mô hình đánh giá tác dụng ức chế enzym xanthin oxidase. .. nhằm đánh giá tác dụng ức chế enzym XO in vitro dịch chiết từ Nở ngày đất 1.4 Các phương pháp đánh giá tác dụng ức chế enzym XO in vitro Để đánh giá tác dụng ức chế XO, cần so sánh hoạt độ enzym. .. ĐẠI HỌC QUỐC GIA HÀ NỘI KHOA Y DƯỢC VŨ THỊ HOA ĐÁNH GIÁ TÁC DỤNG ỨC CHẾ ENZYM XANTHIN OXIDASE IN VITRO CỦA CÂY NỞ NGÀY ĐẤT (GOMPHRENA CELOSIODES MART.) KHÓA LUẬN TỐT NGHIỆP ĐẠI HỌC NGÀNH DƯỢC