Khảo sát khả năng gây ức chế enzym α glucosidase in vitro và hạ glucose huyết in vivo của rễ tơ ké hoa đào (urena lobata l )

• Nghi8n ctim - Ky thuOt

Khao sat kha ding urc che enzym a-glucosidase in vitro
va ha 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:

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 2 treatment.
Keywords: Urena lobata L., hairy root, hydroponic, a-glucosidase, blood glucose.

DOt van d4
Re to duvc tao ra do stir chuyen gen bang cach sir
dyng ha 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 khi A. rhizogenes xam nhiem vao
m6, re hinh thanh 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 ra rang trong qua trinh nuoi cay, re to co
the tao ra 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 do co nhieu
kru diem han cac hinh thirc nuoi cay khac nhu' khd nang
tang truang nhanh va skr de clang trong 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 trong gan va mau [4] . Trong
khi dO ten the giai da cO nhieu nghien cCru chirng minh
hiau qua ho tra dieu trj banh dai thao dverng typ 2 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 nay can cho they re thvong cO hoat tinh tat bon cac
be phan can dai hoac toan cay. Vi vay, nghien cihr nay
dkrac thkrc hian nham chirng minh re to cam Crng tCr cay
ke hoa dao cung CO tiem nang trong viac hO to, dieu trj
22

dai thdo duteng typ 2 thong qua kha nang erc the host
Tang cOa enzym bien &rang carbohydrat a-glucosidase
in vitro va ha 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 do Vien cung cap. ChuOt duvc nuoi
On Binh ey nhiat de ph6ng 3 — 4 ngay Wok khi 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 con ke hoa dao nuoi cay in vitro va bang they
canh sau 15 ngay Wei cluvc tien hanh xam nhiem tao
re to bang vi khuan Agrobacterium rhizogenes ATTC
15834. Re tao thanh 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 ngay 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 thanh bOt kho. Cao chiet ethanol cac
loai re duct dieu che bang k9 thuat chiet ngam dem
(maceration): ngam bOt cay trong 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 trong 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 trong 20 phut, them 40 pl co, chat pNPG
(5 mM), tiep tuc 6 trong 20 phut. arng phan Crng bang
130 pl dung dich Na2CO3 (0,2 M). Do mat do 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& 2 mg/ml, nong de acarbose 20 mg/ml. Cac neng
dO nay duct giarin den mot nira theo thir tkr tang gieng
to ten xuang dip&
Me hinh gay dal thao dtrimg typ 2 ten chuOt
nhat bang alloxan
ChuOt duct cho nhin ddi qua dem tuft khi tien
hanh tiern alloxan. Alloxan duvc pha trong 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 khi 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 trong 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
4 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 1 (16 chOng benh, n = 7): ming nu,ac &rang lieu
4 g/kg the hong.
L6 2 (10 dOi chieu, n = 7): uong acarbose lieu
10 mg/kg the trong ngay trkrac khi uong nkrac &rang.
L6 3 (n = 7): uong cao ethanol re t& nhien lieu
400 mg/kg the trong ngay truck khi ming rxrac &rang.
LO 4 (n = 8): wing cao ethanol re to in vitro lieu
400 mg/kg the tang ngay trkrac khi uong ntrac du'ang.
Lo 5 (n = 7): uong cao ethanol re to they canh lieu
400 mg/kg the trong ngay tnybc khi uong nkrirc duteng.
Tien hanh lay mau to tinh mach dual chuOt vao cac
trial diem 30 va 60 phut sau khi cho uong nkrac dtrang
de xac dinh nOng de glucose huyet trong huyet Krung
bang phu,ang phap enzym mau (thuOc thir glucose
oxydase). So sanh min dq ha du'ang huyet a cac thai
diem 30 va 60 phut sau khi 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/ 4 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, khi 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 trong tu, nhien va re to thuy canh 6, ding nOng dO.
bang 2 va 3. Kat qua cho thay: trong 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 trong bang 4 va stn khac het nay co 9 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 khi 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 4 et cac nong do 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 do 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 nay yen con cao khi
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 9 nghia 5%
Khoo sat tic ding ha glucose huyet in vivo bAng

nghiam phi') dung nap dutong sucrose
Kat qua thin nghiOrn cho they 6 nhOm chirrig bOnh,
nong dO glucose huyet 6, then diem 30 phOt sau khi ueng
raft dveyng cua nhOm chuOt dung thuOc don chang
acarbose thap han co 9 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 trong hinh 1.
Kat qua cho they, 16 chuOt durcyc cho u6ng cao chiet
re to, nhien CO dO bier, thien Wong huyet gram va khac
CO 9 nghia thong ke so vdri nham chCrng bOnh khang
dieu tri (P < 0,05). Them vao do, khi so sanh v6i16 chuOt
CI& chirng du,qc u6ng acarbose thi kung they c0 stn
khdc biet 9 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 2 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 trong 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 trong 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 trong ca the song la enzym
cO met O, to boo niem mac ruet non tham gia x6c tac
butrc cuOi ding trong 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 trong ca the.

Wang loci thao duct tir tu' nhien tir lau de duvc sir
dung do CO kha nang lam ha 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 ngay truck khi
Wong mrac dirtimg de the hien duvc tac dung giam
dirong huyet a thOi diem 30 phut sau khi
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 ngay truttc khi 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 trong 6 tuen
thin nghiem. That vey, dei vai chuet duvc dieu tri Uri
cao chiet re, luting dirtmg giam tir 219,1 con 74,6
mg/100 ml tong khi b cao chiet la luting throng Om
nhe kr 211,5 con 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 do 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 trong re rat CO the
phy thuec vao de tuOi khi cay ngoai tu, nhien co thdi gian
song lau han so vai hai 1'30 re to (30 ngay 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 ha dutyng huyet ten chuet nhom lam
nguen nguyen lieu trong he tra dieu tri benh dai thao
dutmg typ 2.

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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