T R U O N G DAI H O C DONG THAP Tap chf Khoa hoc so 25 (04-2017)
KHAO SAT HO^T TINH CHONG OXY HOA IN VITRO CAO CHIET RAU N G 6 {Enhydra fiuctuans Lour., Asteraceae) TREN HAI THU* NGHIEM DPPH VA MDA
• Huynh Anh Duy'*', Lam Thj Ngoc Giau'"', Bui My Unh'"' Tom t i t
Rau ngd duac dirng nhiiu trong y hgc ddn gian, nhieng cd il nghien ctm vi tdc dung sinh hpc cua lodi cay ndy. Trong bdi bdo ndy, cao chiet cdn 40% cua Rau ngd budc ddu dttac khdo sdt tdc diing chdng oxy hda in vitro tren hai mo hinh thu nghiem DPPH vd MDA, thu dieac cdc gid tri IC^g ldn luat Id 1706,52 \xg/mlvd 1081,17 \xg/ml. Ket qud cho thdy, lodi cdy ndy Id nguyen lieu dim ndng trong viec sudung ldm thuoc chong oxy hoa, dinh hiedng tdc d\ing bdo ve gan.
Tir khoa: Rau ngd, hogt tinh chong oxy hda, thur nghiem DPPH, thu nghiem MDA.
1. D^t van de nghien ciiu, dupe luu tgi Bp mon Dupe lipu, Theo y hpc dan gian, cay Rau ngo dupe sii Khoa Dupe, Dgi hoc Y Duac Thanh pho Ho dung voi nhieu cong dung tri benh nhu cac truong
hop an uong khong tieu, bang huyet, tho huyet, h6 trp gan. Trong nghien ciiu nay, chiing toi khao sat hoat tinh chong oxy hoa huong tac dimg bao ve gan vi day la c6ng dung noi bat ciia dupe lieu thuang dupe nguoi dan sii dyng,
Nghien eiiu buoc dau khao sat hogt tinh chong oxy hoa in vitro cao chiet Rau ngo tren 2 thli nghi?m de xac dinh kha nang d|p tit goc tu do (thli nghi$m DPPH) va kha nang lic che peroxy hoa lipid (thii nghipm MDA).
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2. Nguyen li^u, hoa chat va phucmg phap nghien cuu
2.L Nguyen lieu
Cay Rau ng6 thu hai tai xaHung Hpi, VTnh Lpi, B^c Lieu vao thang 12/2013 MSu dupe dinh danh theo tai lieu tham khao [1]. Sau do, mlu dupe phoi kho, xay tho diing cho qua trinh
" Tnrong Dai hoc Can Thcr.
""••Tniang Cao ding Yt€ B?c Lieu
("•> Trudng Dai hpc Y Duoc Thanh ph6 Ho Chi Minh
Chi Minh.
2.2. Hoa chat
Acid ascorbic (Merck, Dure), silymarin (Sigma Co.Ltd, My), trolox (Calbiochem Ltd.
Co, My), dpm phosphat (pH=^7,2), natri clorid 0,9%, acid thiobarbituric (TBA) (Nacalai Tesque, Nh|t), dpm tris-HCl (Merck, Diic), malonyl dialdehyd ^VIDA) (Merck, Diic), acid tricloacetic (Trung Quoc), DPPH (Sigma Aldrich).
2.3. Phmmg phap nghien cmi Chuan bi djch chiet
Bpt tho duac lipu dupe tien hanh ngam kiet voi con 40% theo ti Ip 1:10, dich chi^ dupe co each thiiy din eao d|c. Cao d^c dung de tien hanh cac thli nghiem chong oxy hoa in vitro. Ckc thu nghipm dupe tien hanh tai Trung tam Sam va Dupe lipu Thanh pho Ho Chi Minh
Thu nghiem xac dinh kha nang dlip tat goc ty do (thu nghiem DPPH) [2], [4]
Nguyen tdc
Ckc chk nghien ciiu co tac dung chong oxy hoa theo co che dap tit goc tu do se lam giam mau cua I, l-diphenyI-2-picrylhydrazyl (DPPH).
Xae dinh kha nang nay bang each do quang o buoc song CO dp hap thu cue dai tai X= 515 nm
Phieang phdp
Lay 0,5 ml mau thu 6 cac nong dp khao sat dupe cho phan iing voi dong lupng dung dich DPPH 0,8 mM pha trong MeOH Hon hap sau khi pha dupe dh trong t6i va o nhiet dp phong 30 phiit. Do quang o buoc song ^=515 nm. Acid 89
TRUdNG DAI HQC DONG THAP Tgp chi Khoa hpc sg 25J04-2017), ascorbic (Merck, Germany) dupe su d^I^ lam
chat doi ehieu.
Thu nghidm xac dinh kha nang uc che peroxy hoa lipid (thu nghiem MDA) [3], [4]
Nguyin tdc
Xac djnh kha nang lic ehe peroxy hoa lipid cua mlu nghien eiiu qua viec xae dinh ham lupng malonyl dialdehyd (MDA). MDA la san phim dupe sinh ra trong qua trinh peroxy hoa lipid mang te bao, khi cho phan iing voi acid tWobarbituric, mot phan tii MDA phan iing voi hai phan tii acid thiobarbituric tao thanh phiic hpp trimethin (mau hdng) c6 dinh hap thu cyc dai a buoc song X,=532 nm. Do cudng dp mau eiia phue hpp suy ra ham lupng MDA co trong mlu Phan iing nay thuc hien trong moi truang pH 2-3, a nhipt do 100°C trong vong 15 phiit.
Phuangphdp
Lay 0,1 ml mau thu o cac nong do thii nghipm dupe cho phan iing v6i 0,5 ml dich dong the nSo va them dem phosphat 50 mM vua du 2 ml. U hon hpp phan irng o 3 7''C trong 15 phiit va dimg phan iing bang 1 ml acid tricloacetic 10%.
Sau khi Iy tam lay dich trong cho phan iing vdi 1 ml acid thiobarbituric 0,8% trong 15 phiit 6 nhipt dp 100°C.Lamlanhvatilnhanh do quang 6 budc song X =532 nm. Trolox duoc sii dyng lam chat d6i chi8u.
Tinh toan ket qua
Phan tram % boat tinh eh6ng oxy hoa (HTCO) dupe tinh theo eong thiie
ODc
ODc M|t dp quang cua mlu doi ehung (dung moi).
ODt: Mat do quang cua mlu thii.
Cdch tinh gid tri IC^^
Ve d6 thi bieu diln ty le % Idia nang dap tat goc tu do theo nong d$ Ichao sat ciia chit cin thli nghidm blng phan mem Excel. Tir d6 thi, npi suy ra gia tri ndng dp dap tit g6c tv dp hay uc ch6 peroxy hoa lipid ICj„ blng phuong trinh hoi quy tuyen tinh c6 dang y = ax + b va thS y = 50 vao de suy ra IC
Cac so lieu ket qua thu nghiem dupe bilu thi blng tri so trung binh cua 3 lln do.
3. Ket qua va thao lu^n 3.1. Chuin bj cao thv nghifm Tiln hanh theo quy trinh, til 1640 g bpt dupe li?u Rau ngo thu dupe 433,8 g cao dac vcri hi$u suat chilt la 26,4% va dp am cao la 14,8%.
3.2. Ket qua thu nghidm ho^t tinh ching oxy hoa in vitro
3.2.1. ThCc nghiem DPPH
Hoat tinh ch6ng oxy hoa tren mo hinh djp tit goc tir do DPPH eiia cap chiet Rau ngo va chit d6i chilu acid ascorbic dupe trinh bay a Bang 1 va Bang 2.
B ^ g 1. Kit qud kfaio sat ho^t tmh ch^ng oxy hoa in vitro ciia cao Rau ngS bSng thi^ Qghi^m DPPH
N6ngd$
Doicbmig 2000 1000 750 500 250 100
OD CRirng binh)
0,7560 0,2830 0,5855 0,6450 0.7005 0,7405 0,7790
%
HTCO 62,57 22,55 14,68 7,34 2,05 -3,04
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DO 1000
R' = 0.98S4 ^
isoD :oa
Hinh 2. Do thi tuong quan giihi nong d$ vi hoat tinb chong oxy hda ciia cao chiet Rau ngS
tr£n thu nghifm DPPH Nhgn xet
Trong thli nghiem DPPH, DPPH co m4u tim nho vao 4i$n tii N chua ghep doi, nhung sau khi phan ling vol oxy nguyen til ciia chit dap tit gle tu do se bi giam mau tim. Hoat tinh chpng oxy 90
TOOONG D A I HOC D 6 N G T H A P Tgp chl Khoa hpc s5 25 (04-2017)
hoa cua cao Rau ngo tfal hien cr vipc lam giam mau DPPH d i n dka gjam dp tuf) thu a buoc soa^
5}Snm.Bang 1 cho th4y boat tinh chat oxy hoa ciia cao Rau ngo tang theo nong dp khao sat va d ^ boat tinh d i d n g oxy hoa cao nh4t a nong dp 2000 pg/ml la 62,57%.
Tinh todn kit qud:
+ Phucmg trinh hoi qui t i : ^ ^ tinh cua acid ascoibic: y = 0,9638x + 7,593 (R? = 0,9812).
+ Phuong trinh hoi qui tuyen tinh cua cao Rau ng6: y = 0,0344x - 8,7044 (R^ = 0,9884).
Biag 2. Ket qaa kbio sat ho^t tmh chong oxy boa in
•ritro CDS chit dSi diiai add ascorbic bing tfav Dalian DPPH
2000 ^g/ml la 67,47%.
Bang 3. K^ qua khio sit ho^t tnih ch6iig oxy hda m viCrtf caa cao Rau DgS bing Uiff i^[hi&n MDA
B & d u i i g 1 0,5 0.25 0,1 0.05 0,01
176,13 88,065 44,032 17,613 8,806 1,761
O D ClksagUaii)
0,913 0,038 0,053 0 3 5 8 0,655 0,760 0,862
HTCO
95,89 94,19 60,S4 28,31 16,76 5,59
Gia tri IC^^ dupe tinh bing phuong trinh hoi qui ti^en tinh sir dung phan mem Excel.
+ Gia tri I C ^ cua acid ascorbic la: I C ^ = 44pg/inL
+ Gia tri ICj, cua cao Rau ngo la : ICj, = 1706^2 pg/ml.
3 3 . T h v nghifm MDA
Hoat tinh chong oxy hoa tren phuong p h ^ xac dinh san pham qua trinh peroxy boa Upid cua cao Rau ngo va chit doi chieu trolox duoc trinh bay o Bang 3 va Bang 4.
Nhdn xet
Trong thii nghiem nay, MDA la chat dugc sinh ra trong qua trinh peroxy boa lipid mang te bao, Idii phan ung voi thuoc tiiu acid tbiobaibitunc tao ra phuc hop c6 mau hong. Hoat tinh chong 0?^ hoa ciia cao Rau ngo the hien qua viec lam giam mau eua phuc hcrp nay do lam giam luong MDA CO trong m l u d i n dkn lam giam dp hdp thu 6 buoc song 532 nm. Bang 3 cho thay kha nang ciiong oxy hoa cua cao chiet Rau ngo 6 nong do
Nong do Qig/ml)
£>6i cluing 2000 1000 500 250 100
OD (IViing binb)
0,332 0,108 0,166 0 J 4 O 0,317 0,361
H T C O %
67,47 50.15 27,86 4,67 -8,73
Hhih 3. Bo thi tnvng qoan giva nong d$ vi hoft tinh chong oxy hoa caa cao chiet Ran ngo
t m i Itadr n^iian MDA Bang 4. K ^ qua Uiao sat boat tmb didng oxy boa in
vitro cua Hmhc d6i c h i ^ tnitox bing thv n ^ « n MDA Nong d9
(mM) 10 5 1 0,5 0,1
OD ( T n i n g binb)
0,124 0,203 0,376 0,445 0,529
HTCO%
77.70 63,49 32,37 19,96 4,86 Tinh todn Mt qud:
+ Phuong trinh logarithm tuyen tinh chat doi chieu trolox:
y = 16,2671n(x) + 36,695 (R^ = 0,9746).
+ Phuong trinh logarithm tuyen tinh ciia cao Rau ngo:
y = 26,6187In(x) -135,9530 (R^ = 0,9821).
Gia tri ICj^ duoc tinh bang phuong trinh phan tich hoi qui tuyen tinh eiia duong cong dap ling li^u tucmg ung, sii dung phSn mem Excel.
9 1
TRUflNG DAI HOC EXSNG THAP T^p chi Khoa hpc so 25 (04-2017) cua trolox la ICj, = 562,72
ciia cao Rau ngo la IC^^ = + Gia tri IC.
^g/ml.
+ Gia trj IC, 1081,17 (ig/mi.
4. Ket luan
Nghien ciiu buoc diu sang lpc hpjt tinh pxy hpa in vitro cao c6n 40% Rau ngo tren hai mp hinh thli nghiem chp gia tri ICj^ lln lugt la 1706,52 ng/ml (thii nghi?m DPPH) va 1081,17 Hg/ml (thli nghi$m MDA).
Ket qua chp thay Rau ngo co hpat tinh chpng pxy hoa in vitro mac du thap hem sp vpi hai chit d6i ehieu la acid ascprbie va trolox.
Tuy nhien, day la mpt loai rau an nen Rau ngo vin CO kha nang gop phin b6 sung nhung chat chong oxy hoa chp eo the. Ngpai ra, nghiSn cihi nay bude diu dinh huong cho nhttng khao sat tiep thep vk Rau ngp tren nhttng mo hinh khac, gop phan chiing minh tac dung dupe ly ciia dupe li?u nay. /.
Tai lifu tham Idiao
[1]. Vo Van Chi (2012), TirdiSn cdy thudc Viet Nam - tdp 2, NXB Y hpe. Ha Npi.
[2]. Pramod, K. S. et al (2012), "Antipxidant activity ot Enhydra fluctuans Lpur. aerial parts", Jaumal of Phytotherapy and Pharmacology, 1 (2), p. 23-34.
[3]. Stroev, E. A. & Makarova, V. G. (1989), Determination of lipid peroxidation rate in tissue homogenate laboratory. Manual in Biochemistry, MPSCPW, p. 243-256.
[4]. Vien Dupe Lieu (2006), Phuangphap nghien cim tdc dung duac ly cua thuoc lii thdo duac, NXB Khpa hpc va Ky thuat Ha Npi.
INVESTIGATING IN VITRO ANTIOXIDANT ACTIVmES OF Enhydrajluctuam Lour. EXTRACT VIA DPPH AND MDA ASSAY
Summary
Enhydra fluctuans Lour, is widely used in folk medicine, but there is still little research found on its biological effects. In this paper, the in vitro antioxidant activity with 40% ethanol extract from Enhydra ftuctuans Lour, is determined by DPPH and MDA assay. The ICjg values are 1706.52 pg/ml and 1081.17 ^ig/ml, respectively. The results show Hiat Enhydra fluctuans Lour, is the potential to be used as an antioxidant and hepatoprotective bioactive-guided medicine.
Keywords'. Enhydra fiuctuans Lour., antioxidant activity, DPPH assay, MDA assay.
Ngdy nhgn bdi: 10/11/2016; Ngdy nhdn lai: 20/12/2016; Ngdy duyet dang: 27/01'2017.
