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• Nghi8n ctim - Ky thuOt Khao sat kha ding urc che enzym a-glucosidase in vitro va glucose huyet in vivo dm re to' ke hoa dio (Urena lobata L.) Quich Ngo Dam Pining', Cao Minh Dal' VG Thi Bach Phtreng', Huynh Ngec Trinh2* 'BM Cong nghe sinh hoc thtrc vett & Chuyeln h6a sinh hoc, Khoa Sinh hoc — Cong nghe sinh hoc, DH KHTN TP Ho Chi Minh 2BO mOn Dtror Ly, Khoa Dam DH Y MAX TP HO Chi Minh 'E-mail: trinhb181@yahoo.com Summary The inhibitory effect in vitro of the Urena lobata L (Malvaceae) on d-glucosidase enzyme was investigated As the root has been known the best part in this term, hypoglycemic activity in vivo of the plant hairy roots produced by coculture with Agrobacterium rhizogenes ATTC 15834 and those cultured in vitro or hydroponically were focused on The ethanol extracts from the obtained hydroponic hairy roots (IC5o = 3.963 pg/m0presented the same value of IC50 as the natural roots (IC50 = 3.851 pg/m0 and so, significantly inferior to the hairy roots in vitro (IC50 = 6.692 pg/m0 Moreover, in alloxan-induced diabetic mice, blood glucose level of the group treated with either of the three tested extracts showed a slight decrease as compared to the untreated group In short, the hairy roots of Urena lobata L proved potential in supporting anti-diabetic type treatment Keywords: Urena lobata L., hairy root, hydroponic, a-glucosidase, blood glucose DOt van d4 Re to duvc tao stir chuyen gen bang cach sir dyng thong vector try nhien ter vi khan dat gram am — Agrobacterium rhizogenes, vao to bao thkrc vat U'u diem cOa re to la sau A rhizogenes xam nhiem vao m6, re hinh va phat then ter vi tri ten thuvng co toc dO Oat then nhanh, khong hinting det, khong phy thuOc vao chat dieu h6a tang trireng ngoai sinh va ben vCrng ye mat di troy& 0] Them vao do, nhieu nghien cCru a chi rang qua trinh nuoi cay, re to co the tao ham lirang cac hap chat thir cep nhieu han so vO'i re try nhien vai thol gian nuoi cay ngan Trong clang ngha nuoi cay re tux vat dac biat la re ta, k9 thuat in vitro va thuy canh thirang dtrac sir dyng co nhieu kru diem han cac hinh thirc nuoi cay khac nhu' khd nang tang truang nhanh va skr de clang thu nhan sinh khei Ka hoa dao (Urena lobata L.) la met 'obi cay thuec hp Bang (Malvaceae) ter lau da duvc sir dyng nhkr met bai thuoc dan gian de dieu tri banh dau khO'p, viem nhiem hay giap Om ma gan va mau [4] Trong dO ten the giai da cO nhieu nghien cCru chirng minh hiau qua ho tra dieu trj banh dai thao dverng typ cOa ke hoa dao th6ng qua kha nang lam Om neng da glucose huyet ten chuOt dai thao &rang thkrc nghiem hay tac dyng chang oxy hem (5.61 Dac brat, cac nghien cCru can cho they re thvong cO hoat tinh tat bon cac be phan can dai hoac toan cay Vi vay, nghien cihr dkrac thkrc hian nham chirng minh re to cam Crng tCr cay ke hoa dao cung CO tiem nang viac hO to, dieu trj 22 dai thdo duteng typ thong qua kha nang erc the host Tang cOa enzym bien &rang carbohydrat a-glucosidase in vitro va glucose huyet in vivo VOt lieu IA phu'ong phap nghien ciru Vet lieu DOng vet thee, nghlOm ChuOt What tang chung Swiss albino giOng dkrc, khOe manh, can nang khoang 25 — 30 g, duvc mua tie Vien Vac xin va Sinh phem y to Nha Trang, &vac nuoi bang thirc an vien Vien cung cap ChuOt duvc nuoi On Binh ey nhiat de ph6ng — Wok bat dau the, nghiem Hari chaff Alloxan (Sigma-Aldrich Chemie Gmbh); Acarbose (Glucobay®, Bayer); Enzym a-glucosidase tie chOng vi khuan Saccharomyces cerevisiae (Sigma, M9); p-nitrophenyl glucopyranosid (pNPG) (Sigma, M9); Thu& thin glucose (ISE, Italy); Dung djch NaCI duvc dyng; Dung mai chi& xuat: can 96% (Trung Quoc) Cao chit ethanol cac lope, re Cay ke hoa dao nuoi cay in vitro va bang they canh sau 15 Wei cluvc tien hanh xam nhiem tao re to bang vi khuan Agrobacterium rhizogenes ATTC 15834 Re tao duvc ben hanh kiem tra hien (AO cua gen chuyen bang phuvng phap PCR sir dyng cap mei dac hiau m Cac mei.' re to in vitro, re to thOy canh 30 tuei va re ke hoa dao thu hai ngoai tkr nhien (phuteng Long Thanh M9, quan 9, TAP CHI DUQC HQC - 2/2017 (SO 490 NAM 57) • Nghien cipu - Ky thuOt Tp HO Chi Minh duvc Kra such, phai kho va cho vao t6 say < 50°C cho den khOi *mg kh6ng del, sau d6 dem meu xay nhuyen bOt kho Cao chiet ethanol cac loai re duct dieu che bang k9 thuat chiet ngam dem (maceration): ngam bOt cay mot binh china bang thily tinh hoac bang thep khong ri, binh nap day R6t ethanol vao binh cho den ngang be mat cua lap bOt cay Giir yen & nhiet dO phong 24 gia de cho dung m6i xuyen them vao cau trim to bao thkrc vat va hoa tan cac hop chat tkr nhien Sau dO, dung dich duvc chiet loc ngang qua met to giay lac, thu dich lox, dem duOi dung m6i se c6 duvc cao chiet Khao sat tic Ong Crc che enzym a-glucosidase in vitro Thi nghiem duct tien hanh theo phkrang phap cua Dong va Huang (2012)181, sir dung dia microliter 96 gieng Hut len luVt vao m6i gieng: 50 pl dung dich cao chiet, 40 pl dung dich enzym a-glucosidase (0,2 Wm!) U a nhiet dc) ph6ng 20 phut, them 40 pl co, chat pNPG (5 mM), tiep tuc 20 phut arng phan Crng bang 130 pl dung dich Na2CO3 (0,2 M) Do mat quang (OD) tai bkrac sang 405 nm; hoat tinh (% uc che) cua meu th& dtrac tinh theo ciOng thirc: (Meu chirng - NAL trang) - (MALI thir - NAL mau) % Crc che = Mau chirng - Meu trang x 100 Trong c16: MALI trgng: khong c6 enzym, khong c6 cao chi& Miu chimg: co enzym, khong c6 cao chgt Mk mat,: c6 cao chi&, khong c6 enzym Mugu fink c6 cao chief, c6 enzym Lap phtrang trinh hOi quy tuyen tinh m6 to scr lien quan gifla log10 cua n6ng de C cua chat khao sat (pg/ml) vai phan tram Crc che enzym a-glucosidase: %Crcche=axlgC+b la niing de cao chiet (pg/ml) uc che 50% host IC50 Ong cua enzym duvc not suy b5ng Gang thin &POI day: 50-b IC, = 10 a Acarbose duvc s& dung lam chimg du'ang Mau trang la mau khong china enzym va meu chi:mg am 16 mau kh6ng china cao chiet Thi nghiem duvc tien hanh ton cac loai cao chi& va chUng &rang Wong de cao chi& mg/ml, nong de acarbose 20 mg/ml Cac neng dO duct giarin den mot nira theo thir tkr tang gieng to ten xuang dip& Me hinh gay dal thao dtrimg typ ten chuOt nhat bang alloxan ChuOt duct cho nhin ddi qua dem tuft tien hanh tiern alloxan Alloxan duvc pha dung dich NaC1 0,9% ve duvc tiern vac) tinh mach duel chuet vai lieu 60 mg/kg (tinh theo alloxan tetrahydrat) Bon mud tam gia sau tiem alloxan, tien hanh dinh luvng nOng de glucose huyet cua chi* nhat Lya chon nhOng chuet c6 nting de glucose huyet z 250 mg/dL va chia ngau nhien vao cac nhOm thin nghiem tac dung hp glucose huyet cua cao chiet to cac lopi re ke hoa deo Khao sat tic Ong hp glucose huyet in vivo bang bien phap dung nap throng sucrose ChM bi dai thao du'ang duvc cho nhin ddi qua dem trkrac thir nghiem Tien hanh lay mau to tinh mach duel chuet de xac dinh nong de glucose huyet ban dau Sau de, chuOt duvc cho uong mac Wang sucrose vai lieu g/kg, co hoac khong uong kern chat Crc che enzym a-glucosidase (acarbose 10 mg/kg the tong hoac cao chiet ethanol to ba loai re 400 mg/kg the trong) Cac 10 chuOt khdo sat nhu, sau: TAP CHI DUQC HQC - 2/2017 (SO 490 NAM 57) LO (16 chOng benh, n = 7): ming nu,ac &rang lieu g/kg the hong L6 (10 dOi chieu, n = 7): uong acarbose lieu 10 mg/kg the trkrac uong nkrac &rang L6 (n = 7): uong cao ethanol re t& nhien lieu 400 mg/kg the truck ming rxrac &rang LO (n = 8): wing cao ethanol re to in vitro lieu 400 mg/kg the tang trkrac uong ntrac du'ang Lo (n = 7): uong cao ethanol re to they canh lieu 400 mg/kg the tnybc uong nkrirc duteng Tien hanh lay mau to tinh mach dual chuOt vao cac trial diem 30 va 60 phut sau cho uong nkrac dtrang de xac dinh nOng de glucose huyet huyet Krung bang phu,ang phap enzym mau (thuOc thir glucose oxydase) So sanh dq du'ang huyet a cac thai diem 30 va 60 phut sau uong ntrirc du'ang cua cac nhOm uong cao so vai nh6m chOng benh hay vai nhOm acarbose de dinh gia hieu qua hp glucose huyet cua cac mau re th& nghiem Lim 9: Trong qua trinh tien hanh thir nghiem dung nap du'ang, chuOt phai bi tech ly vai thirc an Phtrang phap danh gia kat qua Cac s6 lieu thu duvc trinh bay dual dung: Trung binh ± SEM SO lieu &vac x6, 19 bang phan mem SAS 9.1, phan nhOm cac gia tri bang phirang phial) Duncan vai p = 0,05 dOi NAM thi nghiem in vitro va phan mem T-Test (Student test) vai TO tin cay 95% del vai thi nghiem in vivo Mt qua nghien cau Hi.0 sat thu cao ethanol Cac loai re ke hoa deo duvc tier' hanh thu nhAn va x1)19 theo quy trinh de thu duqc cao chiet ethanol HiOu suet thu nhan cao ethanol (bang 1) c6 th6, ty' len luvt to cao den that:, la re ty, nhien, re to thily canh va re to in vitro 23 • Nghien civu - K9 thu4t Bang 1: Hi eu suet thu cao ethanol kr cac log/ ke hoa deo Re Khoi bong &of (g) Kai luong kho (g) Phan tram kai bong kh6 (%) Khat lupng cao (g) Hi6u suit (%) RA tkr nhien 2422,693 536,710 22,153 47,238 8,801 RA to thuy canh 409,633 70,042 17,099 1,455 6,052 RA to in vitro 307,901 19,547 6,349 1,148 5,873 Khoo sat tic dOng Crc ch6 enzym a-glucosidase dpt 54,97% 6' nOng d6 chat 0,c the hiving tty, nOng in vitro dO khao sat gram den mOt ni),a tai 0,061 pg/ml khd nang Kha nang Crc che enzym a-glucosidase in vitro caa Crc che Ian luvt la 34,50; 23,97; 15,79; 14,62; 5,26; 2,34 cao chiet ethanol re ta, nhien, re to, in vitro, they canh va va 1,75%; dOng thdyi cac gid tri deu nh6 han cao chiet re chung &rang acarbose len luvt dined trinh bay tu, nhien va re to thuy canh 6, ding nOng dO bang va Kat qua cho thay: giO1 han cac n8ng Gia trj IC caa cao chi& ethanol re ta, nhien, re to dO khao sat, stn Crc Che enzym a-glucosidase t9 le tuyen thuy canh, re to in vitro va acarbose &rot trinh bay tinh vdri nong dO chirng dvang va cao chiet, ming dO bang va stn khac het co nghia thong ke tang den ten khd nang Crc the enzym a-glucosidase 6, mitt 5% Kat qua cho they kha nang Crc the enzym tang cao (Y neng dO 7,813 pg/ml, cao chiet ethanol a-glucosidase cua ba lopi re deu Ian han chi:mg &rang tir re to, nhien va re to thuy canh c0 khd nang Lk che acarbose tie 68 - 115 len, dac Net re to thuy canh cO a-glucosidase dpt 78,65 va 75,44% Trong d6, cao host tinh tu,ang twang re tu, nhien (6 ding phan hang) chi& re to in vitro chi CO kha nang Crc the a-glucosidase va cao gap 1,7 Ian so videi re to in vitro Bang 2: Str Ou chi a-glucosidase (%) cao chi& ethanol cac loai et cac nong kW() sat Wong de chit at chi Pan tram tic chi a-glucosidase (%) 0,061 0,122 010/m0 0,244 0,488 0,977 1,953 3,906 7,813 tv nhien 2,28 t 1,25 5,74 t 2,49 11,33 t 2,79 15,43 t 2,22 17,18 t 1,65 20,74 t 1,61 47,08 t 0,02 78,65 t 1,59 Re to thuy canh 9,94 t 0,53 14,62 t 0,50 16,37 t 0,49 18,71 t 0,48 18,71 t 0,48 21,05 t 0,46 48,24 t 0,60 75,44 ± 0,14 RA to in vitro 1,75 t 0,57 2,34 t 0,57 5,26 t 0,55 14,62 t 0,50 15,79 t 0,49 23,97 t 0,44 34,50 t 0,38 54,97 t 0,26 Bang 3: Sy, Ou cho a-glucosidase (%) dm acarbose & cac nong khdo sat Nang de chit Or chi Phan tram Ur ca a-glucosIdase (%) 156,25 (Pg/m0 312,5 625 1250 2500 5000 10000 20000 Acarbose 34,30 t 0,41 47,11 t 1,65 51,65 t 2,07 57,02 t 0,83 65,70 t 2,89 69,01 t 2,07 78,51 t 0,83 82,23 t 0,41 Bang 4: Gia tri IC„ caa cao chit cac 100i 16 dOi vbi LO chuOt ueng cao re to thew canh CO dO bien thien a-glucosidase (0,2 Wm!) nong de glucose huyet Om ro ret so vdril6 chi:mg benh nhu'ng chu,a dpt y nghia thong ke (P > 0,05) Rieng 10 Phuong trinh Chit ex chi IC, (pgen0 ueng cao re to in vitro, dO bien thien yen cao hat guy tuyan tinh y = ax + b R6 tv nhien 3,851c t 0,075 a = 20,955 so veril6 chimg benh Theo dO, khd nang lam he &rang b=0 RA to thuy canh 3,963c t 0,027 a = 15,073 huyet caa chuOt bj dai thao dirdyng dpt duvc 6, thdyi diem b=0 to in vitro 6,692B t 0,084 a = 16,001 30 phut theo thir ty, len lu,qt la cao ethanol caa re tv b=0 Acarbose 455,491" t 43,393 a = 18,827 nhien, re to thew canh va re to in vitro b=0 Chit thich: cac cher cai A, B, C thi hien SI,f khac biet vi met th6ng ke bmuc nghia 5% Khoo sat tic ding glucose huyet in vivo bAng nghiam phi') dung nap dutong sucrose Kat qua thin nghiOrn cho they nhOm chirrig bOnh, nong dO glucose huyet 6, then diem 30 phOt sau ueng raft dveyng cua nhOm chuOt dung thuOc don chang acarbose thap han co nghTa so vOi nhOm chi* chu,ng benh (P < 0,05) Nhin chung, ca ba loai cao chi& deu cho they xu hu,o'ng lam Om du,erng huyet so vdri nhom chi:mg benh DO Nen thien du,drng huyet 6, thdri diem 30 phut so yeti ban dau caa cac 16 chuOt thin nghiern duvc trinh bay hinh Kat qua cho they, 16 chuOt durcyc cho u6ng cao chiet re to, nhien CO dO bier, thien Wong huyet gram va khac CO nghia thong ke so vdri nham chCrng bOnh khang dieu tri (P < 0,05) Them vao do, so sanh v6i16 chuOt CI& chirng du,qc u6ng acarbose thi kung they c0 stn khdc biet nghTa ye mat thong ke (P > 0,05) 24 Hlnh 1: DO bgn thien dug hurit (%) b thoi diorn 30 ph& so vbi ban dAu cda cac IS chuOt the, nghiOm Ban Van So vdri cac kat qua nghien ccru ten mOt s6 'obi &roc lieu ye hopt tinh Crc die enzym a-glucosidase (0,2 U/mI), re ke hoa deo, off0C biet la re to thuy canh TAP CHI DUQC HQC - 2/2017 (SO 490 NAM 57) • Nghien clixu - Ky thOt • CO hoot tinh cao han hen so vai 90% cac !obi thut vet da duvc cling bo (gia tri 1C5o nhO han tir den 897 len) [91°1 Re to, von de &rot chirng minh CO kha nang san xuet hap chat try cep CO hopt tinh sinh hoc vai ham luting Wang duvng hoec cao ban Flan so vai re tu, nhien thol gian ngen, vi dy nhu': re to Panax ginseng co kha ()sang tong hap cac saponin twang tu' vai ham luting Op 2,4 In so yeti cay ngoai tu, nhierfl Ben cal-1h dO, vai k9 thuet they canh, viec dieu chinh ham luting dinh du,Ong thich hap m6i trirong co the giup cho cac hap chat thin cep duvc tong hap va tich lOy nhieu han so vai cac k9 thuet tang sinh khoi khacI3I Nhu, vey, re ke hoa deo, dec brat la re to duvc nuoi CAN/ bang k9 thuet thily canh cO the duvc xem la nguen nguyen lieu tiem nang CO hoat tinh uc the a-glucosidase in vitro Enzym a-glucosidase ca the song la enzym cO met O, to boo niem mac ruet non tham gia x6c tac butrc cuOi ding qua trinh thOy phan carbohydrat Khi tinh bet duvc hap thy vao ca the khOng kiem soot, sy, hogt Ong cua a-glucosidase lam cho dutmg huyet tang cao qua milt den den nhieu rOi loan ca the Wang loci thao duct tir tu' nhien tir lau de duvc sir dung CO kha nang lam Wong huyet sau bira an ma it gay cac tac dyng phy Do do, chung t6i de tip dyng nghiem phap dung nap dutyng sucrose de danh gia tac dung Crc the a-glucosidase c6a cao chiet cac lopi re cay ke hoa deo ten chuet bi dai thao dubmg gay b6/i alloxan Ket qua thyt nghiem tren nhom chuet uOng thuOc dOi chi:mg acarbose da chirrig to tinh thich Ong dm nghiem phap dung nap &rang sucrose Acarbose lieu 10 mg/kg the tong duvc cho uOng truck Wong mrac dirtimg de the hien duvc tac dung giam dirong huyet a thOi diem 30 phut sau nOng uOng nu,ac du,Ong th6ng qua de [Dien thien duteng huyet nho han cO jr nghia so vai nhOm chi:mg benh Viec sir dyng cao chiet tir cac loci re, dec biet la re tu, nhien truttc uOng mitt dutmg cho they nhirng 16 chuet th6, nghiem cOng cO xu twang lam giam diking huyet so vai chi:mg benh Twang tu, vai ket qua nay, nghien cCru cua Onoagbe va CS (2010) de chi:mg minh re ke hoa deo co tiem nang hp du,Ong huyet ten chuet dai thao dutmg tuen thin nghiem That vey, dei vai chuet duvc dieu tri Uri cao chiet re, luting dirtmg giam tir 219,1 74,6 mg/100 ml tong b cao chiet la luting throng Om nhe kr 211,5 131,2 mg/100 ml 151 Kha nang hp glucose huyet in vivo dm cao chiet re to they canh kh6ng twang dung so vai thi nghiem in vitro CO the met so nguyen nhan N.Ong de Wong huyet ten chi.* bi benh dai thao dung chiu anh hirOng obi nhieu yeu to nhu, hopt Ong cua enzym a-glucosidase, qua trinh bai tiet insulin cue to boo 13 hay tit-1h nhay cam cOa m6 ngopi bien vai insulin, Ngoai ra, ham luting nhirng hap chat c6 hopt tinh tich luy re rat CO the phy thuec vao de tuOi cay ngoai tu, nhien co thdi gian song lau han so vai hai 1'30 re to (30 tu6i) Do d6, CO the re ty' nhien de tich luny nhu'ng nhom hap chat khac so vai re to they canh mang hopt tinh lam tang tiet insulin W., to boo i3 g6p phan lam Om clang ke glucose huyet so vai re to they canh Ket luen Ket qua thin nghiem in vitro cho they cao chi& ethanol tir cac loci re ke hoa deo, dec biet la re to thOy canh, co tac dyng Crc the enzym a-glucosidase Tuy nhien, ten m6 hinh chuet bi dai thao direrng gay Nil alloxan, tac Ong hp glucose huyet dm re to they canh can thap han so vol re tu, nhien Do dO can CO nhCrn9 nghien cCru sau han nhu, gia tang de tuOi dm re to de cai thien hieu qua dutyng huyet ten chuet nhom lam nguen nguyen lieu he tra dieu tri benh dai thao dutmg typ Tai liOu tham khao 1.Guillon S., Tresmouillaus J., Pati P K., Gantet P (2008), "Bioactive molecules and medicinal plants, Chapter 14: Hairy root: a powerful tool for plant biotechnological advances"; Bio Med Central, 9, (3), pp 271-275 Takafumi Y., Tsutomu F (1987), "Saponin production by cultures of Panax ginseng transformed with A rhizogenes", Plant Cell Reports, 6, (6), pp 449-453 Briskin D P (2000), "Medicinal plants 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Gu X, Xu Q, Huang X (2011), "Antioxidant activities, a-glucosidase inhibitory effect in vitro and antihyperglycemic of Trapa acomis shell in alloxan-induced diabetic rats", J of Medicinal Plants Research, 5, (31), pp 6805-6812 10 Raad A K (2013)., "In-vitro a-glucosidase inhibition and antioxidant activity of methanolic extract of Centaurea calcitrapa from Iraq", American J of Essential Oils and Natural Products, 1, (1), pp 122-125 (Ngay nhan bai: 19/11/2016 - Ngay phan bian: 11/12/2016 - Ngay duyat clang: 08/02/2017) TAP CHI DUQC HQC - 2/2017 (SO 490 NAM 57) 25 ... rhizogenes", Plant Cell Reports, 6, ( 6), pp 449-453 Briskin D P (200 0), "Medicinal plants phytomedicines Linking plant biochemistry and physiology to human health", Plant Physiology, 124, ( 2), pp 507-514... (201 1), "Antioxidant activities, a -glucosidase inhibitory effect in vitro and antihyperglycemic of Trapa acomis shell in alloxan-induced diabetic rats", J of Medicinal Plants Research, 5, (3 1), ... tiern alloxan Alloxan duvc pha dung dich NaC1 0,9% ve duvc tiern vac) tinh mach duel chuet vai lieu 60 mg/kg (tinh theo alloxan tetrahydrat) Bon mud tam gia sau tiem alloxan, tien hanh dinh luvng
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